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Integumentary lesions known as PIX in the American alligator (Alligator mississippiensis)

Permanent Link: http://ufdc.ufl.edu/UFE0022415/00001

Material Information

Title: Integumentary lesions known as PIX in the American alligator (Alligator mississippiensis)
Physical Description: 1 online resource (256 p.)
Language: english
Creator: Townsend, Heather
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: alligator, fungus, histology, hortaea, integument, lesion, pcr, pix, rt, werneckii
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: In the late 20th century in the southeastern United States, a new dermatological condition manifested itself in the alligator farming community. Starting in 1999, unusual pit-scars termed PIX were reported to occur on tanned alligator hides. Tanners have assigned the name PIX to the scars due to their physical appearance resembling a lesion that might have occurred due to an ice pick. During the next years, fresh and tanned hides from Florida were then examined retrospectively; hides from as far back as 1997 had evident PIX marks occurring throughout the skin. PIX scars usually first appear on untanned hides as spherical opaque lesions measuring around 1 mm in diameter. The severity of this problem becomes apparent when the hides are sent for grading, where damaged hides may be severely downgraded. Once the significance of this disease had manifested itself across the southeastern United States, various farms from Louisiana and Florida were reporting the disease outbreak. Our laboratory was the first to study the PIX disease in detail starting in late 1999 and early 2000. Starting in 1999 and continuing throughout 2007, techniques used to determine the causation of PIX have included gross identification, microbiology, histology, polymerase chain reaction, electron microscopy, and virology. All farms that exhibit PIX lesions are suggested to submit samples to be tested through a diagnostic panel that has been designed specifically for this problem. Treatment regimens were also suggested and implemented at some facilities. PIX has been drastically reduced on most farms, and has been eliminated on others.
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 Heather Townsend.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Cardeilhac, Paul T.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022415:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022415/00001

Material Information

Title: Integumentary lesions known as PIX in the American alligator (Alligator mississippiensis)
Physical Description: 1 online resource (256 p.)
Language: english
Creator: Townsend, Heather
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: alligator, fungus, histology, hortaea, integument, lesion, pcr, pix, rt, werneckii
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: In the late 20th century in the southeastern United States, a new dermatological condition manifested itself in the alligator farming community. Starting in 1999, unusual pit-scars termed PIX were reported to occur on tanned alligator hides. Tanners have assigned the name PIX to the scars due to their physical appearance resembling a lesion that might have occurred due to an ice pick. During the next years, fresh and tanned hides from Florida were then examined retrospectively; hides from as far back as 1997 had evident PIX marks occurring throughout the skin. PIX scars usually first appear on untanned hides as spherical opaque lesions measuring around 1 mm in diameter. The severity of this problem becomes apparent when the hides are sent for grading, where damaged hides may be severely downgraded. Once the significance of this disease had manifested itself across the southeastern United States, various farms from Louisiana and Florida were reporting the disease outbreak. Our laboratory was the first to study the PIX disease in detail starting in late 1999 and early 2000. Starting in 1999 and continuing throughout 2007, techniques used to determine the causation of PIX have included gross identification, microbiology, histology, polymerase chain reaction, electron microscopy, and virology. All farms that exhibit PIX lesions are suggested to submit samples to be tested through a diagnostic panel that has been designed specifically for this problem. Treatment regimens were also suggested and implemented at some facilities. PIX has been drastically reduced on most farms, and has been eliminated on others.
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 Heather Townsend.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Cardeilhac, Paul T.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022415:00001


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1 INTEGUMENTARY LESIONS KNOWN AS PIX IN THE AMERICAN ALLIGATOR (ALLIGATOR MISSISSIPPIENSIS) By HEATHER MARIE TOWNSEND 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 2008

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2 2008 Heather Marie Townsend

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3 To my husband, Dr. Forrest Townsend, III. He has given me such inspiration over the years that I owe everything to him. He is the most kind, patient, and loving person that I know.

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4 ACKNOWLEDGMENTS An assorted collection of people had integral parts in helping with the completion of this project. The many hurdles I encountered could not have been overcome without the help of everyone. I came to the University of Florida in 2000 to pursue what I thought would be a 3 year graduate career; 8 years later, I am still dedicating myself to aquatic animal research. The person I would like to thank fully and humbly is my mentor Dr. Paul Cardeilhac. I consider him someone to look up to and a close and personal friend, whom at times I think of as a third grandfather. I have learned many life le ssons through him, and I owe everything that I have accomplished to him. I have had many op portunities working with him on various projects and I am grateful to have come to his laboratory. Whether it was alligators manatees, or bass, he always included me in his projects and I thank him for that. My committee members each have done so mu ch for me over the years and I consider them each dear mentors and friends. Dr. Roger Reep and Dr. Don Samuelson have been helpful in teaching me the great world of histology. I wa s a teaching assistant in histology for Dr. Reep for over 5 years, and I can say that it is one of my favorite aspects of this project. Dr. Samuelson was an integral part in the labor atory preparation of my samples; the electron micr oscopy of this project would not have been possible without him. Dr. Peter McGuire is a molecular expert, and I thank him for all of his insight on the molecula r aspects of this project and for our many office brainstorming sessions. Dr. James Kimbrough is the leading expert in the field of mycology; he personally took me under his wing and taught me all about the wonderful world of fungi. I would not have such a strong interest in this aspect if it werent for him. Pat Lewis, who in 2007 was named histotechnologi st of the year, has been my right hand woman in all of my years at UF. There is not a single stain that she cannot do. She has been a great source of help and support for me and I do not think that I would have left with as much

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5 sanity if she was not there for me. Ann Marie Cl ark was key in developing the PCR protocol for the detection of Hortaea werneckii within alligator integument. The virology portion of this project would not have been possible without the help of Maureen Long and Carlos Romero. Pam Ginn provi ded the pathological re ading of the slides, and I thank her so much for her insight and help Shasta McClenahan and Rebecca Grant helped tremendously with the virology portion of this pr oject. They were also my lunch buddies for many years, and I have had countless laughs w ith them by my side. Maggie Kellogg and Kim Pause were integral with portions of my laboratory work, and I thank them for always helping me when I needed it. I made four great and everlasting friends out of this degree. The whole reason that I even remotely know a nything about alligators is because of Allen Register. I completed an internship with him in 1998 at his alligator farm in Palmdale, Florida and since then I have worked many years with him collecting alligator eggs every summer in Florida. I would not have as much love and appr eciation for the species if it were not for him. I would not be where I am at today if I did not have him to guide me through most of my beginnings. I thank Allen for taking a yankee gi rl in and showing her the ways and for making me a part of his family. Financial support was provided through both the Florida Aqu aculture Review Council and the Louisiana Department of Fisheries. Noel Kinl er and Ruth Elsey had integral parts in helping me with data collection and co mmunication. Numerous travel gr ants were awarded through the University of Florida. I would not have had as many samples over the 7 year period if not for the alligator farmers in Florida and Louisian a. I thank them fo r their sacrifice. My family had an important part in my finishing my PhD through their emotional and financial support through my many years in college. My mother Marie, father Ronald, and

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6 brother Cameron, along with my grandparents Kay, Ernie, and Donald; I thank them for their support. All of my fellow gradua te students: Ann, Anne, and Katie ; I would not have been able to complete portions of my degree if it werent for them. Last, but certainly not least, none of this coul d be possible without th e love that I receive daily from my husband, Forrest Trey Townsend. Not only has he shown support through his love, but pushed me when I needed pushing. I am thankful that he is also a veterinarian, for when it was late at night and I needed help and did not want to wake up any of my committee members, I could always turn to him for advice. I thank him for always planning things when I was starting to get overstressed, and for taking th e dogs out when I was too busy to notice them whining at the door. I owe my heart and soul to him.

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7 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ........11 LIST OF FIGURES.......................................................................................................................12 ABSTRACT...................................................................................................................................15 CHAP TER 1 LITERATURE REVIEW.......................................................................................................17 Introduction................................................................................................................... ..........17 Overview of the Integument...................................................................................................18 Physiology of the Integument.......................................................................................... 18 Protection.................................................................................................................19 Thermoregulation..................................................................................................... 19 Excretion..................................................................................................................20 Reception..................................................................................................................20 Absorption................................................................................................................20 Water Loss................................................................................................................ 21 Immunity..................................................................................................................21 Movement.................................................................................................................21 Macroanatomy of the Integument...................................................................................22 Microanatomy of the Integument.................................................................................... 24 Epidermis.................................................................................................................29 Epidermal cell types................................................................................................. 32 Dermis...................................................................................................................... 35 Dermal ce ll types ...................................................................................................... 37 Development of the Integument............................................................................................. 38 Processes in Reptilian Development............................................................................... 43 Crocodilian Development................................................................................................ 44 Post-hatching Crocodilian Integument..................................................................... 51 Comparisons............................................................................................................. 51 Conclusions..............................................................................................................55 Diagnostic Testing in the Id entification of Pathogens ............................................................56 Visual Identification........................................................................................................56 Environmental Testing.................................................................................................... 57 Microbiology...................................................................................................................57 Histology.........................................................................................................................58 Electron Microscopy....................................................................................................... 60 Polymerase Chain Reaction............................................................................................. 61 Reverse-Transcription-Polymerase Chain Reaction (RT-PCR)...................................... 64

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8 Virology...........................................................................................................................65 In Situ Hybridization....................................................................................................... 66 Use of Antibody Reagents............................................................................................... 69 Review of Fungi.....................................................................................................................72 Eukaryotic........................................................................................................................73 Heterotrophic...................................................................................................................73 Reproduction...................................................................................................................74 Fungal Growth................................................................................................................. 75 Cell Walls..................................................................................................................... ...76 Dimorphic........................................................................................................................77 Deuteromycetes................................................................................................................. .....77 Conidiogenesis................................................................................................................80 Thallic conidiogenesis..............................................................................................81 Blastic conidiogenesis..............................................................................................82 Hortaea werneckii ..................................................................................................................84 Classification and History...............................................................................................84 Morphological Characterization...................................................................................... 85 Epidemiology.................................................................................................................. 86 Human Cases...................................................................................................................87 Diagnosis...................................................................................................................... ...88 Non-Human Animal Cases.............................................................................................. 89 Treatment.........................................................................................................................89 Kochs Postulates....................................................................................................................90 2 HISTORY OF PIX SKIN DISEASE IN THE SOUTHEASTERN UNITED STATES ........ 98 Introduction................................................................................................................... ..........98 Farms Participating in this Study............................................................................................ 99 Farm LA-1..................................................................................................................... 100 Farm LA-2..................................................................................................................... 100 Farm LA-3..................................................................................................................... 100 Farm LA-4..................................................................................................................... 101 Farm FL-1...................................................................................................................... 101 Farm FL-2...................................................................................................................... 101 Farm FL-3...................................................................................................................... 101 Farm FL-4...................................................................................................................... 102 History of Occurrence...................................................................................................102 West Nile Virus and th e Em ergence of PIX......................................................................... 103 Diagnostic Panel to Determine the Causative Agents of PIX.............................................. 105 Gross Identification of Pix Lesions...................................................................................... 105 Discussion.............................................................................................................................106 3 HISTOLOGICAL CHARACTERIZATION OF PIX AND SPHERIC AL OPAQUE LESIONS IN THE AMERICAN ALLIGATOR.................................................................. 115 Introduction................................................................................................................... ........115 Materials and Methods.........................................................................................................116

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9 Results...................................................................................................................................121 Special Stains.................................................................................................................123 Comparison of PIX Lesions Among Farms.................................................................. 124 Histological Examination of the Hinge Regions in Alligators...................................... 124 Discussion.............................................................................................................................125 4 THE USE OF ELECTRON MICROSCOPY IN IDENTIFYING PATHOGENS LOCATED IN PIX LESIONS .......................................................................................... 155 Introduction................................................................................................................... ........155 Materials and Methods.........................................................................................................156 Results...................................................................................................................................157 Discussion.............................................................................................................................157 5 IDENTIFICATION OF MICROBIOLOGICA L ISOLATES FROM PIX LESIONS ......... 165 Introduction................................................................................................................... ........165 Materials and Methods.........................................................................................................166 Results...................................................................................................................................167 Discussion.............................................................................................................................169 6 THE USE OF VIROLOGY IN THE IDEN TIFICATION OF A CAUS ATIVE AGENT OF PIX..................................................................................................................................177 Introduction................................................................................................................... ........177 Materials and Methods.........................................................................................................178 Use of AEF to Determine the Presence of a Virus in SOLs .......................................... 178 Use of AEF to Show Positive CPE due to the Presence of Avian Viruses................... 180 Use of AEF to show CPE using a WNV Chimera........................................................ 181 Use of Real Time-PCR in the Iden tif ication of Viruses within SOLs.......................... 182 Results...................................................................................................................................183 Use of AEF to Determine the Presence of a Virus in SOLs .......................................... 183 Use of AEF to Show Positive CPE due to the Presence of Avian Viruses................... 183 Use of AEF to Show CPE with a WNV Chimera......................................................... 183 Use of RT-PCR in the Identifica tion of a Virus within SOLs ....................................... 184 Discussion.............................................................................................................................184 7 IDENTIFICATION OF HORTAEA WERNECKI I LOCATED WITHIN SPHERICAL OPAQUE LESIONS BY POLY MERASE CHAIN REACTION........................................ 196 Introduction................................................................................................................... ........196 Materials and Methods.........................................................................................................198 Results...................................................................................................................................202 Discussion.............................................................................................................................203

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10 8 TREATMENT REGIMENS AND SUGGESTIO NS FOR THE CONTROL OF PIX ........ 209 Introduction................................................................................................................... ........209 Nutrition........................................................................................................................210 Growth Management.....................................................................................................210 Stocking Densities.........................................................................................................211 Potassium Permanganate............................................................................................... 211 Copper Sulfate...............................................................................................................211 Materials and Methods.........................................................................................................212 Results...................................................................................................................................215 Discussion.............................................................................................................................216 9 EPIDEMIOLOGICAL PROJECTIONS, F UTURE RESEARCH, AND CLOSING DISCUSSION CONCERNING PIX SKIN DISEASE IN THE AMERICAN ALLIGATOR.......................................................................................................................225 Introduction................................................................................................................... ........225 Epidemiological Projections................................................................................................. 226 Problems Encountered.......................................................................................................... 226 Future Research....................................................................................................................227 Summary and Conclusions...................................................................................................228 Closing Remarks...................................................................................................................233 APPENDIX A HORTAEA WERNECKII LINEAGE................................................................................. 235 B DISTRIBUTION OF PIX..................................................................................................... 236 C COMMON SYNONYMS OF HORTAEA WERNECKII .....................................................237 LIST OF REFERENCES.............................................................................................................238 BIOGRAPHICAL SKETCH.......................................................................................................256

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11 LIST OF TABLES Table page 1-1 Summary of the post-laying em bryonic development stages............................................ 96 1-2 Comparison of the terminology of integumentary layers.................................................. 97 6-1 Primers used in the identification of viruse s within PIX lesions. .................................... 195 7-1 Primers used in this study for the identification of H. werneckii within lesions. ............ 208 7-2 GenBank accession numbers for H. werneckii isolates used for com parison.................. 208 8-1 Treatment Groups........................................................................................................... .223 8-2 Suggested solution for the copper sulfate treatment........................................................ 224

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12 LIST OF FIGURES Figure page 1-1 Layers of crocodilian scales............................................................................................... 92 1-2 Scales of lizards (and snakes)............................................................................................ 93 1-3 Chelonian scutes........................................................................................................... .....94 1-4 Scales in Sphenodon punctatus.. ........................................................................................94 1-5 Thallic conidiogenesis..................................................................................................... ..95 1-6 Blastic conidiogenesis..................................................................................................... ...95 2-1 Tanned alligator hide s showing PIX lesions. ...................................................................108 2-2 Florida alligator hide pla ced over a high intensity lam p..................................................109 2-3 Photographs depicting SOLs on a fr esh, unsalted Florida alligator hides. ...................... 110 2-4 Photograph of a Louisiana hi de exhibiting severe SOLs. ................................................111 2-5 Photograph of SOLs before biopsy.................................................................................. 111 2-6 Example of a 6mm biopsy punch with a PIX scar........................................................... 112 2-7 West Nile virus maps reflect surveillan ce repo rts released by st ate and local health departments to CDC's ArboNET system for public distribution..................................... 113 2-8 West Nile virus maps reflect surveillan ce repo rts released by st ate and local health departments to CDC's ArboNET system for public distribution..................................... 114 3-1 Hematoxylin and Eo sin stain (05R-66)............................................................................ 129 3-2 Hematoxylin and Eo sin stain (05R-105).......................................................................... 130 3-3 Hematoxylin and Eo sin stain (05R-267).......................................................................... 131 3-4 Hematoxylin and Eo sin stain (06R-155).......................................................................... 132 3-5 Hematoxylin and Eo sin stain (07R-60)............................................................................ 133 3-6 Hematoxylin and Eo sin stain (07R-73)............................................................................ 134 3-7 Hematoxylin and Eo sin stain (2002R-17)........................................................................ 135 3-8 Hematoxylin and Eo sin stain (2002R-26)........................................................................ 136

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13 3-9 Hematoxylin and Eo sin stain (2002R-166A). .................................................................. 137 3-10 Toluidine Blue stain (07R-20a)....................................................................................... 138 3-11 Periodic Acid Schiff stain (05R-676)..............................................................................139 3-12 Brown and Brennan stain (05R-676)...............................................................................140 3-13 Acid Fast stain (05R-676)................................................................................................ 141 3-14 Giemsa stain (05R-675A)................................................................................................142 3-15 Elastin stain (07R-6)........................................................................................................143 3-16 Grocotts Method for fungi stain (07R-6)........................................................................ 144 3-17 Grocotts Method for fungi stain (05R-676).................................................................... 145 3-18 Old PIX lesion..................................................................................................................146 3-19 Old PIX lesion..................................................................................................................147 3-20 PIX visualization using a confocal m icroscope...............................................................148 3-21 PIX visualization using a confocal m icroscope...............................................................148 3-22 PIX visualization with a confocal m icroscope using a Congo red filter.......................... 149 3-23 PIX visualization using a confocal mi croscope with a yellow filter applied. .................. 149 3-24 Comparison of lesions..................................................................................................... .150 3-25 PIX lesion from a 1999 sample........................................................................................151 3-26 Photomicrograph of hinge region from ventral belly...................................................... 152 3-27 Photomicrograph of the hinge region from the gular area............................................... 153 3-28 Photomicrograph of normal alligator integument............................................................ 154 4-1 Photomicrograph of an SOL located within the superficia l portion of the derm is.......... 159 4-2 Photomicrograph of the periphery of an SOL.................................................................. 160 4-4 Electron micrograph of a fungal element......................................................................... 162 4-5 Electron micrograph of an area within an SOL. ..............................................................163 4-6 Electron micrograph of a fungal element......................................................................... 163

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14 4-7 Electron micrograph of an area within the lesion exhibiting high cellularity. ................. 164 4-8 Electron micrograph of a degenerating fungal element................................................... 164 5-1 Photograph of the Hortaea werneckii growth from a Florida alligator farm in 2002. ... 173 5-2 Photograph of a 2003 Hortaea wern eckii growth from a Florida farm........................... 173 5-3 Photograph of Hortaea werneckii from a farm in Louisiana in 2006.............................. 174 5-4 Photomicrograph of a scraping from the Hortaea werneckii growth in F igure 5-3........175 5-5 Photomicrographs of the Hortaea werneckii growth from Figure 5-3............................ 176 6-1 Samples prepared for SOL inoculation............................................................................186 6-2 Photomicrograph of alligator em bryo fibroblast control plate (50X). .............................187 6-3 Photomicrograph of AE F control plate (100X). .............................................................. 188 6-4 Photomicrograph of AE F control plate (320X). .............................................................. 189 6-5 Photomicrograph of AEF cell culture inoculated with alligator SOLs. ...........................190 6-6 Photomicrograph of AEF cells inocul ated with F owl Pox vaccine strain....................... 191 6-7 Photomicrograph of AEF cells i noculated with pigeon param yxovirus..........................192 6-8 Agarose gel electrophoresis of RT-PCR for pigeon param yxovirus from AEF cell cultures.............................................................................................................................193 6-8 Schematic representation of a modified live Flav ivirus /West Nile virus (WN-FV) chimera deve lopment....................................................................................................... 194 7-1 Visualization of PCR products th rough agarose gel electrophoresis. .............................. 205 7-2 Radial phylogenetic tree..................................................................................................206 7-3 Sequence alignment for positive H. werneckii samples................................................... 207 8-1 Photograph of the housing constructed fo r the treatm ent study at Farm LA-1............... 220 8-2 Example of the treatme nt regim en directions.................................................................. 221 8-3 Approximate size of the alli gators that were used over the two year study for groups one, two and three. ........................................................................................................... 222

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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 INTEGUMENTARY LESIONS KNOWN AS PIX IN THE AMERICAN ALLIGATOR (ALLIGATOR MISSISSIPPIENSIS) By Heather Marie Townsend August 2008 Chair: Paul T. Cardeilhac Major: Veterinary Medical Sciences In the late 20th century in the southeastern United States, a new derm atological condition manifested itself in the alligator farming commun ity. Starting in 1999, unusual pit-scars termed PIX were reported to occur on ta nned alligator hides. Tanner s have assigned the name PIX to the scars due to their physical appearance resembling a lesion th at might have occurred due to an ice pick. During the next years, fresh and tanned hides from Florida were then examined retrospectively; hides from as far back as 1997 had evident PIX marks occurring throughout the skin. PIX scars usually first appear on unta nned hides as spherical opaque lesions measuring around 1 mm in diameter. The severity of this problem becomes apparent when the hides are sent for grading, where damaged hides may be severely downgraded. Once the significance of this disease had manifested itself across the so utheastern United States, various farms from Louisiana and Florida were reporting the diseas e outbreak. Our laborator y was the first to study the PIX disease in detail starting in late 1999 and early 2000. Starting in 1999 and continuing throughout 2007, techniques used to determine the causation of PIX have included gross identification, microbiology, histology, polymer ase chain reaction, electron microscopy, and virology. All farms that exhibit PIX lesions are suggested to subm it samples to be tested through a diagnostic panel that has been designed specifically for this problem. Treatment regimens

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16 were also suggested and implemented at some fa cilities. PIX has been drastically reduced on most farms, and has been eliminated on others.

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17 CHAPTER 1 LITERATURE REVIEW Introduction The Am erican alligator ( Alligator mississippiensis) is the most abundant crocodilian species found in the United States. Coincidently, it is also one of the only re ptilian species that is commercially farmed in large numbers within the southeastern portion of this country. These farms are primarily located in Florida, Georgia, Louisiana, and Texas. Due to the conditions of these farms in relation to the natural habitat of th e alligator, these animals are prone to diseases and husbandry issues that might not affect th eir wild counterparts. One of these disease progressions has arisen in the past decade and has come to be known as PIX skin disease. This integumentary problem is a common occurrence among alligator farms, and it is the basis of this dissertation. The dermatological condition that the researchers studied in th is dissertation had various aspects to be explored and characterized. This literature review will contain a comprehensive look at reptilian integument and disease processes. Development of the integument, comparisons between taxa and a review of crocodilian skin will be examined. The process through which a researcher confirms or refutes detection of a pathogen through diagnosti c testing is important; these tools will be examined, along with the stre ngths and weaknesses of each. The researcher previously wrote a thesis on this integumentary condition (D ickson, 2003), so the literature review presented here will not detail what was cove red in that paper (alligator farming, diseases of reptiles, skin diseases of crocodilians, etc.). Finally, consideration of fungi, in particular the Deuteromycetes will be examined along with a comprehensive review of the fungus Hortaea werneckii a possible etiologic agen t associated with PIX.

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18 Overview of the Integument The integum entary system is comprised of the skin and its associated tissues. The skin invests the entire body, becoming continuous with the mucous membranes of the digestive system, respiratory system, and the urogenital system (Gartner and Hiatt 2001a ). It is the largest organ in the body, often comprising from 6-20% of the bodys weight depending on the species. Skin fits the definition of an organ since it is a group of tissues working together to perform related functions (Marieb et al 2005). The main components of the skin are the epithe lial tissue portion, or the epidermis, and the connective tissue portion, or the dermis, which each have their associated layers. The microand macroanatomy will differ depending on the area s of the body and also between species. The avascular epidermis has many layers, which ar e species specific. Starting from the most superficial layer and going deeper the layers (most commonly na med) are the stratum corneum, stratum lucidum, stratum granulosum, stratum spi nosum and the stratum basale. The dermis is composed of a superficial papillary la yer and the deeper reticular layer. Thickness of the skin depends on its location wi thin the body. The eyelids will have some of the thinnest layers of skin, while the palms and soles of the feet will have the thickest composition (Gartner and Hiatt 2001a). Skin is usually divided into two main categories; glabrous and hairy skin. Glabrous skin is defined as skin that is devoid of hair and is also some of the thickest layers of skin. Physiology of the Integument The skin has m any various anatomical com ponents that relate to its physiological properties. Some of the main functions of th e skin are physical protection, regulation of body temperature, excretion, reception, absorption, wate r loss, immunity, coloration, and movement.

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19 These all also play an important role in the ap pearance of healthy skin (Tortora and Grabowski 1996). Protection Protection is the first f unctional role that skin provides. This is the bodys first line of defense, and is ultim ately the first anatomical f eature that is encountered by external stimuli. The many layers of the integument that compose the framework provide a physical barrier to protect against injury, invasion and desiccation (Gartner and Hi att 2001a). This also employs protection against physical, biological and chemical agents that may play a ro le in destruction of the skin and its components. It protects against abrasions from the exte rnal environment, while at the same time cushioning the internal organs that lie deep to the sk in (Marieb et al 2005). Thermoregulation Skin provides a m eans to regulate body temperature by employing various ways to either keep heat in or let heat out. These include cutaneous vasculature, subcutaneous fat, and the presence of hair. The integument provides a rese rvoir of blood that helps with heat release by narrowing and widening the blood vessels and capillary beds located in the de rmal layers of the integument (Gartner and Hiatt 2001a, Weissengruber et al 20 06). Vasoconstriction and vasodilation allow for varying amounts of blood to flow to a certain area. Blood vessels located in the integument carry 8-10% of the total blood flow in a rest ing adult (Tortora and Grabowski 1996). During moderate exercise, the amount of blood located in the dermis increa ses and aids in heat release, which works in conjunction with the secretion of sw eat. During extreme exercise, these blood vessels will constrict and allo w more blood to circulate thro ugh nearby contracting muscles (Tortora and Grabowski 1996).

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20 Storage of fat is mediated in the body by adipocytes. Adipocytes, or adipose cells, compose adipose tissue and can be classified as either white or brown (Marsella 2005). White adipose tissue acts as the nutrientstoring fat, whereas brown adipos e tissue produces heat. They are specialized in the synthesi s and storage of fat and mainta in proper energy balance. An animals hair coat also plays a role in ther moregulation by preventing heat loss when needed by providing a barrier at the skins surface. Excretion Skin allows for the elim ination of wast es produced by the body by producing sweat. Because of these emunctory, sudoriparous glands the integument is considered an excreting, homeostatic, thermoregulatory organ (Atlee et al 1997, Marsella 2005). Sweat contains ions and organic compound wastes that must be eliminated from the body (Tortora and Grabowski 2005). Producing sweat and excreting it through glands that open at the bodys orifice helps to regulate temperature by cooling down the body in times of extreme heat. Some skin glands also produce pheromones, which will aid in courtship and territ ory disputes in some species if animals. Reception The skin provides reception to the sensations of the external environm ent. This is accomplished by the various networks of nerves that are found within the integument. It completes this task by containing sense organs ca lled sensory receptors that are associated with nerve endings located in the skin (Marieb et al 2005). These ne rve endings detect stimuli related to temperature, touch, pressure and pain (Tortora and Grabowski 2005). Absorption The integum ent aids in absorption by perfor ming a role in calcium homeostasis by the synthesis of Vitamin D. By absorbing UV radiat ion from the suns rays, the skin synthesizes Vitamin D for use by the body. The precursor molecule of Vitamin D is 7-

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21 dehydrocholecalciferol, which is activated within the skin and modified by enzymes in the liver and kidney (Tortora and Grabowski 2005). The modi fied form is converted to cholecalciferol, then finally to calcitriol (calcife rol), the most active form of Vita min D. Vitamin D aids in the absorption of calcium and is also considered a hormone; thus, skin can additionally be considered an endocrine organ. Water Loss The epiderm is aids in protecting the body fr om unnecessary water, electrolyte and macromolecule loss by having waterproof prope rties (Marsella 2005). Water balance is especially important in amphibians, which are gene rally not well adapted to a terrestrial lifestyle and do not have physiologic means to prevent wa ter loss from occurring through their skin. Aquatic species depend on this characteristic gr eatly, since impermeability to water is essential to their survival (Samuelson 2007). Immunity The skin has properties in place that will repel invading microorganisms and defend the body against attack. Vascularization serves a nutritional function along with a pathway for immune cells to travel to the skin in response to microorganism presence (antigenic presence). By having a network of capillary beds connected to lymph vessels, this allows for an effective route for various immune cells to travel to the la yers of the integument. There are a number of cell types that reside in skin and have immune capabilities. These include Langerhans cells, lymphocytes, and macrophages, alon g with other white blood cells. Movement Skin plays a role in m ovement. For instan ce, thickened areas provide a means to help with weight bearing during locomotion; scales and claws additionally help to secure an animal during movement. The foot pads of some lizards allow them the ability to walk up glass or

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22 walls. Feathers found on birds are used in flight. These skin appendages all have an important role in providing an animal with a proper means of locomotion in their specific environments. Macroanatomy of the Integument The anatomy of the integument will vary depend ing on two important factors; the species and location in the body. Focus will lie primarily on two aspects: reptiles, primarily the alligator, and mammals, primarily the glabrous regions. As ther e are many similarities between the skin of mammals and reptiles, there are also a consid erable number of differences. The following section will focus on the macroanatomy of the integument. Hair. The first structure that defi nes macroanatomical visualizati on is hair. Hair is present on all species of mammals, but will vary from spec ies to species. Glabrous skin is formed in areas of the body that do not have hair. These regions include the soles, palms, mucocutaneous areas, hoofs, glans penis, and teats (Marieb et al 2005, Samuelson 2007) of certain mammalian species. Although reptilian skin is considered entirely glabrous, it does not resemble these areas in mammals and has a variety of differenti al characteristics associated with it. Integument thickness. Mammalian glabrous skin is characterized by a thick epidermis, a lack of hair follicles, arrector pilli musc les and sebaceous glands, and the presence of encapsulated sense organs (Gartner and Hiatt 2001a). Localized th ickening of the skin in these areas possess all of the five main skin layers; the presence of the stra tum lucidum characterizes glabrous skin in mammals. Mammals with glabrous skin will have va rying degrees of thickness dependant on body location; thick skin occurs in the areas mentioned above. The r easoning for thicker skin to be located in these areas is because these regions exert the highest degree of pressure over the course of the day (Weissengruber et al 2006). Fo r example, typing on a computer for hours at a

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23 time with glabrous skin will lessen the stress on our fingers. Walking across campus twice a day is not as stressful on our body because of thicke ned soles that absorb some of the shock. Reptilian species will have completely different degrees of skin thickness, for reasons that can be explained by their evolution and adaptation to their environment. Alligators will have the thickest skin on the dorsal region of their thorax and on their foot pads; this is primarily due to the dorsal areas receiving the most abrasions when fighting, and for the foot pads to bear their enormous weight when walking on land. Bony areas. Most turtles have a dorsal carapace and a plastron that consists of dermal plates between their ribs. In crocodilians, os teoderms are found on the dorsal surface, and have also been observed in other animals (Hill 2006, Vickaryous and Hall 2006). These lie under the epidermal scales of the dorsal su rface of the trunk and tail, and in some species (such as caiman) they are even located on regions of the dorsal head. Crocodilian skin on the head is actually fused to the bones of the skull, forming specializ ed osteoderms. They are grouped into two areas known as the nuchal and the dorsal shields. Crocodilians possess osteoderms on their dorsal body region for two main reasons; the first serves as a protective role, and the second as a thermoregulatory role. While an alligator is normally a submissive species, they rely on hun ting and inter-species fighting for survival. When in a territorial battle, the area that is most likely to be bitten or subject to abrasions is their dorsal surface. Osteoderms play a role in lessening the damage that may be inflicted to this area. Osteoderms also act as a solar panel to the anim al, being richly vascularized and aiding in thermoregulation. Alligators are commonly seen ba sking in the sun along the banks of water; because they are ectotherms they must rely on external sources of heat to raise their body

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24 temperature, and will do this through the dorsal oste oderms. Their skin does not posses means in which to insulate themselves or provide a thermoregulatory role (Alibardi 2002a). Microanatomy of the Integument Terminology. In m ost animals (especially mammals), the term stratum is used to distinguish the layers of the integument along with th e corresponding descripti on of the specific layers (ex., stratum basale). When referring to squamates (lizards and snakes) a different terminology is used (Table 1-2). There are six different epidermal layers termed, from superficial to deep: oberhautchen, beta, mesos re gion, alpha, lacunar, and clear layer (Alibardi and Toni 2005). Terminology used to determine crocodilian exoske leton is similar to that used to define mammalian skin. Crocodilians only possess four main layers to their epidermis. The deepest layer is referred to as the stratum basal, much like the stratum basal mammals. Superficial to this, the next layer is termed the suprabasal, which is analogous to the mammalians stratum spinosum. Next, crocodilians have a precorneo us or transitional layer, analogous to the mammalians stratum granulosum layer. The mo st superficial layer in crocodilians is the corneous layer, which is so named in mammals also (Alibardi persona l communication) (Table 1-2). Because mammals and crocodilians possess different types of keratins in their epidermal layers we consider them separate ly, but analogous at the same time. It is important to notice that crocodilians do not possess a stratum lucidum la yer that is commonly found in the glabrous regions of mammals. Glands There are various glands located within the epidermis and dermis to provide a variety of functional roles. The main types of glands are sweat (sudoriferous) glands and sebaceous (oil) glands. In glab rous skin, there are no sebaceous glands present, only sweat glands (Gartner and Hiatt 2001a, Marieb et al 2005, Tortora and Grabowski 1996).

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25 Integumentary glands in reptiles vary. Re ptiles possess only a few skin glands and no sweat glands. Instead of sweat glands, to regulate heat, alligators will lose heat through their oral cavities. Lizards will have femoral glands, and crocodilians and some turtles have scent glands. Crocodilians also posses dermal sense receptors (DSR) which will be discussed in following sections. Scales. Scales cover almost the entire body of reptiles. These scales are virtually waterproof, which makes life on land for these cr eatures a possibility. By forming a continuous barrier evaporation is in hibited and animals can acquire a terres trial lifestyle wit hout desiccation. Scales are formed by a substance that is absent in soft-skinned animals. While alpha-keratin determines soft skin present in glabrous regions of mammals, beta-keratin is what forms scales in reptiles. Beta-keratins produce the hardening of the corneus layer in all reptilian groups and also in birds (Alibardi 2005). The microornamentation in reptiles show separate integumentary layers of alphand beta-keratin s in crocodilians and chelonians, bu t plate-like organization in lizards and snakes. Scales will vary in all rep tiles, such as their shape, thickness, degree of overlapping, absence of overlapping, and exte nsibility (Alibardi 2005). Crocodi lians possess large scales that will overlap infrequently (as in the distal portions of the limbs) or not at all. In chelonians, the scales may be overlapped on the carapace, but not in the plastron. Lizards exhibit a high degree of overlapping scales. Crocodilian scales have a large outer rectangular surface, and the development of scutes that progress from th e outer scale surface (Alibardi and Thompson 2001, Alibardi 2005). These scales are arranged in transverse rows, with the long axis of the scales parallel to the body. They become elevated along the tail forming keels, which gives the tail a larger surface area for improved swimming.

