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Epidemiology of Bovine Anaplasmosis and Babesiosis in Commercial Dairy Farms of Puerto Rico

Permanent Link: http://ufdc.ufl.edu/UFE0021538/00001

Material Information

Title: Epidemiology of Bovine Anaplasmosis and Babesiosis in Commercial Dairy Farms of Puerto Rico
Physical Description: 1 online resource (260 p.)
Language: english
Creator: Urdaz-Rodriguez, Jose Hugo
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: anaplasma, babesia, boophilus, bovis, dairy, ecological, epidemiology, factors, farms, management, marginale, microplus, puerto, rhipicephalus, rico, seroprevalence
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The objectives were (1) to determine seroprevalence of Anaplasma marginale and Babesia bovis in adult lactating dairy cattle (2) to assess associations of geographical and farm management factors with the seroprevalence of A. marginale and B. bovis in commercial dairy farms, and (3) to identify ecologic factors associated with the abundance and spatial distribution of Rhipicephalus (Boophilus) microplus larvae in Puerto Rico (PR). Serum samples were obtained from 2,414 adult lactating cattle from 76 commercial dairy farms between August 2005 and December 2006. Ninety-six sites were sampled for tick larvae during the dry season (March 4-18, 2007) and the rainy season (August 13-26, 2007). Data on farm management factors were obtained by an interviewer-administered questionnaire. Farm seroprevalence for A. marginale ranged from 2.8 to 100% with an overall animal seroprevalence of 27.4%. Risk factors significantly associated with A. marginale included pasture grazing as the main source to feed cattle (OR= 6.4, 95% CI=1.3-32.7), observed monkeys on the premises (OR= 13.7, 95% CI=1.4-137.2), use of 11% permethrin (Atroban?; OR= 13.6, 95% CI=1.9-97.7), farmers who attended an acaricide certification program (OR= 0.17, 95% CI=0.04-0.72), and lack of fly control methods (OR= 5.7, 95% CI=1.3-24.5). Farm seroprevalence for B. bovis ranged from 0 to 51.4% with an overall animal seroprevalence of 25.5%. Risk factors significantly associated with B. bovis included farms located in the north coastal region (OR= 0.21, 95% CI=0.05-0.86), dairy farms with calf raising facilities (OR= 16.0, 95% CI=3.0-86.3), having more than four neighbors with cattle (OR= 16.9, 95% CI=1.6-175.8), same producer owing more than one farm (OR= 7.3, 95% CI=1.7-31.8), and use of government services to apply amitraz on cattle (OR= 5.5, 95% CI=1.5-20.0). Moran's I indicated that the spatial pattern of A. marginale and B. bovis seroprevalence is neither clustered nor dispersed. Risk factors significantly associated with the presence of R.(Boophilus) microplus larvae in PR during the dry season included average wind speed of 2.6 to 10.0 mph (OR= 0.07, 95% CI=0.01-0.63), more than 25% bushes and shrubs on the site (OR= 10.6, 95% CI=1.6-71.1), and presence of cattle on the site (OR= 25.5, 95% CI=3.4-188.2).
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jose Hugo Urdaz-Rodriguez.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Melendez, Pedro.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021538:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021538/00001

Material Information

Title: Epidemiology of Bovine Anaplasmosis and Babesiosis in Commercial Dairy Farms of Puerto Rico
Physical Description: 1 online resource (260 p.)
Language: english
Creator: Urdaz-Rodriguez, Jose Hugo
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: anaplasma, babesia, boophilus, bovis, dairy, ecological, epidemiology, factors, farms, management, marginale, microplus, puerto, rhipicephalus, rico, seroprevalence
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The objectives were (1) to determine seroprevalence of Anaplasma marginale and Babesia bovis in adult lactating dairy cattle (2) to assess associations of geographical and farm management factors with the seroprevalence of A. marginale and B. bovis in commercial dairy farms, and (3) to identify ecologic factors associated with the abundance and spatial distribution of Rhipicephalus (Boophilus) microplus larvae in Puerto Rico (PR). Serum samples were obtained from 2,414 adult lactating cattle from 76 commercial dairy farms between August 2005 and December 2006. Ninety-six sites were sampled for tick larvae during the dry season (March 4-18, 2007) and the rainy season (August 13-26, 2007). Data on farm management factors were obtained by an interviewer-administered questionnaire. Farm seroprevalence for A. marginale ranged from 2.8 to 100% with an overall animal seroprevalence of 27.4%. Risk factors significantly associated with A. marginale included pasture grazing as the main source to feed cattle (OR= 6.4, 95% CI=1.3-32.7), observed monkeys on the premises (OR= 13.7, 95% CI=1.4-137.2), use of 11% permethrin (Atroban?; OR= 13.6, 95% CI=1.9-97.7), farmers who attended an acaricide certification program (OR= 0.17, 95% CI=0.04-0.72), and lack of fly control methods (OR= 5.7, 95% CI=1.3-24.5). Farm seroprevalence for B. bovis ranged from 0 to 51.4% with an overall animal seroprevalence of 25.5%. Risk factors significantly associated with B. bovis included farms located in the north coastal region (OR= 0.21, 95% CI=0.05-0.86), dairy farms with calf raising facilities (OR= 16.0, 95% CI=3.0-86.3), having more than four neighbors with cattle (OR= 16.9, 95% CI=1.6-175.8), same producer owing more than one farm (OR= 7.3, 95% CI=1.7-31.8), and use of government services to apply amitraz on cattle (OR= 5.5, 95% CI=1.5-20.0). Moran's I indicated that the spatial pattern of A. marginale and B. bovis seroprevalence is neither clustered nor dispersed. Risk factors significantly associated with the presence of R.(Boophilus) microplus larvae in PR during the dry season included average wind speed of 2.6 to 10.0 mph (OR= 0.07, 95% CI=0.01-0.63), more than 25% bushes and shrubs on the site (OR= 10.6, 95% CI=1.6-71.1), and presence of cattle on the site (OR= 25.5, 95% CI=3.4-188.2).
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jose Hugo Urdaz-Rodriguez.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Melendez, Pedro.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021538:00001


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1 EPIDEMIOLOGY OF BOVINE ANAPLASMOS IS AND BABESIOSIS IN COMMERCIAL DAIRY FARMS OF PUERTO RICO By JOS HUGO URDAZ RODRGUEZ A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Jos Hugo Urdaz Rodrguez

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3 This dissertation is dedicated to my beautif ul family. My mother and grandmother who listened, supported me through each day of all thes e years of intense work, and always believed in me. My father who gave me courage and pr actical solutions to th e many obstacles I found during this study. To my sisters, Vannesa and Lo rraine, and their husbands, who always looked for bright alternatives to solve my problems, and especially to my w onderful nephew and niece, Lucas Fabin and Victoria Sofa, and to God who guided me throughout all the difficult challenges I faced during this stage of my career. The world is made from the hands of those who have the courage to dream and who are willing to take risks of living these dreams. Paulo Coehlo

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4 ACKNOWLEDGMENTS I would first like to thank my supervisory committee members for their support during my Ph.D. program, Dr. Pedro Melendez, Dr. Owen R ae, Dr. Art Donovan, Dr. Michael Binford, and particularly Dr. Rick Alleman, for hi s strong moral support and expertise in Anaplasma marginale and Dr. Geoff Fosgate, for his guidan ce throughout my research program. Dr. Fosgates thorough epidemiological and statistical approaches, work ethics, and emphasis on clear scientific communication and proper experi mentation have been wonderful examples for me. I would also like to thank Dr. John Fernndez Van Cleve, Dean and Director of the School of Agriculture at the University of Puerto Rico, Mayagez Campus and Dr. Jos R. Latorre, Director of the Department of Animal Scien ce at the School of Agriculture for providing the facilities to carry out the fieldwork in Puerto Ri co. In addition, I want to thank the staff at the Agriculture Experiment Station especially Ma ra de los ngeles Mingue la, Lynette Feliciano, and Silvia Rivera for their unconditional help and support. From the Fondo para el Fomento de la Industria Lechera de Puerto Ri co, I want to thank Luis Tat Cordero, Jos Bentez, and Juan Pedr for their full support during the preparatory stages of the study. Most of all, I wish to tha nk all the dairy farmers w ho participated in this study, without their cooperation this study would not have been possible. I would like to thank the enti re staff from the State Veteri nary Diagnostic Laboratory Dr. Gabriel Gonzlez Caldern and especially Mr. Davi d Vega, the laboratory supervisor, Dr. Hctor Daz Collazo, the state veterinarian, and Angela for kindly allowing me to use their laboratory facilities and equipment to perform the serological analyses for these studies. From the Department of Entomology at the Univ ersity of Florida, I want to thank Dr, Jerry Butler, Dr. Phil Kaufman and Lois Woods for instructing me and providing the necessary

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5 materials and equipment to process tick larvae for identification. In addition, I want to thank Dr. Lance Durden from Georgia Southern University for his help in the confirmation of tick larvae identifications. I would also like to express my sincere appreciation to faculty at Texas A&M, Department of Entomology and Pathobiology. I want to thank Dr. Pete Teel for his excellent recommendations and insights during the deve lopment of the tick co llection study, Dr. Gale Wagner for his support and interest in Puerto Rico, and particularly Dr. Surya Waghela for assisting with serological testing and expertise in Babesia spp. I want to thank Graeme Cumm ing for his patience and his time during the landscape ecology course to help me develop the GIS sampling methodology for the tick survey. From the USDA-APHIS-VS in Puerto Rico, I wa nt to give special thanks to Dr. Carlos Soto, area epidemiology officer, for providing insights and docume ntation about the status of bovine anaplasmosis, babesiosis, and th e tropical cattle tick on the island. From Puerto Rico, I also want to thank Dr. Jos Torrado and Dr. Anabelle Slico for sharing their field experience with me. Dr. S lico provided informati on concerning about the funding opportunities available from the Colegio de Mdicos Vete rinarios de Puerto Rico. Students were also an important component of my experience in this program. I want to thank Mario Rosario, master student at the University of Puerto Rico, School of Agricultural Sciences and Terry Woodland, third year veterinary student at the University of Florida, College of Veterinary Medicine for helping in the collection and processing of data. I also want to mention my fellow graduate students Pablo, Jos Alfr edo, and Katie because they always knew that I would be ab le to finish this dissertation.

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6 I would also want to thank all the administrative, laboratory a nd computer services staff at the University of Florida, College of Veterinary Medicine because withou t them this dissertation would not have been possible. They are Judy Chastain, Sally OConne ll, Frances Edwards, Heather Sorenson, Elizabeth Pritchard, Stepha nie Stein, Megan Bible, Bobby Lee, and Max Donner. I want to thank all the people I contacted for their personal expertise in the field of bovine anaplasmosis, babesiosis, a nd the tropical cattle tick. Special thanks go to a little angel who al ways gave me a hug and made me laugh during the most difficult moments and always saw the light for me, Inez Ellerbe, I will always remembered you. I also want to thank my cousin Alexis B io Zaragoza and my dad for helping me during the tick collections in Puerto Rico. Finally, my dissertation would not have been possible without the unconditional love, support, and understanding from my family at every step of the way. Their efforts and faith in me have meant more than they will ever realize. This material is based on research supported by USDA/CSREES Grant No. 2005-3413518020 in Tropical/Subtropical Agriculure Researc h, Fondo para el Fomento de la Industria Lechera de Puerto Rico, and the Colegio de Mdicos Veterinarios de Puerto Rico.

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7 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ........11 LIST OF FIGURES................................................................................................................ .......13 ABSTRACT....................................................................................................................... ............15 CHAPTER 1 INTRODUCTION..................................................................................................................17 2 LITERATURE REVIEW.......................................................................................................19 Epidemiology of Bovine Anaplasmosis.................................................................................19 Etiologic Agent................................................................................................................19 Clinical Presentation and Pathology of the Disease........................................................19 Antigenic Variation.........................................................................................................20 Antigenic Diversity and Distribution of A. marginale ....................................................23 Host Occurrence and Breed Resistance...........................................................................25 Life Cycle Stages and Development...............................................................................25 Methods of Transmission................................................................................................28 Immune Response and Immunity....................................................................................37 Endemic Stability............................................................................................................38 Treatment and Prevention................................................................................................39 Epidemiology of Bovine Babesiosis.......................................................................................42 Etiologic Agent................................................................................................................42 Clinical Presentation and Pathology of the Disease........................................................43 Antigenic Variation.........................................................................................................45 Antigenic Diversity and Distribution of B. bovis and B. bigemina .................................46 Host Occurrence and Breed Resistance...........................................................................47 Life Cycle Stages and Development...............................................................................47 Methods of Transmission................................................................................................50 Immune Response and Immunity....................................................................................51 Endemic Stability............................................................................................................52 Treatment and Prevention................................................................................................53 Epidemiology of the Tropical Cattle Tick..............................................................................55 Etiologic Agent................................................................................................................55 Clinical Presentation and Pathology................................................................................56 Antigenic Diversity and Distribution of Rhipicephalus (Boophilus) microplus .............57 Host Occurrence and Breed Resistance...........................................................................58 Life Cycle Stages and Development...............................................................................60 Parasitic phase..........................................................................................................60 Non-parasitic phase..................................................................................................61

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8 Immune Response and Immunity....................................................................................64 Endemic Stability............................................................................................................66 Treatment and Prevention................................................................................................66 Economic Impact of Rhipicephalus (Boophilus) microplus Bovine Anaplasmosis, and Bovine Babesiosis.............................................................................................................. .70 Worldwide...................................................................................................................... .70 Caribbean (except Puerto Rico).......................................................................................71 Puerto Rico.................................................................................................................... ..71 History of the tick er adication program in Puerto Rico-reports on Rhipicephalus (Boophilus) microplus bovine anaplasmosis, and bovine babesiosis..............................................................................................................72 Puerto Rico and the importa nce of the dairy industry..............................................76 Importance of the Bovine Anaplasmosis and Babesiosis-Tick-Cattle -Ecological System and Its Implications in Epidemiological Studies.................................................................84 Defining the Bovine Anaplasmosis and Ba besiosis-Tick-Cattle-Ecological System.....85 Key Factors of the Epidemiology A. marginale and Babesia spp. ..................................86 Examples of A. marginale and Babesia spp. and Their Influence on the System...........87 Examples of Arthropod Factors a nd Their Influence on the System..............................87 Examples of Cattle Factors and Their Influence on the System.....................................88 Examples of Farm Management Factors and Their Influence on the System.................89 Examples of Environmental Factors and Their Influence on the System.......................91 The Importance of Reliable Dia gnostics for Identification of A. marginale B. bovis and B. bigemina infections.........................................................................................................91 Microscopic Detection.....................................................................................................92 Polymerase Chain Reaction-Based Techniques (PCR)...................................................93 Serological Tests.............................................................................................................94 Complement fixation test (CF).................................................................................95 Rapid card agglutination test (CAT)........................................................................95 Indirect fluorescent antibody test (IFAT).................................................................96 Enzyme-linked immunosorbent assays (ELISA).....................................................98 3 STUDY 1: SEROPREVALENCE AND MA NAGEMENT FACTORS ASSOCIATED WITH Anaplasma marginale IN COMMERCIAL DAIRY FARMS OF PUERTO RICO........................................................................................................................... ..........107 Introduction................................................................................................................... ........107 Materials and Methods.........................................................................................................109 Study Area.....................................................................................................................109 Study Design and Sample Size Methodology...............................................................111 Questionnaire Development and Administration..........................................................112 Sample Collection.........................................................................................................114 Serological Testing........................................................................................................114 Statistical Analyses........................................................................................................115 Results........................................................................................................................ ...........117 Descriptive.................................................................................................................... .117 Seroprevalence..............................................................................................................118 Risk Factors...................................................................................................................119

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9 Discussion..................................................................................................................... ........120 Conclusions.................................................................................................................... .......123 4 STUDY 2: SEROPREVALENCE AND MA NAGEMENT FACTORS ASSOCIATED WITH Babesia bovis IN COMMERCIAL DAIRY FA RMS OF PUERTO RICO.............137 Introduction................................................................................................................... ........137 Materials and Methods.........................................................................................................138 Study Area.....................................................................................................................138 Study Design and Sample Size Methodology...............................................................140 Questionnaire Development and Administration..........................................................142 Sample Collection.........................................................................................................143 Serological Testing........................................................................................................144 Statistical Analyses........................................................................................................145 Results........................................................................................................................ ...........147 Descriptive.................................................................................................................... .147 Seroprevalence..............................................................................................................148 Risk Factors...................................................................................................................149 Discussion..................................................................................................................... ........149 Conclusions.................................................................................................................... .......153 5 STUDY 3: ASSOCIATION BETWEEN ECOLOGICAL FACTORS AND THE PRESENCE OF Rhipicephalus (Boophilus) microplus LARVAE IN PUERTO RICO......166 Introduction................................................................................................................... ........166 Materials and Methods.........................................................................................................168 Study Area.....................................................................................................................168 GIS Sampling Methodology..........................................................................................170 Field Sampling...............................................................................................................173 Survey Data...................................................................................................................174 Tick Identification.........................................................................................................175 Statistical Analysis........................................................................................................175 Results........................................................................................................................ ...........176 Descriptive.................................................................................................................... .176 Risk Factors...................................................................................................................178 Discussion..................................................................................................................... ........179 Conclusions.................................................................................................................... .......182 A SURVEY FOR FARMERS OR MANAGERS IN SELECTED DAIRY FARMS.............. 195 B CUESTIONARIO OFICIAL PARA GANADERAS PARTICIPANTES EN ESTUDIO SOBRE LA EPIDEMIOLOGA DE LA AN APLASMOSIS Y BABESIOSIS BOVINA EN HATOS LECHEROS EN PUERTO RICO....................................................................207 C TICK SURVEY.....................................................................................................................220

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10 LIST OF REFERENCES.............................................................................................................227 BIOGRAPHICAL SKETCH.......................................................................................................260

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11 LIST OF TABLES Table page 3-1 Population and sample distribution of co mmercial dairy farms by climatological zone and size of the farm to estimate the seroprevalence of Anaplasma marginale in Puerto Rico from August 2005 to December 2006..........................................................125 3-2 Explanatory variables include d in the questionna ire provided to dairy owners or managers for the study of the seroprevalence of Anaplasma marginale in Puerto Rico from August 2005 to December 2006..............................................................................127 3-3 Overall animal seroprevalence and seropr evalence by climatological zone and farm size for Anaplasma marginale in commercial dairy farms of Puerto Rico from August 2005 to December 2006......................................................................................131 3-4 Crude (unadjusted) risk f actor analysis for predicti ng high herd seroprevalence ( 40%) of Anaplasma marginale in 78 commercial dairy farm s in Puerto Rico from August 2005 through December 2006.............................................................................133 3-5 Multivariable logistic regression analysis for predicting high herd seroprevalence ( 40%) of Anaplasma marginale in 78 commercial dairy farms in Puerto Rico from August 2005 through December 2006.............................................................................135 3-6 Crude (unadjusted) general linear analysis for predicting herd seroprevalence of Anaplasma marginale in 78 commercial dairy farms in Puerto Rico from August 2005 through December 2006..........................................................................................136 4-1 Population and sample distribution of co mmercial dairy farms by climatological zone and size of the farm to estimate the seroprevalence of Babesia bovis in Puerto Rico from August 2005 to December 2006.....................................................................155 4-2 Explanatory variables in cluded in the questionnaire prov ided to dairy owners or managers for the study of the seroprevalence of Babesia bovis in Puerto Rico from August 2005 to December 2006......................................................................................157 4-3 Overall animal seroprevalence and seropr evalence by climatological zone and farm size for Babesia bovis in commercial dairy farms of Puerto Rico from August 2005 to December 2006............................................................................................................161 4-4 Crude (unadjusted) risk f actor analysis for predicti ng high herd seroprevalence ( 25%) of Babesia bovis in 78 commercial dairy farms in Puerto Rico from August 2005 through December 2006..........................................................................................163 4-5 Multivariable logistic regression analysis for predicting high herd seroprevalence ( 25%) of Babesia bovis in 78 commercial dairy farms in Puerto Rico from August 2005 through December 2006..........................................................................................164

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12 4-6 Crude (unadjusted) general linear analysis for predicting herd seroprevalence of Babesia bovis in 78 commercial dairy farms in Puerto Rico from August 2005 through December 2006...................................................................................................165 5-1 Explanatory variables incl uded in the field survey to study ecological predictors of R. (Boophilus) microplus in Puerto Rico during the dr y season (March 4-18, 2007) and the wet season (August 16-23, 2007)...............................................................................188 5-2 Identification of Ixodid larv ae collected by site during the dry season (March 4-18, 2007) in Puerto Rico........................................................................................................189 5-3 Identification of Ixodid larv ae collected by site during the wet season (August 13-26, 2007) in Puerto Rico........................................................................................................190 5-4 Crude (unadjusted) risk factor analysis for predicting the presence of R. (Boophilus) microplus on 96 sites in Puerto Rico duri ng the dry and wet seasons in 2007................193 5-5 Multivariable logistic regression for predicting the presence of R. (Boophilus) microplus on 96 sites in Puerto Rico duri ng the dry and wet seasons in 2007................194

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13 LIST OF FIGURES Figure page 2-1 The 78 municipalities of PR............................................................................................103 2-2 A central mountain range extends across the interior of Puerto Rico from east to west. Another set of high alt itude terrain is located in the northeast area near the coast and corresponds to the tropical rainforest known as El Yunque.........................104 2-3 The 8 agricultural regions of PR based on the number of producers and corresponding agricultural commodities in that region...................................................105 2-4 Proposed hierarchical structure fo r the study of the epidemiology of bovine anaplasmosis and babesiosis............................................................................................106 3-1 Geographical dist ribution of the 76 commercial dairy farms ( ) sampled in the 4 climatological zones of Puerto Ri co from August 2005 to December 2006...................126 3-2 Distribution of he rd seroprevalence for A. marginale in commercial dairy farms of Puerto Rico from August 2005 to December 2006..........................................................130 3-3 Proportional distribution of an imal seroprevalence by farm for A. marginale in 78 commercial dairy farms of Puerto Rico from August 2005 to December 2006..............132 4-1 Geographical dist ribution of the 76 commercial dairy farms ( ) sampled in the 4 climatological zones of Puerto Ri co from August 2005 to December 2006...................156 4-2 Distribution of he rd seroprevalence for B. bovis in commercial dairy farms of Puerto Rico from August 2005 to December 2006.....................................................................160 4-3 Proportional distribution of an imal seroprevalence by farm for B. bovis in 78 commercial dairy farms of Puerto Rico from August 2005 to December 2006..............162 5-1 Grid polygon or fishnet of Puerto Ri co used for sample size determination and integration of environmental and animal census data......................................................183 5-2 A) Location of the 102 mete orological stations in Puerto Rico, B) Interpolation for elevation (m), C) Interpolation for averag e precipitation (mm), a nd D) Interpolation for temperature (C).........................................................................................................184 5-3 A) Number of farms per municipality, a nd B) Number of cattle per municipality. Data were obtained from the 2002 Census of Agriculture, Na tional Agriculture Statistics Service, USDA, February 2004........................................................................185 5-4 The 97 clusters based on environmen tal factors and multivariate distances...................186 5-5 Tick drag device used to samp le larvae in Puerto Rico...................................................187

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14 5-6 Final geographical distribution of the 96 ta rget sites sampled in the tick survey in Puerto Rico during the dry season (March 4-18, 2007) and the wet season (August 13-26, 2007)................................................................................................................... ..191 5-7 A) Rhipicephalus (Boophilus) microplus and B) Dermacentor (Anocentor) nitens larvae collected in Puerto Rico dur ing the dry season (March 4-18, 2007)....................192

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15 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EPIDEMIOLOGY OF BOVINE ANAPLASMOS IS AND BABESIOSIS IN COMMERCIAL DAIRY FARMS OF PUERTO RICO By Jos Hugo Urdaz Rodrguez December 2007 Chair: Pedro Melendez Major: Veterinary Medical Sciences The objectives were (1) to determine seroprevalence of Anaplasma marginale and Babesia bovis in adult lactating dairy cattle (2) to as sess associations of geographical and farm management factors with the seroprevalence of A. marginale and B. bovis in commercial dairy farms, and (3) to identify ecologic factors associ ated with the abundance and spatial distribution of Rhipicephalus (Boophilus) microplus larvae in Puerto Rico (PR). Serum samples were obtained from 2,414 adult lactating cattle from 76 commercial dairy farms between August 2005 and December 2006. Ninety-six sites were sampled for tick larvae during the dry season (March 4-18, 2007) and the rainy season (August 13-26, 2007) Data on farm management factors were obtained by an interviewer-administered que stionnaire. Farm seroprevalence for A. marginale ranged from 2.8 to 100% with an overall an imal seroprevalence of 27%. Risk factors significantly associated with A. marginale included pasture grazing as the main source to feed cattle (OR= 6.4, 95% CI=1.3-33), observed mo nkeys on the premises (OR= 14, 95% CI=1.4137), use of 11% permethrin (Atroban; OR= 14, 95% CI=1.9-98), farmers who attended an acaricide certification pr ogram (OR= 0.17, 95% CI=0.04-0.72), a nd lack of fly control methods (OR= 5.7, 95% CI=1.3-25). Farm seroprevalence for B. bovis ranged from 0 to 52% with an overall animal seroprevalence of 26%. Risk factors significantly associated with B. bovis

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16 included farms located in the north coastal region (OR= 0.21, 95% CI=0.05-0.86), dairy farms with calf raising facilities (OR= 16, 95% CI=3.0 -86), having more than four neighbors with cattle (OR= 17, 95% CI=1.6-176), same producer owing more than one farm (OR= 7.3, 95% CI=1.7-32), and use of government services to apply amitraz on cattle (OR= 5.5, 95% CI=1.520). Morans I indicated that the spatial pattern of A. marginale and B. bovis seroprevalence is neither clustered nor dispersed. Risk factors si gnificantly associated with the presence of R.(Boophilus) microplus larvae in PR during the dry seas on included average wind speed of 2.6 to 10.0 mph (OR= 0.07, 95% CI=0.01-0.63), more than 25% bushes and shrubs on the site (OR= 11, 95% CI=1.6-71), and presence of cattle on the site (OR= 26, 95% CI=3.4-188).

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17 CHAPTER 1 INTRODUCTION Today, uncontrolled tick populat ions, bovine anaplasmosis (BA) and bovine babesiosis (BB) in cattle are major concerns among livestock producers in Puer to Rico (PR) (Soto-Alberti, 1999, unpublished data). The livesto ck industry, particularly th e dairy cattle industry, is continuously facing major economic constraints, which are threatening the stability and the economy of the agricultural sect or on the island. Major economic losses include high mortality in adult cattle, abortion, poor grow th performance, reduction in m ilk production, and poor fertility rates. An estimated economic loss of US $20 million was reported in 1989 in PR due to the presence of anaplasmosis, babesiosis, and Rhipicephalus (Boophilus) microplus (Canestrini) (Crom, 1992). Today, it is estimated that cattle operations are facing a yearly deficit of 3,602,873 kg (7,926,321 lbs) of meat and 14,373,315 L (32,274,840 lbs) of milk (S oto-Alberti, 1999, unpublished data). Cattle operations, especially commercial dairy farms, are the most economically important agricultural sector in PR. The dairy industr y produced US $184.8 million (25.6 %) of the gross domestic product in agricultu re in 2006 (PRDA-ASO, 2006). Approximately 25,000 jobs are related to the production, manuf acture, and sales of milk and milk by-products (ORIL-PRDA, 2006). The island has an estimated cattle population of 281,371 of which 153,097 (54%) belong to the dairy industry with 63,181 lactating cows (Planning Boar d of the Commonwealth of Puerto Rico, 2003; NASS-USDA, 20 04). Most lactating cows are raised within 353 commercial dairy farms operating in PR as of 2006. In 2005-06, these dairy farms produced 329 million L (709 million lbs) of milk with an average production of 3,850 L (8,277 lbs) per cow (NASSUSDA and PRDA-ASO, 2005).

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18 As described above, the dairy industry in PR constitutes an important sector for the national economy of the island. Fresh milk is not imported in to the island and the entire commodity is processed for national consumpti on. Demand for milk and milk by-products in the densely populated island (~4 million inhabitants in 9,104 km2) increases almost daily. To sustain this need, it is crucially importa nt to make every effort available to maintain and improve the development of this industry. Although tick-borne hemoparasitic diseases ha ve been reported to cause considerable damage to cattle in PR, the epidemiology of BA and BB in PR has not been thoroughly investigated. To date, no largescale seroprevalence studies or assessment of farm management risk factors have been undertak en in PR. More information is needed concerning the prevalence BA and BB in PR, the abundance and spatial distribution of R. (Boophilus) microplus and the risks of BA and BB within farms in PR befo re making unsubstantiat ed conclusions about appropriate strategies for disease control. Therefore, the objectives of this research program were: To determine the seroprevalence of Anaplasma marginale in adult lactating dairy cattle within the four different climatological zone s of PR and to assess the associations of geographical and farm management factors on the seroprevalence of A. marginale in commercial dairy farms of PR. To determine the seroprevalence of Babesia bovis in adult lactating dairy cattle within the four different climatological zones of PR, to assess the associations of geographical and farm management factors on the seroprevalence of B. bovis in commercial dairy farms of PR, and to document the species of ticks commonly found on cattle among commercial dairy farms in PR. To identify ecologic factors asso ciated with the presence of R. (Boophilus) microplus larvae in PR, and to descri be the seasonal pattern of R. (Boophilus) microplus larvae.

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19 CHAPTER 2 LITERATURE REVIEW Epidemiology of Bovine Anaplasmosis Etiologic Agent Bovine anaplasmosis (BA) is a hemoparasitic disease of cattl e caused by the rickettsiae, Anaplasma marginale (Order Rickettsiales, Family An aplasmataceae) (Dumler et al., 2001; Kocan et al., 2004). These organisms are oblig ate intracellular parasite s that are found within membrane bound vacuoles in the cytoplasm of erythrocytes (Kocan et al., 2004). Four main genera belong to the family Anaplasmataceae: Anaplasma Ehrlichia Wolbachia and Neorikettsia with provisional retention of Aegyptianella (Dumler et al., 2001). Organisms from the genus Anaplasma that affect cattle and small ruminants are A. marginale A. centrale A. ovis A. bovis and A. phagocytophila (Dumler et al., 2001; Kocan et al., 2004). The main structure of the organism consists of 1 to 8 subunits or initial bodies, each being 0.3 to 0.4 m in diameter surrounded by a 1-layer memb rane or vesicle called an inclusion body (0.3 to 1.0 m) (Ristic and Kreirer, 1984). Initial bodies are the infective form of the organism and are composed of primarily DNA and RNA (Ristic and Kreirer, 1984). Clinical Presentation and Pathology of the Disease Bovine anaplasmosis can present two different forms, acute and persistent. The clinical signs of the acute form are consistent with thos e of severe hemolytic anemia, which may include jaundice, weight loss, and sudden death (Ric hey and Palmer, 1990). Other signs include decreased milk production, abortion s, and hyperexitability (due to cerebral anoxia). Differential diagnoses for BA include bovine babesiosis (BB) leptospirosis, erythr ozoonosis, theileriosis, bacillary hemoglobinuria, and postp arturient hemoglobinuria. Anemia results from extravascular hemolysis and usually occurs 1 to 6 days after pe ak rickettsemia and persists for 4 to 15 days.

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20 Rickettsemia during the acute form of disease may reach as high as 109 infected erythrocytes per ml of blood (French et al., 1998; Scoles et al., 2005). Infected erythrocytes are removed from the circulatory system by macrophages in the spleen, and to a lesser extent, the liver and bone marrow. Therefore, neither hemoglobinemia nor hemoglobinuria will develop (Ristic, 1968; Ristic, 1977). On necropsy, splenomegaly, hepato megaly, and pallor and ja undice of the tissues may be observed (Richey and Palmer, 1990). The c onvalescent period may last 1 to 2 months as hematopoiesis brings erythrocytes back to a normal level (Swift and Thomas, 1983). The acute form of the disease may lead to death or recove ry. Recovered animals often become persistently affected carriers of A. marginale for life (French et al., 1998; French et al., 1999). Quantitative polymerase chain reaction (qPCR) analyses have doc umented that persistently affected cattle can have two-week cycles of very low level ricketts emias with the number of infected erythrocytes between 102.5 to 107 per ml of blood (French et al., 1998; Sc oles et al., 2005). Clinical signs of disease are not observed in persistently affected ca rrier animals. However, the persistent form is important for maintenance of A. marginale within a herd. The ticks ar e a biological amplifier of the organism since the rickettsia multiply in tick salivary and gut epithelium, allowing transmission of a large number of organisms fr om a blood meal containing very few parasites. Therefore, chronically infected carriers serve as a reservoir for A. marginale (Richey and Palmer, 1990; Kocan et al., 1992a; Kocan et al., 1993). Antigenic Variation Antigenic variation is the process by which an organism alters its surface proteins in order to evade the hosts immune system. When this process occurs within a population, it is called antigenic diversity. Pathogens us e antigenic variation to prolo ng their circulation in the blood and thus increase the likelihood of transmission (Barbour and Restrepo, 2 000). Initial bodies of A. marginale express numerous proteins in its ou ter membrane that induce production of

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21 antibodies by the immune system of the animal This group of proteins is called major surface proteins or MSPs. These proteins include MSP-1a, MSP-1b, MSP-2, MSP-3, MSP-4, and MSP-5. MSP-1a, MSP-4, and M SP-5 are conserved during replication of the organism whereas MSP-1b, MSP-2, and MSP-3 are highly variable between A. marginale strains (Visser et al., 1992; M underloh et al., 1994; Munderloh et al., 1996; Meeus and Barbet, 2001; Bowie et al., 2002; Kocan et al., 2004). Major Surface Protein-1 is composed of two polypeptides MSP-1a (60-105 kDa) and MSP-1b (100 kDa) (Oberle et al., 1988; Allred et al., 1990; Kocan et al., 2002). MSP-1a is involved in the adhesion process of A. marginale to bovine erythrocytes and Dermacentor tick gut cells (de la Fuente et al., 2001a; de la Fuen te et al., 2001b; de la Fuente et al., 2003b). This is the most important protein used to characterize infectivity of the organism (Brown et al., 2001; de la Fuente et al., 2001b; Kocan et al., 2004). In addition, the gene th at encodes the protein, msp-1 has been used as a genetic marker for differentiation of strains based on geographic location (de la Fuente et al., 2002c). In ende mic areas where multiple genotypes of msp-1 coexist, only one genotype will establish infection per individual animal1 (Palmer et al., 2001). Related studies by de la Fuen te et al. (2002a) described a similar phenomenon in tick cell cultures. However, when distantly related geno types exist in the same region, infection of a single host with multiple A. marginale strains is possible (Palmer et al., 2004). Nevertheless, single strains can be establis hed within geographic areas a nd hinder others from becoming endemic. Despite this fact, ther e is the opportunity for new strain s to be introduced into a region, become established, and reach endemicity (de la Fuente et al., 2002a; de la Fuente et al., 2003a). MSP-1b is encoded by the genes msp-1 1 and msp-1 2 and it is involved in the adhesion process 1 This characterization of the mechanism is known as infe ction exclusion where one genotype is established in the animal excluding other genotypes from becoming established (de la Fuente et al., 2002a; de la Fuente et al., 2003a; Kocan et al., 2004).

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22 of A. marginale to bovine erythrocytes. Unlike MSP-1a, it is not involved in the adhesion to tick gut cells (de la Fuente et al., 2001a). It has high polymorphism among geographic strains (Bowie et al., 2002; Kocan et al., 2003). MSP-2 (36 kDa) is encoded by a multi-gene family ( msp-2 ). This protein demonstrates high polymorphism and plays a major role during cy clic rickettsemia in persistently infected cattle (French et al., 1998; Fr ench et al., 1999; Barbet et al., 2001). New antigenic MSP-2 variants emerge at approximately 5week intervals reach ing peaks of >106 infected erythrocytes per ml, which are then reduced by an effective immune response against that specific variant (Kieser et al., 1990; Palmer et al., 1999). At least 3 different genetic variant types or antigenically different populations are produced within a single peak during these rickettsemic cycles (French et al., 19 98; Barbet et al., 2001). MSP-3 (86 kDa) is the immunodomi nant antigen during natural and experimental infection of cattle. It is encoded by the msp-3 multi-gene family. It demonstrates high polymorphism among geographic strains (Alleman and Ba rbet, 1996; Alleman et al., 1997). In conjunction with MSP-2, it is involved in the cyclic ri ckettsemia observed during infection (Kocan et al., 2003; Brayton et al., 2003). MSP-4 (31 kDa), encoded by the msp-4 gene, is highly conserved during replication. It is also involved in the adhesion of the organi sm like MSP-1a and b and MSP-2 (McGarey and Allred, 1994). The conserved nature of this protein during replication makes msp-4 a stable genetic marker for phylogenetic analyses (de la Fuente et al., 2002c; Kocan et al., 2004). The msp-4 gene provides better phyl ogeographic resolution than msp-1 on a broad geographic scale. The msp-1 gene varies greatly among and within geographic areas but it is useful for

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23 phylogenetic information when there are numerous st rains in a given area (de la Fuente et al., 2002c). MSP-5 (19 kDa) is also highly cons erved during replication. The gene msp-5 is present in A. marginale A. centrale and A. ovis suggesting that this gene is im portant in the lifecycle of the organism, but the true function is unknown (Visser et al., 1992). This protein has a speciesspecific epitope that is recognized by the m onoclonal antibody ANAF16C1. This protein and the monoclonal antibody that recognizes it form the basis for a diagnostic competitive ELISA to diagnose cattle infected with A. marginale (Visser et al., 1992; Know les et al., 1996; Torioni de Echaide et al., 1998). The MSP-5 ANAF16C1 epitope is present during the development of the organism in tick cells and erythrocytes. Recently, 21 more outer membrane proteins ha ve been identified in addition to the MSPs. They are subdominant proteins a nd are classified as type IV s ecretion system proteins. These proteins are highly conserved among geographically distant strains of A. marginale and their potential as antigens for vaccine development have been investigated (Lopez et al., 2007). Of these proteins, Vir89 is capable of inducing exte nsive proliferation of CD 4+ T cells against two strains of A. marginale (Lopez et al., 2007). Antigenic Diversity and Distribution of A. marginale Anaplasma organisms are found in most tropical and subtropical regi ons of the world including Africa, north Australia Asia, southern Europe, and th e Americas (Kocan et al., 2003; Kocan et al., 2004; de la Fuente et al., 2007). Different geographi c areas have different strains, which vary in morphology, protein sequence, anti genic characteristics, and tick transmission characteristics (de la Fuente et al., 2002c; Kocan et al., 2004). Major phy logenetic studies have used the antigenic polymorphism in surface prot eins, especially MSP-4 and MSP-1a, to support

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24 the classification of A. marginale strains into different clades2. There is strong support for clades containing similar strain sequences from Italy, Spain, China, and the Americas (de la Fuente et al., 2007). In the Americas, clades have been described for Mexico, South America, and the United States (US) (de la Fuente et al., 2002c). Within the US, 13 geograp hic strains have been described and grouped into four major clades (d e la Fuente et al., 2002c ). The southern clade includes strains from Virginia, Florida, and Mississippi. The west-central clade consists of strains from California, Idaho, Illinois, Oregon, Mi ssouri, and Texas. Two other clades, the eastcentral and north-central, contain strains from Ok lahoma (de la Fuente et al., 2001c; de la Fuente et al., 2002c; de la Fuente et al., 2003c; de la Fuente et al., 2003d). Studies have described the Virginia, Oklahoma, Idaho, Mississippi, and Ore gon strains as infective and transmissible by ticks whereas the Florida, Okeechobe e, Illinois, and Califor nia strains are not in fective or able to be transmitted by ticks (Smith et al., 1986; Wickwire et al., 1987; de la Fu ente et al., 2001b; de la Fuente et al., 2002a; Kocan et al., 2004). In Australia, few genotypic differences have been reported for strains of A. marginale The msp1genes among Australian strains are la rgely conserved (Lew et al., 2002). Moreover, clinical evidence suggests no signi ficant difference in virulenc e characteristics among strains (Bock and de Vos, 2001). The msp-4 phylogenetic analyses have placed a strain of A. marginale obtained from infected ticks in Puerto Rico (GenBank Acces ion No. AY191827) within the South American clade. This clade also includes strains from Br azil and Argentina (de la Fuente et al., 2003c; Vidotto et al., 2006; de la Fuente et al., 2007) However, multiple if not hundreds of unique strains are likely to be circulati ng in an endemic region such as Pu erto Rico, so one strain should 2 A clade is a taxonomic group of organisms comprising a single common ancestor and all the descendants of that ancestor.

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25 not be considered completely representa tive of the population (Palmer, 2007, personal communication). Host Occurrence and Breed Resistance Clinical disease due to Anaplasma infection is most common in cattle, but other ruminants including water buffalo, bison, white-tailed deer mule deer, black-tai led deer, pronghorn, elk, bighorn sheep, and various African and Asian wild ruminants, can become persistently infected with A. marginale (Kuttler, 1984; Zaugg and Kuttler, 1985; de la Fuente et al., 2003c). A study by Bock et al. (1997) reported th at all breeds of cattle, including pure Bos indicus breeds and pure Bos taurus breeds and their crosses are suscep tible to development of severe disease if exposed to a virulent strain of A. marginale This study evaluated animals inoculated with a strain of A. marginale under laboratory conditions and di d not account for the role of R. (Boophilus) microplus in the transmission of the organisms (Bock et al., 1997). However, a follow-up study using R. (Boophilus) microplus ticks infected with A. marginale reported that innate resistance of purebred Bos indicus and crossbred (50%, F1 generation), cattle was not significantly different. Both breeds were equally susceptible to infection with A. marginale by R. (Boophilus) microplus (Bock et al., 1999a). Even though A. marginale affects both species similarly, under field conditions Bos indicus are not as commonly affected as Bos taurus presumably due to their relative resistance to heavy tick infestation (Bock et al., 1997). Life Cycle Stages and Development The lifecycle of A. marginale in the tick vector and bovine erythrocyte includes a series of similar stages before infection is established. Mo st descriptions are deri ved from observations of Dermacentor andersoni ticks and tick cell cultures. However, the lifecycle of A. marginale in R. (Boophilus) microplus has shown similar characteristics (Ribeiro and Lima, 1996). In the tick vector, A. marginale invades tick mid-gut cells immediately after i ngestion of infected

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26 erythrocytes. The ingested erythr ocytes contain the infective form of the organism known as the dense form. Under electron microscopy, th ese forms are highly dense with individual organisms barely visible (Kocan et al., 1980; Ribeiro and Lima, 1996; Kocan et al., 2004). Based on a cultured tick cell line (IDE8)3, infection of cells occurs within 15 minutes after ingestion by adhesion of the dense form to the host cell membrane (Blouin and Kocan, 1998; Kocan et al., 2004). Projections from the tick mid-gut cells adhere to the outer membrane of A. marginale and invaginate around the organism (Blouin and Kocan, 1998). Subsequently, the organism is completely enclosed and internal ized within a phagosomal vacuole and remains within the vacuole th roughout the lifecycle (Blouin and Kocan, 1998). Once in the vacuole the organism changes into the reproductive form known as the vegetative or reticulated form. This form has large, separated organisms with a light ly stained matrix that is electro-lucent under electron microscopy (Kocan et al., 1992b; Ribeiro and Lima, 1996; Kocan et al., 2004). Intracellular replication of A. marginale by binary fission begins within the mid-gut epithelial cells (Blouin and Kocan, 1998). Within 2 days of ingestion, tick mid-gut cells are infected by many colonies contai ning the reticulated form of A. marginale (Blouin and Kocan, 1998). By day 3 post-infection, there is a comb ination of small dense forms and large round reticulated forms with the latter dividi ng by binary fission (Blouin and Kocan, 1998). On day 4, most of the colonies contain primarily dense fo rms and exocytosis of the organisms is observed. Membranes of colonies of the organism fuse with membranes of the tick mid-gut cell and a channel is opened between the membranes, th at allows the dense forms to exit into the 3 Cultured IDE8 tick cells tick cell line culture system derived from embryos of Ixodes scapularis used extensively in many laboratories to propagate Anaplasma spp. The developmental cycle in these cultured tick cells is similar to naturally infected ticks. A. marginale propagated in this line retained antigenic composition and infectivity for cattle (Munderloh et al., 1994; Munderloh et al., 1996; Blouin an d Kocan, 1998; Blouin et al., 2000). All six MSPs were found to be conserved on the cell culture derived organisms (Barbet et al., 1999). However, A. marginale development within Ixodes ticks raises the possibility that the infec tion stage within the salivary gland might be antigenically different compared to the organisms acquired from infected cattle (Palmer et al., 1999).

