RHODOCOCCUS EQUI</Em> in the FOAL – IMPROVING DIAGNOSTIC

University of Kentucky UKnowledge

Theses and Dissertations--Veterinary Science Veterinary Science

2018

RHODOCOCCUS EQUI IN THE FOAL – IMPROVING DIAGNOSTIC AND PREVENTION MEASURES

Fernanda Bicudo Cesar University of Kentucky, [email protected] Digital Object Identifier: https://doi.org/10.13023/etd.2018.315

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Recommended Citation Bicudo Cesar, Fernanda, "RHODOCOCCUS EQUI IN THE FOAL – IMPROVING DIAGNOSTIC AND PREVENTION MEASURES" (2018). Theses and Dissertations--Veterinary Science. 36. https://uknowledge.uky.edu/gluck_etds/36

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REVIEW, APPROVAL AND ACCEPTANCE

The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above.

Fernanda Bicudo Cesar, Student

Dr. David W. Horohov, Major Professor

Dr. Daniel Howe, Director of Graduate Studies RHODOCOCCUS EQUI IN THE FOAL – IMPROVING DIAGNOSTIC AND PREVENTION

MEASURES

______

DISSERTATION

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A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Agriculture, Food and Environment at the University of Kentucky

Fernanda Bicudo Cesar

Lexington, Kentucky

Director: Dr. David W. Horohov, Professor of Immunology

Lexington, KY

2018

RHODOCOCCUS EQUI IN THE FOAL – IMPROVING DIAGNOSTIC AND PREVENTION MEASURES

Although Rhodococcus equi (R. equi), previously known as Corynebacterium equi, was first isolated from pneumonic foals almost a century ago, it remains the most common cause of subacute or chronic granulomatous bronchopneumonia in foals. While the majority of foals exposed to R. equi develop a protective immune response (regressors), others exhibit a unique susceptibility to infection (progressors). The determinants for either outcome are not completely understood. Therefore, current diagnostic and preventive measures are suboptimal and require betterment. In light of this current need, we hypothesized that immunoglobulin G subisotype T [IgG(T)] against the virulence-associated protein A (VapA) of R. equi, and whole blood cytokine expression profile of foals predict the outcome of infection and can be used as diagnostic markers of clinical disease. Further, we hypothesized that the use of R. equi hyperimmune plasma (HIP) decreases severity of disease in naturally infected foals, playing an important role in disease prevention in the field. Lastly, we hypothesized that specific anti-Rhodococcus equi pili antibodies passively acquired by foals via colostrum after immunization of pregnant mares with a Rhodococcus equi pili-based candidate vaccine will confer protection against induced disease, and therefore have an immediate impact on R. equi pneumonia prophylaxis. The objectives of this study were: (1) to describe the humoral immune response of progressor and regressor foals to R. equi following experimental challenge and natural infection, (2) to compare the cytokine and cell-marker expression profile in whole blood of progressor and regressor foals after challenge, (3) to evaluate the Vap-A specific IgG profile of a commercially available HIP product and its value as a prophylactic tool on an endemic farm, and (4) to evaluate the efficacy of a vaccine based on the Rhodococcus equi pili (Rpl). Although the IgG(T) response of progressor foals after challenge or following natural infection tended to be more pronounced than that observed in regressor foals, its performance as a diagnostic test for predicting disease outcome was poor. Likewise, whole blood cell-marker and cytokine expression profiles of progressor and regressor foals were not significantly different, undermining its reliability as a diagnostic tool. Evaluation of the association of HIP VapA specific IgG profile and rhodococcal disease outcome in the field resulted in the conclusion that progressor foals received significantly less VapA specific IgG, suggesting that HIP may have provided some protection to regressor foals. Although HIP appeared to have provided some protection against clinical pneumonia, Rpl maternally-derived IgG failed to confer any advantage to foals born from vaccinated mares. The Rpl candidate vaccine failed to confer protection to foals after challenge, and did not decrease disease severity in comparison to a control group. In summary, the results of this study do not support the use of VapA specific IgG(T) or whole blood cytokine expression profile as predictors of disease outcome. Further, our results suggest a positive effect of HIP on disease outcome. Lastly, the presence of systemic and local Rpl antibodies was not protective in foals.

