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Regulation of Immune Function in Neonatal Foals

Permanent Link: http://ufdc.ufl.edu/UFE0042093/00001

Material Information

Title: Regulation of Immune Function in Neonatal Foals Responses to Vaccination and Administration of Immunostimulants
Physical Description: 1 online resource (107 p.)
Language: english
Creator: Ryan, Clare
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: cytokine, elispot, equine, foal, immunology, immunostimulant, rhodococcus, vaccine
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: Foals are thought to be susceptible to certain infectious diseases because their immune system is nai umlautve or not as competent as that of adult horses. However, the differences in host defense mechanisms between newborn foals, older foals, and adult horses are poorly understood. The objectives of the first part of this study was to compare the frequency of IFN-gamma and IL-4 secreting cells of newborn foals to that of older foals and adult horses, and to determine the effect of the type of mitogen used for in vitro stimulation on the relative frequency of cells secreting these cytokines. The frequency of IFN-gamma and IL-4 secreting cells was significantly lower in both groups of foals compared to adult horses. Regardless of age, the type of mitogen used for in vitro stimulation had a significant effect on the IFN-gamma/IL-4 ratio. The objective of the second part of this study was to determine the effect of comercially available immunostimulants on neutrophil, macrophage, and lymphocyte function following ex vivo exposure to Rhodococcus equi. Inactivated Propionibacterium acnes (PA), inactivated parapoxvirus ovis (PPVO), or saline (control) was administered to foals on days 0 (7 days of age), 2, and 8. Treatment with PPVO significantly increased phagocytosis of R. equi and oxidative burst activity of neutrophils whereas treatment with PA decreased intracellular proliferation of R. equi within monocyte-derived macrophages, The objective of the third part of this study was to compare serum immunoglobulin concentrations, antigen-specific lymphoproliferative responses, and cytokine profile of proliferating lymphocytes of 3-day old foals, 3-month old foals, and adult horses following vaccination with a killed adjuvanted vaccine. Both humoral and cell-mediated immune responses to the vaccine were modest in 3-day old foals. Although immune responses improved with age, 3-month old foals did not respond with the same magnitude as adult horses. Newborn foals did not have a bias toward a Th2 response following vaccination with the killed vaccine used.
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 Clare Ryan.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Giguere, Steeve.
Local: Co-adviser: Long, Maureen T.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-02-28

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042093:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042093/00001

Material Information

Title: Regulation of Immune Function in Neonatal Foals Responses to Vaccination and Administration of Immunostimulants
Physical Description: 1 online resource (107 p.)
Language: english
Creator: Ryan, Clare
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: cytokine, elispot, equine, foal, immunology, immunostimulant, rhodococcus, vaccine
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: Foals are thought to be susceptible to certain infectious diseases because their immune system is nai umlautve or not as competent as that of adult horses. However, the differences in host defense mechanisms between newborn foals, older foals, and adult horses are poorly understood. The objectives of the first part of this study was to compare the frequency of IFN-gamma and IL-4 secreting cells of newborn foals to that of older foals and adult horses, and to determine the effect of the type of mitogen used for in vitro stimulation on the relative frequency of cells secreting these cytokines. The frequency of IFN-gamma and IL-4 secreting cells was significantly lower in both groups of foals compared to adult horses. Regardless of age, the type of mitogen used for in vitro stimulation had a significant effect on the IFN-gamma/IL-4 ratio. The objective of the second part of this study was to determine the effect of comercially available immunostimulants on neutrophil, macrophage, and lymphocyte function following ex vivo exposure to Rhodococcus equi. Inactivated Propionibacterium acnes (PA), inactivated parapoxvirus ovis (PPVO), or saline (control) was administered to foals on days 0 (7 days of age), 2, and 8. Treatment with PPVO significantly increased phagocytosis of R. equi and oxidative burst activity of neutrophils whereas treatment with PA decreased intracellular proliferation of R. equi within monocyte-derived macrophages, The objective of the third part of this study was to compare serum immunoglobulin concentrations, antigen-specific lymphoproliferative responses, and cytokine profile of proliferating lymphocytes of 3-day old foals, 3-month old foals, and adult horses following vaccination with a killed adjuvanted vaccine. Both humoral and cell-mediated immune responses to the vaccine were modest in 3-day old foals. Although immune responses improved with age, 3-month old foals did not respond with the same magnitude as adult horses. Newborn foals did not have a bias toward a Th2 response following vaccination with the killed vaccine used.
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 Clare Ryan.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Giguere, Steeve.
Local: Co-adviser: Long, Maureen T.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-02-28

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042093:00001


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1 REGULATION OF IMMUNE FUNCTION IN NEONATAL FOALS: RESPONSES TO VACCINATION AND ADMI NISTRATION OF IMMUNO STIMULANTS By CLARE ANN RYAN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FU LFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010

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2 2010 Clare Ann Ryan

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3 To Jillian Adele Stirn

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4 ACKNOWLEDGMENTS I thank the Florida Thoroughbred Breeders and Owner Association, Morris Anima l Foundation, University of Florida College of Veterinary Medicine, and Pfizer Animal Health for their generous support. I thank my committee members (Dr. Jeff Abbott, Dr. Cynda Crawford, Dr. Steeve Gigure Dr. Maureen Long, and Dr. Lori Warren) for thei r tremendous patience and invaluable guidance through out the course of my studies. I thank Dr. Gigure for undertaking the monumental task of being my advisor and for helping me main tain focus. I thank Dr. Long for her constant encouragement technical e xpertise, and sage advice. I thank Dr. Crawford for her assistance with our neutrophil assay. I thank Elise Lee, Linda Lee Ambrose, and Lisa Fultz for their delightful companionship and irreplaceable assistance in the lab. I thank R&D Systems for their assistance with the ELISPOT study. I thank my family for their unconditional support and humor. I thank my husband, Joe, for being my comfort and home, and my daughter, Jillian, for being my greatest joy.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ..................... 9 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 11 2 REVIEW OF THE LITERATURE ................................ ................................ ............ 14 Epidemiology ................................ ................................ ................................ .......... 14 Clinical Disease ................................ ................................ ................................ ...... 15 Treatment ................................ ................................ ................................ ............... 17 Microbiology ................................ ................................ ................................ ............ 17 Mechanism s of Cellular Infection ................................ ................................ ............ 19 Immunology ................................ ................................ ................................ ............ 20 Role of CD4+ T Lymphocytes in Immunity ................................ ....................... 21 Role of CD8+ T Lymphocytes in Immunity ................................ ....................... 22 Neutrophils ................................ ................................ ................................ ....... 23 The Neonatal Immune System ................................ ................................ ......... 23 Foal Immune Responses ................................ ................................ .................. 27 Prevention of Disease ................................ ................................ ............................. 28 Immunostimulants ................................ ................................ ................................ ... 30 3 EFFECT OF AGE AND MITOGEN ON THE FREQUENCY OF INTERLEUKIN 4 AND INTERFERON GAMMA SECRETING CELLS IN FOALS AND ADULT HORSES AS ASSESSED BY AN EQUINE SPECIFIC ELISPOT ASSAY .............. 32 Abstract ................................ ................................ ................................ ................... 32 Introduction ................................ ................................ ................................ ............. 33 Materials and Methods ................................ ................................ ............................ 35 Animals ................................ ................................ ................................ ............. 35 Blood Collection and PBMC Isolation ................................ ............................... 35 ELISPOT Assay ................................ ................................ ............................... 36 Collection of ELISPOT Images and Quantification of Spot forming Cells ......... 37 Western Blotting of Equine IFN and IL 4 Capture Antibodies ........................ 37 Statistical Analysis ................................ ................................ ............................ 38 Results ................................ ................................ ................................ .................... 39 Discussion ................................ ................................ ................................ .............. 40

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6 4 EFFECTS OF TWO COMMERCIALLY AVAILABLE IMMUNOSTIMULANTS ON LEUKOCYTE FUNCTION OF FOALS FOLLOWING EX VIVO EXPOSURE TO RHODOCOCCUS EQUI ................................ ................................ ......................... 47 Abstract ................................ ................................ ................................ ................... 47 Introduction ................................ ................................ ................................ ............. 48 Materials and Methods ................................ ................................ ............................ 50 Animals ................................ ................................ ................................ ............. 50 Blood and BAL Sampling ................................ ................................ .................. 51 Cell Separation ................................ ................................ ................................ 51 Monocyte D erived Macrophage Isolation ................................ ......................... 52 Infection of BAL and Monocyte derived Macrophages ................................ ..... 52 Flow Cytometric Analysis of Neutrophil Phagocytosis and Oxidative Burst in Response to R. equi ................................ ................................ ...................... 53 R. equi Antigen Production ................................ ................................ ............... 55 Proliferation and Cytokine mRNA Expression of PBMC ................................ ... 55 RNA Isolation, cDNA Synthesis, and Quantification of Cytokine mRNA Expression by Real time PCR ................................ ................................ ....... 57 IL 4 and IFN ELISA ................................ ................................ ........................ 58 Statistical Analyses ................................ ................................ .......................... 59 Results ................................ ................................ ................................ .................... 59 Clinical Data and Flow Cytometric Analysis of Neutrophil Phagocytosis and Oxidative Burst in Response to R. equi ................................ ......................... 59 Intracellular Survival and Cytoki ne mRNA Expression in Monocyte D erived and BAL Macrophages Infected with R. equi ................................ ................ 60 Proliferation and Cytokine mRNA Expression of PBMC Stimulated with Mitogens or R. equi ................................ ................................ ....................... 61 IL 4 and IFN in the Supernatants of PBMC Stimulated with CaI PMA ........... 61 Discussion ................................ ................................ ................................ .............. 61 5 EQUINE NEONATES HAVE ATTENUATED HUMORAL AND CELL MEDIATED IMMUNE RESPONSES TO A KILLED ADJUVANTED VACCINE COM PARED TO ADULT HORSES EVEN IN THE ABSENCE OF MATERNAL ANTIBODY INTERFERENCE ................................ ................................ ................. 71 Abstract ................................ ................................ ................................ ................... 71 Introduction ................................ ................................ ................................ ............. 72 Materials and Methods ................................ ................................ ............................ 73 Animals and Experimental Design ................................ ................................ .... 73 Blood Collection and Cell Separation ................................ ............................... 74 Vaccine specific Serum Immunoglobulin Concentrations ................................ 74 Vaccine specific Lymphocyte Proliferations ................................ ..................... 75 Cytokine mRNA Expression ................................ ................................ ............. 76 IFN 4 Concentrations ................................ ................................ ......... 78 Statistical A nalysis ................................ ................................ ............................ 79 Results ................................ ................................ ................................ .................... 79 Vaccine specific Immunoglobulin Concentrations ................................ ............ 79

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7 Vaccine specific Lymphoproliferative Responses and Cytokine Induction ...... 79 Cytokine Induction in Vaccine stimulated PBMCs ................................ ............ 80 Discussion ................................ ................................ ................................ .............. 81 6 SUMMARY AND CONCLUSIONS ................................ ................................ .......... 91 REFERENCE LIST ................................ ................................ ................................ ........ 93 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 107

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8 LIST OF FIGURES Figure page 3 1 Western blot of cellular extract from equine PBMC stimulated for 24 h with CaI PMA. ................................ ................................ ................................ ............ 44 3 2 ELISPOT assay for the quantification of IL 4 and IFN producing cells in equine PBMCs after stimulation for 24 h with calcium ionomycin phorbol myristate acetate (CaI PMA). ................................ ................................ ............. 45 3 3 Least square mean IFN (A), IL 4 (B), and IFN /IL 4 ratio (C) spot forming cells (Log10 SD) in mononuclear blood cells ................................ ................... 46 4 1 Phagocytos is (A) and oxidative burst activity (B) of blood neutrophils after ex vivo exposure to R. equi ................................ ................................ .................... 66 4 2 Intracellular proliferation of R. equi in PBMC derived macrophages (A) and BAL macrophage s (B) of foals prior to (day 0) or after (days 12, 24, 36) administration of a placebo (control; n=6), PPVO (n=5), or PA (n=6) ................. 67 4 3 Fold increase in relative mRNA expression of IL 12p40 in B AL macrophages (A) and of TNF in monocyte derived macrophages (B) 4 h following infection with virulent R. equi ................................ ................................ ............. 68 4 4 Relative IL 10 mRNA expression in PBMC stimulated with R. equi antigen s. Fo als were administered a placebo ................................ ................................ .... 69 4 5 IL 4 concentration in the supernatants of PBMC stimulated with CaI PMA. The cells were collected on day 12 of the study. ................................ ................ 70 5 1 Relative vaccine specific serum total IgG (A), IgM (B), IgGa (C), IgGb (D), and IgG(T) (E) concentrations as determined by capture ELISA ........................ 87 5 2 Me an (SD) vaccine specific lymphoproliferative responses as determined by a colorimetric lymphocyte proliferation assay. ................................ ............... 88 5 3 Mean (SD) concentrations of IFN 4 (B), and IFN 4 ratio (C) in the supernatants of PBMCs stimulated with vaccine antigens as determined by ELISA. ................................ ................................ ......................... 89 5 4 Relative IL 2 (A) and IL 10 (B) mRNA expression in PBMCs stimulated with vaccine antig ens. ................................ ................................ ................................ 90

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9 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy REGULATION OF IMMUNE FUNCTI ON IN NEONATAL FOALS : RESPONSES TO VACCINATION AND ADMI NISTRATION OF IMMUNO STIMULANTS By Clare Ann Ryan August 2010 Chair: Steeve Gigure Major : Veterinary Medical Sciences Foals are thought to be susceptible to certain infectious diseases because the ir immune system is nave or not as competent as that of adult horses. However, the differences in host defense mechanisms between newborn foals, older foals, and adult horses are poorly understood. T he objective s of the first part of this study was to c ompare the frequency of IFN and IL 4 secreting cells of newborn foals to that of older foals and adult horses and to determine the effect of the type of mitogen used for in vitro stimulation on the relative frequency of cells secreting these cytokines The frequency of IFN and IL 4 secreting cells was significantly lower in both groups of foals compared to adult horses. Regardless of age, the type of mitogen used for in vitro stimulation had a significant effect on the IFN /IL 4 ratio. The objective of t he second part of this study was to determine the effect of comercially available immunostimulants on neutrophil, macrophage, and lymphocyte function following ex vivo exposure to R hodococcus equi I nactivated Propionibacterium acnes ( PA ) inactivated parapoxvirus ovis ( PPVO), or saline (control) was administered to foals on day s 0 (7 days of age), 2, and 8. Treatment with PPVO significantly increased phagocyt osis of R. equi and

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10 oxidative burst activity of neutrophils whereas treatment with PA decreased intracellular prolifer ation of R. equi with in monocyte derived macrophages The objective of the third part of this study was to compare serum immunoglobulin concentrations, antigen specific lymphoproliferative responses, and cytokine profile of proliferating lymphocytes of 3 d ay old foals, 3 month old foals, and adult horses following vaccination with a killed adjuvanted vaccine Both humoral and cell mediated immune responses to th e vaccine were modest in 3 day old foals. Although immune responses improve d with age, 3 month old foals did not respond with the same magnitude as adult horses. Newborn foals d id not have a bias toward a Th2 response following vaccination with the killed vaccine used

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11 CHAPTER 1 INTRODUCTION Cell mediated immune responses of murine and human n eonates are generally thought to be biased toward a thymocyte helper type 2 ( Th2 ) response (Adkins, 2000) Several studies have documented that newborn foals are deficient in their ability to induce interferon gamma ( IFN in response to stimulation with mitogens (Boyd et al 2003 ;Breathnach et al. 2006 ) These findings, along with the peculiar susceptibility of foals to infection with Rhodococcus equi a facultative intracellular pathogen known to only cause disease in immunocompetent mice when a Th2 response is experime ntally induced (Kanaly et al. 1995) have led to the hypothesis that T cell responses from newborn foals m ay be biased toward a Th2 cytokine profile. However, experimental infection of neonatal foals with virulent R. equi triggers induction of IFN mRNA transcription in a manner that is similar to that of adult horses, indicating that foals can mount adequate IFN responses providing the proper stimulus (Jacks et al ., 2007a; Jacks et al ., 2007b). There are several gaps in our current understandi ng of the regulation of immune responses in foals. The work presented in this dissertation aims at addressing a few of these gaps. First, a t immune responses also necessitates measurement of Th2 cytokines such as IL 4. Unfortunately, interleukin ( IL ) 4 production has not been investigated in newborn foals. In addition, t he relative Th1/Th2 polarization of equine neonatal immune responses would be better assessed by measuring antigen specific resp onses after vaccination rather than after artificial stimulation with mitogens. There are no studies evaluating cell mediated and cytokine responses of newborn foals in response to vaccination

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12 Finally d ata in human, laboratory animals, and adult hors es suggest that commercially available immunostimulants are potent inducers of IFN and contribute to neutrophil and macrophage activation A product capable of increasing IFN induction and activating neutrophils and macrophages in neonatal foals prior to natural infection may be effective in preventing, or at least curtailing, infec tion with R. equi during the narrow window of susceptibility to this pathogen. The objectives and hypotheses of the first study (Chapter 3) are as follows: 1. T o compare t he frequency of IFN and IL 4 secreting cells of newborn foals to that of older fo als and adult horses The hypothesis was that newborn foals have fewer IFN and IL 4 secreting cells than older foals or adults. 2. To determine t he effect of the type of mitogen used for in vitro stimulation on the relative frequency of cells secreting these cytok ines The hypothesis was that the type of mitogen used to stimulate PBMCs affects the magnitude of the response. The objectives and hypotheses of the second study (Chapter 4 ) are as follows : 1. T o determine t he effect of immunostimulants on in tracellular survival and replication of R. equi in foal macrophages The working hypothesis for this objective is that macrophages obtained from foals pre treated with immunostimulants can kill R. equi more efficiently in vitro 2. T o determine t he effec t of immunostimulants on cytokine induction by R. equi infected macrophages The hypothesis is that mRNA expression of inflammatory (IL 6, TNF Th1 inducing cytokines (IL 12, IL 18) will be significantly higher in macrophages obtained from foa ls pre treated with immunostimulants than in controls. 3. T o determine t he effect of immunostimulants on phagocytic activity and oxidative bu r s t of blood neutrophils of foals The hypothesis is that immunostimulants will significantly enhance phagocytic ac tivity and oxidative burst of peripheral blood neutrophils.