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27 extracellular space without loss of ho st cytoplasm (Blouin and Kocan, 1998). At this stage, dense forms may invade new tick mid-gut cells or penetrate through the gut-wall and enter the hemolymph (Kocan et al., 2004). However, Ribeir o and Lima (1996) did not observed the first colonies of A. marginale in naturally infected R. (Boophilus) microplus ticks until day 19 after detachment of engorged females from experimentally infected calves. In the hemolymph, dense forms of the organism are carried to the ovaries and acinar cells of the salivary glands (Wanduragala and Rist ic, 1993; Ribeiro and Lima, 1996). After invasion of acinar cells, a second round of replication occu rs. Organisms may appear in acinar cells as short as 8 days post-ingestion of infe cted erythrocytes with levels of 104 to 105 organisms per salivary gland at subsequent f eedings (Kocan, 1986; Kocan et al., 1992b; Kocan et al., 1993; Lohr et al., 2002; Futse et al., 2003). Replication within th e tick results in similarly high levels of A. marginale in the acinar cells regardless of the initial rickettsemic levels in the host at the time of feeding (Eriks et al., 1993). When an infected tick bites an an imal host, the infective form of A. marginale is transported from acinar cells, via saliva, into th e animal. The only known s ite of replication of A. marginale in cattle is circulating mature er ythrocytes (Richey and Palmer, 1990). Invasion of mature erythrocytes is similar to that obser ved in tick mid-gut epithelial cells. The cell membrane of mature erythrocytes inva ginates to engulf the dense form of A. marginale within a vacuole (Ristic and Kreirer, 1984). Once in the vacuole of the erythr ocyte, the reticulated form of the organism develops and multiplies by binary fission to produce 2 to 8 new organisms. The reticulated form changes into the dense form to exit the erythrocyte. Pores form within the erythrocyte membrane through which organisms can escape to infect othe r cells. Non-infected

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28 erythrocytes can also be penetr ated by release of hydrolytic en zymes from the organism (Kocan et al., 2004). The number of infected erythrocytes doubles every 24 to 48 hours, and approximately 2 to 6 weeks later an acute infection can develop where 10 to 90% of erythrocytes are infected (>108 infected erythrocytes per ml). Some authors have claimed th at at least 15% of infected erythrocytes are necessary for the animal to show clinical signs, while others suggest only 1% (Richey and Palmer, 1990). Methods of Transmission Anaplasma marginale is biologically transmitted by infect ed ixodid ticks and mechanically by biting flies and contaminated fomites (Ewing, 1981; Kocan, 1986; Kocan et al., 2004). Biological transmission by definition includes mu ltiplication of the organism within the tick vector (McCluskey, 2002). This method is beli eved to be necessary for transmission of A. marginale from persistently infected carriers where there is a low level of rickettsemia. Conversely, mechanical transmission is dependent on the level of rickettsemia during the time at which the vector is feeding. This method of tr ansmission requires higher numbers of infected erythrocytes than biological transmission to es tablish infection. Because of this, mechanical transmission is believed to only occur from acutely infected hosts (Scoles et al., 2005). Seventeen species of ticks have been incr iminated as vectors worldwide (Ewing, 1981; Kocan et al., 2004). The important tick vectors in tropical and subtropical regions include the one-host ticks: Rhipicephalus (Boophilus) microplus R. (Boophilus) annulatus, and R. (Boophilus) decoloratus (Samish et al., 1993; Jongejan and Uilenberg, 2004). To date, R. (Boophilus) decoloratus has not been recognized outside of Africa (Jongejan and Uilenberg, 2004).

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29 Rhipicephalus (Boophilus) spp. have greater vectorial capacity4 for A. marginale when compared to other tick species. Al though these ticks feed on one host for its entire lifetime, some studies have shown that all three stages; larvae, nymphs, and adults can efficiently acquire and transmit A. marginale when they are manually transfe rred from one cattle host to another (Connell and Hall, 1972; Leatch, 19 73; Dalgliesh and Steward, 1982; Aguirre et al., 1994). On the contrary, the three-host ticks of the Dermacentor spp. ( D. andersoni, D. variabilis, D. albipictus, D. occidentalis ) are not as efficient as R. (Boophilus) spp for transmitting the infection because the larval a nd nymphal stages prefer small ma mmals and only adults acquire and transmit A. marginale by feeding on cattle (Futse et al., 2003). Nevertheless, Dermacentor ticks infected with A. marginale as nymphs have shown to transmit the organism in the adult stage (Kocan et al., 1981; Kocan et al., 1986). In situations where R. (Boophilus) spp. have been eradicated, as in United States, Dermacentor spp. are the main vector for A. marginale (Jongejan and Uilenberg, 2004). Lastly, some reports have es tablished the transmission of Anaplasma by Rhipicephalus spp. ( R. evertsi evertsi, R. simus and R. bursa ) but their role in transmission is not as well understood as other specie s (Jongejan and Uilenberg, 2004). Anaplasma marginale can be transmitted transstadiall y and intrastadia lly. Transstadial transmission refers to the capability of immatu re stages (i.e. larva or nymph) to acquire the organism while feeding and then maintain the inf ection through the next molt and transfer it to a new cattle host the next time it feeds as the ne xt immature stage or adult (Bezuidenhout, 1987; Kocan et al., 2003). This method of transmission usua lly occurs in three-host ticks, particularly Dermacentor andersoni and D. variabilis (Kocan et al., 1981). Ho wever, studies on one-host ticks particularly R. (Boophilus) spp. have demonstrated that this method of transmission is 4 Ability to experimentally acquire and transmit A. marginale in all three stages of development; larvae, nymphs, and adults (Futse et al., 2003).

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30 possible by two means. Either by interrupting th e feeding time in one stage and manually relocate the tick into a susceptible calf in the same stage; or by removing the tick in one stage allowing moulting to occur away from the host, and then transfer the next instar into a susceptible calf (Connell and Hall, 1972; Samish et al., 1993). Intrastadial transmission refers to the transfer of A. marginale between cattle hosts by male ticks (Kocan et al., 1992a; Ko can et al., 1992b). Most studies have documented the importance of this method in the epidemiology of BA in US. D. andersoni male ticks become persistently infected with A. marginale and can transmit the organism repeatedly duri ng relocation among cattle hosts (Kocan et al., 1992a; Kocan et al., 1992b). This transmission method has also been implicated as an important mechanism of transmission of A. marginale by R. (Boophilus) spp. (Connell and Hall, 1972; Barbet et al., 2001; Kocan et al., 2003). Trans-ovarial transmission5 of A. marginale has not been well documented in R. (Boophilus) microplus (Connell and Hall, 1972; Leatch, 197 3; Stich et al., 1989). However, studies on Dermacentor variabilis have demonstrated that larvae from infected engorged females do not transmit A. marginale to susceptible cattle (Kocan et al., 1981; Stich et al., 1989). Mechanical transmission occurs when infected blood is transferred to susceptible cattle via blood-contaminated mouthparts of hemat ophagous insects or iatrogenically by bloodcontaminated fomites (Ewing, 1981). In this type of transmission, there is no developmental sequence of the organism; it simply involves th e physical movement of the organism from one animal to another without replication (M cCluskey, 2002). Efficiency of this mechanism depends on the level of rickettsemia during feeding (Scole s et al., 2005). Tabanids, including horse flies ( Tabanus spp. ) and deer flies ( Chrysops spp. ), stable flies ( Stomoxys calcitrans ), horn flies 5 Transovarial transmission occurs when the female tick acq uires the infection while feeding and transfers the agent to the developing ova. In this case, the newly hatched larvae are infected without having to take a blood meal.

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31 ( Hematobia irritans ), and mosquitoes of the genus Psorophora have been implicated as mechanical vectors for the transmission of A. marginale (Ristic, 1968; Ristic, 1977; Ewing, 1981; Potgieter et al., 1981; Hawkins et al., 1982; Foil, 1989). However, Kessler (2001) questioned the capability of hematophagous insects for the transmission of A. marginale and suggested further studies. A recent study by Scoles et al. (2005), estimated the transmission of A. marginale by stable flies ( Stomoxys calcitrans ) and Dermacentor andersoni ticks during the acute and persistent forms of BA. Two different strains of A. marginale were used in that study, St. Maries and Florida. The St. Maries strain is known to be transmitted by Dermacentor ticks whereas the Florida strain is not transmissible by Dermacentor ticks (Wickwire et al., 1987; Eriks et al., 1993; Futse et al., 2003). Stable flies that partially fed on an acutely infected calf and were immediately transferred to susceptible calves to complete their meals failed to transmit the St. Maries strain of A. marginale However, ticks that fed on th e same host after reaching the persistent form of disease (>300-fold lower leve l of rickettsemia) succes sfully transmitted the organism to the same susceptible calves. Therefore, Scoles et al (2005) concluded that biological transmission of A. marginale by ticks is a more efficient met hod of transmission, even at low levels of rickettsemia, than stables flies. This same study documented that a proportion of flies had detectable levels of the two A. marginale strains (Florida and St. Ma ries) in their mouthparts, which increased with increasing percenta ge of parasitized erythrocytes (109 infected erythrocytes (IE)/ml) during acute rickettsemia. At lower leve ls of IE/ml, no mouthparts were found to be contaminated with A. marginale However, the fly transmissi on trial only evaluated the St. Maries strain of A. marginale and the author suggested further studies using othe r strains (Scoles et al., 2005). In the absence of ticks, especially where R. (Boophilus) microplus is not present, hematophagous insects have been incriminated as the major route of infection. In Cuba, tabanids

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32 are the most common hematophagous inse cts suspected to be vectors of A. marginale (Alonso et al., 1992). The only species of tabanids described in Puerto Rico (PR) is Chrysops variegata and it is only present in low numbers (van Volke nberg, 1939). No studies have been performed to assess the feasibility of this ta banid as a potential vector for A. marginale Iatrogenic transmission is believed to occur when blood-contaminated instruments including needles, castrating a nd tattooing tools, dehorning saws, and ear tagging devices spread the organism to susceptible animals (Hilts, 1928; R eeves and Swift, 1977). However, this is only supported by case reports from the 1930s and has not been evaluated experimentally (Stiles, 1936). The first report of an outbreak incr iminating iatrogenic transmission of A. marginale in cattle involved beef stee rs after dehorning (Hilts, 1928). In hi s report, the author determined the cause of the outbreak was the c lippers based on exclusion of ot her major routes of infection including ticks and other biting insects. The ma jor reason for ruling out these routes was the season of occurrence (Hilts, 1928) Most cases of BA attribut able to dehorning have been reported during cold weather (Stiles, 1936). Other outbreaks following dehorning have been reported in California and Oklahoma (Boynton, 1932; Moe et al., 1940). Producers from large dairies in southern California observed th at certain outbreaks o ccurred periodically, approximately 35 to 45 days after the cattle we re bled for Brucellosis test. Those animals immediately bled following carriers contra cted the infection (Boynton, 1932). In 1930, Rees was able to transmit A. marginale and B. bigemina using a contaminated lancet. The procedure consisted of first pricking the ear of two inf ected animals with the lancet and subsequently pricking the ear of two susceptible animals. Th e infected cows were identified by clinical observation of disease and identification of the organism using light microscopy. However, the infection status of the susceptible animals befo re the trial was not reported (Rees, 1930). Almost

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33 50 years later, in a study by Reeves and Swift (1977), 5 beef heifers were inoculated in sequence, using an automatic syringe, which was firs t used on a cow known to be seropositive for A. marginale In this study, only the heifer inoculated immediately after the carrier cow became infected. The infection was confirmed by a positive complement fixation test, the presence of the organisms on the erythrocytes, and a decrease in packed cell volume at day 35 after inoculation. The remaining 4 were free of clinical signs at the end of the 60-day tr ial (Reeves and Swift, 1977). In his conclusion, the author establishe d this procedure as a possible means for transmission of A. marginale Other routes were excluded because of the season and lack of observed ticks on the heifers. The lack of infec tion in the other 4 heifers was attributed to dilution of the blood by a saline so lution within the syri nge. All cows were serologically tested using complement fixation tests before and after the trial. The sensitivity of this assay is reported to be low, varying from 10% to 79% (Gonzalez et al., 1978). Therefore, the identification of infected animals by these tests is unreliable (Alleman, 1995). Based on th ese observations, this transmission method along with me chanical vectors are claimed to play an important role in the epidemiology of A. marginale in enzootic areas in the US a nd other countries (Guglielmone et al., 1997; Kocan et al., 2003). Transplacental transmission of A. marginale has also been reporte d in cattle, but most studies evaluated splenectomized animals. Furthermore, in these studies in utero infection is defined as a calf with a positive card agglutination test (CAT) or ELISA that is maintained for longer than 12 weeks despite the f act that the calves were typical ly allowed to ingest colostrum. However, many authors have documented the presence of in utero infection by the presence of high levels of antibodies in the precolostral se rum of newborn animals (Radostits et al., 1999b;

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34 Tizard, 2004a). Therefore, in utero infection studies on A. marginale should consider precolostral serum samples to assess the risks of transmission in the epidemiology of BA. Potgieter and van Rensburg (1987) allocated 26 calves into 2 groups, those belonging to dams with intact spleens and infected with A. centrale and A. marginale (n=13) and those belonging to splenectomized dams infected only with A. centrale (n=13). In this study, the calves were screened weekly for 12 weeks and monthly for 6 months by CAT. Three calves from the group of splenectomized dams infected only with A. centrale were positive on the CAT at 24, 21, and 25 weeks, but in only 2 were organisms obs erved on blood smears before the ingestion of colostrum. Calves from the group of dams with intact spleens and mixed infections were not tested before the ingestion of colostru m and only in 1 calf from this group was A. marginale recognized on blood smears prior to splenectomy at 30 weeks. This study also monitored a second group of calves (n=51) born from splen ectomized dams. In this part of the study, serological testing was not performed before in gestion of colostrum and only 8 calves (15.7%) were described as having b een infected transplacentally (Potgieter and van Rensburg, 1987). This likely does not represent what would o ccur under field conditions because dams were splenectomized. In adult animals, the immune response depends on 2 systems, the innate immune system, and the acquired immune system. Although these sy stems can act independently more often, they act in combination (Barrington a nd Parish, 2001). The innate immune system plays a crucial part in the initiation and subsequent direction of the acquired immune system (Janeway et al., 2005). The innate immune system is the first line of defe nse against antigens and consists of preexisting or rapidly responding chemical and cellular defense mechanisms that indiscriminately attempt to destroy any antigen (Tizard, 2004b). These in clude macrophages, neutrophils, and the

PAGE 35

35 complement system (Tizard, 2004b). The acquired i mmune system is the second line of defense and consists of antibodies, memory lymphocytes and effector cells (Barrington and Parish, 2001). It provides protection agains t subsequent reinfection with the same antigen (Janeway et al., 2005). This system takes 4 to 7 days to deve lop but is highly specif ic and effective. The acquired immune system is addi tionally divided into humoral immune response (B-cells and antibodies) and cell-mediated immune response (T-lymphocytes or T cells) (Barrington and Parish, 2001; Janeway et al., 2005). The humoral im mune response protects against extracellular or exogenous antigens whereas the cell-mediated is directed against intr acellular or endogenous antigens (Tizard, 2004b). In turn, the humoral i mmune response consists of a primary and secondary response. The primary response to a sp ecific antigen produces a first set of antibodies but in low concentrations whereas the secondary response to the same antigen produces a second set of antibodies at a higher concentration. Each set of antibodies mostly consists of IgM and IgG. IgM is the major antibody produced during a primary response whereas IgG predominates in the secondary response (Tizard, 2004b). The cell-mediated immune response consists of CD8+ T-cells and CD4+ T-cells. The CD8+ T-cel ls control the infection by destroying the infected cells (Janeway et al., 2005 ). The CD4+ T-cells are divided into two subsets, the T helper cells 1 (TH1) and the T-helper cells 2 (TH2). The TH1 cells activate the phagocytic mechanisms of macrophages and release cytoki nes and chemokines that attract more macrophages to the site of infection (Janeway et al., 2005). The TH2 cells stimulate B-cells to produce antibodies (Janeway et al., 2005). In the fetus, the innate and acquired immune systems are not fully functional against infection resulting in severe or fatal damage while the infection in the dam can be mild or

PAGE 36

36 unapparent (Tizard, 2004a). Furtherm ore, the immune response depends on the age of the fetus or calf and the specific infectious organism (W atson et al., 1994; Rados tits et al., 1999b). The new-born calf is able to mount the inna te and acquired immune response. However, the primary response takes longer to develop and the amount of antibody produced is lower compared to adult animals. Therefore, unle ss adequate maternal immunologic assistance via colostrum is provided, calves have an increased risk of infection. Colostrum contains IgG1, IgG2, IgM, IgA, and IgE. The predominant immunoglobulin in colostrum of ruminants is IgG1 (60 to 90%). Commonly, colostrum-fed calves show high levels of immunoglobulins primarily IgG1 after 24 hours of ingestion. The n, these levels st art to decline and reach undetectable values by 60 days (LaMo tte, 1977). The half-life of colostral-derived antibody in the calf is between 11.5 and 16 days (Barrington and Parish, 2001). However, colostral antibodies down regulate the endogenous production of antibodies against infections (Kitching and Salt, 1995; Aldridge et al., 1998; Radostits et al., 1999b). These effects may last for 4 months depending on the infectious orga nism (Barrington and Parish, 2001). Colostrumdeprived calves have higher antibody response wh en compared to colostrum-fed calves and are believed to be immunologically competent at birth wi th respect to most antigens (Aldridge et al., 1998; Radostits et al., 1999b; Barrington and Parish, 2001). Rey et al. (2003) evaluated the presence of A. marginale organisms and antibody response in newborn calves und er field conditions. Fourteen calves born from naturally infected dams were tested at 7 and 30 days afte r birth. Once more, all calves were tested after the ingestion of colostrum, and the pre-colostral status was not reported. At 7 days, all calves were negative for parasitemia based on Giemsa-stained direct smears whereas at 30 days, all calves were parasitemic for A. marginale. However, the second evaluation of parasitemia was measured by a

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37 different staining method other than Giemsa. The authors decided to use acridine orange to stain the smears (Rey-Valeiron et al., 2003). The sensitivity of this method is slightly higher than the Giemsa stain, and results may be biased toward finding positive results. In addition, the authors measure the IgM and IgG antibody response by an ELI SA as a measure of passive transfer via colostrum versus natura l infection, respectively. A cut-off value of 0.4 absorbance at 405 nm was determined for this study based on the averag e absorbance of 3 negative serum samples and 3 standard deviations. Serum samples were tested in triplicates. In this study, all calves had IgM levels below the cut-off value at 7 and 30 days Conversely, only 5 of 14 calves had IgG levels above the cut-off line at 7 days and 4 of 14 had hi gh levels of IgG at 30 days. The low levels of IgM and IgG observed at 7 days were attributed to failure of passive transfer whereas the levels observed at 30 days were attrib uted to normal decline of Ig G (Rey-Valeiron et al., 2003). Therefore, the use of this indirect measure di d not necessarily support the conclusions regarding in utero transmission and further studies are necessary to elucidate the ro le of transplacental transmission in the epidemiology of BA. Potgieter and van Rensburg (1987) did not find a difference in infection risk for A. centrale among the 3 gestation trimesters in the cow wh ereas Zaugg (1985) reporte d that the second and third trimesters were the most common periods for transmission of A. marginale Nevertheless, in this study, only 6 cows were used and only the blood from 2 of the newborn calves successfully transmitted anaplasmosis to splenectomized recipient calves. One calf was born from a cow infected during the second trimester and the other calf was bor n from a cow infected during the third trimester of gestation (Zaugg, 1985). Immune Response and Immunity Infection with A. marginale stimulates a cell-mediated response followed by antibody production against the infected eryt hrocytes. Infected erythrocytes are removed from circulation

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38 by the phagocytic action of macropha ges in the reticuloendothelial system (primarily liver and spleen) (Richey and Palmer, 1990). Based on th e immune response from vaccine trials, infected animals with the acute form of BA develop high levels of IgG2 and IgM antibodies against MSPs of A marginale (Richey and Palmer, 1990; Bock et al ., 1997) (de la Fuente et al., 2002a). Antibody production is enhanced by CD4+ T-cel ls, which secrete interferon gamma (IFN) and tumor necrosis factor (TNF) (Palmer and McElwain, 1995; Brown et al., 1998a; Brown et al., 1998b; Palmer et al., 1999). Interferonactivates macrophages for the phagocytosis of infected erythrocytes and the pr oduction of nitric oxide, a potent molecule for microbial killing (Brown et al., 1998a). Antibodies can also activate macrophages through opsonization of the organism (Brown et al., 1998a). Nevertheless, th ese responses are not cap able of completely eliminating the infection (Palmer et al., 1999). Cattle of all ages can become infected with A. marginale However, mortality and disease severity is greater in adult cattle (Richey and Palmer, 1990). Young calves (less than 6 months of age) are equally susceptible to infection as adults but they seldom devel op clinical signs (Richey and Palmer, 1990; Kocan et al., 2003). Infection between 6 months a nd 2 years of age increases the risk of clinical illness but is rarely fatal. Cattle infected after 2 to 3 years of age are commonly affected by a peracute fatal form of the disease. Incidence of clinical disease (often fatal) increases with age (>3 yrs of age). Ca lves develop persistent infections and lifelong immunity (Kocan et al., 2003). Endemic Stability In areas where the tick vectors are ab undant, prevalence of infection with A. marginale is relatively stable within the cattle population. This situa tion is known as endemic stability and is a form of herd immunity. It is characterized by a high percentage of infect ed cattle but with few clinically affected animals (Coleman et al., 2001; Corona et al., 2005). Two main factors

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39 contribute to this stability: pa ssive immunity provided by colostrum intake and early infection of calves. As mentioned before, young calves typically acquire the infection without demonstrating clinical signs. However, once immunity is establ ished, adult cattle become persistently infected thus ensuring maintenance w ithin the population (Corrier an d Guzman, 1977; Corona et al., 2005). Treatment and Prevention Chemotherapies of choice for A. marginale are tetracycline compounds and imidocarb. Imidocarb is thought to block the entry and preven t the uptake of inositol by erythrocytes thus resulting in the starvation of any hemoparasi te. It has also been proposed that imidocarb combines with DNA causing it to unwind and de nature. Deoxiribonucleic acid damage can inhibit cellular repair and replicat ion (McHardy et al., 1986; Plumb, 1999). Imidocarb is not approved for use in food animals in the US (Ris tic, 1977). The inhibitory effects of tetracyline have been documented for acutely affected calves and IDE8 cell cu lture systems (Simpson, 1975; Blouin and Kocan, 1998; Blouin et al., 200 2). Tetracyclines inhibit replication of A. marginale in cell culture by interfering with the ab ility of the organism to complete its replication cycle within the para sitophorous vacuole in the host cell cytopl asm (Blouin et al., 2002). Nevertheless, in severe clinical cases it is also necessary to provide supportive therapy including electrolytes, antihistamines, and analgesics (Corona et al., 2005). Current BA vaccines employ A. marginale -infected bovine erythrocytes to induce protective immunity. However, these vaccines do not prevent infections and vaccinated animals become persistently infected carriers. Neverthe less, cattle develop sufficient immunity to reduce the severity of disease and prevent clinical signs (Kocan et al., 2003). Live organism vaccines are also used in se veral countries to pr otect cattle against A. marginale infection. The most widely used v accines consist of a strain of A. centrale with low

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40 pathogenicity or attenuated strains of A. marginale (Kocan et al., 2003; Bock et al., 2003). Anaplasma centrale has been used for vaccine production in Australia, the Republic of South Africa, Argentina, Uruguay, Israel, Zimbabwe, a nd Malawi (Tjornehoj et al., 1997; Bock and de Vos, 2001). Studies have shown that A. centrale and A. marginale share surface proteins that account for the cross-protection induced by A. centrale (Shkap et al., 1991; Shkap et al., 2002). However, Shkap (2002) demonstrated that cattle vaccinated with A. centrale could become infected with A. marginale suggesting that further studies in this area are necessary. Recently, a study in Australia has documented the discovery of an isolate of A. marginale (named Dawn), which has a similar or lo wer pathogenicity than A. centrale and may not be transmissible by R. (Boophilus) microplus ticks. This isolate may be useful for the development of a common Anaplasma vaccine for all clades (Bock et al., 2003). Currently, different live vaccines are produced in Australia and provide protection against A. marginale B. bovis and B. bigemina (Kocan et al., 2003). These vaccines are prepared from the blood of infected splenectomized donor calves. Production of these vaccines is expensive and requires strict quality control. Ho wever, the risk of spreading othe r infectious organisms is to all blood-derived vaccines (Rogers et al., 1988). On one occasion, 14,000 vaccine doses were prepared from a single calf infected with B ovine Leukemia Virus (BLV) and distributed to 139 herds. Vaccinated groups had a higher proportio n of BLV-positive cattle when compared to nonvaccinated cattle in the same herds ( 62% versus 6.1%) (Rogers et al., 1988). In addition, there is a risk of vaccinated animals developing th e disease and incomplete protection for the antigenically diverse strains of A. marginale from different geographi c areas (Kocan et al., 2003). Live vaccines given to cattle can also caus e production of antibodies against certain erythrocyte membrane antigens present in th e vaccine. These antibodies are secreted in

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41 colostrum, and when ingested by calves with red blood cells containing the same erythrocyte antigens, the colostral an tibodies will induce neonatal isoeryth rolysis (Dennis et al., 1970; Kocan et al., 2003). Therefore, it has been suggested that the use of blood-derive d live vaccines be restricted to the region where they are produced (Kocan et al., 2003). Killed vaccines can also provide immunological protection (Montenegro-James et al., 1991). To date, killed vaccine formulations have only been proven to provide partial protection against infection with A. marginale and are strain speci fic (Kocan et al., 2004). Similar to the live vaccines, use of a killed va ccine can result in th e lack of cross-protection among geographic isolates and neonatal isoerythrolysis. Currentl y, there are no commercial vaccines available for prevention of BA in the US. Since 1999, two US companies that manufactured and marketed BA vaccines nationwide discontinued pr oduction (Kocan et al., 2003). The first commercial vaccine for cattle in the US was manufactured by Fort Dodge Laboratories (Overland Park, Kansas) and was called Anaplaz. This original vaccine was often contaminated with erythrocyte stroma, which resulted in the development of antibodies against erythrocytes in vaccinated cattle. Consequently, neonatal isoerythrolysis occurred in some calves after the ingestion of colostrum from cows with high antibody titers (Dennis et al., 1970). Later, Mallinkrodt temporarily marketed a vaccine called Plazvax for 3 year s until the company was acquired by Schering Plough (Kenilworth, New Jersey) and the product line was discontinued (Luther et al., 1989; Luther, 2007). Both vaccines protected against A. marginale by similar mechanisms. They used a lyophilized preparation of hemolyzed er ythrocytes obtained from the blood of A. marginale infected animals as an antigen and combined it with an oil-based ad juvant (Brock, 1965). The lack of a commercially available vaccine against BA drove dairy producers in Florida to search out a re-approval of Plazvax from the United States Department of Agriculture

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42 (USDA). The vaccine was not licensed but wa s approved for sale as an Experimental Anaplasmosis Vaccine to veterinarians in Flor ida. Since then, the USDA has approved the use of the vaccine in other states within the US and PR. This vaccine employs the strain of A. marginale and purification procedures used previously in Plazvax. A significant advancement in the study of A. marginale is that it can now be propagated in vitro using cultured-tick cells IDE8. Recently, mo re emphasis has been given to cell culturederived strains of A. marginale for vaccine development (Kocan et al., 2003; Kocan et al., 2004). Anaplasma marginale derived from cell cultures has proven to be an effective antigen for use in vaccine preparations and serologica l tests (Saliki et al., 1998; Kocan et al., 2001; de la Fuente et al., 2002b). Cattle vaccinated with infected bovine erythrocytes produce antibody against MSP-5 and MSP-1a whereas animals immunized with cel l culture-derived antigens produce antibodies predominantly towards MSP-1b (Kocan et al., 2 002; de la Fuente et al., 2002b; Kocan et al., 2003). Commonly, vaccines that induce antibodies specific for MSP-1 confer 100% protection against the acute disease form of BA but studies using cell culture-derived v accines with MSP-1a and b did not induce MSP-1 specifi c responses and disease was not prevented. Alternatives have been recommended to enhance the efficacy of thes e types of vaccines that include addition of tick antigens or different A. marginale clades (Palmer and McElwa in, 1995; Kocan et al., 2003). Epidemiology of Bovine Babesiosis Etiologic Agent Bovine babesiosis (BB) is a hemoparasitic disease of cattle caused by protozoan organisms of the genus Babesia (Phylum Apicomplexa, Order Piroplasmida, Family Babesiidae). Organisms within this genus that affect cattle are B. bovis, B. bigemina B. divergens B. major B. ovata (Japan), and B. occultans (South Africa) (Ohta et al., 1995; Angus, 1996; Bock et al., 2004). Only B. bovis and B. bigemina are considered economically important (Wagner et al.,

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43 2002). The intra-erythrocytic stru cture of these organisms is pyriform in shape and is surrounded by two peripheral membranes within the host cytoplasm (Potgieter and Els, 1977b; Homer et al., 2000). Anterior and posterior ends, termed polar ri ngs, delimit the shape of the parasite. Three major organelles (microtubules, rhoptries, and micr onemes) concentrate in the anterior polar ring and are collectively known as the apical complex (Potgieter and Els, 1977b; Potgieter and Els, 1979; Homer et al., 2000). Babesia bovis is smaller than B. bigemina measuring up to 2 m in length. Under light microscopy, this organism is often found in pairs at an obtuse angle. Conversely, B. bigemina can measure 2 to 5 m in length and extend the full diameter of an erythrocyte. Under light microscopy, B. bigemina is also found in pairs but unlike B. bovis, the angle is acute. Although both organi sms are often found in pairs, single forms of the organism are often found within infected er ythrocytes (Wagner et al., 2002). Clinical Presentation and Pathology of the Disease The incubation period for BB is approximately 7 to 20 days (Radostits et al., 1999a). The disease typically presents in 1 of 3 forms, peracu te, acute, or persistent and chronic. The peracute form usually results in sudden death from severe hemolytic crisis. Disease progression in this form occurs very rapidly and hemoglobinuria is rarely observed. However, it may be observed in domestic ruminants that are exotic to the endemic region (Wagner et al., 2002). In the acute form, the clinical signs include anemia, jaundi ce, and hemoglobinuria. Differential diagnoses for BB include BA, leptospirosis, erythrozoonosis, theileriosis, bacillary hemoglobinuria, and postparturient hemoglobinuria. Other signs incl ude fever, inappetance, depression, increase respiratory rate, weakness, reluct ance to move, muscle wasting, tr emors, and abortions (Callow, 1984; de Vos and Potgieter, 1994). Many severely affected an imals die within 24 hours. On necropsy, splenomegaly, hepatomegaly, and petecchi al hemorrhages are often observed (Bock et

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44 al., 2004). In this form of the disease, the percen tage of circulating eryt hrocytes infected with B. bovis is less than 1% whereas parasitemia with B. bigemina can be as high as 30%. Sequestration of infected erythrocytes in th e microcapillary endothe lia of vital organs (including the brain and lung capi llaries), are of ten observed in B. bovis infections but not in B. bigemina This can cause neurological and respirator y conditions in aff ected cattle known as cerebral babesiosis and respiratory distress syndr ome, respectively. These syndromes result from overproduction of INF, TNF, NO, and other inflammatory cytokines. The coagulation pathway is altered by vasodilati on, increase capillary permeability, edema, endothelial damage, and circulatory stasis, resulting in a hypotensi ve shock syndrome (Brown and Palmer, 1999; Ahmed, 2002). In B. bigemina infection, the predominant clinical si gns include fever, hemoglobinuria, and anemia. As mentioned previously, coagulation di sorders, cytoadherence, and hypotensive states are not observed in B. bigemina infections as in B. bovis (Bock et al., 2004). In B. bigemina infections, the pathologic findings relate more to rapid erythrocyte de struction leading to a massive intravascular hemolysis and hemoglobinuria Clinical signs are not as severe as in B. bovis infections. However, some cattle may die with little warning (Bock et al., 2004). In general, infections with B. bovis are considered more pathogenic than B. bigemina and cause higher morbidity and mortality among su sceptible cattle (Brown et al., 2006b). Cattle affected by the acute form of BB that do not die may take several weeks to regain condition, but complete recovery is typical. These animals will become persistently infected and remain clinically healthy carriers for months to years (Johnston et al., 1978). In endemic situations, protection can be life long. However, this state of sub-clinical infection can be adversely affected by stresses including tran sportation, food deprivation, parturition, or

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45 concurrent diseases. The immune system of thes e animals can readily be disturbed and clinical signs similar to acute disease may re-appear (Radostits et al., 1999a). Antigenic Variation Antigenic variation within the vertebrate host is believed to allow variants of Babesia spp. populations to continue to adhere to endothelial cells a nd be sequestered in tissues thus avoiding splenic passage and clearan ce (Allred, 2001). Extrac ellular merozoites (and tick derivedmerozoites) are coated with surface proteins that facilitate attachment to erythrocytes (Yokoyama et al., 2006). Babesia bovis possess 5 surface proteins know n as variable merozoite surface antigens (VMSA) (Florin-Christensen et al., 2002). These are merozoite surface antigen1 (MSA-1), MSA-2a1, MSA-2a2, MSA-2b, and MSA-2c. These are encoded by two genes msa1 and msa-2 respectively (Yokoyama et al., 2006). These proteins are believed to play a major role in initial erythrocyte atta chment. However, polymorphism in msa-1 and msa-2 genes is common among Babesia strains isolated from endemic re gions worldwide (Suarez et al., 2000). The exception is MSA-2c, which is more hi ghly conserved (Hines et al., 1995; FlorinChristensen et al., 2002; Wilkows ky et al., 2003; Carcy et al., 2006). Another protein extensively studied in Babesia spp is rhoptry-associated protein-1 (R AP-1) (Suarez et al., 1998; Suarez et al., 2003). This protein is locate d in the apical complex of B. bovis merozoites and sporozoites and can elicit a humoral response in the hos t (Dalrymple, 1993; Mosqueda et al., 2002a). This protein is highly conserved among ge ographically diverse isolates a nd plays an important role in erythrocyte invasion (Brown et al ., 1996; Norimine et al., 2002). Babesia bigemina strains also possess similar surface proteins as B. bovis They are designated as gp45/gp55 proteins and express a high degree of antigenic polymorphism among different strains of B. bigemina (McElwain et al., 1991; Madruga et al., 1996; Carcy et al., 2006). Gp45 is expressed by the Mexican strain of B. bigemina but it has not been identified in the

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46 strains from PR and St. Croix (McElwain et al., 1987; McElwain et al., 1991; Fisher et al., 2001). These proteins function in the erythrocyte invasion process. Recent studies have identified a novel protein on the surface of B. bovis -infected erythrocytes infected with mature stages of the parasite (Allred et al., 1993; Allred et al., 1994; Allred and Al Khedery, 2006). This protein is linked to th e process of cytoadhesion of erythrocytes to capillary a nd post-capillary venous endothe lium. Adhesion to capillary endothelium acts to sequester the parasites from the peripheral circulation (Aikawa et al., 1992; O'Connor and Allred, 2000). This protein consists of 2 s ubunits and it is termed variant erythrocyte surface antigen 1 (VESA1) (O'Connor et al., 1997). The larger subunit is referred as VESA1a and undergoes rapid antigenic variat ion. The smaller unit, VESA1b, has similar characteristics but its antigeni c properties have not been de termined (O'Connor et al., 1999) (Allred and Al Khedery, 2006). The MSA and RAP proteins are highly conserved within an infected individual and within a population whereas VESA1 is constantly changing. The rapid changes in VESA1 proteins may c ontribute to the development of chronic infection in cattle by prolonging protozoal survival th rough evasion of the immune system (Allred et al., 1994). The above antigenic properties suggest that thes e surface proteins ar e good candidates for the development of BB vaccines (Mosqueda et al., 20 02a; Mosqueda et al., 2002b; Yokoyama et al., 2006; Carcy et al., 2006). Antigenic Diversity and Distribution of B. bovis and B. bigemina Both parasites, B. bovis and B. bigemina are present in many tr opical and sub-tropical regions of the world including Africa, Asia, Aust ralia, Central and South America, and the West Indies (McCosker, 1981; Hong et al., 1997; Bock et al., 2004). Generally, these parasites have similar distributions that directly relate to th e presence of tick vectors. In Central and South America, B. bovis and B. bigemina are found from Uruguay and northern Argentina to

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47 Guatemala, following the distribution of R. (Boophilus) microplus (Guglielmone, 1995). Rhipicephalus (Boophilus) annulatus is responsible for transmitting BB for the majority of northern Central America. In the US, BB is consider ed an exotic disease since the eradication of R. (Boophilus) microplus in 1961 (Pegram et al., 2000). However, there is recent evidence of B. bigemina infections in cattle infested with R. (Boophilus) microplus in south Texas (Guerrero et al., 2007). Most studies concerning differe ntiation of geographic isolates have been performed in Australia (Bock et al., 2000). The main reason for these studies has be en to determine if outbreaks were due to vaccine reversal s. Thirty different isolates of B. bovis have been reported in Australia alone, including the vaccine st rains, Dixie, T, and K (Lew et al., 1997). Host Occurrence and Breed Resistance Babesia spp. tend to be host-specific. B. bovis and B. bigemina mainly infect cattle. Nevertheless, water buffalo and certain wild rumi nants can be infected with both organisms, but deer and elk are not susceptible to infection. However, deer and elk can act as hosts for the tick vectors (Kistner and Hayes, 1970). There also appears to be a breed susceptibility to infection by Babesia spp. Pure Bos taurus cattle are more susceptible to primary B. bovis and B. bigemina infection compared to pure Bos indicus and their crosses (Bock et al., 1997; Bock et al., 1999b). However, for B. bigemina the susceptibility among individuals varies with the virulence of the field isolate. This fact may explain the differences found among studies concerning the infectivity of B. bigemina in different cattle breeds (Bock et al., 1999c). Life Cycle Stages and Development The discussion of the Babesia life cycle will focus on the life cycle of B. bigemina since more information is available on this species in R. (Boophilus) microplus ticks (Hodgson, 1992; Bock et al., 2004). When the tick vector bites an infected animal, it either ingests merozoite-

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48 infected erythrocytes or directly ingests the gametocytes. If inf ected erythrocytes are ingested, then the merozoites develop into gametocytes (also known as Strahle nkrper or ray bodies) within the tick (Hodgson, 1992). On day 0, gut cells phagocytose the gametocytes (Agbede et al., 1986). By day 1, B. bigemina develop from gametocytes into ga metes (Mackenstedt et al., 1995). Two distinct types of gametes are produced, one with a unique organelle called an arrowhead and one without this organe lle (Rudzinska et al., 1983). This organelle is believed to facilitate the parasite escaping from the phagosome. Subseque nt fusion or fertilization (syngamy) occurs between 2 gametes (one of each type) by day 2 a nd produces a spherical shaped zygote with an arrowhead (Friedhoff, 1988). By day 3 or 4, zygot es invade new epithelial cells, including the basophilic or vitellogenic cells6. By day 5, the zygote develops into multiple fission bodies and after the first round of fission, sporogony occu rs, and kinetes are produced within the vitellogenic cells (Agbede et al ., 1986). By day 6 or 7, kinetes escape into the hemolymph and are released into the hemocoele where they in vade other cells and unde rgo indefinite fission cycles until the female tick dies of natural causes (after oviposition) (Agbede et al., 1986). Other cells invaded by the kinetes include hemocytes, peritracheal cells, nutritive cells of the ovary, and to a lesser extent muscle cells, malpip hian tubules and epidermis (Riek, 1964; Friedhoff, 1988; Hodgson, 1992). Oocytes can become infected a nd the parasite might be transmitted to the next generation of ticks (transova rial transmission). Kinetes invade acinar cells in the salivary glands, where a final fission cycle occurs, a nd kinetes develop into infective forms or sporozoites. Kinetes are multinucleated cel ls that split to form sporozoites. In B. bigemina infective sporozoites take appr oximately 9 days to develop and occur only in the nymphal and 6 Vitellogenic cells are secretory cells pr esent only in the engorged female tick. These cells synthesized egg proteins (vitellogenins) which are secreted into the hemolymph and the eggs (Agbede et al., 1986).

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49 adult stages of the ticks (P otgieter and Els, 1977a). In B. bovis formation of infective sporozoites usually occurs within 2 to 3 days of larval tick attachment (Riek, 1966). In the mammalian host (cattle), organism-contam inated saliva from the tick is inoculated during feeding. Babesia do not parasitized any vertebrate cells other than erythrocytes (Friedhoff, 1988). Invasion of erythrocytes by merozoites is categorized into the two main stages of attachment and reorientation, and penetra tion (no evidence of exoerythrocytic stages) (Yokoyama et al., 2006). First attachment of me rozoites to erythrocytes occurs via multiple adhesive interactions of surface proteins with target receptors on the erythrocyte including sialic acid residues or sulfated glycosaminogly cans (Yokoyama et al., 2006). Recently, a study by Okubo et al. (2007) demonstrated that erythrocyte invasion and replication by B. bovis is also dependent upon the amount of cholesterol in er ythrocyte cell membranes. Merozoites then reorient to bring apical organelles close to the attachment interface (Yokoyama et al., 2006). Penetration occurs with the aid of proteins secreted from the apic al complex (Potgieter and Els, 1979; Blackman and Bannister, 2001; Soldati et al., 2001). Once inside the erythrocyte, merozoites are surrounded by 2 membranes, the pa rasite membrane and the parasitophorous vacuole membrane which originated from the erythrocyte membrane (Igarashi et al., 1988; Hodgson, 1992). However, this surrounding host membra ne starts to disintegrate soon after the parasite enters the erythrocyte and merozoites lie in direct contact with the host cytoplasm (Igarashi et al., 1988). This stag e is known as the trophozoite or feeding stage and during this stage organisms undergo asexual division (merogony ) or binary fission within the erythrocyte (Potgieter and Els, 1977b; Young a nd Morzaria, 1986; Friedhoff, 1988). Trophozoites appear to engulf large portions of the cytoplasm of the er ythrocyte through invagination of the parasite plasma membrane (Potgieter and Els, 1977b). Trophozoite division produces 2 daughter

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50 merozoites. In B. bigemina Mackenstedt et al. (1995), identifie d an ovoid type of merozoite that was termed a gamont, but these structures do not further develop until ingested by ticks. Merozoites escape the host cell a nd invade other erythrocytes. As parasites exit the erythrocytes, they damage the membrane by creating perforati ons, protrusions, and inclusions that leads to intravascular hemolysis. This cycle of asexual division conti nues indefinitely, producing more merozoites until host death or until the immune re sponse is able to eliminate the parasites (Uilenberg, 2006). Development of merozoites into gametocytes can occur either in the mammalian cells or after ingestion by the tick vector. Methods of Transmission As in A. marginale B. bovis and B. bigemina are mainly transmitted by infected ixodid ticks. Four species of the genera Rhipicephalus (Boophilus) have been implicated as the major vectors for these protozoa. R. (Boophilus) microplus and R. (Boophilus) annulatus are the most important and widespread vectors in Central a nd South America and the West Indies whereas, R. ( Boophilus) decoloratus and R. (Boophilus) geigy are the vectors in Africa (Jongejan and Uilenberg, 2004; Bock et al., 2004). Babesia bovis and B. bigemina can be transmitted transovarially in R. (Boophilus) microplus ticks. Babesia bovis and B. bigemina appear to infect female s only during the last day of rapid engorgement whereas other instars appear more refractory to the infection (Callow and Hoyte, 1961; Callow, 1968; Friedhoff and Smith, 1981). Larval progeny from infected R. (Boophilus) microplus ticks can transmit B. bovis after 48 to 72 hours but B. bovis does not persist in R. (Boophilus) microplus beyond the larval stage. Conversely, larval progeny from R. (Boophilus) microplus and R. (Boophilus) decoloratus infected with B. bigemina do not transmit the organism (despite being infected) until they molt into nymphs and adults. Nymphs are the principal infectious stage for B. bigemina (Callow and Hoyte, 1961; Mahoney and Mirre, 1979).