KEY WORDS: Rhodococcus equi, foal, VapA, IgG(T), hyperimmune plasma, vaccine.

Fernanda Cesar

August 1, 2018 RHODOCOCCUS EQUI IN THE FOAL – IMPROVING DIAGNOSTIC AND

PREVENTION MEASURES

Fernanda Bicudo Cesar

Dr. David W. Horohov

Director of Dissertation

Dr. Daniel Howe

Director of Graduate Studies

June 2018

To my husband Patrick and our families, for their endless support.

To my son Benjamin, hopefully as a future example of perseverance.

ACKNOWLEDGEMENTS

I would like to acknowledge and thank the following important people who have been instrumental in the completion of this doctorate. Firstly, I would like to express my gratitude to my advisor, Dr. David Horohov, for his unwavering support, trust and guidance throughout this research project. I would also like to thank the members of my committee for their continuous insight. Special thanks to Dr. John Timoney who carefully reviewed this dissertation, providing very detailed feedback and improvement.

I would like to express my gratitude to all the hands-on help I received from the UK farm personnel, local veterinarians and farms managers. Their contribution and eagerness to aid in answering our research questions drove a significant part of our work.

Finally, I would like to thank my mother-in-law Carolyn Godfrey who has given up so much of her personal time to care for my son Benjamin since he was 7 weeks old. Her generosity has stamped us and definitely made this possible.

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TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... iii LIST OF TABLES ...... vi LIST OF FIGURES ...... vii LIST OF ABBREVIATIONS ...... x CHAPTER 1: LITERATURE REVIEW AND RESEARCH HYPOTHESES ...... 1 Introduction ...... 1 Innate immunity of the healthy foal ...... 2 Phagocyte function ...... 3 Expression of cytokines, activation markers, and cell receptors...... 4 Adaptive immunity of the healthy foal ...... 6 Cell-mediated immunity ...... 6 Humoral immunity ...... 9 Foal’s innate immune response to R. equi ...... 11 Foal’s adaptive immune response to R. equi...... 13 Cell-mediated immunity ...... 13 Humoral immunity ...... 14 Disease prevention strategies ...... 16 Currently used strategies ...... 16 Vaccine ...... 18 Research Hypotheses ...... 19 CHAPTER 2: VAPA SPECIFIC IGG(T) WAS NOT A RELIABLE PREDICTOR OF CLINICAL PNEUMONIA IN FOALS AFTER CHALLENGE OR NATURAL INFECTION WITH R. EQUI ...... 20 Introduction ...... 20 Materials and Methods ...... 21 R. equi challenge ...... 21 Sera and HIP samples ...... 22 Foal evaluation and classification ...... 23 VapA Purification ...... 25 VapA-specific enzyme-linked immunoabsorbent assay ...... 25 Statistical Analysis ...... 26 Results ...... 27 Challenged foals...... 27 Naturally infected foals ...... 34 Discussion ...... 45 CHAPTER 3: WHOLE BLOOD EXPRESSION OF T CELL MARKERS AND CYTOKINES OF PROGRESSOR AND REGRESSOR FOALS CHALLENGED WITH RHODOCOCCUS EQUI ...... 49 Introduction ...... 49 Materials and Methods ...... 50 R. equi challenge ...... 50 Foal evaluation and classification ...... 51 Sample collection ...... 52 Gene expression analysis ...... 52 Statistical Analysis ...... 53 Results ...... 53 Challenged foals...... 53 Gene Expression ...... 53