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13 4. T o determine t he effect of immunostimulants on lymphocyte immunophenotyping, lymphoproliferative responses, and cytokine induction in foals The hypothesis wa s that immunostimulants will signif icantly enhance lymphoproliferative responses and IFN induction by proliferating lymphocytes in foals. The objectives and hypotheses of the third study (Chapter 5) are as follows : 1. T o determine s erum IgM and IgG subclass concentrations of newborn foals older foals, and adult horses following va ccination with a killed vaccine The working hypothesis for this objective is that neonatal foals will produce lower IgGa and IgGb concentrations in response to vaccination compared to older foals or adult horses 2. T o determine a ntigen specific lymphoproliferative responses of newborn foals, older foals, and adult horses following va ccination with a killed vaccine The hypothesis is that newborn foals have decreased antigen specific lymphoproliferative response s compared to older foals or adult horses. 3. T o determine t he cytokine profile of proliferating lymphocytes from newborn foals, older foals, and adult horses following vaccination with a killed vaccine The hypothesis for this objective is that lymphocyte s from newborn foals will produce less IFN and less IL 4 in response to stimulation with the vaccine antigen compared to lymphocytes obtained from older foals or adult horses.

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14 CHAPTER 2 REVIEW OF THE LITERATURE Rhodococcus equi is a facultative intracellular bacterium that causes severe pyogranu lomatous pneumonia in foals. It has also emerg ed as an important opportunistic pathogen in immunosuppressed people and patients infected with the human immunodeficiency virus (Arlotti et al. 1996;Emmons et al. 1991;Prescott, 1991) Although R. equi is found in soils worldwide, it does not uniformly cause disease in all foals exposed to it. The disease mainly affects foals 1 5 months of age. Some farms may have little to no apparent disease, whereas on other farms the disease is endemic There is major economic impact on the equine industry due to the cost of treatment, loss of function, delays in training, and death of some animals. Epidemiology Rhodococcus equi is a saprophytic organism that survives wel l in surface soil (Barton & Hughes, 1984) The bacteria are shed in high numbers in the feces of infected foals, an d persist in the environment. Adult horses, although they do not display clinical signs of the disease, can also be an important source of the bacteria through fecal shedding. However, because it does not replicate within the adult equine intestinal trac t, the amount of bacteria shed in the feces of dams is small (10 1 to10 3 bacteria/gram of feces ) relative to the heavy fecal shedding (about 10 5 bacteria/gram of feces ) of foals, whose intestinal tract allows for replication until about 3 months of age (Takai et al. 1987) The organism is very hardy in the environment, and is able to multiply in f eces on the ground (Hughes & Sulaiman, 1987 ) R. equi is particularly adept at surviving in dry, arid conditions (Barton & Hughes, 1984) The main route of in fection is inhalation of the bacteria. Inoculation of foals with the bacteria intra

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15 bronchially is a consistent experimental method of inducing R. equi pneumonia in foals (Martens et al. 1982) However, ingestion of the bacteria certainly does also occur and may be an alternative route of infection (Johnson et al. 1983) R. equi has a worldwide distribution. Isolates have been identified on most continents, and in several s pecies of animals including pigs, ruminants, dogs, cats and horses, Foals are likely exposed very early in life to Rhodococcus equi based on the ubiquity of the organism in their environment. Clinical Disease The hallmark of Rhodococcus equi infection is severe, chronic, progressive pyogranulomatous pneumonia. Initial signs of the disease may manifest clinically as fever and mild exercise intolerance or elevated respiratory rate. The insidious onset of disease makes detection of early disease a challeng e. More intermediate signs of lethargy, inappe t a nce, increased respiratory rate and effort, and occasionally cough and nasal discharge may occur. Signs may progress rapidly if untreated to severe respiratory distress, cyanosis, and death. Histopathologi c examination of foals that die from, or are euthanized because of the disease, is characterized by large granulomatous, cavitary lesions with intracellular organisms present diffusely throughout the lungs. Infected foals may also frequently develop extra pulmonary lesions including polysynovitis, osteomyelitis, and septic arthritis, which are characterized by varying degrees of lameness and/ or joint effusion. Immune mediated uveitis is also a recognized s equela of the disease. Gastro intestinal lesion s can be found in up to 50% of R. equi patients necropsied (Zink et al. 1986) and can occasionally be associated with the development of diarrhea. Granulomas can often be found in the mesenteric

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16 the colon (Johnson et al. 1983) A variety of methods have been used for t he diagnosis of Rhodococcus equi pneumonia in foals. The gold standard remains culture of the bacteria from transtracheal wash samples along with cytological evidence of septic inflammation Additional diagnotics tools include physical examination, blood work, and imaging studies. Thoracic auscultation may reveal a range of abnormalities including diffuse crackles and wheezes, or simply increased broncho vesicular sounds. Ancillary bloodwork can be helpful in monitoring foals on farms with endemic disease. Specifically, an increase in white blood cell counts has been shown to occur prior to development of clinical disease (Gigu re et al. 2003b) However, these finding s are non and prevalence of R. equi pneumonia on the farm Imaging of the thoracic cavity is a commonly employed diagnostic. Thoracic radiographs are largely reserved for use at refer ral institutions. Most commonly, a prominent alveolar pattern is present. Classical radiographic findings indicating the presence of abscesses are discrete cavitary and nodular lesions. Thoracic ultraso nography is commonly used in the field because of w idespread availability and ease of use Initially, practitioners may detect pleural irregularities as the only abnormality. Progression of the pneumonia may allow for detection of consolidated lung and/or abscesses peripherally. Serological assays have been largely unhelpful in the diagnosis of R. equi pneumonia largely due to the number of foals that are exposed but remain healthy (Gigu re et al. 2003a)

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17 Treatment Currently the mainstay of treatment is antimicrobial therapy with macrolides and rifampin. Although many isolates are sensitive to a wide array of antimicrobials in vitro macrolides and rifampin ha ve much better efficacy in vivo because of their ability to therapy with rifampin and erythromycin was used as the treatment of choice. The treatment regime consisted of a p rolonged course of therapy usually 6 8 weeks, and was successful in decreasing deaths caused by infection. More recently, however, other combinations utilizing newer generation macrolides such as azithromycin and clarithromycin in conjunction with rifampi n, have gained popularity because of better efficacy and the shorter course of therapy required ( Gigure et al. 2004) Simultaneous supportive care may be required in the form of non steroidal anti inflamma tories for analgesic and anti pyretic properties, oxygen supplementation for foals unable to oxygenate properly, fluid therapy for dehydrated animals, and nutritional support in the form of parenteral nutrition for severely debilitated foals. With these drugs and therapies, survival rates have improved. Foals that recover typically do so with no negative impact on performance (Ainsworth et al. 1993;Bernard et al. 1991) Foals recovering from pneumonia enjoy ed similar success at the racetrack compared to cohorts without pneumonia. However, R. equi pneumonia is still very common and remains a tremendous drain on owner and manager resources. Microbiology Rhodococcus equi is characterized as a G ram positive, facultative intracellular bacterium. It is a pleomorphic coccobacillus belonging to the mycolata taxon which also contains the human pathogen Mycobacterium tuberculosis These bacteria

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18 characteristically have a cell envelope rich in high molecular weight branched chain mycolic acids. R. equi has a lipoarabiminomannan (LAM) wall, which is similar to that of M. tuberculosis This wall has been shown to contribute to its immunogenicity, based its ability to increase proinflammatory cytokine expression by e quine peripheral blood mononuclear cells ( PBMC s) exposed to the LAM (Garton et al. 2002) It has also been shown to suppress T cell proliferation and inhibit TNF induced functions including microbicidal activity, induction of cytokines associated with macrophage activation (Chatterjee & Khoo, 1998) Virulent R. equi contains a n 85 90 kilobase plasmid shown to have 69 open reading frames (Takai et al. 2000) The plasmid contains a 27.5 kb pathogenicity island which encod es genes for vir ulence associated proteins (Vap A, and VapC Vap I ) (Takai et al. 2000 ) Regulation of vap gene expression is depende nt on multiple factors including pH temperature, magnesium, and iron (Benoit et al. 2001;Ren & Prescott, 2003;Takai et al. 1996) Vap A and Vap G expression are highly up regulated by exposure to hydrogen peroxide as part of the oxidative burst of activated macrophages (Benoit et al. 2002) and may play a role in mediat ing survival within macr ophages. In vitro infection of murine macrophages with plasmid containing R.equi results in effective replication of the bacterium, whereas macrophages infected with plasmid cured strains successfully clear the bacteria (Gigu re et al. 1999b) Vap A, expressed on the cell surface when R. equi is grown between 34 and 41C (Takai et al. 1996) is associated experimentally and clinically with increased incidence and severity of disease (Takai et al. 1994 ; Takai et al. 1991) Foals infected with R. equi strains containing the virulence plasmid and expressing VapA develop clinical signs of the

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19 bronchopneumonia, while foals infected with plasmid cured strains do not develop disease ( Gigure et al. 1999a;Wada et al. 1997) In one study by Jain et al a mutant strain of R. equi lacking vap A and vapC vapI genes was shown to avirulent ; complementation with va p A restored virulence whereas compl ementation with vapC, vapD, or vapE did not (Jain et al. 2003) Conversely, a recombinant plasmid cured derivative expressing wild type levels of VapA failed to survive and replicate in macrophages and remained avirulent for foals showing that expression of VapA alone is not sufficient to restore the virulence phenotype (Gigu re et al. 1999a) These findings show that although VapA is essential for virulence, other plasmid encoded products also contribute to the ability of R. equi to cause disease Mechanisms of Cellular Infection R equi is a facultative intracellular pathogen. Once inside macrophages live bacteria persist and multipl y within membrane bound phagocytic vesicles (Zink et al. 1987) Approximately 60 75% of R. equi survive after ingestion by macrophages (Zink et al. 1985) Once in the cell, the normal host defenses to bacterial pathogens include fusion of the phagosome and lysosome, and exposure of the pathogen to toxic reactive oxygen intermediate s including oxygen (O 2 ), superoxide anions (O 2 ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical ( OH), and reactive nitrogen intermediates including nitic oxide (NO), and peroxynitrate (ONOO ). Production of these toxic metabolites is increased in macrophage s that have been activated by exposure to the pro inflammatory cytokine IFN R. equi has proven to be resistant to several of these metabolites when exposed in vitro including H 2 O 2 (Benoit et al. 2002) and superoxide (O 2 ) (Brumbaugh et al. 1990) Killing of intracellular R. equi in a murine mac rophage

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20 model is largely depende nt on production of peroxynitrate (ONOO ) by activated macrophages (Darrah et al. 2000 ) R. equi enters the cell via complement mediated mechanisms utilizing the complement type 3 receptor (CR3), also called Mac 1 (Hondalus et al. 1993) Since this receptor is present pr imarily on mammalian monocytes and macrophages, it follows that disease is characterized by R. equi found within macrophages. R. equi is opsonized by the alternative complement pathway (ACP), as demonstrated by fully effective binding to Mac 1 when incuba ted with C2 and C4 deficient serum, and loss of binding with C3 depleted serum (Hondalus et al. 1993) R. equi s ome researchers to speculate that the bac teria prevent fusion of the phagolysosome. Fernandez Mora et al ( 2005) showed that R. equi containing vacuoles completed early endosome stage but did not progress to a fully mature late endosome. In the same study the fusion of the phagolysosome did not occur in murine macrophages infected with plasmid containing R. equi However, another study has demonstrated that R. equi survival in murine J774 macrophages, which occurs in plasmid containing isolates but not plasmid cured isolates, was not due to failure of the phagolysosome, but by suppression of acidification of the phagolysosome (Toyooka et al. 2005) Immunology Crucial to understanding why foals are the s usceptible population of animals is knowledge of unique characteristics of the neonatal foal immune system. First, however, we will examine normal effective adult immune responses. Adult horses are not susceptible to R. equi pneumonia, even when inoculat ed with relatively large numbers of bacteria. T lymphocytes are absolutely required for protection from disease

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21 caused by virulent, plasmid containing Rhodococcus equi as illustrated by studies performed in mice (Kanaly et al. 1995 ;Madarame et al. 1997) Both CD4+ and CD8+ T cell subsets are required for the generation of full protective immunity and will be discussed separately. Role of CD4+ T L ymphocytes in I mmunity CD4+ T lymphocytes also called T he lper cells, can be divided into two main subsets based on their pattern of cytokine secretion and effector fun c tions. The subsets are termed T helper 1 (Th1) and T helper 2 (Th2) subsets. CD4+ Th1 lymphocytes play a critical role in protection against R. equi infection in mice by secreting IFN ( IFN gamma ) (K analy et al. 1993) The production of IFN is both driven by, and helps to sustain, the production of interleukin (IL ) 12 by activated antigen presenting cells (APCs) such as dendritic cells and macrophages. Th1 type responses are required for clea rance of R. equi Conversely, the development of Th2 type cytokine responses, characterized by the production of IL 4, IL 5, and IL 10 promoting humoral immunity, is associated with development of pneumonic lesions following experimental infection of mic e with R. equi (Kanaly et al. 1995 ) The cascade of events leading to the development of adequate Th1 type responses must be initiated by appropriate stimulation of the innate immune system, which then infl uences cytokine and co stimulatory molecule production by antigen presenting cells. Exact specifications for stimulating the innate immune system to support Th1 type responses against R. equi are not yet fully understood As the link between innate and acquired immune responses, dendritic cells likely play a role in the development of Th1 versus Th2 type responses. It has recently been shown that there

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22 are differences in both phenotypic characteristics and cytokine expression of monocyte derived macro phages from foals as compared to adult horses (Merant, et al. 2009). Adult cells had higher numbers of CD1w2 + CD86 + cells in their CD14 monocyte derived macrophages as compared to newborn foals. In the same study, foal cells had lower expression of TNF 10, MCP 1 and TGF cells with LPS resulted in elevation of TNF 10 In studies performed in murine models, the toll like receptor (TLR ) 2 pathway was shown to be crucial for the initiation of intracell ular signaling resulting in increased co stimulatory molecule expression and increased cytokine responses (Darrah et al. 2004) Specifically, up regulation of CD40 molecules on dendritic cells and increased l evels of INF and TNF production by macrophages occurred after exposure of murine cells to VapA, but did not occur in mouse cell lines with knockout TLR 2 receptor. Similarly TLR2 knockout mice failed to clear the bacteria in an in vivo challenge. Based on these f indings, activation of the innate immune system via TLR 2 and subsequent up regulation of cytokine response led to increased macrophage killing of bacteria. Role of CD8+ T L ymphocytes in Immunity CD8+ T lymphocyte subsets also appear to play the major role in immunity through cell mediated defenses (Nordmann et al. 1992) The killing of Rhodococcus equi infected equine peripheral blood monocyte derived macrophages has been investigated (Patton et al. 2004) In this study, R. equi specific CD8+ cytotoxic T lymphocyte activity was present in adult immunocompetent horses, and was carried out in a MHC class I unrestricted fashion. Notably, in a similar study a ssessing the development of CTL activity in foals, R. equi stimulated PBMCs from foals were unable

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23 to lyse R. equi specific target cells at three weeks of age, but gained the ability to lyse by six weeks of age (Patton et al. 2005) Cell mediated killing of R. equi is crucial for clearance of the bacteria and prevention of disease; an inefficacy of CTL response in foals may help explain their unique susceptibility to R. equi pneumonia. Neutrophils It has be en documented that neutrophils from foals exert age related changes in the ir phagocyt ic and oxidative burst activity (McTaggart et al. 2001;Witchel et al 1991) C omparison of neutrophil function between foal s and adults has given contradictory results with some studies showing equal and others showing decreased neutrophil function Both adult horse and foal neutrophils are fully capable of killing ingested Rhodococcus equi although a subset of neonatal fo als may have decreased killing capabilities (Martens et al. 1988 ;Takai et al. 1986a;Takai et al. 1986b) Antibody specific opsonization by neutrophils enhances phagocytosis and killing of the bacteria In a dult horses, stimulation of neutrophils with R. equi leads to increased expression of the pro inflammatory cytokines TNF 12p40, IL 6, IL 8, and IL 23p19 (Nerren et al. 2009b ) Similarly, foal neutrophil s stimulated with R. equi also express pro inflammatory cytokines TNF 12p35, IL 12p40, IL 6, IL 8, and IL 23p19, as well as IFN (Nerren et al. 2009a) In the aforementioned study, expression of IL 6, IL 8, IL 12p40 and IL 23p19 increased with age Ontogeny of the Equine Immune System Development of the equine immune system occurs relatively early during fetal life. Lymphocytes are present in the peripheral blood of the equine fetus by day 120 of ges tation and they proliferate in response to mitogens by day 140 (Perryman et al. 1980) Specific antibody responses to in utero vaccination with coliphage T2 have been