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51 Although R. (Boophilus) decoloratus is an efficient vector of B. bigemina, it does not transmit B. bovis. R. ( Boophilus) annulatus can transmit both organisms, B. bovis and B. bigemina transovarially but this vector doe s not become infected as readily as the other 2. In addition, male ticks of all 3 species can transfer B. bigemina and B. bovis intrastadially amongst cattle in close proximity leading to a shorter prepatent period of approximately 6 to 12 days (Callow, 1979). Other reported species of ticks involved in the transmission of B. bovis and B. bigemina are Rhipicephalus evertsi, R. bursa, Haemaphysalis longicornis and, H. punctata (Buscher, 1988; Jongejan and Uilenberg, 2004). Immune Response and Immunity The immune response against B. bovis and B. bigemina infection involves both cellmediated and humoral immune components. The first line of defense in the cell-mediated response is monocytes, macrophages, and ne utrophils. Under the influence of IFN, these cells produce antimicrobial agents in cluding nitric oxide (NO), supe roxide anion, hydrogen peroxide, and hydroxyl radicals to facilitate phagocytosis of organisms (a live and dead) and infected erythrocytes (Auger and Ross, 1992; Shoda et al., 2001). Infected animals develop antibodies directed against multiple parasitic antigens (Goodger et al., 1985). Antibodies play an important ro le as opsonins that e nhance phagocytosis of extracellular merozoites, sporoz oites, and erythrocytes expr essing VESA1 proteins on their surface (Reduker et al., 1989; Jacobson et al., 1993; Allred et al., 2000). Th e release of NO has a greater role in the pathology of B. bovis than B. bigemina infections. Immune complexes form between babesial antigens and antibodies primarily IgM (to a lesser extent IgG1 and IgG2) and Complement C3. These immune complexes can be deposited within the microvascular system during the acute phase of BB contributing towa rds the intravascular sludging of infected erythrocytes (Goodger et al., 1981). In addition, various studies have shown the involvement of

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52 CD4+ T-lymphocytes in acquiring immunity to B. bovis and B. bigemina (Brown et al., 2006b). T-lymphocytes of the CD4+ class produce IFN, which activates macrophages and further enhances IgG2 production (Homer et al., 2000; Estes and Brown, 2002). Young calves exhibit higher resi stance to infection with Babesia spp. compared to adult cattle, and seldom show clinical disease (Mahon ey et al., 1979a; Mahoney et al., 1979b; Goff et al., 2001). This high resistance is partly due to early expression of sp leen-derived immune mechanisms at the time of in fection (Brown et al., 2006b). Innate immune responses in calves with B. bovis infection involve a rapid inducti on of interleukin-12 (IL-12), IFN, and inducible nitric oxydase synthase (iNos) mRNA by a variety of cells in the spleen including Type 1 CD4+ and CD8+ and natural killer cells (NK cells) (Goff et al., 2001; Brown et al., 2006b; Goff et al., 2006b). Under the influence of IL-12, NK cells produce more IFN, which causes the production of more NO and TNFby macrophages (Goff et al., 1998; Homer et al., 2000). Conversely, adult cattle only respond to th e infection by producing IL-12 and IFN(Brown et al., 2006b; Goff et al., 2006b). Natural innate immunity to Babesia spp. in young animals may only last 3 to 9 months (Riek, 1968; Woodford et al., 1990; Ramirez et al., 1998). Immunity in adults can last over 4 years for tick-transmitted B. bovis but less than 6 months for B. bigemina (Mahoney et al., 1973; Mahoney et al., 1979a; Mahoney et al ., 1979b; Bock and de Vos, 2001). Cattle recovered from B. bigemina infections show some resistance to B. bovis However, the reverse may not be true and the degree of cross-protection is seldom adequate (Wright et al., 1987). Endemic Stability Endemic stability for BB depends upon acquired passive immunity from colostrum intake followed by early infection of calves after waning immunity (~2 months). Colostral antibody confers protection to calves that ma y persist for up to 6 months for B. bovis and 3 to 4 months for

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53 B. bigemina (Wright, 1990). Exposure of young animals to large num bers of ticks infected with Babesia during the first 9 months of life ensures solid long-lasti ng immunity (Dalgliesh, 1993; Bock et al., 2004). However, infect ions after 9 months of age are usually severe and can be fatal (Mahoney, 1974). An endemically st able situation occurs when mo re than 75% of the calves are exposed to B. bovis by 6 to 9 months of age. The resul ting incidence of BB will be low and a state of natural stability will exist with a low risk of clinical disease la ter in life (Mahoney, 1974; Smith et al., 2000; Carrique et al., 2000). Conversely, an unstable situa tion arises when less than 75% of calves develop immunity. These populations can have a greater risk of disease because there are sufficient numbers of susceptible an imals and moderate numbers of infected ticks. Populations with fewer than 10% of calves exposed to Babesia before 9 months of age result in a susceptible herd but with a lower risk of disease. The number of infected ticks is not sufficient to infect many animals later in life (Mahoney, 1974; Carrique et al., 2000). Treatment and Prevention The most commonly used chemotherapy for Ba besia infections in cattle is diminazene aceturate (Berenil, Hoechst Ltd.) It has rapid action against B. bovis and B. bigemina and can protect cattle for 2 to 4 weeks (de Vos, 1979). This product is not approved for use within the US and PR (Kuttler and Johnson, 1986). The drug of c hoice in the western hemisphere is imidocarb dipropionate (Imizol, Shering-Plough). Imidocarb can provide protection from B. bovis for 4 weeks and B. bigemina for 2 months or more (Taylor and McHardy, 1979). It is the only babesiacide that consistently clears the host of parasites. Cattle treated with imidocarb may develop complete immunity without becoming a carrier (Lewis et al., 1981). However, acute forms of babesiosis may not be responsive to tr eatment with imidocarb and resistance has been documented to occur in the laboratory (Zintl et al., 2003). This product may also reduce parasite transmission by tick vectors. It has been reported that the progeny of infected R. (Boophilus)

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54 annulatus ticks failed to transmit infection when placed on animals recently treated with imidocarb (Kuttler, 1975; Kuttler and Johnson, 1986). In the US, this product is labeled for use in dogs only. A major side effect of this drug in la boratory rats is an increase incidence of tumors (Bayley, 2005). Concerns about drug residues in meat a nd milk have led to the withdrawal of imidocarb in many European countries (Zintl et al., 2003). Unlike BA, oxytetracyclines frequently are not able to control virule nt field infections (Bock et al., 2004). Currently, there are no safe vaccines availabl e that protect cattle from BB (de Vos and Bock, 2000; Brown et al., 2006a). As mentioned previously, current live vaccines employ whole blood from infected carriers and may protect 95% of animals for life (Bock and de Vos, 2001; Bock et al., 2004). However, the risk of contam ination of these vaccin es with other disease agents makes post-production quali ty control programs essentia l. Animals used for vaccine production in Australia are claimed to be free of B. bovis B. bigemina A. marginale bovine leukemia virus, bovine repiratory syncytial vi rus, bovine immunodeficiency virus, bovine pestivirus, Nesopora caninum Coxiella burnetti and R. (Boophilus) microplus ticks. Most of the available live vaccines are pr oduced in government-s upported facilities in Australia, Argentina, Republic of South Africa, Israel, and Uruguay and ar e provided in a fresh, chilled form. In Australia alone, 35 million dos es were supplied between 1966 and 2003 (Bock et al., 2004). This vaccine is popular because it is easier and less e xpensive to produce and transport than frozen vaccines. The chilled v accine currently produced in Australia contains 1x107 B. bovis 2.5 x 106 B. bigemina and 1 x 107 A. centrale organism per 2 ml dose (Standfast et al., 2003). Chilled vaccines have a short shelf life of approximately 4 days (Bock et al., 2004). Frozen vaccines have a longer shelf life and have become more marketable during the last decade. Since 2001, the only frozen vaccine from Australia marketed nationally and

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55 internationally has been a vaccine concentrat e registered as Combavac 3-in-1, which uses glycerol as cryoprotectant. It contains bovine erythrocytes infected with the 3 major hemoparasites, B. bovis B. bigemina and A. centrale A frozen bivalent B. bovis and B. bigemina vaccine and a frozen monovalent B. bovis or B. bigemina vaccine using dimethyl sulphoxide (20% DMSO) as the cryo protectant have been produced in South Africa and Israel (Pipano, 1997; de Waal and Combrink, 2006). Australian vaccines have a proven efficacy for BB in many different countries including the Caribbean Islands (Bock et al., 2004). Killed and subunit vaccines are not commercially avai lable for use against any of the bovine Babesia spp. nor has a recombinant vaccine for BB been regist ered for use in any country (Bock et al., 2004; de Waal and Combrink, 2006). Epidemiology of the Tropical Cattle Tick Etiologic Agent The tropical or sout hern cattle tick, Rhipicephalus (Boophilus) microplus (Order Parasitiformes, Suborder Ixodida, Family Ixodidae, Subfamily Rhipicephalinae) is one of the 3 distinct species of ticks present in PR a nd it is considered the principal vector of A. marginale B. bovis and B. bigemina (Crom, 1992). Recent phylogenetic analyses have resulted in changes in the nomenclature that supported the placement of all Boophilus species within the genus Rhipicephalus (Murrell et al., 2000; Beati and Keir ans, 2001; Barker and Murrell, 2002). Nevertheless, because of the global popularity of the previous name and the large amount of literature, the name Boophilus was retained as a subgeneric epithet (Beati and Keirans, 2001; Barker and Murrell, 2002; Horak et al., 2002; Murrell and Barker, 2003). The other 2 tick species found on domestic animals in PR include the tropical horse tick, Dermacentor (Anocentor) nitens and the brown dog tick, Rhipicephalus sanguineous (van Volkenberg, 1939).

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56 Rhipicephalus (Boophilus) microplus is an inornate tick with a hexagonal basis capitulum and very short palps and hypostomes. The eyes ar e present and the anal groove is not distinct. Festoons are absent and the legs are a pale cream color (Arundel and Sutherland, 1988). Morphologically, R. (Boophilus) microplus strains from PR tend to be smaller than strains from other locations. A study by Davey et al. (1984) compared 6 groups of male ticks, 3 from Texas, 1 from northern Mexico, and 1 from PR7 to a laboratory-adapted group. The strains from PR had significantly smaller whole-body surface and cau dal appendages compared to other groups (Davey et al., 1984). However, strains from PR8 exhibited substantial genetic similarity to strains from Mexico and Texas (Sattler et al., 1986). Clinical Presentation and Pathology During heavy infestations, the tick can be f ound anywhere on the host. However, they are more frequently observed in the perineum, tail head, brisket and axillary regions, abdomen, and ears (Radunz, 1997). Direct negativ e effects of tick infestation of cattle are primarily due to irritation and blood loss (Radunz, 1997). Hides can also be damaged by tick bites reducing their value in the market (Arundel and Suth erland, 1988; Jongejan and Uilenberg, 2004). Clinical signs from the irritation and blood loss include d ecreased appetite, loss of body condition, loss of production (weight gain and milk production), an emia, and death in heavily infested cattle (Nunez et al., 1982; Arundel and Sutherland, 1988). A single female can consume 0.5 and 3 ml of blood during its parasitic cycle (Nunez et al., 1982). Other problems associated with the feeding of ticks include dermatos is and related reactions such as inflammation, itching, swelling, and ulcerations at the attachme nt site. Additionally, lesions ca used by tick bites may allow 7 Ticks were collected from Holstein cattle on 3 ranches located in the cent ral mountain region near Utuado on February 1978. 8 Ticks were obtained from an established colony from Gurabo.

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57 secondary invasion by other organi sms including screwworm larvae ( Cochliomyia hominovorax ) or bacteria (Bram, 1978). Despite the direct negati ve effects that ticks pose to cattle, transmission of A. marginale B. bovis and B. bigemina by R. (Boophilus) microplus has the greatest economic importance worldwide. Antigenic Diversity and Distribution of Rhipicephalus (Boophilus) microplus Rhipicephalus (Boophilus) microplus is widely distributed in I ndia, Australia, Africa, Asia, regions of Central and South America (from Me xico to Argentina), and the Caribbean (Teel, 1985). In Australia, the distribution is restricted to the northern part of the country. It is established in most of southern and easter n Africa including Ma dagascar (Arundel and Sutherland, 1988; Walk er et al., 2003). The most common biome in Africa for these ticks is the savannah, where there is wooded grassland us ed to graze cattle (Walker et al., 2003). The eradication of R. (Boophilus) microplus from the continental US was completed in 1943. However, 5 limited outbreaks (1945, 1946, 1948, 1958, and 1960) kept the state of Florida from being officially declared free of the southern cattle ticks until 1961 (George, 1990). Today, fever ticks remain in Texas, where they are conf ined to a quarantine area of approximately 800 km long and 0.4 to 16 km wide, that extends along the Rio Grande from De l Rio to Brownsville, Texas (George et al., 2002). Ticks in this regi on serve as a source of possible re-infestation within the rest of the coun try (Guerrero et al., 2007). Many strains of R. (Boophilus) microplus have been identified primarily by studies of acaricide resistance and vaccine susceptibility (Willadsen, 2004). Most ticks that have been studied are from Australia (Yeerongpilly or Ystrain, N-strain, Ultimo, Biarra, Ridgelands, Mackay, Mt. Alford, Gracemere, Silkwood, Ke nilworth, Pimpana), Mexico (Tuxpan, Mora, Santa Luisa, San Roman, Coatzacoalos, San Felipe), Venezuela (Guaimarito), Brazil (Livramento, Porto Alegre, Alegrete, Camaqua, Mo stardas, Pelotas, Gravati), Argentina (A-

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58 strain, Las guerisas, Goya, Maria Isabel), Cuba (Camcord) and Texas, US (Muoz, La Minita) (Nunez et al., 1982; Passos et al., 1999; Willads en, 2004; Howell et al., 2007; Jonsson et al., 2007). Based on gene sequence similarities of the ticks from Mexico, Venezuela, and Argentina, it has been proposed that in South America there is less intra-continental variation than Australia (Willadsen, 2004). The Yeerongpilly or Y-strain, which is an acaricide-susceptible strain, and the Camcord are used for anti-tick vaccine produc tion in Australia and Cuba, respectively (Willadsen, 2004). The Y-strain, N-strain, and Muoz are standard susceptible reference strains (Jonsson et al., 2007). Host Occurrence and Breed Resistance Rhipicephalus (Boophilus) microplus can parasitize several species including cattle, horses, donkeys, sheep, goats, dogs, pigs, and deer (Tate, 1941; Arundel and Sutherland, 1988; Radunz, 1997). Crom et al. (1989) found a strong associa tion between the presence of cattle on premises in PR and infest ation of the animals with R. (Boophilus) microplus, suggesting a preference of cattle over sheep, goats, and horse s (Crom and Duncan, 1989). Infestation of dogs with these ticks has been considered a potential complication of eradicatio n efforts. However, a study in Australia proved that R. (Boophilus) microplus could be eradicated despite the presence of marsupials, cats, pigs, and domestic dogs (Hoyte, 1964). In PR, Tate (1941) examined different animal species under natura l conditions for infestations with R. (Boophilus) microplus Rhipicephalus (Boophilus) microplus was recovered from 16 of 131 horses (12.2%), 82 of 360 sheep (22.8%), 58 of 375 goats (15.5%), 0 of 383 pigs (0%), and 2 of 180 dogs (1.1%). No ticks were found on 27 bats, 15 mongooses, and 5 rats. The author concluded that dogs, pigs, and other small mammals (bats, mongoose, and rats) have little or no importan ce as natural hosts of the southern cattle tick (Tate, 1941). However, a study by (Corn et al., 1994) in Antigua, West Indies documented a 3.2% infestati on prevalence of mongoose (n=150) by R. (Boophilus)

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59 microplus The author described the presence of this tick in mongoose as inci dental and related it to the presence on cattle. The presence of this tick on dogs is rare or accidental in nature (Tate, 1941). Studies in Australia estimated that only 0.01 % (308 adul t females from 3,000,000 larv ae) of cattle ticks were able to mature on dogs and total egg pr oduction was reduced (Hoyte, 1964). A study in PR documented similar results when only 29 and 35 engorged females developed from an initial inoculation of 2,500 larvae in each of 2 dogs (Tate, 1941). White-tailed deer ( Odocoileus virginianus ) can also act as a host for R. (Boophilus) microplus (George, 1990). The presence of whitetailed deer in Florid a between 1930 and 1950 was considered a serious impediment to the tick eradication campaign (George, 1990). A similar situation developed in the US Virgin Island of St. Croix in 1941 when the tick eradication program (TEP) was suspended due to complications with the deer populations. Various studies in St. Croix demonstrated the relationship between R. (Boophilus) microplus and white-tailed deer. Rhipicephalus (Boophilus) microplus can complete its development on deer. However, engorged females that fed on deer tend to weigh 40% less compared to tick s fed on cattle. The ticks were able to convert approximately 58% of their mass into eggs and 94.8% of the eggs hatched (Diaz et al., 1984). White-tailed deer can exist in the natura l environment as th e sole host and can transport ticks to cattle premises (Kistner and Hayes, 1970; George, 1990). Their susceptibility to infection with of A. marginale and Babesia spp. has also been documented under field and laboratory conditions (Maas et al., 1981; Morley and Hugh-Jones, 1989; Holman et al., 1993; Keel et al., 1995). Bos indicus cattle and their crosses develop a gr eater degree of resistance to tick infestations than Bos taurus breeds (Arundel and Sutherland, 1988; Radunz, 1997). In 1978,

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60 Utech assessed the resistance of R. (Boophilus) microplus among beef cattle and dairy breeds. Resistance levels were measured as the percentage of larval ticks (a 1:1 sex ratio being assumed) that mature into engorged females. Percentage le vels were reported as high (98%), moderate (95 to 98%), low (90 to 95%), or very lo w (90%) resistance. In beef cattle, B. indicus (Brahman) were the most resistant of all cattle, 90% ha d 99% resistance with less than 2% of larvae developing into mature stages. B. indicus and B. taurus crosses (Brahman and Australian Illawarra Shorthorn, Belmont Red, Droughtmaster Santa Gertrudis, Braford, Braford and Hereford, Droughtmaster and Hereford; and Sant a Gertrudis and Hereford) allowed less than 10% of larvae to survive and develop into e ngorged females and an estimated 95 to 97% resistance. Purebred B. taurus (Hereford, Shorthorn, Hereford and Shorthorn Crosses, and Charolais) had the least tick resistance of all ca ttle (85%). Among dairy breeds, Jersey cattle had more resistance, 98% compared to Guernsey (93%) and Friesian (85%) (Utech et al., 1978). Life Cycle Stages and Development Parasitic phase Rhipicephalus (Boophilus) microplus is a one-host tick where all instars; larvae, nymphs, and adults remain on the same animal (Radunz, 1997) The life cycle of this tropical tick consists of 2 phases; the parasitic phase during which th e tick feeds on the cattl e and the non-parasitic phase, which the tick spends on the ground. In the parasitic phase, R. (Boophilus) microplus attaches as a six-legged larvae to cattle, a nd undergoes 2 moultings on the host animal until it becomes a sexually differentiated eight-legged adult (Radunz, 2003). A complete parasitic phase typically requires 25 to 30 days. In PR, a study by Tate (1941) showed a range of 18 to 37 days. However, the majority of specimens had comp leted engorgement and dropped by day 25 (Tate, 1941) (Garris and Popham, 1990). During day 0 to 4, larvae attach to the animals and immediately begin feeding (Nunez et al., 1982). Blood engorgement of the larvae occurs within

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61 the subsequent 4 to 8 days. On day 9, larvae molt and emerge as eight-legged nymphs on day 10. Nymphs find a new site of attachment close to th e previous site and feed for another 5 to 6 days, from day 10 to 16, until fully engorged (Nunez et al., 1982; Popham and Garris, 1991). A second moulting occurs on day 17, and by day 18, adult fe males and males emerge. Adult ticks mate on the host and the fertilized females continue feedin g for two more days until fully engorged. From day 18 to 24, females feed near the site wher e they were nymphs (Nun ez et al., 1982). They engorge with blood and undergo a ra pid final blood uptake 12 hours before detachment. Most of the blood consumed during the rapid stage is fo r the production of vitellogenic cells and egg maturation (Coons et al., 1986). This time is also important because this is when B. bovis infections may occur (Agbede et al., 1986). In PR the larval, nymphal, and adult stages ranged from 7 to 12 days, 5 to 17 days9, and 5 to 23 days, respectively (Tate, 1941). Then, detachment from the animal occured and the non-parasitic phase begins. Most de tachments of engorged females occur in the morning hours rather than in the afternoon (Nun ez et al., 1982). Fully engorged male ticks may remain on the host or detach with the female (Radunz, 1997). Males have been known to survive for 70 days either on the host looking for unfer tilized females or in the vegetation (Arundel and Sutherland, 1988; Radunz, 1997). Non-parasitic phase The non-parasitic phase begins with the detachment of fully engorged females and ends when larvae develop and find a suitable host (R adunz, 1997). This phase consists of 4 main stages, pre-oviposition, oviposit ion, incubation/hatching of eggs, and development of larvae. All processes during this phase depend upon enviro nmental conditions including temperature and humidity. Under favorable conditions, high humidity (greater th an 95%) and optimal 9 In this study, most nymphs molted by day 8 or 9; and most adults engorged and dropped from the host by day 10 (Tate, 1941).

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62 temperatures of 24 to 27C (75.2 to 80.6 F), th e non-parasitic phase can be completed in approximately 2 months. However, it can take up to 6 months if conditions are less than optimal. In PR, the non-parasitic phase observed by Tate (1941) ranged from 89 to 251 days (3 to 8.5 months) and depended upon location. In areas with 935 mm (36.8 inches) of annual rainfall and 22.5 C (72.5 F) mean temperature, the length of the phase was 89 days whereas areas with 4,358 mm (171.6 inches) and 22.5 C (72.5 F) the pha se was 251 days (>8 months) (Tate, 1941). Once detached, female ticks seek a suitable shel ter where they rest for a period before laying eggs (Arundel and Sutherland, 1988). This pre-ovi position period can last 2 to 40 days (Radunz, 1997). The mean pre-oviposition period for strains in PR is 3 to 5 days (Davey et al., 1984). High temperatures and low humidity during the summer months in PR result in the poor survival of females (Garris et al., 1990). High mortality rates were observed in engorged females and eggs in the grass when temperatures were ab ove 30 C (86 F) at the ground surface (Garris et al., 1990). The same study estimated that a single female can produce up to 3,500 eggs with the number depending on location and season. The oviposition pe riod may take 2 to 7 days and the female dies upon completion (Radunz, 1997). The mean oviposition period in PR ranged from 11 to 18 days and females died within 5 to 10 days after laying the eggs (Tate, 1941). Once the eggs have been deposited in the environment, the incuba tion time ranges from 14 to 114 days (Arundel and Sutherland, 1988). In PR, the incubation period range d from 18 to 76 days (Tate, 1941). A study by Davey et al. (1984) compared 6 groups of female ticks, 3 from Texas, 1 from northern Mexico, and 1 from PR to a laboratory-adapted group. The strains from PR produced significantly lighter egg masses a nd showed lower hatchability compare to those from northern Mexico and Texas (Davey et al., 1984).

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63 Free-living larvae are approximately 500 m long and 400 m wide, with a slightly ovoid shape and 3 pairs of legs. They are very activ e and immediately seek suitable hosts by climbing up the grass (Arundel and Suther land, 1988). Larvae congregate in small groups at the tip of grasses or other vertical objects, preferentially on the shaded side (particularly during the hours of strong illumination) (Wilkin son, 1953). In PR, a study by Garris (1990) demonstrated that larvae aggregate at heights between 2.5 to 85.1 cm. They either aggregate in a series of tightly arranged rows with their legs pu lled underneath or by forming a ball that is easily transferred to a passing animal (Garris and Popham, 1990). These a ggregations may consist of approximately 70 to 260 larvae (Wilkinson, 1953). Larvae positioned in rows are not active and they remain immobile until disturbed. When cattle or other animals are near, larvae adopt a questing position. This position consists of waving their front legs in the air and moving towards the animal as it passes (Nunez et al., 1982). These aggregations provide a massive and rapid transfer to a passing host and improve the opportunity for mating on the host (Treverrow, 1980). Larval survival on the ground depends upon ambient temp erature and humidity. Larvae do not feed and can easily become desiccated and die at high temperatures (Arundel and Sutherland, 1988). The only method for survival is by absorbing water vapor through their cuticl e, or drinking water droplets from rain or dew (Wilkinson, 1953; Hitchcock, 1955; Wilkinson and Wilson, 1959; Arundel and Sutherland, 1988; Needham and Teel, 1991). The lowest relative humidity (RH) at which water vapor uptake is possible is 85 to 90% (Bowman and Sauer, 2004). A study by Davey et al. (1991) under laboratory conditions, indicated that at RH 75% the determining factor in larval survival was temperature. At each 5C increase in te mperature, there was a corresponding significant d ecrease in larval survival. Conversely, when RH was 63% the larval survival time was short regardless of temperature and the determining factor of larval

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64 survival was the humidity itself. Shade, tall gr ass, and shrub cover may prolong larvae survival (Arundel and Sutherland, 1988). Data from studies in PR estimated that 10% of hatched larvae will survive in grass for up to 47 to 70 days a nd in shaded or wooded areas for 65 to 72 days (Garris and Popham, 1990; Garris et al., 1990). The life cycle in PR, including both parasitic and non-parasitic phases, may range between 41 to 300 days (1 to 9 cycles a year) (Tate, 1941; de la Fuente et al., 2001c). Rhipicephalus (Boophilus) microplus larvae may disperse on the ground for considerable distances (Arundel and Sutherland, 1988). There is abundant evidence that tick larvae can be carried by wind currents through pastures and by cas ual hosts (or hosts other than cattle) (Lewis, 1968). When larvae are located at the tips of the gra ss they can easily be carried away by wind for several meters because of their light weights. Once in the new location they climb nearby herbage and the process can be repeated. It is possible that some will be carried considerable distances by this method (Arundel and Suther land, 1988). A study by (Lewis, 1968), documented that winds can carry larvae for up to 30 m (100 ft) from the site where engorged females were initially dropped for oviposition. In areas where wind currents were low, dispersal of tick larvae was uniform and never extended beyond 5 m (15 ft ). Moreover, larvae can reach heights above 3 m (10 ft) in the air due to strong winds. Tick la rvae can also be transported by casual hosts and dropped in a viable condition. Le wis (1968) documented that a hor se was able to carry tick larvae for 275 m (900 ft), a rat and cockerels for 30 m (100 ft), a magpie for 183 m (600 ft), and a pigeon for 805 m (0.5 mi). In addition, a horse previously dipped with ethion dropped viable tick larvae after transporti ng them for 46 m (150 ft). Immune Response and Immunity Cattle mount either an innate or an acqui red immune response towards feeding ticks (Brossard and Wikel, 2004). At the attachment lo cation, the host develops a local inflammatory

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65 response recruiting neutrophils under the influe nce of IL-8. In addi tion, activation of the complement pathways (classical and alternative) occurs during tick feeding (Nu ttall and Labuda, 2004; Brossard and Wikel, 2004). Repeated tick infestations of the same host can result in acquired immunity. This resistance can cause reduced engorgement weight, prolonga tion of feeding time, d ecreased production of eggs, reduced viability of eggs, moulting inhi bition, and death of feeding ticks (Wikel, 1996). Repeated tick infestations cause cutaneous infla mmatory reactions at tick attachment sites that activate basophils, eosinophils, and mast cells (Willadsen et al., 1995). This specific type of reaction is termed cutaneous basophil hypersen sitivity (CBH), which is a delayed-type hypersensitivity mediated by T helper cells (Brossard and Wikel, 1997; Brossard and Wikel, 2004). When these granulocytes are activated, th ey release bioactive molecules including histamine, leukotrienes, and prostaglandins that further enhance the immune response. Other mechanisms of action involve antigen-presenting cells (Langerhans and Dendritic cells), T-cells, B-cells, and IgG antibodies (Brossard and Wikel, 2004). Langerhan cells engulf tick salivary gland antigens and migrate to draining lymph node s where they transform into dendritic cells and present antigen to T-cells (Brossard and Wikel, 2004). Not surprisingly, ticks have evolved mechan isms to evade the host immune response. Salivary gland extracts from various species of ticks produce different bi nding proteins to many of the cytokines and antibodies involved in the immune response. These include IL-8, IL-2, histamine, and IgG-binding prot eins (Brossard and Wikel, 2004). Furthermore, anti-complement proteins and prostaglandins have also been identified in tick saliva. These proteins inhibit complement binding and T-lymphocyte prolifera tion, respectively (Brossard and Wikel, 2004).

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66 Endemic Stability Endemic stability for BA and BB can only occur when there are adequate numbers of infected ticks present to e xpose all animals while they ar e young and still possessing ageassociated tolerance (Smith et al., 2000; Jongejan and Uilenberg, 2004). When the numbers of ticks decline because of climatic or farm manageme nt factors, then endemic stability can be lost. This is true during the dry seas on in PR when tick numbers can be drastically reduced affecting parasite transmission rates and produci ng a generation of susceptible cattle. The most common farm management factor influencing this stabil ity is the use of acaricides (Smith et al., 2000). Treatment and Prevention Measures used for the control of R. (Boophilus) microplus include stocking resistant cattle, strategic acaricide treatments, biological contro l, and pasture rotation (Radunz, 1997). Integrated pest management strategies employ a combina tion of these methods (Jongejan and Uilenberg, 2004). Any single approach has disadvant ages if it is used as the so le component of tick control programs (Willadsen, 2004). The use of acaricides has caused tick resistance and can result in drug residues in milk and meat (Willadsen, 2004). Acaricides available in the US and PR and approved for use on cattle include proprietary brands of the organophos phates dioxathion (Delnav), coumaphos (Co-Ral), phosmet (Prolate), and crotoxyphos (Ciodrin)10. Other products approved for tick control include pyrethroids (permetrhin), formamidine (Tactikamitraz), and macrocyclic lactone endentocides (ivermectin, eprinomectin, doramectine, and moxid ectin) (George et al., 1998; George et al., 2002). Ivermectin and moxidectin are highly eff ective acaricides but do not prevent transmission 10 Code of Federal Regulations, Title 9: Animals and Animal Products, Chapter I: Animal and Plant Health Inspection Services, Department of Agriculture; Sunchapter C:Interstate Transportation of Animals (Including Poultry) and Animal Products; Part 72:Texas (Splenetic ) Fever in Cattle; Section 72.13: Permitted Dips and Procedures. (Data current as of May 17, 2007)

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67 of Babesia spp. (Bock et al., 2004). Most of these pr oducts are absorbed th rough the cuticle of the tick and affect the centra l nervous system and inhibit ovi position. However, the mechanism of action varies among products. Organophospha tes bind to and inhibit the enzyme acetylcholinesterase causing excessively high leve ls of acetylcholine. Acetylcholine accumulates at neural synapses that lead to continuous neur onal stimulation and para lysis of the tick. The exact mechanism of action for amitraz is unknown, but it may be metabolized into a toxic form after absorption that interferes with the enzyme monoamine oxidase or the octopamine receptor, which is responsible for metabolism of neurotrans mitter amine in the nervous system of the tick (Jonsson and Hope, 2007). Pyrethro ids exert their action by affec ting voltage-sensitive sodium channels that leads to repetitiv e discharges, producing immediate paralysis and death of the tick. Ivermectins are produced by Streptomyces avermitilis and work by acting as a potent agonist at gamma-aminobutyric acid (GABA) receptors. In tic ks, GABA sends inhibitory signals to motor neurons and ivermectins potentiate these inhibitory signals resulting in the paralysis of the tick. In mammals, GABA is confined to the central ne rvous system (CNS), and ivermectin does not cross the blood brain barrier, so it does not cause paralysis (N unez et al., 1982; Webster, 2001; Blagburn and Lindsay, 2001). Recently, a new insecticide called Elector Sp inosad (2.46% active ingredient; Elanco Animal Health, Division of Eli Lilly and Comp any, Greenfield, IN) was added to the list of products approved for use in tick control programs in PR. This product is derived from a mixture of 2 components, Spinosyn A and Spinosyn D, which are natural metabolites produced by the actinomycete Saccharopolyspora spinosa when grown under specific fermentation procedures. The product is labeled for the control of horn f lies and lice on lactati ng and non-lactating dairy and beef cattle; and stable and houseflies on ag ricultural animal prem ises. The product has good

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68 efficacy against flies and it is safe for the enviro nment and personnel. Its potential for the control of R. (Boophilus) microplus has recently being studied. Davey et al. (2001) estimated acute and residual efficacy by comparing 3 different concentrations (0.0167%, 0.05%, and 0.15%) of Spinosad to a control group. Based on the evaluated concen trations, 0.05% and 0.15% had significantly better resu lts than 0.0167% and the control gr oup. No significant difference was found between the low concentration group a nd the control group or the two highest concentration groups. Treatments appeared to be more effective against nymphs and larvae compared to adult ticks present on animals at the time of treatment (Davey et al., 2001). The product provides 85 to 90% control when used at the highest concentrations and the residual activity can last 1 to 2 weeks. However, the le vel of control is inad equate for tick control procedures at US ports-of-entry. Recently, the majority of biological control research has studied fungi of the genera Beauveria and Metarhizium (Frazzon et al., 2000; Gindin et al., 2001; Benjamin et al., 2002). These fungi are major pathogens of ticks with the ability to penetrate the cuticle at high relative humidities (Samish and Rehacek, 1999). A study by Samish and Rehacek (1999), estimated that when using these fungi, the mortality rate in R. (Boophilus) microplus eggs and engorged females was 96 to 100%. However, few field studie s have been performed concerning their value for commercial tick control (B ahiense et al., 2006). A study by Fernandez-Ruvalcaba et al. (2005), estimated the infectivity of Metarhizium anisoplae among organophosphate susceptible strains and an organophosphate resistant strain of R. (Boophilus) microplus One hundred percent mortality was observed in all tic k strains at 20 days post-infec tion (Fernandez-Ruvalcaba et al., 2005).

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69 Two anti-tick vaccines are curr ently available to control R. (Boophilus) microplus infestation: TickGARD (Hoechst Animal Health, Australia) and GAVAC (Heber Biotec S.A. Havana, Cuba) (de la Fuente et al., 1999). Both vaccines use recombinant Bm86 antigens based on an epitope of R. (Boophilus) microplus gut epithelium (Willadsen et al., 1995; de la Fuente et al., 1999). The vaccines differ only in the cloning method and the type of adjuvant used. TickGARD is expressed in E.coli whereas GAVAC uses Pichia pastoris for expression (de Vos et al., 2001; Willadsen, 2006). These vaccines have been shown to reduce the absolute number of engorging female ticks as well as reducing their weight and reproductive capacity. Direct tick mortality is seldom obser ved (Willadsen, 2006). The primary effect for tick control is the reduction of larval infestation ove r subsequent tick generations (Willadsen et al., 1995; Willadsen, 2006). Even though the mechanism of action is not well understood, recombinant vaccines are believed to elicit antibodies that react with antigen on the surface of gut cells causing lysis. A study by Agbede a nd Kemp (1986) employing a vaccine of whole extracts derived from adult female ticks documen ted shedding of destroyed gut cells into the lumen leaving only the basal lamina and muscle layer in the tick. Following the rupture of the gut, cattle erythrocytes and leucoc ytes entered the hemocoele and a ffected other tissues including the Malpighian tubules. This l eakage of bovine erythrocytes a nd hemoglobin changed the color of the hemolymph, causing the affected ticks to become a dark-red color (Agbede and Kemp, 1986; Angus, 1996). Although these vaccines em ploy an antigen from R. (Boophilus) microplus, some efficacy has been documented for R. (Boophilus) annulatus, R. (Boophilus) decoloratus and Hyalomma spp. (Fragoso et al., 1998; de la Fuente et al ., 1999; de Vos et al., 2001; Pipano et al., 2003). Furthermore, anti-tick vaccine studies in C uba using GAVAC have documented a reduction in

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70 the incidence of BA and BB. This reduction wa s presumably caused by the direct effects on reducing tick numbers (de la Fuente et al., 1999; Pipano et al., 2003; Willadsen, 2004). Despite the documented efficacy, anti-tick vaccines are us ed in combination with conventional control measures and they are not currently available for use in the US and PR11 (de la Fuente et al., 1999; Willadsen, 2004). Economic Impact of Rhipicephalus (Boophilus) microplus Bovine Anaplasmosis, and Bovine Babesiosis Worldwide Bovine anaplasmosis and babesiosis are ec onomically important diseases worldwide, especially in tropical and subtr opical regions. Costs are incurred from mortality, loss of milk and meat production, control measures (acaricide treat ments, vaccines, and chemotherapy), and its impact on international catt le trade (Bock et al., 2004). For example, the US annually imports 1 to 2 million head of Mexican cattle and approximately 90% of apprehended animals are infested with R. (Boophilus) microplus (Wagner et al., 2002). The main concern is the introduction and spread of acaricide-resistant R. (Boophilus) ticks and re-establishing of BB in the US (Wagner et al., 2002). Recent outbreaks in sout h Texas have shown evidence of B. bigemina infections in cattle (Guerrero et al., 2007). The most important economic aspect of BA a nd BB in some countries is mortality in imported cattle (Hong et al., 1997; Bock et al., 2004). Total losses have been estimated at US $0.6 million per year in the United States, US $23.3 million per year for Australia, an average of US $ 9.7m/y for African countries, US $57.2m/ y in India, US $3.1m/y for Indonesia and Philippines, and US $19.4m/y for China (Bock et al., 2004). 11 Veterinary Biological Products, Licensees and Permi ttees December 2006, Center for Veterinary Biologics, USDA-APHIS-VS, Marketing and Regulatory Programs.

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71 Caribbean (except Puerto Rico) In Cuba during 1993, BA was estimated to have cost the cattle industry $2 million US dollars (Corona et al., 2005). However, in 2000 mo rtality due to BA had decreased by 10% and was attributed to selection of more resistant breeds of cows and advances in tick control programs. The latter included the us e of recombinant vaccines against R. (Boophilus) microplus and integrated pest management programs (Corona et al., 2005). In the Lesser Antilles, BA and BB exist in an unstable endemic situation with clinical cases observed mainly in dairy and imported cattle (Camus and Montenegro-James, 1994). Seroprevalence reports ranged from 29 to 71% for A. marginale 22 to 69% for B. bovis and 18 to 58% for B. bigemina (Camus and Montenegro-James, 1994) Overall, economic losses due to ticks on Guadeloupe was estimated to be 13.8 million French Francs (approximately $2.8 million US dollars) in 1995 (Barre, 1997). In Jamaica, BA is widely re cognized and outbreaks of BB are associated with the seasonal recrudescence of ticks between December and March (Bundy and Grey, 1982; McGinnis et al., 1989; Camus and Barre, 1995). Puerto Rico An estimated economic loss of $20 million US dollars was reported in 1989 in PR due to anaplasmosis, babesiosis, and R. (Boophilus) microplus (Canestrini) (Crom, 1992). More recently, it has been estimated that the dairy cattle industry suffers a yearly deficit of 3,602,873 kg (7,926,321 lbs) of meat and 14,373,315 L (32,274,840 lb s) of milk due to these diseases (Soto-Alberti, 1999, unpublished data). A recent su rvey by Cortes et al. (2005) among 261 dairy farmers (47,401 milking cows) reported an estima ted loss of $6.7 million US dollars in 2000 due to cattle mortality caused by these diseases and the tropical cattle tick. Add itional costs related to tick control measures were reported as an averag e per farm per year. These included the price of the acaricides (US $2,882), labor (US $ 1,937), e quipment (US $1,324), and miscellaneous (US

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72 $1,012). A yearly amount of US $7,155 was estimate d per farm for tick control or US $29 per cow per year (Cortes et al., 2005). The total cost including dead animals associated with these diseases in addition to tick control measur es on these 261 farms was approximately $8.6 million US dollars a year. In Puerto Rico, the highest mortality occurs among dairy cattle raised in the US and introduced into PR as adults. Improved breeds of dairy cattle, which have not, been crossed with native stock and which have been in the isla nd for only 1 or 2 genera tions also suffer high mortality when an outbreak occurs (van Volkenberg, 1939). History of the tick eradication pr ogram in Puerto Rico-reports on Rhipicephalus (Boophilus) microplus bovine anaplasmosis, and bovine babesiosis Rhipicephalus (Boophilus) microplus was officially recognized in PR in 1899 and the first observation of BA was reported in the 1930s (van Volkenberg, 1939) However, an official tick eradication program (TEP) was not fully institu ted until 1936 after enac tment of Act Number 10612 (Combs, 1989b; Crom, 1992). This Act described the overwhel ming spread of tick fevers in the Commonwealth and established the requir ement for tick elimination and institution of preventive measures. This act empowered the Secret ary of Agriculture to er adicate tropical cattle ticks by the establishment and enforcement of quarantine zones13. Furthermore, it states that livestock producers have an obligation to provi de systematic acaricide treatments on the dates specified by authorized personnel of the Secretary (Suthern and Combs, 1984). This Act and the TEP in PR was modeled after th e continental US Cattle Fever TEP program (1906) (Pegram et al., 2000). In only 5 years after its establishment (1941), the island was released from US federal 12Act of May 15, 1936, Number 106, Sections 1-17, page 5 43 amended by Act of May 6, 1988, Number 24, Sections 1-14, page 104; the Act is titled: Act to promote the elim ination of the cattle-fever tick; to prevent its propagation and spread and to eradicate it. 13 Act of May 6, 1988, Laws of Puerto Rico Annotated (LPRA), Number 24, Chapter 29: Livestock Disease Control, Sub-Chapter IV: Cattle Tick Fever, Sections 741-756, pages 253-265.

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73 quarantine and was declared a tick-free territory (Suthern and Combs, 1984). Nine years later (1950), ticks were re-introduced to PR from th e US Virgin Island of St. Croix. The TEP was immediately re-activated and afte r 3 years of systematic treatmen t of livestock in dipping vats, eradication was completed (Suthern and Co mbs, 1984; Combs, 1989b; Allred et al., 1990) (Popham and Garris, 1991). The last reported R. (Boophilus) microplus tick associated with the St. Croix introduction was in 1952 (Combs, 1989b) By 1954, PR was once again declared free of the tick (Combs, 1989b). After more than 2 decades, R. (Boophilus) microplus was recognized during January 1978 in a slaughterhouse in Maya gez on the western part of the island. The infested cattle were traced back to a farm lo cated in the north-central municipality of Utuado (Combs, 1989a; Combs, 1989b). The US federal government immediately implemented a quarantine and a third attempt to eradicate R. (Boophilus) microplus was initiated in July 1979 (Suthern and Combs, 1984). Approximately 40% of the total herds were reported infested with the tick (Combs, 1989a; Crom, 1992). During this period, the method of acaricide application consisted of spraying the animals rather than using the dipping strate gy because the 400 swimvats used in the 1950s were in poor condition an d reconstruction costs were prohibitive (Crom, 1992; Pegram et al., 2000). Two areas, northwest and southeast, were selected for eradication efforts due to budgetary constraints and polit ical-social conditions. The Northwest region contained the majority of dairy farms (Pegra m et al., 2000). Two years later, in 1981, the proportion of herd infestations increased to approximately 90% (F ox and Leon, 1983; Combs, 1989b; Pegram et al., 2000). During this time, many economic pr oblems were attributed to increased number of BA cases (Combs, 1989b). In April 1985, the first cases of BB were reported on the island. The disease was first diagnosed in a dairy herd in Hatillo (de Leon et al., 1987). However, cases rapidly spread to areas where R. (Boophilus) microplus was previously

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74 established. By October of the same year, there were 300 premis es under quarantine for BB with a total population of 21,044 head of cattle. A to tal of 118 herds located on 151 premises were positive for BB by complement fixation test. Unfort unately, the program received a poor review in 1984 and funds were reduced the following ye ar (Combs, 1989b; Crom, 1992; Pegram et al., 2000). Problems recognized in the review were hi gh turnover rate of employees, reduced supplies and equipment, and poor produ cer compliance (Pegram et al., 2000). Nevertheless, in 1987, another successful eradication was claimed. The development of a computer database for tracking all premises in the TEP was credited with the success (Crom, 1992). In 1989, the TEP was suspended for 6 months because of the devastation caused when Hurricane Hugo hit the island (Crom, 1992; Bokma, 1996). An alarming number of acute deaths diagnosed as BA and BB were reported within the first month of suspension (Bokma, 1996). Since 1990, efforts were maintained and the entire island is under an acti ve eradication campaign in all 78 municipalities. In 1992, another major outbreak of BA and BB occurred (Crom, 1992). This outbreak led to prompt revisions and strengthening of the control measures (Camus and Barre, 1995; Bokma, 1996). A year after the outbreak almost 50% of the herds on the island were claimed to be free of R. (Boophilus) microplus and it was projected that the current TEP would eliminate BA and BB by 1998 (Crom, 1992). Program inconsistencies and a perceived high risk of failure led the US federal government to veto the program in 1995 and withdraw all funds (Pegram et al., 2000). Currently, the program is voluntary and efforts focus on livestock owners controlling the burden of ticks and tick -borne diseases on their own premises. The PR Department of Agriculture (PRDA) subsidizes th e cost of acaricides, equipment, laboratory, and veterinary and consulting services. The USDA An imal and Plant Inspection Service Veterinary Services (APHIS-VS) is no l onger involved in the program.