Discussion ...... 57 CHAPTER 4: THE IMPACT OF VAPA IGGS FROM RHODOCOCCUS EQUI HYPERIMMUNE PLASMA ON DISEASE OUTCOME AFTER NATURAL EXPOSURE .... 59 Introduction ...... 59 Material and Methods ...... 60 Sera and HIP samples ...... 60 VapA Purification ...... 61 Source of reference control sera...... 61 VapA-specific enzyme-linked immunoabsorbent assay ...... 61 Foal evaluation and classification ...... 62 Statistical Analysis ...... 63 Results ...... 63 Discussion ...... 68 CHAPTER 5: R. EQUI PILI-BASED VACCINE CANDIDATE FAILED TO CONFER PROTECTION TO FOALS AGAINST CHALLENGE ...... 70 Introduction ...... 70 Materials and Methods ...... 72 Study Design ...... 72 Vaccine (provided by Jose Vazquez-Boland’s group) ...... 73 R. equi challenge ...... 74 Foal assessment ...... 75 Outcome evaluation ...... 76 Statistical Analysis ...... 76 Results ...... 77 Discussion ...... 85 CHAPTER 6: CONCLUSIONS AND FUTURE DIRECTIONS ...... 88 APPENDIX ...... 90 REFERENCES ...... 106 VITA ...... 117

LIST OF TABLES

TABLE 2. 1 PHYSICAL EXAMINATION SCORING SYSTEM. F = FAHRENHEIT DEGREES; BPM = BREATHS PER MINUTE; LNS = LYMPH NODES; *TO BE SCORED ACCORDING TO THE SEVERITY OF THE ABNORMAL LUNG SOUNDS.24 TABLE 2. 2 PEARSON CORRELATION COEFFICIENT BETWEEN THE IGG PROFILE OF PROGRESSOR AND REGRESSOR FOALS AND THEIR PHYSICAL EXAMINATION (PE SCORE) AND THORACIC ULTRASOUND (US SCORE) SCORES. * INDICATES SIGNIFICANT CORRELATION BETWEEN VARIABLES...... 30 TABLE 2. 3 MEAN (± SD) VAPA SPECIFIC IGG PROFILE OF PROGRESSOR AND REGRESSOR FOALS BEFORE (PC) AND 2 (2W), 4 (4W), 6 (6W) AND 8 (8W) WEEKS AFTER CHALLENGE...... 34 TABLE 2. 4 PEARSON CORRELATION COEFFICIENT BETWEEN VAPA SPECIFIC IGG PROFILE OF PROGRESSOR AND REGRESSOR FOALS AND THEIR PHYSICAL EXAMINATION (PE SCORE) AND THORACIC ULTRASOUND (US SCORE) SCORES...... 37 TABLE 2. 5 MEAN AND STANDARD DEVIATION (SD) VAPA SPECIFIC IGG PROFILE OF PROGRESSOR, REGRESSOR AND NRA FOALS AT TIMEPOINTS 1-4...... 41 TABLE 2. 6 LOGISTIC REGRESSION OF PROGRESSOR VERSUS REGRESSOR FOALS PREDICTED BY EACH SINGLE AND PAIRED PARAMETER. * INDICATES VARIABLE AS SIGNIFICANT OUTCOME PREDICTOR...... 42 TABLE 4. 1 COEFFICIENTS OF VARIATION (%) DETERMINED FOR VAPA SPECIFIC IGG SUBISOTYPES FOR ALL HIP SAMPLES (OVERALL) AND DIFFERENT LOTS...... 64 TABLE 5. 1 PEARSON CORRELATION COEFFICIENT AND P VALUES BETWEEN THE RPL SPECIFIC IGG TITERS OF MARES AND FOAL’S SERUM, MARE’S COLOSTRUM AND FOAL’S BALF ...... 81 TABLE 5. 2 ULTRASOUND SCORES OF ALL FOALS OF CONTROL AND VACCINATED MARES BEFORE AND AFTER CHALLENGE, AT DIFFERENT EXAMINATION TIMES (1-16). Ε INDICATES EUTHANASIA...... 82 TABLE 5. 3 PHYSICAL EXAMINATION SCORES OF ALL FOALS OF CONTROL AND VACCINATED MARES BEFORE AND AFTER CHALLENGE, AT DIFFERENT EXAMINATION TIMES (1-16). Ε INDICATES EUTHANASIA...... 83 TABLE 5. 4 POST-MORTEM PARAMETERS OF FOALS OF VACCINATED AND CONTROL MARES...... 83 TABLE 5. 5 WHITE BLOOD CELL COUNTS OF ALL FOALS OF CONTROL AND VACCINATED MARES BEFORE AND AFTER CHALLENGE, AT DIFFERENT EXAMINATION TIMES (1-8)...... 84 TABLE 5. 6 FIBRINOGEN OF ALL FOALS OF CONTROL AND VACCINATED MARES BEFORE AND AFTER CHALLENGE, AT DIFFERENT EXAMINATION TIMES (1-8)...... 84