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26 Squamate scales develop first as an epid ermal projection from the body. As the scale continues development, the superficial layer becomes hardened with beta-keratin and the dermal layer recedes. Scales in crocodilians begin as a local thickening of the epidermis with the development of bony plates, or scutes, beneat h the surface on certain areas of the body. Keratins. A chief difference between mammalian glabr ous skin and reptilian skin is the presence of the beta-keratin prot ein that is responsible for the ha rdening of the outer layer of the epidermis. Perhaps the most important and char acteristic difference between scaled animals and non-scaled animals is the presence of this additi onal keratin type. Cyt okeratins, also known as soft keratins or alpha-keratins, are considered the proteins of intermediate filaments and are different from -keratin proteins (Alibar di 2005). Cytokeratins determine the roles that the epidermis will provide in relati on to the environment. Alpha-keratins are strong, inextensible, insoluble, and pliable. In reptiles, the corneocytes unde rgo an alphaand/or beta-keratinization. This results in beta-keratin, which forms the hard, outer surface of all reptiles (and is absent in mammals). Beta-keratin is an extensible, non-pliable protein that aggregates into densely packaged lattices to produce resistant microfibrils (Alibardi 2005). In reptiles, the process of keratin ization varies among the Orders, being more or less continuous in chelonians and crocodilians and cyclic in lepidosaurians (Alibardi and Thompson 2002, Alibardi 2003). In squamates, the epidermis is composed of alternating layers of cell generations produced from the germinal layer (Alibardi and Toni 2005) The oberhautchen and beta layers synthesize beta-keratin, which is deposited after the initial deposition of alpha-kerat in. Beta-keratin is thought of as hard, unelastic and re sistant. Newer epidermal layers are then formed deep to the

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27 older generation of cells, which will eventually be lost in the animals molting period (Alibardi and Toni 2005). This cyclic modality also differs in relation to the keratins that reptiles possess. The alphakeratins are mostly producing the softer mesos, alpha, lacunar and clear layers in reptiles, while the beta-keratins are producing the hard protect ive layers of the oberh autchen and beta-layer (Alibardi and Thompson 2002). The scaly epider mis has additional epidermal layers that are produced underneath the old epidermis, which will ultimately result in shedding In crocodilians, beta-k eratin deposition over al pha-keratin bundles is rapid, and happens within the cells of the intermediate layers. Th e beta-keratins will essentially replace the alphakeratins, whereas in lizards and snakes it is me rely added within the bundles of alpha-keratins (Alibardi 2005, Marsella, 2005, Alibardi and Thompson 1999a). In turtles, the location of alphaand beta-keratins differs within body location. An alpha-keratin epidermis occurs within the neck, head, limbs and tail regions of turtles, whereas the shell will be strictly beta-keratin (Alibardi and Thompson 1999b, Alib ardi and Dipietrangelo 2005). Shedding Complex. Reptiles (in particular lizards and snakes) will shed their skin in one continuous molt. Reptiles will periodically shed their entire epidermis, with a completely new epidermis forming underneath. So theoretically, th ere is a point when the animal wills posses two layers of epidermis. Ecdysis will occur once a fissure zone is formed and the new epidermis is completely formed. Crocodilians do not go through a molt like lizards do; instead, they shed their scales one scale at a time, often at different intervals (not continuously). Mammals will have portions of their epidermis exfoliate when they are regenerating new cells of the skin. In glabrous skin, the stra tum corneum will separate in large amounts and fall

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28 off the underlying areas. Certain conditions of the skin, such as dryness, will facilitate shedding at a higher rate than usual. Pigmentation. Chromatophores are pigment-contai ning cells found only in reptiles, amphibians, and fish (Cooper 2005, Lane 2005). They are responsible in producing the skin and eye color in cold-blooded animals. Reptiles ha ve 6 main types of chromatophores: cyanophores, melanophores, xanathophores, erythophores, irid ophores, and leucophores. Mammals and birds only have one class of a chromatophore-like cell, which is the melanocyte. Mammalian pigmentation is determined by the location of mela nin, rather than to the number of melanocytes (Gartner and Hiatt 2001a). Innervation. Innervation to the integument occurs through sensory cutaneous nerves (Marsella 2005, Marieb et al 2005 ). Mechanoreceptors are sens ory receptors that respond to external stimuli within an animals environment. They are primarily located in the glabrous skin of mammals and are divided into 4 main types. Me rkel discs lie in the epidermis of the skin and respond to light touch. Meissners corpuscles occur in the dermal papillae and respond to light touch and vibrations (Marieb et al 2005). Ruffinis corpuscles ar e located within the dermis and respond to pressure and touch. Pacinian corpuscles are perifollicular nerve endings situated around hair follicles. Typically, nerve endings that respond to cold temperatures are found deep in the dermis, while heat sensitive receptors ar e located in the intermediate and superficial dermis (Tortora and Grabowski 1996). Glabrous skin is known to have an increase in the amount of mechanoreceptors located in the dermal areas (Gar tner and Hiatt 2001a, Marieb et al 2005). Tactile reception in areas of glabrous sk in seems to be heightened (Hamrick 2001, Witter et al 2007) and is especially evident in animals with de xterous use of their hands.

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29 Reptiles contain mechanoreceptors in their skin and have similar functions as in mammals. They have been implicated in sensing vibrations in the water, along with aiding in response to cooling temperatures (Necker 1974, Kenton et al 1971). Some crocodilians have been observed to posse ss integumentary sense organs (ISOs), or dermal pressure receptors (DPRs) in their skin (Jackson et al 1996) These are small sensory pits located along the jaw line in all crocodilians and over the entire surface of the crocodiles body (Jackson et al 1996). These DPRs are bundles of nerve fibers that are able to detect small pressure changes and vibrations in the water. In crocodiles, the DPRs encase the entire body in an effort to detect salinity and chemical recepti on (Jackson et al 1996). These DPRs may play a similar role to manatee vibrissae that are lo cated along their entire body surface (Reep et al 2001, Reep et al 2002). Epidermis The integum entary organizational pattern of sc aled animals is similar to the prototypical animal compositions, but there are many defining characteristic differences that will be discussed. The presence of scales over the enti re body in reptiles is what distinguishes them from other amniotes, birds, and mammals (Alibardi 2005). The epidermis is composed of five chief layers. Focus will start from the deepest layers and proceed superficially, since this is the pa thway that new cells will progress along until they reach the surface. A new epidermis is formed every 35-45 days (Marieb et al 2005). The epidermal skin layers discussed here will re ly on the mammalian classification, since this terminology is used most in the literature. Stratum Basale. The stratum basale, or stratum germinativum, is the deepest layer of the epidermis. This layer is supported by a basement membrane that acts to anchor it to the dermal layers underneath. This basement membrane is composed of a basal lamina and a deeper

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30 reticular layer, which collectivel y act to serve as a filter and support mechanism for regenerating epithelial cells (Marie b et al 2005). This basal layer consists of one layer of mitotically active columnar to cuboidal cells (keratinocytes) that contain a basophilic cytopl asm and a large nucleus (Marsella 2005). About 50% of the cells located in this area are dividing, while the other 50% are in a resting state. These stem cells are involved with tonofila ment production (Samuelson 2007). Melanin is produced in membrane-walled granules, and is transferred to keratinocytes through dendritic processes. These melanin clusters are located on the superficial portion of the cell to shield ultraviolet light from damaging the nucle us (Marieb et al 2005, Marsella 2005). Located along the lateral cell junction are a series of desmosomes that anchor cells to one another. Hemidesmosomes attach cells basally to the underlying basal lamina. Also associated with the hemidesmosomes and desmosomes are tonofilaments, which run throughout the cells and forms a cytoskeleton (Gartner and Hiatt 2001a) Cell regeneration often occurs at night, so mitotic figures are rarely seen here unless sa mpled during night hours. Once mitosis has been completed at this level, the new cells are for ced superficially and furt her compose the stratum spinosum. Stratum Spinosum. The layer directly superficial to the stratum basale is the stratum spinosum. This layer is compri sed of several layers of mitoti cally active cells. It is often referred to as the prickly or spiny layer due to its appearan ce from the spine-like projections formed from its keratinocytes (Marieb et al 2005 ) (that will eventually end as a desmosome). Most often this is the thickest layer in the epid ermis; in thick and hairy skin it ranges from 1-3 layers thick, and in glabrous skin it will be 4-5 layers thick (Samuelson 2007, Marsella 2005). The cells are polyhedral to fla ttened in shape, and contain cytoplasmic secretory units called

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31 membrane-coating granules, also known as La mellar granules or Odland bodies. These are produced in the golgi apparatus and act to house lipid (Gartner and Hiatt 2001a). The keratinocytes located in this layer have more bundles of tonofilaments, sometimes called tonofibrils. Stratum granulosum. The cellular layer directly superficial to the stratum spinosum is the stratum granulosum, or granular layer. Th is layer has 3-5 layers of cells containing basophilic keratohyalin granules, which form keratin in the highe r strata (Marieb et al 2005). Keratohyalin granules consist of pr ofilaggrin, a precursor to filaggri n. Filaggrin is a protein that is responsible for the aggregation of keratin th at leads to subsequent cornification (Samuelson 2007). The flattened keratinocytes still possess a nucleus which they will lose in the next layer. The granular cells located in this layer have increased amounts of lamellar granules (Samuelson 2007). Membrane-coating granules (Odl and bodies) are present in the cells also. These granules are released by exocytosis and fo rm a lipid-rich material into the extracellular space that acts as a waterproof barrier (Gartner and Hiatt 2001a). The stratum granulosum is the last layer that remains alive, since the oldest cells of this layer undergo nuclei degeneration (Samuelson 2007). The death of these cells is a re sult of their distance from the dermal capillary bed where they obtain their nourishment. As they travel further from the vasculature, nutrients do not diffuse as easily across the cell layers and oxygen cannot reach the avascular epidermis. Oxygen is essential in cellular viability. Stratum Lucidum. Glabrous skin is seen to occu r in conjunction with thick skin throughout the body. In areas of thick skin, the epidermis and dermis are considerably larger than in other areas of the body (Atlee et al 1997). Thin skin only has four main epidermal layers; thick skin has an additional integumentary layer, the stratum lucidum. The stratum lucidum is

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32 only present in the body in areas that are subjected to friction (Marse lla 2005). This layer is thin and usually stains clear, which al so gives it the name the clear la yer (Marsella 2005). The cells here have no nuclei or organelles; however they possess a substance known as eleidin. Eleidin is an acidophilic lifeless substance th at is deposited in the form of granules (Gartner and Hiatt 2001a). The cells of this layer have a thickened appearance due to the deposition of the protein involucrin, which forms a portion of the cell envelope. Stratum Corneum. The most superficial layer of the epidermis is the stratum corneum, or the horny layer and is generally thicker in thick skin than thin. This layer is composed of flattened, keratin-containing dead cells (also know n as squames) that have a thick plasmalemma (or cell membrane) (Gartner and Hiatt 2001a). Th e deeper cells (squames) in this layer still contain desmosomes, but the superficial cells will lose theirs and become desquamated, or sloughed off. The keratin and thickened plasmale mma act to protect the skin (Marieb et al 2005). Some areas throughout the body have varying degrees of keratinization; for instance, additional keratin layers are found over the stratum corneum in places where more protection is needed (esophagus, av ian gizzard, etc.) Epidermal cell types Cell types of the epidermis are divided into two main groups: keratinocytes and nonkeratinocytes. Keratinocytes. Keratinocytes m ake up roughly 90% of the epidermis and its layers. They originate from ectoderm within the stratum basal layer of the epidermis and migrate superficially. They exhibit different characteri stics upon this migration in relation to the layer they reside in. Upon differentia tion, they eventually reach the most superficial layer and are shed. Upon migration and cytomorphosis, they accu mulate keratin filaments in their cytoplasm.

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33 The keratinocytes cytomorphosis ev entually results in the five di stinct layers of the epidermis (Gartner and Hiatt 2001a). Keratinocytes contain keratohya lin granules, which include am ino acids, histidine, cystine and profilaggrin. Profillaggrin is a precursor to filaggrin, which causes keratin filaments to aggregate in the process of co rnification (Marsella 2005). Kera tohyalin is the precursor to keratin. Keratin is a tough, fibr ous protein which gives the epid ermis is protective capabilities (Marieb et al 2005). In addition to producing keratin, keratinocytes produce antibodies and enzymes that detoxify harmful products that may enter our skin (Marieb et al 2005). They have been implicated in the production of certain cytokine s, for example, Interl eukin-1 (Marsella 2005), that acts as a growth promoter of cel ls in the skin (Witter et al 2001). Nonkeratinocytes. The other cells located within the epidermis are termed nonkeratinocytes. These cells migrate in from the dermis or the central nervous system, and include Langerhans cells, melanocytes, and Merkel cells. Langerhans cells, also known as dendritic cells, are antigen-presenting cells located in the stratum spinosum. They travel to this layer vi a the blood from their orig ination in bone marrow (Marsella 2005). Their main function is to use receptor-mediated endocytos is to ingest foreign particles that may have found their way into the epidermis (Marieb et al 2005). They have both antibody (Fc) and Complement (C3) receptors attached to their cell membranes (Samuelson 2007). The resulting product of the endocytosis is the formation of membrane-bound Birbeck granules, or vermiform granules. They then travel to a nearby lymph node to begin the process of epitope presentation to T cells (Samuelson 2007).

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34 Characteristically, Langerha ns cells contain a dense, polymorphous nucleus, pale cytoplasm, and long dendrites (Gartner and Hia tt 2001a), while they lack tonofilaments. These cells can be easily damaged by UV light, which may result in some forms of skin cancer (Tortora and Grabowski 1996). Also known as melanin-forming cells, melanocytes are located deep in the epidermis in the stratum basal layer (Samuelson 2007) and sometimes located within the dermis in primates (Marsella 2005). Derived from neural crest, they produce a melanin pigment which gives them their characteristic brown colora tion. These round to column ar cells have long processes (dendrites) that extend from the cell and penetr ates the intercellular spaces of the stratum spinosum (Gartner and Hiatt 2001a). The conversion of a melanin precursor to melanin involves a cascade of events. Tyrosinase is an enzyme that catalyzes the oxidation of phenols and is produced within the rough endoplasmic reticulum of melanocytes. Here it is packaged into oval granules called melanosomes. Tyrosinase converts tyrosine into melanin once activated by UV light. Tyrosinase transforms tyrosine into dihydr oxyphenylalanine (DOPA) and then converts DOPA into dopaquinone, then into melanin. Melanosomes leave the cell body and travel to the tips of their processes; here it penetrat es the cytoplasm of the stratum sp inosum cells (Gartner and Hiatt 2001a). The tips of melanocytes pinch off and de posit the melanosomes into the cells to form a cap over the nucleus (Samuelson 2007). This cap over the nucleus protects the cells DNA from UV damage. One separate melanocyte will serve a number of surrounding keratinocytes; this is otherwise known as an epidermal-mela nin unit (Gartner and Hiatt 2001a). Melanocytes are sometimes found to reside in the dermis; in most species this is viewed as a pathologic finding, but in some species it is norma l. Color difference in an organism is due to

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35 the melanogenic activity of these cells (Samuelson 2007) coupled with secretions of a hormone known as MSH (melanin-stimulating hormone) secreted by the pituitary gland. Melanocyte population increases with increased exposure to UV light (Gartner and Hiatt 2001a). Merkel cells are found scattered among the st ratum basale layer. They are commonly found singly, extending their processes between kera tinocytes and ultimately attaching to them via desmosomes (Gartner and Hiatt 2001a). Merk el cells have close junctions with the nerve endings of Merkel discs to create mechanorecepto rs and relay the sensation of touch (Tortora and Grabowski 1996). Merkel cells characteristica lly have an indented nucleus and contain cytokeratins (Gartner and Hiatt 2001a). In addition to the keratinocytes and nonkerati nocytes, certain cells may also be found to occur within the epidermis. Neutrophils, eosinophils, lymphocytes, and red blood cells may at times be visible in this layer of the integument. Most of these, however are usually present in response to disease and are located ther e due to exocytosis (Marsella 2005). Dermis The derm is, or corium, is the second por tion that composes the main integumentary layers. This dermal area is composed of connective tissue with various fibers that will comprise its background matrix. The main fi ber types of the dermis are elas tic fibers and collagen fibers. Elastic fibers allow the dermis to stretc h, while collagenous fibers allow for strength (Weissengruber et al 2006). The dermis is composed of a dense irregular connective tissue that is strictly derived from mesoderm. It has a rich supply of nerve fi bers, often referred to as corpuscles. The vascularization is abundant, for it needs to suppl y not only itself but the above epidermis. The deepest layer of the dermis (which may or may not be in contact with the underlying hypodermis) is called the reticular layer, which accounts for roughly 20% of the dermis (Marieb

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36 et al 2005). The layer superficial to the reticular layer is called the papillary layer, and is anchored to the stratum basale layer of the ep idermis via the basement membrane. This layer composes almost 80% of the total dermal layers. In haired skin, they are called the superficial and deep dermis (Samuelson 2007). Papillary Layer. This superficial, loose portion of the dermis is separated from the above epidermis by a basement membrane. Collagen type III fibers (reticular fibers) and elastic fibers primarily populate this layer (Gar tner and Hiatt 2001a). Collagen type IV fibers may also be present, and act mainly to extend from the basa l lamina of the above basement membrane into the papillary layer to an chor the epidermis to the dermis. Hemidesmosomes found anchoring to the basal lamina are attached by anchoring fila ments also known as lamina lucida (Samuelson 2007). The papillary layer of the dermis contains capillary loops that play a role in thermoregulation and nourishment to both the derm is and epidermis (Gartner and Hiatt 2001a). A thick basal lamina and an increase in hemide smosomes present in this layer additionally provide a thermoregulatory role (S amuelson 2007). Innervation that is found in this layer is in the form of branching nerve endings (Meissner cor puscles) that provide tactile-type stimulation. Reticular Layer. This layer deep to the papillary layer is composed of a dense, irregular collagenous tissue constituting thick t ype I collagen fibers. These fibe rs run parallel to the skins surface. Animals with thick skin have well form ed reticular layers of the dermis (Samuelosn 2007). Although collagen fibers are predominant here elastic fibers are al so present. Elastic fibers will only be found in abundance in this layer in abnormal formations, known as elastosis (Samuelson 2007). The reticular layer is not name d because of the presence of reticular fibers,

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37 but due to its networks of collagen fibers (ret iculum = network). Th is layer anchors to a hypodermis, deep fascia, or muscle. Sweat glands, sebaceous glands, and hair follic les predominate in this layer (Gartner and Hiatt 2001a). Smooth muscle cells in the from of arrector pili muscles are found lining the outer region of hair follicles. Innervation to this layer includes encapsulated Pacinian (lamellar) corpuscles that are involved mainly with pressure and vibration, and Ruffini corpuscles that are involved mainly in tensile for ces (Gartner and Hiatt 2001a). Dermal cell types Cell types found in the papillary layer of th e derm is include fibroblasts, macrophages, plasma cells and mast cells. In the reticular layer of the dermis, fibroblasts, mast cells, lymphocytes, macrophages, plasma cells and adipose cells are visualized. Fibroblasts. Fibroblasts are the most common cell type of connective tissue in animals. They function in synthesizing a nd maintaining the extracellular ma trix of tissues. They are derived from mesenchyme and prov ide the structural framework for tissues. They secrete the precursors to the extracellular matrix, primarily ground substance and fibers. In skin, they synthesize collagen, reticular and elastic fibers that are located within the dermis (Marsella 2005). Fibroblasts additionall y produce fibronectin. Mast Cells. Mast cells are the resident cell type of areolar conn ective tissue. These cells contain granules rich in histamin e and heparin. They additionally play a role in immunity, being involved in wound healing and pathogen defe nse by binding to antigen (IgE). Lymphocytes. Lymphocytes are a type of white blood cell that is actively involved in immunity. These leukocytes do not function in the bloodstream, but are a primary immune cell of connective tissue (Marieb 2005). There are three types of lym phocytes: natural killer (NK) cells, T lymphocytes and B lymphocytes.

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38 Macrophages. Macrophages are cells that originate fr om monocytes, a specific type of white blood cell. They start their formation as histiocytes and undergo a formation that will direct them to become monocytes. Once act ivated within a tissue, monocytes become macrophages. They have an active role in immunity and are considered phagocytes since they possess the ability to engulf pathogens. Plasma Cells. Plasma cells are involved in immuni ty and have the ability to secrete antibodies. They differentiate from B cells upon antigenic stimulation by CD4+ lymphocytes. Adipose cells. Adipose cells act mainly to store ener gy in the body in the form of fat. Collectively, a group of adipose cells (also known as adipocytes) is referred to as adipose tissue. There are two main types of adipose tissue: wh ite adipose tissue and brown adipose tissue. Vascularization of the Dermis. The dermis relies on many blood ve ssels and vascular plexuses to supply the skin with its nutrients, immune cells, thermo regulatory capabilities, and the exchange of respiratory gases. Most of th e vessels that supply the skin come from musculocutaneous arteries (Mar sella 2005). Microvasculature ple xuses are located frequently in this layer. The two vascular plexuses found in the dermis are referred to as the deep cutaneous plexus and the superficial subpapillary plexus (Marieb et al 2005). In animals that possess dermal papilla, each of these structures possess es one capillary loop that contains a singular arterial and venous vessel; th ese are commonly involved in arteriovenous anastomeses (Marieb et al 2005). The dilati on of this loop plays an active role in thermoregulation. Changes in the amount of blood that flow through these loops help control heat loss thro ugh the skin. These capillary loops also provide the epider mis with the nutrients it requires Development of the Integument In the f ully developed animal, the integument has an important function: to protect the animal from its outside environment. In th e developing animal, the main function of the

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39 integumentary system is to form a protectiv e covering on the surface of the embryo (Carlson 1996); this will aid in the transition from an amni otic lifestyle through th e process of birth and finally subjection to an external environmen t. The development and formation of the integumentary system is a complex process invol ving many interactions be tween cells and tissue layers of the developing fetus. The embryological process varies w ithin the assorted Phyla of the Kingdom Animalia. Developmental processes al so differ within the derivatives of the integument, such as hair, feathers, and scales. In reptiles, a completely formed integumentary system is vital to life. From the moment they hatch from their amniotic egg, reptiles ar e presented with a dangerous environment from which they are shielded from by their integument. Their characteristic scaled skin makes them different from other amniotes. The process of re ptilian and in particular crocodilian integument and scale formation will be discussed further in the following sections. Comparisons will also be made to other species of animals. Once the embryo undergoes gastrulation, germ la yers conform into a three-dimensional Cshaped body (Sweeny 1998). This results in the three main germ layers present in the embryo, which are the ectoderm, endoderm, a nd mesoderm. Germ layers are collections of cells in a forming embryo that develop at the gastrulation stage during embr yogenesis. These germ layers will proceed to develop into the various organs of the animals body during organogenesis (Carlson 1996). Certain germ layers give rise to specific tissue types; in th e formation of the integument the germ layers that are involved are the ectoderm and the mesoderm (endoderm excluding). Ectoderm is the external layer of cells of the gastrula that develops into the superficial layer of the skin, the epidermis (Carlson 1998, M oore and Persand 1993, Maderson 1985). The

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40 mesodermal germ cell layer normally lies between the ectoderm and endoderm in other organs, but in skin development abuts the ectoderm. The pa raxial mesoderm in particular will eventually form the dermal skin layers (Moore a nd Persand 1993, Flaxman and Maderson 1976, Gilbert 2003) which progress from dermatomes in the dorsal body region and from mesenchyme in the remaining body areas. Endoderm is the internal laye r of cells of the gastru la that will develop into the alimentary canal ; it is absent in integument embryogenesis. The early embryo is also covered by a single layer of ectode rmal cells with a distant under growth of mesenchymal cells (Carlson 1996, Flaxman and Maderson 1976, Byrne et al 2003). This area then becomes a bilayered periderm, and eventually differentia tes into a three-layered epidermis. One of the most important events that occu r within the developing integument is the process of cell to cell communica tion, which occurs in response to cell adhesion properties between cells. Tissue and organ formation ar e induced by the action of cell proliferation, extracellular matrix, cell death, cell-cell adhesion, mi gration, polarization, and differentiation (Vleminckx and Kemler 1999, Sawyer 1990). The body s ability to generate signals within a certain cellular population and respond to those si gnals from within other cellular populations is essential in development. Most of these interactions take place within gap junctions between the membranes of two adjoining cells (Carlson 1996a). The properties of the cell surface play an important role in embryonic development. Important features of this phenom enon include selective affinity, histotypic aggregation, and the differential adhesion hypothesis. Cadherins (or desmosomes) play a role in cell to cell recognition, and are divided into three distinct groups; E-cadherins (embryonic), P-cadherins (trophoblast), and N-cadherins (mesodermal). Th ese cell adhesion properties are present in all cells. Cell adhesion molecules play an important role in skin development. They contain long

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41 cytoplasmic extensions that interact with th e cytoskeleton and link cells with either the extracellular matrix or with each other (Vleminckx and Kemler 1999). These transmembrane molecules are essential in tissue formation of the developing fetus. Certain cells within the developing dermis and epidermis will communicate with one another through cytoplasmic extens ions or through the extracellula r matrix that resides between them. The basal lamina (the deepest layer of the epidermis) is a thin sheet-like form of extracellular matrix (ECM) (Car lson 1996a). Extracellular matr ix proteins are a prominent component of all developing systems within the animals formation. ECM mediates certain cellcell communications that will lead to cellular migrations (Carlson 1996a). Examples of ECM components include collagen, tenasc in, and fibronectin (Carlson 1996c). In the integument there are a series of r eciprocal communications between the ectoderm and mesenchyme that lead the way to the continua l development of both of these layers into their respective adult counterparts. There are two types of epithelial-mesenchymal interactions; regional and genetic. The cells of these two la yers and their communication with each other is essential in their development; if ectoderm or me soderm are grown in isolation, then they never develop into epidermis and dermis (Carlson 199 6c). Along with being a regulator of skin development, the ectodermal-mesenchymal communications will also lead in the development and migration of epidermal contained structures such as glands, hair follicles, nerves, blood vessels, etc. (Byrne et al 2003). It is said that epithelium and mesenchyme are inducers and targets of each other (Obinata et al. 2002). Inductive events play significant roles in th e recognition between cells in development. These events represent inducer and responder cells that will play off one another in an effort toward final development. Inductive events may include epithelial-mesenc hymal interactions or

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42 paracrine factors. The signals between the inducer and responder are transmitted in one of two ways; either by juxtacrine interactions (which work between two adjoining cells) or by paracrine interactions (when proteins diffu se over small distances). Paracrine factors can be grouped into four families: the FGF family, the hedge hog family, the Wnt family, and the TGFsuperfamily (Gilbert 2003). The formation of the separate germ layers in the embryo directly re lates to some of the major events that occur at the genetic level (Swe eny 1998). Cells that resi de within these layers go through a series of inductive signal to commit to certain cell and tissu e types. Epithelial derivatives, such as hair, feathers, and scales, form in specific locations from the ectoderm in response to some of the specia lized inductive signals that aris e from the underlying mesoderm. Signals are constantly being generated, transmitted and received to dictate what molecular process will be acted upon (Carlson 1996a). Growth factors will play a role in skin mo rphogenesis within a developing individual. Fibroblast growth factors (FGF) ar e important players in the epith elial-mesenchymal interactions of birds and have also been shown to have an impact on the devel opment of their skin appendages (Fuchs 2007, Widelit et al 1996). Afte r the initial formation of a single layer of neuroectoderm, Wnt signaling will depress FG Fs and allow the cells to express bone morphogenetic proteins (BMP). This series of even ts fate the cells to develop into an epidermis with a periderm overlay (Fuchs 2007). Vertebrate skin appendages appear to va ry morphologically; howev er, developmentally they share very similar pathways that lead to formation. Appendage formation is dictated by a change in ectodermal stem cells that is thought to be programmed by the underlying mesenchyme (Byrne et al 2003, Ob inata et al 2005). Inductive si gnals coming from the dermis

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43 initiate certain epidermal events which, in turn, th en initiate certain derm al events to occur. Scales, hair, feathers and teeth in all animals are all thought to be mediated by common signaling pathways such as Hedgehog, BMP, Notch, betacatenin and Wnt signaling pathways (Chuong et al 1996, Chuong et al 2000, Chuong et al 2003a). These will determine whether cells will have an ectodermal or neural fate. Scale formation in birds is marked by an ex pression of neural cell adhesion molecules (NCAM) and sonic hedgehog (SHH) levels (Chuong et al 2000). Unfortunately at this time, very little is known about inductive events and molecular signaling in reptiles (Alibardi pe rsonal communication). Since bi rd integumentary formation is similar to alligators, and since th ey develop scutate scales that re semble reptilian scales, it can be presumed that events that occur within birds will be shown in the future to occur similarly in reptiles. Researchers have recen tly revealed the first cloning a nd gene structure of crocodilian beta-keratins, which shows their amazing similarity to avian scale and feather keratins (Alibardi, personal communication). Processes in Reptilian Development Em bryonic development of reptilian integument is often visualized as being similar over the various body regions, however variation lies in scale formation and the various stages of development (Maderson 1985). The epidermis is ectodermally derived, a nd its cells lie in a parallel orientation to the body surface at the ti me of development. Epidermal cells become cuboidal with a large nucleus and lie on the basement membra ne, the deepest portion of the epidermis. The epidermis eventually gives rise to a squamous-shaped pe riderm. The periderm forms over the epidermis and allows the epithelium to keep pace with the deeper basal germinal epithelium that is rapidly pro liferating (Alibardi 1996). Epider mal germinal cells then morph into a columnar shape, and cell division leads to the formation of the stratum corneum (beneath the periderm layer at the superficial surface).