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75 The TEP in Puerto Rico was designed to control primarily Rhipicephalus (Boophilus) microplus. Initially, it consisted of dipping entire herds in swim-vat s with an arsenical acaricide (1936 to 1954) at 14-day intervals for 15 cons ecutive treatments. Later in the program, the treatments were reduced to 10 to 12 treatm ents at 21-day interv als and consisted of organophosphates as the ma in acaricide (1979 to 1984) (Suthern and Combs, 1984; Crom, 1992). Coumaphos was used mainly in beef cat tle while crotoxyphos (a lpha-methylbenzyl 3hydroxycrotonate dimethyl phosphate) was em ployed on dairy farms. After July 1984, organophosphates were replaced by formamidines (amitraz) and a thirdgeneration pyrethroid, permethrin (Garris and Zimmerman, 1985; Ga rris and George, 1985; Combs, 1989b; Crom, 1992). Since then, TACTIK 12.5% active ingredient emulsi fiable concentrate of amitraz (Intervet Inc., Milsboro, DE Canada) has been used as the primary acaricide. The most common strategies to treat infested premises in the beginning of the TEP included the block treatment system and island-wide approac h. In the block treatment system, all infested premises in a certain geographical region or block were treat ed at the same time until all were free of R. (Boophilus) microplus The island-wide approach consiste d of treating any single infested premises at different times independen tly of the geographic region (Crom, 1992). During the 1980s, funds for the TEP in PR came from 3 main s ources: USDA-APHIS-VS, PRDA, and the USDA Food and Nutrition Servic es Nutrition Assistance Plan (Food Stamp Program) (Combs, 1989b). Approximately US $11 million were spent yearly on the program from 1984 to 1988. The USDA-APHIS-VS cont ributed US $2 million, PRDA US $1 million, and Food Stamp Program US $8 million (Combs, 1989a; Combs, 1989b). Forty-four million dollars were spent in only 4 years (1984-1988) a nd a reduction in tick infestations were not observed (Combs, 1989a). Lack of success was attributed to illegal cattle movements across

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76 quarantine lines between infested and non-infested premises, lack of quarantine enforcement, and feeding of grass containing ticks (S uthern and Combs, 1984; Combs, 1989b). Most farms had limited pasture that did not a llow for removal of cattle from areas for extended periods of time. Moreover, large numbers of producers kept non-lactating pre-parturie nt cows and heifers outside treatment zones at differe nt premises than the milking cows (Suthern and Combs, 1984). Stray animals were also a concern because they can carry ticks between premises (Suthern and Combs, 1984). Other soci al and ecological impediments include d reluctance of farmers to accept the program and heavy rainfall in treatment areas (Pegram et al., 2000). Puerto Rico and the importance of the dairy industry Puerto Rico is centered in the Caribbean basin (18 N, 66 W). It is the smallest and the most eastern island of the Greater Antilles, east of Hispaniola and northwest of the Virgin Islands. It is bounded on the north by the Atlantic Ocean and south by the Caribbean Sea. Puerto Rico consists of a main island, which is almost rectangular in shape, w ith a length of 180 km, and a width of 65 km. It encompasses an area of 8,870 square kilometers of which 4% is dedicated to agricultural purposes. Six adjacent is lets surround the main island; Mona, Monito, and Desecheo, which are uninhabited and serve as w ildlife reserves; Caja de Muertos; which is a recreational area; and Vieques and Culebra which are inhabited and are considered municipalities. (Figure 2-1) In 1898, after the Span ish-American War, Puerto Rico was ceded to the US. As a commonwealth associ ated with the United States, Pu erto Rico does not have any first-order administrative divisions (i.e. states), as defined by the U.S. Government, but there are 78 second-order municipalities. (Figure 1-1) Ea ch municipality has a mayor and a municipal legislature. Popularly-elected governors have served since 1948. The human population of PR is approximately 3.9 million with an average annual population growth rate of 0.4% percent in 2006. It is the most densely populated island in the world with approximately 430 people per

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77 square kilometer, a ratio higher than within any of the 50 states in the US. Puerto Rico is under US customs jurisdiction and borders are open between the island and the US, allowing movement of people, animals, and me rchandise (NOAA-NCDC, 1982; CIA, 2007). The topography of PR is very rugged, except along the coast, and consists of 3 main physiographic regions: the mountai nous interior, the coastal lowl ands, and the karst area. The mountainous interior is formed by a major mountain range or cordillera which extends across the interior of the island from Mayagez to Aib onito, and transects the is land from east to west. (Figure 2-2) The coastal lowlands extend 13 to 19 km (8 to 12 mi) inward in the north and 3 to 13 km (2 to 8 mi) in the south (NOAA-NCDC, 1982). A series of smaller valleys lie perpendicular near the west and east coasts. The ka rst region is located in the north. This area consists of formations of rugged volcanic rock dissolved by water. This limestone region has numerous mogotes or hayst ack hills (approximately 20% of PR), sinkholes, caves, and limestone cliffs. The karst belt extends from Aguadilla, in the west, to a minor haystack hill formation in Loza, just east of San Juan. The climate is tropical marine with regular temperatures of 26.7 C (80F). In the interior, at higher elevations, the temperature fluctuates between 22.8 to 25.6C (73 to 78F). Mean temperat ures in PR have very small range between the warmest and coldest months. For example, in San Juan the temperature in the warmest month is 27.1C (80.8F) whereas in January and Februa ry it is 23.9C (75.1F). These small annual temperature ranges are due to 2 main factors. Fi rst, PR is an island su rrounded by waters whose temperature changes are minimal from the warm est to the coolest season, 28.1C (82.5F) in September to 26.6C (77.9F) in February. Second, it is also due to the small difference in the energy received form the sun from season to seas on because of the distance from the Equator, which is approximately 1,770 km (1,100 miles) no rth. The difference between the length of the

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78 longest day (13 hours 13 min) a nd the shortest day (11 hours and 2 min) is approximately two hours (NOAA-NCDC, 1982). The majority of PRs rainfall is orographic in nature. Moisture laden air from the ocean is carried by the trade winds inland and forced to ascend over the m ountains, cooling the air, thus causing condensation and rainfall (NOAA-NCDC 1982). The northern half of the island receives more rainfall and has larger rivers th an the drier southern ha lf. The distribution of rainfall over the year does not show an absolu te wet-dry season relations hip, but only a relatively dry season and relatively wet season. The length of the dry season varies by location in the island. In the northern region of th e island, the dry season is genera lly shorter (February April) whereas in the southern region, th e dry season begins in December In both regions of the island, the dry season ends with the onset of the wet season in May, but there is usually a transitional period of approximately 1 month be tween these seasons (NOAA-NCDC, 1982). However, the dry season is not always from December to Apr il nor is the wet season from May to November. Some of the heaviest rainfalls have occurred during the dry seas on. In general, the driest month is either February or March. May is the rainie st month in the northern region and September or October the rainiest month in the south (NOAA-NCDC, 1982). The relative humidity (RH) is approximately 80% over the course of the year The highest RHs are generally found during the night (>90%) when temperatures are the lowest During the day, RH ranges from 60% to 70%. As temperature increases, the RH decreases, reac hing its lowest point at the time of maximum temperatures.

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79 Besides these 3 major physiographic regions, different classification schemes exist for major land resource areas (MLRAs) in Puerto Rico14. One approach is based on a scientific classification by the USDA Natural Resources Conservation Service (NRCS) and can be summarized into 5 general soil types: humid co astal plains, semiarid coastal plains, humid mountains and valleys, and semiarid mountains and valleys. The humid coastal plains have rolling hill topography and an average annual precipitation of 1600 mm (63 in) and annual temperature of 25C (77F). Pasture-based commerc ial dairy farms comprise most of this area. Native and improved tropical gras ses cover approximately 50% of the land, whereas 17% of the acreage is used for a variety of crops. Urban de velopment is significant, especially around the large metropolitan regions. The semiarid coastal pl ains have alluvial flat terrains with an average annual precipitation of 900 mm (35 in) and averag e annual temperature of 26C (78.8F). More than 50% of this area is comprised of native a nd improved grasses, which are mainly used for feeding in beef cattle and racehorses. The humid mountains and va lleys have irregular topography with elevations of 500 to 600 m and av erage annual precipitation of 2100 mm (83 in) and annual temperature of 24C (75. 2F). Approximately 70% of this area is comprised of native and improved grasses, 10% is comprised of coffee plantations, and 7% is covered by forests. The remainder is used for food crops including plaint ains, bananas, and yams. Lastly, the semiarid mountains and valleys have floode d-alluvial plains and an aver age annual preci pitation of 1150 mm (45 in) and average annual temperature of 26C (78.8F). Approximate ly 60% is comprised of native grasses and 35% is c overed by forest (NRCS-USDA, 2007). 14 MLRAs are geographically associated land resource units, usually encompassing several thousand acres, characterized by a particular pattern of soils, geology climate, water resources, and land use. A unit can be a continuous area or several separate nearby areas (NRCS-USDA, 2007).

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80 Another classification by the US Departme nt of Commerce, National Oceanic, and Atmospheric Administration-National Climatic Data Center (NOAA-NCDC ) groups the island's regions according to the rela tive homogeneity of the zone by precipitation and temperature (Guttman and Quayle, 1996)(Stephens, 2007, personnal communication) 15. This approach results in a similar pattern as the one presen ted by the USDA-NRCS, except for 2 additional subdivisions. These are the north coastal regi on, northern slopes region, south coastal region, southern slopes region, eastern interior region, and western interior region. Lastly, a third classification sc heme includes the Holdridge syst em of ecological life zones by Ewel and Whitmore (1973) and subsequently expanded by Helmer et al. (2002) using satellite-image based mapping of fo rest type and landcover of PR. The Holdridge system divides PR into 6 life zones16: subtropical dry forest (13.8% of to tal area), subtropical moist forest (60.5%), subtropical wet forest (2.6 %), subtropical rain fore st (0.1%), subtropical lower montane wet forest (1.2%), and subtropical lowe r montane rain forest (0.1%). The subtropical dry forest is the driest of the 6 zones with a mean annual precipitation range of 600 to 1000 mm (23.6 to 39.4 in). Most of the vegetation consists of grasses, cacti, thor ny legumes, and trees with small and succulent leaves. Fires are common duri ng the dry season. The subtropical moist forest zone is the largest zone in PR with a mean annual precipitation of 1000 to 2200 mm (39.4 to 86.6 in) and mean temperatures of 18 to 24C (64.4 to 75.2 F). This is the most deforested zone partly because of intensive agricultural activity and urbanization. Both native and improved pastures form the dominant landscape in this zone. The subt ropical wet forest zone occupies much of the 15 Scott E. Stephens, Meteorologist, Customer Services /Climate Monitoring NOAAs National Climatic Data Center, 151 Patton Avenue, Asheville, NC 28801-5 001, V: 828-271-4800 F: 828-271-4876, E-Mail: Scott.Stephens@noaa.gov http://www.ncdc.noaa.gov/monitoring 16 Life zones are mapped according to broad bioclimatic units, each of which encompasses a variety of soils, vegetation, microclimates and land use patterns. Each life zone lies within a latitudinal region, an altitudinal belt, and a humidity province. The variables used to delineate any given life zone are mean annual precipitation and mean annual temperature (Ewe l and Whitmore, 1973).

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81 highest parts of the mountains. This zone ha s mean annual precipitation ranges of 2000 to 4000 mm (78.7 to 157.5 in). Because of the abundant mois ture, most of the vegetation consists of epiphytic ferns, bromeliads, and orchids. Most of this life zone, particularly in the western portion of the island is covered by coffee planta tions. The subtropical rain forest zone, the subtropical lower montane wet fore st zone, and the subtropical lo wer montane rain forest zone are the wettest of the life zones, occupying a small area in PR. These 3 zones are mostly located in the Luquillo mountains in the eastern part of the island, particularly in El Yunque National Forest (formerly known as the Caribbean National Fo rest), the only tropical rainforest in the US. Most of the vegetation in the s ubtropical rain forest zone consists of palm trees and epiphytes. The subtropical lower montane wet forest zone is comprised of the Colorado forest type, named for the common palo colorado or swamp cyrilla ( Cyrilla racemiflora L.), and the cloud-forest type (elfin woodland, montane thic ket, or dwarf forest). The vege tation is characterized by short (<7 m tall) gnarled trees with high basal area, small diameter, slow growth rates, and many epiphytes. The subtropical lower montane rain fo rest zone is found only in a narrow band on the windward slopes of the Luquillo Mountains immedi ately above the subtropical rain forest. The mean annual temperature is approximately 18.6C (65.5F), annual precipitation is 4533 mm (178.5 in), and mean relative humidity is 98.5%. Th e vegetation is similar to that of lower montane wet forest, except for greater abundance of epiphytes, palms, and fern trees (Ewel and Whitmore, 1973). Helmer et al. (2002) used Landsat TM im agery data from 1991 to 1992 to develop a detailed map of PRs forest t ypes and landcover. The vegetation classification system used in this scheme required adaptations from different sources including the Ewel and Whitmore life zones. The resulting map has 31 different landcover classes. A pproximately 41.6% of the land is

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82 covered by closed forests (the majority was clas sified as tropical broadleaf evergreen forest) from which only 5% is under protection, 36.7% is covered by pastures and grasslands, 5.9% by agriculture, 2.4% by coffee planta tions, and 10.5% by urban and developed landcover (Helmer et al., 2002). As previously observed, these classification schemes coin cide with similar descriptive patterns for the various zones in the island. Despite the fact that manufacturing (pet rochemicals, pharmaceuticals and technology) accounts for a gross domestic product (GDP) of US $36,555.8 millions, 42.3% in 2006 (total GDP US $86,481.0) (Planning Board of the Commonweal th of Puerto Rico, 2003), agriculture is still an important industrial sector for lo cal economies. Agriculture contributed US $805.6 millions or 1% of GDP in FY 2005 to 2006 17. Production systems on the island vary according to agricultural region. The Agricultural Extensio n Service, established in Puerto Rico in 1934 through an agreement between the United States De partment of Agriculture and the University of Puerto Rico, has establishe d 8 agricultural regions in PR. This subdivision is based on the number of producers and corresponding agricu ltural commodities produced in each region. These regions include Arecibo, Caguas, Lajas, La res, Mayagez, Naranjito, Ponce, and Utuado (Ralat, 2004, personnal communication)18 (Figure 2-3). The dairy industry, mainly loca ted in the region of Arecibo, has been the most important agricultural industry in Puerto Rico since 1948 (Molina-Fern andez, 2001). In 2006, the dairy industry contributed 25.6 % (US $184.8 million) of the GDP in agriculture followed by poultry (US $89.4 millions, 12.4% GDP), and plantain s (US $67.5 millions, 9.4% GDP) (PRDA-ASO, 2006). Producer returns in 2004 averaged $23.90 per hundredweight ($0.516 per quart) whereas 17 FY: Fiscal Year from July 1, 2005 to June 30, 2006. 18 Johnny Ralat, Office for the Agricultural Economic De velopment of Puerto Rico, Department of Agriculture, Commonwealth of Pu erto Rico, 2004.

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83 returns in the US averaged $16.13 per hundredweight. Three percen t of the labor force or 39,000 jobs are dedicated to agriculture. From these, 25,000 are related to the production, manufacture, and sale of milk and milk by-products (ORILPRDA, 2006). The average price of gallon of milk was US $2.17 (1 liter US $1.05) during 2005 to 2006. In Puerto Rico, there are approximately 281,371 cattle. The dair y industry includes 153,097 of these animals of which 88,322 are la ctating and dry cows (NASS-USDA, 2004; PRDA-ASO, 2006). Most cows are located within 353 commercial dairy farms operating in PR as of September 30, 2006. The majority of these farms, 61.8% (218 farms), are located in the agricultural region of Arecibo, 15.3% (54) in Caguas, and 22.9% (81) in Mayagez. During FY 2005 to 2006, dairy herds in PR produced 329 million liters (709 million pounds) of milk. Production per cow averaged 3,850 liters (8,277 pounds). Some of the dairy cattle in PR have been imported from the US a nd the most common breed is Holstein. However, crossbreeding programs with ot her breeds including Jersey a nd Brown Swiss are common. In 2005, the average herd size was 243 cows with an average daily milk production per cow of approximately 16 L. The common dairy ration in PR consists of pelleted feed (concentrates) and tropical grasses. During FY 2005 to 2006 concentr ates represented 47% of the production costs to the producer. Pelleted feed ingredients incl ude grain by-products, mo lasses, vitamins, and mineral supplements imported from US contin ental sources. The most common grasses are bermuda grass ( Cynodon dactylon ), guinea grass ( Panicum maximum ), and pangola grass ( Digitaria decumbens ). The soils in PR do not provide th e optimum environment for legumes to grow and therefore most legumes, especia lly alfalfa, are imported from the US. Demand for milk and milk by-products by the po pulation of PR increases almost daily. To sustain this need, it is important to maintain a nd improve the efficiency of this industry. Fresh

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84 milk is not imported onto the island, the entire commodity must be produ ced locally for national consumption. Puerto Rico is one of most de nsely populated places in the world (~4 million inhabitants in 9,104 km2) with a high demand for milk, and th erefore the dairy industry should be provided with the resources necessary to mainta in a continuous and efficient production system. Importance of the Bovine Anaplasmosis and Babesiosis-Tick-Cattle-Ecological System and Its Implications in Epidemiological Studies Epidemiological studies of BA and BB require focusing on the disease process as a system rather than considering A. marginale and Babesia spp as the sole cause of disease (Melendez, 1998). This system should evaluate how all contribu ting factors affect the presence and level of disease. Environmental factors are composed of mu ltiple variables that have their own scales of space and time, where higher-leve l factors constrain and contro l the lower-level factors to various degrees. Conversely, lowe r-level factors should provide details that may explain the behavior observed at higher-lev el factors (Turner et al., 2001). When using this hierarchical concept, the investigator should always consider at least 3 le vels in any study, the level of interest, one above, and one below (Turner et al., 2001). Studies of BA and BB should not ignore this hierarchical perspective (Figure 2-4). Anaplasma marginale and Babesia spp. are the lowest point of the system; they depend on the R. (Boophilus) ticks and cattle for persistence (Arellano-So ta, 1992; Guglielmone, 1995; Melendez, 1998). Second, they are preceded by R. (Boophilus) ticks, which depend upon the distribution of cattle (and other hosts) to complete their paras itic lifecycle. Third, cattle must be present to support both diseases and the tick vector, but thei r presence is independent of either (ArellanoSota, 1992). Fourth, farm management factors and ecological and geographical conditions influence the organisms, the host, and the lifecycle of R. (Boophilus) ticks. A disturbance of any of these factors at any level may influence the dynamics of the system (Melendez, 1998).

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85 Conventional methods of disease control that include the regular dipping of cattle to control the tick vectors plus chemotherapy and vacc ination to decrease the incidence of clinical disease are based on data from studies in temp erate regions and such results might not be applicable to tropical and subtr opical areas (Wagner et al., 2002). Therefore, studies of BA and BB in tropical enzootic areas should collect info rmation concerning the biology of the diseases, potential carrier hosts, risk factors associated with the transmission of A. marginale and Babesia spp., and geographic locations and local climatic conditions suitabl e for the presence of BA, BB, and tick vectors (Richey and Palmer, 1990; Alons o et al., 1992; George et al., 2002). This information is necessary to develop sustainable ti ck and tick-borne disease control programs in cattle operations (McCluskey, 2002; Jongejan and Uilenberg, 2004). Defining the Bovine Anaplasmosis and Babesiosis-Tick-Cattle-Ecological System The components of the Bovine Anaplasmos is and Babesiosis-Tick-Cattle-Ecological System have a complex interrelationship that keeps the system viable and ensures the propagation of the disease agents and tick (Alonso et al., 1992). This complex relationship is what determines where and when disease will occur (McCluskey, 2002) Alonso et al. (1992) defined the interrelationship for BB only as a clo sed system, where hosts other than cattle and vectors other than ticks are not considered. However, if BA is included within the system, the investigator must overcome 3 major c onstraints of this approach. First, A. marginale Babesia spp., and R. (Boophilus) microplus can survive in association w ith other domestic animals and wildlife hosts (Ristic, 1968; Ristic, 1977). Second, this approach only considers the distribution of Babesia spp. related to the tick vector, excluding the more complex methods of transmission described for A. marginale As mentioned previously, m echanical transmission of A. marginale by biting flies and blood-infected fomites is commonly assumed to be important in the epidemiology of BA in some areas of the US. Howe ver, the scientific lite rature is inconclusive

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86 and further investigations are re quired to completely elucidate their role in the transmission of A. marginale (Scoles et al., 2005). Third, local manage ment procedures and ecological conditions may have important differences among regions, a nd their role in the system should not be ignored. Therefore, all parameters involved in the transmission of A. marginale and Babesia spp. should be studied collectively, to allow correct inferences about the disease processes. Key Factors of the Epidemiology A. marginale and Babesia spp. Anaplasma marginale and B. bovis (B. bigemina) share many important epidemiological similarities along the pathway of disease. The diseases are co-endemic in tropical and subtropical regions and share a similar seasonal pattern. Both are intraerythrocytic parasites that can be transmitted by ixodid ticks, especially R. (Boophilus) microplus at a very low level of parasitemia. They have similar incubation peri ods of approximately 3 to 4 weeks and similar clinical presentations that in clude the potential for survivors to become carriers. Lastly, resistance can be acquired at a young age by pa ssive transfer of antibodies from colostrum (Alonso et al., 1992). Conversely, these organisms also have striking differences in their pathway of disease. Anaplasma marginale is susceptible to antibiotic ther apy whereas few effective drugs are available to control infection with Babesia spp. Trans-ovarian transmissi on in the tick vector is not well documented for A. marginale whereas it is important in the epidemiology of Babesia spp. infections. Infection with A. marginale persists for the life of th e animal, with cycles of rickettsemia occurring every 14 days whereas infection with Babesia spp. is not lifelong. The protozoa can survive up to 6 months in the hos t, and then disappear le aving a sterile immunity persisting for another 6 months. Subsequently, immunity is lost and the animal may become infected again. Lastly, transplacental in fection of cattle has been described for A. marginale but not for Babesia spp.

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87 Examples of A. marginale and Babesia spp. and Their Influence on the System Pathogenic effects due to infection with Babesia spp. have been observed in certain ticks. Rhipicephalus (Boophilus) ticks infected with high numbers of B. bovis or B. bigemina may have shorter duration of oviposition, higher mortality of infected females, decreased egg production, reduced egg hatching proportion, and reduced la rval survival (Samish and Rehacek, 1999; Howell et al., 2007). The severity of these effects is directly related to th e degree of parasitemia and to the susceptibility of the tick for certain Babesia strains. High levels of B. bigemina parasitemias may result in up to 98% mortal ity in female ticks (Hodgson, 1992; Samish and Rehacek, 1999). Conversely, A. marginale infection appears to have no detrimental effects on ticks (Kocan, 1995). Nevertheless, a study by Cen-Aguilar et al. (1998) did not find significant differences among 3 groups (incl uding uninfected) of tic ks infected at di fferent levels of Babesia spp organisms under field conditions in Yucatn, Mexico. The author suggested that an adaptive tolerance of Babesia spp. might develop in naturally infected R. (Boophilus) microplus (CenAguilar et al., 1998). Furthermore, (Oliveira et al., 2005) found that Babesia spp. infection was more frequent in ticks and eggs collected from calves compared to cows but the hatching rate was lower in samples from calves. Examples of Arthropod Factors and Their Influence on the System Understanding arthropod breeding cycles and hab itats is imperative for the development of control strategies (McCluskey, 2002). Vector competence and vectoria l capacity are major factors in the risk of transmission of A. marginale and Babesia spp. Vector competence includes feeding frequency and timing, vector mobility or fli ght ranges, and vector adaptability to biotic (animal) and abiotic (environment) conditions (i ncluding application of insecticides) (Harwood, 1981; McCluskey, 2002). Under natural conditions, mortality proportions in the parasitic phase ranges between 2 to 7% in the larval stage and 20 to 40% in the adult st age. In a study by Nunez

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88 et al. (1972) that experimental ly infested cattle with 20,000 la rvae, only 3,176 engorged females were collected suggesting a mortality proportion of 60%. For example, a large percentage (74 to 90%) of larvae that attach to su sceptible cattle do not complete their cycle. The majority of mortality occurs during the first 24 hours after fi xation on the host. If la rvae have not attached and feed within the first few hours after climbi ng onto a host, the unattach ed larvae will die from the hosts body heat (Roberts, 1971). Fluid (blood ) uptake is sufficient to prevent desiccation of the majority of the attached larvae (Roberts, 1968a; Roberts, 1968b; Koudstaal et al., 1978). Moreover, death seems more rapid in the skin of the host during atmospheric conditions of low humidity (~30%) and high temperatures (32C) (Roberts, 1971) Different tick species and vectors have different vectorial capacity for A. marginale and Babesia spp. The ability of the tick vector to acquire A. marginale from a persistently infected animal is influenced by the level of rickettsemia during feeding, and not all fed ticks will become infected. However, tick infection rates with A. marginale in enzootics areas can reach 100% (Dallwitz, 1987). Vectorial capacity in hematophagous in sects is measured by the proportion of fed flies that carry A. marginale infected blood on their mouthparts and the number of A. marginale infective units maintained in a viable st ate before feeding on a second host (Scoles et al., 2005). Examples of Cattle Factors and Their Influence on the System Bos indicus cattle have shown lower leve ls of parasitemias for B. bovis and B. bigemina when compared to B. taurus (Guglielmone et al., 1989; Aguirr e et al., 1994) In addition, the percentage of R. (Boophilus) microplu s engorged females infected with Babesia spp varied from 0 to 5% in B. indicus herds and 18 to 40% in B. taurus herds. (Guglielmone et al., 1989) A study in Australia reported that onl y 0.04% to 0.05% of tick larvae collected from a grazing paddock occupied by European breeds ( B. taurus) were infected with B. bovis whereas larvae collected

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89 from the same pasture but occupied by Zebu x European cattle ( B. indicus x B. taurus ) failed to demonstrate the presence of B. bovis (Mahoney et al., 1981) In addition, a study by Mahoney and Mirre (1971) documented that R. (Boophilus) microplu s infections with B. bigemina (0.23%) are higher than B. bovis (0.04%) (Mahoney and Mirre, 1971). Therefore, transmission of B. bigemina infections are more likely to develop in those herds where both Babesia species are present (Bock et al., 2004). Cattle behavior is an important factor in the propagation of R. (Boophilus) microplu s. Cattle do not remain passive to attack s of insect vectors (McCluskey, 2002). They protect themselves by tail switching, ear flapping, skin tw itching, immersion in water, coating with mud, or crowding together. In addition, cattle have the ability to detect and avoid areas of high larval density on pastures. Vision appeared to be importa nt in detecting heavily contaminated foci or big clumps of dark brown larvae at the tip of the grass (Sutherst et al., 1986). Studies by Tate (1941) and Koudstaal et al. (1978), demonstrated that cattle inoc ulated with larvae immediately became irritated and began scratching and licking themselves resulting in the removal of a considerable number of larvae. Animals that actively scratch within the fi rst 24 hours maintain a lower number of larvae than those that do not (Koudstaal et al., 1978). Conversely, the nymphs are primarily affected by host resistance. It is speculated that they can be removed by grooming due to higher levels of histamin e resulting from the immune respons e to salivary gland secretions (Doube and Kemp, 1979). Examples of Farm Management Factor s and Their Influence on the System The interactions among R. (Boophilus) A. marginale Babesia spp. and cattle can result in different outcomes due to differences in animal husbandry practices. In e ndemic areas, control of the vector and disease are more difficult and mo re costly. The disease can be controlled by eradicating R. (Boophilus) or by keeping the numbers low to avoid high occurrence of outbreaks

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90 (de Waal and Combrink, 2006). For this strategy to succeed an extremely intensive acaricide program is essential. However, this strategy carri es high risks as regards to the development of acaricide resistance and disruption of endemic st ability (Jonsson et al., 2000; Mekonnen et al., 2002). Outbreaks of BB in dairy cattle have been a ssociated with excessive tick control including in Australia where the intensiv e use of acaricides and pasture spelling brought about enzootic instability (Sing et al., 1983; G uglielmone, 1994; Guglielmone, 1995). In most cases, acaricide resistance depends upon the active ingredient, length of time that the acaricide has been used on the premises, a nd inappropriate use of acaricide (Nunez et al., 1982). Factors related to inappropriate use of acaricides include in adequate time of application, insufficient contact time (i.e. hea vy rains after applications), inco mplete coverage of the animal, and reduced concentration of th e active ingredient (Nunez et al., 1982). For example, amitraz has been used for over 30 years in the control of R. (Boophilus) microplus Its use is common in Australia, Latin America and southern Africa (J onsson and Hope, 2007). It is currently the main acaricide available in Puerto Rico for spray applic ation to dairy cows. Not surprisingly, acaricide resistance has been reported (Jonsson and Hope, 2007). The first reports we re from Australia in 1980 and today there is an estimated resistance prevalence of 11% (11% tick survival). Conversely, amitraz resistance in Mexico was not identified until 2001 but a faster development has been observed since its introduction in 1986 w ith an estimated resistance of 19% (Jonsson and Hope, 2007). Large numbers of B. taurus breeds are imported into tropica l and subtropical countries to improve local livestock industries. Unfortunately, this practice has led to outbreaks of BB, death of animals, and devastating economic losses (Callow, 1977; Bock et al., 2004).

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91 Examples of Environmental Factors and Their Influence on the System Environmental factors have a great in fluence on the non-parasitic phase of R. (Boophilus) microplus tick infestation rates on cattle, and babe sial infection rates for cattle and ticks (Guglielmone, 1995; Jongejan and Uilenberg, 2004). Climate influences the longevity of larvae, number of annual generations of R. (Boophilus) microplus and host seeking-finding activity (Utech et al., 1983). In suitable conditions, heavy rains, tall grass stems (which provide plentiful shade) and high temperatures may shorten the development period and the non-parasitic phase can be less than 3 weeks. Conversely, cold w eather and poor rains can diminish grass quantity and reduce the chances that gravid females and larv ae will find shady sites. Furthermore, larvae that spend 2 to 4 weeks on the ground before infes ting cattle are more likel y to spread babesiosis than newly hatched larvae. It a ppears that babesia need a dormant period within the non-parasitic larvae to complete development. Cool w eather (~14C) enhances this development. Transovarian transmission of Babesia spp. is also affected by climatic factors (Riek, 1964; Dalgliesh and Steward, 1982). In one study, different temperatures influenced the development of Babesia spp. in R. (Boophilus) microplus whereas A. marginale development was not affected (Dalgliesh and Stewart, 1979). In another study, exposure of la rvae at 14C over 65 days increased infection with B. bovis but not B. bigemina However, these larvae were not infective after 76 days at same incubation temp erature (Dalgliesh and Steward, 1982). The Importance of Reliable Diagn ostics for Identification of A. marginale B. bovis and B. bigemina infections Diagnostic methods for BA and BB should be able to accurate ly estimate the prevalence of infection in any geographic loca tion including persistently infected cattle (carriers). As mentioned previously, persistently infected cat tle serve as a reservoir for the spread of A. marginale B. bovis and B. bigemina Therefore, their detection is important for surveillance and

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92 eradication programs. However, all current me thods have limitations in specificity (Sp), sensitivity (Se), or reproducibility (Goff et al., 2006a). Microscopic Detection Direct peripheral blood smears stained with Giemsa stain are the most commonly used technique in developing countries for identifying anaplasma and babesia infections. This test is easy to perform and inexpensive. However, this t echnique is most sensitiv e during the acute form of BA when parasitemia levels of erythrocytes can reach 0.1 to 0.2 % (Eriks et al., 1989). During the persistent form of BA, para sites are seldom detected with this technique because of the extremely low percentage of infected erythr ocytes and hence the method is inappropriate. This technique is also labor-int ensive and is tedious for large numbers of samples. In B. bovis infections, the best results are obtai ned from making thick blood smears of capillary blood obtained, after pr icking the tip of the tail or marg in of an ear (Bock et al., 2004). Thick smears are 10 times more sensitive than thin smears because babesia-infected erythrocytes are more tightly packed together. Analytic Se has been estimated as 1 parasite per 1,000,000 erythrocytes (Bose et al., 1995). The detection limit for thin smears ranges from 10-5 to 10-6 or 1 parasite per 105 to 106 erythrocytes whereas thick smears can detect parasitemias as low as 1 parasite in 107 erythrocytes. Therefore, thick smears ar e more useful for the detection of low levels of B. bovis (Bose et al., 1995). Smears made from blood collected from major veins may contain 20 times fewer B. bovis organisms than capillary bl ood (Callow et al., 1993). This problem does not occur in B. bigemina infections because the para sites are not sequestered but are evenly distributed throughout the circulat ion (Bock et al., 2004). Differentiating between B. bovis and B. bigemina can be very difficult (Bose et al ., 1995). Staining of thick smears with acridine orange is faster and the detection limit is approximately 10-7. However, this technique

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93 requires a fluorescent microscope for ex amination and differentiation between Babesia spp is generally not possible as in Giemsa -stained smears (Bose et al., 1995). Polymerase Chain Reaction-Based Techniques (PCR) Polymerase Chain Reaction-based techniques (PCR) have also been used to identify anaplasma and babesia infections. This techniqu e detects DNA of the parasite and therefore recognizes current infection of th e animal. Moreover, the relative intensity of the DNA amplicon correlates with the numbers of pa rasites and thus the level of pa rasitemia (Wirth et al., 1986; Barbet et al., 1987). In BA, PCR can identify 30 infected erythrocytes pe r ml of blood, which is equivalent to a parasitism of approximately 0.00001% (10-7). This is well below the typical level in carriers (Torioni de Echaide et al., 1998). However, when PCR techniques are used diagnostically for detection of A. marginale infection, a nested reaction is necessary to identify low-level carriers. Nested PCR is more techni cally demanding and imposes significant quality control problems for routine use. Amplified DNA products can be aerosolized and readily contaminate staff, laboratory equipment, and reagents subsequently producing false-positive test results (Bose et al., 1995). Non-specific amplif ication of closely related DNA sequences could also reduce the overall specificity of the test. Th erefore, additional step s such as restriction enzyme analysis, southern hybridization, or se quencing are needed to confirm the positive amplification. Relative to microscopic techniques, PCR is expensive and restricted to wellequipped laboratories with f acilities for molecular biology (Bose et al., 1995). Polymerase chain reaction assays are analytic ally sensitive for dete cting infections and differentiating between B. bovis and B. bigemina in carrier cattle. Detection levels as low as 3 parasitized erythrocytes in 20 ul of packed red blood cells (RBCs) has been reported. The detection limit is approximately 10-8 to 10-9 (Bose et al., 1995). Polyme rase chain reaction assays are a good choice if the inve stigator considers it im portant to differentiate B. bovis which is

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94 claimed to be more pathogenic, from B. bigemina However, PCR assays generally do not lend themselves well to large-scale testing and are un likely to replace serological tests as the method of choice for epidemiological studies. Polyme rase chain reaction assays are useful as confirmatory tests and in some cases for regulatory testing. Recently, a quantitative PCR assay (qPCR) base d on the amplification of the cytochrome b gene (a mitochondrial gene) rath er than the standard techniqu es targeting the 18S rRNA gene, has been developed and is estimated to have high diagnostic Se and Sp (Buling et al., 2007). In addition, this is the first assay reported to quantify B. bovis and B. bigemina DNA in mammalian blood. Validation of this assay was based on 40 co ws and 80 horses from Spain either suspected of suffering from babesiosis or exposed to ti cks (tick species were not described). The tickexposed animals were identified by standard PCR and sequencing of the amplicon. The limit of detection was estimated as 1,000 copies of the ta rget DNA. However, the minimum parasite load was not determined but the authors estimated it to be in the range of 2 to 5 x 10-6. The detection limit is not as high as other PCR-techniques, but the risk of contamination following PCR amplification is considerable reduced because the reaction is carried out in closed system rather than an open system as in standard PCR. Clos ed systems are relatively faster and consume less reagents (Lohret and Kelly, 2004). Serological Tests The accuracy (diagnostic Se and Sp) of serologi cal tests for BA varies based on assay and laboratory. Sensitivities vary from 20% for complement fixation tests to 100% for the competitive ELISA (cELISA) in detecting carrie r animals. The small numbers of known positive and negative animals for these studies results in a large amount of statistical uncertainty. An exception is the new MSP-5 cELISA (VMRD, Pullm an, Washington) for diagnosis of BA. This test has been extensively validated using positive and negative animals as defined by nested PCR

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95 (nPCR) relative accuracy. Serological tests for diagnosing infection with Babesia spp. (especially B. bigemina ) are not considered as sensitive and specific as tests for anaplasmosis (Bose et al., 1995). However, recent advances in cELISA development for B. bovis and B. bigemina infections could prove to be promisi ng (Molloy et al., 1998a ; Molloy et al., 1998b). Complement fixation test (CF) The complement fixation test (CF) primarily detects IgM antibodies, which are produced during acute infections. Ther efore, the Se of the test is lowest for persistently infected animals (Bose et al., 1995). False positives have been repo rted and are considered to be due to the presence of normal bovine erythrocyte stroma in the CF antigen (Bradway et al., 2001). Another disadvantage of the CF test, is that it is highly complex and diffi cult to perform (Bradway et al., 2001). The antigen employed in the CF for BA is a crude mixture of A. marginale and erythrocyte proteins. This CF test is still widely used in the field to detect animals infected with A. marginale Gonzalez et al. (1978) estimated a Se of 79% and Sp of 100% of the CF for 82 A. marginale -infected cattle and 48 non-infected catt le. Conversely, Bradway et al. (2001) evaluated the sera of 15 0 infected cattle and 82 non-infected cattle reporting Se and Sp values of 20% (95% CI 14 to 26%) and 98% (95% CI 95 to 100%), respectively. Rapid card agglutination test (CAT) The rapid card agglutination test (CAT) is a common diagnostic method for BA used in the US. The CAT antigen is a suspension of intact A. marginale parasites, which have been separated from erythrocytes by lysi s in a French pressure cell a nd stained with fast green dye (Amerault and Roby, 1968; Amerault and Roby, 1977; Wright, 1990). After mixing standard quantities of antigen and serum or plasma on a test area of the card, the card is tilted to and from and the degree of agglutination visibl y assessed after a set time (Wright, 1990). Gonzalez et al. (1978) estimated a Se of 84% and Sp of 98% when evaluated in 82 A. marginale -infected cattle

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96 and 48 non-infected cattle. In a more recent study, the CAT was compared to the ELISA within 208 sera from Anaplasma free herds, 86 experimentally infected cattle, and 757 sera from cattle in areas endemic for A. marginale The Se and Sp of the CAT in this study were 98% (95% CI of 95.5 99.6) and 98.6 % (95% CI of 88.0 99.9) respectively (Molloy et al., 1999). Based on these results, the CAT could be considered the test of choice. In addition, this method is easily performed in the laboratory or field and result s are obtained within 10 minutes. However, it is sensitive to variations in test conditions, particularly temper ature. For optimal results, the temperature should be between 19 to 30C (65 to 85 F) (Amerault and Roby, 1968). Results are also subjective, labor intensive, and not easily automated (Molloy et al., 1999). Indirect fluorescent antibody test (IFAT) The indirect fluorescent antibody test (IFAT) is the most widely accepted technique for the serological diagnosis of B. bovis and B. bigemina infections (Bose et al., 1995). The antigen is derived from B. bovis or B. bigemina in vitro cultures of erythrocytes at parasitemia levels of approximately 8%. Infected erythrocytes are fi xed to a microscope slide where reaction with sample serum is performed. Recognition of antibody in the sample serum is accomplished by addition of an enzyme conjugate, usually an ti-bovine IgG antibody labeled with fluorescein isothiocyanate at 1:80 dilution, and examinati on with a fluorescent microscope. (Torioni de Echaide et al., 1995). Unlike other tests, IFAT are not standardized. Different serum or conjugate dilutions in IFAT among studies have shown mark edly different results on Se and Sp values. Johnston et al. (1973) evaluated the IFAT for antibodies to B. bovis by using a 2-fold dilution of the test serum on 290 unexposed animals from n on-endemic areas of Australia and 98 exposed animals suffering from acute and persistent infe ctions. The Se and Sp were reported as 99% and 96%, respectively (Johnston et al ., 1973). Conversely, Hadani et al. (1983) evaluated the IFAT for B. bovis on brain smears stratified by sex and age. Se ranged from 76.5% to 100% and Sp

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97 ranged from 30.8% to 66.7%. The pooled values for Se and Sp were 85.9% and 46.6%, respectively. Cattle in this study (n=213) originat ed from abattoirs in the northwest region of Argentina and all serum samples were tested at a dilution of 1:60 with a conjugate dilution of 1:80 (Hadani et al., 1982; Hadani et al., 1983). C onversely, a study by Torioni de Echaide et al. (1995) using the same dilution for serum and c onjugate reported a Se range of 92.8% to 100% for B. bovis Most studies report kappa agreement values 90% between the IFAT and an indirect ELISA test for B. bovis infections (Torioni de Echaide et al., 1995; Molloy et al., 1998a). The IFA for detection of antibodies against B. bigemina have been reported to have poor specificity. (OIE, 2004) Serol ogical cross-reactions with B. bovis make differentiation between these 2 species difficult, particul arly in regions where the 2 para sites coexist. The IFAT is the test of choice if the goal of the invest igator is to establish a prevalence of Babesia spp., considering that both species may pose similar ri sks for disease and species differentiation is not important for regulation and control measures. In BA, the test is performed as for Babesia spp. except that A. marginale -infected blood is used for preparation of antigen smears, at approx imately 5 to 10% parasitemias. The Se of IFAT for A. marginale persistently infected cattle is report ed to be 97% (Gonzalez et al., 1978). The IFAT does not require high technical skills. Howe ver, it has the disadvantages of being labor intensive and causing exte nsive operator fatigue. Usually, no more than 70 to 90 samples per day should be examined. In addition, results are su bjective and depend upon the experience of the operator. An important issue is background or non-specific fluorescence, which can cause sample misclassifications at different serum titrations (Duzgun et al., 1988; Jongejan et al., 1988).

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98 Enzyme-linked immunosorbent assays (ELISA) A competitive ELISA for BA has been developed, extensively validated, and commercialized for the detection of antibodies specific for Anaplasma in bovine serum19 (Knowles et al., 1996; Torioni de Echaide et al., 1998; Reyna-B ello et al., 1998). The assay utilizes a recombinant E. coli expressed MSP-5 (rMSP-5) protein, which contains an epitope that is defined by the monoclonal antibody, ANAF16C1. This ELISA kit uses an avidin alkaline phosphatase/p-nitrophenyl phosphate dete ction system (Palmer et al., 1994). As discussed previously, MSP-5 is hi ghly conserved among most Anaplasma species (Knowles et al., 1996). Positive and negative A. marginale -infection status was determined in 235 randomly selected cattle using a nested PCR (nPCR) coupled with msp5 sequence analysis and hybridization (Torioni de Echaide et al., 1998) At a cutoff point of 28% inhi bition, the rMSP5 cELISA had a relative Se of 96% (95% CI of 91 to 98%) and a Sp of 95% (95% CI of 88 to 98%). The validity of the assay for identifying cattle infected wi th BA has been tested for 12 strains of A. marginale ; Florida, Washington-Clarkston, Washington-Ok anogan, South Idaho, Virginia, North Texas, Missouri, Mississippi, Africa (Zimbabwe), Hawaii, Canada, Israel, and Vene zuela (Visser et al., 1992; Palmer et al., 1994; Knowles et al., 1996). This assay is highly accura te and is therefore recommended as the test of choice for epidem iologic studies, erad ication programs, and international movement of animal re gulation (Palmer and McElwain, 1995). An indirect ELISA kit for B. bovis has been developed and valid ated in Mexico, Cuba and Brazil20 (Waltisbuhl et al., 1987; Bose et al ., 1990; Torioni de Echaide et al., 1995; FAO/IAEA/SIDA, 1997; Molloy et al., 1998a). Originally, the antigen consisted of an 19 Anaplasma Antibody Test Kit, cELISA VMRD, Inc. PO Box 502 Pullman, WA 99163 U.S.A. USDA Product Code 5002.20. 20 Manual for Babesiosis Indirect ELISA Kit, Bench Protocol Version BBO 2.1, by the Joint Food Animal Organization International Atomic Energy Agency (FAO-IAEA) Program, Vienna (1993).