LIST OF FIGURES

FIGURE 2. 1 AVERAGE PHYSICAL EXAMINATION SCORES OF PROGRESSOR AND REGRESSOR FOALS BEFORE AND AFTER R. EQUI CHALLENGE AT PHYSICAL EXAMINATION TIMES 1-16. * INDICATES STATISTICAL DIFFERENCE BETWEEN GROUPS...... 28 FIGURE 2. 2 AVERAGE LUNG ULTRASONOGRAPHIC SCORES OF PROGRESSOR AND REGRESSOR FOALS BEFORE AND AFTER R. EQUI CHALLENGE AT PHYSICAL EXAMINATION TIMES 1-16. * INDICATES STATISTICAL DIFFERENCE BETWEEN GROUPS...... 29 FIGURE 2. 3 TOTAL VAPA SPECIFIC IGG OF PROGRESSOR AND REGRESSOR FOALS BEFORE (PC = PRE-CHALLENGE), AND 2 (2W), 4 (4W), 6 (6W), AND 8 (8W) WEEKS AFTER CHALLENGE. BARS REPRESENT SAMPLE STANDARD DEVIATION. *INDICATES STATISTICAL DIFFERENCE BETWEEN GROUPS; LETTER “A” INDICATES STATISTICAL DIFFERENCE BETWEEN PRE-CHALLENGE VALUES WITHIN PROGRESSOR GROUP...... 31 FIGURE 2. 4 VAPA SPECIFIC IGGA OF PROGRESSOR AND REGRESSOR FOALS BEFORE (PC = PRE-CHALLENGE), AND 2 (2W), 4 (4W), 6 (6W), AND 8 (8W) WEEKS AFTER CHALLENGE. BARS REPRESENT SAMPLE STANDARD DEVIATION. *INDICATES STATISTICAL DIFFERENCE BETWEEN GROUPS; LETTER “A” INDICATES STATISTICAL DIFFERENCE BETWEEN PRE-CHALLENGE VALUES WITHIN PROGRESSOR GROUP...... 32 FIGURE 2. 5 VAPA SPECIFIC IGGB OF PROGRESSOR AND REGRESSOR FOALS BEFORE (PC = PRE-CHALLENGE), AND 2 (2W), 4 (4W), 6 (6W), AND 8 (8W) WEEKS AFTER CHALLENGE. BARS REPRESENT SAMPLE STANDARD DEVIATION. *INDICATES STATISTICAL DIFFERENCE BETWEEN GROUPS; LETTER “A” INDICATES STATISTICAL DIFFERENCE BETWEEN PRE-CHALLENGE VALUES WITHIN PROGRESSOR GROUP...... 33 FIGURE 2. 6 VAPA SPECIFIC IGG(T) OF PROGRESSOR AND REGRESSOR FOALS BEFORE (PC = PRE-CHALLENGE), AND 2 (2W), 4 (4W), 6 (6W), AND 8 (8W) WEEKS AFTER CHALLENGE. BARS REPRESENT SAMPLE STANDARD DEVIATION...... 34 FIGURE 2. 7 VAPA SPECIFIC IGGA IN HIP RECEIVED BY NATURALLY EXPOSED FOALS (PROGRESSOR, REGRESSOR, NRA). BARS REPRESENT SAMPLE STANDARD DEVIATION *INDICATES STATISTICAL DIFFERENCE IN COMPARISON TO THE NRA GROUP...... 35 FIGURE 2. 8 VAPA SPECIFIC IGGB IN HIP RECEIVED BY NATURALLY EXPOSED FOALS (PROGRESSOR, REGRESSOR, NRA). BARS REPRESENT SAMPLE STANDARD DEVIATION *INDICATES STATISTICAL DIFFERENCE IN COMPARISON TO THE NRA GROUP...... 36 FIGURE 2. 9 TOTAL VAPA SPECIFIC IGG OF PROGRESSOR, REGRESSOR, AND NRA FOALS OVER TIME (1,2,3 AND 4 SAMPLE COLLECTION TIME POINTS). BARS REPRESENT SAMPLE STANDARD DEVIATION. LETTER “A” INDICATES STATISTICAL DIFFERENCE BETWEEN PRE-CHALLENGE VALUES WITHIN NRA GROUP...... 38 FIGURE 2. 10 VAPA SPECIFIC IGGA OF PROGRESSOR, REGRESSOR, AND NRA FOALS OVER TIME (1,2,3 AND 4 SAMPLE COLLECTION TIME POINTS). BARS REPRESENT SAMPLE STANDARD DEVIATION. *INDICATES STATISTICAL DIFFERENCE BETWEEN PROGRESSOR AND REGRESSOR GROUPS; LETTERS “A” AND “B” INDICATE STATISTICAL DIFFERENCE BETWEEN PRE-CHALLENGE VALUES WITHIN NRA AND PROGRESSOR GROUP, RESPECTIVELY...... 39 FIGURE 2. 11 VAPA SPECIFIC IGGB OF PROGRESSOR, REGRESSOR, AND NRA FOALS OVER TIME (1,2,3 AND 4 SAMPLE COLLECTION TIME POINTS). BARS REPRESENT SAMPLE STANDARD DEVIATION. *INDICATES STATISTICAL DIFFERENCE BETWEEN PROGRESSOR AND REGRESSOR GROUPS; LETTERS “A”