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44 The dermis, derived from mesenchymal ce lls, shows no organizational patterns with relation to the epidermis. During development of the skin, the mesenchyme originates from mesoderm and migrates toward the surface of the body where it will meet with the epidermal anlagen (Chuong et al 2003a); the mesenchymal cells will then signal the epidermis to form skin and skin appendages. The mesenchymal cells become rounded and densely packed among each other. Dermal structure present under the epider mis also varies according to the region; this has been shown to influence the types of skin append ages that are produced, such as scales, hairs or feathers (Alibardi 1998). In the hinge region of crocodilians, there is also a close contact between the dermis and the epidermis; this clos e contact is not seen in developing areas that occur deep to undulations that will eventu ally develop into th e animals scales. Scale differentiation begins in the neck region and proceeds ventrally and caudally throughout the animal. The reptilian scale form s shortly after the formation of a primordial epidermis and dermis (Maderson 1985). The body surface will st art development into what appears to be wavy contours (undulations) and w ill accumulate more cellular processes in the presumptive stratum corneum. This cellular aggregation will not be as pronounced in the hinge regions of the animal. Crocodilian Development Crocodilian development almost fully occurs wi thin the egg during the post-laying stages. The developmental processes are easily categor ized by placing them into a post-laying embryonic development stages; these begin at st age 1 and continue until hatch, which usually occurs between stages 25-28 (Ferguson 1985, Fer guson 1987). A summary of these stages and their corresponding days in developm ent is located at the end of this chapter (Table 1-1). For the purpose of this dissertation, the numbered em bryonic stages (ES) will be used, not the morphological age. In addition, refe rence will be made according to the Alligator

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45 mississippiensis stages. Crocodile developmental stages will differ because their hatch occurs at ninety days, whereas alligator incubation time is sixty days. Gross morphology. The epidermis starts as a loose ep ithelium at ES13 and develops into the rigid epidermis by ES25. At ES13 through ES16, the integument of alli gators is smooth and contains no pigmentation (which will not start until ES21 and will be complete by stage ES23). At ES18, small undulations are visualized and it is evident where scale formation will eventually occur (Alibardi and Thompson 2001) ES21 also marks the first appearance of recognizable scalation on the tail and the most dor sal aspect of the back. This area is where the first scales will eventually form. Scales are absent at this time from the gular, head, jaw, flank, distal limbs, and digit areas (Alibardi and Thompson 2001, Alibardi and Thompson 2000). By ES24, the body is almost fully scaled and pigmented, and w ill resemble that of a hatchling (Alibardi and Thompson 2001). At ES25 (hatch) scale formati on is complete and no further integumentary development occurs. Microscopic observations. Development of the integument is studied from ES13 onward due to its incomplete formation prior to this stag e. At ES13, the epidermis consists of two layers of flattened cells with varying de grees of intercellular spaces that separate them (Alibardi and Thompson 2001) along with a periderm superficiall y. The periderm will ev entually differentiate into a primary and a secondary periderm (Sawyer and Knapp 2003). This layer has been shown to contain coarse filament and reticulate bodies, which are both associated with mucus and lipid substances (Alibardi 2002b). Reticulate bodies ha ve also been shown to be associated with desmosomes and in mammals have been considered an embryonic form of keratohyalin (Alibardi and Thompson 1999b). At some areas of the animal (head), there still remains only a single layer of ectoderm. The basement membrane is discontinuous and the dermal mesenchyme is

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46 loose. There is no evidence of strong dermal-epi dermal communication betwee n the cells, as this will appear later in development. ES16 shows no signs of melanogenic cells, as co rrelates with gross observations at this stage. Basal cells are still irregular with the same broad extracellular spaces present as in the previous stages. Mesenchymal arms are beginni ng formation under the epidermis; however, the dermal-epidermal contacts still remain sparse. At ES18 the basal cells become more column ar and the epidermis is thickening with progression. Mitoses are commonl y seen within the flat areas of epidermal formation, which will foreshadow the development of the first scal es within this animal (Alibardi and Thompson 2001). Small areas of glycogen formation are starting to appear, along with the presence of sparse melanogenic cells. Glycogen is an essential molecule that is needed for cell proliferation to occur. The cytoplasm contains the usual ce ll organelles with lipid droplets occasionally observed (Alibardi and Thompson 2 001). Micropinocytic vesicles are present in the cytoplasm, but no hemidesmosomes are seen. Intracellular spaces are broad and small projections are seen between basal and peridermal cells. Desmosom es are common between the peridermal cells only. The lamina densa is apparent in the baseme nt membrane of the epidermis, and the dermis is still homogeneously mesenchymal at this time (Alibardi and Thompson 2001, Alibardi and Thompson 2000). Dermal-epiderm al anchoring complexes are b ecoming more apparent, and are usually more frequent within the undulations rather than the hinge region of the scutes. At ES 19-20, the epidermis is still visualized as being bilayered. Th e basal cells of the epidermis are becoming columnar and the presence of glycogen is readily vi sible, especially in the undulated areas where scale formation is be ginning to occur. The dorsal, caudal and trunk regions have prominent scale formation and taller cells are accumulating within the undulations

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47 where the outer scales will subsequently deve lop (Alibardi and Thompson 2001). Small bundles of intermediate filaments are visible in the suprab asal cells that are present in the epidermis, and desmosomes are common between the epidermal and peridermal cells. The dermis at this point starts to form two layers (superficial dense and deep reticular) in most body regions. Collagen fibrils start to appear in the superficial dermis in the tail, belly, and areas of the back (Alibardi and Thompson 2000). In other areas of the body the dermis remains mesenchymal with no separation of layers. ES21 marks an increase in glycogen distribut ion among the cells of the periderm and suprabasal cells. In the future hinge regions, glycogen will be pronounced but will disappear in some areas of the body upon further development. Keratin bundles are seen more frequently in the basal cells, and hemidesmosom es are still rare. The dermis is now comprised of three layers in certain areas of the body (A libardi and Thompson 2001). The three layers of the dermis are the loose superficial dermis, intermediate dens e dermis, and a loose-re ticular dense subdermis (subcutis) (Alibardi and Thompson 2001, Alibardi and Thompson 2000). Chromatophores start to appear within the dermal and epidermal areas. Mesenchymal fibroblasts will contact the basement membrane, as their processes are l ong and continue development. Collagen and reticular fibrils also start to contact the epidermis at this stage. A flat subperidermal layer is sometimes noted at ES22 and will be located superficial to the suprabasal layer in the tail, dorsal and ve ntral regions of the thorax and the neck. Melanogenic cells are still not prominent at this stage in development. Intracellular spaces become reduced, hemidesmosomes become ap parent, and glycogen distribution increases particularly in the outer scale surface. This is the first stage at which the peridermis will begin to

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48 flake off. Collagen fibrils are more numerous in the dermis, a nd more mesenchymal fibroblasts contact layers of the epidermi s (Alibardi and Thompson 2000). At ES 23, the forming scales are composed of columnar cells, while the hinge region will be populated by cuboidal cells. Peridermal cells start to become extremely flattened and will continue to flake off (Alibardi and Thompson 2001). The thickness of the epidermis at this time depends on the body region of association. There are two remarkable events that occur at ES23. The first is the appearance of -keratin deep to the subperiderm is, primarily in the back, neck, tail, and ventral scales. The accumulation of glycoge n has a direct correlati on to the presence of keratin, for this precedes -keratin production (Alibardi and Thompson 2001). Glycogen is now prominent in the basal and suprab asal layers, but sparse in the hinge regions and the peridermis, the digits and the head (Alibardi and Thompson 2001). The second is the appearance of pigment cells in the epidermis along various regions in the animal. The subperidermal cells contain coarse filaments and will aggregate into reticulate bodies. Also at this stage, the dermis is almost completely differentiated. In certain areas, dermal tissue will follow the upbending of the epidermis into the core of the scale (Alibardi and Thompson 2000). This does not include all layers of the dermis, and will only have a sl ight invagination dependi ng on scale type and location. Dermal-epidermal anchoring complexes are almost fully complete and functioning, but are still less numerous in the hi nge regions of the animal. ES24 is remarked by heavy pigmentation of the epidermis, and the animal will start to exhibit the banding pattern that is evident upon ha tching. Suprabasal layers are thickened along the center of the scale (three layers) and thinne r along the hinge regions (two layers). The superficial peridemis is now flattened and vac uolated more frequently with pyknotic nuclei (Alibardi and Thompson 2001). Me lanocytes are readily visible in the basal layers, and their

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49 arms extend upward through the suprabasal layers. -cells start formation over the pre-layer that is located beneath the subperiderm al layers (Alibardi and Thompson 2001). -keratin is seen as it was in ES23 and is continuing its development. Many desmosomes link the subperidermal layers, and the periderm becomes mo re detached than the previous stages. Large fibrils are apparent in the derm is, and chromatophores are usually lost from this area (Alibardi and Thompson 2000) due to the fact that th e dermis does not show much pigmentation. ES25 is the last stage that is normally studied ; this is due to the subsequent hatching of alligators at this stage. Norm al incubation times usually termin ate at day 60, but alligators will hatch anywhere from day 50 until day 70. Pigmen t, melanocytes, and glycogen are all apparent in the epidermis (Alibardi and Thompson 2001). Gl ycogen is now confined to the basal layers, which appear conformally cuboidal. Dermal melanocytes are less abundant than epidermal populations, but are present nonethele ss (Alibardi and Thompson 2001). Scale formation. Embryogenesis in reptilians culminat es with the generation of a scaled integument (Lillywhite and Maderson 1982). The dor sal and ventral scales of crocodilians and birds are mainly formed of -keratin, while the hinge region is comprised mainly of -keratin (Maderson 1972, Chuong et al 2003b). Squamate scal es develop first as an epidermal projection from the body. As the scale continues development, the superficia l layer becomes hardened with beta-keratin and the dermal layer recedes. A charac teristic of crocodilian skin is that it does not overlap as seen in many other reptilian species. Scales begin as a local thickening of the epidermis with the development of bony plates, or scutes, beneath the surface on certain areas of the body. Fish also possess scales, but these are de rmal in origin, whereas in reptiles they are epidermal in origin.

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50 In alligators, the transforma tion of the skin to produce epidermal scales takes place between ES19 and ES23, and the development of scales varies according to body regions along the animal (Alibardi and Thompson 2000). In the early stages (ES13), th e skin is smooth and starts to form epidermal undulations by stage ES 16. At ES18, initial scale formation is marked by an increase in undulations, alt hough these do not represent true scales due to the lack of a hinge regions between them. Starting at ES19, recognizable scalation is present only on the dorsal tail in addition to some areas of the dorsal thorax (Alibardi and Thompson 2000). Consequen tly, these areas of the integument are the toughest regions in the adult al ligator and have the largest scutes associated with them (Alibardi and Thompson 2002). At ES20, scale formation has spread to the neck and tail, and by ES21 developing alligat or scales are prominent on the head, neck, abdomen and back and have started to be visible in the flank re gion. By ES22, they are better defined and have easily identifiable shapes in some regions; plat e-like dorsal scutes, sma ll roundish flank scales, or quadrangular ventral scales (Alibardi and Thompson 2001). ES23 marks the first time that scale formation extends over most of the body areas, except for the distal limbs, digits, and some regions of the head. Asymmetrical true overlapping scales are formed, but overlapping is not extensive. By ES24, the body is almost completely pigmented and the claws are well developed. ES25 is the last studied embryonic stage. At this time, the animal is as close to its complete transformation and ready for hatching. Pigmentation and scalation are complete, and the only variable anatomical feature is the si ze of the yolk (Alibardi and Thompson 2000). At hatch the integument in fully formed a nd no additional development occurs.

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51 Post-hatching Crocodilian Integument At hatch, the epiderm is consists of one layer of basal cells, 3-6 cell layers in the suprabasal layers, 1-2 pseudostratified transitional or precorneous layers, and a thick variable stratum corneum (Alibardi and Toni 2006). The localization of melanosomes present within beta-cells of certain scutes will determine a crocodilians coloration pattern. Scales are separated by narrow hinge regions from other scal es and do not exhibit high degrees of overlapping (Figure 11). The hinge region of crocodilians exhibits a thinning of the precorneous keratinocytes beneath layers of na rrow corneocytes (Alibardi and Toni 2006). The decrease in the thickness of the epidermis a nd the thinner corneocyte s in the hinge region resemble (sebo)keratinocytes of avian apteri c epidermis (Alibardi and Toni 2006). During development the outer scale surface will expand into larger scutes, however this formation and its cellular mechanisms remain unknown. The shedding of the outer layer of the scal e is hypothesized to occur due to gradual environmental wearing that slowly consumes the co rneous layer of scutes. A continuous loss of superficial corneocytes persists (Alibardi and Toni 2006). There is a differences in integument characte ristics between dorsal and ventral regions in crocodilians (along with other species). The dorsal region tends to be thicker than ventral regions; susceptibility to injury in the dorsal region is key to th is variation. Pigmentation is different on the dorsal regions also, playing a role in camouflage. Comparisons Crocodilian integum ent formation resembles formation that is evident during embryonic stages in birds. The shedding of the peridermis prior to hatching is one of these similarities. Periderm will detach due to desmosome degradation between the periderm and the -keratin layer (Alibardi and Thompson 2002).

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52 In turtles, the plastron and carapace form from an epidermal placode, similar to how birds generate their feathers (Figure 1-3). This is no t seen in crocodilians. The anlagen epidermis is represented in the developing alligator by only one or two layers, which is similar to that in other lizards. In contrast, turtles embryonic epidermis is made of 45 layers (Alibardi and Thompson 2001). With regards to the number of cell-ce ll interactions, there are a small number of intercellular interactions in the early stages of development in th e alligator. This small number is also seen in avian and mammalian skin embryogene sis. Anchoring desmosomes are lost at ES23 in the alligator, and they subsequently lose their function as peridermal sloughing starts. An increase in the amount of glycog en prior to the formation of the -keratin layer is comparable to similar developments found in bi rds, lizards and snakes (Alibardi and Thompson 2001). Glycogen is associated commonly with lipids and lipids are the main source of energy in the formation of keratin, as has been describe d in turtles (Alibardi and Thompson 2001). In crocodilians, this layer functions to prevent wate r loss, whereas in other reptiles this layer is strictly mechanical. Maturation of the epidermis in alligators is a regional phenomenon; it spreads throughout the body and areas develop at different times. This has only been seen in rodents (Alibardi and Thompson 2001). Epidermal ma turation in crocodilians spreads from the dorsocaudal areas to the lateroventral areas (Alibardi and Thompson 2001). Mammal epidermal embryogenesis occurs in four main periods, and development occurs conformally at these stages. In all mammals that have been studied, their epidermis and dermis development undergoes a sequence of proliferation; these events occur equally over all pa rts of their body (Carlson 1996c). This differs greatly from crocodilians, who exhi bit varying degrees of formation throughout their body development.

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53 Superficial dermal fibroblasts in mammalian skin produce molecules (such as collagen type VII and cytokines) that play a role in the proliferation of the overlying epidermis (Alibardi and Thompson 2000). In crocodilians, these same cells have not been shown to produce these molecules. Unlike reptiles, snakes and turtles, crocodilians do not form dermal aggregations within the scale anlagen (Alibardi and Th ompson 2000, Alibardi and Toni 2005, Alibardi 2002c). At times, there may be slight aggregations of dermal mesenchymal cells in some areas, but does not happen at the rate or maturity as seen in other reptiles. It is suggested that in formation the dermis is pulled down and is evident upon microsc opic evaluation by the distortion of this layer around the hinge regions of the skin. Mammals fo rm epidermal ridges during their development, which reptilians lack (Moore and Persand 1993) The dermis remains mesenchymal under the outer scale surface as in lizards in turtles, and the reasons fo r this are unknown (Alibardi and Thompson 2002). Scale formation resembles other lepidosaurians; they occur at different rates in different body regions (Alibardi and Thompson 2000). The onl y major difference is that aquatic species scales have very minimal overlapping. This decr ease in the amount of overlapping scales is most likely related to the animals ability to thrive in an aquatic environment (swimming). In development, both feathers and scales are formed due to the interactions that occur between the epithelium and underlying mesenchyme (Chuong at al 2000). Feather/scale location and size are relative to the underlying mesenchyme, whereas the orientation is a result of the epithelium (Chuong et al 2000). Birds form their main scutate feathers from an epidermal placode, which is an aggregation of columnar ectodermal cells (Sawyer and Knapp 2003).

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54 It is hypothesized that at some poi nt reptiles and birds evolved the -keratin genes that are involved in the production of cl aws and scales (Sawyer and Kna pp 2003). Also, the ancestors of crocodilian and birds at some point acquired the ability to express the feather-type -keratin; this lead to the ability to form feathers or scales. Alligator tissues have b een shown to cross react with antibodies against -keratin in chick scales, which show s that they must share conserved epitopes (Alibardi and Thompson 2002). This additional factor links the evolution of crocodilians and birds to gether (Alibardi, personal communication). At hatch, snakes and lizards do not have a complete -keratin layer, whereas the -keratin in crocodilians is complete (F igure 1-2) (Alibardi and Toni 2005, Alibardi and Thompson 2002). Turtles possess -keratin in their neck and leg regions (Alibardi and Thompson 1999b, Alibardi and Dipietragelo 2005) where rigidity is not needed, but will have -keratin mainly located within their shells scutes (know n as a horizontal differential di stribution). The presence of keratin aids in an epidermal barrier that prev ents water-loss in the hatched animal. These -cells represent the future stratu m corneum (Alibardi and Thompson 2001) which is a major component in their ability to prev ent desiccation. This is opposite to lepidosaurians, which have a water barrier present in their meso-layer (Alibardi and T hompson 2001). The presence of lipid in -cells in alligators among the keratin networ k also aids in water proofing (Alibardi and Thompson 2002). Squamates possess overlapping scales over their entire body. -keratin and -keratin form layers, known as vertical disposition. The oberhautc hen layer is filled only after fusion with the -layer. They have six epidermal layers because of the cyclic modality of keratins. Another comparison between the species is the terminology of the layers that compose the epidermis, which will vary between species (Figure 1-1, Figure 1-2, Figure 1-3, Figure 1-4). In

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55 mammals they are commonly referred to as: stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and the stratum basa le. Lepidosaurian reptiles (lizards, snakes) have major differences compared to the archosauri ans (birds and crocodilia ns). In relation to terminology, there are six different epidermal laye rs termed superficial to deep: oberhautchen, beta, mesos region, alpha, lacunar, and clear la yer (Alibardi and Toni 2005). Crocodilians only possess four main layers to their epidermis (Table 1-2). The deepest layer is referred to as the stratum basal, much like the stratum basal in the mammal. Superficial to this, the next layer is termed the suprabasal, which is analogous to the mammalians stratum spinosum. Next, crocodilians have a precorneous or transitional layer, analogo us to the mammalians stratum granulosum layer. The most superficial layer in crocodilians is the corneous layer, which is so named in the mammal also (Alibardi 2007). Conclusions W hile there are many similarities between the gl abrous skin of mammals and reptiles, their differences are vast and play an important role in the skins physio logy in those species. Mainly, glabrous skin is thicker, richly innervated, a nd devoid of certain glands. Reptilian skin is generally thick in all areas, thicker on th e dorsal surface, and lacking many glands. In conclusion, comparisons among the reptiles vary accordingly. Chelonians exhibit a keratinization replacing -keratin in the scales. -keratin predominates in the shell, while keratin is present on the neck, legs and head. They shed their scutes one at a time, and shed their soft skin all at once. Squamates possess overl apping scales, with their obertuhautchen layer filling only after fusion with the -layer. -keratin and -keratin will form a vertical disposition, which produces a shedding complex th at facilitates molting. The presence of 6 layers is due to the cyclic modality of keratins within their layers. Finall y, crocodilians exhibit a phenomenon

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56 where -keratin replaces -keratin altogether. They shed only one scale at a time, and possess minimal overlapping between scales. Diagnostic Testing in the Id entification of Pathogens The diagnosis and identification of m icroorganisms located with in tissue is essential when disease processes are involved. When an animal is compromised with dis ease, one of the first processes involves performing dia gnostic tests for the detection of causation. Primary diagnosis is usually accomplished through culture isolat ion and morphological traits; however, these methods are commonly laborious and may take se veral weeks for growth to occur (Rodriguez 1997, Sterflinger et al 1998). Since some species may not be culturable on media, and contamination is commonly a problem, diagnosis based solely on culture techniques should be avoided when possible (Sterflinger et al 1998, Nardoni et al 2007, Jaco bson et al 2000). Diagnostic techniques used to determine the causation of disease vary depending on the presumptive causative agent and the clinical progre ssion of the disease. Pa nels are developed to test for and detect the presence of pathogens th at may be implicated in the outbreak of the disease. Significance will rely primarily on test s that have been prove n through the ability to detect causation on numerous occasions. The utility of the various tests will be discussed, along with their disadvantages. Visual Identification The first tool em ployed by researchers when id entifying causation of disease in an animal is gross evaluation, commonly referred to as a presumptive diagnosis, or outbreak observation. This entails the examination of the whole animal prior to death or n ecropsy to identify gross characteristic flaws that may be indicative of certain diseases. Clinical symptoms, abnormal behaviors, uncharacteristic lesi ons or discharge may be indicator s of certain disease processes that occur in that species of animals.

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57 Upon the necropsy of animals, other important disease characteristics can be noted. Abnormal bleeding, inflammation, tumors, and sk in pathologies can be visualized through careful dissection. For animals s acrificed for their hides, placi ng the skin over a high intensity lamp may show imperfections. Advantages to this procedure is that valuable information will be learned from an animal that may have not expired yet; for example, head tilting and neurological signs when an animal is still alive may lead a researcher to examine for West Nile Virus. The disadvantages with using this tool are usually due to visual errors since some differential diagnoses are not easily discerned from one another. Further diagnostic tests are additionally needed. Environmental Testing Environm ental testing is a ke y factor in identifying pat hogenic organisms. Employing culture techniques in a laborator y setting may reveal microorganism s located within the diseased animals environment. If an animal is kept in an aquatic environment, water testing is essential (McCoy and Seidler 1973) in determ ining potential infectious agents that may be thriving in the location. Soil samples, swabs of the animals pe ns, and air testing are all common techniques in identifying the pathogen count of an enclosure and surroundings. As this technique has the adva ntage of culturing potential viable pathogens, contamination is a major disadvantage and will commonly overc ome any results. Many organisms that may culture are considered normal flora and are not indicative of disease. Microbiology Testing tissue for the presence of m i croorganisms is commonly done through microbiological techniques. Culture is the only diagnostic test available that will detect completely viable organisms (Lane 2003). My cology, bacteriology, virology, and parasitology are all performed as part of a routine microbiological panel.

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58 As microbiology is considered the gold stan dard in the identific ation of a viable organism, it also has limitations. Contamina tion may be a problem when working with infectious organisms. This is kept to a minimum by using varying concentrations of antibacterial and antifungal ag ents added to the agar to inhibit the grow th of other microorganisms. Another limitation is there may only be a slight amount of pathogen present in the tissue sample submitted. This coupled with prolong incubation time required by some organisms may cause other fungi and bacteria to overcrowd the plat e and inhibit certain important microorganisms from be ing able to culture on media. Histology Histological techniques in th e iden tification of microorganisms located within animal tissues is an important tool used in diagnostic panels. The use of pathological examination has always been one of the ideal methods in determ ining disease progression microscopically. In order to examine tissues and their cells structur ally, tissue needs to be preserved and stained. This will show their extr acellular components by the use of colo r reactions to the stains and dyes used (Samuelson 2007). Histopathology is commonl y used when demonstrating the invasion of tissues by pathogenic organisms (Lane 2003). For the identification of normal cellular elements, a Haematoxylin & Eosin (H&E ) stain is used. Periodic acid-Schiff (PAS) stain will show nor mal continuity of tissue, and will sometimes detect the presence of fungal hyphae (Gartner and Hiatt 2001b). For the detection of microorganisms within a tissue sections, many va rious stains can be employed to show positive or negative reactions Bacteria are single-celled organisms that ar e able to metabolize, grow and reproduce within certain hosts. Bacteria take the form of cocci, bacilli, spirochetes, rickettsiae, chlamydiae and mycoplasmas (Carson 1997c). While most bact eria are not pathogenic, some are harmful to

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59 an organism. This makes species identification an important tool. A Giemsa stain is used for the detection of parasites and certain bacteria. A Ziehl-Neelsen (acid fast) stain is performed on sections to show the presence of acid-fast bac illi Acid fast organisms will show up red while non-acid fast bacteria will show up blue. The waxy coats on their cell walls will prevent them from taking dye up, hence, the co lor differential. A Brown and Brennan's (B&B) Gram stain is performed to show gram negative and gram positive bacteria. Other stains include the Fite acidfast for leprosy organisms and the Brown-Hops modification. Acid-fast stains allow the organism to be classified into acid-fast and non-acid-fast groups (Carson 1997b). Gram stains allow the microorganisms to be classified as gram positive or negative. Gram-positive bacteria have a thicker wall than gram-negative bacteria, and gram-negative bacteria also contain a layer of lipopolysaccharides exte rnal to the cell wall. In the cases of fungal invasion, a Grocott meth enamine-silver nitrate (GMS) fungal stain is the preferred staining method to identify such infections. Chromic aci d treated fungi possess aldehydes which will reduce the hexamine-silver mix to produce a black deposit; hence, fungi will show up black upon a green background. A GMS stains the fungus cell wall black, hence the dark characteristic of a positive tissue. Wh ile this is an excellent method to detect the presence and location of the microorganism within the tissue, it is almost impossible to identify the causation to the genus or species level (Nard oni et al 2007), and some fungi will not exhibit mycelia in tissue section (Almeida et al 2003). A Verhoeff-Van Giesons st ain is performed in conjunction with a GMS to show the presence of el astic fibers in order to distinguish them from fungal hyphae. Other pathogens can be visualized in tissu e through the use of histology. Viruses found within tissue either contain DNA or RNA coupled with a protein coat called a capsid (Carson

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60 1997b). Most viruses cannot be visualized w ithout electron microscopy; however, some will form inclusion bodies that may be seen with regular staining. Protoz oans are single-celled organisms that are classified depending on their primary means of locomotion. Parasites are often identified within tissue using normal staining techniques (H&E). Advantages of histology are the ability to visualize tissues an d their cells at a microscopic level. With the use of special stains, additi onal microscopic elements are visualized through reactions with the dyes used. The main disadvantage of histol ogy is the interpretation of the stains and their reactions. Depending on the pathologist or interpreter that may be reading the slides, some will have different opinions of what the re sults show. Care must also be taken to follow the directions exactly when performing some of the special stai ns used in this study. For example, the silver that is used for the GMS will commonly overs tain a section. The presence of undesirable precipitate may form, causing ar tifacts that may resemble pathogenic elements such as hyphae (Speranza and Fail 2005). Additionally, it is im possible to make an identification down to the species level if pathogens are present. Electron Microscopy While histo logy will show many of the cells a nd cellular components that are needed to identify a disease, there are some benefits to viewing tissue at a much higher resolution and magnification. The advantage to using an electron microscope allows for the evaluation of the topography, morphology and composition of the cells that are being examined (Gartner and Hiatt 2001b). Cellular organelles, cytoplasm structures, and nuclear elements can be seen. Many viruses are not discernable unless seen at the electron microscopy level. Limitations of electron microsc opy are numerous; first, a great deal of time is needed to examine a very minute piece of tissue. Electr on microscopes are very expensive to buy and

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61 maintain, so this would not be a very time and cost efficient method of diagnosis. The samples have to be prepared in ways that will give prope r detail, which may lead to artifacts that are the result of the treatments th at the tissue must go through in order to be preserved. Polymerase Chain Reaction When diagnosis by culture m ethods cannot be achieved, molecular methods can be employed to achieve the identifi cation of a pathogen. One of the most common identification methods for microorganisms is through molecula r diagnostic tools (Makimura et al 1994), primarily the polymerase chain reaction (PCR). P CR is a rapid way to detect the DNA of certain microorganisms that may be located within th e tissue of an animal (Wu et al 2003, Erlich 1989, Rodriguez 1997, Lischewski et al 1997, Lindsley et al 2001). It will allow for the potential analysis of many microorganisms of importance in a single technique (Rodriguez 1997, Kordick et al 1999), which makes this be neficial if minimal amounts of a sample are available (Ouenzar et al 1998). This is especially importa nt since small numbers of many pathogenic microorganisms cause infections and pathology. PCR is the process in which DNA is amplified re peatedly so as to become detectable in a sample of tissue. The utility of PCR in identi fying and detecting a pathogen in animal tissue is proven through its common use in molecular diagnostic testing. Laboratory tests of viruses ar e extremely slow and insens itive (Rodriguez 1997), whereas PCR allows for a rapid, extremely sensitive method for the identification of more than one viral infection found within an animal. Animal viral pathogens have been identified in a wide range of species. Viruses such as adenovirus, epizootic haemorrhagic disease virus, herpesvirus, and rabies are all examples of di seases that are commonly identif ied through the process of PCR (Rodriguez 1997, Stroop et al 2000, Schrenzel et al 2005).

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62 Rabies has been reported to occur in all of the contin ents throughout th e world (WHO, http://www.who.int/en/ ). W ith the use of P CR, this zoonotic pathogen can be detected quickly and efficiently to avoid spread. One study showed the reliability of PCR in diagnosis of rabies in animal brains and human saliva collected antemort em (Nadin-Davis et al 2007). Applications of such uses are widespread and beneficial, main ly in the prevention of further cases and the diagnosis of rabies in new areas. Bacterial diseases affecting animals face the sa me problems of identification as viruses, including prolonged incubation time a nd also contamination of culture media. Bacteria that are difficult to culture in vitro have been widely detected via PCR (Makin et al 1994). Bacterial pathogens such as staphylococcus, anthrax, and Escherichia coli have all been detected by PCR (Rodriguez 1997). A rickettsia-like bacterium has been identified through this molecular technique using extracted DNA from infected tissue as a template for primers (Nunan et al 2003). This was then confirmed using the am plicon and employing in situ hybridization. Mycobacteria commonly cause granuloma formation in animals, and are detected through the use of staining reagents (Ziehl-Neelsen). This does not accurately confirm the presence of a specific DNA sequence from an infectious microorganism, so PCR is needed to confirm this diagnosis. Mycobacteriosis in fresh water crocodiles ( Crocodylus johnstonii ) has been diagnosed through PCR with confirmation by gene sequencing (Soldati et al 2004). While histology will show only the presence of a micr oorganism type, PCR will confirm the specific species The identification of parasites within animal s has also made use of the polymerase chain reaction technique. Leishmaniasis, trypanosoma, toxoplasma, cryptosporidium, and trichinella

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63 are all caused by protozoa and have been detected and identified through the applications of PCR (Rodriguez 1997). Most fungal pathogens identified today have specific primer se ts designed for use so the application of PCR is as fast as it is reliable. In phyloge netic determination, ribosomal RNA genes (rDNA) located at chromosomal sites ar e popularly targeted within the fungus for identification (Guarro et al 1999). The intenti on is that rDNA (which codes for rRNA) is a multi-copy, non-protein-encoding gene Fungi contain an 80S ribosomes that consist of large (Jensen et al 2001) and small (Wu et al 2003) subunits. Each of these subunits contains proteins; the 60S contain the 25S to28S, 5.8S, and 5S rRNA molecules (Lopez et al 2007). The 40S contains the 18S rRNA molecule. By targeting ge nes that encode for ribosomes (present in all living organisms), this molecule is highly conserved and is used in evolutionary studies (Guarro et al 1999). Located between 18S and 28S are transcribed spacer regions, the in ternal and external transcribed spacers (ITS and ETS). The ITS region includes two spacer s, ITS-1 (located between 18S and 5.8S) and ITS-2 (located betw een 5.8S and 28S) that are separated by a 5.8S conserved region (Abliz et al 2003, Guarro et al 1999). The ITS regions are used due to the higher degree of variation than the small a nd large RNA subunits. Universal PCR primers designed from highly conserved regions flanking the ITS region will allow for the easy amplification of this area becau se it has a high copy number of r DNA repeats (Lopez et al 2007). This ITS region is now the most widely used sequenced DNA region that is found within fungi. Universal ITS1 and ITS4 PCR primers are used to broaden the range of det ectable fungi within a sample (Abliz et al 2003) and ha ve been regarded as being funga l-specific (Cubero et al 1999).

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64 Along with the universal ITS primers that ha ve been made, taxon specific primers have also been formulated to target selective amp lification of certain sequences located within a species. Designing oligonucleotide primer sets that are based on the sequences of ITS of rDNA will allow for the evaluation and detection of specific DNA sequences of fungal pathogens (Abliz et al 2003). Limitations of using PCR as a diagnostic tool vary. In some cases, the animals DNA will overpower the microorganisms DNA, rendering it unde tectable. In these instances, care must be taken to eliminate as much animal tissue as po ssible while preserving th e microorganisms DNA. Some pathogens have hard cell walls that must be disrupted prior to DNA isolations. When performing DNA isolations on pathogens such as fungi it is essential to di srupt the cell wall as efficiently as possible, as this may inhibit the use of PCR in detecting fungal pathogens (Lugert et al 2006). This is accomplishe d by grinding the sample in liqui d nitrogen to allow for the most consistent isolation (Griffin et al 2002, Cubero et al 1999). Cont amination may also prove to be a factor in PCR, so positive and negative controls should always be used at various stages in testing (DNA isolations, PCR, etc.). Reverse-Transcription-Polymera se Chain Reaction (RT-PCR) Reverse-transcription-polym erase chain reac tion (RT-PCR) is a molecular diagnostic technique that amplifies a specifi c segment of an RNA molecule. Certain culture techniques can be time consuming, and RT-PCR is a rapid and hi ghly sensitive technique (Steininger et al 2002). RT-PCR works by transcribing an R NA strand into a complementary DNA (cDNA) strand and then amplifying the re sults using PCR techniques. RT-PCR has been used in a variety of appli cations. Most commonly it is used in the detection of viruses. Pigeon paramyxovirus fi rst was seen in European pigeons and had developed as a variant to Newcastle disease virus. It was first reported in racing pigeons in

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65 1981, and has spread worldwide (Toro et al 2004, Zanetti et al 2000, and Barbezange and Jestin 2002). RT-PCR is successfully employed in the detection of th is fatal disease. Virology The use of cell cu ltures in microbial detecti on is a very useful and routine laboratory technique. Cell culture employs the maintenance of cells in a specialized medium after their removal from the body. Fibroblast cell culture monolayers have proven to be an extremely satisfactory system in which to grown and repl icate various diseases, most commonly viruses (Colwell et al 1974). By developi ng an undifferentiated cell line us ing fibroblasts, it is possible to synthesize and maintain the extracellular matrix of many animal tissues. Once fibroblasts have the chance to grow confluently, addition of samples (that contain a virus) would induce cytopathic e ffects (CPE). CPEs are defined as any damage to infected host cells that may be caused by the presence of infect ious viruses. Morphological changes in the host cell include altered shape, detachment from substrate, ly sis, membrane fusion, altered membrane permeability, inclusion bodies, and ap optosis (Colwell et al. 1974, Hunt and Brown 2005). Disadvantages include contamination, error, and no pathogen present. The use of some fibroblast cell lines have not b een proven, and some viruses do not cause CPEs. Also, media changes and cell splitting must be performed at essential times to ensure that the cells are confluent and nourished. Media changes will replenish nutrients and avoid build up of harmful byproducts as a result of dying and dead cells. Just as with PCR, positive and negative controls should be performed at various stages in diagnosis. Overall, the use of cell lines in the diagnosis of viral presence is very sensitive and useful.

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66 In Situ Hybridization While PCR can detect th e presence of DNA in a sample, one of the main disadvantages is the absence of locality with diagnosis. Some pathogens are cons idered secondary invaders; PCR can not distinguish between a primary and seco ndary invader since there is no way to show where the DNA that it is amplifying is located wi thin a tissue sample. PCR simply confirms or denies the existence of the DNA se quence in a submitted sample. In situ hybridization (ISH) is a rapid and sens itive molecular technique that allows for the detection of a targeted piece of DNA within a tissue (Samuelson 2007), while at the same time showing its specific location within the tissue sa mple. With the use of a nucleic acid labeled probe, the targeted DNA sequence is detected in situ (within tissue) Pathogens that may infect animal tissue can be visualized via in situ hybridization and be a useful diagnostic tool in the detection of infection. ISH uses processes of sp ecific annealing of labeled nucleic acid probes to complimentary sequences (Jin and Lloyd 1997). With the use of ISH for pathological diagnostics, infectious agents th at commonly affect animals are available to be identified down to the species level, while at the same time s howing locality within the tissue sample. Foreign genes that are commonly identified through the use of ISH are bacteria, fungi, and viruses (Jin and Lloyd 1997). One of the main reasons to perform in situ hybridization on a sample is maintaining the morphological relationship between the organism and the surroundi ng tissue. ISH will produce results that will determine if th e pathogen present is merely a contamination or the result of a true infection (Hayden et al 2001 ). Molecular based assays hol d many advantages to detecting pathogenic organisms. By using a probe specific to a species, this will allow the detection of various species within a sample of tissue (Jeyanathan et al 2006) if a mixed microorganism infection is suspected (Hayden et al 2003). The use of rRNA ta rgeted oligonucleotide probes is

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67 an increasingly valuable tool in the identification and location of specific microorganisms (Lischewski et al 1997). It is also useful for identification in the dimorphic fungi, since they are slow growing and require a prolonged iden tification time (Stockman et al 1993). Fungal probes have been designed to detect th e location of specific fungi located within tissue sections. The most common probes bind to and target a specific nucleotide region in the 18S rRNA, an ITS region that is specific for a fungus (Lischewski et al 1 997, Jensen et al 2001). A pan-fungal probe has been used against a common 18S rRNA sequence in all fungi (Hayden et al 2001, Haytden et al 2003). Us ing a probe to target common seque nces in all fungi is useful to determine if a fungus is even present in a tissue specimen (Abbott et al 2006, Brookman et al 2000). Some universal fungal primers commonly used are ITS1 and ITS4, which are directed at the conserved regions of rRNA. The rRNA gene is a common target of fungi because this area of the genome contains both unique and conser ved regions (Sandhu et al 1995, Lindsley et al 2001). Using probes that hybridize with species-spec ific sequences of DNA or RNA will provide the most accurate results when using ISH (Abbottt et al 2006). For example, when detecting the fungus Candida albicans within a tissue, oligonu cleotide O20 is used; this is a probe that is complimentary to a sequence within the 18S rRNA of this species (Lischewski et al 1997). Many dimorphic fungi have been identified within a tissue secti on through in situ hybridization. These fungi are sometimes easily conf used with each other, especially if conidial formation are similar between the species. Through ISH, specific DNA probes have been formulated and made readily available for thei r detection. AccuProbes are used in clinical microbiology to test for fungi such as Blastomyces dermatitidis Coccidiodies immitis, Cryptococcus neoformans, and Histoplasma capsulatum (Stockman et al 1993).