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24 detected in equine fetuses as early a s day 200 of gestation (Martin & Larson, 1973) In other studies, administration of a V enezuelian equine encephalomyelitis antigen to equine fetuses between 232 and 283 days of gestational age resulted in higher serum neutralization titers than that elicited by the same preparation in adult horses (Mock et al ., 1978; Morgan et al ., 1975) Recent work supports these findings, showing that active B cell development and immunoglobulin isotype switching occur during equine gestation and the neonatal period (Tallmadge et al ., 2009) Proliferation of peripheral blood lymphocytes in response to mitogens is slightly reduced at birth but rapidly increases to adult level s (Flaminio et al ., 2000;Sanada et al ., 1992) Foals also have normal lymphokine activated killing (LAK) cell activity of peripheral blood lymphocytes at birth and during early life (Flaminio et al. 2000) The Neonatal Immune System For neonates, exposure to a large number o f infectious organisms occurs after birth. For the first few months, the maternal transfer of antibodies to the newborn may protect to some degree against potential pathogens until immune responses can be initiated. Intracellular organisms, such as Rhodo coccus equi present a particular challenge to foals because of their exposure at a very young age, and because passive immunization does not completely control infection. In addition, the neonatal immune system has many differences compared with the adul t immune system that may make control of viral and intracellular bacterial infection more difficult. Differences in the neonatal immune system of mice and human neonates versus adults have been well documented (Adkins et al. 2004 ;Siegrist, 2001) Neonatal responses to agents that normally provoke strong immune responses in adults are in many cases dampened, which may lead to lack of protection from pathogens. Until fairly recently, neonatal

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25 immune responses w ere considered to be deficient or immature. However, it is becoming apparent that the immune system of neonates is competent, but that their immune cells are under different regulation than adult immune cells on many levels. Many studies have illustrate d these differences of the neonatal immune system. For example neonatal mice have been found to produce less I L 2 and have less T cell proliferation in response to certain stimuli when com pared to adult murine responses Inoculation of neonatal mice re sults in a Th2 biased response, as opposed to the Th1 type response that adult mice develop. However, this Th2 bias is not absolute; neonatal agents that are able to pr omote strong Th1 responses. Examples of these agents include some DNA vaccines, bacillus Calmette Guerin (BCG), and oligonucleotides containing CPG motifs (Hussey et al. 2002;Ito et al. 2005) Additionally, other factors such as the dose of antigen and the pre existing cytokine/environmental milleu, may (Power et al. 1998) For example, exp osure to a relatively low inoculum of BCG in neonatal mice leads to development of an almost exclusively Th1 type response and cell mediated immunity, whereas a higher inoculum of BCG resulted in a mixed Th1/Th2 response (Power et al. 1998) This stratification of responses based on dose of antigen exposure is also important in foals and will be discussed below. Expression of cell surface receptors also varies between neonates and adults. T Cell Receptor ( TCR ) complex density is lower on human and mouse neonatal cells versus adults (Harris, 1992) which may suggest a requirement for higher levels of receptor agonist to initiate intracellular signaling i n neonatal cells. Additionally, the

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26 level of expression of CD40 ligand, which assists in antibody production, class switching of antibodies by produced by B cells, and memory B cell responses, by neonatal T lymphocytes is controversial. One study indi cated that CD40 ligand expression in T cells stimulated by anti CD3+ antibody isolated from human cord blood was similar to that of adults (Splawski et al. 1996) In the aforementioned study it was note d that up regulation of expression was decreased from adult levels when exposed to the phorbol myristate acetate (PMA) and ionomycin. However, other studies have reported a consistent an d substantial deficiency in expression of CD40 ligand by neonatal T cells after stimulation with ionomycin, PMA, or by engagement of the TCR receptor (Jullien et al. 2003) Human neonatal T cells have a vari ety of mechanisms for altered responsiveness when compared to adults which may explain their tendency away from developing Th1 responses. The expression of IFN by CD4+ T lymphocytes is decreased in neonates. This has been associated with hypermethylation of CpG and non CpG sites in or proximal to the IFN promoter region (White et al. 2002) Hypermethylation of IFN promoter sites in disease states such as infection with HIV has been shown to decreased expression of IFN on cytokine production (Mikovits et al. 1998) One study investigating other mechanisms of control over cell responsiveness described decreased levels of nuclear factors of activated transcription (NFAT) in un stimulated human stem cells versus adult cells (Kadereit et al. 1999;Kadereit et al. 2003) In the study, decreased NFAT1 levels correlated well with decreased expression of cytokines TNF and IFN In a follow up to this study, the same investigators were able to increase the production of

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27 IFN by stem cells. This was accomplished by exposure to IFN and antigen presenting cells, which in turn causes positive feedback and up regula tion of NFATc2 driven IFN production (Kadereit et al. 2003) Two important concepts are highlighted here. The essential requirement for initial stimulation of the innate immune system is highlighted because, in vitro, the product ion of IFN begins with recognition of a Adequate stimulation of these cells is mandatory for mounting Th1 responses; they create the environment that lymphocytes reside in and re spond to. Secondly, under the correct environmental conditions (which are not yet fully defined but include certain cytokines and co stimulatory factors), neonatal T cells are fully capable of producing cytokines that can lead to an effective Th1 type im mune response. Once established, the specific T helper profile of cytokines tends to persist because of positive feedback and inhibition of the opposite type response. These principals become important when manipulations of the neonatal immune system, i n the form of immunomodulators and vaccines, are attempted. Foal Immune R esponses The immune defenses of neonatal foals are similar in many ways to that of mice and humans. In contrast to adults, neonatal foals are considered to have poor ability to mount T h 1 responses, and are generally considered inefficient killers of intracellular organisms. The production of IFN by equine neonatal cells in response to PMA and ionomycin stimulation has been shown to be markedly decreased in the first several weeks of life (Breathnach et al. 2006 ) A study by Boyd et al (2003) identi fied an increase in the production of RNA expression of IFN TGF 1, and IL 1 by PBMCs

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28 over the first 4 weeks of life. In contrast, under different conditions, the foal immune system can be stimulated to produce levels of Th1 cytokines comparable to th at of adults Infection of neonatal foals with a low dose of virulent R. equi results in higher production of IFN mRNA than adult horses receiving the same inoculum (Jacks et al. 2007a) In another study, the size of the R. equi inoculum was shown to modulate the IgG subisotype response and possibly the cytokine profile of foals. A recent study by Liu et al ( 2009) shows that foal PBMCs stimulated with R. equi o r CpG ODN have increased expression of IL 6 and IL 8, and that the magnitude of this increased expression was greater in older foals versus newborn foals In the same study, foals had a differential response to the stimulus used; stimulation with R. equi led to increased expression of IL 23p19/p40, whereas stimulation with CpG OPN led to increased expression of IL 12p35/p40. Taken cumulatively, studies of the equine neonat al immune system indicate that there are difference between neonates and adults, wit h neonates being deficient in their ability to produce IFN However, when exposed to the right environment, foals appear capable of mounting Th1 responses o f the same magnitude as adults. Prevention of Disease Several strategies have been employed in an attempt to prevent or decrease the severity of R. equi pn eumonia in foals. Humoral immunity appears to play a role in host defense against the organism as indicated by results of passive immunization studies. In an effort to prevent disease, hyperimmune plasma has been administered to foals on farms with endem ic R. equi pneumonia. There has been a range of effect, but on some farms with endemic R. e qui pneumonia there was a significant decrease in incidence of

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29 disease when foals received hyperimmune plasma (Madigan et al. 1991 ;Martens et al. 1989; Prescott et al. 1997a) Other studies have been inconsistent in identifying significant effects of passive immunization (Gigu re et al. 20 02; Hurley & Begg, 199 5 ) These s tudies indicate that antibody plays a partial role in clearance but is not completely protective. Foals that are present on farms that have endemic Rhodococcus equi are exposed to high concentrations of bacteria from very early on in life. However, most foals on endemic farms do not develop severe pneumonia. Also, previous studies on the effect of oral inoculation with live virulent R. equi indicate that oral immunization provides protection against heavy challenge in foals (Chirinotrejo et al. 1987;Hooper McGrevy et al. 2005) These findings illustrate the potential utility in vaccinating foals against key antigens of Rhodococcus equi Several active vaccination strategies have been investigated in mice, a nd both foals and their dams, and have been met with varying degrees of success. In 1991 studies by Martens and Madigan investigated the utility of vaccinating mare s to enhance colostral R. equi specific antibody and increase passive immunity. In both st udies, vaccination of the mares was non protective. Vaccination of mares with VapA in a water base nanoparticle adjuvant may have provided a degree of protection against a small number of naturally challenged foals (Cauchard et al. 2004) Recently, a DNA vaccine expressing VapA was used successfully in mice to enhance clearance of virulent R. equi (Haghighi & Prescott, 2005) Concomitant increases in IgGa, indicative of a type 1 based immune response, were also noted. In the same study, the addition of IL 12 to the VapA DNA vaccine resulted in even more marked effect on clearance of

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30 R. equi by mice. However, a similar DNA vaccine induced strong cell mediated responses in adult horses but poor responses in immunized foals (Lopez et al. 2003) Although success has not yet been achieved, active immunization is still likely to become an important part of R. equi prevention and further studies are ongoing. Immunostimulants An immunostimulant can be defined as effector cells such as macrophages, lymphocytes, and neutrophils, which subsequently activate one or more terminal immune responses such as antigen uptake, cytotoxicity, phagocytosis, cytokine release, and antibody (Flaminio et al. 1998) Several immunostimulants are commercially available for use in equids. One product, EqStim ( Neogen Corporation ), is composed of inactivated Propionibacterium acnes It has been used for the treatment of non specific respiratory disease in horses This preparation has been used with favorable results in clinical trials for prevention of stress induced respirat ory infection in adult horses (Nestved, 1996) I t has also been investigated as an adjunct to conventional therapy in the treatment of horses with equine respiratory disease complex (Vail et al. 1990) Results showed that 96% of horses treated with traditional therapy in combination with P. acnes recovered from disease in 14 days versus only 35% recovery of horses treated with traditional therapy alone. Proposed mechanism of action is activatio n of macrophages, which contributes to enhanced pathogen killing. In h orses Davis et al (2003) id entified increased gene expression of the type 1 cytokines IFN and NK lysin as determined by RT PCR in adult horses treated with inactivated P. acnes Flaminio et al (1998) showed that administration of inactivated P acnes to healthy weanling (aged 6 8 months) horses

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31 resulted in increased CD4+ T lymphocytes and lymphocyte activated killer activity in peripheral blood and BAL fluid, increased nono psonized phagocytosis in peripheral blood leukocytes, and decreased pulmonary cellularity. Administration of EqStim to foals starting at age 2 3 days of age failed to have an effect on the production of IFN by PMA stimulated mononuclear cells, as de termined by flow cytometric analysis, from PBMC or BAL samples (Sturgill, 2006) Another immunomodulator available fo r use in horses is inactivated paprapoxvirus (Orf virus). This viral product is able to stimulate the innate immune system. It has been shown to cause an up regulation in human immune cells of both pro inflammatory (Th1 type) cytokines such as IFN and TNF followed by anti inflammatory (Th2 type) cytokines such as IL 10 and IL 4 (Friebe et al. 2004a) In porcine leukocytes it has increased the release of IFN IFN and IL 2, and has resulted in i ncreased proliferation, while failing to increase phagocytosis, oxidative burst, or NK activity (Fachinger et al. 2000) Inactivated parapoxvirus induce s some protection against the intracellular viral pathogens hepatitis B and herpes simplex virus infections in mice (Weber et al. 2003 ) In this study there was an initial up regulation of the production of Th1 helper type cytokines I L 12, IL 18, and IFN which was followed by a down regulation of the same cytokines and production of IL 4. IL 10 expression was also up regulated in livers of hepatitis B virus infected mice, which may have prevented excessive tissue damage.

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32 CHAPTER 3 EFFECT OF AGE AN D MITOGEN ON THE FREQUENCY OF INTERLEUKIN 4 AND INTERFERON GAMMA SECRETING CELLS IN FOALS AND ADULT HORSES AS ASSESSED BY AN EQUINE SPECIFIC ELISPOT ASSAY 1 Abstract P eripheral blood mononuclear cells (PBMCs) were obtained f ro m 6 foals < 1 week of age, 6 f oals between 3 and 4 months of age and 10 adult horses. PBMC were stimulated with concanavalin A (ConA) or calcium ionomycin phorbol myristate acetate (CaI PMA) and the frequency of interferon IFN and IL 4 secreting cells was measured using an equine specific ELISPOT assay. The number of IFN secreting cells was significantly lower in both groups of foals than in adult horses regardless of the mitogen used for stimulation. The number of IFN secreting cells was significantly higher in cells stimulated with CaI PMA than in cells stimulated with ConA. In cells stimulated with CaI PMA, the number of IL 4 secreting cells was significantly lower in both groups of foals compared to adult horses. I n adult horses only, CaI PMA stimulation resulted in significantly more IL 4 secreting cells than ConA stimulation. Regardless of age, the ratio of IFN /IL 4 spot forming cells (SFC) was significantly higher in cells stimulated with CaI PMA than in cells stimulated with ConA. These findings indicate that the frequency of IFN and IL 4 secreting cells is lower in foals than in adult horses and that the type of mitogen used has a profound effect on the relative production of both cytokines. 1 Reprinted with permission from Ryan, C., Gigure, S., Hagen, J., Hartnett, C. & Kalyuzhny, A. E. (2010).Effect of age and mitogen on the frequency of interleukin 4 and interferon gamma secreting cells in foals and adult horses as assessed by an equine specific ELISPOT assay. Vet Immunol Immunopathol 133, 66 71.

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33 Introduction Neonates are particularly susceptible to infectious agents which rarely affect or only cause mild disease in adults. The susceptibility of newborn s to infectious diseases may be partly explained by their lack of pre existing immunological memory as well as by the relatively small number of immune cells in peripheral lymphoid tissues in early life (Fadel & Sarzotti, 2000) Additionally, many studies in both humans and mice have demonstrated that lymphocytes from neonates are qualitatively distinct from adult cells. Neonatal responses in both humans and mice are often deficient in their ability to mount protective Th1 responses characterized by interferon (IF N ) production. While T cell responses in neonatal mice are typically biased towards the Th2 cell lineage distinguished by producing primarily interleukin (IL) 4, this clear skewing is not always readily apparent in newborn humans (Adkins et al. 2004) In human neonates, a Th2 bias has been demonstrated in some studies (Prescott et al. 1998;Ribeiro do Couto et al. 2001) while other studies have show n that both Th1 and Th2 responses are decreased in magnitude compared to the responses of adults (Adkins et al. 2004 ;Xainli et al. 2002) Newborn foals are also deficient in their ability to produce IFN in response to stimulation with mitogens (Boyd e t al. 2003 ;Breathnach et al. 2006 ) These findings, along the peculiar susceptibility of foals to infection with R hodococcus equi a facultative intracellular pathogen known to only cause disease in immunocompetent mice when a Th2 response is experimen tally induced (Kanaly et al. 1995) have led to the hypothesis that T cell responses from newborn foals ma y be biased toward a Th2 cytokine profile. However, in recent studies, IL 4 mRNA expression in response to

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34 stimulation of bronchial lymph node mononuclear cells with various R. equi antigens was significantly lower in foals than in adult horses (Jacks et al. 2007a ;Jacks et al. 200 7b ) Collectively, the studies summarized above do not support the theory of a Th2 bias and suggest that young foals may be deficient in their intrinsic ability to produce both IL 4 and IFN Studies performed in humans indicate that the type of mitogen used for in vitro stimulation of lymphocytes may have a profound effect on the relative production of IL 4 and IFN (Brown et al. 2003;Gonzale z et al. 1994;Stolzenburg et al. 1988) A variety of mitogens have been used to stimulate cytokine production by equine lymphocytes in vitro the type of mitogen used on IFN and IL 4 secretion in foals. Until fairly recently, most studies evaluating cytokine induction in horses have relied on bioassays or methods assessing mRNA expression. The development of immunoassays has been delayed by the lack of species specific anti bodies and cytokine standards. Recently, measurement of equine IFN and IL 4 by flow cytometric analysis, enzyme linked immunosorbent assay (ELISA) and enzyme linked immune spot (ELISPOT) assay has been reported using mouse anti bovine antibodies that cr oss react with equine IFN and IL 4 (Breathnach et al. 2006;Hamza et al. 2007;Paillot et al. 2005;Pedersen et al. 2002) The ELISPOT assay offers several advantages over other immunoassays. It is approxim ately 10 200 times more sensitive than ELISA, allowing detection of cytokines that are released at low concentrations or by a small frequency of cells (Tanguay & Killion, 1994)

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35 Thus, the objective s of this study were to compare the frequency of IFN and IL 4 secreting cells of newborn foals to that of older foals and adult horses and to determine the effect of the type of mitogen used for in vitro stimulation on the relative frequency of cells secreting these cytokines. To achieve these objectives we developed a ready to use ELISPOT assay using polyclonal antibodies against equine IFN and IL4 Materials and Methods Animals Twelve healthy Thoroughbred or Thoroughbred Quarter Horse crossed foals, and ten Thoroughbred or Quarter Horse adult horses (seven mares and three geldings) were used in this study. Mares and foals were housed together on pasture with ad libitum access to grass hay and water, and supplemented with sweet feed twice daily. Animals were considered healthy on the basis of physica l examinations and daily observation. Adequate passive transfer of immunoglobulin was confirmed in foals 12 to 24 h after birth using an immunoassay for measurement of total IgG ( DVM Stat, VDx Inc, Belgium, WI ). Additionally, all animals had normal CBC a nd plasma chemistry profile values at time of sampling. Blood samples from adult horses were collected at one time point. Blood from foals was collected at between day 1 and day 5 of age (n=6) or between 3 and 4 months of age (n=6). All procedures were approved by the University of Florida Institutional Animal Care and Use Committee. Blood C ollection and PBMC I solation Heparinized blood (15 ml) was collected by jugular venipuncture and stored at room temperature until processing. Peripheral blood monon uclear cells (PBMC) were separated by density gradient centrifugation using endotoxin free Ficoll Paque (Amersham Biosciences, Pittsburgh, PA). PBMCs were washed twice with PBS and