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AND “B” INDICATE STATISTICAL DIFFERENCE BETWEEN PRE-CHALLENGE VALUES WITHIN NRA AND PROGRESSOR GROUPS, RESPECTIVELY...... 40 FIGURE 2. 12 VAPA SPECIFIC IGG(T) OF PROGRESSOR, REGRESSOR, AND NRA FOALS OVER TIME (1,2,3 AND 4 SAMPLE COLLECTION TIME POINTS). BARS REPRESENT SAMPLE STANDARD DEVIATION. # INDICATES STATISTICAL DIFFERENCE BETWEEN PROGRESSOR AND NRA GROUPS; LETTER “B” INDICATES STATISTICAL DIFFERENCE BETWEEN PRE-CHALLENGE VALUES WITHIN PROGRESSOR GROUP...... 40 FIGURE 2. 13 ROC CURVE ANALYSIS OF VAPA SPECIFIC IGGA AT TIMEPOINT 2 AS A PREDICTOR OF CLINICAL DISEASE (PROGRESSOR) AMONG R. EQUI INFECTED FOALS (PROGRESSORS AND REGRESSORS). THE AREA UNDER THE CURVE IN INDICATED ON THE GRAPH...... 43 FIGURE 2. 14 ROC CURVE ANALYSIS OF VAPA SPECIFIC IGGB AT TIMEPOINT 2 AS A PREDICTOR OF CLINICAL DISEASE (PROGRESSOR) AMONG R. EQUI INFECTED FOALS (PROGRESSORS AND REGRESSORS). THE AREA UNDER THE CURVE IN INDICATED ON THE GRAPH...... 44 FIGURE 2. 15 ROC CURVE ANALYSIS TOTAL VAPA SPECIFIC IGG AND IGG SUBISOTYPES SERIALLY MEASURED OVER THE FIRST 3 MONTHS OF LIFE AS PREDICTORS OF CLINICAL DISEASE (PROGRESSOR) AMONG R. EQUI INFECTED FOALS (PROGRESSORS AND REGRESSORS). THE AREAS UNDER THE CURVE (A) ARE INDICATED ON THE GRAPH...... 45 FIGURE 3. 1 GENE AMPLIFICATION MEASURED BY RT-QPCR DENOTED AS LOGARITHMIC TRANSFORMED RELATIVE MEASURE OF MRNA EXPRESSIONS (RQ) OF IFN-Γ , IL-4, CD4, MHCII, FOXP3, AND CD25 AT 2 (2W) AND 6 WEEKS (6W) IN ALL FOALS AFTER CHALLENGE. BAR WITH ASTERISK DENOTES SIGNIFICANT DIFFERENCE BETWEEN TIMEPOINTS...... 54 FIGURE 3. 2 GENE AMPLIFICATION MEASURED BY RT-QPCR DENOTED AS LOGARITHMIC TRANSFORMED RELATIVE MEASURE OF MRNA EXPRESSIONS (RQ) OF IFN-Γ , CD4, MHCII, FOXP3, AND CD25 AT 2 (2W) AND 6 WEEKS (6W) IN REGRESSOR (R) AND PROGRESSOR (P) FOALS AFTER CHALLENGE. LETTERS “A” AND “B” DENOTE SIGNIFICANT DIFFERENCE BETWEEN TIMEPOINTS WITHIN REGRESSOR AND PROGRESSOR GROUPS, RESPECTIVELY...... 56 FIGURE 4. 1 TOTAL VAPA SPECIFIC IGG CONCENTRATION IN 20 LOTS OF HIP. THE OVERALL DIFFERENCE WAS SIGNIFICANT (P=0.025)...... 65 FIGURE 4. 2 VAPA SPECIFIC IGGA CONCENTRATION IN 20 LOTS OF HIP. THE OVERALL DIFFERENCE WAS SIGNIFICANT (P<0.001)...... 65 FIGURE 4. 3 VAPA SPECIFIC IGGB CONCENTRATION IN 20 LOTS OF HIP HIP. THE OVERALL DIFFERENCE WAS SIGNIFICANT (P<0.001)...... 66 FIGURE 4. 4 VAPA SPECIFIC IGG(T) CONCENTRATION IN 20 LOTS OF HIP. THE OVERALL DIFFERENCE WAS SIGNIFICANT (P<0.001)...... 66 FIGURE 4. 5 TOTAL VAPA SPECIFIC IGG CONCENTRATION IN THE HIP RECEIVED BY NRA, REGRESSOR AND PROGRESSOR FOALS. ASTERISK DENOTES SIGNIFICANT DIFFERENCE BETWEEN THE REGRESSOR AND THE OTHER GROUPS...... 67 FIGURE 4. 6 TOTAL VAPA SPECIFIC IGG CONCENTRATION IN THE HIP RECEIVED BY REGRESSOR AND PROGRESSOR FOALS. ASTERISK DENOTES SIGNIFICANT DIFFERENCE BETWEEN GROUPS...... 68 FIGURE 5. 1 OVERVIEW OF RPL SUBUNIT RECOMBINANT VACCINE PRODUCTION...... 73 FIGURE 5. 2 BASELINE SERUM RPL SPECIFIC IGG TITERS OF PREGNANT MARES APPROXIMATELY 2 MONTHS PRIOR TO FOALING. EACH BAR REPRESENTS A CONTROL (BLUE) AND A VACCINATED (RED) MARE RANKED FROM LOWEST TO HIGHEST ANTIBODY TITER...... 77