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68 Adenovirus infections are found in over 12 species of reptiles, a nd often results in death of the infected animal (Leigh Perkin s et al 2001). Fresh tissues ar e often unavailable, and viral testing can be long and tedious. DNA ISH with th e use of an adenovirusspecific oligoprobe has been routinely used to detect the presence or abse nce of this virus in animal tissue (Ramis et al 2000, Leigh Perkins et al 2001). It is important to use additional techniques with ISH to detect viruses, such as electron microscopy. In 1996, the sudden death of 72 Northern aplo mado falcons raised concerns as to the causative disease process. These animals are scarce in nature and causation was needed to be determined and to prevent further die-offs. Mol ecular analysis determined that a new species of adenovirus was the cause (Schrenzel et al 2005). ISH demonstrated that the virus was present, and its location within areas of inflammation in tissue sectio ns confirmed this diagnosis (Schrenzel et al 2005). Bacteria, such as Chlamydia have the potential to cause zo onotic infections in various species. Availability of rapid dia gnostic tests that work in various species is needed to detect this pathogen; ISH probes have been used for this purpose. The use of multiple probes (both universal and species-specific) will verify the utility of ISH in the identification of infections caused by chlamydiae (Chae et al 1999, Pollman et al 2005) in animal tissue. Rickettsia are gram-negative bacteria that grow within a cells cytoplasm, sometimes within the nucleus (Nunan et al 2003). Ri ckettsia infects a large population of aqua tic species, such as crabs, crayfish and prawns. The detection of this bacterial infection is important, especially if consumption of the affected species is a possibi lity. ISH has effectively identified rickettsia infections in these species of aquatic organisms (Nunan et al 2003) Utility was confirmed when the DNA probes designed failed to hybridize with other bacteria l infections.

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69 One of the main advantages (in addition to lo cation of a gene) is th at hundreds of samples can be made with a small amount of tissue. This allows for a large number of possible procedures to be performed even if samples are scarce (Wilcox 1993). Another advantage to performing in situ hybridizati on with a sample of tissue is maintaining the morphological relationship between the organism and the surrou nding tissue. ISH will produce results that will determine if the pathogen present is merely a contamination problem or if it is the result of a true infection (Hayden et al 2001). Histology is usually a very sensitive technique; however, identification down to the species level is often impossible. Molecular based assays hold many advantages to detecting pathogenic organisms. Using a specific probe specific will allow the detection of various species within a sample of tissue (Jeyanathan et al 2006). The use of rRNA targ eted oligonucleotide probes is an increasingly valuable tool in the identification and locati on of specific microorganisms (Lischweski et al 1997). ISH is limited where tissue necrosis may have taken place; this will degrade the rRNA signal and will not yield a strong positive reaction, if any at a ll (Abbott et al 2006). Necrotic tissue may interfere with nucleic acid hybridiza tion, and may even resu lt in the release of endogenous nucleases (Hayden et al 2003). ISH shoul d also be limited to cases where there is a positive histological diagnosis. Th e use of DNA eliminates possibi lities for error that may be caused by antigenic cross-reactivity or al tered binding sites as commonly seen in immunohistochemistry (IHC) (Chae et al 1999). In addition, the incor poration of appropriate controls minimizes the error that ma y be seen within this technique. Use of Antibody Reagents Using DNA to detect and locate a pathogen with in a sam ple places li ttle doubt as to the validity of the procedure. In addition to these diagnostic tools, there are methods to detect

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70 pathogens within a tissue using the binding prope rties of antibodies to antigens. In these techniques, commonly referred to as immunostaining, antibody-antige n binding is used to detect specific proteins (epitopes) in tissue samples. While polyclonal antibodies raised to specific antigens are sometimes used, monoclonal an tibodies are usually employed. Monoclonal antibodies are more specific and their production does not require the use of purified antigen. The main techniques that employ antibody reagen ts are immunohistochemistry, enzyme-linked immunosorbent assay, immuno-electron microscopy, and western blot. Immunohistochemistry. One of the most commonly a pplied immunostaining techniques is immunohistochemistry (IHC). IHC is a histol ogical-like pro cedure that entails processing, embedding, sectioning, and adherenc e of tissue to slides. This process works by localizing proteins within the cells of tissues by binding antibodies to antigens (Samuelson 2007). There are two main approaches to IHC; the direct and indirect method. Following disruption of the cell membrane, the direct met hod would employ a single step staining method which involves a labeled antibody that reacts direct ly with a specific anti gen located within a tissue sample (Carson 1997a). The indirect method employs a primary and secondary labeled antibody, which is typically a more sensitive pro cedure. The visualization of IHC employs the use of antibodies tagged to fluorescent dyes (imm unofluorescence) (Wood, et al 1997, Theis et al 2003, Carson 1997a) or non-fluorescent methods th at employ the use of enzymes such as peroxidases (immunoperoxida se staining) (Carson 1997a, Fukuzawa et al 1995). The use of IHC to detect pathogens in animal tissues is useful and commonly employed in scientific diagnosis. Primary antibodies raised in rabbits immuni zed with the pathogen are used for the detection in infected ti ssue. Anti-rabbit Ig antibody will then be used to detect the antigen-antibody complex (Ells et al 2003, Kaufman et al 1997).

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71 Many microorganisms possess proteins and struct ural elements different than those found in animals that help in the production of antibodies to be used in IHC. For example, most of a fungus cell wall is composed of polysaccharides, most importantly -glucan, mannan, galactomannan, and chitin (Ishibashi et al 2005 ). These are not found within the plasma membrane of animal cells. Probes may be synthesized that use antibodies specific to these elements to detect the absence or pr esence of fungus within tissue sections. The use of polyclonal and monoclonal antibodies is used in IHC to detect complement antigens (Kaufman et al 1997, Jacobson et al 200 2, Ishibashi et al 2005, Kaufman et al 1995). For the detection of species-sp ecific antigens within tissue, po lyclonal antibodies are raised against the species-specific fungus, usually in a rabbit (Kaufman et al 1997). Monoclonal antibodies are developed with the use of a hybridoma, and usually a more specific method. Enzyme-linked immunosorbent assay. Enzyme-linked immunosorbent assay (ELISA) has been used to detect the presence of either an tigens or antibodies with relation to infectious diseases (Martinelli et al 2003, Marshall et al 1997). ELISA quantitatively determines protein concentrations from various ti ssues, a major advantage over othe r molecular techniques. ELISA is performed by adsorbing proteins in solution (blood plasma, serum, tissue extracts) to plates specific for ELISA. Antibodies specific for the protein are then added to the plates, and the secondary antibodies are added that will bind to the antigen-antibody complex that had been formed. Immuno-electron microscopy. Electron microscopy (EM) is used to study the microarchitecture and specific details of tissues and cells. Immuno-EM is a molecular technique using the detailed analysis of EM c oupled with the specific detec tion of certain proteins using antibodies to detect the intracellu lar location of structures (Marsh all et al 1997). Antibodies are

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72 labeled with heavy metal particles to be visualiz ed. Electron-dense labels, such as ferritin and colloidal gold, absorb electrons and manifest at the cellular laye r as black dots (Goldsby et al 2003). Particles of various sizes can be used to illustrate va rious antigens within a cell. Antibodies can be made against cer tain cellular components of a mi croorganism that are specific to that pathogen. The advantag es and disadvantages to this te chnique are the same for electron microscopy. Western Blot. A western blot (also called immunoblot) is a method using the properties of antibodies to detect specific organisms with in a tissue sample. Us ing antibodies, this technique detects proteins in a sample of tissu e. Gel electrophoresis (s ometimes an SDS-PAGE) separates the proteins based on shape and size, and the proteins are then transferred onto a membrane and probed. By using a primary and secondary antibody to probe a specific protein, the size and amount of the protein in a sample can be determined. When attempting to detect a certain protein within a tissue, antibodies must be raised against that protein to elic it a response for examination. Fungal metabolites, proteins, polysaccharides, or secreted proteinases are all co mmon target areas for the detection of a fungal pathogen within tissue (Pisa et al 2007). Differences among speci es of fungi have also been identified using western blot anal ysis (Lilley et al 1997). Western blotting has been a very useful technique in detecting microorgani sms within the blood (Pisa et al 2007, He et al 2004, Pizzini et al 1999) and urine (Zaragoza et al 2003) of patients. It has proven to be just as sensitive as other commonly used detection methods. Review of Fungi Fungi are a very diverse and non-classic King dom of species. Some estimate that there are over a million species of f ungi in existence today, so understandably they comprise an important Kingdom within phylogenetics (Kendrick 2000) Originally, fungi were classified as

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73 plants because they are immotile and possess cell walls. Fungi represen t one branch of the phylogenetic tree called the Eukarya domain (Deacon 1997). Currently these diverse organisms have been placed into the Kingdom Fungi. Fungi have a multitude of characteristics that separates them from the other Kingdoms and will be reviewed in the following sections. Eukaryotic Fungi, like plants and anim als, are eukaryotes ; their chromosomes ar e contained within a nucleus, usually referred to as a true nucleus (Kendrick 2000). In addition to a membranebound nucleus, eukaryotic cells are usually larger than prokaryotes (which lack nuclei). They contain various cytoplasmic organelles locate d within their cells such as mitochondria, endoplasmic reticulum, etc. that are not found in prokaryotes (Deacon 1997). Eukaryotic DNA is arranged into bundles called chromosomes, whic h contain the genetic makeup of the organism. Eukaryotes can reproduce via mitosis (asexual division) or meiosis (sexual reproduction). Heterotrophic As opposed to autotrophic organism s wh ich can produce their own food, fungi are classified as heterotrophs (chemo-organotrophs ) since they cannot synt hesize organic, carbon based compounds in order to produce food (Kendr ick 2000). Hence, a heterotroph is known as a consumer in the food chain due to the charac teristic that they c onsume food products and energy from sources other than itself. Fungi, as carbon heterotrophs, require preformed organic compounds as carbon sources. Fungi al so perform what is termed digest, then ingest, which is the mechanism in which they digest food firs t, then consume food (Deacon 1997). This is a characteristic form of an absorptive classification of food consumption. Heterotrophs can be subclassified as to the relationship that they have with their food source. Fungi exhibit three main classificati ons of the methods in which they obtain their nutrition. Saprophytes are organisms that obta in their nourishment fr om their surroundings,

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74 usually through dead organic matter. Others or ganisms are classified as mutualistic, having a mutually beneficial relationshi p with another organism (Alexopoul os et al 1996). Some fungi can also be parasitic and cause serious da mage to their host while obtaining their food. Fungi produce exoenzymes, a mechanism in which they digest food into smaller molecules. This allows them to absorb and use the nutrients for survival (Campbell and Reece 2002). Fungi will store their food as glycogen until needed, much like the way that animals store glucose (Kendrick 2000). Reproduction Reproduction in various fungal species is done ei ther sexually by m eiosis or asexually via mitosis. A spore (conidium) is a dispersal agent that is capable of furt her development into an adult, sometimes without fusing with another cell (Kendrick 2000). Spores are usually enclosed by cell walls and function in dissemination and survival (Kimbrough 2006). Conidia are nonmotile, asexual spores that are borne by a co nidiophore or conidiogenous cell and dispersed either actively through wind or water in aqua tic fungi with zoospores (Dugan 2006), or passively. Sexual reproducti on utilizes male and female parts for reproduction. Sexual Reproduction. Fungi are divided into gr oups depending on specific environmental conditions, which will further result in the type of repr oduction that the fungus undergoes. The teleomorph describes fungi that are capable of repr oducing sexually, anamorph describes the state of the fungus when reproducing asexually, and holomorph is used when describing the whole fungus, or teleomorph plus anamorph stages (Kendrick 2000). Fungi sexually reproduce through meiosis. Th e sexual state of a fungus (telomorph) is completed by the union of two nuclei followed by meiotic division yielding a recombinant progeny. The cycle includes plasmogamy, karyog amy and meiosis. The sexual organs, or gametangia, produce gametes required for se xual reproduction (Campbell and Reece 2002).

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75 The main phyla of sexual fungi include: the Chytridomycota (Chytrids), which are a class of fungi that possess flagella; the Zygomycota which produce spores identified as zygospores; the Basidiomycota, which possess meiosporangi a called basidia; and the Ascomycota, which contain meiosporangiums called asci (Kendrick 2000). Asexual Reproduction. Asexual reproduction in fungi requi res only a single individual, and spores produced are genetically identical to the parental genetics through mitosis. The asexual fungal state (anamorph) reproduce by conidia, which are exogenous asexual spores that are formed within a conidiophore. This type of reproduction is characteristic of mitosporic fungi that belong to the Ascomycetes and the Basidiom ycetes. The types of conidiogenesis will be discussed further in th e subsequent sections. Yeasts can reproduce asexually by means of budding. Budding occurs when a small outgrowth or bud on the cells surf ace appears that increases in size until a cell wall forms to separate the outgrowth into a completely new in dividual (Kendrick 2000). Some yeasts may also exhibit reproduction by binary fiss ion, involving one cell dividing into two completely new cells. Asexual fungi are placed within th e group Deuteromycota, a nonphylogenetic (artificial; not a true phylum) group which includes fungi that reproduce asexua lly. Many Ascomycetes seem to have lost the ability to reproduce sexua lly and therefore only reproduce via conidia. The Glomeromycota, which are characterized by the production of blastospores, only produce asexually. Asexually produced sporangiospore s are endogenous spores formed within a sporangium of fungi belonging to the Chytridiomycota, Oomycota and Hyphochytridiomycota. Fungal Growth Fungi grow and gather food by tubular elem ents called hyphae. Hyphae are the prim ary body of the fungus composed of cytoplasm and nuc lei along with a chiti nous cell wall. Each hypha is one cell that co ntinually grows and branches. Mass es of thread-like, branching hyphae

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76 form a feeding thallus called a mycelium, which is an aggregation of hyphae. This thallus, the vegetative body of a fungus (Dugan 2006), is th e actively growing portion of the hyphae. The apical cell of the mycelium and the few cells th at lie immediately behind it are the absorptive portions of the fungus, which will drive the organi sm to further growth and further digestion (Campbell and Reece 2002). Growth is dependant on a number of different principles, most importantly moisture, temperature, pH, nutrients and oxygen (Alexopou los 1996). Fungi require a high moisture content in their environment for favorable grow th, however they also grow in environmental extremes in which their spores can survive desiccation (Kendrick 2000). Cell Walls The cell walls of fungi are com posed of a co mplex fibrillate material along with an amorphous matrix. The fibrils are composed of beta-linked glucans and a polymer of Nacetylglucosamine called chitin (Alexopoulos 1996) This long-chain polymeric polysaccharide forms a hard material that links together to cons truct the fungus cell wall. Chitin is also found in various other species, such as th e exoskeleton of insects and the shells of crustaceans. The fungal cell wall acts as a barrier between the fungus and its envir onment, while at the same time maintaining the shape and rigid ity of the fungus itself (Kendr ick 2000). Most hyphal walls will contain two primary layers of polymeric fibrils and a matrix. Some hyphal cells within a hyphal filament ar e separated from one another by internal cross walls called septa. Septa contain pores to allow for the m ovement of cytoplasm within the hyphae, and thus the movement of nutrients thro ughout the mycelium. The septa will also act to limit the loss of cytoplasm when and if the cell wall is compromised in some fashion, such as damage to the cell wall or through conidial liberation (Kendrick 2000). When hyphae form a mycelium, the cytoplasm is permitted to move throughout the entire structure. These hyphae are

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77 considered septate; examples of which include the ascomycetes and basidiomycetes (Kendrick 2000). Hyphae that are without septa are termed aseptate, or coenocytic; these include the chytrids and zygomycetes. Unicellular vegetative bodies of fungi that are found in moist and humid habitats are called yeasts (Alexopoulos 1996). Unlike most fungi, yeast cell walls are composed of linked fibrils of mannans (instead of glucans), specif ically beta 1-6 linked polymers. Dimorphic Som e fungi are capable of exis ting in two forms; a mycelial growth and a unicellular yeast phase. Dimorphism is common in fungi that cause disease. The form that they exhibit is often dependant on the environmental conditions that they are subjected to (Alexopoulos 1996). These fungi will commonly grow as hyphae outside of their hosts, but once they have effectively entered their hosts they will assume a yeast-like appearance. This switch ultimately depends on variations in temperature. Deuteromycetes There are five m ain phyla of represented fungi that are recognized today. There are the Basidiomycota, the Ascomycota, the Zygomycot a, the Chytridiomycota, and finally the Deuteromycota. The Deuteromycota (also know n as anamorphic fungi, conidial fungi, fungi imperfecti, and mitosporic fungi) comprise a very diverse group of asexually producing organisms. A key attribute of fungi is th e ability to reproduce both sexua lly and asexually. However, over 20,000 species of fungi have no known sexual state. For clas sification purposes, scientists once believed that certain fungal species did not have a sexual phase associat ed with them at all (Baird 2003). The Ascomycetes and the Basidi omycetes are phyla in which sexual reproduction is sometimes not noted; these fungi were viewed only to produce as exual spores, or conidia, and

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78 were placed into a nonphyletic (artificial) group (Dugan 2006). F ungi classified in this phylum were placed there due to an im perfect knowledge of their existe nce and the absence of taxonomic status. The phylum Deuteromycota, or Fungi imperfecti, was therefore formed. The Deuteromycetes collectively represent conidial stages from members of phyla whose fruiting bodies are rarely found, have not been found, or perhaps have been deleted all together throughout evolution (Gua rro et al 1999). While the asexual phase may be the only phase th at is reported, this does not lead to the assumption that no known sexual phase exists, for it is quite the opposite. Some sexual fungi may only produce sexually on occasions (Alexopoulos 1996), while others that never exhibited sexual reproduction are slowly being reported to have sexual phases. The Deuteromycetes produce asexual spores, kno wn as conidia, in a variety of ways. A conidium (plural: conidia) is a nonmotile, asexual spore that is not formed within a sporangium (Kendrick 2000) and is identical to the parental fungi due to th e absence of meiosis. Spore development within a sporangium is distinctive to the Zygomy cetes (Kendrick 2000). Conidia come in a variety of shapes, colors and septations and sometimes are classified as such. Growth (usually always occurs) at the apical hyphal ti p of the mycelium (Barnett and Hunter 1998). Other characteristics of the De uteromycota include the presen ce of a conidiogenous cell, or conidiophore. A conidiophore is a specialized portion on the hyphae in which the conidia are produced (Baird 2003). They may occur singly or in organized groups. If conidiophores are formed individually, then the fungi are called Hyphomycetes, which are further divided into groups based on the color of their hyphae and spores (such as dematiaceous, or darkly pigmented) (Baird 2003). If the conidiophores are clustered, then th ey are formed within structures called conidiomata. Sporodochia and synnemata conidiomata are assigned under

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79 specialized subgroups of the hyphomycetes (Baird 2003). The Hyphomycetes conidiophores are always free and do not occur below the surf ace of the host tissue. Sporodochia resemble acervuli with the exception that the clusters of conidiophores form a layer on the surface of the host tissue, not contained within. Finally, synn emata are conidiophores that are fused together and will form conidia at the apex of the structure (Baird 2003). There are three main Deuteromycete form Orde rs that are divided according to the means in which they produce their spores. The Order Coelomycetes form their spores in a sealed conidiomata that are located on or just below the surface of a host. Those conidiomata are referred to as acervuli and pycnidia. Members of the Melanconiales produ ce acervuli, which are conidiophores that are formed just below the epidermal layer of plant tissues. Members of the group Sphaeropsidales produce pycni dia (Dugan 2006). Pycnidia normally form a flask-shaped or globose structure composed of fungal tissue th at encloses the conidi ophores and all of the conidia being produced. The Order Moniliales w ill form either sporodochia or synemmata and can either be described as being hyaline or pigmen ted. Mycelia Sterilia is an Order that does not produce conidia at all. Mechanisms of how conidia are borne or reproduced on the conidiophore are also classified in distinctive ways. If the conidia ar e arranged in a chain in which the new spores are formed at the base and the oldest conidia are apical, the term basi petal is used. If the conidia are formed in an opposite fashion (new spores at apex, old spores at base) then the chain is considered to be acropetal (Kendrick 2000). Conidia germinate by germ tube formation a nd then further produce mycelium; this then leads to the formation of more conidia a nd so on (Alexopoulos 1996). Conidia (also called mitospores due to the method in which they are generated thr ough mitosis) vary from one-

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80 or two-celled, cell shape, and can be classified according to thei r pigmentation of either hyaline (hyalo-) or colored (phaeo-) (Kimbrough 2002). Morphological groups of conidia include amerospores (single conidium), botryose (grape-l ike clusters), blastospore (budding), ellipsoid (oval), helicospores (coiled), allantoid (sau sage shaped), peniciulus (broom shaped), staurospores (with projections), dictymospores (one cross wall), scolecospores (worm-like), verucose (smooth), and velvutinous (velvety) (Kendrick 2000). How the septa occur within the conidia is anothe r technique in classification; if the spores have horizontal septa only, they are referred to as phragmospores, wher eas spores that have vertical and horizontal septa are te rmed dictyospores (Kendrick 2000). The division of the Deuteromycetes that comp rise the aganomycetes, or Mycelia Sterilia, do not produce any conidia, and thus are different from the other t ypes of asexual fungi. These fungi will only be present in the mycelial or hyp hal form (Dugan 2006), and are thus also termed sterile hyphae. Conidiogenesis Deuterom ycota can be classified based on th eir conidiogenesis. Conidiogenesis is the manner in which conidia are produced, along with the involvement of th e wall layers of the conidiogenous cell after liberati on (Kendrick 2000). The various techniques that asexual fungi employ to produce their conidia can be divided into two major patterns of development, either thallic or blastic. There are eight subcat egories within the two major groupings of conidiogenesis in which to cla ssify asexually reproducing fungi. There are six divisions under the blastic development and two categorized und er the thallic form of conidial ontogeny. It is first important to make certain distinct ions regarding the cell wall of the conidiophores and conidiogenous cells. The walls that compose these conidial bearing st ructures are composed of two layers and are termed de pending on the interacti on and involvement of these layers in the

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81 production of conidia (Kimbrough 2002). Hologenous wall development is evident when both of the cell wall layers of the conidiophore or conidi ogenous cell are involved in the development of conidia. Both the cell wall layers of the coni diogenous cell and the coni dia are continuous with one another (Alexopoulos 1996). Enterogenous wall de velopment occurs when the outer wall of the conidium is continuous with the inner layer of the conidiophore (as seen in anneloconidia that will frequently leave scars in their development). Last, endogenous ontogeny occurs when the walls of the conidium are not continuous with any layers of the conidiophore (Kimbrough 2002). The liberation of conidia is given two nomenc latures: rhexolytic and schizolytic. In rhexolytic dehiscence, the outer wall of the cel l between conidia will br eak down and eventually rupture, setting the spore free. Schizolyti c dehiscence includes double septation between the cells which will split apart by the degeneration of a middle lamella, thus once again setting the spores free (Kendrick 2000). Conidiogenesis is important in the evoluti on of fungal categorization; many fungi that were previously placed into certain genera were reclassified due to their conidiogenesis. Thallic conidiogenesis Thallic conidiogenesis is the first of the two basic m ethods of conidial development (Figure 1-5). In this arrangeme nt, the conidia will develop after and only once the cross-wall or septum is laid down; it is thought of as deve loping from a conversion of hyphal elements previously in existence (Kendrick 2000). The co nidias cell walls are al ready defined before it actually swells and liberates its elf (Kimbrough 2002). The division s that fall under this heading are thallic-arthric, or arthroconidial devel opment, and thallic-solitary, or aleuroconidial development.

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82 The first division that falls under thallic develo pment is thallic-arthri c, or arthroconidial development (Figure 1-5A). During the development process, hyphae will cease development and become divided into shorter segments by ir regularly arising septa (Kimbrough 2002). Once the septations are formed, they will break apart an d give a chain of arthro conidia that appear to be joined (Kendrick 2000). If a spore somewhere in the chain eventually dies, this will set some of the arthroconidia free. The second form of thallic conidiogenesis is th allic-solitary, or aleuroconidial development (Figure 1-5B). Conidia are form ed and broken off singly and are not involved in a chain-type of arrangement as in the previous thallic conidiogenesis. Blastic conidiogenesis In the second m ethod of conidial development, blastic conidiogenesis, the conidia enlarges and starts formation prior to th e septation of the cell wall, othe rwise known as budding (Figure 1-6) (Kendrick 2000). Along with the conidial el ongation, the conidia us ually originate at a narrow and fixed point in the conidiogenous cell (Alexopoulos 1996). If the conidiogenous cell is loose or not a portion of the mycelial system, th en it is considered to be a true form of budding (de Hoog et al 2000). It is evid ent upon development that there is a spore present even before it separates from the hyphae. The six divisions th at fall under this heading are blastic-acropetal, blastic-synchronous, blastic-sympodial, blastic-anellidic, blastic-phialidic, and blastic-basauxic. The first blastic conidium development is bl astic-acropetal (Figur e 1-6A). In this conidiogenesis, conidia are produced in single chains (Kimbr ough 2002) by apical budding. The youngest conidia are located at the tip of the chain (Kendrick 2000). In blastic-synchronous conidiogenesis, conidia are produced simultaneously in many clusters on a single conidiogenous cell (Kim brough 2002) (Figure 1-6B). Some fungi will

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83 develop further and form acropetal chains of se condary conidia, and ot hers do not (Kendrick 2000). Blastic-sympodial conidiogenesis is distinct in the effect th at spore production has on the conidiophore (Figure 1-6C). After conidia are produced, the conidiophore will continue with its development to produce new spores (Kim brough 2002). Each new apex will extend sympodially, and will further develop into a coni dium. As more spores are produced, then the conidiogenous cell becomes longer also (Kendrick 2000). The fourth type of blastic conidiogenesis is blastic-anellidic (Figure 1-6D). With each development of spores, a ring-like scar is left around the elongating conidiogenous cell, called annellation (Kendrick 2000, DeHoog et al 2000). When the spore pushes through the inner wall of the conidiophore, this leaves a scar that remains on the outer wall of the structure (Kimbrough 2002). The conidiogenous cell will have a scar for each conidium that is produced and subsequently liberated. In blastic-phialidic conidiogenesis, spores ar e produced from the open end of a specialized conidiogenous cell called a phial ide (Kendrick 2000) and pushed out from within (Campbell 1972) (Figure 1-6E). A phialide is a conidioge nous cell that has an open end through which conidia will develop in a basipetal succession (A lexopoulos et al 1996). The conidia will be only partly continuous with the conidiogenous cel l (DeHoog et al 2000). Only one phialid will produce new spores, and new conidiogenous cells are not formed. The last type of blastic conidia development is blastic-basauxic, al so known as meristem arthrospore development (Figure 1-6F). In this conidiogenesis, the ch ain of spores becomes progressively longer with development (Kimbr ough 2002). The conidia will gradually mature,

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84 which puts the oldest conidia at the tip and the youngest at the hyphal cell just below (Kendrick 2000). Hortaea werneckii Hortaea werneckii is a dimorphic fungus found throughout tropical areas of the world. It is the etiological agent of the condition in m an known as tinea nigra. Since this fungus was a focus of this research, it is important to cons ider the occurrence of its life cycle, location, classification, and morphological ch aracteristics in deta il. The discovery of this fungus in a reptilian species by our laboratory is the first reported occurrence. Classification and History Mycologis ts today have accepted the name Hortaea werneckii as the fungus that causes the disease in man called tinea nigra. It has taken many years for th e classification to remain as such, as it changed generic divisions many times si nce its initial discovery. Tinea nigra was first reported by Alexandre Cerqueira in 1891 as a chronic fungal infection found in the stratum corneum of the epidermis. He named the fungus Keratomycosis nigricans palmaris after the condition keratophytosis negra (Diniz 2004). Year s later (1921), Parreiras Horta isolated the fungus and re-classified it as Cladosporium werneckii due to the acropetal type conidiogenesis. The fungus was then reclassified as Exophiala werneckii in 1970 by von Arx. In 1985, two researchers, McGinnis and Schell, on ce again reclassified the fungus as Phaeoannellomyces werneckii primarily due to the discovery that the fungus could travel through th e air. Later that same year, the fungus found its place in the genus that it remains in today, Hortaea, due to its combination of sympodial and anellidic conidiogen esis (Abliz et al 2003). Nishimura and Miyaji rendered this name from one of the original discoverers, Horta. This genus until recently was comprised of only one species, Hortaea werneckii (Abliz et al 2003). In 2004, Holker et al

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85 discovered a new black yeast th at has been classified as Hortaea acidophila Appendix A has a complete classification of H werneckii and all known names. When characterizing an organism special consideration is given regarding where to place the organisms phyletically. Mammals are classifi ed from other animals due to the presence of hair and mammary glands. Bacter ia are classified based on their ability to stain. With fungus, a great deal of characterization lies on the presen ce of an asexual and se xual phase, as discussed earlier. Hortaea werneckii has only been identified in the asexual phase; there have been no reports of a sexual phase for this fungus. He nce, it is placed into the Deuteromycete classification. Morphological Characterization There are various charac ter istics that make H. werneckii very distinct in classification. One of these features is a two celled annelidic yeast form that may give rise to oneand twocelled conidia (de Hoog et al 1992, Mok 1982). This fungus is also classified as dimorphic, in that it can be found in two different forms; a yeastlike form and a hyphal form (Hardcastle 1974). It presents itself as being a dematiaceous black fungus, which will manifest mostly in the yeast phase. The ability to synthesi ze dihydroxynaphthalene melanin cla ssifies this fungus further as dematiaceous (Tetsch et al 2005, Kogej et al 2004). Hyphae appear to be thick walled and da rkly brown pigmented, however younger cells may be colorless (Ng et al 2005) Conidia are septate, light brown, elliptical, and bicellular (Diniz 2004). H. werneckii grown on Sabouraud agar medium re veals an initial colony growth that seems shiny, smooth, and spherical. It will la ter reveal an olive-grey growth with a black reverse and will grow to its maximum size after 25 days. Of great importance to our research is that is characterized as growing very slowly. It grows optimally at 29oC (Diaz-Munoz et al 2005). Microscopically, yeast-lik e cells may be seen, and typically grow by polar budding

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86 (Harcastle 1974). Yeasts that do not enter the budding sequence will conv ert directly to hyphae by elongation. Most dimorphism in fungi is mediated by nutrition, a low oxidation -reduction potential, or elevated carbon dioxide c ontent (Hardcastle 1974). Histoplasma capsulatum conversion from mycelial growths to a yeast phase has been accomplished by using media including blood agar, egg-potato flour medium, and cystei ne-blood medium (Hardcastle 1974). Mucor rouxii yeast development has been shown to depend on the presence of carbon dioxid e and the absence of oxygen. However, in the case of H. werneckii temperature has an important role in the switch to a mycelial form from a yeast-like form. In natu re, the yeast phase has been noted to occur in watery environments, while the thick-walled hyphal stage would be suited for survival after desiccation (de Cock 1994). This desiccated state may aid in its airborne dispersal and in its possible dormancy. A secondary metabolite produced by H. werneckii was discovered by Br auers et al in 2001. Isolates from the Mediterranean sponge, Aplysina aerophoba, exhibited a phenolic natural product characterized by a unique ri ng structure not described in nature. This product, named hortein, is unique to this fungus. Epidemiology H. werneckii has a predilection for tropical and s ubtropical areas around the world. The warm th and humidity of these environments a llow this fungus to thrive. Cases have been reported in Asia, Africa, Centra l and South America, and North America (Diniz 2004, Schwartz 2004). Most of the cases in North America have been reported from the Atlantic southern coastal areas (McKinlay et al 1999) such as in Florida, Texa s, Alabama, Louisiana, Virginia, Georgia, and North Carolina (Schwart z 2004). It is a saprobic fungus that is found in a variety of

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87 environments that include soil, plants, beach sand, air, decomposing fish, decaying wood material and normal skin (de Hoog and Gerrits van den Ende 1992). Since cases frequently occur in coastal regions, researchers began looking into H. werneckii s capability of inhabiting areas of high salt. It is extremely halophilic, and clinical growth has focused on media containing concentrations of salt. In nature, it is considered one of the most halotolerant fungal spec ies, with reports of tolerating up to 30% NaCl (Petrovic 2006). Human Cases Clinical fungal infections are regularly divi ded into four categorie s based on infectious location. These include superficial, cutaneous subcutaneous, and system ic (Schwartz 2004). Tinea is a general name for many different kinds of superficial fungal in fections of the skin, hair, and nails. Tinea nigra has most notably been categorized as a superficial fungal infection until recently. Recently, H. werneckii was isolated from blood and sple nic abscesses in humans. Traditionally invasive fungal infestations ar e rare and are often f ound in conjunction with neutropaenia and therapy with high doses of corticosteroids which commonly suppress the immune system (Ng et al 2005). Fungi not previously noted as human pathogens are seen to occur more frequently. Many of these are regularly seen as opportunistic infections, until recent cases such as this one. Two patients with acu te myeloid leukemia exhibited colony growth arising from clinical samples of blood and splenic abscesses. Both growths were subjected to genetic testing, and the identities of 306-bp fragme nts were confirmed by sequencing (Ng et al 2005). These were the first cases of human systemic infections of H. werneckii to date, which demonstrates that superficial mycoses have the ability to become systemic. H. werneckii affects both males and females, and ma ny various age groups. The period of incubation of tinea nigra varies. Cases have re ported incubation to be between two to seven

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88 weeks (Diniz 2004, Schwartz 2004), however, one report has shown an incubation period of 20 years (McKinlay, et al 1999). Tinea nigra is often found on the stratum corneum of the epidermis. An H. werneckii infection will clinically appear as a dark asymptomatic brown to black pigmented macules on the palms or soles that enlarge centrifugally (Uezato et al 2006). The non-sc aly lesions will grow from one to five centimeters, and may at time s connect to one another (Diniz 2004). There appears to be minimal desquamation, and the borders are well delimitated. It s appearance is seen as a non-elevated macule (Perez et al 2005) a nd is usually located on the palmar regions and along the fingers, but has also manifested on the dorsal region of the hands, the inferior portion of the feet, and the b ack (Diniz 2004). Diagnosis Diagnosis of H. werneckii can be m ade in a variety of wa ys. Depending on the availability of testing techniques, direct my cological examination is the pref erred method. A scraping of the lesion incubated with 20% potassium hydroxide and dimethyl sulfoxide reveals multiple characteristic hyphae. A downfall of the tradi tional methods of morphological identification is that they are both time consuming and require highl y trained personnel. Col onies may be sent to the Fungus Testing Laboratory at the University of Texas Health Sc ience Center in San Antonio, Texas for complete examination. Polymerase chain reaction-based methods of identification have become popular and can efficiently diagnose various pathogenic and morphol ogically similar species (Abliz et al 2003). Specific oligonucleotide primers sets based on th e sequences of internal transcribed spacer regions (ITS) of ribosomal DNA (rDNA) have been designed by Ab liz et al. These primers successfully identify H. werneckii by PCR. PCR is a practical technique to diagnose H. werneckii in various clinical cases.