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36 live cells were counted using trypan blue exclusion. The cells were resus pended at low (2 x 10 5 cells/ml) and high (1 x 10 6 cells/ml) concentrations in RPMI 1640 (Gibco BRL, Grand Island, NY) supplemented with 10% fetal bovine serum, 25 mM HEPES, 55 mM 2 mercaptoethanol, and gentamicin (0.5 g per ml) ELISPOT A ssay Ready to use ELISPOT kits for the detection of equine IL 4 and IFN were developed (R&D Systems, Minneapolis, MN) for this project. Both assays were optimized according to R&D Systems guidelines and performed as recommended by the manufacturer. Each kit included a dry polyvinylidene difluoride backed 96 well plate pre coated with the respective capture antibody (goat anti horse specific for equine IFN or IL 4), a concentrated solution of detection antibodies, a concentrated solution of streptavidin conjugated alkaline phosphatase, BCIP/NBT substrate, as well as wash and dilution buffers. A ntibodies were raised against recombinant equine IL 4 and IFN proteins and then affinity purified using recombinant equine IFN and IL 4 immunogens, respectively. Assays were performed according to the protocols included with each ELISPOT kit. Briefly, plates were saturated with 200 l of RPMI 1640 and incubated for 20 min at room temperature. Culture media was aspirated and 100 l o f low ( 2 x 10 5 cells/ml) or high (1 x 10 6 cells/ml ) concentration PBMCs was added to triplicate wells. Cells were stimulated with concanavalin A (ConA; 4 g/ml or with phorbol 12 myristate 13 acetate (PMA; 0.05 g/ml) and calcium ionomycin (CaI; 0.5 g/ml ) for 24 h at 37C in the presence of 5% CO 2 The ideal concentration of each mitogen was established based a dose response curve in preliminary experiments. Recombinant equine IL 4 or IFN was used in triplicate wells as positive control

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37 whereas unstim ulated cells and sterile culture medium were used as negative control or background control, respectively. After incubation, PBMCs were removed from wells by washing the plates 4 times. Diluted biotinylated goat anti horse IFN or IL 4 antibody was adde d (100 l) and the plates were incubated at 4C overnight. After 4 washes, 100 l of diluted (1:120) alkaline phosphatase conjugated to streptavidin was added to each well and the plates were incubated at room temperature for 2 hours. Unbound enzyme was removed with 3 successive washes and 100 l of BCIP/NBT chromogen solution was added to each. Plates were incubated in the dark at room temperature for 1 h. Wells were then rinsed with distilled water and dried before employing image analysis to quantify spots on the membranes. Aluminum foil was used as previously described to reduce background staining and minimize well to well variation (Kalyuzhny & Stark, 2001) Collection of ELISPOT Images and Quantification of Sp ot forming C ells The cytokine releasing activity of PBMCs was evaluated by quantifying spot forming cells (SFC). SFC were determined by counting colored spots distributed over the entire area of the membrane backing each well assuming that one cell will p roduce one spot. Images from the developed ELISPOT plates were collected and quantified using semi automated QHub ELISPOT reader (MVS Pacific, St. Paul, MN). Western Blotting of E quine IFN and IL 4 Capture A ntibodies Specificity of capture antibodies us ed in IFN and IL 4 ELISPOT assays was tested on l ysates of PBMCs from adult horses. PBMCs were stimulated with CaI PMA as described above, lysed in RIPA buffer (Thermo Scientific, Waltham, MA ) and 25 ng of protein from lysed cells per lane was run on 5 2 0 % gradient Tris HCl SDS

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38 polyacrylamide mini gels ( Bio Rad Laboratories, Hercules, CA ). The proteins were then transferred to a nitrocellulose membrane in a semi dry blot chamber ( Bio Rad Laboratories ). After transfer the membranes were blocked with 5% n on fat milk in PBS with 0.1% Tween 20 (TTBS) for 1 h at 4C After 3 washes with TTBS, membranes were incubated with either 0.7 g/ml of anti IFN or 0.3 g/ml anti IL 4 antibodies overnight on a plate shaker at 4C. Membranes were washed 3 times in TTBS and then incubated at room temperature for 1 h with HRP conjugated donkey anti goat secondary antibodies diluted 1:1000 in TTBS containing 2 % non fat milk M embranes were washed in TTBS and then incubated with ECL reagent for 2 min and developed using Konica SRX 101A radiograph processor (Konica Minolta Medical Imaging, Wayne, NJ). Western blot absorption controls were done by in cubating blots with the solution of primary antibodies mixed with their cognate recombinant protein at 1:10 molar ratio. Statistical Analysis Normality and equality of variance of the data were assessed using the Kolmogorov Smirnov and Levene's tests, res pectively. All data were log transformed to meet the assumptions of the ANOVA. A 2 way ANOVA for repeated measurements was used to determine the effects of age (neonates, older foals, adult horses), type of mitogen (ConA, CaI/PMA), and the interaction be tween age and type of mitogen on the frequency of cells secreting IFN or IL 4. When appropriate, multiple pairwise comparisons were done using the Student Newman Keuls test. For all analyses, a value of P < 0.05 was considered significant.

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39 Results The IFN and IL 4 capture antibodies used in the ELISPOT assays appear ed to be specific, producing single bands of expected (~10 14 kDa) sizes ( Figure 3 1) based on previous reports (Dohmann et al. 2000;Wu et al. 2002) SFC were not detected in unstimulated cells. Wells conta ining mitogen stimulated PBMCs from adult horses at a concentration of 1 x 10 6 cells/ml had too many merged spots for accurate quantification. As a result, only data from wells containing 2 x 10 5 cells/ml were analyzed statistically. There was a visible difference in the number of SFC between foals and adult horses for both IL 4 and IFN ( Figure 3 2). There was a significant effect of age ( P < 0.001) and type of mitogen ( P < 0.001) on the number of IFN SFC was significantly lo wer in both groups of foals than in the adult horse group regardless of the mitogen used for stimulation ( Figure 3 3). The number of IFN SFC was significantly higher in cells stimulated with CaI PMA than in cells stimulated with ConA, regardless of age ( Figure 3 3 ). A significant effect of age ( P = 0.004) and a significant interaction between age and type of mitogen ( P = 0.026) was seen on the number of IL 4 SFC. In cells stimulated with CaI PMA, the number of IL 4 SFC was significantly lower in both g roups of foals compared to adult horse group ( Figure 3 3). In cells stimulated with ConA, the number of IL 4 SFC was significantly lower in 3 to 4 month old foals compared to newborn foals or adult horses ( Figure 3 3). In adult horses only, CaI PMA stimu lation resulted in significantly more IL 4 SFC than ConA. There was a significant ( P < 0.001) effect of the type of mitogen on the ratio of IFN 4 SFC. Regardless of age, the ratio of IFN 4 SFC was significantly higher in cells stimulated with C aI PMA than in cells

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40 stimulated with ConA ( Figure 3 3). There was no effect of age ( P = 0.299) on the IFN 4 ratio regardless of the type of mitogen used. Discussion produce I FN in response to stimulation with mitogens (Breathnach et al. 2006;Boyd et al. 2003) responses also necessitates measurement of Th2 cytokines such as IL 4. The present study is th e first to document a significantly lower frequency of IL 4 secreting cells in foals compared to adults following stimulation with CaI PMA and to document a significant effect of the type of mitogen used on the Th1/Th2 cytokine balance both in foals and in adult horses. In one study, mRNA expression of IFN with ConA increased by an estimated 2.5 fold during the first month of life (Boyd et al. 2003 ) In another study, peripheral blood and bronchoalveolar lavage ( BAL ) mononuclear cells from newborn foals were deficient in their ability to produce IFN following stimulation with CaI PMA (Breathnach et al. 2006) Similarly, the present study documented a significantly lower frequency of I FN secr eting c ells in foals less than one week of age compared to that of adult horses regardless of the mitogen used for stimulation. However, as opposed to the results of Breathnach et al (2006), the frequency of IFN secreting cells in 3 to 4 month old foals in the present study was not significantly different from that of newborn foals. This may be a reflection of the environment of the foals and exposure to different antigenic stimuli. Alternatively,

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41 differences in methodologies and the small sa mple sizes in both studies may be the origin of this inconsistency. Collectively, the two studies referenced above and the results of the present study clearly indicate that young foals, like neonatal mice and humans, are deficient in their baseline abil ity to produce IFN However, multiple studies have shown that, although their default response is of the Th2 phenotype, murine neonates can mount Th1 responses providing the right antigen, dose of antigen, costimulatory signal, or type of adjuvant (Adkins, 2005;Barrios et al. 1996;Forsthuber et al. 1996;Martinez et al. 1997;Sarzotti et al. 1996) Human neonates also have the ability to mount strong Th1 responses providing the right circumstances. For ex ample, vaccination of infants with Mycobacterium bovis BCG, a microorganism closely related to R. equi induces IFN production of a similar magnitude to that produced by adults (Marchant et al. 1999;Ota et al. 2002;Vekemans et al. 2001) In a recent study, young foals experimentally infected with R. equi were shown to mount strong Th1 based immune responses as evidenced by their significantly higher IFN mRNA expression in bronchial lymph nodes cells following stimulation with R. equi antigens and significantly higher IFN /IL 4 ratio compared to that of adult horses (Jacks et al. 2007 a ;Jacks et al. 2007 b ) These findings suggest that, like human and murine neonates, foals have the ability to mount adult like Th1 based responses providing an appropriate stimulus Much le ss is known regarding the ability of foals to produce IL 4. In one study, mRNA expression of IL change significantly during the first month of life (Boyd et al. 2003 ) In another study, IL 4 mRNA expression was significantly lower in health y or R. equi infected foals

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42 compared to normal or R. equi infected adult horses (Jacks et al. 2007 a ) Similar to results obtained w ith IFN the frequency of IL 4 secreting cells in response to stimulation with CaI PMA in the present study was significantly lower in both groups of foals than in adults. The difference in the number of IL 4 secreting cells between newborn foals and ad ult horses following stimulation with ConA was not significant. This was because significantly lower numbers of IL 4 producing cells in adult horses responded to stimulation with ConA compared to CaI PMA, not because a greater number of ConA stimulated f oal PBMCs produced IL 4. T cell mitogens such a s PMA, ConA, phytohemagglutinin and pokeweed mitogen are commonly used in immunological assays to induce cell proliferation and cytokine production. Studies performed in humans indicate that the type of mi togen used for in vitro stimulation of lymphocytes may have a profound effect on the relative production of IL 4 and IFN (Brown et al. 2003;Gonzalez et al. 1994;Stolzenburg et al. 1988) In one study, stim ulation of human PBMCs w ith CaI PMA, phytohemagglutinin and ConA resulted in IFN /IL 4 ratios of 58, 14 and 6, respectively (Gonzalez et al. 1994) The present study indicates that the type of mitogen used has a profound effect on the relative frequency of IFN and IL 4 secretion in horses also. CaI PMA induced primarily pattern (negative IFN /IL 4 ratio). As a result, general conclusion s regarding Th1 or Th2 bias in ho rses should not be made based on stimulation with a single mitogen. In the present study, there was no significant effect of age on the IFN /IL 4 ratio This would contradict the theory of a Th2 bias in newborn foals. However, these results

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43 must be int erpreted with caution because the small sample size resulted in a low statistical power for the effect of age. Flow cytometry had been used previously to detect intracytoplasmic equine IFN in response to antigenic or mitogenic stimulation (Breathnach et al. 2006;Hines et al. 2003;Paillot et al. 2005) The disadvantage of flow cytometry is that it only measures intracytoplasmic cytokines produced over a small window of time Studies assessing the kinetics of intracytoplasmic Th1 and Th2 cytokines in humans have shown that flow cytometry is a useful tool providing that multiple time points are examined. For example, intracytoplasmic IFN peaks at 12 h, decreases around 24 h, and peaks again after 36 h of culture following in vitro cell stimulation with CaI PMA (Rostaing et al 1999) In contrast, IL 4 peaks at 4 and 36 h (Rostaing et al 1999) The ELISPOT assay used in the present study provides the advantage of detecting cytokines produced during the entire incubation period, hence eliminating the need for sampling at multiple time points (Kalyuzhny, 2005) ELISPOT assays can detect cytokine release by as little as 10 100 cells per well compared to the minimum of 10,000 needed in an ELISA (Tanguay & Killion, 1994) The ELISPOT assays for the detection of equine IFN and IL 4 described in the present study can now be applied to investigate both pathogen specific immune responses a nd immune responses following vaccination.

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44 Figure 3 1. Western blot of cellular extract from equine PBMC stimulated for 24 h with CaI PMA.

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45 Figure 3 2 ELISPOT assay for the quantification of IL 4 and IFN producing cells i n equine PBMCs after stimulation for 24 h with calcium ionomycin phorbol myristate acetate (CaI PMA). (A) IL 4 SFC in an adult horse. (B) IL 4 SFC in a 2 day old foal. (C) IFN SFC in an adult horse. (D) IFN SFC in a 5 day old foal. A B C D

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46 Figure 3 3. Lea st square mean IFN (A), IL 4 (B), and IFN /IL 4 ratio (C) spot forming cells (Log10 SD) in mononuclear blood cells of 6 foals < 1 week of age, 6 foals between 3 and 4 months of age, and 10 adult horses following in vitro stimulation with concanavallin A (ConA) or calcium ionomycin phorbol myristate acetate (CaI PMA). 1,2 Different letters indicate a statistically significant difference between the 2 mitogens within a given age group ( P < 0.05). a,b Indicates a statistically significant difference betwee n foals and adult horses within a given mitogen ( P < 0.05)

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47 CHAPTER 4 EFFECTS OF TWO COMME RCIALLY AVAILABLE IM MUNOSTIMULANTS ON LEUKOCYTE FUNCTION O F FOALS FOLLOWING EX VIVO EXPOSURE TO RHODOCOCCUS EQUI Abstract The objective of this study was to deter mine the effect of immunostimulants on neutrophil, macrophage, and lymphocyte function following ex vivo exposure to R. equi Eighteen foals were randomly assigned to one of 3 treatment groups. Treatment consisted of inactivated Propionibacterium acnes ( PA), inactivated parapoxvirus ovis virus (PPVO), or saline (control) administered on days 0 (7 days of age), 2, and 8. Bronchoalveolar lavage (BAL) fluid and blood were collected on days 0 (baseline), 12, 24 and 36. Intracellular replication of R. equi i n macrophages, cytokine induction by R. equi infected macrophages, phagocytic and oxidative burst activity of neutrophils, lymphoproliferative responses, and cytokine induction of proliferating lymphocytes were measured. Neutrophils from foals treated wit h PPVO had significantly greater ability to phagocytize R. equi and undergo oxidative burst on day 12 and day 24 compared to baseline values. On day 24, foals treated with PPVO had significantly greater phagocytosis and oxidative burst than foals treated with PA. Treatment with PA resulted in significantly less intracellular proliferation of R. equi within monocyte derived macrophages on day 12 compared to control foals. The ability of R. equi to replicate in BAL macrophages decreased significantly with time with lower replication in BAL macrophages of older foals compared to younger foals, regardless of treatment. On day 12, TNF derived macrophages and IL 12 p40 induction in BAL macrophages infected with R. equi was significantly higher in foals treated with

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48 PPVO than in controls. Lymphoproliferative responses and IFN induction were not significantly di fferent between groups. Introduction Rhodococcus equi a facultative intracellular pathogen that replicates in macrophages, is one of the most important causes of pneumonia in foals and has a major financial impact on the equine industry. Control of R. eq ui infections on farms where the disease is endemic currently relies on early detection of disease using thoracic ultrasonography and initiation of treatment with antimicrobial agents prior to development of clinical signs Although this approach has decre ased mortality (Slovis et al. 2005) mass antimicrobial treatment is not without risking reported sequelae of macrolide associated hyperthermia and diarrhea in treated animals, and may lead to development of antimicrobial resistance Previous immunoprophylactic strategies for th e prevention of R. equi pneumonia by vaccination of foals, vaccination of the dam, or administration of hyper immune plasma have not consistently decreased the incidence of disease ( Gigure et al. 2002b;Hurley & Begg, 1995;Martens et al. 1991;Prescott et al. 1997b) Epidemiological evidence suggests that foals become infected with R. equi early in life (Horowitz et al. 2001) while a dult horses are typically resistant to R. equi infections. Neutrophils are critical for host resistance against infection with virulent R. equi in mice (Martens et al. 2005) In addition, s tudies in adult hor ses and mice have clearly shown that a T lymphocyte helper type 1 (Th1) response characterized by IFN induction is essential for macrophage activation and protection against infection with R. equi whereas IL 4 is detrimental (Darrah et al. 2000;Kanaly et al. 1996;Lopez et al.