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FIGURE 5. 3 VACCINATED AND CONTROL MARE’S RPL SPECIFIC IGG TITERS OVER TIME. ASTERISK INDICATES SIGNIFICANT DIFFERENCE FROM BASELINE. LETTER “A” INDICATES SIGNIFICANT DIFFERENCE BETWEEN GROUPS...... 78 FIGURE 5. 4 TITERS OF RPL SPECIFIC IGG IN COLOSTRUM OF CONTROL AND VACCINATED MARES. ASTERISK INDICATES SIGNIFICANT DIFFERENCE BETWEEN GROUPS...... 79 FIGURE 5. 5 RPL SPECIFIC IGG SERUM TITERS OF FOALS OF CONTROL AND VACCINATED MARES 3-7 DAYS AFTER BEING BORN (BEFORE EXPERIMENTAL INFECTION). ASTERISK INDICATES SIGNIFICANT DIFFERENCE BETWEEN GROUPS...... 80 FIGURE 5. 6 RPL SPECIFIC IGG BALF TITERS OF FOALS OF CONTROL AND VACCINATED MARES 3-7 DAYS AFTER BEING BORN (BEFORE EXPERIMENTAL INFECTION). ASTERISK INDICATES SIGNIFICANT DIFFERENCE BETWEEN GROUPS...... 81