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89 Non-Human Animal Cases In addition to the discovery of H. werneckii in hum ans, there has until recently been one additional report of its oc currence in an animal. This fungus was identified as the causative agent of a superficial mycotic infection of a guinea pig in Japan (Abliz et al 2003, Sharmin et al 2002). The male animal was born in February of 2001 in Japan. The animal remained in Japan upon diagnosis of the disease. The animal had b een scratching vigorously and was noted to have formed dark lesions on the dorsal surface of the thorax. Upon microbiologi cal isolation, colonies formed from scrapings of the le sions revealed a shiny black yeas t-like growth. This growth was identified as H. werneckii and further molecular testing confirmed this diagnosis. H. werneckii first appeared in Japan in 1983 as reported by Nakama et al. Roughly 28 human cases have been reported in Japan to date (Uezato et al 2006). Proposed spread of this fungus is hypothesized based on climate change due to global warming and increase in imports from other countries. How the guinea pig cont racted the fungus is unknown, since the animal was kept indoors at all times. In addition to the above guinea pig, H. werneckii has been identified in several American alligators in the southeastern United States (Dicks on and Cardeilhac 2001, Dickson et al 2002a, Dickson et al 2002b, Townsend et al 2006). This w ill be the focus of this dissertation, and will be discussed in future chapters. Treatment Fungal diseases are routinely treated with or al antifungal m edication. However, in the cases of H. werneckii topically administered antifungals are all that is required. Topical use of imidazol antifungal agents or bifonazole cream us ually takes four weeks to clear the infection (Uezato et al 2006). Keratolytic agents such as 2 to 4% salicylic acid may have an effect on the fungus (Diniz 2004). Ketoconazole, miconazole, terbinafine, and cicl oporox olamine have all

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90 been successfully used. Many of the oral antifungals have not been effective in treating tinea nigra. Kochs Postulates Robert Koch was a Germ an physician is known not only for his isolation of Bacillus anthracis but for the development of Koch postulates. These set of criteria were created to determine whether a microorganism is the primar y cause of a presented disease (Fredricks and Relman 1996, Grimes 2006). At the time, these criteria were considered in establishing causation of microorganisms. These postulates were originally created for skeptics who did not believe that microbes cause diseas e. Presently, many continue to hold Kochs postulates as the standard for causation, yet recent findings based on new technologies, for example, that nucleic acids are the source of genetic information, provi des a new method to view these guidelines. A summary of Kochs original postulates are as follows: 1) the microorganism must be found in all organisms suffering from the diseas e, but not in healthy organisms, 2) the microorganisms must be isolated from a diseased organism and grown in pure culture, 3) The cultured organism should cause disease when in troduced into a healthy organism, and 4) the microorganism must be reisolated from the inoc ulated, diseased animal (Fredricks and Relman 1996, Grimes 2006). While at the time Koch thought that these cr iteria were unarguable, he quickly amended certain portions of his postulates. For example, from the first postulate he abandoned but not in a healthy organism when he discovered as ymptomatic carriers of disease. Some microorganisms may also be opportunistic, and cause disease only once an individual is immunocompromised. For postulate number two, it states that the mi croorganism must be grown in pure culture; however viruses cannot grow in artificial culture. Some pathogens, such as Mycobacterium

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91 leprae have never grown in laboratory settings. Other bacteria are considered somnicells, dormant cells that become stressed and cannot be cultured (Grimes 2006). Postulate number three is scru tinized since some pathogens e xhibit disease differently in various subjects. Not all subjects exposed to an infectious agent will acq uire the infection. In addition, some cases that have been confined to humans make it difficu lt to verify the third postulate in an animal model (Grimes 2006). No rmal flora may cause disease in one host and not another (Fredricks and Relman 1996, Grimes 2006). As quoted by Fredricks and Relman (1996), bio logical relationships can only be inferred from observationscausation is not observable, only the events that suggest a link between cause and effect are observable. This sugge sts that although most microbes fit Kochs postulates in relation to dis ease causation, inability to fulf ill Kochs postulates does not necessarily mean that it is not an agent of the disease. Causation theories have slowly moved away from Kochs postulates and towards Alfred Evans proposed Elements of Immunological Proof of Causation. This uses the detection of antibodies to certain microorganisms. A flaw with this design was encountered with CreutzfeldJakob disease, which could not be seen, grown in a laboratory, or serologically detected (Fredricks and Relman 1996, Grimes 2006). While many of Kochs principl es are still relied upon today, it may not be correct to create unconditional statements regarding forms of proof. Strict observan ce of these postulates may not be needed if enough diagnostic evidence lead s to a certain microorganism. Even though additional causation models have been proposed, there are flaws identified with in most of them.

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92 Figure 1-1. Layers of crocodilian scales. In crocodilians, -keratin replaces -keratin; there is minimal overlapping of scales and scales ar e shed one at a time. Note the hinge region, where there is a close relationshi p between the epidermis and dermis, along with the presence of only -keratin. Picture reprinted from Alibardi and Toni 2006 with permission from the author.

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93 Figure 1-2. Scales of lizards (and snakes). These species will have overlapping scales that they will shed all at the same time. The oberhau tchen layer is filled only after fusion with the layer. -keratin and -keratin will form alternating layers with one another, known as vertical disposition. This is what produces the shedding complex that facilitates molting. They will ultimately have si x total layers as a result of this cyclic modality of keratins. Picture reprinted fr om Alibardi and Toni 2006 with permission from the author.

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94 Figure 1-3. Chelonian scutes. In chelonians, -keratin replaces -keratin. -keratin is found mostly on the shell, while -keratin is found primarily on the neck, legs, and head. These species will shed scutes one at a time, and shed their soft skin all at once. Picture reprinted from Alibardi and T oni 2006 with permission from the author. Figure 1-4. Scales in Sphenodon punctatus. Picture reprinted from Alibardi and Toni 2006 with permission from the author.

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95 A B Figure 1-5. Thallic conidiogenesis. Reprinted with permission from the author (Kendrick 2000). A) Thallic arthric. B) Thallic solitary. A B C D E F Figure 1-6. Blastic conidiogenesis. Reprinted with permission from the author (Kendrick 2000). A) Blastic acropetal. B) Blastic sync hronous. C) Blastic sympodial. D) Blastic annelidic. E) Blastic phialidic. F) Blastic basauxic.

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96 Table 1-1. Summary of the post-la ying embryonic development stages. Stage Days (post laying) Morphological age 1 0-1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10-11 11 12 12 13-14 13 15 14 16-17 15 18-20 16 21 17 22-23 18 24-26 19 27-28 20 29-30 21 31-35 22 36-40 23 41-45 24 46-50 25 51-60 26 Absent 27 60-63 28 64-70 Note: Alligator development ends at day 60. Incubation time usually last until day 60, however hatch will occur at anytime between day 50 a nd day 70. Crocodiles have a 90 day incubation period.

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97 Table 1-2. Comparison of the terminology of integumentary layers Mammalian Crocodilian Chelonian Squamata Stratum corneum Corneus layer Beta-compact Oberhautchen Stratum lucidum Beta-keratin layer Stratum granulosum Transitional layer Beta-cellular Mesos Stratum spinosum Suprabasal Suprabasal Alpha-keratin layer Stratum basal Stratum basal Basal Lacunar C l e a r Note: The layers progress from superficial at the top of the chart and proceed deeper. The layers are entered as they are analogous to each of the different groups

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98 CHAPTER 2 HISTORY OF PIX SKIN DISEASE IN THE SOUTHEASTERN UNITED STATES Introduction Starting in the fall of 1999, unusual pit-like scar s term ed PIX were reported on tanned alligator hides. Tanners had assigned the name PIX due to the scars physical appearance resembling an indentation that might have occurr ed due to an ice pick (Figure 2-1). During subsequent years, fresh and tanne d hides from Florida were examin ed retrospectively; hides from as far back as 1997 had evident PIX marks occurring throughout the skin (Figure 2-1). One of the first observations made was that PIX was evident as localized pit-like marks on a tanned hide, however on a fresh hide these mark s were not always seen. Instead these marks manifested as spherical opaque lesions, termed SO Ls (Figure 2-3). It is important to note that the term SOL is used when visualizing the lesi ons on a fresh hide placed over a high intensity lamp, whereas the term PIX is used when re ferring to the indentations/marks on a tanned alligator hide. It is now known that the SOLs will eventually lead to PIX depending on eruption of the lesion. The significance of this problem became appare nt when hides were sent for grading, where damaged hides were severely downgraded if thes e lesions were evident. More recently, hides were branded as total rejects (no value). Hides above four feet in leng th seemed to have the highest incidence, which suggested that an increase in occurrence a ppeared to be associated with an increase in size and probably age. PIX poses a hazard to alligator farming by significantly reducing the value of hides produced by the farms. If the SOLs are not seen within a group of animals previously identified to have PIX, the suggestion is that the lesions may have time to regress from their first observed state. As few as five SOLs on an individual hi de can cause down-grading and a higher incidence

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99 of SOLs may reduce the value of the hide by more than 50%. After discus sing the situation with several farmers, it seemed that the tanneries would downgrade a hide that may have very few SOLs, even though they may never deve lop into PIX scars once tanned. The incidence and prevalence of PIX scars on hides from Florida farms appears to be associated with the occurrence of stress in the pens. This may be caused by the presence of disease, overcrowding, unsanitary conditions, or a failure of the heating system. Once treatment and prevention plans were implemented to decrease stress among animals, PIX regressed on some farms and was nearly eradicated on other fa rms. Louisiana farms have experienced similar progression and regression of th e disease (Figure 2-4). Once the significance of this disease had manife sted itself across the southeastern United States, various farms from Loui siana and Florida reported the di sease outbreak. Our laboratory was the first to study the PIX skin disease in detail starting in 1999. The progression of PIX has seen a spread from a few Florida farms in 1998 across the southeaste rn United States to encompass most alligator farms in this region. While PIX is not cons idered a fatal disease process, the downgrading of the hides has been a significant fina ncial problem for the economy. Farms Participating in this Study The research perform ed for this dissertati on was completed over a seven year period and included a total of four farms in Louisiana and four farms in Florida. Hides exhibiting SOLs were also examined from wild animals along with alligators from Texas and Georgia farms, but no samples were taken. The farms that participated in this study have been kept anonymous and referred to as Farms LA-1, LA-2, LA-3, LA-4, FL -1, FL-2, FL-3, and FL-4. The farms located in Louisiana are depicted as LA, and the farms sites sampled in Florida will be depicted as FL. The majority of the current study was completed from 2003 to 2008; samples prior to this have

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100 also been included, however comple te diagnostic testing and comp lete records were not always available. Farm LA-1 This f arm was the primary location where the treatment study of this project was performed. This farm started to demonstrate cases of PIX in 2001, and has seen periods of regression and emergence over the last six years. Animals located on this farm were collected from the alligator egg collection every year in July. In this collection, 100% of the nests from approved areas was collected, and roughly 16% of the hatch rate wa s returned to the wild after two years. This information could be important when determining the transmission route of this disease. Samples collected from this farm and incl uded in this study were obtained by repeated visits over the course of five year s. Samples were also sent via ma il if visits could not be made. At the current time, this farm has seen a dr astic reduction in the occurrence in PIX and has almost completely eradicated this disease on the farm. Farm LA-2 Farm LA-2 was not visited over the course of this study; samples were sent via mail and examination and biopsies were performed immedi ately upon arrival. An imals at this farm exhibited varying outbreaks of SO Ls throughout the current years. Farm LA-3 Farm LA-3 was also not visited over the course of this study; samples were sent via mail and examination and biopsies were performed im mediately upon arrival. Animals at this farm exhibited varying outbreaks of SO Ls throughout the current years.

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101 Farm LA-4 Farm LA-4 has three primary locations, howev er the main farm used for this study is located in Louisiana. Alligators were transporte d from farm to farm which may have a role in transmission of this disease. This farm ha s had a PIX problem steadily since 2001 and is still exhibiting the problem currently. Samples from LA-4 have been s poradic over the years of this study. Frequent changes in management and owne rship and destruction due to hurricane Katrina have made frequent sampling here difficult. Farm FL-1 Farm FL-1 was one of the first farms to repo rt PIX, in addition to being the first farm studied by the researchers. Their first repo rted outbreak occurred in November of 1999, however, an examination of hides previous to this year have shown occurrences of PIX. This farm has also seen a reduction in PIX over the past seven years. Farm FL-2 Farm FL-2 (located across the street from Farm-1) has reported PIX over the past few years (1999-present). This farm was additionally one of the first farms to report PIX, in addition to being one of the first farms studied by the researchers. Farm FL-1 and Farm FL-2 have in the past traded animals. This may have played a role in the transmission of PIX between the two farms. In recent years however, they no longer tr ade animals. Farm FL-1 is not observing SOLs or PIX currently, whereas Farm FL-2 has seen the greatest incidence of SOLs in years. The first PIX lesions were seen to appear at this farm in September of 1999. Farm FL-3 Farm Fl-3 previously reported occurrences of PIX. In recent years, very few SOLs and subsequent PIX has been observe d in relation to other farms. The occurrence of PIX on this farm is unknown at this time.

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102 Farm FL-4 Upon retrospective analysis, this farm was noted to have PIX dating back to 1997, however samples were not collected until 1999. Tanned hides were examined and showed multiple PIX lesions per hide. Samples obtained from this farm in 2002 exhibited multiple erupted PIX marks that were examined histolog ically and will be discussed further in the histology chapter. This farm is currently not producing hide s for commercial sale, and SOL occurrence is presently unknown. History of Occurrence PIX was first reported at FL-2 in Septem ber 1999. At the time, roughly 30% of hides had SOLs at the time of first raw crus t inspection. SOLs increased to more than 50% in six months. Of one lot (360 hides) examined, 70% of the hide s had some SOL occurrence. Hides with five or more SOLs per hide were termed significant PIX. Forty seven percent of the 260 hides exhibited this classification. Some hides examined were observed to have nine SOLs in a single scale. In November of 1999, FL-1 started to report PIX lesions on their tanned hides. Analysis of their salted and fresh hides revealed SOLs. In the spring of 2000, their occurrence per 1000 hides was 20%, but steadily incr eased to 28% over the next 10 months. After 18 months, PIX increased to 100%, indicating that every hide examined had some degree of SOLs. Seventy five percent of these hides were serious, with 10 or more SOLs per hide. In the spring of 2001, FL-3 and FL-4 PIX occu rrence were at lower levels than the other Florida farms, but were still evident. One lot (193 hides) examined at FL-4 showed an 86% significant PIX lesions (5 or more per hide). Samples from farms in Louisiana were collect ed starting in 2001, ev en though lesions were observed late in 2000. The majority of samples were submitted between 2002 and 2007.

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103 West Nile Virus and the Emergence of PIX While this study relied prim arily on a fungal a ssociation, researchers at another institution were focusing on viral associations to this disease. Researchers at Louisiana State University began studying the disease in 2003 and focused on a viral etiology of PIX di sease (Nevarez et al 2007). Researchers at LSU have renamed this condition in Louisiana Lymphohistiocytic Proliferative Syndrome of Alligators (LPSA). For the purpose of this study, this name is not used; LPSA is used by the researchers at LSU for animals that were seen only in Louisiana and not in Florida. Since this proj ect includes animals from both states and samples from as far back as 1999, the term PIX will still be used. Alligators can become infected with West Nile virus (WNV), and cases have been reported in Florida, Georgia and Louisian a. Not only do alligators become infected, but they also have the ability to amplify the disease (Klenk et al 2004). Steps were taken to detect any viral presence, and epidemiological maps were anal yzed for the emergence of both PIX and WNV. West Nile Virus (WNV) is an arthropod-bor ne virus that commonl y causes neurological problems in the species that it infects. WNV is a positive sense, single stranded RNA virus that is located within the Flavivirus genus of the family Flaviviridae (Long et al 2007, Wellehan and Johnson 2005) This family has over 70 known pathogenic viruses that are common throughout the world (Long et al 2007). The virus was first isolated in 1937 from a wo man in the West Nile Providence of Uganda (Long et al 2007). Initially only birds, horses and man could become infected with the disease. WNV progressed across the world and eventually came to the United States in 1999 where it was identified in birds and horses in Long Island, New York (Long et al 2007). In 2001, the first case of WNV infection in reptiles was noted.

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104 WNV was initially isolated from farmed a lligators that were exhibiting neurological problems. Alligators first exhibiting the disease we re presented with star gazing behaviors that are commonly indicative of a vi ral infection (Miller et al 2003 ). Upon death, tissues were submitted for histological identification and molecu lar testing. No observable changes within the integument were noted. In most cases of WNV in other species, in tegumentary lesions are not a common occurrence. The Center for Disease Control first reported WNV in the United States in 1999. It was initially seen in the New York City area, and su bsequently spread throughout the United States (Figure 2-7 and Figure 2-8). Th e Florida Department of Health Bureau of laboratories began screening samples for WNV in 2000. The first po sitive case in Florida was detected in 2001. Retrospective studies performed by the state of Fl orida on St. Louis encepha litis-positive sentinel chicken and human sera were c onducted and were not positive for antibodies to WNV (personal communication with the Florida Department of H ealth Bureau). Based on these findings, WNV was not present in Florida pr ior to 2001. PIX has been observed in Florida since 1999, and retrospectively since 1997. PIX was first detected in 2000 in Louisiana, whereas they did not have WNV in the state until 2001. Based on these epidemiological maps, we do not believe that WNV and PIX have a high association with one another. While alligators exhibiting PIX ma y also test positive for WNV, the absence of WNV during peak PIX outbreak s does not match and it was concluded that alligators can have PIX without a WNV infection. PIX skin disease in alligators was here prior to the emergence of WNV, so emphasis is placed on this data in form ing a correlation between the two diseases. The two diseases have been s hown to occur concurrently in animals, however the hypothesis that PIX is caused by WNV is inconsiste nt with the divergent years of emergence.

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105 Diagnostic Panel to Determine the Causative Agents of PIX Diagnostic techniques used to determ ine the causation of disease vary depending on the presumptive causative agent and the clinical progression of the disease. The processes that a researcher takes in identifying the problem also varies according to the host species that is harboring the disease or pathogen. For exampl e, a specific species of fungus may exhibit different characteristics in mamm als than it does in reptiles. To identify the fungus, steps must be performed differently based on host animal and fungal species. This holds true for most diagnostic procedures an d pathogen detection. PIX is a skin disorder in alligators that is primarily identified within the farming industry throughout the southeastern United States. The high moisture and humidity where these animals are housed may play a role in the spread and main tenance of this disease. For years, various techniques and diagnostic methods have been employed to establish causation of this disease. Since the alligator is an aquatic ectothermic an imal, disease may manifest differently in this species than would in a terrestrial endothermic animal. Animals kept in farming conditions are subjected to extrinsic factors and sometimes are viewed as having an increase in their susceptibility to certain diseas es (Miller et al 2003). Star ting in 1999 and continuing throughout 2007, techniques used to determine the causation of PIX have included gross identification, microbiology, histology, polymerase chain reac tion, electron microscopy, and virology. The following chapters will discuss the diagnostic panel that has been designed specifically for this problem and its use in identifying an d characterizing PIX skin disease in the American alligator. Gross Identification of Pix Lesions Once tanners notified alligator farm ers about th e occurrence of PIX, farmers started to notice the presence of SOLs while the hides were be ing graded. The SOLs appeared to be in an arrangement that resembled PIX scar patterns. Gr ading is done by placing the hide over a table

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106 with a strong light illuminating from the bottom. The light shines th rough the hide and any imperfections can be visualized such as bite marks, umbilical scars, and SOLs. Once the skin is examined over a high intensity lamp (such as spot light), PIX is then evident as SOLs that are approximately 1mm in diameter (Figure 2-2). These SOLs appear as a transluminant area on the hide that usually occu rs on the ventral region of the animal, primarily along the abdomen and most anteri or neck regions. Lesions ha ve not been observed along the dorsal surface of the animal, most likely because this region is highly pigmented and also contains bony areas fused to the skin (osteoderms). The primary diagnostic method for the gross id entification of SOLs is the use of a high intensity lamp commonly embedded into a gradi ng table. Placement of fresh, unsalted hides on the table will allow for the lesions to be visua lized. Depending on the severity of PIX, anywhere from one to more than ten lesions been observed occurring on a single scale. Discussion Although PIX was first reported in Florida in 199 9, hide analysis of previous years have shown PIX lesions as far back as 1997. The avai lability of fresh sam ples from those years could not be obtained, so one can only speculate about past outbreaks. Once PIX was evaluated and examined at the initial farms (FL-1 and FL -2), samples were r outinely collected. PIX is regarded as being the major problem in the alligator industr y due to the economic loss endured by the farmers. Certain farms were almost forced to cl ose due to significant economic losses from PIX. If even one hide from a farm is seen to have SOLs, the graders and tanners will downgrade the entire lo t of hides. The need for erad ication and characterization of the disease led to the research reported here. While PIX may be correlated with the onset of West Nile virus, simple demographics do not support these two diseases oc curring together in the years previous to 2001. Reports from

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107 the CDC and Florida Department of Health Bure au of Laboratories are confident that WNV was not in the United States prior to 1999. Maps of WNV activity show that the virus did not reach Florida and Louisiana until 2001. Overlapping time lines and demographics could allow one to be caused by the other; that was not the cas e regarding WNV and PIX. There may be an association currently, but PIX occu rred before WNV had made its entrance into the United States does not support this association. It may be pos sible that WNV enhances the virulence of PIX. One of the hypotheses on the spread of PIX is the transport of animals from farm to farm. If there is a pathogenic microorga nism causing this disease, then it could be capable of traveling within the animal to other farms. Since ma ny of the farms have in the past traded and transported animals, transmissibility of this disease could have been increased.

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108 A B Figure 2-1. Tanned alligator hides showing PIX lesions. Arrows pointing to the PIX lesions. Note the appearance of the ice pick-like mark. A) Alligator hide in the crust tanned stage. B) Alligator hide in the tanned hide stage.

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109 A B Figure 2-2. Florida alligator hide placed over a high intensity la mp. Upon closer examination, SOLs will appear on the most mid-ventral areas of the skin. Note the light that is illuminating through the layers of the alliga tors integument. This makes it easy to visualize imperfections that might be eviden t. A) Whole alligator hide picture. B) Higher magnification of image of A with arrows pointing to lesions.

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110 A B Figure 2-3. Photographs depicting SOLs on a fresh, unsalted Florida alligator hides. A) Alligator hide showing multiple lesions; this would be a case of an extreme outbreak. The boxed area depicts clusters of SOLs. B) Allig ator hide showing only a few lesions per scale; this would be a less extreme case than A. Arrows pointing to the lesions

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111 Figure 2-4. Photograph of a Louisiana hide exhibiting severe SOLs. This section is from the ventral gular region (neck) region. Figure 2-5. Photograph of SOLs be fore biopsy. The circle repr esents an area where the 6mm biopsy punch would be made. Biopsies would then be submitted according to each diagnostic tests protocol.

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112 Figure 2-6. Example of a 6mm bi opsy punch with a PIX scar.

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113 A B C Figure 2-7. West Nile virus maps reflect surveillance reports released by state and local health departments to CDC's ArboNET system for public distribution. Map shows the distribution of avian, animal, or mosqu ito infection occurring during 1999 with number of human cases, if any, by state. Reprinted with permission from the CDC. A) 1999. B) 2000. C) 2001.

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114 A B C Figure 2-8. West Nile virus maps reflect surveillance reports released by state and local health departments to CDC's ArboNET system for public distribution. Map shows the distribution of human neur oinvasive disease (encepha litis and/or meningitis) incidence occurring during 1999 with number of human cases shaded and any WNV activity (human, mosquito, veterinary, avia n and sentinel data). Reprinted with permission from the CDC. A) 1999. B) 2000. C) 2001.

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115 CHAPTER 3 HISTOLOGICAL CHARACTERIZATION OF PIX AND SPHERIC AL OPAQUE LESIONS IN THE AMERICAN ALLIGATOR Introduction The use of histopathology and various staining techniques are ideal m ethods in detecting lesions and disease progression wi thin tissue. In order to examine tissues and their cells structurally, samples need to be preserved and stained. This allows their extracellular components to be viewed in response to various dyes used. In addition to general characteristics of tissues, histopathology is used when demons trating the invasion of pathogenic organisms in tissues. Early in the process of the char acterization of PIX, histology was the first procedure to be added to the diagnostic panel developed by the res earchers. At the gross level, only the general appearance of the lesions could be noted, wherea s cellular and morphological data were needed. Special consideration was to be placed on staining techniques used in the detecti on of pathogenic organisms. Since PIX could have many causes, various stains were performed to detect the presence of any microorganisms that may be loca ted within the integument of the PIX infected animals. Histology is important in the ch aracterization of a disease to show the progression and the presence of suspected pathogens. For the curre nt study, histology was vita l since this was a new disease that had not been previ ously described. A baseline was needed for comparison to other diseases, as well as for a comparison of PIX between farms. Preliminarily, it was not known whether the spherical opaque lesions (SOLs) evident upon light identification were similar among the farms as seen in multiple states and multiple facilities. Examination of normal integument was perf ormed as a comparison. Specifically, the hinge region of the alligators skin was ex amined as a possible transmission route for

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116 integumentary pathogens. This area in crocod ilians exhibits a thi nning of the precorneous keratinocytes beneath layers of narrow corneocyte s (Alibardi and Toni 2006). The decrease in the thickness of the epidermis and the thinne r corneocytes in the hinge region resembles (sebo)keratinocytes of avian ap teric epidermis (Alibardi and T oni 2006). Since this area is thinner than the middle portion of scales, this area was suspected as an entry route for microorganisms. The goal of this portion of the study was to use histology as an effective tool in the identification of SOLs, to properl y characterize the disease, and to detect any pathogens located within the lesions. Employing various sectioning and staining tec hniques has allowed the SOLs to be microscopically examined and viewed for continuity among samples and a comparison among farms. Materials and Methods Sterile latex gloves were worn during collec tions and changed if multiple hides were examined. Samples were taken for hist ology using a Keyes 6mm biopsy punch. A new instrument was used for each animal/hide. Biopsie s were placed in 10% buffered formalin for 24 hours to allow for proper fixation. In prepar ation for embedding, samples were dehydrated by using ethyl alcohol as follows: 70%, 80%, two changes of 90%, three changes of 100%. The tissue was then cleared with tw o changes of xylene. The tissu es were infiltrated with two changes of paraffin (Fisher Tissue Prep) at 570C. Tissues were embedded using Fisher Tissue Prep T565 in metal molds and allowed to harden for at least two hours. Blocks were sectioned using a Reichert-Jung 203 0 microtome. Tissues were cut in serial sections at a thickness of 5 microns (um) Tissue sections were placed in a 600C water bath and then placed on Superfrost microscope slides and in some cases on Superfro st Plus slides (Fisher Scientific, Pittsburg, Pennsylvania). Each slide c ontained at least 2 sections of tissue. Slides

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117 were labeled according to the research succession number, order of section, and later the stain used. Slides were placed in a sl ide rack and set in an oven at 60oC overnight to dry and allow the tissue to adhere to the slides. The slides were then deparaffinized and stained. In most cases, sagittal sections were performed to show the epidermis and dermis relationship to each other (Figure 3-1). It was sometimes difficult to find the lesions depending on where the SOLs were located within th e 6mm biopsy punch, so further histology was performed using an alternative method. A face -on approach was employed, sectioning through the entire epidermis first and proceeding thr ough the dermis where the lesions have been commonly located (Figure 3-2 and Figure 3-5). This frontal (or coronal) section yielded many lesions in one viewing field, allowing them to be examined in rela tion to one another. Sectioning was additionally performed on the hi nge region from the ventral abdominal area and from the ventral gular (cer vical) region. This was done to determine a possible mode of transmission for potential infectious microorganisms within SOLs. Integument thickness in these regions would be compared to normal animals not exhibiting SOLs or PIX (found within the literature). Since many tissue components have approximately the same densities, it was necessary for them to be stained (Gartner and Hiatt 2001). Once sections adhered to slid es, various stains were used to show tissue organization, along with iden tifying a possible causative agent or agents. With all staining methods, controls were employed. A positive control was used to show what the microorganism should resemble in a tissue section. A negative cont rol consisted of normal alligator skin without SOLs to ensure that tissu e elements stained within the skin samples were considered normal (Figure 3-28).

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118 For the identification of normal tissue struct ures, a hematoxylin & eosin (H&E) stain was used (Figure 3-3 and Figure 3-4). This is the most commonly used stain in histology (Gartner and Hiatt 2001). H&E is used for nuclear and cy toplasmic staining. Hematoxylin is a base that reacts with the acidic portions of the cell; this is advantageous because DNA and ribosomes are acidic in nature. Eosin is an acid that dyes the basic components of the cell; these components include the cytoplasm and extracellular components. Nuclei will stain blue, while the cytoplasm and other tissue elements will stain various shades of pink (Carson, 1997c). This contrast makes viewing various tissue elements possible. Periodic acid-Schiff (PAS) was used for th e demonstration of polysaccharides and basement membranes. The reaction here is base d upon oxidation of tissue elements to aldehydes by periodic acid (Carson 1997c). Basement memb ranes will stain bright fushia, and background tissue will stain various shades of pink. This stai n was used for normal continuity of the alligator skin and for identifying the location of the lesi ons within the dermis. PAS can sometimes show hyphal elements (Carson 1997c), which stain bright rose. Toluidine blue staining is routinely used to de monstrate the presence of mast cells within tissue; however it was used in the current study as a rapid way to distinguish the normal continuity of sections. Mast cells and their gr anules will stain blue and the background will be various shades of blue (Carson 1997c). This stain was employed to show whether we had reached the point of the dermis and the subsequent lesions. Giemsa stains are typically performed on he matopoietic tissue to differentiate cellular elements such as DNA regions with large amounts of adenine-thymine bonding in chromosomes. It can additionally be used to demonstrate the pr esence of certain microorganisms. The principle behind this stain uses combinations of the basic dye methylene blue and the acidic dye eosin. In

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119 this staining technique, nuclei will stain blue, bacteria will exhi bit blue, and cytoplasm and other elements will be shades of blue and pink (Car son 1997b). For the purpose of the current study, a Giemsa stain was used for the detection of pa rasites and various bacterial agents. Commonly, reptilian nodules and granulom as are caused by the bacteria Mycobacterium A Ziehl-Neelsen method was completed on sect ions to show the pr esence of acid-fast bacilli. The principle behind this stain is based on the lipoid cap sule of the acid-fast organism absorbing the carbol-fuchsin stain, while at th e same time resisting decolorization (Carson 1997b). The stain will be removed from bacteria that lack the waxy coat. Acid-fast bacteria stains bright red, and the background is light blue. A Brown and Brennan's (B&B) Gram stain wa s used to detect th e presence of gram negative and gram positive bacteria in tissue. The concept behind this stain is that both gram positive and gram negative bacteria have peptidogly cans located in their cell walls. This enables both the gram positive and negative bacteria to take up crystal violet. Gram negative bacteria have an additional layer composed of lipopolysaccharide external to the peptidoglycan layer; this additional layer allows the gram-negative bacter ia to take up the basic fuchsin stain (Carson 1997b). The gram positive bacteria stains blue and the gram negative bacteria are red. Additional tissue elements are yellow. Grocotts methenamine-silver nitrate (GMS) fungal stain was used on sections containing lesions to detect the presence of fungal organisms. The principl e behind the GMS stain is that the polysaccharides located within the fungal cell wall are oxi dized by the chromic acid used in this procedure. If fungus is present, they will appear black (Carson 1997b). Additional tissue elements are green.

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120 The Verhoeff-Van Giesons stain was used for th e demonstration of elastic fibers within tissue. The principle of this st ain uses an overstaining method that is then differentiated. Since elastic tissue has a strong affinity for the iron-hematoxylin complex that is used in the method, it demonstrates these fibers in tissue (Carson 1997b). The decision was made to perform this stain to show the presence of elastic fibers within the dermis to distinguish them from pathogenic (fungal) elements that may be present. Trichrome stains are regularly used to de termine the difference between collagen and smooth muscle in tissue. This stain employs the use of three dyes, which are used to bind specifically to the collagen in tissue as well as different types of cells (Carson 1997c). Nuclei will stain black, cytoplasm, keratin and muscle fi bers will stain red, and collagen will stain blue. A Massons trichrome was performed on the PIX samples to distinguish skeletal and smooth muscle fibers form other pot entially pathogenic elements. After slides were stained, a coverslip was placed over the samples to protect the tissue from damage during visualization under th e light microscope (Carson 1997c) and for preservation. Coverslips were adhered using synthetic xylene based mounting media (Eukitt, Hatfield, Pennsylvania). Photomic rographs were taken of slides to characterize the lesions, and record the presence of any inflamma tory or abnormal tissue reactions. Since PIX was detected on a vari ety of farms from different states, an accurate comparison of the lesions was performed. Samples containi ng SOLs were processe d and stained with H&E from most of the farms for the comparison portion of this study. Once lesions were identified from each farm, they were evaluated to observe any similarities or differences among the farms; this was an important aim, primarily because th ere had been some speculation that the lesions

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121 might vary from state to state. Farms used in this portion of the histological study were Louisiana farms LA-1, LA-2, LA-3, LA-4, and Florida farms FL-1 and FL-2. Selected slides were taken to the University of Floridas Brain Institute to view the lesions with a confocal microscope. This microscope us es a set of fluorescent be ams of laser to increase contrast by using a special pinhol e to concentrate on specific areas and acts to center out-offocus tissues and allow for better visualization of certain elements (Prasad et al 2007). By filtering different beams, color differentials were noted. Specifically, a green filter (Figure 321), congo red filter (Figure 3-22), and a ye llow filter (Figure 3-23) were applied. Results Upon staining of the SOLs, m ost of the lesions were visualized as being non-necrotizing and composed primarily of lymphocytes; these were preliminarily identified to be the cause of the same SOLs identified upon initial gross examin ation. Various stains were used to show the progression and contents of the lymphoid aggregations. Once the lesions were examined, it was determined with the consultation of a pathologist that they were lymphocytic in origin. The lymphocytic nodules started their progression from the dermal regions of the integument and proceeded superficially toward the epidermis. The lesion starts to progress towards the surface of the epithelium, and in some cases has been shown to actually break through the epidermal surface (Figure 3-7, Figure 3-8, Figure 3-9); (this occurrence is believed to cause the PIX marks on a tanned hide). Wounds and infections in the dermis commonly migrate towards the epidermis as they clear up during the healing pr ocess. Using the face-on approach proved to be successful for lesion identific ation (Figure 3-2, Figure 3-5). These lymphoid nodules are well delimitated (F igure 3-4, Figure 3-6) and range in size from 0.5 mm to 1mm. Smaller nodul es are suspected to be SOLs in early development. Larger

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122 nodules are most likely in the la ter stages of development. The nodules are found within the papillary dermis most often and occasionally re cede into the reticular dermis (Figure 3-1). These nodules appear to be non-necrotizing a nd become contained at the end stages of progression. Some sections of skin contained more than one lesion, which directly correlates with the gross identification that sometimes show s more than one SOL per scale. None of the sections appeared to show any pathogens within them for this particular study; however, one of the GMS slides was inconclusive for fungal elements within the lesion from older samples. Fungi have been previo usly identified through histological procedures. Most of the lesions consiste d of areas of lymphoid aggreg ates, with small numbers of macrophages and dendritic cells seen. Focal ar eas of epidermal erosion were visualized overlying a dermal aggregate of primarily lymphocytes. In some specimens, a small number of lymphocytes surrounding dermal vessels associated in close proximity to the lymphocytic aggregate were observed. Also commonly, eosinophilic, granular, and nuclear debris seen on H&E stain were found. A peripheral rim of sm all lymphocytes was evident, and mild to moderate apoptosis of lymphocytes was sometimes present (Figure 3-8). Other cell types seen included Russell body plasma cells and cells havi ng segmented nuclei (neutrophils). There was abundant eosinophilic granular and nuclear debris in the aggregates, some of which may have been phagocytised by macrophages. Pigmentation and melanocytes were readily found in the dermis (Figure 3-4), which was consistent with the literature. Melanin accumulation was seen within the dermis, which was considered normal for the alligator. Most of th e samples available from farm FL-4 contained old PIX scars that had ruptured. U pon histological examination, areas of scar tissue were seen deep to areas of epidermal regeneration (Figure 318, Figure 3-19). The scar tissue was more

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123 concentrated around a spherical area where an SO L previously was and had erupted. The area appeared different than the normal irregular dense connective tissue of the dermis surrounding the lesion. Viruses are difficult to examine at the light microscopy level, however sometimes inclusion bodies are visible. None were se en in any of the lesions or surrounding areas. Using the confocal microscope, a green filter (Figure 3-21), congo red filter (Figure 3-22), and a yellow filter (Figure 3-23) were applied wh en visualizing slides. Well demarcated nodules were visualized using this techni que (Figure 3-20). Confocal microscopes are also used in the detection of microorganisms and viral inclusion bodies. No inclusion bodies were seen, and pathogen detection was inconclusive. Special Stains Hae matoxylin & eosin (H&E) (Figure 3-3), tolu idine blue (Figure 310), and periodic acidschiff (PAS) (Figure 3-11) stains proved to be paramount at locating and characterizing the SOLs. The lymphoid nodules best appeared with these three stains. A Verhoeff-Van Gieson (Figure 3-15) stain and Massons trichrome stain were perfor med to differentiate elastic, collagen and muscle fibers within the tissue and within the lesions. Thes e both proved beneficial in the detection of these fibers allowing them to be distinguish ed from possible pathogens. The Giemsa stain (Figure 3-14) Ziehl-Neelsen stain (Figur e 3-13), Brown and Brennan's gram stain (Figure 3-12), and the Grocotts me thenamine silver stain (Figure 3-16, Figure 3-17) were all considered to be negative. No organisms were present in any of the samples submitted for this portion of pathologic examination. While fungal elements had been visualized previously, the current set of slides examined did not yield any positive results. Hypotheses regarding this are based on the degenerating fungal elements seen upon electron microscopy identification (discussed in the following chapter).