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99 oxyhemoglobin-free distilled lysate of B. bovis infected erythrocytes bu t it was further modified using a recombinant antigen that reacts to a monoclonal antibody designated as W11C5. This ELISA kit use horseradish peroxidase as the enzy me conjugate (Waltisbuhl et al., 1987; Bose et al., 1990). The test was validated in Australia usi ng 158 experimentally infected cattle and 318 B. bovis free cattle and Se and Sp were estimate d as 100% (95% CI of 97.7 to 100%) and 99.4% (95% CI of 97.5 to 99.0%), respec tively (Molloy et al., 1998a). In Africa, validation was based on 328 sera from B. bovis free herds in Kenya and South Africa and Sp was estimated as 99.7%. Another study in Argentina estimated Se and Sp as 98% (95% CI of 96 to 99%) and 95% (95% CI of 93 to 97%), respectively. In this study, the test was eval uated using 500 sera from known infected animals and 500 sera from regions of Ar gentina free of the tic k vector (Torioni de Echaide et al., 1995; Molloy et al., 1998a). Cross-reactions in sera from B. bigemina and A. marginale infected animals were reported in 6% and 8% of th e cattle, respectively (Waltisbuhl et al., 1987; Torioni de Echaide et al., 1995). Recently, 2 cELISAs have been developed and validated for the detection of antibodies specific for B. bovis Both techniques utilize a recombinan t rhoptry-associated protein 1 (rRAP1) antigen (Boonchit et al., 2002; Goff et al ., 2003; Boonchit et al., 2004; Goff et al., 2006a). The first one was developed by Boonc hit et al. (2002) who employed th e full-length rRAP-1 as the antigen. However, this assay had problems with cross-reactions in sera from B. bigemina infected cattle and subsequently modified usi ng more specific recombinant antigens from the Cterminus of rRAP-1 (rCT1, rCT2, and rCT3). An evaluation was performed on sera from 14 B. bovis -infected cattle, 12 B. bigemina -infected cattle and 30 non-infect ed cattle. At cut-off values of 0.140 and 0.136 (OD at wavelength of 450 nm) both, the Se and Sp of this cELISA with rCT1 and rCT2 were reported as 100% Conversely, the assay based on rCT3 had Se and Sp of 92.8%

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100 and 100%, respectively, at a cut-off value of 0.133 (Boonchit et al., 2004). However, sera from cattle in other geographic regions were not evaluated. The second B. bovis cELISA employs a B-cell epitope within the C terminus of rRAP-1 (Goff et al., 2003). This antigen reacts with the monoclonal antibody, BABB75A4. Initial validation were based on 227 randomly select ed samples from the US, 135 samples from experimentally infected cattle in the US, and 131 samples from Ar gentina (1), Bolivia (20), PR (38), and Morocco (72). Specificity was repor ted as 98.7% (95% CI of 98.75 100%). Further validation within 135 sera from known positive animals and 141 sera from known negative animals estimated a Se and Sp of 100% a nd 91%, respectively. The known positive sera were from experimentally infected animals (n=119) and animals from an endemic region of PR (n=16). These samples were determined to be positive by a B. bovis -specific nPCR and confirmed by sequence analysis. The known negativ e sera were from the northwest US. This assay has good reproducibility with agreement betw een four laboratories in different countries estimated between 88% and 94% wi th kappa values all above 0.721 (Goff et al., 2006a). Conversely, attempts to develop a reliable ELISA for detection of B. bigemina infections have not been as successf ul. ELISAs for detecting B. bigemina infections have poor specificity. In a study by eI Ghaysh et al. (1996), the presence of bovine fi brinogen contributed to more positive ELISA results than the presence of B. bigemina specific antibodies. In addition, crossreactivity with antibodies specific for B. bovis complicate diagnosis in regions where the 2 parasites coexist (el Ghaysh et al., 1996). A cELISA for detection of antibodies against B. bigemina in cattle has been developed in Australia (Molloy et al., 1998b). This assay uses a 58-kDa B. bigemina merozoite protein as the 21 Concordance among laboratories was established using C ohens kappa values, Hartleys test for homogeneity and one-way analysis of variance of ODs among the four laboratories (Goff et al., 2006a).

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101 antigen. Two monoclonal antibodies have been developed (B9 and D6) against independent epitopes of the protein (Molloy et al., 1998b). This antigen was chosen because it is conserved among geographically diverse isolates of B. bigemina including Mexico (3 isolates), Texas, Puerto Rico, St. Croix, and Co sta Rica (Figueroa et al., 1990). This assay was evaluated in 70 known positive sera from experimentally infected cattle and 166 antibody ne gative sera collected from non-endemic areas of Australia. Using a cut-off of 30% inhibition, the Se and Sp of this test was reported as 95.7% (95% CI of 87.2 98.9%) and 97.0% (95% CI of 92.7 and 98.9 %), respectively. The test is recommended for areas where B. bovis and A. marginale have overlapping distributions with B. bigemina because false-positive reactions do not occur in the presence of antibodies to B. bovis and A. marginale (Molloy et al., 1998b). Nevertheless, further validation is required to a ssess performance in the field (Molloy et al., 1998b). Recently, Boonchit et al. (2006) evalua ted the potential of a full-length B. bigemina RAP-1 and a truncated C-terminal RAP-1 fo r use in an indirect ELISA for B.bigemina The assay was evaluated using sera from 14 B. bovis -infected cattle, 13 B. bigemina -infected cattle, and 30 noninfected cattle. The cut-off va lue at a wavelength of 415 nm wa s set as the mean OD of the 30 negative bovine sera plus 3 sta ndard deviations. This resulted in cut-off values of 0.12 and 0.11 for the full rRAP-1 and the truncated C-termin al RAP-1, respectively. High levels of crossreactivity between B. bigemina and B. bovis were found with the full version of RAP-1 whereas the truncated C-terminal RAP-1 was more specific for sera from B. bigemina -infected cattle. All sera from B. bovis -infected cattle and non-infected cattle were below the cut-off value whereas all sera from B. bigemina infected cattle were above the cut-off. This technique offers some advantages over other assays because it uses reco mbinant antigen rather than antigen from live animals (Boonchit et al., 2006). This makes it less expensive and highly reproducible (Bose et

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102 al., 1995). In summary, ELISA techniques are objective a nd have a higher capacity to test a large number of samples in a short time compared to IFAT. ELISA allows for better standardization because results are computed automatically unlike th e reliance of IFAT on a technician to read the slides microscopically. For this reason, ELISA is considered a useful technique for screening large numbers of samples.

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103 Figure 2-1. The 78 municipalities of PR; eac h municipality has a mayor and a municipal legislature. Six adjacent islets surroun d the big island; Mona, Monito, Des echeo, Caja de Muertos, Vieques and Isla de Culebra.

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104 Figure 2-2. A central mountain range extends across the interior of Puerto Rico from east to west. Another set of high altitude terrain is located in the northeast area near th e coast and corresponds to the tropica l rainforest known as El Yunque.

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105 Figure 2-3. The 8 agricultural regions of PR based on the number of producers and corresponding agricultural commodities in tha t region. Common commodities include milk, da iry replacement heifers, hay (Arecibo ); broiler chickens (Ponce); coffee, plantains, bananas, sugar, livestock (L ares); and recao and peppers (Caguas).

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106 Figure 2-4. Proposed hierarchical structure for the study of the epidemiology of bovine anaplasmosis and babesiosis. Adapted from Turner M.G. et al., 2001. GEOGRAPHIC LOCATION OF THE REGION (Tropical, Sub-tropical, Temperate) ECOLOGICAL CONDITIONS WITHIN A REGION (Seasonality, Landscape, Elevation, Temperature, Rainfall, Relative Humidity) FARM MANAGEMENT (Nutrition, Use of Vaccines, Use of Acaricides, Chemotherapy, Origin of replacement animals, AnimalMovements ) CATTLE (Other hosts) (Breed, Age, Immune Status) TICK (Other vectors) (Stage, Species) BOVINE ANAPLASMOSIS BOVINE BABESIOSIS A. marginale B. bovis B. bigemina Components (explanation) Level of Interest Constraints

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107 CHAPTER 3 STUDY 1: SEROPREVALENCE AND MANA GEMENT FACTORS ASSOCIATED WITH Anaplasma marginale IN COMMERCIAL DAIRY FARMS OF PUERTO RICO Introduction Bovine anaplasmosis (BA) is a hemoparasi tic disease of great importance in cattle production systems in the tropical and subtropica l regions of the world (Kocan et al., 2003), including the island of Puerto Rico (PR) (v an Volkenberg, 1939; Crom, 1992; Bokma, 1996). The disease is caused by the rickettsiae, Anaplasma marginale (Order Rickettsiales, Family Anaplasmataceae) (Dumler et al., 2001; Kocan et al., 2004) and the only recognized vector in PR is the one-host tick, Rhipicephalus (Boophilus) microplus (Tate, 1941; de Leon et al., 1987; Crom, 1992; Bokma, 1996). The disease is charac terized by severe anemia and jaundice without hemoglobinemia and hemoglobinuria (Richey and Palmer, 1990). Other clinical signs include weight loss, decreased milk produ ction, abortions, hyperexitability (due to cerebral anoxia), and sudden death (Richey and Palmer, 1990). Recovered animals become persiste ntly infected with A. marginale for life (French et al., 1998) and serv e as a reservoir for the maintenance of A. marginale within a herd (Richey and Palmer, 1990; Kocan et al., 1992; French et al., 1999). The first studies of BA in PR were reporte d by van Volkenberg in the late 1930s. Since then the disease has been considered ende mic on the island (Crom, 1992). An estimated economic loss of US $20 million was reported in 1989 in PR, attributable to the presence of BA, bovine babesiosis, and R. (Boophilus) microplus despite an ongoing tick eradication program (1936-1995) (Crom, 1992). A recent survey by Co rtes et al. (2005) am ong 261 dairy farmers (47,401 milking cows) in PR reported an estimated loss of US $6.7 million in 2000 due to these diseases and R. (Boophilus) microplus infestation. An annual loss of US $7,155 was estimated per farm for tick control or US $29 per cow per year (Cortes et al., 2005).

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108 Cattle operations, especially commercial dairy farms, are the most economically important agricultural sector in PR. The dairy industr y produced US $184.8 million (25.6%) of the gross domestic product in agricultu re in 2006 (PRDA-ASO, 2006). Approximately 25,000 jobs are related to the production, manuf acture, and sale of milk and milk by-products (ORIL-PRDA, 2006). The island has an estimated cattle population of 281,371 of which 153,097 (54%) belong to the dairy industry with 63,181 lactating cows (Planning Boar d of the Commonwealth of Puerto Rico, 2003; NASS-USDA, 20 04). Most lactating cows are raised within 353 commercial dairy farms operating in PR as of 2006. From 2005-06, these dairy farms produced 329 million liters (709 million pounds) of m ilk with an average production of 3,850 liters (8,277 pounds) per cow (NASS-USDA and PRDA-ASO, 2005). Although tick-borne hemoparasitic diseases ha ve been reported to cause considerable morbidity and mortality to cattle in PR, the ep idemiology of BA in PR has not been thoroughly investigated. Successful management of BA depends on adequate knowledge concerning seroprevalence estimates for A. marginale risk factors associated w ith the transmission of BA, and geographic locations and local climatic condi tions suitable for the distribution of BA and possible vectors (Richey and Palmer, 1990; Alonso et al., 1992). The transmission of A. marginale is not determined exclusivel y by the distribution of the tick vector. In some regions of Latin America, the geographical distribut ion of BA is more widespread than R. (Boophilus) spp. indicating the existence of other vectors or different m odes of transmission (Anziani, 1979; Alonso et al., 1992). Mechanical transmission of A. marginale by biting flies and bloodcontaminated fomites is considered to be impor tant in the epidemiology of BA in areas of Central and South America, Africa, and th e US (Ewing, 1981; Foil, 1989; Morley and HughJones, 1989; Scoles et al., 2005).

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109 Several factors such as type of livestock, herd size, interaction with wildlife, ecological and socio-economic factors may play important roles in the epidemiology of BA, and their role in the system should not be ignored (Alonso et al., 199 2). Therefore, all parameters involved in the transmission of A. marginale should be studied collectively, to allow correct inferences about the disease processes. The objectives of the present study were (1) to determine th e seroprevalence of A. marginale in adult lactating dairy cattle in the 4 diffe rent climatological zones of PR, and (2) to assess the associations of geographical and farm management factors on the seroprevalence of A. marginale in commercial dairy farms of PR. Materials and Methods Study Area Puerto Rico is centered in the Caribbean basin between the coordinates 17 N and 18 N, and its longitude ranges from 65 W to 67 W. It is the sm allest and easternmost island of the Greater Antilles, east of Haiti and Dominican Republic and northwest of the Virgin Islands. It is bounded on the north by the Atlantic Ocean and on the south by the Caribbean Sea. It encompasses an area of 8,870 km2 from which 41.6% is closed forests, 36.7% pastures and grasslands, 5.9% crop agriculture land, 2.4% coff ee plantations, and 10.5% urban and developed landcover (Helmer et al., 2002). Elevations range from sea level to 1338 m. The climate is predominantly tropical maritime. Average temperat ures in PR have very small range between the warmest and coldest months, but decrease mark edly with increasing elevation (NOAA-NCDC, 1982). In the high mountainous interior, the temp erature fluctuates between 22.8-25.6C (7378F). The northern half receives mo re rainfall and has larger rivers than the drier southern half. The distribution of rainfall follows a relative wet-dry seasonal pattern. The relative humidity (RH) is approximately 80% over the course of th e year with the highest RHs generally found at

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110 night (>90%) when temperatures are the lowest During the day, RH ranges from 60% to 70% (NOAA-NCDC, 1982). Puerto Rico is classified into 4 main climatological zones according to soil type, vegetation, agricultural developmen t, precipitation and temperature indices: humid coastal plains (north coastal region, NC), semiarid coastal plai ns (south coastal region, SC), humid mountains and valleys (western interior, WI), and semiarid mountains and valle ys (eastern interior, EI). The NC zone has rolling hill topography and an aver age annual precipitation of 1600 mm (63 in) and an average annual temperature of 25C (77F) Pastured-based commercial dairy farms are common in this area. Native and improved tropic al grasses cover approximately 50% of the land, whereas 17% of the acreage is used for a variety of crops. This is the most deforested zone partly because of intensive agricultural activities and urban development. Both native and improved pastures form the dominant landscap e in this zone. The SC zone has alluvial flat terrains and is the driest of the 4 zones with an average annual precipitation of 900 mm (35 in) and an average annual temperature of 26C (78.8F). More than 50% of this area is comprised of native and improved grasses, which are mainly used for f eeding beef cattle and ra cehorses. The remainder of the landscape consists of cacti, thorny legum es, and trees with small and succulent leaves. Fires are common during the dry season. The WI z one has irregular topog raphy with elevations of 500-600 m and an average annual precipita tion of 2100 mm (83 in) an d an average annual temperature of 24C (75.2F). A pproximately 70% of this area is native and improved grasses, 10% coffee plantations, and 7% forest. The remainde r is used for food crops including plantains, bananas, and yams. Because of the abundant moisture most of the original landscape consists of epiphytic ferns, bromeliads, and orchids. Lastly, the EI zone is comprised of very steep floodedalluvial plains and has an average annual precip itation of 1150 mm (45 in) and an average annual

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111 temperature of 26C (78.8F). Approximately 60% is native grasses and 35% is covered by forest (Ewel and Whitmore, 1973; Guttm an and Quayle, 1996; NRCS-USDA, 2007). The NC zone contains 62% of the dairy herds that have 68% of the adult lactating cattle population in PR. The predominant dairy production system is semi-intensive, which is based on concentrates and year-round grazing on improved pastures including African bermuda grass ( Cynodon nlemfuensis var. robustus ), pangola grass ( Digitaria eriantha ), and guinea grass ( Urochloa maxima ). Concentrates typically consist of grain by-products, molasses, vitamins, and mineral supplements imported from continental US sources. Most dairy replacement heifers are raised in PR using crossbreeding programs with Holstein-Friesian, Jersey and Brown Swiss, but some purebred Holstein-Friesians are imported from the US. Study Design and Sample Size Methodology A cross-sectional study with a multi-stage desi gn was carried out over 18 months between August 23, 2005 and December 4, 2006. The reference population for this study was adult lactating cows (>2 years of age) from commer cial dairy farms. Comme rcial dairy farms were defined as those farms having an active license in the 2004 Annual Inventory for Grade A dairies list provided by the Office for the Regulation of the Dairy Industry of Puerto Rico (La oficina de la reglamentacin de la industria lechera de Puerto Rico, ORIL) and the State Veterinarian from the Department of Agriculture, Commonwealth of Puerto Rico, in conjunction with the United States Department of Agriculture (USDA). The sample size for the number of farms wa s calculated assuming a total population of 362 farms, an expected herd ser oprevalence of 50% (the seroprev alence was unknown), a confidence level of 95%, and an acceptable margin of error of 10% with available software (Win Episcope 2.0, Easter Bush, Roslin; Zaragoza, Spain; Utre cht and Wageningen, Netherlands). To calculate the proportional distribution of farms to be sa mpled, the total number of farms in PR was

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112 stratified by climatological zone and farm size. Farm size was divided into approximately 3 equal groups using percentiles of the total number of farms (small, 36-109 animals; medium, 110-196; and large, 197-1000). An estimated pe rcentage was obtained by dividing the total number of farms within each stratum (e.g. WI-s mall) by the overall total number of farms (n=362). Each percentage was then multiplied by the necessary sample size. Study farms were randomly selected from all farms within each of the 12 strata. The sample size was estimated to be 76 farms and within each climatological zone was NC=51, WI=13, EI=8, and SC=4 (Table 31). Location of farms within each climatol ogical zone is presented in Figure 3-1. The sample size per herd was calculated using the number of lactating cows listed in the most recent ( 1 month) Dairy Herd Improvement Asso ciation (DHIA, Verona, WI) records of each farm visited. Number of animals per farm in this study ranged from 12 to 850 adult lactating cows. The minimum samp le size to detect disease was estimated assuming an expected prevalence of at least 8% at 95% level of confidence (S urvey Toolbox, version 1.0 beta, Wentworth Falls, NSW, Australia). Calculations were not adjusted for imperfect sensitivity and specificity of the diagnostic te st. Cows were chosen by either simple random selection (n=53 farms) using ear tag identification numbers fr om DHIA records (random number generator from MicrosoftOffice Excel, 2003) or systematically sa mpled through the chute based on the lactating herd size and the target number of samples in the herd (n=23 farms). Questionnaire Development and Administration A questionnaire (Appendix A) was administered to dair y owners and managers of participating farms from August 16, 2005 to Nove mber 29, 2006. The questionnaire consisted of 80 questions on 8 pages (printed on both sides). The questionnaire includ ed both open and closed questions with directions to either mark those answers that applied, choo se yes or no, or provide a free text response. When applicable, data that pertained to the preced ing 12-month period were

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113 collected. Dairy owners and managers answered que stions about type of facilities, common farm husbandry practices, past and recent experiences with ticks and use of acaricides, past and recent experiences with BA, experience with treatmen t protocols and vaccines, knowledge of disease diagnosis, and experiences with flies and fly control methods (T able 3-2). The questionnaire was first written in English and then translated in to Spanish (Appendix B). The English version was pre-tested on 5 faculty members and 6 ve terinary students from the Food Animal and Reproduction Medicine Service at the University of Florida, Co llege of Veterinary Medicine. The Spanish version was pre-tested at a 3-hr group discussion with 13 dairy farmers from the ORIL Executive Board 1 month before the first interview. Information concerning management factors was obtained before the seroprevalence of the farms was determined. Each participating farm was visited twice. Fo r the first visit, dairy owners (or managers) were contacted by phone 1 week in advanced of the expected visit to set up an appointment. During this visit, a brief summary of the study, an administrative order from the ORIL, and a consent form (including the purposes of the surve y, the procedures for part icipating in the study, and an assurance of confidentiality) were provided with the inte rviewer-administered questionnaire. The dairy owner (or manager) provid ed the most recent DHIA list of the lactating cows, returned the signed consent form, and se t up the appointment for the second visit. All interviews were conducted in Spanish by the first author. To standardize the interview process, the investigator was trained to ask all participan ts the same questions in the same manner. The questions with dichotomous response options that were not answered we re classified as no response. XY-coordinates from each participating dairy farm were collected using the milking parlor of the farm as the point of refere nce by a global positioning system (GPS) (Garmin e Trex Legend, Garmin International Inc., Olathe, KS, USA).

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114 Sample Collection All procedures were approved by the Inst itutional Animal Care and Use Committee (IACUC # D806 and #E151) at the Un iversity of Florida. A blood sample (~10 ml) was collected from each selected animal (n=2,414) during the s econd visit. Selected cows were either grouped by the farmers immediately after milking or were bled individually after leaving the milking parlor. Samples were collected aseptically via coccygeal venipuncture into 10-ml uncoated red top tubes (Vacutainer, Fisher Scie ntific, Pittsburgh, PA, USA) usi ng disposable needles. Samples were labeled and transported on ice to the laboratory facilities at the Agricultural Experiment Station, University of Puerto Rico (Mayagez Campus). In the laboratory, serum samples were centrifuged at 3700 rpm for 15 min at room temperat ure, serum separated, and frozen in 1 ml aliquots at -25 C (-13 F) until analyzed. Samp les were transported to the State Veterinary Diagnostic Laboratory Dr. Gabriel Gonzlez-Cald ern located in Dorado, Puerto Rico for serological testing. All tests were performed by a trained technician and the first author. Serological Testing Sera were tested for antibodies against A. marginale using a commercia lly available MSP5 competitive ELISA (cELISA) ( Anaplasma Antibody Test Kit, c ELISA, Catalog No. 282-5, VMRD, Inc., Pullman, WA, USA). Test procedur es were performed according to guidelines provided by the manufacturer. Briefly, 96-well flat -bottom plates coated with recombinant MSP5 were incubated with the sample sera for 1 hour at room temperature (21-25 C, 70-77 F) and washed 2 times with a wash solution. The n, monoclonal antibody ANAF16C1 conjugated to horseradish peroxidase was added to each well and incubated for an additional 20 minutes at room temperature. After incubation, slides were washed 4 times and o -phenylenediamine dihydrochloride was added to the plates and in cubated for an additi onal 20 minutes. A stop solution was added before the plate was rea d. An ELISA microplate reader ELX800 (BIOTEK

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115 Instruments Inc., Winooski, Vermont, USA) a nd a computer software program IDEXX xChek 3.2TM Software (IDEXX Laboratorie s, Inc., Westbrook, Maine, USA) were used to measure and record optical density (OD) at 650 nm. Three know n negative controls were included on each plate to determine the mean OD of the negative controls (ranged from 0.40 to 2.10). Percent inhibition (% I) was calculated as 100[(Sample OD x 100) / (Mean Negative Control OD x 100)]. Two known positive controls were also included on each plat e. A cutoff of 30% inhibition for the MSP-5 cELISA was used to classify samp les (Torioni de Echaide et al., 1998; Molloy et al., 1999). Samples with <30% inhibition were co nsidered negative, and samples with greater than or equal to 30% inhibition were considered positive. The MSP-5 cELISA has been reported to have a Se of 96% (95% CI of 91 to 98%) a nd a Sp of 95% (95% CI of 88 to 98%) at this cutoff (Torioni de Echaide et al., 1998). Statistical Analyses The overall and stratified anim al seroprevalence by each climat ological zone in PR was calculated as the proportion of animals positive on the MSP-5 cELISA divided by the total number of cows tested, weighted by the pr oportion of the total cattl e population in each climatological zone (STATA 10.0 for Windows, StataCorp LP, College Station, TX, USA). Student t tests using Tukeys adjustment for multiple pairwise comparisons were used to compare the mean prevalences among the 4 climatological zones and 3 farm sizes. The coordinates and survey data for each farm were loaded into a geo-database (ArcGIS 9.1TM, ESRI, Redlands, CA, USA) for mapping and cluster analys es. To standardize the GIS analyses, all data were collected under the sa me projection parameters, North American Datum 1983 (NAD83) and a coordinate system correspondi ng to Universal Transverse Mercator (UTM) Zone 19 North. Spatial autocorrelation of the observe d seroprevalence was estimated using Morans I (ArcGIS 9.1TM).

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116 The associations between the explanatory variables collect ed via the questionnaire and outcome response variab le (seroprevalence to A. marginale ) were estimated using logistic regression (SPSS 14.0 for Windows, SPSS Inc. Headquarters, Chicago, IL, USA). The unit of analysis for assessing risk factors was the farm The outcome variable was dichotomized using a natural break in the distribution of the observed herd seroprevalence for A. marginale (Figure 32). The break was set at the boundary where a re latively large change in the distribution occurred. Those herds with a seroprevalence 40% were considered hi gh prevalence and those farms with a seroprevalence 39.9% were considered low prev alence. Variables that were significantly associated with high/lo w seroprevalence at a Wald P value 0.20 in a bivariable screening model were retained for further ev aluation in multivariable models. A backward stepwise multivariable logistic regression m odel was built starting with a complete model containing all main effects identified in the sc reening models and terms were removed one-byone based on likelihood ratio tests. The main eff ect terms remaining in the model were evaluated for effect modification by adding all possibl e 2-way interactions between main effects individually and testing for significance using Wald tests. The fit of the final multivariable model was assessed using the Hosmer-Lemeshow test. St atistical significance was assessed at the 5% level. Continuous independent variables in logistic models were ev aluated for linearity in the log-odds. These variables were categorized to pr ovide an indication of how the risk of the outcome changes with the variable and have bett er specification of the functional form of the equation (Cheung and Smith, 1981; Hosmer and Lemeshow, 2000). Initia l categorization was performed either by using 3 or more natural breaks of the data or by equally dividing the data in

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117 quartiles. If the continuous inde pendent variables were not lin ear in the log-odds, they were retained as categorical variables. The response (seroprevalence to A. marginale ) was also analyzed as a continuous dependent variable and the associ ations between the explanatory variables and the response were estimated using linear regre ssion. The associations of coefficients to the response were compared to the odds ratio of the variables that were significantly a ssociated with high/low seroprevalence at a Wald P value 0.20 in the bivariable logi stic regression analyses. Results Descriptive The number of randomly selected farms sampled was 76 and 2 additional farms volunteered to be in the study, one in the SC zone and one in the EI zone. An average of 32 cows was selected per farm for a total sample size of 2,414 from the 76 farms and 61 additional sampled cows from the 2 volunteer farms. Th e 2 volunteer farms were not included in the analyses for prevalence, but were included for the logistic regression analys es of risk factors. Premises size ranged from 16 to 600 cuerdas (6 to 236 ha), with an average of 150 cuerdas (59 ha). One cuerda is equivalent to 0.97 acres. Stocking density ranged from <1 to 11 animals per cuerda with an average of 4 animals per cuerda The time elapsed between the beginning of the interview to the end averaged 50 min (ra nge 20 min to 95 min) am ong dairy owners and managers and the average time between the first a nd second visit was 7 days (range 0 d to 75 d). Forty (51.3%) of the sampled farms had semi -confined facilities where cattle spent approximately 12 hours in open pasture grazing and 12 hours under a metal roof with concrete floors (ranchn); 31 (39.7%) had 24 hours open pa sture grazing as the main source of feed; 4 (5.1%) had dry lots for their ca ttle; and 3 (3.9%) had free-stalls The herds were comprised of 92.3% Holsteins and Holstein crosses; and 7.6% Jersey and Brown Swiss breeds and their

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118 crosses. Fifty-five (70.5%) of the farms were e quipped with operational ir rigation systems. Most pastures were irrigated with water from oxida tion lagoons. Only 2 (2.6%) of the farmers reported the presence of deer on their premises, whereas 7 (9.0%) reported the presence of monkeys and 18 (23.1%) reported seeing green iguanas in the surroundings of their premises. Seventy-three (93.6%) of the farmers used amitraz as the primary acaricide for tick control. Fifty-nine (75.6%) of the dairy farmers applie d amitraz every 20 to 30 days; 12 (15.4%) every 7 to 14 days; and 7 (9.0%) every 40 to 60 days. Twenty-two (28.2%) of the dairy farmers did not utilize any methods for fly control in the farms. Thirty-two (41.0%) of the farmers used eprinomectin (Eprinex Pour-On, Merial); 63 (81.0%) used ivermectin (Ivomec, Merial); 22 (28.2%) used met homyl (Z)-9-tricosene (Golden Malrin Fly Bait with Muscamone, Wellmark International); 20 (25.6%) used 1% piperonyl butoxide (P.B.O) and 1% permethrin (KattleGuard, Dairy Solutions, Inc.); 11 (14.1%) used 5% piperonyl butoxide (P.B.O) and 5% permethrin (ULTRA BOSS Pour-On, Schering-Plough); 9 (11.5%) used 11% permethrin (Atroban EC Insecticide, Schering-Plough); and 5 (6.4%) used dust bags containing coumaphos (Co-Ral, Bayer Animal Health). The use of these fly control methods was not exclusive and most farmers used more than 1 method. Fifty-six (71.8%) of the dairy farmers in th is survey participated in the certification program titled: Guidelines for use and application of insecticides from the Puerto Rico Department of Agriculture (PRDA), represente d by Agricultural Serv ices and Development Administration (ASDA) whereas 22 (28.2%) did not have the certifi cation or were not aware of the program. Seroprevalence The MSP-5 cELISA for A. marginale antibodies was positive for 743 of 2,414 (30.8%) sampled adult lactating cows (Table 3-3). Anim al seroprevalence within farms ranged from 2.8

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119 to 100.0% (Figure 3-3). The overall animal seroprevalence of antibodies against A. marginale was 27.4% (95% CI = 25.3-29.5). The NC zone had the lowest seroprevalence of the 4 zones, 25.8% (95% CI = 23.3-28.2) followed by the WI zone 28.1% (95% CI = 22.4-33.9) and the EI zone 31.6% (95% CI = 24.4-38.9). The SC had the highest seroprevalence for A. marginale of the 4 zones, 40.6% (95% CI = 30.9-50.3). However, no statistically signifi cant differences at the 5% level were found among the 4 climatological zone s and the 3 sizes of farms. Spatial analysis for A. marginale seroprevalence did not suggest any gl obal spatial autocorrelation (I = -0.03; Z score = -0.4; P value = 0.34). Risk Factors Twenty farms had high animal-level seroprevalence for A. marginale ( 40%) and 58 had low seroprevalence ( 39.9%). During the initial bivariable screening, 21 management factors were found to be significantly associated with high A. marginale seropositivity at P 0.20 (Table 3-4). The final multivariable logistic re gression model (Table 3-5) demonstrated that pasture grazing as the main source to feed cattle (OR= 6.4, 95% CI=1.333), observed monkeys on the premises (OR= 14, 95% CI=1.4137), use of 11% permethrin (Atroban), (OR= 14, 95% CI=1.9-98), farmers who attended an acaricid e certification program (OR= 0.17, 95% CI=0.040.72), and lack of fly control methods (OR= 5.7, 95% CI=1.3-25) had a significant association with high BA seropositivity. Other variables incl uded in the final model were use of irrigation systems and application of amitraz at 14-day intervals. Although these had a significant likelihood test and were retained in the model, they were not significant based on the Wald test. No other main effects or 2-way interaction terms were significant. The Hosmer-Lemeshow goodness-of-fit test demonstrated that the model was a good fit to the data (Chi = 2.346, df = 7, P-value = 0.938). Logistic regression analyses identified the same mode l with and without the two volunteer farms. The coefficients in the linear regression had the same

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120 direction of association as the odds ratios for all variables asso ciated with high/low seroprevalence at a Wald P value 0.20 in the bivariable logistic regression analyses (Table 36). Discussion The present study is the first in PR to assess the overall seroprevalence for A. marginale and identify farm management factors significa ntly associated with high seropositivity. The study sample was limited to adult lactating cattle from commercial dairy farms. Therefore, inferences about the present study might not re present other cattle populations. Serological results indicated that A. marginale is common and widely distributed in PR. Overall seroprevalence for A. marginale in PR was within the lower ra nge documented for other islands in the Caribbean region (1 to 71%) (Camus a nd Montenegro-James, 1994). Spatial distribution was not clustered at the scale of the entire island suggesting limited influence by geographic predictors. Therefore, management factors ap pear to be of primary importance in the epidemiology of BA on the island. In contrast to Swai et al.( 2005a) and Swai et al. (2005b), we found a strong and significant association between pasture gr azing and high seropositivity for A. marginale on dairy farms in PR (OR=6.4). Similarly, Rubaire-Ak iiki et al. (2004), found higher risk for tick infestation and increased seroprevalence for A. marginale in herds using pasture gr azing than those herds with partial or no access to pasture grazing. Pastur e grazing may increase the spread of ticks among herds, particularly in hi gh stocking density areas. Those dairy farms where monkeys were observe d on the premises had higher odds of high seropositivity for A. marginale (OR=14). The presence of monkeys might be confounded by some other ecological or management factors that were not adequately measured by the questionnaire. However, transmission of inf ectious agents between wildlife and domestic

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121 livestock has been documented, particularly in areas where free-ranging wildlife and livestock share common grazing grounds (Chomel et al ., 1994; Bengis et al., 2002). Wildlife are commonly considered vectors, rese rvoirs, or primary targets of infectious diseases (Trainer, 1970). Two introduced species of non-human primates, rhesus macaques ( Macaca mulatta ) and patas monkeys ( Erythrocebus patas ) have well established colonies primarily in the south-west region of PR (Jensen et al., 2004). From 1990 to 1993, the macaques population consisted of 45 to 85 individuals and the pata s population of more than 120 in dividuals (Gonzalez-Martinez, 2004). Today, it is estimated that more th an 1,000 individuals free-range the area (Commonwealth of Puerto Rico, 2007). These species were originally imported to a facility located on 2 cays off the southwest coast PR in the 1960s and 1970s with support from the National Institutes of Health (NIH) and the Food and Drug Administration (FDA). These facilities were administered by the Caribbean Pr imate Research Center of the University of Puerto Rico-Medical Sciences Campus from 1970 until its closure in 1982. Monkeys began migrating from the cays onto the mainland soon after the initial stocking of animals but intensified from 1974 to 1981 (Gonzalez-Martin ez, 1998; Gonzalez-Martinez, 2004). After 30 years, these monkeys have become a public nuisan ce to farmers and public health officials of the area (Jensen et al., 2004) and efforts to trap and remove them have been unsuccessful (Wikipedia contributors, 2007). Patas appear to live commensally with cattle in the area. They have been observed foraging for dung beetles ( Carabidae ) in and under dry cow manure on pastures on a daily basis (Gonzalez-Martinez, 199 8). Results of the present st udy raises concerns about the influence of these monkeys on the seropositivity of A. marginale in cattle and further serologic studies in monkeys and livestock should be considered. No natural infections of Anaplasma spp. in rhesus and patas monkeys have been documen ted in the literature. However, under laboratory

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122 conditions rhesus monkeys can be infected with Anaplasma spp., which raises speculations of rhesus being reservoirs for Anaplasma spp. (Foley et al., 1999; Munderloh et al., 2004). The use of 11% permethrin (Atroban) was positively associated with high seroprevalence of A. marginale (OR=14). Possible reasons for this effect may include the effectiveness and the price of the product. If this product is less expensiv e, the chances are that the farmer may select it over other more effective products. In addition, the use of Atroban or any other products might be influenced by the time treatment in terval and methods for application. Lack of a fly control program was asso ciated with a high seroprevalence for A. marginale (OR=5.7). Many hematophagous insects have been incr iminated as the major route of infection. Tabanids, including horse flies ( Tabanus spp. ) and deer flies ( Chrysops spp. ), stable flies ( Stomoxys calcitrans ), horn flies ( Hematobia irritans ), and mosquitoes of the genus Psorophora have been implicated as mechani cal vectors for transmission of A. marginale (Ristic, 1968; Ristic, 1977; Ewing, 1981; Potgiete r et al., 1981; Hawkins et al ., 1982; Foil, 1989). In the US, tabanids are the most common hematophagous insects suspected to be vectors of A. marginale (Morley and Hugh-Jones, 1989). Chrysops variegata has been the only species of tabanids described in Puerto Rico (PR) and in the 1930s it was only present in low numbers (van Volkenberg, 1939). In Cuba, tabani ds and mosquitoes including Psorophora confinnis Mansonia titillans and Culex nigripalpus are considered to play an important role in the transmission of A. marginale in the region (Postoian et al., 19 77; Alonso et al., 1992). No studies could be identified that described the current status of tabanid sp ecies in PR. Therefore, further studies on the species of hematophagous insects a ffecting commercial dairy farms and their role in the transmission of A. marginale in Puerto Rico are warranted.

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123 Farmers certified in the use of insecticides we re less likely to have high seroprevalence for A. marginale (OR=0.17). Participation in this prog ram was required from 2003-2004 to obtain a license to use trademarks bel onging to the PRDA, including amitraz at no cost or subsidized from designated farmers centers (PRDA-ASDA, 2007). Farmers spent 2 half days (08:00-12:00 hours) in seminars on the use of insecticides and were required to pass a final exam. After this time, the use of amitraz was no longer restricted and participation in the seminars was no longer required. Since then, farmers were able to obtain amitraz by simply showing picture identification (Maldonado-Somera, 2007, personal communication). The results of this study have provided base line prevalence information and identified some key risk factors that can guide local aut horities and policymakers to implement strategic control measures to decrease the seroprevalence of A. marginale in PR. However, this is a crosssectional design and there are important limitati ons that must be considered. For example, measured variables in the pres ent study might not have had th e same value as when cattle became seropositive. The findings of this study are also limited to the 18-month period 20052006 when data were collected. Hi gh seroprevalence does not necessa rily equate to more disease and higher economic losses. A different farm ser oprevalence cut-off could have yielded different results; however, a linear modeling of the seropr evalence rather than high/low dichotomization demonstrated similar findings and overall conclusi ons. Lastly, testing did not attempt to adjust prevalence for sensitivity and speci ficity of the test and might ha ve resulted in misclassification of the farms (e.g. high seroprevalence not due to high BA prevalence). Conclusions Management factors appear to be of primar y importance for understanding and control BA in PR. We suggest that at the time designers of tick and tick-borne disease control programs in

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124 PR implement measures to control BA they s hould considered the biol ogy of the tick and the current knowledge of the epidemiology of BA.

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125Table 3-1. Population and sample distributi on of commercial dairy farms by climatological zone and size of the farm to estimat e the seroprevalence of Anaplasma marginale in Puerto Rico from August 2005 to December 2006. Climatological zone Farm sizea No. of farms No. of adult lactating cows Percent farm needed Farm sample needed North coastal Small 705310 1915 Medium 8211916 2317 Large 9128154 2519 Western interior Small 312336 97 Medium 243556 75 Large 71778 21 Eastern interior Small 9710 22 Medium 152364 43 Large 134109 43 South coastal Small 10759 32 Medium 5684 11 Large 51505 11 Totalb 36263181 76 a Farm size: Small (36-109), Medium (110-196), Large (197-1000). b Data obtained from the 2004 Annual Inventory for Grade A dairies list provided by the Office for the Regulation of the Dairy Industry of Puerto Rico (ORIL) and the State Veterinarian from the Department of Agriculture, Commonwealth of Pu erto Rico, in conjunction w ith the United States Departme nt of Agriculture (USDA).

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126 Figure 3-1. Geographical di stribution of the 76 commercial dairy farms ( ) sampled in the 4 climatological zones of Puerto Rico from August 2005 to December 2006. NC: North Coasta l Zone (n=51); WI: Wester n Interior Zone (n=13); EI: Eastern Interior Zone (n=8); and SC: South Coastal Zone (n=4).

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127 Table 3-2. Explanatory variable s included in the quest ionnaire provided to dairy owners or managers for the study of the seroprevalence of Anaplasma marginale in Puerto Rico from August 2005 to December 2006. Explanatory variable s Categories Climatological zone North coastal (NC), western interior (WI), eastern interior (EI), and south coastal (SC) UTM coordinatesa Easting ( x -coordinate) and Northing ( y coordinate) Type of operation Dairy farm with lactating herd onlyb, dairy farm with lactating herd and calf-raising, dairy farm with lactating herd and heiferraising facilities, dairy farm with lactating herd, calf, and heifer-raising facilities, mixed (beef and dairy) Herd size Number of bulls, milking cows, dry cows, heifers (1-2 years), and calves (<1 year), total Premises (property) sizec Continuous Stocking densityd Continuous Breed(s) of cows and percent of breed in herd Holstein, Jersey, Brown Swiss, Cross-bred Presence of horses in the facilities Yes/no Number of horses Continuous Presence of sheep in the facilities Yes/no Number of sheep Continuous Presence of goats in the facilities Yes/no Number of goats Continuous Presence of pigs in the facilities Yes/no Number of pigs Continuous Presence of poultry in the facilities Yes/no Number of poultry Continuous Main type of pasture grown on the farme African bermuda grass ( Cynodon nlemfuensis var. robustus ), guinea grass ( Urochloa maxima ), pangola grass ( Digitaria eriantha ), and bermuda grass ( Cynodon dactylon ) Type of facility Confined, semi-confined, dry-lot, pasture grazing Observed stray cattle on the premises Yes/no Presence of dogs in the facilities Yes/no Number of dogs Continuous Presence of cats in th e facilities Yes/no Number of cats Continuous Presence of adjacent farms Yes/no Number and type of adjacent farms Total, dairy farms, beef farms, heifer farms, and crops Observed deer on the premises Yes/no Observed monkeys on the premises Observed ostriches on the premises Yes/no Yes/no

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128 Table 3-2. Continued Explanatory variables Categories Observed green iguanas on the premises Yes/no Observed mongooses on the premises Yes/no Observed rats on the premises Yes/no Use of irrigation systems Yes/no History of flooding Yes/no Own more than one farm Yes/no Number of farms owned Continuous Origin of replacement heifers Farm-raised, buy from other farm, buy from local market, import from the US Purchase of animals in last 12 months Yes/no Number of animals bought in the last 12 months Continuous Use of quarantine area on the premises Yes/no Origin of hay, silage, green chop Same farm, other dairy farm, feed store, commercial forage farm Use of any vaccination protocol Yes/no Dehorning Guillotine, paste Use of needles Single animal use, multiple animal use Use of palpation sleeves Single animal use, multiple animal use Use of veterinary services Yes/no Problems with ticks Yes/no Use of acaricides Yes/no Type of acaricide Amitraz (Taktic), 11% permethrin (Atroban), 1% piperonyl butoxide and 1% permethrin (KattleGuard), ivermectins (Ivomec), eprinomectin (Eprinex), use of dust bags containing coumaphos (Co-Ral) Amitraz application method Spray races, handspraying Personnel in charge of applying amitraz Self, private, government Certification programf Yes/no Concentration of amitraz for applicationg Continuous Volume of amitraz applied to each cowh Continuous Time interval between amitraz treatments 7d, 14d, 21d, 30d, 40d, 50d, >60d Season of amitraz application Thr oughout the year, wet season (MayNovember), dry season (December-April) History of treatment for BA Yes/no Number of suspected BA cases in last 12 months Continuous Number of suspected BA cases diagnosed by laboratory means Continuous Number of suspected BA cases that died during the last 12 months Continuous Group of cattle most affected by BA Bulls, lactating cows, dry cows, heifers, calves

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129 Table 3-2. Continued Explanatory variables Categories Origin of BA affected animals Raised on-farm, bought in PR, imported from US Treatment protocol for BA clinical cases Only short-acting tetracycline (100mg/ml), only long-acting tetracycline (200mg/ml), combination of short and long-acting tetracycline Use of BA vaccine Yes/no Years since last BA vaccine Continuous Use of fly control Yes/no a Universal Transverse Mercator. b A herd with only lactat ing and dry cows on-farm. c Number of cuerdas (1 cuerda = 0.97 acres = 0.4 ha). d Total number of animals divided by total number of cuerdas. e Scientific names were obtained from th e USDA NRCS Plant Database. Available at: http://plants.usda.gov accessed 25 September 2007. f Certification program of the Puerto Rico Department of Agricultur e, agricultural extension service titled Guidelines for use and application of insecticides. g Number of cans (760 ml) per gallons (3.8 L) of water. h Number of gallons divided by total number of cows sprayed.

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130 0 2 4 6 8 10 12 14 16 18 20 102030405060708090>90 Prevalence (%)Count Figure 3-2. Distribution of herd seroprevalence for A. marginale in commercial dairy farms of Puerto Rico from August 2005 to December 2006.