LIST OF ABBREVIATIONS

(Alphabetical order)

ANOVA ...... Analysis of variance

AUC ...... Area under the curve

BALF...... Bronchoalveolar lavage fluid

CBC...... Cell blood count

CD ...... Cluster of differentiation cfu ...... Colony-forming units

CI...... Confidence Interval

CpG-ODNs ...... Cytosine-phosphate guanine oligodeoxynucleotide

CR3 ...... Complement receptor 3

CTLs ...... Cytotoxic T lymphocytes

CV ...... Coefficient of variation

DCs ...... Dendritic cells

ELISA ...... Enzyme-linked immunosorbent assay

EU ...... ELISA units

Fcγ ...... Fragment crystallizable receptor gamma

FES ...... Fetal equine serum

FoxP3 ...... Forkhead box P3

HIP ...... Rhodococcus equi-specific hyperimmune plasma

HR ...... Heart rate

HRP ...... Horseradish peroxidase

IFN-γ ...... Interferon gamma

IgA ...... Immunoglobulin A

IgG ...... Immunoglobulin G

IgM ...... Immunoglobulin M

IgGa...... Immunoglobulin G subisotype A

IgGb...... Immunoglobulin G subisotype B

IgG(T) ...... Immunoglobulin G subisotype T

IL ...... Interleukin

KY ...... Kentucky

MHC ...... Major histocompatibility complex

MHz ...... Megahertz mRNA ...... Messenger ribonucleic acid nm ...... nanometer

NRA ...... No respiratory abnormality

OD ...... Optical density

PAMPs...... Pathogen-associated molecular patterns

PBMCs...... Peripheral blood mononuclear cells

PBS ...... Phosphate buffered saline

PCR ...... Polymerase chain reaction pVAP ...... plasmid for Vap

R. equi ...... Rhodococcus equi

ROC ...... Receiver-operating characteristic

Rpl ...... Rhodococcus equi pilus

Rpl specific IgG...... Immunoglobulin G specific for Rpl protein

RR ...... Respiratory rate

SD ...... Standard deviation

SDS-PAGE ...... Sulfate-polyacrylamide gel electrophoresis

Th1...... T-helper lymphocyte type 1

Th-2 ...... T-helper lymphocyte type 2

TLRs ...... Toll-like receptors

TNFα ...... Tumor necrosis factor alpha

TNFβ ...... Tumor necrosis factor beta

Tregs ...... T regulatory cells

TSAYE ...... Tryptic soy agar yeast extract

TSBYE ...... Tryptic soy broth yeast extract

TTW ...... Transtracheal wash

UKVDL...... University of Kentucky Veterinary Diagnostic Laboratory

Vap ...... Virulence associated protein

VapA specific IgG ...... Immunoglobulin G specific for VapA protein

VapA+ ...... R. equi that carries gene encoding for Vap

WBC ...... White blood cells

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CHAPTER 1

LITERATURE REVIEW AND RESEARCH HYPOTHESES

Introduction

Rhodococcus equi is considered one of the most important pathogens of early life in horses worldwide 1. In infected foals, a wide range of respiratory disease can be observed varying from subclinical lung lesions to life-threatening suppurative bronchopneumonia that may develop within the first months of life 1,2. Endemically infected farms experience morbidity of 70% or greater, and case fatalities range from 13% to 40% for R. equi pneumonia 3–6. This disease has a major negative financial impact in the equine industry due to the ubiquitous pathogen distribution, high cumulative disease incidence on affected farms, and costly preventive and treatment measures. Although R. equi is most often recognized as an etiologic agent of bronchopneumonia in foals, other body systems can be infected, as well as immunocompromised humans and other animal species1,7.