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124 Comparison of PIX Lesions Among Farms Sa mples were taken from many of the alligato r farms to determine the connection of SOLs from farm to farm and from stat e to state (Figure 3-24). The lesi ons were all characteristically similar upon examination with H&E staining. Each of the lymphatic nodul es exhibited similar progression, sharp demarcated borders, and ce ll populations. All nodul es were located in roughly the same areas in the dermis, with some starting to erupt and located closer to the epidermis. In addition, a sample from the 1999 outbreak wa s compared to the newer lesions examined (Figure 3-25). This sample from Farm FL-1 wa s taken in 1999 when PIX was first visualized in Florida. The epidermal layers pushed outwa rdly where the lesion was located deeper. Eventually, the nodule pushed through all of the in tegumentary layers and eruption ensued. This eruption has been hypothesized to cause the PIX marks evident upon tanning of the hide. This was used as a comparison to the newer samples to determine if the lesions appeared the same now as they had previously. Examination has led to the conclusion that they are in fact the same lesions. Histological Examination of the Hinge Regions in Alligators The hinge regions found in typi cal reptilian integum ent were generally thi nner than the other regions of the integument, specifically the scales themselves. Examination of this region was important to test the hypothe sis regarding possible transmissi on route of dermal pathogens into the integument. Sections of these tissue samples were examined from two areas that commonly contain PIX lesions; the mid-ventral region (Figure 3-26) and the ventral gular region (Figure 3-27). Samples that had SOLs upon visual identification were examined so a relationship between SOLs and the hinge region could be examined.

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125 Upon examination, the hinge areas (in complian ce with the literature) were much thinner than the medial areas of the scale. In the se ction taken from the gular region, the epidermis was substantially thinner in the hinge region than the other portions of th e scale. The dermis directly below the epidermis was denser than the areas of dermis located in the middle of the scale. The results were consistent with the literature and th ere were no differences in the thickness of this region in animals with SOLs. Discussion A setback to the histological exam ination of the SOLs and PIX lesions was the absence of a firm diagnosis of and fungi or other pathogens via staining. While intr alesional diagnosis of a fungus could not be definitively made, this did not rule out the possibility. Some fungi in occasionally do not show up in stains (Almeida et al 2003). Since this is the first case of Hortaea werneckii in a reptilian species, no comparisons can be made for exactly what the disease process should resemble (see chapter 5). The fungal structures may have regressed and therefore not be available for visualization vi a histopathology. The al ligators integument reacted to some infectious damage; other dia gnostic testing performed in this study presents evidence that a fungal etiology is th e most logical explanation. Samples from farm FL-4 showed extensive sc arring and seemed to have lost the presence of nodules. Eruption may have ensued upon further development and scar tissue formed in its place. The histological examina tion of ruptured and old PIX lesi ons shows the ability of these nodules to regress or be pulled fr om the integument duri ng the staining process. After either of these events, the area that once contained these lesions seems to undergo repairs as indicated by the persistence of fibrous connective tissue and scar formation. Ou r study indicates that this scar formation may result in a PIX scar on a tanned hide. This is not a common occurrence because animals are usually killed before this type of lesi on is noted. Animals are not kept over four feet,

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126 whereas this type of scarring may take several mo nths to manifest and is evident in older and larger animals. While the lesions may not be a granuloma by de finition, it is important to note that birds and reptiles appear to re act with granulomas to a wider ra nge of infectious agents than do mammals (Montali 1988). The lesions seen were all non-necrotizing with a diverse population of lymphoid cells. Since this is the first identification of H. werneckii in a reptilian species, there is no data in the literature fo r comparison. Development of granulomas in reptiles is usually temperature-dependant and begins as a central ma ss of necrotic heterophils that will stimulate a foreign body response, thus attr acting macrophages to the lesion (Mader 2005). There were some macrophages seen in the SOLs, but not to th e extent that would be expected of a typical granuloma. These lymphoid nodules are well delim itated, which is an additional characteristic of granulomas. As noted previously, we do not consider this comparable to normal mammalian granulomas, but similarities are seen. Giant cells were seen in a few of th e early lesions, but not in the recent samples. The immune system provides defense agains t infections caused by foreign organisms (Cooper et al 1985). Foreign materials will then elicit an immune response by the formation of antibodies and will ultimately be destroyed by ac tivated cells. Most reptile immune response appear to be T-cell dependant, with a primary response of IgM and a secondary response to IgY (similar to IgM) (Tizard 2000). The main problem in reptile immunity is that most of the mechanisms of immunity are still unknown. It is known that there is a temperature dependence of antibody synthesis in reptiles. Of the cell types visualized in the nodules, lymphocytes seem to be predominant. It is not known what type of lymphocyte is visualized, ho wever T-cells would be a possible candidate for

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127 the nodules appear to be a cell-mediated response. Also seen were macrophages and neutrophils. Macrophages and neutrophils are essentially phagoc ytic, and neutrophils a dditionally cause rapid elimination of microorgani sms (Cooper et al 1985). The stains performed were necessary in orde r to detect the lesions and the presence of pathogenic organisms within tissue. While histology is often regard ed as very sensitive, it does have the potential to overlook microorganisms w ithin tissue samples. Most of the results obtained from the stains used for the current st udy were beneficial. Ma ny microorganisms were ruled out, and continuity of the nodules was visualized. The absence of positive staining is not viewed as being unsupportive towards the results. The lesions all resemble one another when compared between farms and states, which argues that these nodules are most likely cause d by the same pathogen or disease process. Duplicate results between states and farms reveal the likeli hood of continuity between areas. The comparison portion of the curre nt histology was useful in dete rmining the association of PIX skin disease between farms and states. The ol der samples from 1999 that were examined also had the same characteristics as the newer samples. This leads to the conc lusions that not only are the nodules between farms the same, nodules from almost ten years ago are the same nodules seen currently. The histology of the hinge region was performe d to speculate as to a possible transmission site between animals. Another possibility in the transmission of pathogens to these sites is through the vasculature located within the derm is. Since dermal vessels were seen upon examination by a pathologist, assumptions can be made as to the mode an organism may take to reach the integument. Alligators have a high amount of cutaneous vasculature when kept at high temperatures (Smith et al 1978 and Smith 1976) Cutaneous heat flow during warming far

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128 exceeded heat flow during periods of cooling. Si nce these animals are kept at high temperatures, it can be assumed that cutaneous blood flow is working to capacity and blood flow through the animal is high. Pigmentation and melanocytes were readily f ound in the dermis (Figure 3-4), which was consistent with the literature. Most animals onl y have melanocytes in the dermis in response to disease, however in the reptiles it is considered normal. There seems to be a varying degree of how many melanocytes are found, as it seems to vary between animals

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129 A B Figure 3-1. Hematoxylin and Eosi n stain (05R-66). This sec tion was completed using the sagittal sectioning technique. Note the epidermis located superficially with the lesion located deeper in the dermis. Lesions extend from the papillary dermis into the reticular dermis. A) 4X. B) 10X.

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130 A B C Figure 3-2. Hematoxylin and Eosin stain (05R-105). This is an example of the face-on, or coronal section. This allowed the lesions to be viewed more effec tively than with the sagittal sections. This type of sectioni ng enabled the lesion to be viewed from its most superficial aspects. A) 2X. B) 10X. C) 25X.

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131 A B C Figure 3-3. Hematoxylin and Eosin stain (05R-267). Note the encapsulation of the lesion and the lymphoid cells (arrows). A) 2X. B) 10X. C) 40X.

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132 A B C Figure 3-4. Hematoxylin and Eosin stain (06R-155). This lesion is well delimitated; in addition, areas in the periphery contain a new deve loping lesion. Arrow pointing to darkened areas consistent with melanin accumu lation. A) 2x. B) 10x. C) 25X.

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133 A A B Figure 3-5. Hematoxylin and Eosin st ain (07R-60). This is an example of the face-on approach that allows for improved visualization. So me animals scales will possess more than one lesion. A) 2X. B) 10X. C) 25X.

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134 A B C Figure 3-6. Hematoxylin and Eosin stain (07R-73). Note the one encapsulated lesion (left) situated next to a developing lesion (r ight). A) 2X. B) 10X. C) 25X

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135 A B C Figure 3-7. Hematoxylin and Eosin stain (2002R-17). The lesion is starting to erupt through the epidermal layers. A) 2X. B) 10X. C) 25X.

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136 A B C Figure 3-8. Hematoxylin and Eosin stain (2002R-26). Rupturing lesi on. Note the pale area that may be the lymphocytes undergoing apopt osis. A) 2X. B) 10X. C) 40X.

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137 A B C Figure 3-9. Hematoxylin and Eosi n stain (2002R-166A). Rupturi ng lesion affecting the outer epidermal area. Possible eruption will ensue upon continued development. This is the proposed hypothesis as to the causation of PIX marks on tanned hides. A) 2X. B) 25X. C) 40X.

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138 A B C Figure 3-10. Toluidine Blue stain (07R-20a). Th is section illustrates the ability for multiple lesions to be located within a single scale. A) 2X. B) 2X. C) 10X.

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139 A B Figure 3-11. Periodic Acid Schiff stain (05R -676). PAS was done to show normal tissue elements of the alligators integument and nodules. No fungal elements were visualized. A) 4X. B) 10X.

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140 A B Figure 3-12. Brown and Brennan stain (05R-676) B&B was performed to distinguish the presence of gram negative and gram positive bacteria. The stain was considered negative for the presence of bacteria. A) 4X. B) 10X.

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141 A B Figure 3-13. Acid Fast stain (05R-676). An aci d fast was used to determine the presence of Mycobacterium Commonly reptilian integumentary lesions are caused by this bacterium, however none were observed in the lesion. A) 4X. B) 10X.

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142 A B Figure 3-14. Giemsa stain (05R-675A). Giemsa stains were performed to show the presence of bacteria. None were visu alized. A) 4X. B) 10X.

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143 A B Figure 3-15. Elastin stain (07R-6). This st ain was performed to determine the difference between elastic fibers and possible fu ngal hyphae present. A) 2X. B) 10X.

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144 Figure 3-16. Grocotts Method for fungi stain (07R-6). This wa s performed to determine the presence of fungal hyphae. Of the lesi ons obtained recently, none were positive for fungal elements. A) 2X. B) 10X.

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145 A B Figure 3-17. Grocotts Method for fungi stain (05R-676). A) 4X. B) 10X.

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146 A B C Figure 3-18. Old PIX lesion. This is an example of a lymphatic nodule that has either regressed or erupted. A) 2X. B) 10X. C) 25X. Note the tissue difference between the developed scar (left) and the normal dermal tissue (right). The normal dermal tissue is usually an irregular dense connective tissu e, while the area containing the scar is infiltrated by scar tissue.

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147 A B C Figure 3-19. Old PIX lesion. This is an example of a lymphatic nodule that has either regressed or erupted. The collagen structure is not organized in th e scar tissue/remodeled area and therefore is not as strong. A) 2X. This area has a portion where epidermal skin is evident in the middle of the scar. This is due to regeneration afte r eruption. B) 2X. A section deep to that in picture A. C) 10X. Note the area on the left that has a high degree of collagenous connectiv e tissue compared to the relatively normal dermal tissue on the right.

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148 Figure 3-20. PIX visualization using a confocal microscope. This was performed for better visualization of the nodules and to determin e the presence of mi croorganisms. PIX lesion from a 1999 sample (pl ease refer to figure 3-25). Figure 3-21. PIX visualization us ing a confocal microscope. A gr een filter was used to attempt microorganism visualization. PIX lesion fr om a 1999 sample (please refer to figure 3-25).

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149 Figure 3-22. PIX visualization wi th a confocal microscope usi ng a Congo red filter. PIX lesion from a 1999 sample (pleas e refer to figure 3-25). Figure 3-23. PIX visualization us ing a confocal microscope with a yellow filter applied. PIX lesion from a 1999 sample (pl ease refer to figure 3-25).

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150 A B C D E F Figure 3-24. Comparison of lesions. Note that all of the lesi ons have similar cell populations (with lymphocytes predominating), demar cation develops at the end stages of development, and similar locations within th e integument. A) 10X. Farm LA-1. B) 2X. Farm LA-2. C) 10X. Farm LA-3. D) 2X. Farm LA-4. E) 2X. Farm FL-1. F) 25X. Farm FL-2.

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151 Figure 3-25. PIX lesion from a 1999 sample. This sample is from Farm FL-1 and was taken in 1999 when PIX was first visualized in Flor ida. Note the epidermal layers pushing outward where the lesion is located deeper Eventually, the nodule will push through all of the integumentary layers and erupt. This eruption is hypothesized to cause the PIX marks evident upon tanning of the hide. Note the similarity between this lesion and lesions from current years (Figure 3-24).

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152 A B C Figure 3-26. Photomicrograph of hinge region from ventral belly. A) 2X. B) 2X. C) 10X. Note the thinner areas within this region compared to the peripheral portions of the scale (arrow).

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153 Figure 3-27. Photomicrograph of th e hinge region from the gular ar ea. A) 2x. B) 10X. C) 10X.

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154 Figure 3-28. Photomicrograph of normal alligator integument. 10X.

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155 CHAPTER 4 THE USE OF ELECTRON MICROSCOPY IN ID ENTIFYI NG PATHOGENS LOCATED IN PIX LESIONS Introduction While histo logy will demonstrate th e cellular population that is needed to identify disease progression, there are some benefits to vi ewing tissue at a much higher resolution and magnification. Use of an electron microscope allows evaluation of the topography, morphology and composition of the cells that are being exam ined (Gartner and Hiatt 2001b). Additionally, many pathogens (such as viruses) are not dis cernable unless visualized at high resolution. Electron microscopy (EM) is a useful tool for viewing and identifying cellular components within tissue. There are two basic types of el ectron microscopes used in research: transmission and scanning. The use of a transmission elect ron microscope has made it possible to view structures such as organelle s and pathogens that might otherwise go undetected. Scanning electron microscopes are generally used to view su rface structures and will elicit a useful threedimensional view. For the purpose of this study, transmission el ectron microscopy was used to view the spherical opaque lesions (SOLs) found in the alligators skin along with the cellular components within the surrounding area of the nodules. This technique made it possible to view microorganisms or viral particles that may be present within the tissue. While electron microscopy was added to the diagnostic panel to establish causation, its use on every specimen is both impractical and costly. Although only two lesions were examined by electron microscopy, this proved to be sufficient for characterization purposes. Emphasis was placed on cell types that were located within the nodules, both infl ammatory and pathogenic in origin.

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156 Materials and Methods Gloves were worn during all co llections, and initial biopsies were obtained with a sterile 6mm biopsy punch. Samples taken for electron microscopy were fixed in gluteraldehyde upon collection and refrigerated until processing. It was important to expose the samples to fixative as soon as possible after blood disrupt ion. Gluteraldehyde is a fixativ e often used for visualization of cellular structures (Carson 1997). This dialdeh yde preserves these structures to resemble the organization the cells had when still living (Sam uelson 2007). The specimens were postfixed in osmium tetroxide. Samples were added to equal amounts of osmium (final 1% concentration) and sodium cacodylate buffer. Once sections were prepared for processing, after incubation fo r one hour, they were washed w ith two additional changes of sodium cacodylate buffer and then dehydrated in a graded manner in EtOH and then acetone. The samples were then placed into an acetone Ep on-Araldite mixture (plastic) and finally 100% Epon-Araldite. Epoxy resins are required as embedding media when ultrastructural visualization is desired. Samples were left overnight in a Roto-torque. Once the samples were fixed in the plastic bl ocks, they were trimmed using glass knives and examined. Selected blocks were sectioned on an ultramicrotome at 1m and stained with toluidine blue to target specific areas of interest Tissue was visualized using a light microscope, giving researchers an opportunity to view the structures of the lesion and to determine if the block was suitable for EM viewing. These area s of interest were further examined for subsequent ultramicrotomy. Once 1m sections were selected, ultra-thin sections (80-90 nm thick) were made to distinguish the cells w ithin the lesion and th e areas surrounding the le sions (Figure 4-1 and Figure 4-2). Sections were trim med with a razor blade to reduce the area of interest to a size suitable for ultramicrotomy. Sections were gras ped using copper formvar-coated grids. These

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157 sections were stained with Reynolds lead citrate and uranyl aceta te. Coated grids allowed for better stabilization of the tissues to the grids. Examination was limited to areas within the lesions themselves. Sections were viewed with a Hitachi H-7000 transmission electron microscope located at the Ultrastructural Core Facility at the University of Florida. Results Electron m icroscopy revealed a variety of ce llular components (Figure 4-5 and Figure 4-7). Most notably, fungal elements with their distinc tive cell walls were visualized within the nodule (Figure 4-4 and Figure 4-6). U pon consultation with a specialis t (Dr. James Kimbrough), these hyphae were seen to branch in places which is a characterist ic of filamentous fungi. In some sections, the fungi were seen to degene rate (Figure 4-8). Cell walls appeared to be double layered, which is also a t ypical characteristic of fungal ce ll walls (Boeger et al 2005). Neither viral particles nor bacteria were en countered within the viewed tissues. Discussion While histo logy will show many of the cells a nd cellular components that are needed to identify a disease, there are some benefits to viewing tissue at a much higher resolution and magnification. Many viruses are not discernable unless seen at the elec tron microscopy level, and many diseases and fungal infections have been properly diagnosed and viewed using electron microscopy (Boeget et al 2005, Reddacli ff et al 1993, Mittag 1993, and Holker et al 2004). The characteristic fungal elemen ts visualized within the SOLs are consistent with results from microbiology and molecular testing performe d (results in following chapters). While intralesional diagnosis was not made using histol ogy, the presence of fungi within the lesions at the electron microscopy level is concrete evidence of a correla tion between PIX, SOLs, and a fungal etiology. Multiple br anching elements with characteris tic double cell walls were seen in

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158 many sections. Since animal cells do not have a cell wall, the presence of this structure in the lesion is consistent with the presence of fungi. The degeneration of some of the fungal elements may explain why we do not see them in histology; the fungus may die and di sintegrate prior to visualizati on and staining. A few of these degenerating elements were seen in the samples. Since viruses and viral particle s are more readily visualized by electron microscopy, their absence within the lesions examined suggests th at PIX is not caused by a virus. Alligator samples obtained for characterization in th is study did not cont ain viral elements.

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159 A B Figure 4-1. Photomicrograph of an SOL located w ithin the superficial portion of the dermis. There is some artificial t earing within the lesion, however, these sections were performed only to locate the lesions within the block. The arrows are indicating the peripheral surface of the lesions. 1m pl astic section, Toluidine Blue. A) 10X magnification. B) 20X magnification.

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160 Figure 4-2. Photomicrograph of th e periphery of an SOL. Note the possible fungal elements evident (arrow). 1m plastic section, Toluidine Blue. 40X magnification.

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161 Figure 4-3. Electron micrograph depicting an overview of an area within the SOL. Arrow is pointing to a structure with char acteristic fungal elements. This is most likely the mycelial phase of a fungus, not a yeast phase.

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162 A B Figure 4-4. Electron micrograph of a fungal element. Higher magnifi cations of Figure 4-3. A) A fungus found in an SOL nodule (arrow). B) Greater magnification of A. Note the presence of double cell walls aroun d the periphery of the cell.

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163 Figure 4-5. Electron micrograph of an area within an SOL. Arro w is pointing to another fungal element. 3000X. Figure 4-6. Electron micrograph of a fungal element. This is a higher magnification of the above figure. Note the branching that is ch aracteristic of fungal morphology (arrow). 20,000X.

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164 Figure 4-7. Electron micrograph of an area within the lesion exhibi ting high cellularity. Arrow is pointing to a degenerating fungal element. 3500X. Figure 4-8. Electron micrograph of a de generating fungal element. 20,000X.

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165 CHAPTER 5 IDENTIFICATION OF MICROBIOLOGICAL ISOLAT ES FROM PIX LESIONS Introduction Microb iological culturing is a diagnostic tool used to detect viable pathogenic microorganisms within tissue. Upon some of the primary samples taken from alligator skin containing spherical opaque lesions (SOLs), it wa s unclear whether the SOLs were due to an infectious response. Infectious agents that might have been causi ng the SOLs were fungi, bacteria, virus, and parasites. Organisms that are considered nonpathogenic or normal flora to the alligator were ruled out ear ly in the preliminary diagnosis. Microbiological techniques focused on methods that would accurately detect pathogenic microorganisms located within the SOLs in the alligator skins. One organism isol ated from SOLs that is not considered normal flora for the alligator was Hortaea werneckii H. werneckii is a dimorphic fungus found throug hout humid regions of the world. Dimorphism allows the fungus to have both mycelia l and yeast stages. This is usually dependant on environmental temperatures. Th e yeast phase of fungus tends to occur at higher temperatures (37oC), whereas the filamentous phase is found at lower temperatures (25oC). H. werneckii is the etiological agen t of the condition in man known as tinea nigra. H. werneckii has a predilection for tropical and subtr opical areas around the world. The warmth and humidity of these coastal envi ronments lend a role in the abil ity of this fungus to thrive. Cases of tinea nigra have been reported in Asia Africa, Central and South America, and North America (Diniz 2004, Schwartz 2004). Most of the cases in North America have been reported from the Atlantic southern coastal areas (McKin lay et al 1999), such as in Florida, Texas, Alabama, Louisiana, Virginia, Georgia, and Nort h Carolina (Schwartz 20 04). It is a saprobic fungus (derives its nutrition from dead organic ma tter) that is found in a va riety of environments

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166 that include soil, plants, beach sand, air, de composing fish, and decaying wood material (de Hoog and Gerrits van den Ende 1992). Various environmental conditions are preferre d by fungi depending on the species. Most fungi favor environments that are moist, humid, and dark. The housing situations in which most farmed alligators are kept resemble conditions th at are conducive for mycological growth. One major factor is environmental temperature. In a farm setting, alligators are kept at higher temperatures (roughly 37oC) to promote rapid growth and development, which are coincidently temperatures that are also conducive for mycotic growth. Along with elevated temperatures, these animals are housed in aquatic enclosures with high humidity that allows fungi to thrive. The purpose of this portion of this study was to use microbiological techniques to accurately detect possible pathogenic microorganism (s) that may be causing SOLs to form in the integument of farmed American alligators. The addition of microbiol ogical culture to the diagnostic panel was needed to detect viable pathogenic species that may be present. Once a possible causative agent was identified, techniques focused on cultivation of that species. Materials and Methods Ani mals exhibiting SOLs were sampled for the complete diagnostic panel. Sterile gloves were worn during all collections and changed between each animal being examined. New 6mm Keyes punch biopsies were used on each sample ta ken for microbiology. Prior to collection, the area was cleaned with alcohol and sterile gauze to kill superficial microbes that may be present. This would ensure that any growth taking plac e was a result of the mi crobial population within the lesion and not merely a contaminant on the su rface of the alligators skin. Biopsies were placed into sterile vials and labe led accordingly. Each biopsy was placed into a separate vial. Samples were submitted to the Clinical Microb iology Service Center in the University of Floridas Veterinary Medical Center. Samples we re macerated with sterile blades and placed

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167 onto selective media. The sample was embedded in to the agar in specific places to allow for optimal nutrition. Sabouraud dextrose agar wa s used for the cultivation and isolation of pathogenic fungi, specifically dermatophytes. Brai n heart infusion agar was used as a general purpose medium suitable for cultivating bacteria, yeas ts and molds. This agar uses additions of 10% sheep blood for the isolation of fungi. Alligat or skin with no visible SOLs were used as negative controls. Agar plates exhibiting growth were initially left to incuba te for 25 days. Plates were checked every few days and any growth was noted. A dematiaceous fungus was noted during one of the submissions. Dematiaceous parasitic fungi typically require longer growth periods than other non-pathogenic species, so plates were th en left to incubate past 35 days for growth to be observed. Results An unidentif ied fungus was initially cultured di rectly from a sample containing an SOL, however identification could not be made at the UF laboratory. The fungus was a medium brown growth that appeared to turn dark black and shiny upon development (Figures 5-1 and 52). The reverse of the colony was black to olivaceous. The reverse is seen when the Petri dish is turned over to examine the back of the colony. Microscopically, the gr owth was filamentous with darkly pigmented cells (Fi gures 5-4 and 5-5). The growth was sent to the Fungus Testing Laboratory at the University of Texas Health Sc ience Center in San Antonio, Texas. Fungi are commonly identified based on their morphological characteristics, specifically their colony morphology and hyphal distinctions. Upon examination, the fungus was identified as H. werneckii a pathogenic fungus found in the epidermis of humans and causing the condition tinea nigra. The Fungus Testing Laboratory has identified this f ungus previously. The criteria that the laboratory used to

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168 definitively identify the growth as H. werneckii were as follows: initial shiny, black colony grown on sabouraud dextrose later revealing an olive-grey growth with black reverse, anellidic conidiogenesis, dimorphism, dematiaceous, and septate conidia. The first of these growths was identified in 2002 from a farm in Florida; since then, the fungus has been cultured on a total of 3 occasi ons from farms in both Florida and Louisiana (Figure 1-1, Figure 1-2, and Fi gure 1-3). There were two additional dematiaceous fungal cultures isolated during the study, however comple te diagnosis down to the genus or species could not be made. Based on other diagnostic tools, no known bacteria viruses, or parasites were seen within the lesions. Microbiological growth focused prim arily on the mycological characteristics of the SOLs. Since H. werneckii causes skin lesions in humans (a nd a single case of a guinea pig in Japan), similarities with disease processes in the alligator were seen. The H. werneckii growths in all cases pr ogressed directly from the lesion embedded in the agar (Figure 5-3). It is important to note that in all of the instances that H. werneckii was cultured the growths came direc tly from where the tissue contai ning the SOLs were embedded in the agar. This illustrates that the fungus grown was not as a result of error or contamination. By growing outward from the lesion, this proves that the fungus is located within the SOL found in the alligator hides. Characteristics were typical of H. werneckii as described by the Fungus Testing Laboratory. Colonies took longer than 25 days to grow, and often did not become evident until after day 28. Isolates were grown at room temperature (about 29oC); this fungus does not grow at temperatures (37oC) used to culture other fungi. Some of the initial sa mples submitted to

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169 microbiology were not incubated fo r that length of time, which ma y explain why there were not more positive growths noted. None of the negative controls exhibited any H. werneckii growth. Discussion Hortaea werneckii the causative agent of tine nigra, affects both m ales and females and many age groups. Tinea is a general name for superficial fungal infectio ns of skin, hair, and nails. The period of incubation of tinea nigra varies. Cases ha ve reported incubation to be between two to seven weeks (Diniz 2004, Schw artz 2004), however one report has shown an incubation period of 20 years (McKinlay, et al 1999). In humans, tinea nigra is often found on the stratum corneum of the epidermis. An H. werneckii infection will clinically appear as dark asyptomatic brown to black pigmented macules on the palms or soles of the extremities that en large centrifugally (Uezato et al 2006). The nonscaly lesions will grow from one to five centimeters and may at times connect to one another (Diniz 2004). There appears to be minimal de squamation, and the borders are well delimitated. Its appearance is seen as a non-el evated macule (Perez et al 2005) and is usually located on the palmar regions and along the fingers, but has also manifested on the dorsal region of the hands, the inferior portion of the feet, and the back (Diniz 2004). Recently, H. werneckii was isolated from blood and sple nic abscesses in humans. Traditionally invasive fungal in festations are rare, and are of ten found in conjunction with neutropenia and high doses of corticosteroids which will suppress the immune system (Ng et al 2005). Two patients with acute myeloid leukemia e xhibited colony growth arising from clinical samples of blood and splenic abscesses. Both gr owths were subjected to genetic testing, and the identity of a 306-bp fragments were confirmed by sequencing (Ng et al 2005). These were the first cases of human systemic infections of H. werneckii to date, which demonstrates that

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170 superficial mycoses have the ability to become sy stemic in immune suppressed patients. In the present study, no alligator with SOLs exhibited systemic illness. In addition to being found in humans, H. werneckii has been identified in one additional animal species. This fungus was identified as the causative agent of a superficial mycotic infection of a guinea pig in Japan (Abliz et al 2003, Sharmin et al 2002). The animal had been scratching vigorously and was noted to have formed dark lesion s on the dorsal surface of the thorax. Upon microbiological isol ation, colonies formed from scra pings of the lesions revealed a shiny black yeast-like growth. Th is growth was identified as H. werneckii and further molecular testing confirmed this diagnosis. Based on gross observations, histological appearance, and microbiological studies on farms in Louisiana and Florida, we have hypothesized that many SOLs are caused by fungi with some of them caused by H. werneckii There is a suspected relati onship between the appearance of PIX, SOLs, and the presence of this fungus. The size of the SOL and nodule incidence may ultimately depend on the number of fungal spores th at initiated the infecti on, the virulence of the organism, and the status of the immune system of the animal. Size of inoculum or number of spores causing infection likely depends on the nu mber of spores in the environment of the alligator. The virulence of the organism depends on the variety of the fungal pathogen present on the farm, while the ability for the fungus to grow may depend on the immune status of the alligator. A relationship betw een the ambient temperature, body temperature, and seasonality may also be contributors. Dematiaceous parasitic fungi typically requi re longer growth periods than other nonpathogenic species. In the beginni ng of the study, samples were not left to incubate past 25 days; since this fungus typically requ ires a longer incubation time the plates were left for a longer

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171 period of time. This may be why the research ers did not visualize mi crobiological growth of H. werneckii more often in earlier isolates. Microbiological cultures are not the only di agnostic test used as a positive detection method for this fungus. The presence of a known dermal pathogen located within the SOLs may lead to the development of these lesions (since no other etiological agents ha ve been identified). H. werneckii is likely to have a direct association with PIX. One hypothesis is that the fungus may travel throughout the bloodstream to the surr ounding tissues, where the alligator then elicits an immune response to the fungus. Histological ly, dermal vessels were sometimes noted around the lesions. However, no blood borne microorganisms were seen. The thinner areas of the hinge region of the alligators or small abrasions on the skin coupled with immune suppression may lead to an opportunistic infection. Additionally, the ventral area of the body is most sus ceptible to injury since this po ssesses the thinnest areas of the integument. The animals are regularly kept in ho uses that contain concre te floors which lead to minor abrasions on the skin which might make the animal more susceptible to entering pathogens. The question remains whether H. werneckii is the main cause of the SOLs, or just an association with the problem. Diseases caused by microorganisms will manifest differently depending on the species in which they are found. Since this is the first case of the fungus seen in a reptilian species, there are no comparisons to be made. The data here presents a direct association between H. werneckii and the SOLs. There may be additional accelerators, promoters, enhancers, etc.; however this dermatol ogic fungus is seen as being one of the primary causes of PIX skin disease in the alligator.

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172 Fungi are sometimes regarded as a pathogeni c or opportunistic infection depending on its virulence. A viral infection, bacterial inf ection, or immunosuppression may be the primary initiation in a disease. We ar e not ruling out that there may be other underlying conditions that make the alligator susceptible to SOLs. At th e current time, the identif ication of a pathogenic integumentary fungus isolated in multiple cases leads to th e association of H. werneckii and PIX. No other pathogens have been cultured mi crobiologically or seen histologically. In the future, agar that is specific to H. werneckii can be constructed and used for identification. The literature discusses such media. Since H. werneckii has a high tolerance for salt and has been found in halosa ltern areas, selected media containing concentrations of NaCl can be prepared (Kane and Summerbell 1987).

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173 Hortaea werneckii Figure 5-1. Photograph of the Hortaea werneckii growth from a Florida alligator farm in 2002. This shows the characteristic olive to black growth that this species exhibits. This culture was sent to the Fungus Testing Labor atory at the University of Texas Health Science Center in San Antonio, Texas. Figure 5-2. Photograph of a 2003 Hortaea werneckii growth from a Florida farm. Hortaea werneckii

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174 Figure 5-3. Photograph of Hortaea werneckii from a farm in Louisiana in 2006. This culture also exhibited the characteristic growth of the species on reverse examination. Note that the growth seen here is coming di rectly from where the macerated SOL was embedded into the agar.

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175 Figure 5-4. Photomicrograph of a scraping from the Hortaea werneckii growth in Figure 5-3.

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176 A B Figure 5-5. Photomicrographs of the Hortaea werneckii growth from Figure 5-3. A) 40X with slight magnification. B) Single celled chains.