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49 2002) Neonatal foals show a marked decrease in their ability to produce IFN in response to mitogens in the first few weeks of life (Boyd et al. 2003;Breathnach et al. 2006;Ryan et al. 2010) Recent data demonstrate that experimental infection of neonatal foals with a low inoculum of virulent R. equi triggers induction of IFN mRNA transcription in a manner that is similar to that of a dult horses (Jacks et al. 2007 a ;Jacks et al. 2007 b ) These findings indicate that foals are capable of inducing IFN providing the proper stimulus. A product capable of increasing I F N induction and activa ting neutrophils and macrophages in neonatal foals prior to natural infection may be effective in preventing or at least curtailing, infection with R. equi during the narrow window of susceptibility to this pathogen. Immunostimulants are products that i nduce non antigen specific enhancement of innate or adaptive defense mechanisms. Most commercially available veterinary immunostimulants are relatively crude preparations derived from bacteria, plants, or viruses. Data in human s laboratory an imals, and adult horses suggest that commercially available immunostimulants such as inactivated Propionibacterium acnes (PA) or parapoxvirus ovis (PPVO) are potent inducer s of IFN and contribute to neutrophil and macrophage activation (Davis et al. 2003b;Flaminio et al. 1998b;Friebe et al. 2004b;Weber et al. 2003) Randomized clinical trials in adult horses and weanling foals indicate that these immunostimulants significantly decrease the incidence, duration and sev erity of undifferentiated respiratory disease (Cormack et al. 1991;Nestved, 1996;Vail et al. 1990;Ziebell et al. 1997) However, the exact effects of these products on foals in relation to host defense again st R. equi have not been studied.

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50 As a basis for this study, it was hypothesized that commercially available immunostimulants enhance equine phagocytic cell function against ex vivo exposure to R. equi The objectives of this study were to determine the effect of immunostimulants on intracellular survival and replication of R. equi i n foal macrophages cytokine induction by R. equi infected macrophages, phagocytic activity and oxidative burst of blood neutrophils, and the lymphoproliferative responses an d cytokine induction in foals. Materials and Methods Animals Eighteen healthy Thoroughbred or Thoroughbred cross foals were used. The foals were consider ed healthy on the basis of physical examinations adequate transfer of passive immunity, and complete blood cell counts on day one of life. Foals were randomly assigned to one of 3 treatment groups and treatment s began at one week of age (day 0) Six foals received 1 ml of inactivated PA (EqStim Neogen C orporation Lexington, KY ) intravenously on day s 0, 2, and 8 of the study Six foals received 2 ml of PPVO (Zylexis Pfizer Animal Health, New York, NY ) intramuscularly on day s 0 2, and 8 Six foals served as controls and received 2 ml of physiological saline (0.9% NaCl) intramuscularly on day 0, 2 an d 8 of the study. Foals were managed under normal pasture conditions and overall health monitored daily for the duration of the project. Starting at day 0 (prior to treatment) and f ollowing the initial injection of immunostimulant or placebo rectal tem peratures were recorded twice daily for 14 days Blood and bronchoalveolar lavage (BAL) fluid samples w ere collected from each foal on day 0 (prior to treatment) and again on day s 12 (4 days after the last treatment) 24, and 36 of the study. The study w as approved by the Institutional Animal Care and Use Committee of the University of Florida.

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5 1 Blood and BAL S ampling A 16 gauge teflon catheter (Abbocath, Abbott Labs, Abbott Park, IL) was placed in a jugular vein and 250 mL of blood was collected into a g lass collection bottle that contained sodium heparin as the anticoagulant. Foals were sedated intravenously with xylazine (0.5 mg/kg) and butorphanol (0.04 mg/kg) prior to BAL fluid collection. A 1.8 m bronchoalveolar lavage catheter (Jorvet, Loveland, CO ) was passed via nasal approach until wedged into a bronchus. The lavage solution consisted of 4 aliquots of 50 mL physiologic saline (0.9% NaCl) solution infused and aspirated immediately. Cell S eparation Mononuclear cells were harvested from both blo od and BAL samples by density gradient centrifugation ( Ficoll Paque, Amersham Biosciences, Pittsburgh, PA ), washed 3 times with phosphate buffered saline (PBS), and counted using a hemacytometer. Aliquots of peripheral blood mononuclear cells (PBMCs) were used to generate monocyte derived macrophages (see below). The resulting monocyte derived and BAL macrophages were used for infection with virulent R. equi Aliquots of 3 10 7 PBMCs or BAL mononuclear cells were cryogenically preserved in 90% fetal bov ine serum and 10% DMSO in liquid nitrogen until used for lymphocyte proliferation assays (see below). For the neutrophil phagocytosis and oxidative burst assay, freshly drawn w hole blood was aliquoted into 50 m L c onical tubes and allowed to settle for 30 40 min until a visual separation of plasma and the erythrocytes occurred without formation of an obvious buffy coat. Th is leukocyte rich plasma was removed without disruption of the erythrocyte layer placed in a clean centrifuge tube, and centrifuged at 150 g for 10 min at room temperature to pellet the cells After centrifugation, the plasma was decanted from the pellet, the cells were washed twice by resuspending in PBS and

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52 counted to determine the number of R. equi to add in the phagocytosis assay (see below) Monocyte D erived Macrophage I solation Monocyte derived macrophages were obtained using the procedure previously described by Raabe et al (1998) Gelatin coated plates were incubated for 1 hour a t 37C in 6% CO 2 with 15 ml of donor horse serum, and washed 3 times with PBS prior to plating the blood mononuclear cells. Briefly, PBMCs were suspended in Minimum Essential Medium of 4 10 6 cells/ml and incubated for 18 hours at 37C in 6% CO 2 After incubation, non adherent and loosely adherent cells were removed by a series of washes and the remaining cells were harvested by eluting with a 1: 1 mixture of 10 mM EDTA and MEM + 10% HS medi um for 5 10 min at 37C Cells were pelleted by centrifugation at 200 g for 10 min at 4C. Cells were then resuspended in media for infection with virulent R. equi Infection of BAL and Monocyte derived M acrophages Monocyte derived and BAL macrophage s were incubated in two chamber glass slides (Nalgene Nunc International, Rochester, NY) at a concentration of 10 5 cells/ml in one ml of culture media ( MEM with 10% HS and 2 mM glutamine). Cells were incubated at 37C in a humidified atmosphere containing 6% CO 2 for four hours to allow macrophages to adhere to the glass slide. Media was removed and macrophages were then infected with virulent R. equi (ATC C #33701, Rockville, MD). source of complement. The bacterial suspension was added to the monolayers at a ratio of five bacteria to one macrophage. The slides were incubated for 40 min utes at

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53 37 C to allow phagocytosis. Noninfected macrophage monolayers cultured under the same conditions were used as controls. The slides were washed 3 times and incubated te per ml to kill remaining extracellular bacteria and to prevent extracellular growth with continuous re infection of macrophages (Hondalus & Mosser, 1994) At time 0 (immediately post infection) and 24 h post infection, monolayers were fixed with methanol and stained with Wright G iemsa stain to enumerate R. equi (Gigu re & Prescott, 1998) The number of bacteria associated with 200 macrophages was determined using light microscopy. Because of the d ifficulty in reliably counting large numbers of bacteria in a macrophage, any cell containing 10 or more bacteria was scored as having 10 bacteria (Hondalus & Mosser, 1994) Each macrophage infection assay was performed in triplicate. In parallel monolayers containing 1 10 6 cells/ well, the supernatants were removed and the cells were lysed in denaturing solution (RNeasy kit, QIAGEN Inc., Valencia, CA ) 4 h post infection for mRNA extraction and quantification of IL 1 IL 6, IL 8, IL 10, IL 12 p35, IL 12 p40, and TNF mRNA expression b y real time PCR as described below. For each cytokine in a given animal the results were reported as the ratio of mRNA expression in infected macrophages to that of uninfected control monolayers. Flow Cytometric Analysis of Neutrophil Phagocytosis and Oxidative Burst in R esponse to R. equi Flow cytometric analysis of neutrophil phagocytosis and oxidative burst activity was performed as previously reported except that R. equi (ATCC 33701 ) was used as the test microorganism instead of Staphylococcus aureus (Flaminio et al. 2002) Bacteria were enumerated by colony forming unit (CFU) counting and subsequently

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54 suspended in PBS at a concentration of 7 10 9 CFU /ml A suspension of killed R equi was prepared by exposure to heat (90C for 45 min) prior to the assay. Heat killed R. equi was then added at a 1:10 ratio to a propidium iodide (PI) working solution (200 mg/ml in 0.1M carbonate buffer ) The bacteria and PI dye were incubated at room temperature overnight, in the dark, on a rocking plate to ensure mixing. The stained bacteria were then centrifuged at 14,000 g for 20 min the supernatant was removed, and the pellet was resuspended in PBS to maintain a concentration of 7 x 10 9 CFU /ml. The solution was stored in the dark until the assay was performed. On the day of blood collection the heat killed, PI labeled R. equi was opsonized by incubating 1 part of bacterial suspension with 1 part of commercial donor horse serum (Invitrogen, Eugene, OR) for 30 min at 37C in the d ark on a rocking plate. The same source of adult horse serum was used on all samples to eliminate the possibility of variations in serum opsonic activity. After processing, 1 ml of the neutrophil suspension containing 1 10 7 cells/ml was added to a 5 ml Falcon tube (Becton Dickinson, Franklin Lakes, NJ). Twenty eight dihydrorhodamine ( DHR ) working solution was added to each tube gently mixed and then incubated with rotation at 37C for 10 min in the dark. Heat killed PI labeled R. equi were a dded to each sample at a ratio of 10 bacteria per cell Phorbol myristate acetate (PMA [1 mg/ml], Sigma Aldrich Biochemika, St. Louis, MO) was used as a positive control to stimulate neutrophil oxidative burst. Non opsonized R. equi was used as a negative control. The samples were then incubated at 37C for 40 min in the dark, with constant rotation. The tu bes were then immediately placed on ice to stop

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55 to quench extracellular fluorescence. The sample was analyzed for phagocytosis and oxidative burst activity using a flow c ytometer ( FacScan Becton Dickenson, San Jose, CA). Forward scatter and side scatter were used to identify the granulocyte population and to gate out other cells and debris as previously described (Richardson et al. 1998) Twenty thousand events were counted per sample. Mean fluorescence intensity of PI and DHR were used to quantify the phagocytic an d oxidative burst responses, respectively as previously described (Flaminio et al. 2002) R. equi A ntigen P roduction The antigen used for stimulation of cells in proliferation assays was prepared as previously described (Lopez et al. 2002) Briefly, R. equi ATCC 33701 was grown in brain heart infusion (BHI) for 48 h at 37C with agitation. The bacteria were harvested by centrifugation at 3,840 g for 10 min and washed with sterile PBS. Two ml of the bacterial pellet were resuspended in 10 ml of PBS and the bacteria were disrupted by three cycles of freezing at 20C and thawing in a water bath at 37C. The sample was centrifuged at 12,000 g for 15 min at 4C to separate the pellet of intact bacteria and debris. The resulting supernatant was further centrifuged at 25,000 g for 20 min at 4C to obtain the soluble antigens. The supernatant was tested for protein concentration using a BCA protein assay kit (Thermo Scientific Pierce, Rockford, IL) according to the Proliferation and Cytokine mRNA E xpression of PBMC Immediately afte r thawing, PBMCs were washed twice and placed in MEM supplemented with 10% H S, 2 mM glutamine, and penicillin streptomycin (100 U and

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56 100 g per ml, respectively). More than 8 0% of the cells were viable after thawing as assessed by trypan blue (Mediatech Herndon, VA ) exclusion. Proliferative responses were assessed using a non radioactive colorimetric assay. This assay has been shown to correlate closely with conventional radioactive [ 3 H] thymidine incorporation in many species, including the horse (Ahmed et al. 1994;Witonsky et al. 2003) and has been used previously in R. equi experiments (Jacks et al. 2007a ;Jacks et al. 2007b ) 6 cells/ml) were placed in triplicate wells of 96 well black plates with flat, clear bottom wells (Corning Inc., Corning, NY). C ells were separately ConA, positive control), or 10 g/ml of soluble R. equi antigen. Optimal concentrations of antigens and mitogen were determined in previous studies (Jacks et al. 2007a;Jacks et al. 2007b) The cells were sti mulated at 37C for 72 h in 6% CO 2 Eighteen hours before the end of (Accumed International Inc, Westlake, O H ) was added to each well and fluorescence was determined with a fluoromete r (Synergy HT, BioTek Instruments Inc., Winooski, VT) using an excitation wavelength of 530 nm and emission was measured at 590 nm Change in fluorescence was calculated as the mean of the stimulated cells minus the mean of the cells without antigen or mitogen (blank). Blood mononuclear cells used for quantification of mRNA expression were prepared as described previously with the exception that the cells were stimulated with the soluble R. equi antigen or with PMA (0.05 g/ml) and calcium ionomycin (CaI; 0.5 g/ml) for 24 h. Expression of mRNA for IL 2, IL 4, IL 10, and IFN was quantified by real time RT PCR as described below.

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57 RNA Isolation, cDNA Synthesis, and Quantification of Cytokine mRNA Expression by R eal time PC R Isolation of total RNA was performed with the RNeasy kit (QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions. The RNA concentration was measured by optical density at 260 nm (OD 260 ). All RNA samples were treated with amplification g rade DNase I (Gibco BRL, Rockville, MD) to remove any trace of genomic DNA contamination. Briefly, 1 U of DNase I and 1 l of 10x DNase I reaction buffer were mixed with 1 g of total RNA for a total volume of 10 l. The mixture was incubated for 10 min at room temperature and then inactivated by the addition of 1 l of 25 mM of EDTA and incubating at 65C for 10 min. A commercial kit was used to synthesize cDNA (Advantage RT for PCR kit, Clontech, Palo Alto, CA) according to the protocol of the manufactur er. Briefly, 1 g of DNase treated total RNA was mixed with 1 l of oligo(dT) 18 primer (20 M) and heated at 70C for 2 min. After cooling to room temperature, the following reagents were added: 4 l of 5x reaction buffer, 1 l of deoxynucleoside triphosph ates (10 mM each), 0.5 l of RNase inhibitor, (40 U/l) and 1 l of Moloney murine leukemia virus reverse transcriptase (200 U/l). The mixture was incubated at 42C for 1 h, heated at 94C for 5 min, diluted to a final volume of 100 l, and stored at 70 C until being used for PCR analysis. Gene specific primers and internal oligonucleotide probes for equine G3PDH (glyceraldehyde 3 phosphate dehydrogenase), IL 1 IL 2, IL 4, IL 8, IL 10, IL 12p35, IL 12p40, and IFN have been previously reported (Ainsworth et al. 2003;Garton et al. 2002b) The internal probes were labeled at the 5' end with the reporter dye 6 carboxyfluorescein and at the 3' end with the quencher dye 6 carboxytetramethyl

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58 rhodamine. Amplification of 2 l of cDNA was performed in a 25 l PCR mixture containing 900 nM concentration s of each primer, 250 nM TaqMan probe, and 12 l of TaqMan Universal PCR Mastermix (Applied Biosystems). Amplification and detection were performed with the ABI Prism 7 9 00 Sequence Detection System (Applied Biosystems) with initial incubation steps at 50C for 2 min and 95C for 10 min followed by 40 cycles of 95C for 15 s and 60C for 1 min. Serial dilutions of cDNA from equine blood mononuclear cells stimulated for 24 h with ConA were used to generate a standard curve for relative quantification of each gene of interest. Each sample was assayed in triplicate, and the mean value was used for comparison. Samples without cDNA were included in the amplification reactions to determine background fluorescence and to check for contamination. To account for varia tions in the amount and quality of the starting material, all results were normalized to G3PDH expression. IL 4 and IFN ELISA Supernatant of PBMC stimulated with PMA CaI for 72 h was concentrated using a centrifugal filter device (Amicon Ultra, Millip ore, Billerica, MA) according to the 4 and IFN proteins in the concentrated supernatant was measured using a commercially available equine cytokine ELISA kit (R&D Systems, Minneapolis, MN) according to instructions. For each assay, a seven point standard curve using 2 fold serial dilutions of recombinant equine IFN or IL 4 (starting at 1000 pg/ml) was used for quantification and as positive controls. The lower detection limits wer e 30 and 16 pg/ml for IFN and IL 4 respectively.