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131Table 3-3. Overall animal seroprevalence and ser oprevalence by climatological zone and farm size for Anaplasma marginale in commercial dairy farms of Puerto Ri co from August 2005 to December 2006. Category No. of farms No. of cattle sampled No. seropositive Prevalencea SEb 95% CIc North coastal 511646466 25.81.223.3-28.2 Western interior 13390122 28.12.922.4-33.9 Eastern interior 8253105 31.63.724.4-38.9 South coastal 412550 40.64.930.9-50.3 Large 24837215 25.11.621.9-28.2 Medium 23759205 26.71.723.3-30.1 Small 29818215 38.11.734.6-41.5 Overall 762414743 27.41.125.3-29.5 a Weighted by sampling fractions for climat ological zone and size of the farm (%). b SE = standard error (%). c CI = confidence interval (%).

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132 Figure 3-3. Proportional dist ribution of animal ser oprevalence by farm for A. marginale in 78 commercial dairy farms of Puerto Rico from August 2005 to December 2006. Farms with higher anim al seroprevalence are denot ed by larger circles.

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133Table 3-4. Crude (unadjusted) risk factor analysis for pred icting high herd seroprevalence ( 40%) of Anaplasma marginale in 78 commercial dairy farms in Puerto Rico from August 2005 through December 2006. 95% CIa Exposure variable Comparison No. of farms exposed Odds ratio Lower Upper Wald P value Easting ( x -coordinate) Continuous 0.990.991.000.373 Northing ( y -coordinate) Continuous 0.970.931.010.089 Total number of animals in the herd Continuous 0.99 0.991.000.025 Guinea grass in major grazing areas of the farm Otherwise 16 0.15 0.021.230.077 Pasture grazing Otherwise (confined, semiconfined, dry lot) 31 2.32 0.836.530.110 Presence of cats in the facilities No cats in the facilities 45 0.280.100.820.020 Presence of adjacent beef farms No adjacent beef farms 15 3.371.0311.000.045 Observed monkeys on the premises Monkeys not observed on the premises 7 9.331.6552.960.012 Observed rats on the premises Rats not observed on the premises 55 0.390.131.130.083 Use of irrigation systems No irrigation systems 55 0.390.131.130.083 Own more than one farm Otherwise 53 0.470.161.330.155 Percent replacement heifers bought from another farm Continuous 4.630.7229.780.107 Use of any vaccination protocol No vaccination protocol 35 0.43 0.151.270.126 Use of guillotine for dehorning Paste exclusively 52 0.480.171.370.168 Use of a single sleeve for multiple animals Use of a single sleeve per animal 34 2.46 0.876.950.091 Use of 11% permethrin (Atroban) Otherwise 9 2.650.6411.060.181 Spray race for application of amitraz Handspraying for application of amitraz 30 0.44 0.141.370.157 Participated in certification pr ogram Did not participate in certification program 56 0.260.090.770.015 14 d time interval between amitraz treatments 7 d, 21d, 30d, 40d, 50d, >60d time interval between amitraz treatments 11 2.890.7710.800.115

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134Table 3-4. Continued 95% CIa Exposure variable Comparison No. of farms exposed Odds ratio Lower Upper Wald P value Use of 1% piperonyl butoxide and 1% permethrin (KattleGuard) Otherwise 20 0.250.051.180.079 No fly control methods Use of fly control methods 22 2.830.978.300.058 a CI = confidence interval.

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135Table 3-5. Multivariable logistic regression anal ysis for predicting high herd seroprevalence ( 40%) of Anaplasma marginale in 78 commercial dairy farms in Puerto Rico from August 2005 through December 2006. 95% CIa Exposure variable Comparison No. of farms exposed Odds ratio LowerUpper Wald P value Pasture grazing Confined, semi-confined, dry lot 31 6.42 1.2632.670.025 Observed monkeys on the premises Monkeys not observed on the premises 7 13.72 1.37137.210.026 Use of irrigation systems No irrigation systems 55 0.25 0.061.090.065 Use of 11% permethrin (Atroban) Otherwise 9 13.59 1.8997.660.010 Participated in certification pr ogram Did not participate in certification program 56 0.17 0.040.720.016 No fly control methods Use of fly control methods 22 5.71 1.3324.510.019 14 d time interval between amitraz treatments 7 d, 21d, 30d, 40d, 50d, >60d time interval between amitraz treatments 11 5.09 0.7534.730.097 a CI = confidence interval.

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136Table 3-6. Crude (unadjusted) general linear an alysis for predicting herd seroprevalence of Anaplasma marginale in 78 commercial dairy farms in Puerto Rico from August 2005 through December 2006. 95% CIa Exposure variable Comparison Coefficient ( ) Lower Upper P value Easting ( x -coordinate) Continuous -0.009-0.0360.0190.545 Northing ( y -coordinate) Continuous -.0.259-0.6040.0850.137 Total number of animals in the herd Continuous -0.030 -0.049-0.0110.002 Guinea grass in major grazing areas of the farm Otherwise -14.437 -25.200-3.6740.009 Pasture grazing Confined, semi-confined, dry lot 7.256 -1.88316.3950.118 Presence of cats in the facilities No cats in the facilities -10.285-19.180-1.3890.024 Presence of adjacent beef farms No adjacent beef farms 2.8980.4345.3630.022 Observed monkeys on the premises Monkeys not observed on the premises 19.9144.67535.1530.011 Observed rats on the premises Rats not observed on the premises -9.053-18.8040.6990.068 Use of irrigation systems No irrigation systems -6.547-16.4023.3090.190 Own more than one farm Otherwise -4.974-14.6484.6990.309 Percent replacement heifers bought from another farm Continuous 13.586-4.24431.4170.133 Use of any vaccination protocol No vaccination protocol -8.032 -16.9850.9210.078 Use of guillotine for dehorning Paste exclusively -3.588-13.3886.2130.468 Use of a single sleeve for multiple animals Use of a single sleeve per animal 5.135 -3.95614.2270.264 Use of 11% permethrin (Atroban) Otherwise 2.113-12.10616.3320.768 Spray race for application of amitraz Handspr aying for application of amitraz -10.044 -19.064-0.9450.031 Participated in certification program Did not participate in certification program -9.221-19.1000.6580.067 14 d time interval between amitraz treatments 7 d, 21d, 30d, 40d, 50d, >60d time interval between amitraz treatments 5.177-7.82918.1830.430 Use of 1% piperonyl butoxide and 1% permethrin (KattleGuard) Otherwise -14.361-24.240-4.4820.005 No fly control methods Use of fly control methods 15.8706.44225.2980.001 a CI = confidence interval.

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137 CHAPTER 4 STUDY 2: SEROPREVALENCE AND MANA GEMENT FACTORS ASSOCIATED WITH Babesia bovis IN COMMERCIAL DAIRY FARMS OF PUERTO RICO Introduction As in many tropical and subtr opical countries, cattl e operations in Puerto Rico (PR) are affected by the economic burden of tick-borne hemoparasitic diseas es caused by protozoan organisms of the genus Babesia (Phylum Apicomplexa, Order Piro plasmida, Family Babesiidae), particularly Babesia bovis and Babesia bigemina (de Leon et al., 1987). The disease is commonly characterized by outbreaks of sudden death that may be accompanied by clinical signs including hemoglobinuria, anemia and jaundice (Wagner et al., 2002). The first cases of bovine babesiosis (BB) in PR were reported in April 1985 (Combs, 1989). By October of the same year, 118 he rds were found to be seropositive for Babesia spp. by the complement fixation test. Babesia spp. are considered endemic in most parts of the island, especially those areas where the tropical cattle tick, Rhipicephalus (Boophilus) microplus is established (de Leon et al., 1987). An estimated economic loss of US $20 million was reported in 1989 in PR, attributable to the pres ence of bovine anaplasmosis, BB, and R. (Boophilus) microplus despite an ongoing tick eradication program (1936-1995) (Crom, 1992). A recent survey by Cortes et al. (2005) among 261 dairy fa rmers (47,401 milking cows) in PR reported an estimated loss of US $6.7 million in 2000 due to these diseases and the tropical cattle tick infestation. A yearly amount of US $7,155 was estimat ed per farm for tick control or US $29 per cow per year (Cortes et al., 2005). Cattle operations, especially commercial dairy farms, are the most economically important agricultural sector in PR. The dairy industr y produced US $184.8 million (25.6%) of the gross domestic product in agricultu re in 2006 (PRDA-ASO, 2006). Approximately 25,000 jobs are related to the production, manuf acture, and sale of milk and milk by-products (ORIL-PRDA,

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138 2006). The island has an estimated cattle population of 281,371 of which 153,097 (54%) belong to the dairy industry with 63,181 lactating cows (Planning Boar d of the Commonwealth of Puerto Rico, 2003; NASS-USDA, 20 04). Most lactating cows are raised within 353 commercial dairy farms operating in PR as of 2006. From 2005-06, these dairy farms produced 329 million liters (709 million pounds) of m ilk with an average production of 3,850 liters (8,277 pounds) per cow (NASS-USDA and PRDA-ASO, 2005). Although tick-borne hemoparasitic diseases ha ve been reported to cause considerable morbidity and mortality to cattle in PR, the ep idemiology of BB in PR has not been thoroughly investigated. Successful management of BB depends on adequate knowledge concerning seroprevalence estimates for Babesia spp., risk factors associated with the transmission of BB, and geographic locations and local climatic condi tions suitable for the distribution of BB and the tick vectors (Alonso et al., 1992; Ge orge et al., 2002). Several factor s such as type of livestock, herd size, interaction with wildlife, ecological and socio-economic fact ors may play important roles in the epidemiology of BB (Melendez, 1998). This information forms the basis for sustainable tick and tickborne disease control progra ms in cattle operations. The objectives of the present study were (1) to determine th e seroprevalence of B. bovis in adult lactating dairy cattle in the 4 different climatological zones of PR, (2) to assess the associations of geographical and farm ma nagement factors on the seroprevalence of B. bovis in commercial dairy farms in PR, and (3) to documen t the species of ticks infesting cattle within commercial dairy farms in PR. Materials and Methods Study Area Puerto Rico is centered in the Caribbean basin between the coordinates 17 N and 18 N, and its longitude ranges from 65 W to 67 W. It is the sm allest and easternmost

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139 island of the Greater Antilles, east of Haiti and Dominican Republic and northwest of the Virgin Islands. It is bounded on the north by the Atlantic Ocean and on the south by the Caribbean Sea. It encompasses an area of 8,870 km2 from which 41.6% is closed forests, 36.7% pastures and grasslands, 5.9% crop agriculture land, 2.4% coff ee plantations, and 10.5% urban and developed landcover (Helmer et al., 2002). Elevations range from sea level to 1338 m. The climate is predominantly tropical maritime. Average temperat ures in PR have very small range between the warmest and coldest months, but decrease mark edly with increasing elevation (NOAA-NCDC, 1982). In the high mountainous interior, the temp erature fluctuates between 22.8-25.6C (7378F). The northern half receives mo re rainfall and has larger rivers than the drier southern half. The distribution of rainfall follows a relative wet-dry seasonal pattern. The relative humidity (RH) is approximately 80% over the course of th e year with the highest RHs generally found at night (>90%) when temperatures are the lowest During the day, RH ranges from 60% to 70% (NOAA-NCDC, 1982). Puerto Rico is classified into 4 main climatological zones according to soil type, vegetation, agricultural developmen t, precipitation and temperature indices: humid coastal plains (north coastal region, NC), semiarid coastal plai ns (south coastal region, SC), humid mountains and valleys (western interior, WI), and semiarid mountains and valle ys (eastern interior, EI). The NC zone has rolling hill topography and an aver age annual precipitation of 1600 mm (63 in) and an average annual temperature of 25C (77F) Pastured-based commercial dairy farms are common in this area. Native and improved tropic al grasses cover approximately 50% of the land, whereas 17% of the acreage is used for a variety of crops. This is the most deforested zone partly because of intensive agricultural activities and urban development. Both native and improved pastures form the dominant landscap e in this zone. The SC zone has alluvial flat terrains and is

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140 the driest of the 4 zones with an average annual precipitation of 900 mm (35 in) and an average annual temperature of 26C (78.8F). More than 50% of this area is comprised of native and improved grasses, which are mainly used for f eeding beef cattle and ra cehorses. The remainder of the landscape consists of cacti, thorny legum es, and trees with small and succulent leaves. Fires are common during the dry season. The WI z one has irregular topog raphy with elevations of 500-600 m and an average annual precipita tion of 2100 mm (83 in) an d an average annual temperature of 24C (75.2F). A pproximately 70% of this area is native and improved grasses, 10% coffee plantations, and 7% forest. The remainde r is used for food crops including plantains, bananas, and yams. Because of the abundant moisture most of the original landscape consists of epiphytic ferns, bromeliads, and orchids. Lastly, the EI zone is comprised of very steep floodedalluvial plains and has an average annual precip itation of 1150 mm (45 in) and an average annual temperature of 26C (78.8F). Approximately 60% is native grasses and 35% is covered by forest (Ewel and Whitmore, 1973; Guttm an and Quayle, 1996; NRCS-USDA, 2007). The NC zone contains 62% of the dairy herds in PR that have 68% of the adult lactating cattle population. The predominant dairy production system is semi-intensive, which is based on concentrates and year-round grazing on improved pastures including African bermuda grass ( Cynodon nlemfuensis var. robustus ), pangola grass ( Digitaria eriantha ), and guinea grass ( Urochloa maxima ). Concentrates typically consist of grain by-products, molasses, vitamins, and mineral supplements imported from continental US sources. Most dairy replacement heifers are raised in PR using crossbreeding programs with Holstein-Friesian, Jersey and Brown Swiss, but some purebred Holstein-Friesian are imported from the US. Study Design and Sample Size Methodology A cross-sectional study with a multi-stage desi gn was carried out over 18 months between August 23, 2005 and December 4, 2006. The reference population for this study was adult

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141 lactating cows (>2 years of age) from commer cial dairy farms. Comme rcial dairy farms were defined as those farms having an active license in the 2004 Annual Inventory for Grade A dairies list provided by the Office for the Regulation of the Dairy Industry of Puerto Rico (La oficina de la reglamentacin de la industria lechera de Puerto Rico, ORIL) and the State Veterinarian from the Department of Agriculture, Commonwealth of Puerto Rico, in conjunction with the United States Department of Agriculture (USDA). The sample size for the number of farms wa s calculated assuming a total population of 362 farms, an expected herd ser oprevalence of 50% (the seroprev alence was unknown), a confidence level of 95%, and an acceptable margin of error of 10% with available software (Win Episcope 2.0, Easter Bush, Roslin; Zaragoza, Spain; Utre cht and Wageningen, Netherlands). To calculate the proportional distribution of farms to be sa mpled, the total number of farms in PR was stratified by climatological zone and farm size. Farm size was divided into approximately 3 equal groups using percentiles of the total number of farms (small, 36-109 animals; medium, 110-196; and large, 197-1000). An estimated pe rcentage was obtained by dividing the total number of farms within each stratum (e.g. WI-s mall) by the overall total number of farms (n=362). Each percentage was then multiplied by the necessary sample size. Study farms were randomly selected from all farms within each of the 12 strata. The sample size was estimated to be 76 farms and within each climatological zone was NC=51, WI=13, EI=8, and SC=4 (Table 41). Location of farms within each climatol ogical zone is presented in Figure 4-1. The sample size per herd was calculated using the number of lactating cows listed in the most recent ( 1 month) Dairy Herd Improvement Asso ciation (DHIA, Verona, WI) records of each farm visited. Number of animals per farm in this study ranged from 12 to 850 adult lactating cows. The minimum samp le size to detect disease was estimated assuming an expected

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142 prevalence of at least 8% at 95% level of confidence (S urvey Toolbox, version 1.0 beta, Wentworth Falls, NSW, Australia). Calculations were not adjusted for imperfect sensitivity and specificity of the diagnostic te st. Cows were chosen by either simple random selection (n=53 farms) using ear tag identification numbers fr om DHIA records (random number generator from Microsoft Office Excel, 2003) or systematically sa mpled through the chute based on the lactating herd size and the target number of samples in the herd (n=23 farms). Questionnaire Development and Administration A questionnaire (Appendix A) was administered to dair y owners and managers of participating farms from August 16, 2005 to Nove mber 29, 2006. The questionnaire consisted of 80 questions on 8 pages (printed on both sides). The questionnaire includ ed both open and closed questions with directions to either mark those answers that applied, choo se yes or no, or provide a free text response. When applicable, data that pertained to the preced ing 12-month period were collected. Dairy owners and managers answered que stions about type of facilities, common farm husbandry practices, past and recent experiences with ticks and use of acaricides, past and recent experiences with BB, and knowledge of disease diagnosis (Table 4-2). The questionnaire was first written in English and then translated in to Spanish (Appendix B). The English version was pre-tested on 5 faculty members and 6 veterina ry students from the Depa rtment of Food Animal Medicine at the University of Florida, College of Veterinary Medicine. The Spanish version was pre-tested at a 3-hr group discussion with 13 dairy farmers from the ORIL Executive Board 1 month before the first interview. Informati on concerning management factors was obtained before the seroprevalence of the farms was determined. Each participating farm was visited twice. Fo r the first visit, dairy owners (or managers) were contacted by phone 1 week in advanced of the expected visit to set up an appointment. During this visit, a brief summary of the study, an administrative order from the ORIL, and a

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143 consent form (including the purposes of the surve y, the procedures for part icipating in the study, and an assurance of confidentiality) were provided with the inte rviewer-administered questionnaire. The dairy owner (or manager) provid ed the most recent DHIA list of the lactating cows, returned the signed consent form, and se t up the appointment for the second visit. All interviews were conducted in Spanish by the first author. To standardize the interview process, the investigator was trained to ask all participan ts the same questions in the same manner. The questions with dichotomous response options that were not answered we re classified as no response. XY-coordinates from each participating dairy farm were collected using the milking parlor of the farm as the point of refere nce by a global positioning system (GPS) (Garmin e Trex Legend, Garmin International Inc., Olathe, KS, USA). Sample Collection All procedures were approved by the Inst itutional Animal Care and Use Committee (IACUC # D806 and #E151) at the Un iversity of Florida. A blood sample (~10 ml) was collected from each selected animal (n=2,414) during the s econd visit. Selected cows either were grouped by the farmers immediately after milking or were bled individually after leaving the milking parlor. Samples were collected aseptically via coccygeal venipuncture into 10-ml uncoated red top tubes (Vacutainer, Fisher Scie ntific, Pittsburgh, PA, USA) usi ng disposable needles. Samples were labeled and transported on ice to the laboratory facilities at the Agricultural Experiment Station, University of Puerto Rico (Mayagez Campus). In the laboratory, serum samples were centrifuged at 3700 rpm for 15 min at room temperat ure, serum separated, and frozen in 1 ml aliquots at -25 C (-13 F) until analyzed. Samples were sent to the Department of the Veterinary Pathobiology at Texas A&M University (Texas, USA) for serological testing. All sampled animals were examined for the presence of ticks. If observed, ticks were removed from the tail-head and flanks, but other parts of the body were also examined. The ticks

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144 from each farm were placed in separate labe led glass vials containing 70% alcohol and were subsequently counted. All ticks were examined under a dissecting microscope and categorized into species, sex, and feeding status (unfed, semi-engorged, and fully engorged) according to identification keys by Keirans and Litwak (1989). A sub-sample wa s sent for verification to Dr. Lance Durden, Assistant Professor, Departme nt of Biology, Georgia Southern University (Statesboro, GA, USA). Serological Testing Sera were tested for antibodies against B. bovis using an indirect fl uorescent antibody test (IFAT). The test was conducted as initially de scribed by Todorovic and Long (1976) with minor modifications. Briefly, the antigen slides were prepared from in vitr o cultures of B. bovis at 3-5% parasitemia levels. Cultures from 12 individual we lls of a 25-well culture plate were pooled and centrifuged at 1000 xg for 10 min. The culture pelle ts were re-suspended in cold phosphate buffered saline (PBS) buffered to a pH of 7.2, ce ntrifuged, and re-suspended again in an equal volume of PBS containing 1% purif ied egg albumin to produce a packed cell volume of 20-30%. Ten l of the red blood cell suspension was placed on individual clean frosted-end microscope slides and evenly dist ributed thick films were prepared. Slides with the antigen were dried and placed smear-down on 1-inch masking tape and kept at -20 C until use. Before use, slides were removed from the fr eezer and allowed to warm at r oom temperature for 10 minutes. After removal of masking tape, 3 rows of 11 squa res each (3-4 mm in width) were drawn using fingernail polish contained in a tuberculin syringe fitted with a blunt 22-G needle. Nail polish was allowed to dry before testing was performed. Sample sera were thawed and d iluted 1:80 with cold PBS. Seven l of diluted sample sera were added to each square on the slide and incubated for 60 min at 37 C in a moist chamber. One known weak positive control, one known nega tive control sera, and one PBS control were

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145 included on each slide. After inc ubation, smears were washed by spra ying the slides with cold PBS using a wash bottle. The wash step was performed 3 times. After washing, smears were allowed to partially dry. Ten l of a commercial Protein G conjugated with fluorescein isothiocyanate (Sigma-Aldrich, ST. Louis, MO, USA) diluted 1:300 with cold PBS was added to each square and incubated for 60 min at 37 C in a moist chamber. After incubation, slides were washed as done previously and allowed to dry. Slides were examined using an epi-fluorescence microscope (Olympus America Inc., Center Valley, PA, USA) with 100X oil-immersion objective. Results for serum samples were reported as positive or negative. Statistical Analyses The overall and stratified anim al seroprevalence by each climat ological zone in PR was calculated as the proporti on of animals positive on the IFAT divided by the total number of cows tested, weighted by the proportion of the tota l cattle population in each climatological zone (STATA 10.0 for Windows, StataCorp LP, College Station, TX, USA). St udent t tests using Tukeys adjustment for multiple pairwise comparisons were used to compare the mean prevalences among the 4 climatol ogical zones and 3 farm sizes. The coordinates and survey data for each farm were loaded into a geo-database (ArcGIS 9.1TM, ESRI, Redlands, CA, USA) for mapping and cluster analys es. To standardize the GIS analyses, all data were collected under the sa me projection parameters, North American Datum 1983 (NAD83) and a coordinate system correspondi ng to Universal Transverse Mercator (UTM) Zone 19 North. Spatial autocorrelation of the observe d seroprevalence was estimated using Morans I (ArcGIS 9.1TM). The associations between the explanatory variables collect ed via the questionnaire and outcome response variab le (seroprevalence to B. bovis ) were estimated usi ng logistic regression (SPSS 14.0 for Windows, SPSS Inc. Headquarters, Chicago, IL, USA). The unit of analysis for

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146 assessing risk factors was the farm. The outcome variable was dichotomized using a natural break in the distribution of the observed herd seroprevalence for B. bovis (Figure 4-2). The break was set at the boundary where a re latively large change in the di stribution occurr ed. Those herds with a seroprevalence 25% were considered high prev alence and those farms with a seroprevalence 24.9% were considered low prevalence. Variables that were significantly associated with high/low seroprevalence at a Wald P value 0.20 in a bivariable screening model were retained for further evaluation in multivariable models. A backward stepwise multivariable logistic regression model was built starting with a complete model containing all main effects identified in the screening mode ls and terms were removed one-by-one based on likelihood ratio tests. The main ef fect terms remaining in the m odel were evaluated for effect modification by adding all possible 2-way interac tions between main e ffects individually and testing for significance using Wald tests. The f it of the final multivariable model was assessed using the Hosmer-Lemeshow test Statistical significance was assessed at the 5% level. Continuous independent variables in logistic models were ev aluated for linearity in the log-odds. These variables were categorized to pr ovide an indication of how the risk of the outcome changes with the variable and have bett er specification of the functional form of the equation (Cheung and Smith, 1981; Hosmer and Lemeshow, 2000). Initia l categorization was performed either by using 3 or more natural breaks of the data or by equally dividing the data in quartiles. If the continuous inde pendent variables were not lin ear in the log-odds, they were retained as categorical variables. The response (seroprevalence to B. bovis ) was also analyzed as a continuous dependent variable and the associations between the explan atory variables and the response were estimated using linear regression. The associations of coefficients to the response were compared to the

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147 odds ratio of the variables that were significantly associated with high/lo w seroprevalence at a Wald P value 0.20 in the bivariable logi stic regression analyses. Results Descriptive The number of randomly selected farms sampled was 76 and 2 additional farms volunteered to be in the study, one in the SC zone and one in the EI zone. An average of 32 cows was selected per farm for a total sample size of 2,414 from the 76 farms and 61 additional sampled cows from the 2 volunteer farms. Th e 2 volunteer farms were not included in the analyses for prevalence, but were included for the logistic regression analys es of risk factors. The time elapsed between the beginning of the interview to the end averaged 50 min (range 20 min to 95 min) among dairy owners an d managers and the average time between the first and second visit was 7 days (range 0 d to 75 d). Ticks were collected from animals on 7 (9%) of the 78 participating commercial dairy farm s. The only ticks recognized infesting cattle (n=87) were R. (Boophilus) microplus Cattle were found to have nymphs (n=12), unfed females (n=5), engorged females (n=52), and males (n=18). Eighteen (23.1%) of the sampled farms were co mmercial dairies with only lactating and dry cows on the premises; 14 (17.9%) were commercia l dairy farms with calf raising facilities in addition to lactating and dry co ws; 6 (7.7%) were commercial dair y farms with heifer raising facilities in addition to lact ating and dry cows; and 40 (51.3% ) were commercial dairy farms with calf and heifer raising facilities in addi tion to lactating and dry cows. The herds were comprised of 92.3% Holsteins and Holstein cros ses; and 7.6% Jersey and Brown Swiss breeds and their crosses. Although 74 (94.9%) of the farmers had wellconstructed fences surrounding their premises, 19 (24.4%) of the farmers reported that stray cattle had entered their premises on

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148 limited occasions and 8 (10.8%) reported frequent incursions. Eleven (14%) of the farmers had more than 4 adjacent neighbors with cattle and 53 (67.9%) of the farmers owned more than one farm. Seventy-three (93.6%) of the farmers used amitraz as the primary acaricide for tick control. Amitraz was applied either by a re-circulating spray race (38.5%) or by handspraying (61.5%). The typical spray race consisted of an approximately 6m long a nd 1m wide tunnel with masonry sidewalls and a concrete floor. A spray pipe system spanning 3 to 3.5m of the tunnel has 25 to 30 nozzles placed in the walls, ceiling and floor, a nd discharges amitraz at a high pressure. Cattle are exposed to a dense spray as they pass thr ough the tunnel. The disc harged amitraz collects on the floor and a draining race leads to a sump wher e a pump is used for re-circulation. Fifty-nine (75.6%) of the farmers applied amitraz every 20 -30 days whereas 12 (15.4%) applied amitraz every 7-14 days and 7 (9.0%) applied the acarici de every 40 days. Within the 73 farmers that used amitraz, 68 (93.2%) applied am itraz year-round and 5 (6.8%) appl ied it either in the dry or rainy season alone. Amitraz application was pr ovided either by the same owner (61.5%), a private contractor (6.4%), or th e government services (32.1%). Seroprevalence The IFAT for B. bovis antibodies was positive for 619 of 2,414 (25.6%) sampled adult lactating cows (Table 4-3). An imal seroprevalence within farms ranged from 0 to 54.1%; only 2 farms were seronegative for the organism (Figur e 4-3). The overall animal seroprevalence of antibodies against B. bovis was 25.5% (95% CI = 23.4-27.6). Th e EI zone had the lowest seroprevalence of the 4 zone s, 19.0% (95% CI = 13.3-24.7) followed by the NC zone 25.3% (95% CI = 22.8-27.8). Conversely, the SC and WI zones had the highest seroprevalence for B. bovis 29.0% (95% CI = 19.8-38.1) and 31.8% (95% CI = 25.4-38.2), respectively. However, no statistically significant differen ces at the 5% level were found among the 4 climatological zones

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149 and the 3 sizes of farms. Spatial analysis for B. bovis seroprevalence did not suggest any global spatial autocorrelation (I = -0.02; Z score = -0.1; P value = 0.46). Risk Factors Thirty-four farms had high animal-level seroprevalence for B.bovis ( 25%) and 44 had low seroprevalence ( 24.9%). During the initial bivariable sc reening, 15 management factors were found to be significantly associated with high B. bovis seropositivity at P 0.20 (Table 4-4). The final multivariable logistic regression model (Table 4-5) demonstrated that farms located in the north coastal region (OR= 0.21, 95% CI=0.05-0.86), da iry farms with calf ra ising facilities (OR= 16, 95% CI=3.0-86), having more than 4 neighbors with cattle (OR= 17, 95% CI=1.6-176), same producer owing more than 1 farm (OR= 7.3, 95% CI=1.7-32), and use of government services to apply amitraz on cattle (OR= 5.5, 95% CI=1.5-20) had a significant asso ciation with high BB seropositivity. No other main eff ects or 2-way interaction terms were significant. The HosmerLemeshow goodness-of-fit test demons trated that the model was a r easonably good fit to the data (Chi = 2.590, df = 7, P-value = 0.920). Logistic regression analyses identified the same model with and without the 2 volunteer farms. The coefficients in the linear regression had the same direction of association as th e odds ratios for all but 1 variab le associated with high/low seroprevalence at a Wald P value 0.20 in the bivariable logistic regression analyses (Table 46). Discussion The present study is the first in PR to assess the overall seroprevalence for B. bovis and identify farm management factors significantl y associated with high seropositivity. The study sample was limited to adult lactating cattle from commercial dairy farms. Therefore, inferences about the present study might not re present other cattle populations.

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150 The number of ticks observed on animals was lo wer than expected with only 7 of the 78 participating commercial dairy farms having animals infested with R. (Boophilus) microplus The apparent reduced population of ticks on the farms can result from intensive use of acaricide treatments that might keep the number of ticks at very low levels. Nevertheless, farmers that interrupt the amitraz treatments for 30 to 60 days have anecdotally reported an increase in tick numbers. Serological results indicated that B. bovis is common and widely distributed in PR. Overall seroprevalence for B. bovis in PR was within the lower range documented for other islands in the Caribbean region (22 to 69%) (Camus and Mont enegro-James, 1994). This might be explained by the low numbers of R. (Boophilus) microplus observed in sampled dairies. The frequent use of amitraz may maintain low tick populations on the premises (Sutherst et al., 1979). Spatial distribution was not clustered at the scale of the en tire island suggesting limited influence by geographic predictors. Therefore, management fact ors appear to be of primary importance in the epidemiology of BB on the island. In the present study, 2 dominant factors were associated with increased odds of high seropositivity for B. bovis : farm having more than 4 neighbori ng premises with cattle (OR= 17) and a farm having calf-raising faci lities (OR=16). The association of a farm having more than 4 neighboring premises may be explai ned in part by inadequate tick control measures by neighbors and the possible spread of infected ticks, which can be a contributing cause of high seropositivity (Callow and Dalgliesh, 1980; Bock et al., 2004). In addition, the high number of neighbors in a limited space may increase the stocking density in the area increasing the probability of R. (Boophilus) microplus tick larvae meeting a susceptible an imal (Solorio-Rivera et al., 1999). Most dairy farmers in PR do not apply amitraz to 4 month and older calves raised in groups or

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151 colonies (Torrado and Cordero, 2007, personal communica tion). These calves may be exposed to tick infestations, increasing the risk of B. bovis infection (Perez et al., 1996). Therefore, this particular group could serve as a constant sour ce of infection on certain farms (Howell et al., 2007). Owning more than 1 farm was also associ ated with increased odds of having high seropositivity (OR=7.3). Most dairy farmers in PR split their herds in 2 groups. At the main dairy facility, they only have the milking cows whereas dry cows and replacement heifers are usually kept in premises away from the dairy (Suthe rn and Combs, 1984). M ovement of cattle among these different premises is very common in da iry farmers that owned more than one farm (Torrado, 2007, personal communication). Some of these premises are geographically close to the main facilities but in many situations these pr emises are located a moderate distance from the dairy (e.g. different municipality). As heifers or dry cows freshen, the dairy farmer immediately moves these groups between premises. Therefor e, cattle can be moved from high seropositive areas to low seropositive areas. Dairy farms located in the NC zone had d ecreased odds of having high seropositivity for B. bovis when compared to farms located in the WI EI, and SC zones (OR= 0.21). It is possible that this association is relate d to spatial or unmeasured manage ment factors. Although Morans I suggested that the spatial pattern for B. bovis was neither clustered nor dispersed, it is a global indicator of spatial autocorrelati on and finer local differences might not be detected. Therefore, local clusters may have been missed. The positive association between the use of government servi ces for amitraz application with high seroprevalence farms (OR= 5.5) may s uggest a general misuse of the acaricide and reduced efficacy. Previously and during the st udy period, the policy on tick control methods

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152 from the Division of Crop Protection and Tick C ontrol (Divisin de proteccin de cultivos y control de garrapatas) of the PR Department of Agriculture consisted of providing dairy farmers with the acaricide (amitraz) and assisting with the mixing and applica tion of amitraz every 30 days by handspraying (PRDA-ASDA, 2007). Factors limiting the effectiveness of this service may include the timing and frequency of amitraz treatments and the method of preparing and applying the amitraz (Jonsson, 1997; George et al ., 2004). The 30-day interval is based on the duration of the parasitic phase of R. (Boophilus) microplus (approximately 25 days) (Tate, 1941) and the duration of the residual acaricide activity (approximately 1 week) (Smith et al., 2000). If the treatment intervals are longer than the paras itic cycle of the tick, then it might not interrupt the cycle and prevent the tick on the host from reach ing the stage of engorged female, drop to the ground, lay eggs, and consequently developmen t into larvae. Larvae are responsible for producing new re-infestations. Incorrect amitraz c oncentrations will reduce the dose of the active ingredient. This reduced dose may not be suffi cient to kill most ticks, allowing a larger percentage of ticks to continue with the para sitic lifecycle and develop amitraz resistance (Georghiou and Taylor, 1977; Suth erst and Comins, 1979; George et al., 2004). One of the key points with spraying equipment is that applicatio n is only as thorough as the operator. Operators tend to inadequately apply acaricide s, particularly in areas that are difficult to reach including the abdomen, axillary regions, and inside of the ears (Jonsson, 1997). Failure to completely cover the animal with acaricide minimizes the quality of tick control programs (George et al., 2004). The results of this study have provided base line prevalence information and identified some key risk factors that can guide local aut horities and policymakers to implement strategic control measures to decrease the seroprevalence of B. bovis in PR. However, this is a crosssectional design and there are important limitati ons that must be considered. For example,

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153 measured variables in the pres ent study might not have had th e same value as when cattle became seropositive. The findings of this study are also limited to the 18-month period 20052006 when data were collected. Hi gh seroprevalence does not necessa rily equate to more disease and higher economic losses. A different farm ser oprevalence cut-off could have yielded different results; however, a linear modeling of the seropr evalence rather than high/low dichotomization demonstrated similar findings and overall conclusi ons. Lastly, testing did not attempt to adjust prevalence for sensitivity and speci ficity of the test and might ha ve resulted in misclassification of the prevalence (e.g. high seropr evalence not due to high BB prevalence). Unlike other tests, IFAT is not standardized. Diffe rent serum or conjugate dilutions in IFAT among studies have shown markedly different Se and Sp values. Johnston et al. (1973) evaluated the IFAT for antibodies to B. bovis by using a 2-fold dilution of the test serum on 290 unexposed animals from non-endemic areas of Australia and 98 exposed animals sufferi ng from acute and persistent infections. The Se and Sp were reported as 99% and 96%, respectivel y (Johnston et al., 1973). Conversely, Hadani et al. ( 1983) evaluated the IFAT for B. bovis on brain smears stratified by sex and age. Se ranged from 76.5% to 100% and Sp ranged from 30.8% to 66.7%. The pooled values for Se and Sp were 85.9% and 46.6%, respec tively. Cattle in this study (n=213) originated from abattoirs in the northwest region of Arge ntina and all serum samples were tested at a dilution of 1:60 with a conjugate dilution of 1:80 (Hadani et al., 1982; Hadani et al., 1983). Conversely, a study by Torioni de Echaide et al. (1995) using the same dilution for serum and conjugate reported a Se ra nge of 92.8 % to 100% for B. bovis Conclusions Management factors appear to be of primar y importance for understanding BB in PR. We suggest that at the time designers of tick and tick-borne disease control programs in PR

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154 implement measures to control BB they should cons idered the biology of th e tick and the current knowledge of the epidemiology of BB.

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155Table 4-1. Population and sample distributi on of commercial dairy farms by climatological zone and size of the farm to estimat e the seroprevalence of Babesia bovis in Puerto Rico from August 2005 to December 2006. Climatological zone Farm sizea No. of farms No. of adult lactating cows Percent farm needed Farm sample needed North coastal Small 705310 1915 Medium 8211916 2317 Large 9128154 2519 Western interior Small 312336 97 Medium 243556 75 Large 71778 21 Eastern interior Small 9710 22 Medium 152364 43 Large 134109 43 South coastal Small 10759 32 Medium 5684 11 Large 51505 11 Totalb 36263181 76 a Farm size: Small (36-109), Medium (110-196), Large (197-1000). b Data obtained from the 2004 Annual Inventory for Grade A dairies list provided by the Office for the Regulation of the Dairy Industry of Puerto Rico (ORIL) and the State Veterinarian from the Department of Agriculture, Commonwealth of Pu erto Rico, in conjunction w ith the United States Departme nt of Agriculture (USDA).

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156 Figure 4-1. Geographical di stribution of the 76 commercial dairy farms ( ) sampled in the 4 climatological zones of Puerto Rico from August 2005 to December 2006. NC: North Coasta l Zone (n=51); WI: Wester n Interior Zone (n=13); EI: Eastern Interior Zone (n=8); and SC: South Coastal Zone (n=4).

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157 Table 4-2. Explanatory variables included in th e questionnaire provided to dairy owners or managers for the study of the seroprevalence of Babesia bovis in Puerto Rico from August 2005 to December 2006. Explanatory variable s Categories Climatological zone North coastal (NC), western interior (WI), eastern interior (EI), and south coastal (SC) UTM coordinatesa Easting ( x -coordinate) and Northing ( y coordinate) Type of operation Dairy farm with lactating herd onlyb, dairy farm with lactating herd and calf-raising, dairy farm with lactating herd and heiferraising facilities, dairy farm with lactating herd, calf, and heifer-raising facilities, mixed (beef and dairy) Herd size Number of bulls, milking cows, dry cows, heifers (1-2 years), and calves (<1 year), total Premises (property) sizec Continuous Stocking densityd Continuous Breed(s) of cows and percent of breed in herd Holstein, Jersey, Brown Swiss, Cross-bred Presence of horses in the facilities Yes/no Number of horses Continuous Presence of sheep in the facilities Yes/no Number of sheep Continuous Presence of goats in the facilities Yes/no Number of goats Continuous Presence of pigs in the facilities Yes/no Number of pigs Continuous Presence of poultry in the facilities Yes/no Number of poultry Continuous Main type of pasture grown on the farme African bermuda grass ( Cynodon nlemfuensis var. robustus ), guinea grass ( Urochloa maxima ), pangola grass ( Digitaria eriantha ), and bermuda grass ( Cynodon dactylon ) Type of facility Confined, semi-confined, dry-lot, pasture grazing Observed stray cattle on the premises Yes/no Presence of dogs in the facilities Yes/no Number of dogs Continuous Presence of cats in th e facilities Yes/no Number of cats Continuous Presence of adjacent farms Yes/no Number and type of adjacent farms Total, dairy farms, beef farms, heifer farms, and crops Observed deer on the premises Yes/no Observed monkeys on the premises Observed ostriches on the premises Yes/no Yes/no

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158 Table 4-2. Continued Explanatory variables Categories Observed green iguanas on the premises Yes/no Observed mongooses on the premises Yes/no Observed rats on the premises Yes/no Use of irrigation systems Yes/no History of flooding Yes/no Own more than one farm Yes/no Number of farms owned Continuous Origin of replacement heifers Farm-raised, buy from other farm, buy from local market, import from the US Purchase of animals in last 12 months Yes/no Number of animals bought in the last 12 months Continuous Use of quarantine area on the premises Yes/no Origin of hay, silage, green chop Same farm, other dairy farm, feed store, commercial forage farm Use of any vaccination protocol Yes/no Dehorning Guillotine, paste Use of needles Single animal use, multiple animal use Use of palpation sleeves Single animal use, multiple animal use Use of veterinary services Yes/no Problems with ticks Yes/no Use of acaricides Yes/no Type of acaricide Amitraz (Taktic), 11% permethrin (Atroban), 1% piperonyl butoxide and 1% permethrin (KattleGuard), ivermectins (Ivomec), eprinomectin (Eprinex), use of dust bags with coumaphos (Co-Ral) Amitraz application method Spray races, handspraying Personnel in charge of applying amitraz Self, private, government Certification Programf Yes/no Concentration of amitraz for applicationg Continuous Volume of amitraz applied to each cowh Continuous Time interval between amitraz treatments 7d, 14d, 21d, 30d, 40d, 50d, >60d Season of amitraz application Thr oughout the year, wet season (MayNovember), dry season (December-April) History of treatment for BB Yes/no Number of suspected BB cases in last 12 months Continuous Number of suspected BB cases diagnosed by laboratory means Continuous Number of suspected BB cases that died during the last 12 months Continuous Group of cattle most affected by BB Bulls, lactating cows, dry cows, heifers, calves

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159 Table 4-2. Continued Explanatory variables Categories Origin of BB affected animals Raised on-farm, bought in PR, imported from US a Universal Transverse Mercator. b A herd with only lactating and dry cows on-farm. c Number of cuerdas (1 cuerda = 0.97 acres = 0.4 ha). d Total number of animals divided by total number of cuerdas. e Scientific names were obtained from th e USDA NRCS Plant Database. Available at: http://plants.usda.gov accessed 25 September 2007. f Certification program of the Puerto Rico Department of Agriculture, agricultural exte nsion service titled G uidelines for use and application of acaricides. g Number of cans (760 ml) per gallons (3.8 L) of water. h Number of gallons divided by total number of cows sprayed.

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160 0 2 4 6 8 10 12 14 16 18 5101520253035404550>50 Prevalence (%)Count Figure 4-2. Distribution of herd seroprevalence for B. bovis in commercial dairy farms of Puerto Rico from August 2005 to December 2006.

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161Table 4-3. Overall animal seroprevalence and ser oprevalence by climatological zone and farm size for Babesia bovis in commercial dairy farms of Puerto Rico from August 2005 to December 2006. Category No. of farms No. of cattle sampled No. seropositive Prevalencea SEb 95% CIc North coastal 511646419 25.31.322.8-27.8 Western interior 1339098 31.83.325.4-38.2 Eastern interior 825370 19.02.913.3-24.7 South coastal 412532 29.04.719.8-38.1 Large 24837198 23.91.620.8-27.1 Medium 23759204 28.31.824.7-31.9 Small 29818217 26.11.623.0-29.2 Overall 762414619 25.51.123.4-27.6 a Weighted by sampling fractions for climat ological zone and size of the farm (%). b SE = standard error (%). c CI = confidence interval (%).

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162 Figure 4-3. Proportional dist ribution of animal ser oprevalence by farm for B. bovis in 78 commercial dairy farm s of Puerto Rico from August 2005 to December 2006. Farms with higher animal seroprevalence are denoted by larger circles.

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163Table 4-4. Crude (unadjusted) risk factor analysis for pred icting high herd seroprevalence ( 25%) of Babesia bovis in 78 commercial dairy farms in Puerto Rico from August 2005 through December 2006. 95% CIa Exposure variable Comparison No. of farms exposed Odds ratio Lower Upper Wald P value NC WI, EI, and SC 51 0.48 0.181.230.124 Dairy farm with calf-raising facilities Otherwise 14 6.54 1.6525.850.007 Dairy farm with calf and heiferraising facilities Otherwise 43 0.45 0.181.130.088 Guinea grass in major grazing areas of the farm Otherwise 16 0.36 0.101.220.101 More than 4 adjacent neighbors with cattle None to 3 adjacent neighbors 11 17.922.16148.580.008 Observed monkeys around the farm Monkeys not observed 7 3.62 0.6619.950.140 Own more than 1 farm Otherwise 53 2.67 0.967.450.061 Buy hay in feed store Otherwise 9 2.93 0.6812.700.151 Use of any vaccination protocol No use of vaccination protocol 35 2.22 0.895.530.088 Use of guillotine for dehorning Paste exclusively 52 1.97 0.725.350.185 Use of a single sleeve per animal Use of a single sleeve for multiple animals 34 0.55 0.221.370.196 Spray race for application of amitraz Handspraying for application of amitraz 30 0.39 0.151.030.059 Government administration of amitraz Private contractor or same producer applying amitraz 25 2.10 0.805.520.132 Use of 1% piperonyl butoxide and 1% permethrin (KattleGuard) Otherwise 20 0.460.161.360.160 Use of dust bags with coumaphos (Co-Ral) Otherwise 5 5.730.6153.870.127 a CI = confidence interval.