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177 CHAPTER 6 THE USE OF VIROLOGY IN THE IDENTIFI CATION OF A CAUS ATIVE AGENT OF PIX Introduction The use of cell cu ltures in identifying causative agents of disease is a useful and routine laboratory technique. Fibroblast ce ll culture monolayers have proven to be a satisfactory system in which to grow and replicate various pathoge ns, most commonly viruses. Cell culture is a process that allows cells to be grown and ma intained under controlled conditions after their removal from the body or embryologic tissues. A cell line acquires the ability to proliferate exponentially with the continued nourishment of the cells and subsequent passing (splitting the cells and transferring them to additional plates). By developing an undifferentiated cell line using fibroblasts, it is possibl e to synthesize and maintain th e extracellular matrix of many animal tissues. While other diagnostic tests used in this st udy identified that a pa thogenic fungus of the integument was associated with the lymphoc ytic nodules found in the alligators skin, experiments were performed to identify any additi onal pathogens that may also be present. A study using cell cultures was perf ormed to identify a possible vi ral association of PIX. The use of fibroblast cell lines to detect viruses has been an important tool in the identification of many infectious diseases. A known cell line of alligator embryo fibroblasts (AEF) was being maintained at the University of Floridas Depart ment of Pathology and Infectious Disease. This is one of the first studies using th is cell line in the detection of pathogenic microorganisms within tissue samples. Since many viruses are species specific, it was important to use a known alligator cell line to determine the presence or absence of a viru s within alligator SOLs. Once fibroblasts have grown to confluency, addition of samples that contain a virus should induce cytopathic effects

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178 (CPE). CPEs are defined as any damage to infected host cells that may be caused by the presence of infectious viruses. Morphological changes in the host cell include altered shape, detachment from substrate, lysis, membrane fusion, altered membrane permeability, inclusion bodies, and apoptosis (Colwell et al 1974, Hunt and Brown 2005). The goal in using virology in this project was to determine if there was evidence for a virus causing the SOLs and subsequent PIX lesions. In addition, since CPE had not been previously shown to occur within the AEF cell cultures, an additional study was performed to show what positive CPE would look like within these cells; thes e could then be used as a positive control. Recently, researchers at LSU suggested an a ssociation between PIX and West Nile virus (Nevarez 2007) so additional steps were taken to examine viral presence within SOLs. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to specifically analyze SOLs for the presence of common viruses (St. Louis ence phalitis, or SLE, Eastern equine encephalitis, or EEE, and West Nile virus, or WNV) that may be present. Materials and Methods AEF cell cultures were p reviously created and maintained in a laboratory at the College of Veterinary Medicine (Dr. Carlos Romero, Principal Investigator). Earlier, researchers in the laboratory had developed this cell line in vari ous growth media containing concentrations of dimethyl sulfoxide (DMSO) and then cryopreser ved the AEF cells in liquid nitrogen at -196oC. DMSO is used as a cryoprotec tant to preserve organs, tissu es, and cell suspensions (Lynn 1996). Use of AEF to Determine the Presence of a Virus in SOLs AEF cells were taken from liquid nitrogen and thawed in a 37oC water bath. All of the following methods were performed within a bio-safety level 2 hood. Sterile gloves were worn during all procedures. The hood was disinfect ed with 70% ETOH during and in between procedures to prevent contamination. Cells were placed immediately in labeled 75cm2 culture

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179 flasks and cultured with 20 ml of DMEM (Dul beccos Modified Eagles medium) supplemented with 5% fetal bovine serum (FBS) to allow the fi broblasts to grow to confluency. Antibiotics such as penicillin, streptomycin and fungizone were added to suppress an y microbial growth. The flasks (Figure 6-1) were placed in an incubator at 27oC with a constant influx of 5% carbon dioxide. The cells grew best when this percentage of CO2 was used. On the second day, cell growth was assessed. If confluent, they were trypsinized, counted, and re-plated. Each new dish contained 1x106 cells per 35mm dish. Two ml of DMEM with 5% FBS were added and cells were incubated overnight at 27oC. Splitting cells is important for cell viability. On the third day, cells growth was determined. Alligator SOL biopsies were processed for inoculation on the AEF cultures. The 6mm biopsy punch sample was weighed and diluted to a 5% sample volume in DMEM. The tissue was homogenized with a sterile glass mortar and pestle to disrupt the lesion components. Th e samples were then clarified at 3000 rpm (Jouan, CR3i, St-Herblain, France) for 5 minutes at 4oC. A 0.5ml digest of the supernatant solution was used to inoculate AEF cells. The dish was incubated for 1 hour at 27oC, and then the inoculum was removed. Two ml of DMEM supplemented with 2% FBS was added and the cells were placed in the incubator at 27oC. A negative control with no inoculum was used to assess any CPE due to contamination. The dishes were examined daily for seven days to detect any CPE by comparing th e inoculated with the control dishes. Media was replaced daily to replenish nutrients and avoid accumulation of harmful byproducts and dead cells. After seven days, the cell cultures were passaged into new 25mm dishes. The medium from the first cell culture was removed and 1ml of trypsin was added. The trypsin was left on

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180 the cell cultures for approximately one minute a nd removed. The dishes were incubated at 37oC for five minutes, after which the detached cells were resuspended in 1ml DMEM with 2% FBS. One half ml of the resuspende d cells was transferred to a new 35mm dish with 2ml DMEM containing 2ml FBS and incubated at 27oC. The passaged cells were monitored daily for another seven days to note any CPE. Use of AEF to Show Positive CPE due to the Presence of Avian Viruses While the A EF cell line proved to be an eff ective tool, a positive control was needed to determine what viruses would e licit CPE on these cells to compar e to cells inoculated with SOLs. Since West Nile virus started primarily as an avian virus which eventually spread to alligators, it was decided to use a variety of avian viruses to tr y to elicit CPE within the AEF cells. West Nile virus itself could not be used since a level three biosaf ety laboratory was not available. The following viruses were used: Mareks disease frozen virus, Mareks disease lyophilized vaccine strain, fowl pox lyophilized vaccine strain, bronchitis virus lyophilized vaccine strain, pigeon paramyxovirus, koi herpesviru s, and infectious bursal disease virus. These viruses were being maintained at the laboratory where the SOL inoculations were performed. AEF cells were plated in 35mm dishes, as described above, and inoculated with 0.5ml of each virus. The viral inoculum was incuba ted with the AEF cells for one hour at 27oC and then replaced with 2ml DMEM containing 2% FBS a nd antibiotic. The cell cultures were incubated at 27oC and monitored daily for CPE for a total of se ven days. After seven days the cell cultures were passaged into new dish es as described above. If CPE from the avian viruses was det ected on the AEF cell cultures, RT-PCR was completed to determine if the CPE was caused by th e virus replicating in the AEF cell cultures. Total RNA was extracted from the first and s econd passages of the AEF cell demonstrating CPE

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181 with Trizol LS (Invitrogen, Carlsbad, CA) accordi ng to the manufacturers instructions. Briefly, the cell pellet was lysed with Trizol reagent for 10 min, and then chloroform was added for phase separation. The aqueous phase was transferred to a new tube, and the RNA was precipitated with an equal volume of isopropyl alcohol. Th e RNA was pelleted at 12,000 rpm for 10 min at 8C, washed one time with 70% ethanol, air dried, and resuspended in RNase free water. The RNA was reverse transcribed into complementary DNA (cDNA) using random primers (Invitrogen) and reverse transcriptase (New England Biolabs, Ipswich, MA). The cDNA was then tested by RT-PCR using virus specifi c primers (Table 6-1). The RT-PCR for the pigeon paramyxovirus targeted a 254 basepair (bp) fragment of the fusion gene. The RT-PCR products were resolved by gel el ectrophoresis in a 1% agarose ge l. Positive RT-PCR products were processed and sequenced directly to confirm that the expected viral gene product was amplified. Use of AEF to show CPE using a WNV Chimera A chim era was used to ascertain the infectivity of the AEF with a vi rus that has been found specifically to infect alligators. A chimera composed of the WNV envelope and premembrane genes (preME) and yellow fever genes (YF-17D) for nonstructural (NS) and capsid (C) proteins was developed by Long et al. ( 2007) (Figure 6-8). The constr uction involved replacing the structural genes encoding for the premembrane (p rM) and envelope (E) proteins of West Nile virus with the same genes in the yellow fever 17D virus genome (Long et al 2007). This attenuated West Nile virus vaccine (live Flavivirus ) chimera was designed for horses to vaccinate against WNV; its safety has been proven. This chimera was chosen due to the presence of WNV genes. This chimera would be used as a positive control to compare to samples of SOLs that may have caused CPE within the AEF.

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182 AEF cells in three inch round Petri dishes at 90% confluency were i noculated with 100l of the WNV-Chimera virus. The cells were in cubated for two days and examined for CPE. Use of Real Time-PCR in the Identi fication o f Viruses within SOLs SOL samples were collected from alligators at farm FL-2. These animals all had over 50 SOLs per hide and the farmer reported at the time of collection that most animals on the farm had similar conditions. For collection, 6 mm pun ch biopsies were collected. Biopsies were placed on sterile Petri dishes and the tissue was dissected to the SOLs to eliminate as much alligator DNA as possible. The objective was to use the tissue for RT-PCR from only the lesion itself. Unaffected areas were submitted as negati ve controls. Samples we re placed in RNAlater for processing (Dr. Maureen L ong, Principal Investigator). Tissues were minced and extracted by a commercial kit (RNAEasy) using the manufacturer protocol for fr esh and fixed tissue. All RNA samples were treated with amplification grade DNAse I and dissolved in di-e thyl-propyl carbonate (DEP C) treated water. cDNA was synthesized from 1ug of RNA with a commercial RT-for PCR kit (Advantage, Clontech, Mountain View, CA) using the manufacturer s protocol. Amplification of 2 uL of cDNA was performed in a 25 uL PCR reacti on using TaqMan Universal PCR Mastermix (Applied Biosystems, Foster City, CA), with 0.9 uM forward and reverse primers, and 0.25 uM probe. At least two primers were used on every samp le, while some of the samples used three primers (Table 6-1). The primers amplify the nonstructural protein 5 (NS-5) region of the WNV genome and the E protein of the SLE, WNV, EEE viral genomes (Briese et al, 2002). Amplification and detection was performed using the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster C ity, CA) and included 40 cycles at 94oC for 45s, and 60oC for 45s with a final hold step at 4oC (Tewari et al, 2004)

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183 Results Use of AEF to Determine the Presence of a Virus in SOLs No CPE was observed w hen AEF cells were inoc ulated with SOL material. Plates were examined daily and grew confluently for two w eeks with no visible cell death (Figure 6-5). Media were changed routinely to allow for pr oper nutrient concentrations; however the AEF cells continued to grow to confluency. Plates inoculated with SOL samples were compared to the negative control plates (Figures 6-2, 6-3, a nd 6-4) and no detectable difference was observed. Use of AEF to Show Positive CPE due to the Presence of Avian Viruses Of the avian viruses used to test for CPE on AEF cells, the two viruses that were shown to be positive were pigeon paramyxovirus and fo wl pox vaccine strain. Of the two, pigeon parymyxovirus was chosen as a representative avia n virus that elicited CPE on the AEF cells. Cytopathic effects consistent with a para myxovirus were observed after seven days of infection of the AEF cell cultures with the pige on paramyxovirus (Figure 6-7), and again within one day after passage of the cells. RT-PCR was performed to illustrate that the CPE occurred due to the presence of the virus and not due to the conditions of the culture. A 254-bp fragment was amplified by RT-PCR using paramyxovirus primers from the second passage of the AEF cell culture infected with the pigeon paramyxovirus (Fi gure 6-8). The RT-PCR product was sequenced directly, and a 254-bp sequence was obta ined that shared 100 % nucleotide identity with the original paramyxovirus isolated from pigeons. Use of AEF to Show CPE with a WNV Chimera The results of the use of AEF to show CPE with a W NV chimera were inconclusive. CPE was noted at day 2-3, but the cells lifted from the plate around day 4-5. Supernatant from the Petri dishes was harvested and i noculated into Vero cells cultur ed with serum plaque forming units (PFU). Even though there was possible CPE, the virus could not be passed. PCR was

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184 performed for the E protein gene on both the supernatant and the cells, but there were no detectable products. Use of RT-PCR in the Identification of a Virus within SOLs Of the sa mples submitted for RT-PCR, none show ed a detectable presence of West Nile virus, Eastern Equine encephalitis, or St. L ouis encephalitis within SOL samples. Multiple samples were tested, and a variety of primers used, none of which detected any evidence for viral presence. Discussion Fibroblas t cell cultures are a useful system in which to grow and replicate various viruses (Colwell et al 1974). The development of such a specific system (using AEF cells) is an important tool for anyone studying crocodilian viruse s. The results from this portion of the study have led to some important conclusions concer ning the SOLs. While some researchers have speculated that a virus causes PIX, no eviden ce for virus was found by cell culture or RT-PCR in the current study. These results suggest that a viru s is not likely to be the cause of SOLs in the samples analyzed here. When samples collected from alligators with SOLs were placed on confluent AEF cells, no detectable CPE was visualized on an alligator-s pecific cell line. The positive CPE from the pigeon paramyxovirus in the AEF cell cultures, along with positive RT-PCR result indicate that this virus can replicate in the alligator cell cu ltures. The implications are far reaching when showing that a known avian virus can cause CP E on the AEF cells. Once positive cell death was noted, the first conclusion made was that these AEF cells do have the ability to exhibit CPE. Second, a known avian virus could elicit CPE, a llowing for proper comparison of other plates inoculated with SOLs. This can be used as a positive control for future studies.

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185 The Chimera experiment did show CPE occurring in the AEF cells; however it may not have been due to the Chimera. There was no positive PCR from the cells or the supernatant for the Chimera. The cells could have just lifted fr om the plates, which may have been due to the high acidity of the liquid or perh aps increased temperature. The Chimera is a very slow forming virus, so the short CPE time may not have a correlation to the Chimera. The absence of any detectable product fr om the RT-PCR diagnostics using primers for WNV, EEE, and SLE further indicates the absence of a virus from most of the lesions tested. RT-PCR is a very sensitive technique in the id entification of RNA viruses and has been used successfully in the detection of these three viruse s in the past. If one of the viruses had been located within the samples with many SOLs, th en bands would have been visualized and sequencing could have been performed. Epidemiological charts that first depicted the entry of WNV into both the United States and the Southeastern portion of the United States do not overlap the period when PIX was first seen (Figure 2-6 and Figure 2-7). WNV did not enter into the United States until 1999, and did not reach Florida until 2001. Historically PIX was first noted in 1999, and has been retrospectively seen since 1997. These dates do not match or correlate. A virus may be associated in some cases of PIX; however it is not visualized as the major cau se of this disease.

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186 Figure 6-1. Samples prepared for SOL inoculation. Alligator embr yo fibroblasts were allowed to become confluent, then inoculated with lesions and left to incubate. All experiments used negative controls during the process.

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187 Figure 6-2. Photomicrograph of alligator embryo fibroblast control plate (50X). Note that the cells are confluent and cellular growth is still continuing.

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188 Figure 6-3. Photomicrograph of AE F control plate (100X). Cells are growing confluently and no CPE is noted.

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189 Figure 6-4. Photomicrograph of AE F control plate (320X). Cells are growing confluently and no CPE is noted. At this stage, SOL inoculation is performed.

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190 Figure 6-5. Photomicrograph of AEF cell culture inoculated with allig ator SOLs. Note that there is no evidence of CPE when compared to controls. All samples exhibited similar confluency.

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191 Figure 6-6. Photomicrograph of AEF cells inoculated with Fowl Pox vaccine strain. Note the occurrence of CPE in the cells (arrows). This would be later used as a positive control to determine what CPE looked like on AEF cell lines.

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192 Figure 6-7. Photomicrograph of AEF cells inoculated with pi geon paramyxovirus. Note the occurrence of CPE in the cells (arrows). This virus elicited a positive RT-PCR sequence analysis.

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193 Figure 6-8. Agarose gel electrophoresis of RT -PCR for pigeon paramyxovirus from AEF cell cultures. Avian viruses were inoculated onto AEF cell cultures and monitored for cytopathic effects for two passages of the cell cultures. RNA was extracted from the infected cells after the first and seco nd passages and tested by RT-PCR using paramyxovirus specific primers targeting the fu sion gene. Lane 1molecular weight marker, Lane 2AEF cell control, Lane 3infectious bursal disease virus (IBDV) first passage (negative viral control) Lane 4pigeon paramyxovirus, first passage, Lane 5IBDV second passage, Lane 6pigeon pa ramyxovirus second passage 30 hrs postinfection, Lane 7pigeon paramyxovirus s econd passage 56 hr post-infection, Lane 8PCR negative control, no cDNA added. 1 2 3 4 5 6 7 8 300-bp

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194 Figure 6-8. Schematic represen tation of a modified live Flavivirus /West Nile virus (WN-FV) chimera development. Reprinted with perm ission from the author (Long et al. 2007).

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195 Table 6-1. Primers used in the identification of viruses within PIX lesions. Name of Virus Name of Primer Used Probe Sequence (5-3) West Nile Virus 10668wnv-p1 Cag acc acg cta cgg cg West Nile Virus 10770cwnv-p2 Cta ggg ccg cgt ggg West Nile Virus PRO WN 10692 O ct g cgg aga gtg cag tct gcg at 8 West Nile Virus WN 1160 Tca gcg atc tct cca cca aag West Nile Virus WN 1229 Ggg tca gca cgt ttg tca ttg West Nile Virus PRO WN 1186 O tgc ccg acc atg gga gaa gct c 8 West Nile Virus WN NS5 F Gct ccg ctg tcc ctg tga West Nile Virus WN NS5 R Cag tct cct cct gca tgg atg West Nile Virus WN NS5 Probe 6fam-tgg gtc cct acc gga aca accacg tt-tamra-3 Eastern Equine Encephalitis EE 1898 Acc ttg ctg acg acc agg tc Eastern Equine Encephalitis EE 1968 Gtt gtt ggt cgc tca atc ca Eastern Equine Encephalitis PRO EE 1919 O ctt gga agt gat gca aat cca act cga ca 8 Eastern Equine Encephalitis EE1858 Aca ccg cac cct gat ttt aca Eastern Equine Encephalitis EE 192 Ctt cca agt gac ctg gtc gtc Eastern Equine Encephalitis PRO EE 1881 O tgc acc cgg acc atc cga cct 8 St. Louis Encephalitis SLE 264 Gtt gct gcc tag cat cca tcc St. Louis Encephalitis SLE 195 Gaa aac tgg gtt ctg cgc a St. Louis Encephalitis PRO-SLE 218 O tgg ata tgc cct agt tgc gct ggc 8 St. Louis Encephalitis SL 1781 Ggc tgt cgg agg aat tct ca St. Louis Encephalitis SL 1848 Ggt caa ttg cac atc ccg ag St. Louis Encephalitis SL 1805 O tc t ggc aac cag cgt aca agc cg 8 Pigeon paramyxovirus CR-242 ccttggtgaitctatccgiag Pigeon paramyxovirus CR-243 ct gccactgctagttgigataatcc

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196 CHAPTER 7 IDENTIFICATION OF HORTAEA WERNECKI I LOCATED WITHIN SPHERICAL OPAQUE LESIONS BY POLYMERASE CHAIN REACTION Introduction While histology is useful in the identifica tion of m icroorganisms within specific tissue locations, some microorganisms are present in su ch minute amounts that de tection is not always possible. Additionally, it is unfeasible with light microscopy to identify specific species of microorganisms that may be present in a ti ssue. Limitations are further seen with microbiological techniques because not all funga l species are easily cultured. Mycological isolation may not always prove useful since some fungal species are slow growing and plates are often overgrown by fast growing non-target species (Sterflin ger et al 1998). Molecular techniques have been developed to de tect nucleic acid sequences within tissues. Molecular testing is regarded as a valuable tool in determining the presence of a microorganism located within tissue. The most common molecu lar test employed today is the polymerase chain reaction (PCR). In performing PCR, DNA is re plicated many times to ensure the optimal amount of DNA is available for detection and later sequencing. PCR is regarded as the molecular gold standard in the identification of microorganisms (Sterfli nger et al 1998). PCR should be used in addition to other diagnostic methods when possible. These combinations will lead to improved results. In the case of PIX, the dematiaceous fungus Hortaea werneckii was microbiologically isolated from alligator farms in both Louisiana a nd Florida. Traditional methods of identification based on morphological characteristics were perfor med; however proper mo lecular detection was needed to confirm the findings. Since this f ungus has a suspected association with PIX at the time of isolation, PCR was employed to accurately detect any H. werneckii DNA that would be

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197 present in the tissue and subsequently the spherical opaque lesions (SOLs). This is the first case of H werneckii identified within a non-mammalian species, specifically a reptilian species. PCR has been widely employed to determine causative agents for many fungal infections due to its high reproducibility (Uezato et al 2006). Ideal primers should target specific markers that are present within all f ungal genera, but possess enough varia tion to have the ability to define a given species (Ferre r et al 2001). Regions of ri bosomal DNA (rDNA) have been identified as highly specific for fungal identificat ion; more specifically, th e internal transcribed spacer (ITS) regions of rDNA. This ITS region is located between the 18S and 28S region of rDNA and is the most widely sequenced DNA regi on of fungi. This makes interindividual variations in rDNA minute (Laure nco et al 2003). Primers used for this purpose were first described by White et al 1990. Abliz et al. (2003) reported methods for the accurate identification of H. werneckii in tissue via PCR. They successfully designed an oligonucleotide primer set for H. werneckii based on the nucleotide sequence of the ITS region found in rDNA. This region proved to be highly conserved in intraspecies detection. Primers were tested against other dematiaceous fungal species, and primer detection of H. werneckii was specific. The primers developed were from the ITS1-5.8S-ITS2 sequence data (Abliz et al 2003). By using a species specific primer, detection can identify the exact causative microorganism. The purpose of this portion of the study was to detect the presence of H. werneckii within SOLs located in the alligators integument. By developing a forensic protocol, the researchers would accurately target specific regions of the fungus DNA and locate the fungus within the SOLs. The presence of the DNA of this fungus within the SOLs would lead to a direct correlation between nodule formation and pathogen detection within.

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198 Materials and Methods Upon obtaining alligator hide s exhibiting SOLs, sam ples were collected using the following protocol. Sterile gloves were worn during all collections and changed between animals. Areas with SOLs were disinfected with alcohol to minimize contamination. A 6mm biopsy of the SOL was made and the sample was placed in 95% ethanol or frozen until DNA isolations were performed. If the sample b ecame lodged in the biopsy punch, a sterile needle was used to extract it. Each samp le was placed into separate vials. In a laminar-flow (sterile) hood, 2 mm biopsies containing the SOLs were excised from the larger 6mm alligator tissue bi opsies to minimize the presence of host DNA and to increase the accessibility of the causative organisms DNA. Sterile needles were used if the sample became lodged in the biopsy punch. The foreseeable prob lem in the start of the project was that the reaction may be overwhelmed by the quantity of alligator DNA present, hence making the H. werneckii DNA inaccessible during PCR. The lesion wa s then macerated to further optimize microorganism accessibility. Isolations were performed to optimize th e amount of DNA from the microorganisms in question. The DNA was first extracted by grinding th e samples in liquid nitrogen with a sterile mortar and pestle to disrupt the fungus hard chitinous cell walls and to make the fungal DNA available for isolation. This method has previously been described (Griffin et al 2002, Cubero et al 1999). The DNeasy Plant Mini kit was used for DNA isol ations (Qiagen, Valencia, CA). This kit is designed to isolate DNA from plants, which al so possess a hard cell wa ll much like fungi. The protocol for isolation was as follows: four hundred l of Buffer AP1 and 4 l of RNase A stock solution were added to collection tubes containi ng the samples. Samples were vortexed until opaque and then incubated for 10 minutes at 650C, vortexing three times during the incubation

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199 period. Buffer AP2 was added to the mixture (1 30 l), vortexed, and then placed in ice for 5 minutes. The mixture was placed in a centrifuge on high speed for 5 minutes, at which time the lysate was then added to QIA shredder spin column s and then further placed in the centrifuge for 2 minutes. The flow-through was carefully transfe rred to a new tube without disrupting the celldebris pellet. Buffer AP3/E at 675 l was added and mixed. Six hundred fi fty l of the mixture was placed into a DNeasy mini spin column a nd centrifuged for 1 minute. After the flowthrough was discarded, the remainder of the mixtur e was subjected to the same step, discarding the flow-through and collection tube The DNeasy mini spin columns were placed into a new collection tube and 500 l of Bu ffer AW were added. The mixt ure was centrifuged for 1 minute, and flow-through was discarded. This step was repeated once more. The DNeasy column was placed into a new 1.5 ml tube and 100 l of heated Buffer AE were added and left to incubate at room temperature for 5 minutes. After centrifugi ng for 1 minute, samples were either prepared for the PCR reaction or frozen until ready. Positive and negative controls were always used when performing isolations. Positive controls were needed to an alyze any amplifications of H. werneckii for comparison. DNA was isolated from a known culture of H. werneckii which was obtained from the American Type Culture Collection (ATCC, Manassas, VA) and used as the positive control. Collection tubes with no sample were used as negative contro ls and were subjected to the same extraction procedures as the specimens to ensure that there was no contamination when performing isolations. Regions in the alligato rs hide that did not show any SO Ls were also used as negative controls. PCRs were then completed to amplify any H. werneckii DNA that was present. The protocol for the identification of this fungus was developed and pe rformed in the University of

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200 Florida Interdisciplinary Cent er for Biotechnology Research (UF ICBR) Genetic Analysis Laboratory. Two PCR primer sets were selected for use in this study to iden tify the presence of H. werneckii within the lesions. The reactions we re conducted using previously optimized conditions that produced amplified regions of H. werneckii (Abliz et al. 2003). Amplification attempts were initially made using the unive rsal ITS primers (ITS 4 and ITS5) described by Abliz et al (2003) (Table 7-1). Although these prim ers are fungal specific, th ey span a very large area in the genome and are not species specific. This may lead to weak amplification and in some cases no detectable product. H. werneckii specific primers (Abliz et al. 2003) were also used to attempt detection of specific gene fragments. H. werneckii specific primers Hor-F (5TGGACACCTTCATAACTCTTG-3) and Hor-R (5-TCACAACGCTTAGAGACGG-3) from the ITS1-5.8S-ITS2 sequences were optimized for use in this study (Table 7-1). PCR was performed in 25 l reactions. The PCR cockta il contained 1X Sigma PCR buffer, 800 uM dNTP, 3 mM MgCl2, 1 unit JumpStart Taq DNA polymerase (Sigma, St. Louis, MO), 0.4 uM forward and reverse primers (Hor-R and Hor-F), and 1.0 l of the template were used for each reaction. Negative PCR controls were also used at this step to detect contamination in the PCR reaction. After initial denaturation of DNA for one cycle at 95C for 4 minutes, 30 cycles of amplification was performe d. Each cycle consisted of a denaturation at 91C for one minute, annealing at 58C for one minute, and an extension step at 72C for one minute. A final extension step at 72C for 5 minutes was performed. Agarose gel electrophoresis was performed as directed to show the presence of H. werneckii specific ITS1-5.8S-ITS2 PCR product. Five l of the amplicons were run on an agarose gel containing ethidium bromide and view ed using ultraviolet illumination. If a product

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201 was visualized, sequences were then subjected to another cycle of P CR to create an ample quantity of DNA for sequencing. Following th e second PCR amplification, products were cleaned and prepared for sequencing with the Qiaprep PCR Clean-up Kit following the manufacturers protocol (Qiage n, Valencia, CA). All sequencing was performed at the UF ICBR Sequencing Core Laboratory. DNA sequences were analyzed using the National Center for Biotechnology Information (NCBI) and Basic Local Alignment Search Tool (BLAST) program ( http://www.ncbi.nlm.nih.gov/BLAST ) to determine their relationship to other H. werneckii sequences. Sequences were used to determine phylogenetic positioning with respect to similar strains of H. werneckii found in GenBank. A multiple ali gnment of accession numbers GenBank DQ168665 and EF060816, and alligator samples F11, HW310, HW38, HW12, and G11 (Table 7-2) were completed with Megalign (DNA Star, Madison WI). The nucleotide sequences of F11, HW310, HW 38, HW12, and G11 and those of other homologues from the GenBank database were aligned using Clustal X slow and accurate function. Neighbor-joining phyloge netic trees were constructed using PAUP version 4.0b10 (Sinauer Associates, Sunderland, MA) and draw n with TreeView software (Page 1996). GenBank accession numbers for sequences us ed in phylogenetic analyses were: DQ168665, EF060816, AB079595, and DQ519102. The above GenBank strains DQ168665 and EF060816 were used for positive ATCC H. werneckii culture comparisons. An H. werneckii strain isolated by Sharmin et al. (2002) from a guinea pig (accession no. GenBank AB079595) along with the other member of the Hortaea genus, Hortaea acidophila (accession no. GenBank DQ519102) were additionally used for comparisons (Table 7-2).

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202 Results The prim ers first used for the identification of H. werneckii in this study were previously defined by White et al. 1990. The ITS4 and ITS5 primers are fungal specif ic; however they span a very large area in the genome and are not species specific. Initially these demonstrated a very weak amplification and in some cases no detectable product. With the species specific primers, the PCR product was more detectable and yielded stronger amplifications. These primers (HorR and Hor-F) were used for the remainder of the study. H. werneckii was positively identified via PCR in lesions from both Florida and Louisiana alligator farms. PCR was successfully performe d on a total of five sa mples and the expected 306-bp fragment was observed initially with gel el ectrophoresis (Figure 7-1). The identity of the fragments was confirmed by automated DNA sequenc ing. The sequences identified with Blast matched the sequence of H. werneckii in GenBank confirming that H. werneckii was present in the SOLs. The sequences were determined to be H. werneckii with a 100% match to other H. werneckii strains in most of the cases, while othe r samples showed between a 95% and a 98% match. A multiple sequence alignment was performed on the positive alligator samples and compared with H. werneckii samples found in GenBank (Figures 7-3). The alignment was a complete match (95% 100%) for most of the base s that were sequenced. The differences in the 278 and 279 base region may have been due to bases th at did not amplify, or that were out of the amplification region. An unrooted phylogenetic tree was constr ucted based on the positive sequences of H. werneckii from farms, positive ATCC sequences, the guinea pig sequence, and the sequence from H. acidophila (Figure 7-2). This was prepared to illustrate similarities and differences between all of these sequences. The focus point for the alligator samples and GenBank

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203 accession numbers DQ68665 and EF060816 all radiated fr om the same area. This implies that these sequences are all directly related and are considered the same species. The accession numbers AB079595 and DQ519102 were focused from a different point on the trees center, implying that these sequences ar e related, but no t directly. Discussion Genetic testing with PCR is of ten critical for confirming the presence of target microorganisms within tissues. This process has been perfected throughout the years and is very reliable for the identification of DNA sequences within tissue samples. A variety of pathogenic microorganisms, especially fungi, have been accur ately detected through this method. By using PCR to target specific regions of a microorgani sms genetic code, direct links can be identified between microorganisms and their ho sts, leading to postulations of causative agents of disease and disease processes. The ge netic results presented here pr ovide some insight as to an association between PIX a nd a causative fungal pathogen. Hortaea werneckii has been positively detected within tissues of humans guinea pigs and now alligators. In all cases, the subjects were exhibiting an integumentary phenomenon and this fungus was either cultured or sequenced in all of the cases. PCR has been proven repeatedly as an accurate way to detect H. werneckii within tissues (Diniz 2 004, de Cock 1994, Perez et al 2005, Gunde-Cimerman and Plemenitas 2006, Uezato et al 2006, Ng et al 2005, and Abliz et al 2003). While H. werneckii was not cultured or sequenced in all of the samples submitted in the current study, this was not visualized as a pr oblem when defining an association between PIX and the microorganism. The PCR developed for the current study can be further modified to become more sensitive to amplification. Since th is is the first case of this fungus identified

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204 within a reptilian species, ther e are additional modifications that can be attempted to make the PCR used in this study a better diagnostic tool. The positive H. werneckii alligator sequences were BLASTed against other positive Hortaea samples found in GenBank. They matc hed at between 95% and 100%; this is considered to be a positive set of samples. Upon sequence alignment, there was a 100% match to ATCC H. werneckii strains located in GenBank. The presence of this fungus within the alligato r samples is seen to di rectly correlate with the presence of SOLs and is not present due to contamination. H. werneckii is not commonly seen as a contaminant within samples at the labo ratory, and has not been previously reported as such. The multiple instances of both micr obiological growth and DNA sequencing are not viewed as possible contaminations or coincidental but as direct correlat ions between SOLs and H. werneckii The DNA isolation blanks and the PCR bl anks tested negative for the fungus. One explanation as to why the H. werneckii found within the al ligator samples was different on the radial tr ee from the guinea pig H. werneckii sequence was possibly due to the organisms that were infected (mammal verse reptile). Additionally, di fferent regions of the world (Florida verse Japan) may yield diffe rent strains of this particular fungus. The dermal lesions that present themselves upon histological examination have been implicated as the primary causation of SOLs se en upon light identification, and the subsequent PIX marks that appear upon tanning of the alligator hides. Considering that H. werneckii had a predilection for the integument, the initial gr owth and succeeding PCR positive results, it is concluded that this fungus has a strong association with PIX skin disease in the American alligator.

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205 Figure 7-1. Visualization of PCR products through agarose gel el ectrophoresis. Lanes 1-12 are the result of a set of reactions using the H. werneckii specific primer sets (Hor-R and Hor-F). Lane 11 is the positive H. werneckii control obtained from an ATCC culture. Products were observed in lanes 3 and 5. Lane 12 is the PCR Blank.

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206 Figure 7-2. Radial phylogenetic tree This tree depicts the sequen ce variability and consistency of certain DNA regions. Accession numbers GenBank DQ168665 and EF060816 are H. werneckii strains obtained that were a 95% and 100% match, respectively. Accession number GenBank AB079595 was fr om the case of tinea nigra in the guinea pig from Japan. Accession no. GenBank DQ519102 is from the additional species located within the Hortaea genus ( H. acidophila ). The other sequences on the tree are from the PIX samples obtained from farms in Florida and Louisiana (F11, HW310, HW38, HW12, G11). G11 EF060816 HW12 HW38 HW310 F11 DQ68665 A B079595 DQ519102

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207 Figure 7-3. Sequence alignment for positive H. werneckii samples. Note that there is almost a complete match between the farm samples and the ATCC samples. Similarities are represented by a red bar, and differences are represented with a yellow bar. The slight differences in the 278 and 279 base region is likely due to bases that were undetectable in the alligator samples (did not amplify for some reason). Accession nos. GenBank DQ168665 and EF060816 are H. werneckii strains obtained that were a 95% and 100% match, respectively. The other sequences on the tree are from the SOL samples obtained from farms in Florida and Louisiana (F11, HW310, HW38, HW12, G11).

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208 Table 7-1. Primers used in this study for the identification of H. werneckii within lesions. Name of Primer Primer sequence ITS4 (5-TCCTCCGCTTATTGATATGC-3) ITS5 (5-GGAAGTAAA AGTCGTAACAAGG-3) Hor-R (5 -TCACAACGCTTAGAGACGG-3 ) Hor-F (5 -TGGACACCTTCATAACTCTTG-3 ) Note: ITS4 and ITS5 detects a wide range of fungi (White et al. 1990), while Hor-R and Hor-F are species specific primers developed for the identification of H. werneckii (Abliz et al. 2003). These proved to be the most efficient primers for the current study. Table 7-2. GenBank accession numbers for H. werneckii isolates used for comparison. Source GenBank Accession Number H. werneckii ATCC strain DQ68665 H. werneckii strain EF060816 H. werneckii from Guinea pig AB079595 H. acidophila DQ519102 Note: DQ68665 and EF060816 were used for positive comparisons in this study. These both aligned close to the alligator samples.