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59 Statistical A nalyses Normality of the data and equality of variances were analyzed using the Kolmogorov Smirnov and Levene's tests, respectively. A 2 way ANOVA for repeated measures was used to evalua te the effect of treatment (control, PPVO PA ), time (0, 12, 24, and 36 days), and the interaction between treatment and time on neutrophil phagocytosis and oxidative burst, survival of R. equi into monocyte derived and BAL macrophages, lymphoproliferative responses, and cytokine induction by PBMCs in response to stimulation by PMA CaI or R. equi Variables that did not meet the assumptions of the ANOVA were log transformed prior to analysis. Cytokine mRNA expression in monocyte derived and BAL macrophage s infected with R. equi as well as IFN and IL 4 in the supernatant of proliferating PBMC were compared between treatment groups at each time point using a 1 way ANOVA or a Kruskal Wallis ANOVA on ranks. When indicated, multiple pairwise comparisons were done using the Student Newman Keuls test. Significance was set at P < 0.05. Results Clinical Data and Flow Cytometric Analysis of Neutrophil Phagocytosis and Oxidative Burst in R esponse to R. equi One foal randomly assigned to the PPVO group was removed from the study because of an illness unrelated to the study protocol. No adverse effects of immunostimulant administration were noted. Rectal temperature of foals receiving PPVO or PA was not significantly different from that of controls. Neutrophils f rom foals treated with PPVO had significantly greater ability to phagocytize opsonized R. equi and undergo oxidative burst on day 12 and day 24 compared to baseline values ( Figure 4 1 ). On day 24, foals treated with PPVO had significantly greater phagocyt osis and

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60 oxidative burst than foals treated with PA (Figure 4 1) There was no significant effect of time on phagocytosis or oxidative burst in control foals a nd in foals treated with PA The effect of PPVO on neutrophil function was no longer detectable on day 36. Intracellular Survival and Cytoki ne mRNA Expression in Monocyte D erived and BAL Macrophages I nfected with R. equi Treatment with PA resulted in significantly less intracellular proliferation of R. equi within monocyte derived macrophages on d ay 12 compared to control foals but not compared to foals treated with PPVO (Figure 4 2A). There was no significant effect of treatment on day 24 or day 36 of the study. There was no effect of treatment on intracellular proliferation of R. equi within B AL macrophages at any time point. However, the ability of R. equi to replicate in BAL macrophages decreased significantly ( P = 0.005) with time (Figure 4 2B) with lower replication in BAL macrophages of older foals (day 24 and 36 of the study) compared to younger foals (day 0), regardless of treatment. Expression of IL 1 IL 6, IL 8, IL 10, and IL 12 p35, IL 12 p40, and TNF mRNA by both monocyte derived and BAL macrophages infected with R. equi was not significantly different between groups at time 0 (data not shown) On day 12 only, TNF derived macrophages and IL 12 p40 mRNA expression in BAL macrophages infected with R. equi was significantly higher in foals treated with PPVO than in controls (Figure 4 3) There was no significant difference in cytokine mRNA expression between groups on days 24 and 36 of the study (data not shown)

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61 Proliferation and Cytokine mRNA Expression of PBMC Stimulated with M itogens or R. equi T here was no significant difference in lymphoproliferative responses to ConA or R. equi between groups at any time (data n ot shown) Treatment with PA significantly increased expression of IL 10 mRNA on day 24 in PBMCs stimulated with R. equi antigens compared to that of control foals or f oals treated with PPVO (Figure 4 4) Expression of mRNA for IL 2, IL 4, and IFN in P BMCs stimulated with R. equi was not significantly different between experimental groups (data not shown) Similarly, expression of mRNA for IL 2, IL 4, IL 10 and IFN following stimulation with ConA was not significantly different between experimental groups regardless of the time point (data not shown) IL 4 and IFN in the S upernatants of PBMC S timulated with CaI PMA Concentration of IFN secreted by R. equi or CaI PMA stimulated PBMCs was below the limit of detection for the assay (30 pg/ml) at a ll time points. Concentration of IL 4 in the supernatant of PBMCs stimulated with CaI PMA was significantly higher in foals treated with PPVO compared to controls and foals treated with PA on day 12 only (Figure 4 5). Discussion Immunostimulants have been defined as agent s that stimulate the response of effector cells such as macrophages, lymphocytes, and neutrophils, which subsequently activate one or more terminal immune responses such as antigen uptake, cytotoxicity, phagocytosis, cytokine release, and antibody response (Rush & Flaminio, 2000) Th e present study was the first to assess the effect of commercially available

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62 immunostimulant s on immune function of foals following ex vivo stimulation of effector cell populations with R. equi Neutrophils are absolutely essential in host resistance against infection with virulent R. equi in mice (Martens et al. 2005) In addition to their dir ect microbicidal effects, equine neutrophils are capable of producing pro inflammatory cytokines such as IFN TNF 12, IL 6, IL 8 and IL 23p19 when stimulated with R. equi (Nerren et al. 2009b) Decrea sed neutrophil function against R. equi has been documented in some neonatal foals and this has been proposed as one of the possible mechanism s for the peculiar susceptibility of foals to this pathogen (Martens et al. 1988) The significant enhancement of phagocytosis and oxidative burst activity of neutrophils in foals pre tre ated with PPVO ( inactivated parapoxvirus ovis ) was not unexpected in the present study. In one study, inactivated parapoxvirus ovis stimulation enhanced the phagocytotic activity of canine neutrophils and monocytes in a dose dependent manner (Schutze et al. 2010) Similarly, data in humans have demonstrated enhanceme nt of both phagocytosis and oxidative burst following in vitro exposure to inactivated parapoxvirus ovis (Forster et al. 1994) In one study, a dministration of PA ( inactivated Propionibacterium acnes ) to healthy weanlings horses result ed in significantly increased non opsonized phagocytosis and oxidative burst in peripheral blood leukocytes following ex vivo exposure to Staphylococcus aureus (Flaminio et al. 1998b) In the present study, treatment of neonatal foals with PA had no significant effect on neutrophil pha gocytosis and oxidative burst These different findings might be related to time of sampl ing, the age of the animals, or the different bacterial species used to measure phagocytosis ( R. equi versus S aureus ). Given the importance of neutrophils in early

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63 host defense against infection with R. equi a product capable of directly or indirectly enhancing neutrophil function might be useful in preventing disease associated with R. equi during the window of susceptibility to this pathogen. Additional studies will be necessary to determine if the positive effects of PPVO observed ex vivo will resul t in enhanced host defense against R. equi under natural challenge in a field situation. Several significant effects of immunostimulant on macrophage function were demonstrated in the present study. Treatment with PA resulted in significantly less intrace llular proliferation of R. equi within monocyte derived macrophages on d ay 12 compared to control foals but not compared to foals treated with PPVO. However, this effect could not be documented in alveolar macrophages. Interestingly, replication of virul ent R. equi in BAL macrophages was significantly higher on day 0 of the study compared to day 24 or 36, regardless of treatment. These results suggest that R. equi replicates to a greater extent in the BAL macrophages of 1 week old foals compared to that defect in BAL macrophage function of neonatal foals following infection with R. equ i In one study, the migrational activity of alveolar macrophages was significantly impair ed in foals 2 3 days of age compared to 2 week old foals and adult horses (Liu et al. 1987) In another study, newborn foal alveolar macrophages had a lesser ability to phagocytize and kill S. aureus than peripheral blood neutrophi ls from the same an imals (Fogarty & Leadon, 1987) In the present study, TNF derived macrophages and IL 12 p40 mRNA expression in BAL macrophages infected with R. equi were significantly higher in foals treated with PPVO than in controls on study day 12 only (4 days following the last treatment). Interleukin 12 is essential for development

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64 of the Th1 responses necessary for clearance of R. equi (Kanaly et al. 1996;Trinchieri & Gerosa, 1996) Similarl y, TNF is also essential for host defense against infection caused by R. equi in mice (Kasuga Aoki et al ., 1999) Previous studies have documented the ability of immunostimulants at induc ing IFN expression in adu lt horses. In one study administration of PA resulted in a significant increase in mRNA expression of the Th1 cytokines IL 2 and IFN in P BMCs 1 week after administration (Davis et al. 2003) Similarly, adm inistration of PPVO to adult horses in another study resulted in a significant increase in IFN mRNA expression (Horohov et al ., 2008) In the present study there was no difference between treatment groups in the induction o f IFN mRNA following ex vivo stimulation with R. equi or a mitogen Consistent with our findings administration of PA to foals starting at 2 3 days of age failed to increase production of IFN stimulated mononuclear cells (Sturgill & Horohov, 2006) These finding might be due to the fact that neonatal foals typically ha ve lower IFN induction than adult horses in response to mitogens (Boyd et al. 2003;Breathnach et al. 2006;Ryan et al. 2010) Alternatively, the lack of IFN induction in the present study may have been in fluenced by the sampling time selected In one study, peak IFN induction following administration of PPVO to adult horses occurred 24 h following administration (Horohov et al ., 2008) The first sampling tim e in the present study (study day 12) was 4 days after administration of the last dose. This time point was selected based on prior studies demonstrating enhancement of various measures of immune function in adult horses and weanlings following administra tion of immunostimulants (Davis et al. 2003;Flaminio et al. 1998) A transient increase in cytokine mRNA expression might have been

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65 missed. Additional studies including more time points would be needed to ch aracterize the kinetics of cytokine mRNA expression following administration of immunostimulants. In the present study, administration of PPVO resulted in significantly increased concentration of IL 4 in the supernatant of PBMCs stimulated with PMA on day 12 of the study. This effect is not completely unexpected based on studies in other species. In humans, inactivated parapoxvirus ovis has been shown to cause up regulation of pro inflammatory (Th 1 type) cytokines such as IFN and TNF followed by anti inflammatory (Th2 type) cytokines such as IL 10 and I L 4 (Friebe et al. 2004) Inactivated parapoxvirus ovis has been shown to induce some protection against hepatitis B and her pes simplex virus infections in mice (Weber et al. 2003 ) In the aforementioned study, there was initial up regulation of the production of Th1 cytokines such as I L 12, IL 18, and IFN followed by a down regu lation of the same cytokines and production of IL 4 (Weber et al. 2003) Neither PA nor PPVO increased relative expression of IFN in PBMCs from neonatal foals in this study IFN concentration s in the su pernatant of PBMCs were below the threshold of detection in this study, despite concentrating the supernatant samples. To address this issue of relatively low IFN levels in PBMC supernatants, increased numbers and concentrations of cells should be used for stimulation in future studies. In conclusion, the two commercially immunostimulants evaluated in the present study significantly enhanced phagocytic cell function upon ex vivo exposure to virulent R. equi The clinical relevance of these findings in the prevention of R. equi infections on endemic farms remains to be determined in a prospective randomized clinical trial.

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66 Figure 4 1 Phagocytosis (A) and oxidative burst activity (B) of blood neutrophils after ex vivo exposure to R. e qui Neutrophils were collected for baseline (day 0) neutrophil function testing prior to administration of a placebo (control; n=6), PPVO (n=5), or PA (n=6). Neutrophil function was reassessed on day 12, 24, and 36 of the study. The results are display ed as least square mean fluorescence intensity SD. 1,2,3 Different numbers within a given treatment indicate a significant difference in neutrophil function between sampling days (P < 0.05). a,b Different letters between experimental groups within a gi ven day indicate a statistically significant difference in neutrophil function (P < 0.05).

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67 Figure 4 2. Intracellular proliferation of R. equi in PBMC derived macrophages (A) and BAL macrophages (B) of foals prior to (day 0) or after ( days 12, 24, 36) administration of a placebo (control; n=6), PPVO (n=5), or PA (n=6). The results are displayed as mean fold change ( SD) in intracellular bacterial number over a 24 h period. a,b Different letters between experimental groups within a gi ven day indicate a statistically significant difference (P < 0.05). 1,2,3 Different numbers within a given treatment indicate a significant difference in neutrophil function between sampling days (P < 0.05).

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68 Figure 4 3. Fold i ncrease in relative mRNA expression of IL 12p40 in BAL macrophages (A) and of TNF in monocyte derived macrophages (B) 4 h following infection with virulent R. equi The cells were collected on day 12 of the study. Foals were administered a placeb o (control; n=6), PPVO (n=5), or PA (n=6). Each symbol represents an individual foal a nd the m edian value from each group is represented as a horizontal line I ndicates a statistically significant difference in mRNA expression compared to controls ( P < 0.05).

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69 Figure 4 4. Relative IL 10 mRNA expression in PBMC stimulated with R. equi a ntigens. Foals were administered a placeb o (control; n=6), PPVO (n=5), or PA (n=6). Each symbol represents an individual foal and the m edian value from each group is represented as a horizontal line I ndicates a statistically significant difference in mRNA expression at 24 days compared to control foals and foals treated with PPVO ( P < 0.05).

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70 Figure 4 5. IL 4 concentration in the supernatants of PBMC stimulated with CaI PMA. The cells were collected on day 12 of the study. Foals were administered a placeb o (control; n=6), PPVO (n=5), or PA (n=6). Each symbol represents an individual foal and the m edian value from each group is represented as a horizontal line I ndicates a statistically significant difference compared to controls ( P < 0.05).

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71 CHAPTER 5 EQUINE NEONATES HAVE ATTENUATED H UMORAL AND CELL MEDIATED IMMUNE RESPONSES TO A KILLED ADJUVANTED VACCINE COMPARED TO ADULT HORSES Abstract The objectives of this study were to compare relative serum immunoglobulin concentrations, antigen specific lymphoproliferative responses, and cytokine profile of proliferating lymphocytes between 3 day old foals, 3 month old foals, and adult horses following vaccination with a bovine killed adjuvanted vaccine. Horses were vaccinated intramuscul arly twice at 3 week intervals with a vaccine containing antigens from bovine viral respiratory pathogens to avoid interference from maternal antibody. Both groups of foals and adult horses responded to the vaccine with a significant increase in relative vaccine specific IgGa and IgG(T) concentrations. In contrast, only adult horses and 3 month old foals mounted significant total IgG, IgGb and IgM responses. Relative c oncentrations of IgM and IgG(T) were significantly different between all groups with t he highest concentrations in adult horses followed by 3 month old foals and finally 3 day old foals. Only the adult horses mounted significant vaccine specific lymphoproliferative responses. Baseline IFN and IL 4 concentrations were significantly lower in 3 day old foals than in adult horses. Vaccination resulted in a significant decrease in IFN concentrations in adult horses and a significant decrease in IL 4 concentrations in 3 day old foals. After vaccination, the ratio of IFN /IL 4 in both gro ups of foals was significantly higher than that of adult horses. The results of this study indicate that the humoral and lymphoproliferative immune responses to this killed adjuvanted vaccine are modest in newborn foals. Although immune responses

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72 improve with age, 3 month old foals do not respond with the sa me magnitude as adult horses Introduction Development of the equine immune system occurs relatively early during fetal life. Lymphocytes are present in the peripheral blood of the equine fetus by day 120 of gestation and they proliferate in response to mitogens by day 140 (Perryman et al. 1980) Specific antibody responses to in utero vaccination with coliphage T2 have been detected in equine fetuses as early as day 200 of gestation (Martin & Larson, 1973) In other studies, administration of a V enezuelian equine encephalomyelitis antigen to equine fetuses between 232 and 283 days of gestational age resulted in higher serum neutralization titer s than that elicited by the same preparation in adult horses (Mock et al ., 1978;Morgan et al ., 1975) Recent work supports these findings, showing that active B cell development and immunoglobulin isotype switc hing occur during equine gestation and the neonatal period (Tallmadge et al ., 2009) Proliferation of peripheral blood lymphocytes in response to mitogens is slightly reduced at birth but rapidly increases to a dult levels (Flaminio et al ., 2000;Sanada et al ., 1992) Foals also have normal lymphokine activated killing (LAK) cell activity of peripheral blood lymphocytes at birth and during early life (Flaminio et al. 2000) Although these findings suggest that newborn foals should b e able to mount adequate immune responses at birth, maternal antibodies acquired through ingestion of colostrum have been shown to exert a considerable suppressive effect on antibody production (Jeffcott, 1974) In addition, the recognized Type 2 bias in immune responses of murine and human neonates, along with the recent findin g that young foals are deficient in their ability to produce IFN in response to stimulation with

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73 mitogens, has led to the widespread hypothesis that foals are born with an inherent inability to mount strong cell mediated immune response (Boyd et al. 2003;Breathnach et al. 2006) Recent ly we have demonstrated that newborn foals can produce robust IFN responses and high concentrations of IgG subclasses when challenged with intrabronchial ly with virulent R. e qui (Jacks et al. 2007a ;Jacks & Gigure 2010) However, there are no studies comparing primary humoral and cell mediated immune responses of newborn foals to that of older foals and adult horses following va ccination in the absence of vaccine specific maternal antibody interference. A thorough understanding of immune responses of newborn foals following vaccination would be essential for the future development of rational vaccination strategies against patho gens likely to infect foals early in life. The objectives of this study w ere to compare serum IgM and IgG subclass concentrations, antigen specific lymphoproliferative responses, and cytokine profile of proliferating lymphocytes of newborn foals, older f oals, and adult horses following vaccination with a killed adjuvanted vaccine. A killed adjuvanted vaccine was selected because most vaccines commercially available for use in horses currently are of this type. The central hypothesis for the present stud y was that newborn foals mount inferior immune responses to a killed vaccine compared to adult horses. Materials and Methods Animals and Experimental D esign Thirty two healthy Thoroughbred or Thoroughbred cross foals were used. The foals were considered healthy on the basis of adequate transfer of passive immunity, and complete blood cell counts on day one of life physical examinations, and daily monitoring. Healthy adult horses (n=6) were also used. Foals were randomly assigned

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74 to one of 2 age groups; 3 day old (n=11) and 3 month old (n=15). Each animal received a series of 2 intramuscular injections of a killed adjuvanted cattle vaccine (Triangle4, Fort Dodge Animal Health, Fort Do d ge IA) at 3 week intervals. This vaccine included antigens from typ e II bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, parainfluenza 3 virus, and bovine respiratory syncytial virus. The vaccine was selected based on the lack of detectable serum antibody to these antigens in horses as well as demons trated safety and immunogenicity in horses (Slack et al ., 2000) Blood was collected from each animal on day 0 (prior to vaccination), 21 ( dose 1; prior to administration of the second dose), and 42 days (dose 2) after initiation of immunization. Blood Collection and Cell S eparation One hundred ml of blood was collected by jugular venipuncture using heparin as the anticoagulant. Blood (10 ml) was collected without anticoagulant for separation of serum. Pe ripheral blood mononuclear cells (PBMCs) were harvested from blood samples by density gradient centrifugation (Ficoll Paque, Amersham Biosciences, Pittsburgh, PA), washed 3 times with phosphate buffered saline (PBS), and counted using a hemacytometer. Ali quots of 3 10 7 PBMCs were cryogenically preserved in 90% fetal bovine serum and 10% DMSO in liq uid nitrogen until used for lymphocyte proliferation and cytokine assays (see below). Serum was stored at 80C until used for measurement of vaccine specific immunoglobulin concentrations. Vaccine specific Serum Immunoglobulin C oncentrations Vaccine specific relative IgM, total IgG, IgGa, IgGb, and IgG(T) concentrations in serum were determined by ELISA as previously described (Jacks et al. 2007 a ) Optimal dilutions of reagents were determined by checkerboard titration. Briefly, wells

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75 in 96 well microtiter plates (Immulon II, Thermo Fis her Scientific, Waltham, MA) were coated at 4C overnight with whole vaccine (Triangle 4, Fort Dodge Animal Health) diluted 1:250 in carbonate bicarbonate buffer (pH 9.6; total volume 100 l/well). Plates were washed 4 times with PBS 0.05% Tween 20 between each of the following incubations. Plates were blocked with PBS 1% BSA for 1 h at room temperature. Serum from each experimental animal was diluted 1:100 and 100 l were added to each well for 1 h of incubation at room temperature. To determine isotype specific responses, 100 l of peroxidase conjugated goat anti equine IgGa (1:5000), IgGb (1:5000), IgG(T) (1:1000), and IgM (1:2500) (Serotec Raleigh, NC ) were added to the wells for 1 h incubation at room temperature. After addition of substrate (ABTS, Roche Diagnostics, Indianapolis, IN), plates were incubated for 45 min in the dark at room temperature and the OD was measured at 405 nm. For each immunoglobulin subisotype measured, s erum from a high responder was serially diluted to generate a standard curve for relative quantification of immunoglobulin concentration in the experimental animals. The standard curve was run on each plate to correct for interplate variability. Wells i ncubated without serum were used as blank to subtract out the background absorbance. Each sample was run in triplicate and the mean OD was used. Vaccine specific Lymphocyte P roliferations Immediately after thawing, PBMC s were washed twice and placed in ME supplemented with 10% horse serum, 2 mM glutamine, and penicillin streptomycin (100 U and 100 g per ml, respectively). More than 80 % of the cells were viable after thawing as assessed by trypan blue (Mediatech Herndon, VA) exclusion.