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164Table 4-5. Multivariable logistic regression anal ysis for predicting high herd seroprevalence ( 25%) of Babesia bovis in 78 commercial dairy farms in Puerto Rico from August 2005 through December 2006. 95% CIa Exposure variable Comparison No. of farms exposed Odds ratio LowerUpper Wald P value North coastal WI, EI, and SC 51 0.21 0.050.860.030 Dairy farm with calf-raising facilities Otherwise 14 15.98 2.9686.270.001 More than 4 adjacent neighbors with cattle None to 3 adjacent neighbors 11 16.90 1.63175.850.018 Own more than 1 farm Own only one farm 53 7.281.6731.770.008 Government administration of amitraz Private contractor or same producer applying amitraz 25 5.50 1.5120.000.010 a CI = confidence interval

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165 Table 4-6. Crude (unadjusted) general linear analysis for pr edicting herd seroprevalence of Babesia bovis in 78 commercial dairy farms in Puerto Rico from August 2005 through December 2006. 95% CIa Exposure variable Comparison Coefficient ( ) Lower Upper P value NC WI, EI, and SC -1.025 -7.1595.1080.740 Dairy farm with calf-raising f acilities Otherwise 10.527 3.30817.7460.005 Dairy farm with calf and heifer-raising facilities Otherwise -5.111 -10.8640.6430.081 Guinea grass in major grazing areas of the farm Otherwise -2.329 -9.5414.8830.522 More than 4 adjacent neighbors with cattle None to 3 adjacent neighbors 10.3472.29718.3970.012 Observed monkeys around the farm Monkeys not observed 8.804 -1.21318.8210.084 Own more than 1 farm Otherwise 7.646 1.63713.6540.013 Buy hay in feed store Otherwise 5.621-3.42814.6700.220 Use of any vaccination protocol No use of vaccination protocol 6.095 0.39111.7980.037 Use of guillotine for dehorning Paste exclusively -0.258 -6.5636.0460.935 Use of a single sleeve per animal Use of a single sleeve for multiple animals -1.813 -7.6874.0620.541 Spray race for application of amitr az Handspraying for application of amitraz -7.342 -13.105-1.5790.013 Government administration of amitr az Private contractor or same producer applying amitraz 3.944 -2.24810.1360.208 Use of 1% piperonyl butoxide and 1% permethrin (KattleGuard) Otherwise -2.768-9.4253.8900.410 Use of dust bags with coumaphos (CoRal) Otherwise 10.685-0.98422.3550.072 a CI = confidence interval.

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166 CHAPTER 5 STUDY 3: ASSOCIATION BETWEEN ECOL OGICAL FACTORS AND THE PRESENCE OF Rhipicephalus (Boophilus) microplus LARVAE IN PUERTO RICO Introduction The tropical or sout hern cattle tick, Rhipicephalus (Boophilus) microplus (Canestrini) (Order Parasitiformes, Suborder Ixodida, Family Ixodidae, Subfamily Rhipicephalinae) is one of the 3 species of ticks present in Puerto Rico (P R) and it is considered the principal vector of Anaplasma marginale Babesia bovis and Babesia bigemina (Crom, 1992). The other 2 tick species found on domestic animals in PR include the tropical horse tick, Dermacentor (Anocentor) nitens and the brown dog tick, Rhipicephalus sanguineous (van Volkenberg, 1939). An estimated economic loss of US $20 million was reported in 1989 in PR due to anaplasmosis, babesiosis, and R. (Boophilus) microplus (Crom, 1992). It has been estimated that the cattle industry suffers a yearly defi cit of 3,602,873 kg (7,926,321 lbs) of meat and 14, 373,315 L (32, 274,840 lbs) of milk due to these dis eases (Soto-Alberti, 1999 unpublished data). A recent survey by Cortes et al. (2005) among 261 dairy farmers (47,401 milking cows) reported an estimated loss of US $6.7 million in 2000 due to these diseases and the tropical cattle tick. A yearly amount of US $7,155 was estimated per farm for tick cont rol or US $29 per cow per year (Cortes et al., 2005). Rhipicephalus (Boophilus) microplus is a one-host tick where each instar (larvae, nymphs, and adults) remain on the same animal (Radunz, 1997). The life cycle of this tropical tick consists of a parasitic phase and a nonparasitic phase. In the parasitic phase, R. (Boophilus) microplus attaches as a 6-legged larvae to cattle, and undergoes 2 moultings on the host animal until it becomes a sexually different iated 8-legged adult (Radunz, 2003). The complete parasitic phase in PR typically requires 18 to 37 days (T ate, 1941). However, the majority of ticks will complete engorgement and drop by day 25 (Tate, 1941).

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167 The non-parasitic phase begins when the fully engorged female ticks detach and drop onto the ground and ends when larvae develop and fi nd a suitable host (Radunz, 1997). This phase consists of 4 main stages: pr e-oviposition, oviposi tion, incubation and hatching of eggs, and development of larvae. Approximately 72-89% of the life cycle consists of the non-parasitic phase. The effects of temperature, relative humid ity, type of vegetation, and seasonality on the biology and survival of th e non-parasitic phases of R (Boophilus) microplus are well documented in the literature (Hit chcock, 1955; Harley, 1966; Utech et al., 1983; Wilson and Sutherst, 1990). In PR, the length of this phase ranges from 89 to 251 days (3 to 8.5 months) and depends upon location. In areas with 935 mm (36.8 inches) of annual rainfall and 22.5 C (72.5 F) mean temperature, the length of the phase is approxi mately 89 days whereas in areas with 4,358 mm (171.6 inches) and 22.5 C (72.5 F) the phase last approximately 251 days (Tate, 1941). The larvae are the non-parasitic stage of R. (Boophilus) microplus that infests the cattle host and can be subjected to environmental c onditions and vegetation stresses for a relatively long period of time. Larvae seek suitable hosts by climbing a nd aggregating at the tip of vegetation or other vertical object s, preferentially on the shaded side during the hours of strong illumination (Wilkinson, 1953; Arundel and Sutherland, 1988). They either aggregate in a series of tightly arranged rows with their legs pulled underneath or by formi ng a ball that is easily transferred to a passing animal (Garris and Popham, 1990). A study by Wilkinson (1953) estimated that these aggregations could consist of 70 to 260 larvae. Larvae positioned in rows are not active and they remain immobile until dist urbed. When cattle or other animals are near, larvae adopt a questing position. This position cons ists of waving their first pair of legs in the air and moving towards the animal as it passes (Nunez et al., 1982). These aggregations provide

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168 a massive and rapid transfer to a passing hos t (Treverrow, 1980) and provide an excellent opportunity for sampling larvae in the fi eld using the tick drag technique. Some researchers have documented that many species of ticks are influenced more by environmental and vegetation factors than by hos t availability (Cummi ng, 1999). However, these effects may vary according to th e spatial scale at which the st udy is performed varying within countries, regions, and even within a given ha bitat (Randolph, 2000). Certain factors may be associated differently in differe nt scenarios and conclusions cannot be immediately extrapolated without considering local conditions. Therefore, th ese factors must be evaluated and applied to the area of interest (Teel, 1984; Panda et al., 1992). Local microclimate depends upon topography and the type and amount of vegetation. An understanding of the effects of ecological factors and host availability on the tropical cattle tick in PR is essential to establish more effective control measures and may have an impact on current tick eradication methods. The objectives of the present study were, (1) to identify ecologic fact ors associated with the presence of R. (Boophilus) microplus larvae in PR; and (2) to desc ribe the seasonal pattern of R. (Boophilus) microplus larvae. Materials and Methods Study Area Puerto Rico is centered in the Caribbean basin between the coordinates 17 N and 18 N, and its longitude ranges from 65 W to 67 W. It is the sm allest and easternmost island of the Greater Antilles, east of Haiti and Dominican Republic and northwest of the Virgin Islands. It is bounded on the north by the Atlantic Ocean and on the south by the Caribbean Sea. It encompasses an area of 8,870 km2 from which 41.6% is closed forests, 36.7% pastures and grasslands, 5.9% crop agriculture land, 2.4% coff ee plantations, and 10.5% urban and developed landcover (Helmer et al., 2002). Elevations range from sea level to 1338 m. The climate is

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169 predominantly tropical maritime. Mean temperatur es in PR have a very small range between the warmest and coldest months, but decrease mark edly with increasing elevation (NOAA-NCDC, 1982). In the high mountainous interior, the temp erature fluctuates between 22.8-25.6C (7378F). The northern half receives mo re rainfall and has larger rivers than the drier southern half. The distribution of rainfall follows a relative wet-dry seasonal pattern. The relative humidity (RH) is approximately 80% over the course of th e year with the highest RHs generally found at night (>90%) when temperatures are the lowest During the day, RH ranges from 60% to 70% (NOAA-NCDC, 1982). There are 6 ecological life z ones in PR based on the Holdridge classification system: the subtropical dry forest (df-S, 13.8% of total area), subtropical moist forest (mf-S, 60.5%), subtropical wet forest (wf-S, 2.6%), subtropical rain forest (rf-S, 0.1%), subtropical lower montane wet forest (wf-LM, 1.2%), and subtropi cal lower montane rain forest (rf-LM, 0.1%) (Ewel and Whitmore, 1973). The subtropical dry fore st is the driest of th e 6 zones with a mean annual precipitation range of 600 to 1000 mm (23.6 to 39.4 in). Most of the vegetation consists of grasses, cacti, thorny legumes, and trees wi th small and succulent leaves. Fires are common during the dry season. The subtropical moist forest zone is the largest zone in PR with a mean annual precipitation of 1000 to 2200 mm (39.4 to 86.6 in) and mean temperatures of 18 to 24C (64.4 to 75.2F). This is the most deforested zone partly because of intens ive agricultural activity and urbanization. Both native and improved pastures form the dominant landscape in this zone. The subtropical wet forest zone occupies much of the highest parts of the mountains in PR. This zone has mean annual precipitation ranges of 2000 to 4000 mm (78.7 to 157.5 in). Because of the abundant moisture, most of the vegetation consists of epiphytic ferns, bromeliads, and orchids. Most of this life zone, particularly in the western portion of the island is covered by coffee

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170 plantations. The subtropical rain forest zone, th e subtropical lower montan e wet forest zone, and the subtropical lower montane rain forest zone are the wettest of the life zones, occupying a small area in PR. These 3 zones are mostly located in the Luquillo mountains in the eastern part of the island, particularly in El Yunque National Forest (formerly known as the Caribbean National Forest), the only tropical rainforest in the US. Most of the vegetation in the subtropical rain forest zone consists of palm trees and ep iphytes. The subtropical lower montane wet forest zone is comprised of the Colorado forest type named for the common palo colorado or swamp cyrilla ( Cyrilla racemiflora L.) and the cloud-forest type (elf in woodland, montane thicket, or dwarf forest). The vegetation is characterized by short gnarled tr ees (<7 m tall) with high basal area, small diameter, slow growth rates, and ma ny epiphytes. The subtropical lower montane rain forest zone is found only in a narrow band on th e windward slopes of the Luquillo Mountains, at a higher elevation than the subtropical rain fore st. The mean annual temperature is approximately 18.6C (65.5F), annual precipitation is 4533 mm (178.5 in), and mean relative humidity is 98.5%. The vegetation is similar to that of lower montane wet forest, except for having a greater abundance of epiphytes, palms, and fe rn trees (Ewel and Whitmore, 1973). GIS Sampling Methodology A GIS-based sampling methodology was developed to determine the number and selection of sites for evaluation. Sample size was calculate d for a grid polygon shapef ile (fishnet) created and georeferenced for PR in a GIS software program (ArcGIS 9.1TM, ESRI, Redlands, CA, USA) (Figure 5-1). The polygon consisted of 9,010 grid cells each measuring 1-km2. Grid cells along the coastal areas included water resulting in a greater total area of grid cells compared to reported land area of PR. The sample size to estimate the proportion of tick positive sites was calculated using the total number of grid cells assuming a 50% probability of finding the tick larvae at each sampled site (presence/absence), a confidence level of 95%, and an acceptable margin of error of

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171 10% with available software (Win Episcope 2.0, Easter Bush, Roslin; Zaragoza, Spain; Utrecht and Wageningen, Netherlands). The samp le size was estimated to be 97. Initial data for selection of sample sites we re obtained from 25 different raster files containing information related to environmental parameters (elevation, average precipitation, and average temperature) and animal census data by m unicipality within PR. To standardize the GIS analyses, all raster files were projected to the same datum and coordinate system, North American Datum 1983 (NAD83) and Universal Tr ansverse Mercator (UTM) Zone 19 North, respectively. Data on elevation, average temperature, and av erage precipitation were obtained from the records of 102 meteorological sta tions located in Puerto Rico. Files were downloaded from the Southeast Regional Climate Center website available at http://www.sercc.com/ and manipulated in Microsoft Office Excel 2003. The spreadsheet consisted of 102 rows and 5 columns including information on each stations geographic coordinates, elevation (m), average temperature (C), and average precipitation (mm). The data pertained to the period from January 1 to December 31, 2002. A vector shapefile was cr eated from this table in a GIS software program (ArcGIS 9.1TM). Consequently, 3 raster files with a continuous surface were created from the information on elevation, average temper ature, and average precipitation from each of the 102 meteorological stations using an inverse distance weighted (IDW) interpolation technique within the GIS soft ware program (Figure 5-2). Data on livestock and farm inventory per municipality were obtained from the 2002 Census of Agriculture of Puerto Rico (NASSUSDA, 2004). Files were downloaded in digital format and manipulated in Microsoft Office Excel 2003. The animal census data included farm density (number of farms/total area of the munici pality), cattle density (n umber of cattle/total

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172 area of the municipality), number of farms, number of cattle, number of cow farms, number of cows, number of heifer farms, number of heifer s, number of bull farms, number of bulls, number of dairy farms, number of dairy cattle, numbe r of dairy cows, number of dairy cow farms, number of dairy heifers, number of dairy heifer farms, number of beef farms, number of beef cattle, number of beef cows, number of beef cow farms, number of beef heifers, and number of beef heifer farms. This table consisted of 78 rows corresponding to municipalities and 22 columns corresponding to each census variable. This table was joined to an attribute table of a polygon shapefile of PR using municipality as the unique identifier. Subse quently, 22 raster files with continuous surfaces were created from the animal census data using an inverse distance weighted (IDW) interpolati on technique (Figure 5-3). Data from all raster files were extracted using the grid polygon a nd tabulated using the summarize zones option within a GI S software program (ArcView 3.3, ESRI, Redlands, CA, USA). Each variable was quantif ied within all grid cells. The final output table contained 9,010 rows consisting of 2 columns for the XY-coordinates of the centroid of each grid cell, 25 columns for the 3 environmental paramete rs and 22 animal census variables. Satellite pictures for each site were ob tained from Google Earth 2007 and used to identify key features (e.g. buildings, water bodies) for site identific ation. A handheld global positioning unit (GPS) with an accuracy of 4 m (Garmin e Trex Legend, Garmin International Inc., Olathe, KS, USA) was used to locate the se lected sampling sites in the field. Daily records for average precipitation corres ponding to the dates of collect ion were obtained from the National Climatic Data Center, NOAA-Satellite and Information Center, US Department of Commerce website available at http://lwf.ncdc.noaa.gov/oa/ncdc.html

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173 Continuous variables were stan dardized prior to analysis by subtracting the mean and dividing by the standard deviati on. Principal component analysis (PCA) was used to summarize the variability of the 25 continuous variables into 3 components that explained 80% of the variability. The resulting table containing the 9,010 georeferenced rows covering PR and the 3 principal components were partitioned into cl usters by using a K-mean multivariate cluster analysis (SPSS 14.0 for Windows, SPSS Inc. Headquarters, Chica go, IL, USA). Clusters were grouped by membership and distance from the cl uster multivariate center recorded for each row (or grid). The number of groups was based on the sa mple size calculation and the output table of these analyses was loaded into GIS software (ArcGIS 9.1TM). The 3 components that explained 80% of th e variance were a dairy component, a beef component, and a component consisting of averag e precipitation, elevation, and temperature. The multivariate cluster analysis consisted of 97 clusters based on these components (Figure 54). The most representative grid cell of the multivariate cluster was used for the sample location (based on measured variables and not location). If the most representative site was inaccessible then the second best grid cell or point was used as the sampling site. One cluster was excluded from sampling because it was comprised exclusivel y of water and a small flat cay off the east coast of the main land. Field Sampling The procedure used to sample larvae from th e field was based on the tick drag technique presented by Sonenshine et al (1966) with minor modifications The sampling device consisted of a sheet of white flannel 1 m long by 1 m wide we ighted with 5 reusable split shot lead weights (3.61g 0.03) at the trailing edge and attached to a 1.2 m woode n pole along the leading edge by 4 plastic clamps. A 2 m cord was attached to bo th ends of the pole to form a loop, which was used to pull the drag (Figure 5-5). The sampli ng for larvae was performed by holding the flannel

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174 cloth outstretched and walking with it in contact with the ground and vege tation in one direction along a 50 m straight walk course at each select ed site (Wilkinson, 1961). The drag was held on the right side as the investigator walked at a normal pace. The number of paces and timing in the 50 m walk was similar at all collection sites. At the end of the dra g, the flannel cloth was examined for larvae and if positive, the entire cl oth was folded and placed in a sealed 3.8-liter plastic bag for later removal. Each bag was labe led with the site number. A single new sheet of flannel was used for each site because disrup ted fibers may reduce collection numbers (Teel, 2005, Personal communication). Ticks were collected twice from designated collection sites. The first collection was obtained during the peak of th e dry season, March 4 to April 1, 2007 and the second collection was obtained during the peak of the wet season, August 13 to 26, 2007. In the laboratory, cloths were unfolded on white fiberglass pans measuring 121 cm (47 and 5/8 in) in length, 60.6 cm (23 and 7/8 in) in width, and 10.2 cm (4 in) deep. All larvae found clinging to the cloth were counted, removed with forceps, and placed in gl ass vials containing 70% alcohol for preservation prior to identification. Vi als were labeled with the date of co llection, site number and number of larvae. Ticks were shipped to the University of Florida and identified by the investigator at the Department of Entomology. Survey Data Information on geographic and climatic parame ters, major landscape characteristics, type of grass, and cattle availability was recorded for each sample site (Table 5-1). Site coordinates and elevations were recorded by a handheld global positioning system (GPS) (Garmin e Trex Legend, Garmin International Inc., Olathe, KS, US A). Coordinates were collected under the North American Datum 1983 (NAD83) and the c oordinate system corresponding to Universal Transverse Mercator (UTM) Z one 19 and 20 North. The distan ce between target and actual

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175 sampling site was calculated. Data on wind spee d (WS), air temperature (T), wind chill (WC), relative humidity (RH), heat index (HI), and dew poi nt temperature (DP) were recorded for each site by a handheld anemometer (Kestrel3000 Pocket Weather Meter, Nielsen-Kellerman, Chester, PA). Grass classification was perfor med using an identification scheme by Mas and Garcia-Molinari (1990). All gr ass samples were cross-refere nced with Alexis Zaragoza, agronomist and expert in tropical forage s (High Quality Silage, Arecibo, PR). Tick Identification Three larvae were mounted per slide. Specime ns were placed on the center of a 75 mm (3 in) x 25 mm (1 in) glass microscope slide and a clean glass rod was used to place a drop of Hoyers medium on the larvae. Hoyers medium cons ists of 50 ml of distilled water, 30 g of crystalline gum arabic, 200 g of chloral hydrate, and 20 ml of gly cerin. A clean pair of angular forceps was used to apply an 18 mm, 1 mm thic kness cingular coverslip at the opposite edge of the droplet of Hoyers medium, which was ge ntly lowered onto the larvae. A diamond tipped inscriber was used to number the slides. The mo unted slides were placed in a drying oven at 36C for approximately a week. After drying, na il polish was used to seal the coverslip. All slides were examined under a compound light mi croscope at 40X and 100X. The tick larvae from each site were categorized into species according to identific ation keys by Clifford et al. (1961). Randomly selected slides containing the la rvae were sent to Dr. Lance Durden, Assistant Professor, Department of Biology Georgia Sout hern University (Statesboro, GA, USA) for confirmation of identification. Statistical Analysis The proportions of positive sites were estima ted and exact 95% confidence intervals (CI) were calculated in available software (Epi Info Version 6.04, CDC, Atlant a, GA). Paired t-tests were used to compare means between the e nvironmental factors dur ing the dry and rainy

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176 seasons. The number of sites where R. (Boophilus) microplus larvae were recovered between seasons was compared using the Fisher sign te st. The associations be tween the explanatory variables collected vi a the survey and outcome respons e variable presence/absence of R. (Boophilus) microplus larvae were estimated us ing logistic regression. Th e unit of analysis for assessing risk factors was the sampled site. Vari ables that were significantly associated with presence/absence at a Wald P value 0.20 in a bivariable screening model were further evaluated in multivariable models. A backward st epwise multivariable logistic regression model was built starting with a complete model containing all main effects identified in the screening models and terms were removed one-by-one base d on likelihood ratio tests. The main effect terms remaining in the model were evaluated for effect modification by adding all possible 2way interactions between main e ffects and individually evaluated us ing Wald tests. The fit of the final model multivariable logistic model was assessed using the Hosmer-Lemeshow test. Continuous independent variable s in logistic models are a ssumed linear in the log-odds. Therefore, these variables were categorized to evaluate how the risk of the outcome changed with the variable. Initial categor ization was performed either by using 3 or more natural breaks of the data or by equally dividing data into qua rtiles. If the continuous independent variables were not linear in the log-odds th en variables were reta ined as categorical predictors. Analyses were performed in commercially av ailable software (SPSS 14.0 for Windows, SPSS Inc. Headquarters, Chicago, IL, USA) and statistica l significance was assessed at the 5% level. Results Descriptive Ninety-six sites were sampled and the mean distance between the target and actual sampling sites was 0.6 km (range 0.0 km to 5.0 km). An average of 6 sites was visited per day. Collection times were from 8:00 to 17:00 hours. Av erage collection time was 6 minutes per site

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177 including collection of survey data. Passing of the tick drag ove r vegetation was approximately 1 minute per site. Elevation ranged from 0.30 m to 776.6 m (1 ft to 2,548 ft), with an average of 184.7 m (606 ft). Forty-two of the sites were located on a sl oped surface, 48 on a flat surface, and 6 on a ridge. Forty-eight were located in grasslands, 45 in areas covered by bushes and shrubs, and 3 in forests. Thirty sites had trees on the sampling location. The predominant grasses included guinea grass ( Urochloa maxima ) and broadleaf carpet grass ( Axonopus compressus ). Thirty-six sites were covered by guinea grass, 18 by broadleaf carpet grass, 8 by African bermuda grass ( Cynodon nlemfuensis var. robustus ), 5 by Angleton grass ( Dicanthium annulatum ), and 29 sites contained other grasses incl uding tropical signal grass ( Urochloa distachya ), Indian goose grass ( Eleusine indica ), and Mexican crown gra ss or Venezuelan grass ( Paspalum fasciculatum ). Average grass height was 38.1 cm (15 in), mini mum 5.1 cm (2 in), and maximum 152.4 cm (60 in). Thirty-two sites had cattle present at the si te with 13 having dairy ca ttle and 19 beef cattle. Sixty-six sites were located in th e subtropical moist forest, 16 in the subtropical dry forest, and 14 in the subtropical wet forest. Tick larvae were collected from 24 (25%, 95% CI=0.17-0.35) of the sampled sites during the dry season (Table 5-2) and 9 (9.4%, 95% CI=0.04-0.17) during the wet season (Table 5-3). Figure 5-6 presents the locations of positive site s in relationship to the 6 ecological zones. Difference in the seasonal pattern for presence of R. (Boophilus) microplus was significant (P value = 0.031). Significant differences were found in all measured environmental factors between the 2 seasons. The average values fo r the dry season were 3.7 km/h (2.4 mph 0.215 S.E.) for wind speed, 28.4 C (83.2 F 0.538) for environmental temperat ure, 28.6 C (83.4 F 0.439) for wind chill, 19.4 C (66.9 F 0.415) for dew point temperature, 57.8% 0.937 for

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178 relative humidity, 31.3 C (88.3 F 0.650) for h eat index, and 1.36 mm (0.054 in 0.006) for average precipitation. The aver age values for the wet season were 2.9 km/h (1.8 mph 0.181) for wind speed, 30.9 C (87.6 F 0.494) for envi ronmental temperature, 30.9 C (87.6 F 0.498) for wind chill, 23.9 C (75.1 F 0.343) for dew point temperature, 66.6% 0.881 for relative humidity, 37.3 C (99.1 F 0.907) for h eat index, and 6.96 mm (0.274 in 0.020) for average precipitation. During th e dry season 15 sites (16%, 95% CI=0.09-0.24) were identified with R. (Boophilus) microplus larvae (n=606) and 9 (9.4%, 95% CI=0.04-0.17) had Dermacentor (Anocentor) nitens larvae (n=779) (Figure 5-7). Duri ng the wet season 5 sites (5.2%, 95% CI=0.02-0.12) were identified with R. (Boophilus) microplus larvae (n=94), and 5 (5.2%, 95% CI=0.02-0.12) also had Dermacentor (Anocentor) nitens larvae (n=275). One site had a mixed population of both larvae. Risk Factors During the initial bivariable screening, 7 f actors were found to be significant at P 0.20 for the dry season and 2 for the wet season (Table 5-4). The final multivariable logistic regression model for the dry season (Table 5-5) demonstrated that average wind speed of 4.2 to 16.1 km/h (2.6 to 10.0 mph) (OR= 0.07, 95% CI= 0.01-0.63), more than 25% bushes and shrubs on the site (OR= 11, 95% CI=1.6-71), and presence of any cattle (beef or dairy) on the site (OR= 26, 95% CI=3.4-188) in the dry season were sign ificantly associated with presence of R. (Boophilus) microplus larvae. Shade had a significant likeliho od ratio test and was retained in the model, but it was not significant based on the Wald test. During the wet season, the only risk factor significantly associated with the presence of larvae was the presence of only beef cattle on the site (OR= 7.0, 95% CI=1.1-46). No other main effects or 2-way interactions terms were significant. The Hosmer-Lemeshow goodness-of-fit test demonstrated that the model for the dry

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179 season was a reasonably good fit to the data (Chi = 2.814, df=7, P-value = 0.902). The HosmerLemeshow goodness-of-fit test for the final model in the wet season could not be performed. Discussion Host availability and environm ental factors are thought to be important for the detection of tick species. In the present study, the predominan t factor associated with the presence of Rhipicephalus (Boophilus) microplus larvae during the dry season was the presence of any cattle (beef or dairy) on the site (OR=25). A study by Crom and Duncan (1989) in PR found a strong association between the presence of any cattle (beef or dairy) on premises and infestation of the animals with R. (Boophilus) microplus, suggesting a preference of cattle over sheep, goats, and horses. More than 25% bushes and shrubs on the s ite was also significantly associated with the presence of R. (Boophilus) microplus in the dry season (OR=11). Larvae do not feed and can easily become desiccated and die at high temper atures (Arundel and Sutherland, 1988). Larvae survive by absorbing water vapor through their cuticle or by drinki ng water droplets from rain or dew (Wilkinson, 1953; Hitchcock, 1955; Wilkin son and Wilson, 1959; Arundel and Sutherland, 1988; Needham and Teel, 1991). The lowest relative humidity at which water vapor uptake is possible is 85 to 90% (Bowman and Sauer, 2004). Tall grass and shrub cover may provide these humidity levels and prolong larvae survival (A rundel and Sutherland, 1988). Shade is also an important factor prolonging the survival of tick larvae (Wilkinson and Wilson, 1959; Cerny and de la Cruz, 1971). Data from studies in PR estim ated that 10% of hatche d larvae survive in grass for up to 47 to 70 days whereas larvae in shad ed or wooded areas survive for 65 to 72 days (Garris and Popham, 1990; Garri s et al., 1990). The highest temp eratures and lowest relative humidities were recorded in open grass plots. Average wind speed of 4.2 to 16.1 km/h (2.6 to 10.0 mph) was negatively associated with the presence of R. (Boophilus) microplus. Rhipicephalus (Boophilus) microplus larvae may

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180 disperse through pastures and on the ground by wind currents for considerable distances (Arundel and Sutherland, 1988). When larvae are located at the tip s of the grass they can easily be carried away by wind for several meters. Once in the new location they climb nearby herbage and the process can be repeated. A study by Le wis (1968), documented that winds can carry larvae for up to 30 m (100 ft) from the site wh ere engorged females were initially dropped for oviposition. In areas where wind currents are low, dispersal of tick larvae is uniform and does not extend beyond 5 m (15 ft). Moreover, larvae can reach heights above 3 m (10 ft) in the air due to strong winds. There is a possibility th at strong wind currents may reduce the number of tick larvae aggregations in th e pastures by dispersing the larv ae through a wider range. This condition decreases the oppor tunities of finding larvae if the tick drag is performed in a single 50 m by 1 m transect. Fewer numbers of site s were positive for R. (Boophilus) microplus larvae during the wet season (n=5) and it was not possible to obtain a Hosmer-Lemeshow test. Therefore, the final model may not be a good fit for predicting positive locations. Ho wever, these results are in accordance with previous reports from Cuba, Ja maica, and Costa Rica, which found a negative impact of heavy rain showers over the populatio n of tick larvae (Cerny and de la Cruz, 1971; Rawlins, 1979; Alvarez et al., 2003). Seasonal rain may influe nce the environment for short periods of time by creating a marginal habitat fo r tick survival (Suthers t et al., 1983). Larvae can be dislodged from vegetation by the physical effects of the ra in (Wilkinson and Wilson, 1959). If trapped in a water droplet, larv ae are too small to break the surf ace tension of the droplet and can drown (Allan, 2007, Personal Communication). This and the limitations of the tick drag may explain the limited number of positive sites and reduced tick larvae found per site during the wet season when compared to the dry season.

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181 Zimmerman and Garris (1985) found that tick drags were the most efficient method available to sample free-living populations of R. (Boophilus) microplus larvae in Puerto Rico. However, only 10% of tick larvae were recovere d from artificially infested plots (Zimmerman and Garris, 1985). The number of small aggreg ations of larvae and the location of these aggregations on plants affects the efficacy of th e sampling device. Some of these aggregations may not be on the vegetation surfac e that will have direct contact with the flannel cloth (Garris and Popham, 1990). In PR, a study by Garris and Popham (1990) documented that most R. (Boophilus) microplus tick larvae aggregated at heights betw een 2.5 to 85.1 cm (1 to 34 inches). In addition, some tick larvae that are picked up by the flannel cloth are subsequently knocked off by vegetation as the drag is pulled forwar d (Wilkinson, 1961; Zimmerm an and Garris, 1987). Seasonal rain can also interfere with dragging procedures. Wet vegetation markedly decreased tick collections as high water content in the fl annel cloth may reduce the attachment of larvae (Gladney, 1978). This study provided information concerning the effects of ecol ogical factors on the presence of R. (Boophilus) microplus larvae. However, this is a cr oss-sectional design and there are important limitations that must be considere d. The findings of this study are limited to a 2week period in March and A ugust 2007. Results may have been different during another timeframe. Lastly, misclassification of sampling sites as tick nega tive must be considered. The distribution of R. (Boophilus) microplus larvae is known to be extremely patchy within the environment. Commonly, larvae do not migrate a ppreciable distances and large concentrations are frequently found only where the gravid adult female detached from the host and deposited the eggs (Gladney, 1978). Therefore, some locations may have shown negativ e results in spite of being tick positive within the general area. In ad dition, larvae within the path of the tick drag

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182 might also have failed to attach or were subsequently knocked off during the collection procedure. This misclassification might have bias ed point estimates for evaluated risk factors. Lastly, high numbers of tick larvae sampled does not necessarily equate to high tick density. Conclusions Accurate assessments of tick presence ar e necessary to understand the variation of R. (Boophilus) microplus larvae in the environment. The presence of R. (Boophilus) microplus larvae in PR is determined by the presence of an y cattle (beef or dairy) and the landscape habitat conditions. Bushes and shrubs particularly at the boundary between graz ing areas and the haystack hills mogotes can be cleared and access of cattle to these areas should be controlled. However, the practical value of these sugges tions should be assesse d in further studies.

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183 Figure 5-1. Grid polygon or fishnet of Puer to Rico used for sample size determina tion and integration of environmental and an imal census data. Each grid cell was geor eferenced and consists of a 1-km2.

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184 Figure 5-2. A) Location of the 102 me teorological stations in Puerto Rico, B) Interpolation for el evation (m), C) Interpolation for average precipitation (mm), and D) Interpolation for temperature (C). A B D C

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185 Figure 5-3. A) Number of farms per munici pality, and B) Number of cattle per munici pality. Data were obtained from the 2002 Cen sus of Agriculture, National Agriculture St atistics Service, USDA, February 2004. A B

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186 Figure 5-4. The 97 clusters based on environm ental factors and multivariate distances. Each cluster represents 3 major componen ts that explain 80% of the variabilit y. The components consisted of dair y, beef, and climatologic variables.

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187 Figure 5-5. Tick drag device used to sample larvae in Puerto Rico. A) Reusable split shot lead weights (3.61g 0.03) used at the tr ailing edge of the flannel cloth. A

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188 Table 5-1. Explanatory variables included in the field survey to study ecological predictors of R. (Boophilus) microplus in Puerto Rico during the dr y season (March 4-18, 2007) and the wet season (August 16-23, 2007). Explanatory variables Measurements Duration of collection (mins) Continuous UTM coordinatesa (m) Continuous Elevation (m) Continuous Maximum wind speed (km/h) Continuous Average wind speed (km/h) Continuous Ambient temperature (C) Continuous Wind chillb (C) Continuous Relative humidity (%) Continuous Heat indexc (C) Continuous Dew point temperatured (C) Continuous Location of plot Ridge, slope, flat Steepness of slope Continuous Main landcover type Grassla nd, bushes and shrubs, forest Percent landcover type Continuous Ground cover Herbaceous, litter, soil, rock, crops Presence of trees Yes/no Grass height (in) Continuous Presence of shade Yes/no Cloud cover Continuous (0% no clou ds, blue sky; 100% sky fully covered by clouds) Type of grasse Guinea grass ( Urochloa maxima ), broadleaf carpet grass (Axonopus compressus ), pangola grass ( Digitaria eriantha ), elephant grass ( Pennisetum purpureum ), tropical signal grass ( Urochloa distachya ), African bermuda grass ( Cynodon nlemfuensis var. robustus ), buffel grass ( Pennisetum ciliare ), Angleton grass ( Dicanthium annulatum ), Indian goose grass ( Eleusine indica ), Mexican crown grass or Venezuelan grass ( Paspalum fasciculatum ), and pitted beardgrass or huracan grass ( Bothriochloa pertusa ) Presence of any cattle (beef or dairy) Yes/no Type of cattle present Bos taurus or Zebu (Brahman, Bos indicus ) Manure on the site without presence of any cattle (beef or dairy) Yes/no Life zone Mf-S, wf-S, df-Sf a Universal Transverse Mercator Easting ( x -coordinate) and Northing ( y -coordinate). b This parameter combines the effects of air temperature and wind speed. c This parameter combines the effects of relative humidity and air temperature. d This parameter indicates the temperature at which the air must be cooled to observed liquid condensation or dew. e Scientific names were obtained from the USDA Natural Resources Cons ervation Service Plant Database. Available at http://plants.usda.gov/, accessed 25 September 2007. f Subtropical moist forest (mf-S), subtropical wet forest (wf-S), a nd subtropical dry forest (df-S).

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189 Table 5-2. Identification of I xodid larvae collected by site du ring the dry season (March 4-18, 2007) in Puerto Rico. The larger of 6 or 10% of the larvae coll ected per site were identified. In sites where 6 larvae or less were collected, all the specimens were identified. Site no. No. of collected larvae No. of identified larvae Genus 67 11 Rhipicephalus microplus 53 22 Rhipicephalus microplus 85 22 Rhipicephalus microplus 13 33 Rhipicephalus microplus 60 33 Rhipicephalus microplus 12 44 Rhipicephalus microplus 77 44 Rhipicephalus microplus 86 44 Rhipicephalus microplus 63 86 Rhipicephalus microplus 20 136 Rhipicephalus microplus 90 136 Rhipicephalus microplus 21 186 Rhipicephalus microplus 43 296 Rhipicephalus microplus 5 838 Rhipicephalus microplus 28 41942 Rhipicephalus microplus 44 11 Dermacentor nitens 56 55 Dermacentor nitens 1 66 Dermacentor nitens 3 126 Dermacentor nitens 46 486 Dermacentor nitens 10 546 Dermacentor nitens 59 586 Dermacentor nitens 47 17618 Dermacentor nitens 9 41942 Dermacentor nitens

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190 Table 5-3. Identification of I xodid larvae collected by site du ring the wet season (August 13-26, 2007) in Puerto Rico. The larger of 6 or 10% of the larvae coll ected per site were identified. In sites where 6 larvae or less were collected, all the specimens were identified. Site no. No. of collected larvae No. of identified larvae Genus 12 11 Rhipicephalus microplus 73 11 Rhipicephalus microplus 26 106 Rhipicephalus microplus 63 176(2) Rhipicephalus microplus and (4) Dermacentor nitens 13 658 Rhipicephalus microplus 9 11 Dermacentor nitens 85 226 Dermacentor nitens 60 10912 Dermacentor nitens 56 14316 Dermacentor nitens

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191 Figure 5-6. Final geographical distri bution of the 96 target sites sampled in the tick survey in Puerto Rico during the dry sea son (March 4-18, 2007) and the wet season (August 13-26, 2007). Each si te represents the grid cell w ith the nearest distance to the multivariate cluster center.

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192 Figure 5-7. A) Rhipicephalus (Boophilus) microplus and B) Dermacentor (Anocentor) nitens larvae collected in Puerto Rico during the dry season (March 4-18, 2007). A B

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193Table 5-4. Crude (unadjusted) risk factor analysis for predicting the presence of R. (Boophilus) microplus on 96 sites in Puerto Rico during the dry and wet seasons in 2007. 95% CI Season Exposure variable Comparison/categories No. of sites exposed Odds ratio Lower Upper Wald P value Dry Average wind speed 0 = 0.0-1.9 km/h 1 = 2.1-4.0 km/h 2 = 4.2-16.1 km/h 32 32 32 referent 0.37 0.17 0.10 0.03 1.34 0.87 0.065 0.129 0.033 Slope Flat surface, ridge 423.060.969.790.059 More than 25% bushes and shrubs on the site Zero to 20% bushes and shrubs 585.201.1024.550.037 Grass height 0 = no grass 1 = 2.5-25.4 cm 2 = 27.9-152.4 cm 6 45 45 referent 0.38 0.16 0.08 0.03 1.93 0.95 0.119 0.244 0.043 Presence of shade Absence of shade 272.670.868.290.090 Presence of any cattle (beef or dairy) on the site Absence of cattle on the site 323.781.2111.830.022 Mf-sa Otherwise (Wf-S and df-S) 663.430.7216.300.121 Wet Presence of trees at sampled site Absence of trees at sampled site 193.560.5622.500.178 Presence of only beef cattle on the site Absence of beef cattle 197.031.0945.570.041 a Subtropical moist forest (mf-S), subtropical wet forest (wf-S), a nd subtropical dry forest (df-S).