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209 CHAPTER 8 TREATMENT REGIMENS AND SUGGEST IONS FOR THE CONTROL OF PIX Introduction During the study period of the current proj ect, PIX skin disease rapidly progressed throughout the southeastern United S tates. A treatment regimen was recommended to prevent further spread of the disease. The PIX problem at alligator farms in Florida and Louisiana dramatically increased after the initial characterizati on of the disease in 1999. By the year 2003, the incidence and prevalence of PIX had increased on nearly every farm th at initially exhibited an outbreak of PIX, and had additionally spread to new farms throughout the region. The most effective treatment is prevention, something that can be accomplished by reducing stress to these animals. It is susp ected that animals with PIX lesions are immunocompromised (in a farm setting most notably) due to unnatural physiologic stress imposed on them. To optimize growth rate, these animals are grouped in high numbers together with above normal temperatures. Overcrowding in a farm setting has been proven detrimental in pig and poultry farms (Funk et al 2007, Leman and Cutler 1985) because it increase s the occurrence of disease. Part of the treatment protocol for this study was the reduction of animals in a single enclosure to reduce the effect of overcrowding. Other manageme nt applications were proposed for the general maintenance of alligators in a farming situation and included a proper diet and growth management. Potassium permanganate treatments and a copper sulfate solution are effective for the eradication of pathogenic orga nisms in an aquaculture setting (Francis-Floyd and Klinger 1997, Cardeilhac and Whita ker 1988, Buenviaje et al 1994). Antifungal drugs were proposed in the initial st ages of diagnosis. However, one of the major problems when using antifungal drugs is that fungal infections (that may result in the formation of nodules) make it extremely difficult fo r the drugs to reach the site of infection.

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210 Additionally, the alligators located at these farm s are used for their meat, therefore systemic antifungal drugs are restricted for use by the United States Department of Agriculture. Methods were employed that would attempt to limit the spr ead of disease rather than eradicate the fungus through the use of drugs. Along with the use of topical antifungals, s upportive care must be employed along with good husbandry practices. Nutrition Proper nutrition for any farm -raised animal is esse ntial in the management of the stock. In the case of alligators, their size and growth potential has a direct correlation with the quality of feed used. Improper nutrition can contribute an adverse effect on a farm raised animal. Improper nutrition may also potentiate the manifestati on of disease. The diet used needs to be at optimal quality levels in order to ensure healthy disease-free animals. Commercial alligator feed is available and has been optimized to meet the alligators nutritional needs. Essentia l vitamins, minerals and nutrients shoul d be contained within the feed. Growth Management Growth m anagement is a practice that is frequently overlooked in good alligator farming management practices. Keeping animals at equiva lent sizes in enclosur es helps reduce fighting and competition. Housing larger animals with sma ller ones will lead to an increase in hostility, food competition, and struggle for dry space. Disr uption of the integument due to fighting can give opportunistic organisms a chance to thrive. Maintaining similar size classes of animals should result in even growth rates among animals. A second factor in growth management is th e culling of overly aggressive animals. Alligators that are more prone to fight and are extremely aggressive towards other animals (regardless of the size ratios) shoul d be removed at once and placed in separate pens. This will

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211 decrease the appearance of scars and potentially secondary infections from fighting that will be evident upon tanning. Stocking Densities Stocking densities is a key fact or in the m anagement of alli gators, specifically to produce high quality hides. High stocking densities will not only lead to aggression within animals, but will have a factor in the spread of disease. Stoc king densities for alligators less than four feet in length should be within the limits of one square f oot per linear foot of an imal (i.e., a three foot animal should have three square fe et all to itself. Ten, three foot animals should be in a tank that is at least 30 square feet). It is recommended th at pens contain no more than fifty alligators that are less than four feet in length. Potassium Permanganate Potassium permanganate (KMnO4) has been used to treat exte rnal infections of aquatic organisms in farm settings. It is an oxidizing agent that is able to chemically dissolve organic material. Potassium permanganate is used as a control of bacterial, parasitic and fungal infections prior to these pathogenic microor ganisms becoming systemic (Francis-Floyd and Klinger 1997). The main limitation for its use in alligators is that this treatment has not been extensively used previous ly in farm situations. Copper Sulfate The use of copper sulfate (CuSO4) as a fungicide has been used previously in farming situations (Cardeilhac and Whitaker 1988, Buenviaje et al 1994). Copper ci trate (citrated copper sulfate) added to pen water to give a copper c oncentration of about 0.25 ppm each time the water is changed is an effective treatment against fungal pathogens. Copper may have some side effects, so it is not always a recommended tr eatment for daily use. Copper potentiated with formalin may be more effective.

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212 The goal of this portion of the project was to develop a proposed protocol for the control of fungal SOLs (and subsequently PIX) and to evalua te different treatment regimens at farms that were experiencing outbreaks. Stocking densities a nd husbandry practices were suggested to each farm. One farm participated in and completed a treatment regimen that included a copper sulfate stock solution to decrease the amount of pathogens in the animals pen water. This treatment regimen is suggested for any farm that is encountering animals with SOLs. Materials and Methods For the con trol of the PIX skin disease in alligators, a copper sulfate solution was suggested as one of the primary tools for an e ffective treatment regimen. Copper sulfate alone added directly to the pens can be detrimental in the wrong concentra tions, so a solution must first be made. By using citric acid monohydrate as a ch elator to hold the copper in solution, this effectively produces a solution that is less harm ful than copper alone, while at the same time increases its solubility in a stock solution (wate r). This treatment has been approved by the US Environmental Protection Agency for the use in w ildlife, so its use in a farming situation is considered within the limits of its efficacy. Copper-based fungici des have been shown to control various diseases in other species, so its use here in a farming situation is reliable. For the treatment of Brown Spot, copper sulfate has b een used previously in crocodilian farming operations (Cardeilhac and Whitaker 1988, Buenviaje et al 1994). A control group of hatchling alligators were us ed in this study to ensure that animals placed in the treatment groups were disease free. Alligator eggs were collected in July from Louisiana and transported to the researchers laboratory. Eggs we re disinfected and placed into sterile enclosures. The old nest ing material was discarded and autoclaved moss was used as a substrate. Animals were incubated and hatche d roughly 60 days later. Animals were removed upon hatch and placed into disinfected holding ta nks until distributed to the various farms

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213 involved in the treatment study. These animals were termed Specific Pathogen Free (SPF) hatchlings. Two separate hatch ye ars were used in this study. Initially there were three farms that agreed to participate as treatment sites for this portion of the study. For circumstances ou t of the researchers control, only one of the farms (LA-1) was able to complete the treatment protocols and can be included in these results. One treatment site was destroyed in hurricane Katrin a, and the other farm experienced financial hardship prior to the collection of the data. Housing was constructed for the treatment study at Farm LA-1. The houses were constructed out of concrete and had ventilated tops to keep animal s isolated (Figure 8-1). Each pen had its own drainage system and pipes for water cleaning. These houses were used for treatment groups one, two and three. The assigned treatment regimen for the pen was attached to the top of the tanks to allow for easy directions at treatment times. The pen num ber was put at the top of the page, the control group designation in the middle, and the directions underneath (Figure 8-2). This was used for groups one, two and three. The approximate size of the alligators that were used over the two year study for groups one, two and three starte d at around 12 inches (F igure 8-3). By the completion of the treatment study the an imals were roughly 3 feet in length Animals in the treatment study were placed in one of three treatment groups. Each of the pens were disinfected with each water change us ing the farms usual disinfection protocol, which included chlorine additions. Each of the three gro ups had a total of nine pens with nine alligators per pen (Table 8-1). This amounted to a to tal of 81 animals per treatment group, and 243 animals total. The study lasted from September 2005 until November 2007.

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214 Animals in group 1 were placed in pen numbers one through nine. The treatment protocol used for these pens consisted of control hatchlin gs and a control regimen. Hatchlings obtained from the farm were placed through standard farm procedures that are used for grow-out. This treatment group was never subjected to the copp er sulfate treatment regimen (Table 8-1). Group 2 had the designation of pens ten thr ough eighteen (Table 8-1). The treatment protocol used for these pens consisted of disi nfected pens, control hatchlings, and a treatment regimen. Normal hatchlings from the farm were placed in pens and treated with Clorox (6% sodium hyperchlorite) to the pen water each time the wate r was changed and the pen was disinfected. This equaled one tablespoon or fluid ounce of Cl orox. In January and May, the chlorine treatment was interrupted for two weeks, at which time Copper citrate or citrated copper sulfate was added to the pen water each time th e water is changed over the 14 day period. The chlorine treatment was resumed after the 14 day period. Group 3 had the designation of pens nineteen through twenty seven (Table 8-1). The treatment regimen for these pens consisted of disinfected pens, PIX-free hatchlings (SPF alligators), and the treatment regimen. SPF hatch lings were treated with additions of Clorox to the pen water each time the water was changed and the pen disinfected. This equaled one tablespoon or fluid ounce of Clorox (6% sodi um hyperchlorite). In January and May, the chlorine treatment was interrupted for two weeks, at which time Copper citrate or citrated copper sulfate was added to the pen water each time th e water is changed over the 14 day period. The chlorine treatment was resumed after the 14 day period. An additional group that was not part of th e above groups was studied. Group 4 consisted of 2 year old SPF hatchlings w ith no specific treatment group. These animals were placed on the farm after hatch and kept together, however they received no treatment regimen. They were

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215 subjected to the normal daily farm operations and kept in normal farm housing. These would be used to compare what SPF hatchlings hides woul d look like after a coup le of years in normal farm husbandry and operations. The copper treatments are performed twice in a years time for a duration of two weeks. On the day the copper treatments are started, the pools must be drained and no other treatments should be used (salt, chlorine, etc.). Copper treatment must be added on the days that the pools are drained in that 2 week period; this should be done daily. It is importa nt to drain the pools so the concentration isnt multiplied in the water. After the 2 week copper treatment is over, normal farm operations and treatments can be restarte d (chlorine, salt, etc.). A summary of the directions for the solution are outlined in Table 8-2. Most animals were sacrificed at the end of the study and examined. Animals from Group 4 were sacrificed at three time in tervals; once in the beginning, middle, and then again at the completion of the study. Alligators were exam ined and detection of SOLs was noted when visible. Animals with SOLs were sampled and tr ansported to our laborator y at the University of Floridas College of Veterinary Medicine. Results In Novem ber 2005, forty animals from Farm LA-1 were examined for the presence of SOLs. These animals were obtained from normal farming operations prio r to the start of the study. Seven animals had some degree of evident SOLs, ranging from two to more than ten SOLs per hide. In November 2006, forty animals taken from normal farming operations that were not part of the study were examined, and te n animals had evident SOLs. At the same time, twenty animals from Group 4 were examined; none had any evidence of SOLs. In July 2006 twenty animals from the normal farming operatio n with no treatment were examined along with

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216 ten Group 4 animals, and no SOLs were visualize d. This may have been due to low stocking densities. In July 2007, the treatment study was comple ted and all four treatment groups were examined. Twenty animals from each group from a representative number of tanks were examined. Out of twenty animals, Group 1 had a total of three animals that had evidence of SOLs, however the number of lesions were less that two per animals. Of the twenty animals, the remainder seventeen did not have any evidence of SOLs. Group 2 only had one animal of the twenty exam ined that had evident SOLs. The animal had less than three SOLs visualized on its hide Of the remainder groups examined, Groups 3 and 4 did not have any animals with SOLs out of the twenty that were observed. The most efficient techniques in the reduction of PIX were the combination of the copper sulfate solution, the use of SPF hatchlings, and controlling the stocking densities. By decreasing the number of animals housed in a single enclos ure, fighting was reduced and animals appeared subjectively to be less stressed. This ultimately le d to fewer bite scars and possible entry routes for pathogenic microorganisms. By using the co pper sulfate solution, this should lead to a decrease in the amount of pat hogenic microorganisms present with in the animals water based on the literature and its proven results in other aquatic farming situations. Discussion Based on the proposed treatm ents, the combination of SPF hatchlings, general husbandry considerations and the copper sulf ate solution have been shown to reduce the incidence of SOLs on alligator farms. At time of slaughter, treatment animals have a lower rate of SOLs than animals that did not receive any treatment. This, coupled with the reduction in stocking densities and proper nutrition, has all but er adicated PIX on some farms. If farmers adhere to these

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217 suggestions and continue to carry out these be st management practices, PIX should be lowered among farms that have this disease and a trend s hould manifest toward er adication all together. A limitation seen with the treatment porti on of the study was that farm LA-1 was experiencing a decrease in the prevalence of SO Ls when this study was taking place. It would have been ideal to perform this study on a fa rm that was experiencing an outbreak for the duration of the study, but there wa s still valuable information learned. This farm was having problems with PIX in the past, and it was out of the researchers control exactly when an outbreak would occur. The total number of animals that had evident SOLs was very small in comparison to previous years. Farm FL-1 had been experiencing a high rate of SOLs and PIX on their farm at the start of this study. By simply reducing stocking densities, performing daily water changes, and treating with Clorox and copper, the presence of SOLs was reduced. A ll animals examined from this farm were exhibiting over twenty SOLs per hide in the beginning of the study. Data was obtained for a portion of the study, however a co mplete set of data was not available for evaluation at the end of the study (for reas ons out of our control). Through personal communication with the owner and ma nager of farm FL-1, they have reported that their animals are having the lowest amount of SOLs and PIX in years due to the complete treatment regimen recommendations. The information obtained from th is farm and others that have had a decrease the prevalence of SOLs a nd PIX leads to the conclusion that the proposed treatment protocol is effective. Even though there were no complete re sults, this information is still valuable and has led to the conclusions presently noted. Copper comes in many forms that are commercia lly available and can be added to water. It dissolves easily in water and is effective in controlling problems in freshwater aquaculture

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218 farms. It should not be used in areas that contain fish or other i nvertebrates, for it is extremely toxic to these species. It is often difficult to implement a standard m eans of disease control because of the diverse number of farm designs and husbandry practices used by farmers (Buenviaje et al. 1994). Each farm must establish farming procedures that the entire staff will continue to perform, and goals should be set for disease eradicat ion. A person at each farm shoul d be assigned as the lead to allow for the proper implementation of the treatm ents, along with suitable record keeping on the prevalence of PIX and other diseases. It is further advised that an imals are not moved from farm to farm; this could potentially lead to further transmission of the disease. Since some farms own multiple locations, those farms with animals showing PIX at slaughter should not transport any alligators to other farms. People that move from farm to farm (tanners skinners, etc.) should completely shower and change clothes to prevent cross contamination. The fact that Louisiana also releases 16% of their hatch back into the wild may al so play a role in transmission. Keeping animals at optimal husbandry conditio ns should lower the incidence of SOLs on animals. A theory as to why alligators are so susceptible to this disease may be answered by looking at their immunosuppressive states. These animals are kept in enclosures with minimal light exposure, a low amount of dry land, and no stimulation. Proper nutrition is an essential factor in a successful alligator farm. Nutritional dermatoses have been implicated when animals do not get the correct concentra tions of critical nutrients and vitamins (Harkewicz 2001). Overall, the proposed treatments and good mana gement practices implemented at alligator farms experiencing varying degrees of SOLs and PIX will decrease the occurrence of this

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219 integumentary condition. Farms that have put in to practice the recommended regimens have had success in the reduction and near eradication of SOLs and PIX.

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220 Figure 8-1. Photograph of the housi ng constructed for the treatment study at Farm LA-1. Note that the assigned treatment regimen was atta ched to the top of the tanks (arrow) to allow for easy directions at treatment tim es. The houses were constructed out of concrete with ventilated t ops. Each pen had its own drainage system and pipes for water additions. These houses were us ed for groups one, two and three.

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221 Figure 8-2. Example of the treatment regimen direct ions. These were attach ed to the top of the pens that housed the treatment groups. No te the pen number (19) at the top of the page, the control group designation, and the di rections underneath. This was used for groups one, two and three.

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222 Figure 8-3. Approximate size of the alligators that were used ove r the two year study for groups one, two and three. The treatment direction sheet is a standard 8.5 x 11 inch size. Animal were roughly 10 to 12 inches in le ngth. By the completion of the treatment study the animals were about 3 feet in length.

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223 Table 8-1. Treatment Groups. Group Number Pen Number Hatch lings Used Treatment Used 1 Pen 1 Control hatchlings Control regimen 1 Pen 2 Control hatchlings Control regimen 1 Pen 3 Control hatchlings Control regimen 1 Pen 4 Control hatchlings Control regimen 1 Pen 5 Control hatchlings Control regimen 1 Pen 6 Control hatchlings Control regimen 1 Pen 7 Control hatchlings Control regimen 1 Pen 8 Control hatchlings Control regimen 1 Pen 9 Control hatchlings Control regimen 2 Pen 10 Control hatchlings Treatment Regimen 2 Pen 11 Control hatchlings Treatment Regimen 2 Pen 12 Control hatchlings Treatment Regimen 2 Pen 13 Control hatchlings Treatment Regimen 2 Pen 14 Control hatchlings Treatment Regimen 2 Pen 15 Control hatchlings Treatment Regimen 2 Pen 16 Control hatchlings Treatment Regimen 2 Pen 17 Control hatchlings Treatment Regimen 2 Pen 18 Control hatchlings Treatment Regimen 3 Pen 19 SPF hatchlings Treatment Regimen 3 Pen 20 SPF hatchlings Treatment Regimen 3 Pen 21 SPF hatchlings Treatment Regimen 3 Pen 22 SPF hatchlings Treatment Regimen 3 Pen 23 SPF hatchlings Treatment Regimen 3 Pen 24 SPF hatchlings Treatment Regimen 3 Pen 25 SPF hatchlings Treatment Regimen 3 Pen 26 SPF hatchlings Treatment Regimen 3 Pen 27 SPF hatchlings Treatment Regimen Note: All groups had their pens disinfected at each water change. Each pen had a total of 9 animals. There was also an additional group (Group 4) that was composed of SPF hatchlings that underwent normal farm operation (Control regimen).

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224 Table 8-2. Suggested solution fo r the copper sulfate treatment Solution Directions Copper sulfate solution Dissolve 6 grams of citric acid monohydrate in one quart of water. Add 9 grams of copper sulfate pentahydrate. Dissolve all of the copper sulf ate in this solution and add water to give a total of one gallon of stock copper sulfate solution. Adding solution to pens Add 2 quarts of the stock copper solution to each 1,200 gallon tank (0.25 ppm Cu) each time that the water is changed. Treatment should not last for more than 15 days for each treatment period. Animals must be off copper treatments at least 30 days prior to slaughter. Add roughly 8 ounces of the st ock copper solution to each 88 gallon tank (0.21 ppm) each time that the water is changed. Treatment should not last past day 15 for each treatment period. Animals must be off copper treatments at least 30 days prior to slaughter.

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225 CHAPTER 9 EPIDEMIOLOGICAL PROJECTIONS, F UTURE RESEARCH, AND CLOSING DISCUSSION CONCERNING PIX SKIN DISEASE IN THE AMERICAN ALLIGATOR Introduction The Am erican alligator has survived evolut ion and time by morphing into a well designed species. Crocodilians belong to th e clade archosaurs, which include s birds, chelonians (turtles and tortoises) and lepidosaurs (lizards, snak es, and Tuatara). Among the living reptiles, crocodilians seem to be closest to birds phyl ogenetically (Cooper et al 1985, Alibardi 2004). Alligators belong to the class Sauropsida, which encompasses all reptiles. Crocodilians include 28 total species and subspeci es that are divided into four subfamilies: Alligatorinae, Crocodylinae, Gavialinae, and To mistominae (Lane 2005). Alligatorinae includes two species of alligators and eight species of caimans. While the American alligator (Alligator mississippiensis ) was once on the endangered species list, its cousin the Chinese alligator ( Alligator sinesis ) is severely endangered at this time. Alligator farming dates back to the 1800s, where in 1891 the first reported alligator farm in Florida was started in th e city of Jacksonville (Woodward 1981). As hunting began decreasing the natural stock, the alligator was placed on the endangered species list in 1973, but its classification was reduced to threatened in 1975. Since this time, alligator farming has increased exponentially. Currentl y, there are over 200 alligator farm s located in the southeastern United States. Texas and Florida each have approximately 40 farms, Louisiana has 122, and Georgia, Mississippi and Alabama have a combin ed 14 farms. Florida, Louisiana and Texas produce a combined annual total of around 45,000 wild and farmed alligator hides per year. This study was completed to effectively characterize the condition known as PIX to alligator farmers and tanners. The connec tion between PIX and SOLs has led to the development of a diagnostic panel to illustrate possible causative agents of this integumentary

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226 condition. In addition, a treatment regimen was fo rmulated for farmers to use on their farms to limit the presence of SOLs and subsequent PIX. Epidemiological Projections From the initial discovery of the alligator skin disease that has been termed PIX, there have been many changes in its epidemiology and progression. Since its first general occurrence in 1999, the disease progressed steadily from Florida to the other s outheastern states that have alligator farms: Georgia, Louisi ana, and Texas have all reported the dermatological anomaly since 2000. One of the questions is why cer tain farms will exhibit constant outbreaks of SOLs and PIX for years while others do not. The treatment regimens that have been proposed have lent a hand in the diseases submission. Adhering to the re searchers suggestions on co ntrol will lead to its regression. Although many farms were not put thr ough a complete treatment study, they were all given the suggestions that they then followed; this seems to have worked, since many of the farms that took the suggestions now have a low if not nonexistent SOL problem. Problems Encountered Initially, there were three farm s that agreed to have the treatment study carried out at their facility. Unfortunately, one of these facilities was destroyed in hurricane Katrina. The second farm did participate in the study for a brief period of time, however, due to financial hardship they were forced to withdraw from the pr oject and no additional samples were obtained. One of the Louisiana farms that were incl uded in this study went bankrupt and samples came intermittently throughout the du ration. Since the farm was in financial turmoil, hides were sold no matter what their condition and farming ope rations were mediocre. This is what we hypothesize might be one of the reasons for their lo w hide quality and high incidence of SOLs. A study was also started at this farm that enta iled marking SOLs prior to tanning to get an

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227 accurate measurement of how many of those lesions will actually turn into PIX lesions. The study was never completed Since Hortaea werneckii is commonly transmitted through water and soil, a study was conducted to perform environmental testing on both substrates. This proved to be time consuming and most of the colonies that were re covered was not considered to be pathogenic to the alligator (normal organism s of their environment). A study was performed to test alligator nests from 30 different sites in Florida for any pathogenic microorganisms. We had hoped to provide insight into the transmissibility of PIX, perhaps that it is something that they can cont ract in the wild. Samples were placed on specialized agar that contained varying perc entages of NaCl to promote the growth of H. werneckii No pathogens were cultured, and H. werneckii was not found. This may be in part due to the overgrowth of fungi and bacteria within the Petri dishes despite the presence of salt. The study was stopped immediately. Future Research W hile diagnostic testing was performed within th e scope of this project, there are areas that have yet to be examined. In s itu hybridization would be benefici al in studying the SOLs and the fungal association of these nodules. By detectin g a targeted piece of DNA within a tissue and showing its specific location within the tissue sa mple, specific microorganisms can be targeted. The researchers did attempt to st art ISH and wanted to develop a probe specifically designed to target Hortaea werneckii within the SOLs, however time c onstraints and lack of financial support for this portion of the study prevented this molecular test from being conducted. Immunohistochemistry would be beneficial in determining the presence of specific microorganisms within histological sections. Th ere are a number of localizations that can be performed with immunohistoche mistry. The cells that are located within the nodules are

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228 lymphoid in origin, but exactly what origin is not known. Phenotypically identifying the cells as B-cell, T-cell or dendritic would be interesting. At this time, there are no markers available for this type of characterization in reptiles. Scanning electron microscopy of both the ep idermal surface above a PIX nodule and the nodule itself may lend some insight into morphologi cal characteristics of the SOLs. By using this high powered microscope to distinguish surface features of SOLs and PIX, better characterization might be available. Summary and Conclusions Alligator farm ing relies on the production of high quality hides to supply leather to an ever-growing market. Any marks or imperfecti ons will severely downgrade a hide to almost no value. The fact that alligators live in an aqua tic environment seems to allow for the growth of microorganisms that may affect the skin, and any skin problems that may ensue will be hard to cure based on their environment. One of the primary observations made was that PIX was evident as localized pit-like marks on a tanned hide, however on a fresh hide these marks were not always seen. Instead these marks manifested as spherical opaque lesions, termed SOLs by the researchers. It is important to note that the term SOL is used when visualizin g the lesions on a fresh hide placed over a high intensity lamp, whereas the term PIX is used when referring to the indentations/marks on a tanned alligator hide. It is now known that the SOLs will eventually lead to the PIX marks upon development and tanning. At the present time, alligator farms have seen a decrease in the prevalence of PIX in the southeastern United States. Most of the farms included in this research have dramatically reduced the occurrence of SO Ls on their farms also.

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229 Upon histological staining of the SOLs, non-necrotizing lesions composed primarily of lymphocytes were preliminarily identified to be the cause of the sa me SOLs identified upon initial gross examination. Various stains were us ed to show the progression and contents of the lymphoid aggregations. The lymphocytic nodules appear to start progr ession in the dermal regions of the integument and proceed superficially towards the epidermis. The lesion will start to progress towards the surface of the epithelium, and in some cases has been shown to actually break through the epidermal surface. This is wh at we believe causes the PIX scars in a tanned alligator hide. These lymphoid nodules are well delimitated and range in size from 0.5 mm to 1mm. Smaller nodules are suspected to be SOLs in early development. Larger nodules are most likely in the later stages of development. Some sectio ns of skin contained more than one lesion, which directly correlates with the gross identificati on that sometimes shows more than one SOL per scale. None of the sections appeared to show any presence of pathogens within them for this portion of the study. Viruses are difficult to ex amine at the light microscopy level, however sometimes inclusion bodies are visible. None were seen in any of the lesions or surrounding areas The histological examination of ruptured and old PIX lesions shows the ability of these nodules to regress or be pulled fr om the integument duri ng the tanning process. After either of these events, the area that once contained these le sions seems to infiltrate with dense connective tissue and scar formation persists. Our study indicat es that this scar form ation will result in a PIX lesion on a tanned hide. Samples were taken from some of the sample sites to determine the similarities of SOLs from farm to farm and from state to state. The lesions all resemble one another when compared,

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230 which lead to the presumption th at these nodules are most likely caused by the same pathogen or disease process. Each of the lymphatic nodules exhibited similar progression, sharp demarcated borders, and cell populations. All nodules were lo cated in roughly the same areas in the dermis, with some located closer to the epidermis and st arting eruption. Duplicate results between states and farms reveal the likelihood of continuity between areas. The examination of a sample from the 1999 outbreak was compared to the newer lesions and they appear to be the same lesions. This sample from Farm FL-1 was taken in 1999 wh en PIX was first visualized in Florida. Electron microscopy revealed a variety of cellular components; most notably fungal elements were visualized to occur within the nodule. The character istic fungal elements visualized within the SOLs are consistent with results from microbiology and molecular testing. While intralesional diagnosis was not made usi ng histology, the presence of fungi within the lesions at the electron microsc opy level is evidence of a correlation between the SOLs, PIX, and a fungal etiology. Multiple branch ing elements with ch aracteristic double cell walls were seen in many sections. The degeneration of some of the fungal elements seen using electron microscopy may explain why we do not see them in histology; the fungus may die and di sintegrate prior to visualization and staining. Sin ce viruses and viral part icles can be visualiz ed with the use of electron microscopy, their absence within the lesions examined lends some insight as to whether or not PIX is caused by a virus. Alligator samp les obtained for characteri zation in this study did not contain viral elements. Upon examination of microbiological sa mples, growths were identified as Hortaea werneckii a pathogenic fungus found in the epider mis of humans causing the condition tinea nigra. Based on other diagnostic tools, no known bacteria, viruses, and parasites were seen

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231 within the lesions. Since H. werneckii causes skin lesions in humans, similarities with disease processes in the alligator were significant. The H. werneckii growths in all cases pr ogressed directly from the lesion embedded in the agar. This is important to note for this confirms that the cultured fungus is directly related to the lesion itself and not a contaminant. Growth characteristics were typical of H. werneckii Based on gross observations, histologi cal appearance, and microbiol ogical studies on farms in Louisiana and Florida, we have hypothesized that many if not all SOLs are caused by fungi with some of them caused by H. werneckii While no other etiological agents have been identified in the lymphoid aggregations, the presence of a known dermal pathogen may lead to the development of the SOLs. Since H. werneckii is the only organism that the researchers have evidence for and since it causes dermatologic problems in other species, it is belie ved that there is a dire ct association between H. werneckii and the SOLs. The results from the virology aspect of the study have led to some important conclusions concerning the SOL lesions. While some research ers have speculated as to a viral causation of PIX, not one viral particle was noted, cultured, or detected via PCR w ithin the current study. Through the viral diagnostic testi ng results, a virus is not likely the cause of SOLs that were collected and submitted for the current study. Of the SOLs submitted for RT-PCR, none show ed a detectable presence of West Nile virus, Eastern Equine encephalitis, or St. Louis encephalitis. The absence of any detectable product from the RT-PCR diagnostics using prim ers for WNV, EEE, and SLE further confirms the absence of a virus from most of the lesions tested. RT-PCR is a very sensitive technique in

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232 the identification of RNA viruses. If one of the viruses had been located within the samples then banding and sequencing would have been positive. Epidemiological charts that first detected th e entry of WNV into both the United States and the Southeastern portion of the United States do not overlap when PIX was first seen. WNV did not enter into the United States until 1999, and did not reach Flor ida until 2001. Historically PIX was first noted in 1999, and has been retrospe ctively seen since 1997. These dates do not correlate and a direct association between PI X and WNV can not be made. A virus may be associated in some cases of PIX; however it is not visualized as the major cause of this disease H. werneckii was positively identified via PCR in SOLs from both Florida and Louisiana alligator farms. The identity of the fragments was confirmed by automated DNA sequencing. The sequences identified with BLAST matched the sequence of H. werneckii in GenBank confirming that H. werneckii was present in the lesions. The sequences were determined to be H. werneckii with a 100% match to other H. werneckii strains in most of the cases; other samples showed between a 95% and a 98% match. The phyl ogenetic tree constructed clearly shows the relationship of the H. werneckii DNA obtained from within the lesions to other H. werneckii sequences available from the NCBI database. Based on the proposed treatments, using th e combination of SPF hatchlings, general husbandry considerations, and the copper sulfat e solution, it has been shown to reduce the incidence of SOLs. By decreasi ng the number of animals housed in a single enclosure, fighting was reduced and animals appeared subjectively to be less stressed. This ultimately led to fewer bite scars and possible entry routes for pathogenic microorganisms. At time of slaughter, treatment animals have a lo wer rate of SOLs than animals that did not receive any treatment. This, coupled with th e reduction in stocking densities and proper

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233 nutrition, has all but eradicated PI X on some farms. If farmers adhere to these suggestions and continue to carry out these best management practices, PIX should be lowered among farms that have this disease and a trend should manifest toward eradication all together. Through this research, the connection be tween PIX and the SOLs seen upon light identification has made it possible to complete the diagnostic panel formulated. Histology, electron microscopy, microbiology, virology, and PCR have all proven to be very useful techniques in the characterization of PIX. This research lends st rong evidence into the classification of this integumentary disease. Closing Remarks PIX skin disease has been an economically detr im ental problem that has lead to financial downfalls of many farms across the United States. It is our opinion that this problem has been around prior to the emergence of West Nile viru s in the United States. The location of the nodules and the occurrence througho ut many farms leads to the suggestion that it is caused by the same pathogen. In addition, since PIX was not seen prior to 1997, it is probable that the nodules are caused by a microorganism due to its ab sence in previous years. Alligator farming has been around since the early 1900s and if this condition wa s a normal immune response in alligators, it would have been noted previously. We believe that this is a conditi on that is limited to farmed anim als. This is a disease that is a result of keeping these animals in unna tural conditions conduciv e for microbiological growth. While observations of SOLs have been made on wild animals, an absence of diagnostic testing on those lesions can only lead to assumptions. We have seen trends within the past 6 years that show crocodilians ar e far more intelligent than previously reported. The fact that they are the only reptilian spec ies that cares for and nurtures their young should lead us to believe that their intellectual reach is far greater than we

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234 realize. They are a very tactile and responsive species that has been s hown to enjoy interacting with objects placed in their enclos ures. Perhaps if enrichment was added to the pens at farms, stress could be reduced and bette r quality of hides could be produ ced. Also, the addition of more dry land and proper lighting (since almost all animals are kept in the dark) might lend a role in reducing stress. Our final explanation on the pr esence of SOLs and PIX skin disease is that these nodules are caused by a pathogenic microorganism. The subsequent culture and PCR positive results of the fungus H. werneckii located within SOLs leads to a dire ct correlation between the two. The absence of any detectable viruses or bacteria within the lesions lends strong evidence to their nonexistent role in correlating with PIX.

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235 APPENDIX A HORTAEA WERNECKII LINEAGE Table A-1. Hortaea werneckii lin eage Domain Eukarya Kingdom Fungi Phylum Ascomycota Class Euascomycetes Order Dothideales Family Dothioraceae Genus Hortaea species werneckii

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236 APPENDIX B DISTRIBUTION OF PIX Table B-1. Distribution of PIX as observed by our laboratory State PIX Observed Year First Seen Florida 1999* Louisiana 2000 Georgia 2002 Texas 2002 Note: The observed PIX lesions in Florida occu rred prior to 1999, however fresh samples that could be examined with histology were only available in 1999.

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237 APPENDIX C COMMON SYNONYMS OF HORTAEA WERNECKII Table C-1 Hortaea wern eckii synonyms Fungus name in current use Synony m of names used previously Hortaea werneckii Keratomycosis nigricans palmaris Cladosporium werneckii Exophiala werneckii Phaeoannellomyces werneckii Note: Table adapted from McGinnis, et al 1999.

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256 BIOGRAPHICAL SKETCH Heather Townsend was born Heather Dickson in W esterly, Rhode Island in 1977. She received her Bachelor of Science degree from th e University of Rhode Island in Kingston, Rhode Island in May 2000 with a concentr ation in animal and veterinary sciences. She entered the College of Veterinary Medicines graduate program at the Univer sity of Florida in August 2000. She received her Master of Science degree in A ugust 2003 in veterinary medical sciences. After completion of her masters degr ee, Heather enrolled in the docto r of philosophy program in the department of Small Animal and Clinical Scienc es in the University of Floridas College of Veterinary Medicine. She addi tionally obtained a masters cert ificate in environmental policy and management from the College of Engineering in 2006. Heather was hired as a visiting lecturer in human anatomy at the Community College of Rhode Island in 2006 and a zoology instructor at Bridgewater Stat e College in 2007. She has carried out teaching responsibilities as well as working towards completing her degree. Upon completion of her degree, Heather will continue her career in teaching.