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76 Lymphoprolife rative responses were assessed using a non radioactive colorimetric assay. This assay has been shown to correlate closely with conventional radioactive [3H] thymidine incorporation in many species, including the horse (Ahmed et al ., 1994; Witonsky et al ., 2003). In preliminary experiments, the adjuvant of the killed vaccine was found to exert mitogenic effects on equine lymphocytes, thereby preventing our ability to detect antigen specific lymphoproliferative responses. A modified live vaccine (Pyramid 5 Fort Do d ge Animal Health) containing the same viral agents as the killed adjuvanted vaccine was used as a source of antigen for the lymphoprolifertive assay. The advantage of the modified live vaccine was that antigen and adjuvants were provided in sepa rate vials. Vaccine antigen inactivated by heating at 60C for 1 h. The optimal concentration of antigen (1:7000) was determined 6 cells/ml) were placed in triplicate wells of 96 well black plate s with flat, clear bottom wells (Corning Inc., Corning, NY). pokeweed mitogen (positive control), or vaccine antigen. The cells were stimulated at 37C for 72 h in 6% CO 2 Eighte blue (Accumed International Inc, Westlake, OH) was added to each well and fluorescence was determined with a fluorometer (Synergy HT, BioTek Instruments Inc., Winooski, VT) using an excitation wavelengt h of 530 nm and emission was measured at 590 nm. Change in fluorescence was calculated as the mean of the stimulated cells minus the mean of the cells without antigen or mitogen (blank). Cytokine mRNA E xpression by Real time PCR PBMC s were cultured in tr iplicate wells for 24 h in the presence of the vaccine antigen as described above. Time of stimulation (24 h) was selected based on peak

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77 mRNA expression in preliminary experiments with adult horse PBMC s ( Gigure & Prescott, 1999 b ;Jacks et al. 2007b ) Isolation of total RNA was performed with the RNeasy kit (QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions. The RNA concentration was measured by optical density at 260 nm (OD260). All RN A samples were treated with amplification grade DNase I (Gibco BRL, Rockville, MD) to remove any trace of genomic DNA contamination. Briefly, 1 U of DNase I and 1 l of 10x DNase I reaction buffer were mixed with 1 g of total RNA for a total volume of 10 l. The mixture was incubated for 10 min at room temperature and then inactivated by the addition of 1 l of 25 mM of EDTA and incubating at 65C for 10 min. A commercial kit was used to synthesize cDNA (Advantage RT for PCR kit, Clontech, Palo Alto, CA) according to the protocol of the manufacturer. Briefly, 1 g of DNase treated total RNA was mixed with 1 l of oligo(dT)18 primer (20 M) and heated at 70C for 2 min. After cooling to room temperature, the following reagents were added: 4 l of 5x reac tion buffer, 1 l of deoxynucleoside triphosphates (10 mM each), 0.5 l of RNase inhibitor, (40 U/l) and 1 l of Moloney murine leukemia virus reverse transcriptase (200 U/l). The mixture was incubated at 42C for 1 h, heated at 94C for 5 min, diluted to a final volume of 100 l, and stored at 70C until being used for PCR analysis. Gene specific primers and internal oligonucleotide probes for equine G3PDH (glyceraldehyde 3 phosphate dehydrogenase), IL 2, and IL 10 have been previously reported (Garto n et al ., 2002). The internal probes were labeled at the 5' end with the reporter dye 6 carboxyfluorescein and at the 3' end with the quencher dye 6 carboxytetramethyl rhodamine. Amplification of 2 l of cDNA was performed in a 25 l

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78 PCR mixture containi ng 900 nM concentrations of each primer, 250 nM TaqMan probe, and 12 l of TaqMan Universal PCR Mastermix (Applied Biosystems). Amplification and detection were performed with the ABI Prism 7900 Sequence Detection System (Applied Biosystems) with initial incubation steps at 50C for 2 min and 95C for 10 min followed by 40 cycles of 95C for 15 s and 60C for 1 min. cDNA from equine blood mononuclear cells stimulated for 24 h with ConA was used as positive control and run on each plate as a calibrator samp le. Each sample was assayed in triplicate, and the mean value was used for comparison. Samples without cDNA were included in the amplification reactions to determine background fluorescence and to check for contamination. DNase treated RNA samples were s ubjected to PCR using the G3PDH primers to confirm the absence of genomic DNA contamination. Relative gene expression was calculated using the method described by Pfaffl et al. (2001) IFN and IL 4 C oncentrations Supernatants of PBMCs stimulated as described above for 72 h were collected and stored at 80 C until use. Supernatants were concentrated using a centrifugal filter device (Amicon Ultra, Millipore, Billerica, MA) according to the instruction. The concentration of IL 4 and IFN proteins in the concentrated supernatant was measured using commercially available equine cytokine ELISA kits r each assay, a seven point standard curve using 2 fold serial dilutions of recombinant equine IFN or IL 4 (starting at 1000 pg/ml) was used for quantification and as positive controls. The lower detection limits were 30 and 16 pg/ml for IFN and I L 4, respectively.

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79 Statistical A nalysis Normality and equality of variance of the data were assessed using the Kolmogorov Smirnov and Levene's tests, respectively. Variables that were not normally distributed were log or rank transformed prior to analysis. A two way ANOVA for repeated measurements was used to determine the effects of vaccination (baseline, dose 1, and dose 2), experimental group (3 day old, 3 month old, and adult horses ), and the interactions between vaccination and group on antibody conce ntration, lymphoproliferative responses, and cytokine induction. When appropriate, multiple pairwise comparisons were done using the Holm Sidak test A value of P < 0.05 was considered significant. Results Vaccine specific Immunoglobulin C oncentrations Both groups of foals and adult horses responded to the vaccine with a significant increase in relative IgGa and IgG(T) concentrations (Figure 5 1) In contrast, only adult horses and 3 month old foals mounted significant total IgG, IgGb and IgM responses Relative c oncentrations of IgGb were significantly higher in adult horses than in both groups of foals. Relative c oncentrations of IgM and IgG(T) were significantly different between all groups with the highest relative concentrations in adult horses f ollowed by 3 month old foals and finally 3 day old foals (Figure 5 1) Relative c oncentrations of IgGa were not significantly different between groups. Vaccine specific Lymphoproliferative Responses and Cytokine I nduction Adult horses had significantly greater lymphoproliferative responses to the vaccine antigen after the second dose of vaccine compared to that measure d at baseline or after a single dose (Figure 5 2). In both groups of foals, vaccine induced

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80 lymphoproliferative responses after vaccina tion were not significantly different from baseline values (Figure 5 2). Lymphoproliferative responses of adult horses after administration of 2 doses of vaccin e were significantly higher than that of either group of foals (Figure 5 2). Three month old f oals had significantly greater lymphoprolifertive responses after 2 doses of vaccine than 3 day old foals. There was no significant effect of vaccination on lymphoproliferative responses to pokeweed mitogen but there was a significant effect of group ( P = 0.016). Lymphoproliferative responses to pokeweed mitogen in adult horses (1,470 1058) were significantly higher than that of 3 day old foals (387 533) and 3 month old foals (806 1608). Lymphoproliferative responses to pokeweed mitogen were not si gnificantly different between the 2 groups of foals. Cytokine I nduction in V accine stimulated PBMCs Baseline IFN concentrations were significantly lower in 3 day old foals than in 3 month old foals and adult horses (Figure 5 3A). Vaccination resulted in a significant decrease in IFN concentrations after the first dose of the vaccine in 3 day old foals and after both doses in 3 month old foals and adult horses. After 2 doses of the vaccine, IFN concentrations were significantly lower in adult horses than in both groups of foals (Figure 5 3A). Baseline IL 4 concentrations were significantly lower in both groups of foals compared to adult horses (Figure 5 3B). Concentrations of IL 4 significantly decreased after the second vaccination compared to base line in 3 day old foals. Concentrations of IL 4 after the second vaccination were significantly lower in 3 day old foals than in 3 month old foals and adult horses (Figure 5 3B). Baseline IFN /IL 4 ratio was significantly higher in 3 month old foals than in 3 day old foals (Figure 5 3C). There was a significant increase in the IFN /IL 4 ratio after the second dose of

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81 vaccine in 3 day old foals and a significant decrease in the same ratio in adult horses. The IFN /IL 4 ratio of both groups of foals was significantly higher than that of adult horses after the second dose of the vaccine (Figure 5 3C). Baseline relative IL 10 mRNA expression was not significantly different between groups Ther e was a significant increase in relative IL 10 mRNA expression after the first dose of the vaccine in 3 day old foals only but expression returned to baseline after the second dose (Figure 5 4 B ). Relative IL 10 mRNA expression after the first dose of the vaccine was significantly higher in 3 day old foals than in adults (Figure 5 4 B ). Relative IL 10 mRNA expression did not change significantly with vaccination in 3 month old foals and in adults. There was a significant effect of group ( P = 0.033) on rela tive IL 2 mRNA expression but the effect of vaccination ( P = 0.17) and the interactions between group and vaccination ( P = 0.97) were not statistically significant. Relative IL 2 mRNA expression in adult horses was significantly higher than that of both g roups of foals (Figure 5 4 A ). Discussion Although vaccination of horses is widely practiced and forms an important part of infectious disease control programs, very little is known regarding development of immune responses following vaccination in newborn foals. Response to vaccination is generally assessed in a population of healthy adult horses. However, age has a profound effect on immune responses as evidenced by the fact that old horses have decreased antibody production and lymphoprolifera tive respo nses to some vaccines (Horohov et al. 2010) Current equine vaccination guidelines state that vaccination of foals should begin between 3 and 6 months of age, depending o n the specific vaccine.

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82 This is because maternal antibody acquired through ingestion of colostrum has been shown to exert a considerable suppressive effect on antibody production. This is substantiated by the fact that the onset of antibody production is advanced in colostrum deprived foals compared to foals with adequate transfer of passive immunity (Jeffcott, 1974) The rate of decline of maternal antibodies varies depending on the nature of the antigen. For many pathogens, the concentration of maternal antibody in foals falls to non protective levels by 2 3 months of age. For equine influenza and tetanus, maternal antibodies in foals born from mares vaccinated in the last 2 months of pregnancy can persist until approximately 6 months of age and prevent adequate immune responses in foals vaccinated prior to reaching that age (Wilson et al. 2001) There are no studies comparing primary humoral and cell mediated immune responses of newborn foals to that of older foals and adult horses following vaccination in the absence of antigen specific maternal antibody interference. A thorough understanding of the default immune response of newborn foals following vaccination is essential for the future development of rational vaccination strategies for pathogens expected to cause disease ear ly in life. The killed adjuvanted vaccine selected for use in the present study was well tolerated and invoked robust humoral and lymphoproliferative responses in adult horses. Relative s erum concentrations of IgM, IgG(T), IgG(a), IgG(b) and total IgG a ll increased following vaccination. Total IgG, IgM, IgG(T), and IgG(b) concentrations in adult horses were highest after the second vaccination. In contrast, IgGa concentrations increased after the first vaccination but a considerable decrease in IgGa co ncentrations was observed after the second vaccination in most horses. These

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83 results are in accordance with those of Slack et al (2000) who reported a peak in IgGa concentrations 2 weeks following administrati on of the second dose of the same vaccine to adult horses with a substantial decrease in IgGa concentrations at 3 weeks. Although newborn foals mounted statistically significant IgGa and IgG(T) responses, the magnitude of these responses was modest compar ed to that of older foals and adult horses. In one study, vaccination of 8 to 15 day old foals with 2 doses of a DNA vaccine expressing the vapA gene of Rhodococcus equi f ailed to elicit a measurable antibody response while the same vaccine elicited robu st antigen specific IgG responses in adults (Lopez, et al. 2003) In contrast, infection of 7 day old foals with live virulent R. equi resulted in a significant increase in IgGa, IgGb, IgGc, and IgM concentrations compared to pre infection values (Jacks & Gigu re, 2010 ; Jacks et al. 2007a) In the same study, post infection IgGa and IgGb concentrations in infected foals were significantly higher than those achieved following administration of the same inoculum to adult horses. (Jacks et al ., 2007a) Administration of a higher inoculum of th e same virulent R. equi resulted in significantly higher IgG(T) and IgM responses (Jacks & Gigu re, 2010) Collectively, these findings indicate that newborn foals can mount adequate humoral immune responses providing the right stimulus. However, the nature and dose of antigen and possibly the type of adjuvant have a profound effect on the magnitude and IgG subclass of the respon se in newborn foals. In this study, relative immunoglobulin concentrations were measured by an ELISA technique utilizing the whole vaccine. Therefore, immunoglobulin responses to the entire vaccine, not just the viral antigens, were measured. Immunoglob ulin responses

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84 to other components of the vaccine such as adjuvant or additives (as opposed to vaccine antigen specific responses) cannot be ruled out. The present study showed that the antigen specific lymphoproliferative responses of adult horses are su bstantially greater than those of foals. Similarly, 7 day old foals infected with virulent R. equi had decreased lymphoproliferative responses to R. equi antigens when compared to adult horses (Jacks et al. 200 7a) Limited antigen specific lymphoproliferative responses in foals are unlikely to be a result of impaired proliferative ability of neonatal lymphocytes because several studies have indicated that neonatal foals and adult horses have similar lymphoprol iferative responses in response to stimulation with various mitogens (Flaminio et al. 2000;Jacks & Gigure 2010;Sanada et al. 1996) The decreased antigen specific lymphoproliferative responses in neonatal foals may be the result of impaired or immature function of antigen presenting cells. Recent studies have shown that monocyte derived dendritic cells from foals are phenotypically different from that of adult horses having decreased MHC class II and CD86 expression (Flaminio et al. 2009;Merant et al. 2009) Cell mediated immune responses of murine and human neonates are generally thought to be biased toward a Th2 response (Adkins, 2000) Several studies have documented that newborn foals are deficient in their ability to i nduce IFN in response to stimulation with mitogens (Boyd et al. 2003;Breathnach et al. 2006) These findings, along with the peculiar susceptibility of foals to infection with R. equi a facultative intrace llular pathogen known to only cause disease in immunocompetent mice when a Th2 response is experimentally induced (Kanaly et al. 1995) have led to the hypothesis that T cell responses from newborn foals may be biased toward a Th2

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85 cytokine profile. However, experimental infection of neonatal foals with virulent R. equi triggers induction of IFN mRNA trans cription in a manner that is similar to that of adult horses, indicating that foals can mount adequate IFN responses providing the proper stimulus (Jacks et al ., 2007 a ;Jacks et al ., 2007 b ) Thorough assessmen t of the Th2 cytokines such as IL 4. Recent data demonstrate that foals are also deficient in their ability to produce IL 4 in response to stimulations with mitogens, sug gesting that a clear cut polarization towards a Th2 response in unlikely in neonatal foals. (Ryan et al. 2010;Wagner et al. 2010) The relative Th1/Th2 polarization of equine neonatal immune responses would be better assessed by measuring antigen specific responses after vaccination rather than aft er stimulation with mitogens. the present study is the first to measure Th1 and Th2 cytokines in response to vaccination of newborn foals with a killed adjuvanted vaccine. Consistent with studies using mitogens, baseline IFN and IL 4 concentrations in the present study were significantly lower in 3 day old foals compared to that measured in adult horses. However, the IFN /IL 4 ratio after vaccination was significantly higher in both groups of foals compared to adult horses These results indicate that, although basal cytokine secretion on neonatal foals may be considerably dampened, there is not a clear bias towards a Th2 response to the vaccine used in the present study. In the current study, we found that foal and adu lt horse PBMCs produced IFN and IL 4 when cultured in the presence of vaccine antigen. Surprisingly, the amount of IFN produced by these antigen stimulated 3 month old foal and adult PBMCs decreased after vaccination, while production from 3 day old foals was unchanged. It is

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86 possible the use of a killed (rather than live) vaccine led to a diversion away from an IFN dominated response, towards a more prominent Th2 type response. Although this theory is cannot be supported by a simultaneous rise in IL 4 production, the increa sed mRNA expression of IL 10 in 3 month old foals could fit with this scenario. In conclusion, the present study demonstrated considerably decreased humoral and cell mediated immune responses in newborn foals after vaccination with a killed vaccine compar ed to that of adult horses even in the absence of vaccine specific maternal antibody interference. Although immune responses to the vaccine improved with age, 3 month old foals did not respond with the same magnitude as adult horses. However, newborn fo als do not have a bias toward a Th2 response in response to vaccination. Additional studies are needed to determine the effects of type of antigen, dose of antigen, and form of adjuvant on induction of robust humoral and cell mediated immune responses in foals.