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194Table 5-5. Multivariable logistic regr ession for predicting the presence of R. (Boophilus) microplus on 96 sites in Puerto Rico during the dry and wet seasons in 2007. 95% CI Season Exposure variable Comparison/categories No. of sites exposed Odds ratio LowerUpper Wald P value Dry Average wind speed 0 = 0.0-1.9 km/h 1 = 2.1-4.0 km/h 2 = 4.2-16.1 km/h 32 32 32 referent 0.40 0.07 0.09 0.01 1.80 0.63 0.050 0.233 0.018 More than 25% bushes and shrubs on the site Zero to 20% bushes and shrubs 5810.641.5971.090.015 Presence of shade Absence of shade 275.400.9530.760.058 Presence of any cattle (beef or dairy) on the site Absence of cattle on the site 3225.453.44188.160.002 Wet Presence of only beef cattle on the site Absence of beef cattle 197.031.0945.570.041

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195 APPENDIX A SURVEY FOR FARMERS OR MANAGERS IN SELECTED DAIRY FARMS For Official Use Only Time started: Time finished: Agricultural Region: Ques tionnaire Id: Interviewer: Municipality: Farm ID: Date: Barrio: GPS UTM Coordinates: Northing _______________, Easting ___________________ Number of Samples: BA serological prevalence: BB serological prevalence: Farm Picture Taken: YES / NO Main Grazing Area Picture Taken: YES / NO Section A. Farm demographic information Thank you for participating in this survey. In this first set of questions, we will be asking about general information of your farm. 1. Which of the following categories best describe s the type of operation you own or manage? (CIRCLE THE LETTER OF ALL THAT APPLY) a. Commercial dairy farm only b. Commercial dairy farm w ith calf-raising facilities c. Commercial dairy farm with heifer-raising facilities d. Commercial dairy farm with cal f and heifer-raising facilities e. Mixed (beef and dairy) f. Other (please explain) ____________________ 2. Please indicate the total number of animals in the herd: ____________________ 3. Please indicate the number of animals for each category: a. Number of Bulls: ____________________ b. Number of Milking and Dry Cows (over two years of age): ____________________ c. Number of Heifers (one to two years): ____________________ d. Number of Calves (less than one year): ____________________ 4. Which of the following best describes the size of your farm (size of main grazing area in cuerdas)? [1 cuerda = 0.39303956 hectares] a. 1-50 b. 51-100 c. 101-150 d. 151-200 e. >200

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196 5. What type of dairy cattle do you have on the farm? (CIRCLE THE LETTER OF ALL THAT APPLY) If you have more than one breed, please i ndicate the percentage for each breed in the herd. Percentage (0-100%) a. Holstein-Friesian _________ b. Jersey _________ c. Guernsey _________ d. Ayrshire _________ e. Brown Swiss _________ f. Criollo _________ g. Other (please explain) _________ _________ 6. Are there other farm animals on your dairy? a. Yes (If Yes, please continue with question 7) b. No (If No, skip to question 8) 7. If the answer to question 6 is Yes, what t ype of farm animals besides cattle do you have? (CIRCLE THE LETTER OF ALL THAT APPLY) Number of Animals a. Pigs ________________ b. Sheep ________________ c. Goats ________________ d. Horses ________________ e. Other (please explain) ________________ 8. What type of pasture is found in the main grazing areas of the farm? (CIRCLE THE LETTER OF ALL THAT APPLY) a. Star grass ( Cynodon nlemfuensis ) b. Guinea grass ( Panicum maximum ) c. Pangola grass ( Digitaria decumbens ) d. Bermuda grass ( Cynodon dactylon ) e. Other (please explain) ____________________ Section B. General Descriptions of Pr emises and Surrounding Areas In this section, we would like to know what are the features that best describe your premises. 9. Which of the following categories best describe s the type of housing facilities on your farm? a. Tie-stall b. Free-stall c. Loose housing shed d. Dry-lot e. Open pasture grazing f. Other (please explain): ____________________ 10. Are there fences demarcating the boundaries of your farm? a. Yes b. No

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197 11. Are there loose cattle other than yours on the premises? a. Yes b. No 12. Are there stray/owned animals other than livestock on the premises? a. Yes (If Yes, please cont inue with question 13) b. No (If No, skip to question 14) 13. If answer to question 12 is Yes, what kind of stray/owned animals other than livestock are on your farm? Number of animals a. Dogs only _______________ b. Cats only _______________ c. Other (please explain) _______ _______________ 14. Are there adjacent farms to your farm? Number Use a. Yes ___________ ____________ b. No (If No, skip to question 15) 15. Have you ever observed wild deer wandering your premises? a. Yes b. No 16. Besides deer, are any other wild animals commonly seen wandering your premises? a. Mongoose b. Rodents c. Monkeys d. Ostriches e. Other (please explain) ____________________ 17. Is the farm equipped with an irrigation system? a. Yes b. No 18. Do you have flooding problems in your farm? a. Yes (If Yes, please cont inue with question 19) b. No (If No, skip to Section C) 19. If your answer in question 18 is Yes, please indicate the ma in reasons for flooding and the main areas where the problem occur on your farm. (For example, the farm becomes easily flooded when it rains) ______________________________________________________________________________ ______________________________________________________________________________

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198 Section C. General Farm and Feed Management Practices In this section, we would like to know which are the most common management practices used in your farm. 20. Do you owned more than one farm? If the answ er is Yes, please indicate how many farms you owned. a. Yes, _____________ b. No 21. Which of the following categories best descri bes the main source of cattle replacement on your farm? (CIRCLE THE LETTER OF ALL THAT APPLY) If more than one answer is selected, please indicate the approximate per centage (0-100%) for each choice? Percentage (0-100%) a. You raise your own cattle _____________ b. You raise your own cattle in a another of your farms _____________ c. Buy from another farm _____________ d. Buy from a market _____________ e. Imported _____________ f. Other (please explain) __________ _____________ 22. Did you purchase any livestock during the last 12 months? a. Yes (If Yes, please continue with questions 23 and 24) b. No (If No, skip to question 25) 23. If the answer to question 22 is Yes, please i ndicate the type of animals and number of animals purchased: Class of animal Number of animals __________________________ _______________________________ __________________________ _______________________________ 24. If you purchased animals from a local farm, local market, or directly imported from United States in the last 12 months, please indicate th e name of the farm, market, or main dealer where you made the purchase. ________________ 25. How frequently do you purchase animals? ______________ 26. Do you have a quarantine zone (isolation ar ea within your farm where new animals are separately located for a specific amount of time before being introduced to the main herd) on your farm? a. Yes (If Yes, please cont inue with question 27) b. No (If No, skip to question 28) 27. If the answer to question 26 is Yes, please i ndicate how long (days) purchased animals are kept in this quarantine zone? ____________, where in the farm is this quarantine zone located in respect to the existent he rd (e.g. distance from main herd area)

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199 _________________________________________, and who takes care of this animals___________________________________. 28. Do you allow other farmers to use your pastures to graze their animals? a. Yes b. No 29. Which of the following categories best describe s the main source of hay, haylage, or green chop fed on your farm? a. Grow your own hay, haylage, green chop b. Buy hay, haylage, or green chop from another dairy c. Buy hay, haylage, or green chop fr om a commercial feed company d. Buy hay, haylage, or green chop fr om a commercial forage company e. Other (please explain) ____________________ 30. If you buy hay from another source, please indi cate if it is local or imported: _____________ 31. Are the bulls on your farm shared w ith other farms for breeding purposes? a. Yes b. No 32. Is rotational grazing practiced on your farm? a. Yes b. No 33. Which of the following procedures are performed on the farm? (CIRCLE THE LETTER OF ALL THAT APPLY) Please indicate ( ) if the equipment is disinfected between animals, after being used in all animals, or not disinfected. Disinfected between After Not disinfected a. Dehorning ________________ ________ ____________ b. Castration ________________ ________ ____________ c. Vaccinating ________________ ________ ____________ d. Multiple use of needles22 ________________ ________ ____________ e. Single use of needles23 ________________ ________ ____________ f. Tattooing ________________ ________ ____________ g. Ear tagging ________________ ________ ____________ h. Multiple use of palpation sleeves24________________ ________ ____________ i. Single use of palpation sleeves25 ________________ ________ ____________ 22 Multiple use of needles: same needle is used in several cows 23 Single use of needles: only one needle is used per cow (one time use) 24 Multiple use of palpation sleeves: same sleeve is used in several cows 25 Single use of palpation sleeves: only one sleeve is used per cow (one time use)

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200 34. What disinfectant do you use? _______________ (pl ease include concentration and duration of application) 35. How often do you carry out these routine husbandry procedures? 36. Are veterinary services used in the farm? a. Yes (If Yes, please cont inue with question 37) b. No (If No, skip to Section D) 37. How often did your veterinarian attend the herd during the last 12 months? a. 1 b. 2 c. 3 d. 4 e. 5 f. >6 Please indicate main reas ons for the visit/s___________________________________________. Section D. Ticks and Acaricides (type, application method, frequency, and season of application) In this section, we would like to know a bout the use of acaricides in your farm. 38. Do you consider having a problem with ticks? a. Yes b. No Please indicate what animals ar e most commonly affected: ______________________________ 39. Are acaricides used in the farm? a. Yes (If Yes, please cont inue with question 40) b. No (If No, skip to question 49) 40. Which of the following categories best describe s the type of acaricide used in your farm? (CIRCLE THE LETTER OF ALL THAT APPLY) a. Coumaphos (Co-Ral) b. Crotoxyphos (Cio-Rid) c. Formamidines: Amitraz (Taktic) d. Synthetic pyretroid: Permethrin (Atroban, Ectiban, Permethrin, Insectrin) e. Avermectins: Ivermectin (Ivomec) f. Synthetic pyretroid: Fenvalerate (Ectrin) g. None used h. Other (please explain) ____________________ 41. Which of the following categories best descri bes the application method commonly used for the application of amitraz? (CIRCLE THE LETTER OF ALL THAT APPLY) a. Dipping

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201 b. Spray race c. Hand spraying d. Pour-on e. Injecting f. Other (please explain) ____________________ 42. Please indicate the name of the equi pment use to apply the amitraz: ____________________ 43. Who applied the amitraz? a. By owner b. By private contractor c. By use of government personnel d. Other (please explain) _______________ 44. Have you participated in the certification pr ogram of the Puerto Rico Department of Agriculture, agricultural extension service ti tled Guidelines for use and application of acaricides? a. Yes b. No c. I was not aware of the program 45. If you have to mix the amitraz for applica tion, which of the following categories best describes the concentration (or mixing protoc ol) of the amitraz currently used? Give an approximate ratio of amitraz to water. a. :1 b. 1:1 c. 1:2 d. 1:3 e. 1:4 f. 1:5 g. Other (please explain) ____________________ ______________ (pint, oz, lb, gallon) per _________________ gallons of water 46. If spraying is used, what volume do you apply? ___________ 47. Which of the following categories best describe s the frequency of amitraz when applied? a. Every 7 days b. Every 14 days c. Every 21 days d. Every 30 days e. Every 40 days f. Every 50 days g. More than 60 days

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202 48. Which of the following categories best describes the time of the year when the amitraz is commonly applied? a. Throughout the year b. December to March (dry season) c. June-November (rainy season) d. Other (please explain) ____________________ 49. Which of the following categories best desc ribes the problem you have with ticks? a. Not a problem b. Minor c. Moderate d. Serious Section E. Treatments: type, application method, and frequency In this section, we would like to know about drugs you use to treat anaplasmosis or babesiosis on your farm. 50. Do you consider having a problem with bovine anaplasmosis in your herd? a. Yes (If Yes, please cont inue with question 51) b. No (If No, please contin ue with question 57) 51. What are the most common clinical signs of bovine anaplasmosis observed in your herd? Mention the 3 most commonly observed signs. ____________________ ____________________ ____________________ 52. Do you treat clinical cas es of anaplasmosis? a. Yes b. No 53. Which of the following categories best de scribes the type of treatment used? (CIRCLE THE LETTER OF ALL THAT APPLY) a. Short-acting oxytetracycline (50-100mg/ml) b. Long-acting oxytetracycline (L A-200, 200mg/ml) Liquamycin c. Long-acting oxytetracycline (300 mg/ml) Tetradure LA-300 (Merial) d. Chlortetracycline in feed (oral) e. Other (please explain) ____________________ 54. Please indicate the dose of oxyt etracycline offered to the c linically affected animal: ______________________ cc/100lb of ___________________ cc total lbs or _____________________ total ccs

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203 55. Please indicate the route of oxytetracycline use in the clinically affected animal: a. Intravenously (IV) b. Intramuscular (IM) c. Subcutaneous (SC) d. Other (please explain) ____________________ 56. Please indicate how often treatm ents are applied (days): _____________________ 57. Do you consider having a problem with bovine babesiosis in your herd? a. Yes (If Yes, please cont inue with question 58) b. No (If No, skip to Section F) 58. What are the most common clinical signs of bovine babesiosis observe in your herd? Mention the 3 most commonly observed signs. ____________________ ____________________ ____________________ 59. Do you treat clinical cases of babesiosis? a. Yes b. No 60. What do you use to treat bovine babesiosis? ______________________________________________________________________________ ______________________________________________________________________________ Section F. Vaccines: type, application method, frequency, and season In this section, we would like to know about products you use to vaccinate your herd against anaplasmosis. 61. Do you vaccinate the herd for anaplasmosis? a. Yes (If Yes, please cont inue with question 62) b. No (If No, skip to question 67) 62. What animals do you vaccinate for anaplasmosis? a. All animals in the herd b. Sick Animals only c. Specific age group (please indicate) ____________________ d. Other (please explain) ____________________ 63. What type of vaccine is commonly used on your farm? (CIRCLE THE LETTER OF ALL THAT APPLY) a. Anaplaz (killed) b. AM-VAXTM (killed) c. Plazvax (Malincro) d. COMBAVAC (modified live) e. Other (please explain) ____________________

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204 64. How many doses are administered to each cow? _______________________ 65. How often are the doses applied per cow? a. Every two weeks b. Every month c. Every other month d. Other (please explain) ____________________ 66. Which of the following categories best descri bes the last time you vaccinated your cows against anaplasmosis? a. Six months b. One year c. Two years d. Three years e. More than four years f. Other (please explain) ____________________ 67. Do you vaccinate the herd against ticks? a. Yes (If Yes, please cont inue with question 68) b. No (If No, skip to Section G) 68. What brand and type of tick v accine you commonly us e? (i.e. GAVAC) ____________________ Section G. Clinical cases of bovine anaplasmosi s and or babesiosis during last year 69. Please indicate the number of bovi ne anaplasmosis or babesiosis cases observed during last 12 months (if not sure pleas e indicate total cases): ____________________ cases of anaplasmosis ____________________ cases of babesiosis ____________________ total cases 70. From cases in question 69, please indicate th e number of cases diagnosed by laboratory means in the last 12 months: ____________________ 71. Please indicate the number of animals that b ecame affected and survived in the last 12 months: _______________ 72. Please indicate the number of animals that beca me affected and died in the last 12 months: ____________________ 73. Please indicate the age group of th e animals that were affected: (CIRCLE THE LETTER OF ALL THAT APPLY) a. Milking and Dry Cows (over two years of age) b. Heifers (one to two years) c. Calves (less than one year) d. Bulls

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205 74. From the affected animals, how many were rece ntly purchased from a source in Puerto Rico (other than your farm), imported, or born in the farm. Please indicate the approximate number of animals for each category. Recently Purchased __________ Imported __________ Born in Your Farm __________ 75. Which of the following categories best de scribes the problem you have with bovine anaplasmosis and/ or babesiosis? a. Not a problem b. Minor c. Moderate d. Serious Section H. Flies and Fly Control 76. Do you consider having a problem with flies on your farm? a. Yes (If Yes, please cont inue with question 77) b. No (If No, skip to Section I) 77. Please indicate the type of fl y control methods (name of product and method) used on your farm. _________________________ 78. Which of the following categories best desc ribes the problem you have with flies? a. Not a problem b. Minor c. Moderate d. Serious Thank you for participating in our survey. Your an swers will be very helpful as we evaluate the need for and feasibility of designing new contro l programs for bovine anaplasmosis, babesiosis, and ticks in Puerto Rico. Section I. Interviewee address information and assessment of the survey We would like to send you the resu lts and general statistics of this survey once it is analyzed. Please provide the necessary information. These w ill be separated from the rest of the survey answers and will only be used to contact you with the results obtained from the analyses. 79. Name of the farmer: __________________________________________________________ 80. Farmers Address: ___________________________________________________________ 81. Farm Address:_______________________________________________________________ 82. Phone Numbers:_____________________________________________________________ 83. Fax: ______________________________________________________________________

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206 84. E-Mail: ____________________________________________________________________ 85. Name of Veterinarian in Charge: ________________________________________________ 86. Name of Person in Charge: ____________________________________________________ 87. Name of Interviewee: _________________________________________________________ 88. Overall, how would you rate th is survey in terms of c oncepts, clarity, and purpose? a. Poor b. Fair c. Good d. Excellent e. No opinion 89. Please feel free to indicate any additional comm ents you may have about any of the topics covered in this survey. ______________________________________________________________________________ ______________________________________________________________________________

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207 APPENDIX B CUESTIONARIO OFICIAL PARA GANADERAS PARTICIPANTES EN ESTUDIO SOBRE LA EPIDEMIOLOGA DE LA ANAPLASMOS IS Y BABESIOSIS BOVINA EN HATOS LECHEROS EN PUERTO RICO For Official Use Only Time started: Time finished: Agricultural Region: Ques tionnaire Id: Interviewer: Municipality: Farm ID: Date: Barrio: GPS UTM Coordinates: Northing _______________, Easting ___________________ Number of Samples: BA serological prevalence: BB serological prevalence: Farm Picture Taken: YES / NO Main Grazing Area Picture Taken: YES / NO Seccin A Informacin demogrfica de la finca Una vez ms gracias por participar en esta investigacin. En esta primera seccin, le preguntaremos informacin general acerca de su finca. 1. Cul de las siguientes categoras mejor descri be el tipo de operac in que usted tiene o maneja? (CIRCULE LA LETRA DE TODAS LAS OPCIONES QUE APLIQUEN) a. Ganadera commercial solamente b. Ganadera comercial con facilidades de crianza de becerras c. Ganadera comercial con facilidades de crianza de novillas d. Ganadera comercial con facilidades de crianza de becerras y novillas e. Mixto (carne y leche) f. Otro (por favor explique) ____________________ 2. Por favor indique el nmero total de animales en su finca: _______________________ 3. Por favor indique el nmero de animales dentro de las siguientes categoras: a. Nmero de toros: __________________ b. Nmero de vacas en ordeo y horras (mayor de 2 aos de edad): _______________ c. Nmero de novillas (1 a 2 aos de edad): __________________ d. Nmero de becerras (menos de 1 ao de edad): __________________ 4. Cul de las siguientes opciones mejor describe el tamao de su finca en cuerdas? [1 cuerda = 0.39303956 hectreas] a. 1-50 b. 51-100 c. 101-150 d. 151-200 e. >200

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208 5. Qu raza de ganado lechero usted tiene en su finca? (CIRCULE LA LETRA DE TODAS LAS OPCIONES QUE APLIQUEN) Si tiene una mezcla de razas en su finca, por favor indique el porcentaje aproximado (0-100%) de cada raza. Porcentaje (0-100%) a. Holstein-Friesian _________ b. Jersey _________ c. Guernsey _________ d. Ayrshire _________ e. Brown Swiss _________ f. Criollo _________ g. Otro (por favor explique) _________ _________ 6. Usted posee en su finca otros animales de granja que no sean vacas? a. S (Si la respuesta es S, por fa vor contine con la pregunta 7) b. No (Si la respuesta es No proceda a la pregunta 8) 7. Si la respuesta a la pregunta 6 es S, entonces qu clase de animales de granja usted posee que no sean vacas? (CIRCULE LA LETRA DE TODAS LAS OPCIONES QUE APLIQUEN) Por favor indique el nmero aproximado de animales de granja para cada categora seleccionada: Nmero de animales a. Cerdos ________________ b. Ovejas ________________ c. Cabras ________________ d. Horses ________________ e. Otro (por favor explique) ________________ 8. Qu clase de pasto o forraje predomina en la s reas de mayor pastoreo del ganado en su finca? (CIRCULE LA LETRA DE TODAS LAS OPCIONES QUE APLIQUEN) a. hierba estrella ( Cynodon nlemfuensis ) b. hierba guinea ( Panicum maximum ) c. hierba pangola ( Digitaria decumbens ) d. hierba bermuda ( Cynodon dactylon ) e. Otro (por favor explique) ____________________ Seccin B. Descripciones generales de las facilidades y reas alrededor En esta seccin, estamos interesados en las cara ctersticas que mejor de scriben sus facilidades. 9. Cul de las siguientes categoras mejor descri be el tipo de facilida des de su finca? a. Confinado (Free-stall) b. Semiconfinado (Loose housing shed) c. Chiquero en barro con r ea de comer (Dry-lot) d. Pastoreo e. Otro (por favor explique): ____________________

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209 10. Su finca tiene verjas a su alrededor que de marquen y separen esta de los alrededores? a. S b. No 11. Existe en sus facilidades otro ganado suelto (ejemplo: realengos, invasin) que no sea el suyo? a. S b. No 12. Existen otros animales que no sean ganado en sus facilidades? a. Si (Si la respuesta es S, por fa vor contine con la pregunta 13) b. No (Si la respuesta es No proceda a la pregunta 14) 13. Si la contestacin a la pregunta 12 es S, qu clase de animales que no sea ganado existen en su finca? Por favor indique el nmero de animales por cada cate gora seleccionada: Nmero de animales a. Perros solamente ________________ b. Gatos solamente ________________ c. Otro (por favor explique) _______ ________________ 14. Tiene usted otras fincas adyacen tes a su finca? Si la contesta cin es S, por favor indique el nmero de fincas adyacentes y el uso principal de esa/s finca/s: Nmero Uso a. S __________ __________ b. No (Si la respuesta es No proceda a la pregunta 15) 15. Usted ha observado venados cerca de sus facilidades? a. S b. No 16. Usted ha observado otros animales salvajes adems de venados en sus facilidades? a. mangostas b. roedores c. monos d. avestruces e. Otro (por favor explique) ____________________ 17. Est su finca equipada con sist emas de riego funcionales? a. S b. No 18. Tiene usted problemas de rea s inundables en su finca? a. S (Si la respuesta es S, por fa vor contine con la pregunta 19) b. No (Si la respuesta es No, proceda a la Seccin C)

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210 19. Si la respuesta en la pregunta 18 es S, por favor indique las reas donde ms frecuentes ocurre las inundaciones y las raz ones principales por las cuales estas reas son inundables. ______________________________________________________________________________ ______________________________________________________________________________ Seccin C. Prcticas generales del manejo de la finca y la alimentacin En esta seccin, nos interesa conocer cuales son la s prcticas de manejo ms comnmente utilizadas en su finca. 20. Usted tiene ms de una finca? Si la respuest a es S, por favor indique cuantas fincas usted tiene. a. S, _____________ b. No 21. Cul de las siguientes categoras mejor describe la mayor fuente de reemplazo de ganado en su finca? (CIRCULE LA LETRA DE TODAS LAS OPCIONES QUE APLIQUEN) Si ms de una respuesta es seleccionada, por favor indique el porcentaje aproximado (0-100%) para cada opcin. Porcentaje (0-100%) a. Usted cra su propio ganado en la misma finca _____________ b. Usted cra su propio ganado en otra de sus fincas _____________ c. Compra de otra finca (que no es suya) _____________ d. Compra de un mercado local _____________ e. Importado _____________ f. Otro (por favor explique) _____________ _____________ 22. Usted ha comprado ganado durante los ltimos 12 meses? a. S (Si la respuesta es S, por favor contine con las preguntas 23 y 24) b. No (Si la respuesta es No proceda a la pregunta 25) 23. Si la respuesta en la pregunta 22 es S, por favor indique la clase de animales y el nmero que compr: Clase de Animal Nmero __________________________ _______________________________ __________________________ _______________________________ 24. Si usted ha comprado animales de una finca local, un mercado local, o directamente importado de los Estados Unidos durante los ltimos 12 meses, por favor indique el nombre de la finca, mercado o importador prin cipal donde usted hizo su compra. ____________________________________ 25. Cun frecuente usted compra animales? ____________________________________

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211 26. Usted tiene en su finca una zona de cuaren tena? (rea de aislamie nto dnde los animales recientemente comprados estn separados por un tiempo determinado antes de introducirlos al rebao existente) a. S (Si la respuesta es S, por fa vor contine con la pregunta 27) b. No (Si la respuesta es No proceda a la pregunta 28) 27. Si la respuesta en la pregunta 26 es S, por favor indique por cuanto tiempo (das) los animales recientemente comprados son mantenidos en esta zona __________________, dnde en la finca se encuentra esta zona de cuarentena en respecto al rebao existente (ejemplo: la distancia del rea de mayor pastoreo) _________________________________________, y quin esta a cargo de estos animales __________________________________. 28. Usted permite que otros ganaderos utilicen los pa stos de su finca para pastorear los animales de estos? a. S b. No 29. Cul de las siguientes categoras mejor describe la mayor fuente de heno, ensilaje o pasto chopeado utilizado para alimentar sus animales? a. El heno, ensilaje, o pasto chopeado es producido en su propia finca. b. El heno, ensilaje, o pasto chopeado es comprado de otra finca. c. El heno, ensilaje, o pasto chopeado es comprado de una compaa comercial de alimentos. d. El heno, ensilaje, o pasto chopeado es comp rado de fincas de produccin de forraje comercial. e. Otro (por favor explique) ____________________ 30. Si usted compra el heno de otra fuente, por favor indique si este es local o importado:______ 31. Los toros existentes en su finca son comp artidos con otras fincas con el propsito de empadronar vacas? a. S b. No 32. El pastoreo rotacional es practicado en su finca? a. S b. No 33. Cul de los siguientes procedimient os son efectuados en su finca? (CIRCULE LA LETRA DE TODAS LAS OPCIONES QUE APLIQUEN) Por favor si la opci n aplica, indique ( ) si el equipo es desinfectado entre animal es, luego de terminar con todos los animales, o no son desinfectados. Entre Luego No es a. Descornar ______ ______ ______ b. Castracin ______ ______ ______

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212 c. Vacunacin ______ ______ ______ d. Uso multiple de agujas26 ______ ______ ______ e. Uso sencillo de agujas27 ______ ______ ______ f. Tatuaje ______ ______ ______ g. Identificar la vaca con un nme ro en la oreja ______ ______ ______ h. Uso mltiple de guantes de palpacin28 ______ ______ ______ i. Uso sencillo de guantes de palpacin29 ______ ______ ______ 34. Cul desinfectante usted u tiliza? _______________ (por favor indique la concentracin y la duracin de la aplicacin) _____________________ ___________________ 35. Cun seguido usted lleva a cabo estos procedimientos? _______________________________________________ 36. Usted utiliza servicios veterinarios en su finca? a. Si (Si la respuesta es S, por fa vor contine con la pregunta 37) b. No (Si la respuesta es No proceda a la seccin D) 37. Cun seguido su veterinario atendi su finca durante los ltimos 12 meses? a. 1 b. 2 c. 3 d. 4 e. 5 f. >6 Por favor indique las razones pr incipales de la/s visita/s_________________________________. Seccin D. Garrapatas e insecticidas (clase, mtodo de ap licacin, frecuenci a, y temporada de aplicacin) En esta seccin, nos interesa conocer ms sobre el uso de in secticidas en su finca. 38. Usted considera haber tenido o te ner problemas con garrapatas? a. S b. No Por favor indique cules de los an imales son los ms afectados: __________________________ 26 Uso mltiple de agujas: la misma ag uja es utilizada en varias vacas 27 Uso sencillo de agujas: slo una aguja es utilizada por vaca (se usa una sola vez) 28 Uso mltiple de guantes de palpacin: el mismo guante es utilizado en varias vacas 29 Uso sencillo de guantes de palpacin: solo un guante es utilizado por vaca (se usa una sola vez)

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213 39. Usted utiliza insecticidas en su finca? a. S (Si la respuesta es S, por fa vor contine con la pregunta 40) b. No (Si la respuesta es No, proceda con la pregunta 49) 40. Cul de las siguientes categoras mejor describe la clase de insecticidas utilizados en su finca? (CIRCULE LA LETRA DE TODAS LAS OPCIONES QUE APLIQUEN) a. Coumaphos (Co-Ral) b. Crotoxyphos (Cio-Rid) c. Formamidines: Amitraz (Taktic) d. Synthetic pyretroid: Permethrin (Atroban, Ectiban, Permethrin, Insectrin) e. Avermectins: Ivermectin (Ivomec) f. Synthetic pyretroid: Fenvalerate (Ectrin) g. No uso h. Otro (por favor explique) ____________________ 41. Cul de las siguientes categoras mejor desc ribe el mtodo de aplicacin mas comnmente utilizado cuando ap lica el amitraz? (CIRCULE LA LETRA DE TODAS LAS OPCIONES QUE APLIQUEN) a. Sumergir b. Bao de vacas Roceador automtico (estacionario) TNEL c. Fumigar Roceador manual d. Pour-on POSS e. Inyectable f. Otro (por favor explique) ____________________ 42. Por favor indique el nombre el equipo u tilizado para aplicar el amitraz: _________________ 43. Quin se encarga de aplicar el amitraz? a. Por cuenta propia b. Contrata personal privado c. Agente de gobierno d. Otro (por favor explique) _______________ 44. Usted ha participado en el programa de cer tificacin del Departamento de Agricultura de Puerto Rico del servicio de extensin agrco la titulado Guas para el uso y aplicacin de insecticidas? a. S b. No c. No estaba al tanto del programa 45. Si usted tiene que mezclar el amitraz para ap licarlo, cul de las siguientes categoras mejor describe la concentracin (direcciones pa ra la mezcla) del amitraz que usted utiliza actualmente? Por favor describa la pr oporcin aproximada de amitraz a agua. a. :1 b. 1:1 c. 1:2 d. 1:3

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214 e. 1:4 f. 1:5 g. Otro (por favor explique) ____________________ ______________ (pinta, onza, libras, gallone s) por _________________ gallones (dron) of agua. 46. Si utiliza baos de vaca o fumiga, indi que que volumen es aplicado por vaca. __________________________________________ 47. Cul de las siguientes categoras mejor desc ribe la frecuencia con que usted aplica el amitraz? a. Cada 7 das b. Cada 14 das c. Cada 21 das d. Cada 30 das e. Cada 40 das f. Cada 50 das g. Ms de 60 das h. Otro (por favor indique)________ 48. Cul de las siguientes categoras mejor descri be la temporada del ao que ms comnmente el amitraz es aplicado? a. durante todo el ao b. diciembre a abril (temporada seca) c. mayo a noviembre (temporada de lluvia) d. Otro (por favor explique) ____________________ 49. Cul de las siguientes categoras mejor describe el problema que uste d ha tenido o tiene con garrapatas? a. no es problema b. mnimo c. moderado d. serio Seccin E. Tratamientos: clase, mtodo de aplicacin, y frecuencia En esta seccin, nos interesa conocer acerca de lo s medicamentos que usted utiliza para tratar la anaplasmosis y babesiosis en su finca. 50. Usted considera haber tenido o tener problem as con anaplasmosis bovina en su finca? a. S (Si la respuesta es S, por fa vor contine con la pregunta 51) b. No (Si la respuesta es No proceda a la pregunta 57)

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215 51. Cules son los sntomas ms comunes de la anaplasmosis bovina observados en sus animales? Mencione los 3 sntomas ms comunes. ____________________ ____________________ ____________________ 52. Usted trata o ha tratado los casos de anaplasmosis? a. S b. No 53. Cul de las siguientes categoras mejor describe la clase de tratamie nto que usted utiliza? (CIRCULE LA LETRA DE TODAS LAS OPCIONES QUE APLIQUEN) a. oxytetracyclina de corta duracin (50-100mg/ml) b. oxytetracyclina de larga duraci n (LA-200, 200mg/ml) Liquamycin c. oxytetracyclina de larga duracin (3 00 mg/ml) Tetradure LA-300 (Merial) d. chlortetracyclina en el alimento (oral) e. Otro (por favor explique) ____________________ 54. Por favor indique la dosis de oxytetracyclin a administrada a los animales afectados: ______________________ cc/100libras de ___________________ cc total lbs _____________________ total ccs 55. Por favor indique la ruta util izada para administrar la oxytetracyclina en los animales afectados: a. Intravenoso (IV) b. Intramuscular (IM) c. Subcutneo (SC) d. Otro (por favor explique) ____________________ 56. Por favor indique cun frecuente estos tratamientos son administrados (das): _____________________ 57. Usted considera haber tenido o tener problem as con babesiosis bovi na en su finca? a. S (Si la respuesta es S, por fa vor contine con la pregunta 58) b. No (Si la respuesta es No proceda a la seccin F) 58. Cules son los sntomas ms comunes de la babesiosis bovina observados en sus animales? Mencione los 3 sntomas ms comunes. ____________________ ____________________ ____________________

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216 59. Usted trata o ha tratado los casos de babesiosis? a. S b. No 60. Qu usted utiliza para tratar animales con babesiosis bovina? ______________________________________________________________________________ ______________________________________________________________________________ Seccin F. Vacunas: clase, mtodos de apl icacin, frecuencia, y temporada En esta seccin, nos interesa c onocer ms sobre los productos que usted utilice para vacunar sus animales contra anaplasmosis. 61. Usted vacuna sus animales contra la anaplasmosis? a. S (Si la respuesta es S, por fa vor contine con la pregunta 62) b. No (Si la respuesta es No, proceda a la pregunta 67) 62. Cules son los animales que usted vacuna contra la anaplasmosis? a. Todos los animales en la finca b. Slo los animales afectados c. Un grupo en especfico (por favor indique) ____________________ d. Otro (por favor explique) ____________________ 63. Cul vacuna es la ms comnm ente utilizada en su finca? (CIRCULE LA LETRA DE TODAS LAS OPCIONES QUE APLIQUEN) a. Anaplaz (killed) b. AM-VAXTM (killed) c. Plazvax (Malincro) d. COMBAVAC (modified live) e. Otro (por favor explique) ____________________ 64. Cuntas dosis son administradas por cada vaca? _______________________ 65. Cuntas veces son las dosis aplicadas por vaca? a. Cada dos semanas b. Cada mes c. Un mes s y otro no d. Otro (por favor explique) ____________________ 66. Cul de las siguientes categoras mejor descri be la ltima vez que usted vacun sus vacas contra la anaplasmosis? a. Hace seis meses b. Hace un ao c. Hace dos aos d. Hace tres aos e. Hace ms de cuatro aos f. Otro (por favor explique) ____________________

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217 67. Usted vacuna o ha vacunado sus anim ales en contra de la garrapata? a. S (Si la respuesta es S, por fa vor contine con la pregunta 68) b. No (Si la respuesta es No, proceda a la seccin G) 68. Qu marca o clase de vacuna contra las garr apatas usted utiliza o ha utilizado? (ejemplo: GAVAC) ____________________ Seccin G Casos clnicos de anaplasmosis o babe siosis bovina durante el ltimo ao 69. Por favor indique el nmero animales afect ados por anaplasmosis o babesiosis bovina durante los ltimos 12 meses (si no est seguro por favor indique el nmero total de casos): ____________________ Casos de anaplasmosis ____________________ Casos de babesiosis ____________________ Total de casos 70. De los casos en la pregunta 69, por favor in dique el nmero de casos diagnosticados por anlisis de laboratorio en los ltimos 12 meses: ____________________ 71. Por favor indique el nmero animales que se afectaron y sobrevivieron en los ltimos 12 meses: ____________________. 72. Por favor indique el nmero animales que se afectaron y murieron en los ltimos 12 meses: ____________________. 73. Por favor indique el grupo de animales que fueron afectados: (CIRCULE LA LETRA DE TODAS LAS OPCIONES QUE APLIQUEN) a. Toros b. Vacas en ordeo y horras (mas de 2 aos) c. Novillas (1 a 2 aos) d. Becerros (menos de 1 ao) 74. De los animales afectados, cuntos fueron recientemente comprados de una fuente en Puerto Rico (que no sea su finca), importados, o nacido s y criados en su finca. Por favor indique el nmero aproximado de animal es para cada categora. Puerto Rico _________ Importados _________ Nacidos y criados en su finca __________ 75. Cul de las siguientes categoras mejor describe el problema que uste d tiene o ha tenido con la anaplasmosis o babesiosis bovina? a. no es problema b. mnimo c. moderado d. serio

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218 Seccin H. Moscas y control de moscas 76. Usted considera haber tenido o tener problemas con moscas en su finca? a. Si (Si la respuesta es S, por fa vor contine con la pregunta 77) b. No (Si la respuesta es No proceda a la seccin I) 77. Por favor indique el mtodo de control de mos cas (incluyendo nombre del insecticida) que usted utiliza en su finca. _________________________ 78. Cul de las siguientes categoras mejor describe el problema que uste d tiene o ha tenido con las moscas? a. no es problema b. mnimo c. moderado d. serio Gracias por participar en nuestra investigacin. Sus contestaci ones sern de mucha ayuda para evaluar las necesidades, diseos y realizacin de nuevos programas para el control de la anaplasmosis, babesiosis, y las garrapatas en Pu erto Rico. Una vez ms, quiero recordarle que este cuestionario estar bajo estricta conf idencialidad y su informacin personal no ser divulgada, slo las estadsticas generales resultantes sern utilizadas para publicaciones cientficas y la tesis de mi estudio. Seccin I. Informacin del entrevistado y evaluacin del cuestionario Nos interesa enviarles sus result ados y las estadsticas generale s de esta investigacin una vez sea analizada. Por favor proceda a llenar la siguiente informacin. Esta forma ser separada del resto del cuestionario y slo se utilizar para c ontactarlo con los resultad os finales obtenido de los anlisis. 79. Nombre del ganadero: ________________________________________________________ 80. Direccin del ganadero: ______________________________________________________________________________ ______________________________________________________________________________ 81. Direccin de la finca: ______________________________________________________________________________ ______________________________________________________________________________ 82. Nmero de telfono: _________________________________________________________ 83. Fax: ______________________________________________________________________ 84. E-Mail: ________________________________________________________ ____________

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219 85. Nombre del veterinario a cargo: ________________________________________________ 86. Nombre de la persona encargada:________________________________________________ 87. Nombre del entrevistado: ______________________________________________________ 88. En general, cmo usted cataloga este cuestiona rio en trminos de conceptos, claridad, y propsito? a. Pobre b. Justo c. Bueno d. Excellente e. Sin opinin 89. Por favor sintese libre de indi car algn comentario adicional que usted tenga respecto a los temas presentados en este cuestionario. ______________________________________________________________________________ ______________________________________________________________________________

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220 APPENDIX C TICK SURVEY Today's Date: _____________________ Collection Site No. or Farm No.________________ Collectors Name: __________________________ University of Florida, College of Veterinary Medicine, Large Animal Clinical Sciences Spatial Epidemiology of Bovine Anapla smosis and Babesiosis among Dairy Cattle in Puerto Rico, FORM D: TICK SAMPLE COLLECTIONS Time beginning of collection: Time end of collection: Sunrise Time: Sunset Time: Day Length Within-site observation Edge observation Vantage observation GPS MEASUREMENTS (GPS Unit Garmin Etrex Legend) Units sets at UTM Zone 19 Q (Position Fo rmat: UTM UPS, Map Datum: WGS 84, distance: metric, elevation: feet, vertical speed: ft/min, depth: metric) UTM Northing (X): ____________________ UTM Easting (Y): ______________________ Accuracy: ___________________ ____ meters Number of Satellites: ___________________ Elevation: _______________________ feet above sea level ANEMOMETER MEASUREMENTS Wind Speed MAX: _______________mph Wind Chill30: ______________________F Wind Speed AVG: _______________ mph Relative Humidity : ________________% Temperature: _______________ F Heat Index31: ______________________ Dew Point Temp32: ___________________F 30 How cold it feels given the combined effects of the actual air temperature and the wind speed. 31 How hot it feels when the effects of relative humid ity are combined with th e actual air temperature.

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221LANDSCAPE TOPOGRAPHY AND CHARACTERISTICS Location of Plot (circle one): Ridge Slope Flat Steepness of Slope: 0 45 90 Ground Cover Estimate: _____________% Herbaceous _____________% other _____________% Litter _____________% crop _____________% Soil _____________% Rock Main landcover type: grassland shrubs and bushes forest matorral Trees Present: Yes No Where? __________ ______________________ Average Grass Height: Presence of shade: Yes No Canopy cover over the sample site (percentage): 0 % (none) --------------------------------------------------------------------------------100% (fully covered by trees) Cloud Cover: 0 % (no clouds, blue sky) --------------------------------------------------------100% (fully covered by clouds, no blue) 32 Temperature to which the air must be cooled to observe liquid condensation, or dew, at high RH DP is only a little lower than T, at low RH DP is markedly low than T.

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222Type of Grass (See drawings below): Yerba Guinea/ Urochloa maxima/ Guinea Grass Yerba Alfombra/ Axonopus compressus /Carpet Grass Yerba Pangola/ Digitaria eriantha/ Pangola grass Yerba Elefante, Napier Pennisetum purpureum/ Elephant grass

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223 Yerba Signal/ Urochloa brizantha/ Signal grass Yerba Estrella Cynodon nlemfuensis var. robusta/ Star Grass Yerba Buffel/ Pennisetum ciliaris/ Buffel grass Pajn/ Dicanthium annulatum /Angleton grass

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224 Pata de gallina/ Eleusine indica/ Goose grass Yerba venezolana/ Paspalum fasciculatum Yerba Huracan/ Bothriochloa pertusa

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225GENERAL DIAGRAM OF SAMPLE SITE (include landmarks, north arrow, scale bar, and yourself) Aerial View Profile Diagram (overall draw of vegetation and slope, include vertical scale) COMMENTS

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226TICK NUMBER AND SPECIES LARVAE Number: Genus: Species: NYPMHS Number: Genus: Species: ADULTS Number: Genus: Species:

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227 LIST OF REFERENCES Agbede, R.I., Kemp, D.H., 1986. I mmunization of cattle against Boophilus microplus using extracts derived from adult female ticks: hi stopathology of ticks feed ing on vaccinated cattle. Int. J. Parasitol. 16, 35-41. Agbede, R.I., Kemp, D.H., Hoyte, H.M.D., 198 6. Secretory and digest cells of female Boophilus microplus : Invasion and development of Babesia bovis ; light and electron microscope studies. In: Sauer, J.R., Hair, J.A. (Eds.) Morphology, Physiology, and Behavioral Biology of Ticks. Ellis Horwood Li mited, Chichester, pp. 457-471. Aguirre, D.H., Gaido, A.B., Vinabal, A.E., de Echaide, S.T., Guglielmone, A.A., 1994. Transmission of Anaplasma marginale with adult Boophilus microplus ticks fed as nymphs on calves with different levels of rickettsaemia. Pa rasite 1, 405-407. Ahmed, J.S., 2002. The role of cytokines in immunity and immunopathogenesis of piroplasmoses. Parasitol. Res. 88, S48-S50. Aikawa, M., Pongponratn, E., Tegoshi, T., Nakamura K., Nagatake, T., Cochrane, A., Ozaki, L.S., 1992. A study on the pathogenesis of human cerebral malaria and cerebral babesiosis. Mem. Inst. Oswaldo Cruz 87, 297-301. Aldridge, B.M., McGuirk, S.M., Lunn, D.P., 1998. Effect of colostral ingestion on immunoglobulin-positive cells in calves Vet. Immunol. Immunopathol. 62, 51-64. Alleman, A.R., 1995. Evaluation of Anaplasma marginale major surface protein 3 (MSP3) as a diagnositc test antigen. Ph.D dissertation. Univer sity of Florida, United States, Florida, pp. 1123. Alleman, A.R., Barbet, A.F., 1996. Evaluation of Anaplasma marginale major surface protein 3 (MSP3) as a diagnostic test antig en. J. Clin. Microbiol. 34, 270-276. Alleman, A.R., Palmer, G.H., McGuire, T.C., McElwain, T.F., Perryman, L.E., Barbet, A.F., 1997. Anaplasma marginale major surface protein 3 is encoded by a polymorphic, multigene family. Infect. Immun. 65, 156-163. Allred, D.R., 2001. Antigenic variation in babesios is: is there more than one 'why'? Microbes Infect. 3, 481-491. Allred, D.R., Al Khedery, B., 2006. Antigenic varia tion as an exploitable weakness of babesial parasites. Vet. Parasitol. 138, 50-60. Allred, D.R., Carlton, J.M., Satcher, R.L., L ong, J.A., Brown, W.C., Patterson, P.E., O'Connor, R.M., Stroup, S.E., 2000. The ves multigene family of B. bovis encodes components of rapid antigenic variation at the infected erythrocyte surface. Mol. Cell. 5, 153-162.

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228 Allred, D.R., Cinque, R.M., Lane, T.J., Ahrens K.P., 1994. Antigenic variation of parasitederived antigens on the surface of Babesia bovis -infected erythrocytes. Infect. Immun. 62, 91-98. Allred, D.R., Hines, S.A., Ahrens, K.P., 1993. Is olate-specific parasi te antigens of the Babesia bovis -infected erythrocyte surface. Mo l. Biochem. Parasitol. 60, 121-132. Allred, D.R., McGuire, T.C., Palmer, G.H., Lei b, S.R., Harkins, T.M., McElwain, T.F., Barbet, A.F., 1990. Molecular basis for surface antigen size polymorphisms and conservation of a neutralization-sensitive epitope in Anaplasma marginale Proc. Natl. Acad. Sci. U. S. A. 87, 3220-3224. Alonso, M., Arellano-Sota, C., Cereser, V.H., Cordoves, C.O., Guglielmone, A.A., Kessler, R., Mangold, A.J., Nari, A., Patarroyo, J.H., So lari, M.A., 1992. Epidemiology of bovine anaplasmosis and babesiosis in Latin Amer ica and the Caribbean. Rev. Sci. Tech. 11, 713733. Alvarez, V., Bonilla, R., Chacon, I., 2003. Relative frequency of Boophilus microplus (Acari: Ixodidae) in bovines ( Bos taurus and B. indicus ) in eight ecological zones of Costa Rica. Rev. Biol. Trop. 51, 427-434. Amerault, T.E., Roby, T.O., 1968. A rapid card aggl utination test for bov ine anaplasmosis. J. Am. Vet. Med. Assoc. 153, 1828-1834. Amerault, T.E., Roby, T.O., 1977. Card test : an accurate and simple procedure for detecting anaplasmosis. World Anim. Rev. 22, 34-38. Angus, B.M., 1996. The history of the cattle tick Boophilus microplus in Australia and achievements in its control. Int. J. Parasitol. 26, 1341-1355. Anziani, O.S., 1979. Anaplasmosis en areas libre s de garrapatas. Memoria de la Reunion Anual de Informacion Tecnica, Instituto Naci onal de Tecnologia Agropecuaria. Estacion Experimental Regional Agr opecuaria Rafaela, pp. 63-68. Arellano-Sota, C., 1992. Epitozoology of bovine an aplasmosis and babesiosis in Latin America and the Caribbean. Report FAO Library Fiche AN: 327229, Food and Agriculture Organization of the United Nations, Santiago, Chile. Arundel, J.H., Sutherland, A.K., 1988. Animal Hea lth in Australia: Ectopa rasitic Diseases of Sheep, Cattle, Goats, and Horses. Australian Government Publishing Service, AGPS Press, Canberra. Auger, M.J., Ross, J.A., 1992. The biology of the macrophage. In: Lewis, C.E., McGee, J.O.D. (Eds.), The Macrophage. IR L Press, Oxford, pp. 1-57. Bahiense, T.C., Fernandes, E.K., Bittencour t, V.R., 2006. Compatibility of the fungus Metarhizium anisopliae and deltamethrin to control a resistant strain of Boophilus microplus tick. Vet. Parasitol. 141, 319-324.

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260 BIOGRAPHICAL SKETCH Jos Hugo Urdaz Rodrguez was born on A ugust 16, 1973 in Arecibo, Puerto Rico. He graduated from Colegio San Felipe in 1991 and bega n his studies in the fi eld of Biology at the University of Puerto Rico, Mayagez Campus. Afte r two years, he transferred to The Ohio State University and earned his B.S. in Agriculture with a Major in Animal, Dairy, and Poultry Science and a Minor in Life Sciences in 1995. Im mediately after graduation, he was accepted at the University of Florida, College of Veterina ry Medicine, where he earned his D.V.M. in 1999. Upon graduating in May 1999, Jos did a one-y ear internship program in Food Animal Medicine, Surgery, and Production Me dicine at the University of Missouri-Columbia, College of Veterinary Medicine in 2000. U pon completion of the internshi p, Jos started a three-year residency program in Dairy Production that incl uded a Masters in Preventive Veterinary Medicine (M.P.V.M.) at the University of Calif ornia-Davis, School of Ve terinary Medicine. In 2003 after finishing his residency program, Jos was awarded a four-year Alumni Fellowship and Grinter Assistantship from the University of Flor ida, College of Veterinary to pursue a Ph. D. in the Department of Large Animal Clinic al Sciences. Joss Ph.D. program has afforded him wonderful opportunities includi ng working with the agricultura l community in Puerto Rico and presenting his research in vari ous prestigious scientif ic meetings in other countries including England, Australia, France, and Italy. Upon completion of his Ph.D. program, Jos w ill be working as a specialist in animal health and food safety for the In ter-American Institute for Cooperation on Agriculture (IICA) in San Jos, Costa Rica. The IICA is a specialized agency of the Inter-American System, and its purposes are to encourage and support the efforts of 34 countries in Central and South America and the Caribbean to achieve agricultural development and well-being for rural populations.