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87 Figure 5 1. Relative vaccine specific serum total IgG (A), IgM (B), IgGa (C), IgGb (D), and IgG(T) (E) concentrations as determined by capture ELISA. Adult horses, 3 day old foals, and 3 month old foals were vaccinated with a killed adjuvanted vaccine twice with 3 weeks between administrations. Serum was collected prior to vaccination as well as 3 weeks after administration of each dose of the vaccine. Symbols represent individual data points. Horizontal bars indicate median values. 1,2,3 Di fferent numbers within an age group indicate significant differences between sample time points. a,b,c Different letters within a time point indicate significant differences between age groups. Significance was set at P < 0.05.

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88 Figure 5 2. Mean (SD) va ccine specific lymphoproliferative responses as determined by a colorimetric lymphocyte proliferation assay. Adult horses, 3 day old foals, and 3 month old foals were vaccinated with a killed adjuvanted vaccine twice with 3 weeks between administrations. PBMCs were collected prior to vaccination as well as 3 weeks after administration of each dose of the vaccine. a,b,c Different letters within a time point indicate significant differences between age groups (P < 0.05)

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89 Figure 5 3 Mean (SD) concent rations of IFN (A) and IL 4 (B), and IFN /IL 4 ratio (C) in the supernatants of PBMCs stimulated with vaccine antigens as determined by ELISA Adult horses, 3 day old foals, and 3 month old foals were vaccinated with a killed adjuvanted vaccine twice with 3 weeks between administrations. PBMCs were collected prior to vaccination as well as 3 weeks after administration of each dose of the vaccine 1,2,3 Different numbers within an age group indicate significant differences between sample time points. a,b,c Different letters within a time point indicate significant differences between age groups. Significance was set at P < 0.05

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90 Figure 5 4. Relative IL 2 (A) and IL 10 (B) mRNA expression in PBMCs stimulated with vaccine antigens. Adult horses, 3 d ay old foals, and 3 month old foals were vaccinated with a killed adjuvanted vaccine twice with 3 weeks between administrations. PBMCs were collected prior to vaccination as well as 3 weeks after administration of each dose of the vaccine. Symbols represe nt individual data points. Horizontal bars indicate median values. 1,2,3 Different numbers within an age group indicate significant differences between sample time points. a,b,c Different letters within a time point indicate significant differences betwee n age groups. Significance was set at P < 0.05.

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91 CHAPTER 6 SUMMARY AND CONCLUSIONS The research p resented in this body of work focused on examining the differences in immune system function bet ween newborn foals, older foals, a nd adult horse s The impor tance of this work lies in our ability to use this information to design fut ure preventative and treatment strategies for foal diseases, such as R. equi pneumonia. It has become clear in recent years that neonates of many species, including equids, do not mount immune responses in the exact same way or magnitude as compared to adult s Although f about 1 year of age there is still a large window of time during which foals are suscept i ble to infections that are easily controlled by adults For these reasons, foals to view their responses as nave. Some authors have suggested that a predisposition of foals to develop Th2 type responses may be responsible for their suscept i bility to intracellular infections such as R. equi However, only a sm all percentage of foals exposed to R. eq ui responses can and does occur. Our research is focused on the unique aspects of the foal immune system which might allow them to become infected with an organism that is non pathogenic to adults. The main objectives of this project were threefold. First, the effect of age and different mitogens on the ability of PBMCs to secrete cytokines was determined. It was shown that foals are not just deficient in their abilit y to produce IFN They are also deficient in their ability to produce IL 4. As a result, it may not be appropriate to state that the

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92 immune system of equine neonates is biased towards a Th2 response. In addition, the present study demonstrated that regardless of age, the type of mitogen used for in vitro stimulation had a significant effect on the IFN /IL 4 ratio. Therefore, it will be important to take the type of cellular stimulation in consideration in future studies aiming at assessing regulation of immune respon ses in horses. Second the effects of two commercially available immunostimulant s on phagocytic cell function a fter ex vivo exposure to R. e q ui were assessed The two commercially immunostimulants evaluated significantly enhanced phagocytic cell function u pon ex vivo exposure to virulent R. equi The clinical relevance of these findings in the prevention of R. equi infections on endemic farms remains to be determined in a prospective randomized clinical trial. Finally the effects of age on humoral and ce ll mediated immune responses to a killed adjuvanted vaccine were examined H umoral and lymphoproliferative immune responses to the killed adjuvanted vaccine used were modest in newborn foals consisting mainly of a weak IgGa and IgG(T) response Althou gh immune responses improve d with age, 3 month old foals d id not respond with the same magnitude as adult horses. The post vaccination IFN /IL 4 ratio of both groups of foals was significantly higher than that of adult horses Collectively, the work presented in this dissertation indicate that, although basal cytokine secretion in neonatal foals may be considerably dampened, newborn foals d o not exhibit clear bias towards a Th2 response

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96 Garton, N. J., Gilleron, M., Brando, T., Dan, H. H., Gigure S., Puzo, G., Prescott, J. F. & Sutcliffe, I. C. (2002) A novel lipoarabinomannan from the equine pathogen Rhodococcus equi Structure and effect on marcophage cytokine production. Journal of Biological Chemistry 277 31722 31733. Gigu re, S., Gaskin, J. M., M iller, C. & Bowman, J. L. (2002 ). Evaluation of a c ommercially available hyperimmune plasma product for prevention of naturally acquired pneumonia caused by Rhodococcus equi in foals. Journal of the American Veterinary Medical Association 220 59 63. Gigu re, S., Hernandez, J., Gaskin, J., Prescott, J. F., Takai, S. & Miller, C. (2003a). Performance of five serological assays for diagnosis of R hodococcus equi pneumonia in foals. Clin Diagn Lab Immunol 10 241 245. Gigu re, S., Hernandez, J., Gaskin, J. M., Miller, C. & Bowman, J. L. (2003b). Evaluation of WBC concentration, plasma fibrinogen concentration, and an agar gel immunodiffusion test for early identification of foals with Rhodococcus equi pneumonia. J Am Vet Med Assoc 222 775 781. Gigu re, S., Hondalus, M. K., Yager, J. A., Darrah, P., Mosser, D. M. & Prescott, J. F. (1999 a ). Role of the 85 kilobase plasmid and plasmid encoded virulence associated protein A in intracellular survival and virulence of Rhodococcus equi Infection and Immunity 67 3548 3557. Gigu re, S., Jacks, S., Roberts, G. D., Hernande z, J., Long, M. T. & Ellis, C. (2004). Retrospective comparison of azithromycin, clarithromycin, and erythromycin for the treatment of foals with Rhodococcus equi pneumonia. Journal of Veterinary Internal Medicine 18 568 573. Gigu re, S. & Prescott, J. F. (1998). Cytokine induction in murine macrophages infected with virulent and avirulent Rhodococcus equi Infect Immun 66 1848 1854. Gigu re, S. & Prescott, J. F. (1999 b ). Quantitation of equine cytokine mRNA expression by reverse transcription competitive po lymerase chain reaction. Vet Immunol Immunopathol 67 1 15. Gigu re, S., Wilkie, B. N. & Prescott, J. F. (1999c). Modulation of cytokine response of pneumonic foals by virulent Rhodococcus equi. Infection and Immunity 67 5041 5047. Gonzalez, S., Beck, L., Wilson, N. & Spiegelberg, H. L. (1994). Comparison of interferon gamma and interleukin 4 production by peripheral blood mononuclear cells and isolated T cells after activation with polyclonal T cell activators. J Clin Lab Anal 8 277 283.

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97 Haghighi, H. R. & Prescott, J. F. (2005). Assessment in mice of vapA DNA vaccination against Rhodococcus equi infection. Veterinary Immunology and Immunopathology 104 215 225. Hamza, E., Doherr, M. G., Bertoni, G., Jungi, T. W. & Marti, E. (2007). Modulation of allergy i ncid ence in I celandic horses i s a ssociated with a change in IL 4 p roducing T c ells. Int Arch Allergy Immunol 144 325 337. Harris, D. (1992). Phenotypic and functional immaturity of human umbilical cord blood T lymphocytes. 89 edn, 10006 10010. Edited by Schum acher, M., Locascio, J., Besencon, F., Olson, G., DeLuca, D., Shenker, L., Bard, J. & Boyle, E. Hines, S. A., Stone, D. M., Hines, M. T., Alperin, D. C., Knowles, D. P., Norton, L. K., Hamilton, M. J., Davis, W. C. & McGuire, T. C. (2003). Clearance of viru lent but not avirulent Rhodococcus equi from the lungs of adult horses is associated with intracytoplasmic gamma interferon production by CD4(+) and CD8(+) T lymphocytes. Clin Diagn Lab Immunol 10 208 215. Hondalus, M. K., Diamond, M. S., Rosenthal, L. A. Springer, T. A. & Mosser, D. M. (1993). The intracellular bacterium Rhodococcus e qui requires Mac 1 to bind to mammalian cells. Infection and Immunity 61 2919 2929. Hondalus, M. K. & Mosser, D. M. (1994). Survival and replication of Rhodococcus equi in ma crophages. Infect Immun 62 4167 4175. Hooper McGrevy, K. E., Wilkie, B. N. & Prescott, J. F. (2005). Virulence associated protein specific serum immunoglobulin G isotype expression in young foals protected against Rhodococcus equi pneumonia by oral immuniz ation with virulent R equi. Vaccine 23 5760 5767. Horohov, D. W., Adams, A. A. & Chambers, T. M. (2010). Immunosenescence of the Equine Immune System. Journal of Comparative Pathology 141 S78 S84. Horohov, D. W., Breathnach, C. C., Sturgill, T. L., Rashid C., Stiltner, J. L., Strong, D., Nieman, N. & Holland, R. E. (2008). In vitro and in vivo modulation of the equine immune response by parapoxvirus ovis. Equine Vet J 40 468 472. Horowitz, M. L., Cohen, N. D., Takai, S., Becu, T., Chaffin, M. K., Chu, K. K., Magdesian, K. G. & Martens, R. J. (2001). Application of Sartwell's model (lognormal distribution of incubation periods) to age at onset and age at death of foals with Rhodococcus equi pneumonia as evidence of perinatal infection. J Vet Intern Med 15 1 71 175. Hughes, K. L. & Sulaiman, I. (1987). The ecology of Rhodococcus e qui and physicochemical influences on growth. Veterinary Microbiology 14 241 250. Hurley, J. R. & Begg, A. P. (1995). Failure of hyperimmune plasma to prevent pneumonia caused by Rhodo coccus equi in foals. Aust Vet J 72 418 420.

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98 Hussey, G. D., Watkins, M. L. V., Goddard, E. A., Gottschalk, S., Hughes, E. J., Iloni, K., Kibel, M. A. & Ress, S. R. (2002). Neonatal mycobacterial specific cytotoxic T lymphocyte and cytokine profiles in resp onse to distinct BCG vaccination strategies. Immunology 105 314 324. Ito, S., Ishii, K. I., Gursel, M., Shirotra, H., Ihata, A. & Klinman, D. M. (2005). CpG oligodeoxynucleotides enhance neonatal resistance to Listeria infection. Journal of Immunology 174 777 782. Jacks, S. & Gigu re, S. (2010). Effects of inoculum size on cell mediated and humoral immune responses of foals experimentally infected with Rhodococcus equi : A pilot study. Veterinary Immunology and Immunopathology 133 282 286. Jacks, S., Gigu r e, S., Crawford, P. C. & Castleman, W. L. (2007a). Experimental infection of neonatal foals with Rhodococcus equi triggers adult like gamma interferon induction. Clinical and Vaccine Immunology 14 669 677. Jacks, S., Gigu re, S. & Prescott, J. F. (2007 b ). I n vivo expression of and cell mediated immune responses to the plasmid encoded virulence associated proteins of Rhodococcus equi in foals. Clin Vaccine Immunol 14 369 374. Jain, S., Bloom, B. R. & Hondalus, M. K. (2003). Deletion of vapA encoding virulence associated protein A attenuates the intracellular actinomycete Rhodococcus equi Molecular Microbiology 50 115 128. Jeffcott, L. B. (1974). Studies on passive immunity in the foal. 1. Gamma globulin and antibody variations associated with the maternal tra nsfer of immunity and the onset of active immunity. J Comp Pathol 84 93 101. Johnson, J. A., Prescott, J. F. & Markham, R. J. F. (1983). The pathology of e xperimental Corynebacterium e qui i nfection in foals f ollowing i ntragastric c hallenge. Veterinary Path ology 20 450 459. Jullien, P., Cron, R. Q., Dabbagh, K., Cleary, A., Chen, L., Tran, P., Stepick Biek, P. & Lewis, D. B. (2003). Decreased CD154 expression by neonatal CD4(+) T cells is due to limitations in both proximal and distal events of T cell activa tion. International Immunology 15 1461 1472. Kadereit, S., Junge, G. R., Kleen, T., Kozik, M. M., Tary Lehmann, M. & Laughlin, M. J. (2003). Deficient IFN gamma expression in umbilical cord blood (UCB) T cells can be rescued by IFN gamma mediated increase in expression of Nuclear Factor of Activated T Cells 1 (NFAT1). Faseb Journal 17 C293. Kadereit, S., Mohammad, S. F., Miller, R. E., Woods, K. D., Listrom, C. D., McKinnon, K., Alali, A., Bos, L. S., Iacobucci, M. L., Sramkoski, M. R., Jacobberger, J. W. & Laughlin, M. J. (1999). Reduced NFAT1 protein expression in human umbilical cord blood T lymphocytes. Blood 94 3101 3107.

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99 Kalyuzhny, A. & Stark, S. (2001). A simple method to reduce the background and improve well to well reproducibility of staining in EL ISPOT assays. J Immunol Methods 257 93 97. Kalyuzhny, A. E. (2005). Chemistry and biology of the ELISPOT assay. Methods Mol Biol 302 15 31. Kanaly, S. T., Hines, S. A. & Palmer, G. H. (1993). Failure of pulmonary clearance of Rhodococcus equi infection in Cd4+ T lymphocyte deficient transgenic mice. Infection and Immunity 61 4929 4932. Kanaly, S. T., Hines, S. A. & Palmer, G. H. (1995). Cytokine modulation alters pulmonary clearance of Rhodococcus equi and development of granulomatous pneumonia. Infect Immu n 63 3037 3041. Kanaly, S. T., Hines, S. A. & Palmer, G. H. (1996). Transfer of a CD4+ Th1 cell line to nude mice effects clearance of Rhodococcus equi from the lung. Infect Immun 64 1126 1132. Kasuga Aoki, H., Takai, S., Sasaki, Y., Tsubaki, S., Madarame H. & Nakane, A. (1999). Tumour necrosis factor and interferon gamma are required in host resistance against virulent Rhodococcus equi infection in mice: cytokine production depends on the virulence levels of R. equi. Immunology 96 122 127. Liu, I. K., Wa lsh, E. M., Bernoco, M. & Cheung, A. T. (1987). Bronchoalveolar lavage in the newborn foal. J Reprod Fertil Suppl 35 587 592. Liu, T., Nerren, J., Liu, M., Martens, R. & Cohen, N. (2009). Basal and stimulus induced cytokine expression is selectively impaire d in peripheral blood mononuclear cells of newborn foals. Vaccine 27 674 683. Lopez, A. M., Hines, M. T., Palmer, G. H., Alperin, D. C. & Hines, S. A. (2002). Identification of pulmonary T lymphocyte and serum antibody isotype responses associated with pro tection against Rhodococcus equi Clin Diagn Lab Immunol 9 1270 1276. Lopez, A. M., Hines, M. T., Palmer, G. H., Knowles, D. P., Alperin, D. C. & Hines, S. A. (2003). Analysis of anamnestic immune responses in adult horses and priming in neonates induced b y a DNA vaccine expressing the vapA gene of Rhodococcus equi Vaccine 21 3815 3825. Madarame, H., Takai, S., Matsumoto, C., Minamiyama, K., Sasaki, Y., Tsubaki, S., Hasegawa, Y. & Nakane, A. (1997). Virulent and avirulent Rhodococcus equi infection in T ce ll deficient athymic nude mice: Pathologic, bacteriologic and immunologic responses. Fems Immunology and Medical Microbiology 17 251 262.

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107 BIOGRAPHICAL SKETCH Clare Ryan attended the University of Wisconsin Madison where she ma jored in biochemistry. She received her Doctor of Veterinary Medicine from the University of Wisconsin Madison School of Veterinary Medicine in 2002. She completed a large animal medicine and surgery internship at the Ontario Veterinary College in Ontario, Canada in 2003. S for a residency in l arge a nimal i nternal m edicine, which she completed in 2006. She also completed her requirements to become a d iplomate of the American College of Veterinary Medicine at this time. She then began her graduate studies at the University of Florida. She rec e ived her Ph.D. from the University of Florida in the summer of 2010.