R. equi belongs to an extended genus of ubiquitous Actinobacteria with more than 35 recognized species, among which R. equi remains the species with the most pathogenic potential for humans and animals. Rhodococcus spp. are obligatory aerobic, non-motile, non-spore-forming gram-positive coccobacilli. They are part of the phylogenetic group described as mycolata, characterized by a unique cell envelope that consists of mycolic acids covalently linked to the arabinogalactan-peptidoglycan layer of the cell wall8. This thick hydrophobic outer membrane-like sheath protects the actinomycetes from environmental and host-derived assaults 9. R. equi is primarily a soil saprophyte, where it uses herbivore manure as a growth substrate, explaining its widespread distribution in horse farms where the infection is primarily contracted by inhalation of aerosolized R. equi in dust10.

R. equi is a facultative intracellular pathogen, which parasitizes macrophages causing granulomatous inflammation1. In affected foals, alveolar macrophages are the primary survival and replication niche for the inhaled R. equi 11. Macrophage uptake is mediated either via complement receptor 3 (CR-3 or Mac-1) or mannose receptor, which recognizes residues of the bacterial cell

1 envelope12,13. Entry through these receptors bypasses the antibody-mediated Fcγ receptor pathway and may play a major role in promoting macrophage colonization by R. equi, while uptake of opsonized R. equi with specific antibodies against unkown surface antigens is likely to result in intracellular killing 14. Overwhelming intracellular replication of R. equi results in cell necrosis, release of a multitude of pro-inflammatory signals, culminating in tissue abscessation and destruction15. The ability to survive intracellularly in macrophages is conferred to Rhodococcus by a family of plasmid encoded determinants, known as virulence associated proteins (Vap) 16.

Virulence associated proteins are small, secreted proteins that weigh 17-20 kDa and are 164-202 amino acids long. Gene deletion and complementation studies have shown that VapA, one of the nine Vap encoded in the bacterial plasmid, is the primary R. equi virulence determinant17. Virulent

R. equi are able to modify the phagocytic vacuole of host macrophages to prevent acidification and subsequent fusion with lysosomes18, by molecular mechanisms yet to be determined. A second key

R. equi virulence determinant discovered during the analysis of the R. equi genome is a chromosomal horizontally acquired island that encodes for cytoadhesive Flp type IVb subfamily pili 19. The R. equi pili (Rpl) form long, rigid appendages, typically 2-5 per bacterial cell19 that mediate attachment to epithelial cells and macrophages and are essential for lung colonization in live mice 20.

Epidemiological evidence from field and challenge studies indicates that foals are most susceptible to rhodococcal infections shortly after birth 21–24, when exposure to ubiquitous R. equi is likely to occur. While the majority of foals infected with R. equi shows spontaneous resolution of lung lesions, others exhibit a unique susceptibility to infection. The determinants for either outcome are not completely understood.

Innate immunity of the healthy foal

The innate immune system plays a critical role in protecting neonates against infections since antigen-specific immune responses require previous exposure and time for development. A critical determinant for an evolutionary conserved immature innate immune response in young

2 mammals is the high antigenic pressure that follows birth, posed not only by a wide variety of pathogens, but also by commensals, as well as harmless environmental and food antigens. This high load of novel antigens requires well-established immune regulatory mechanisms in order to allow concomitant protective responses and the development of immune self-recognition and tolerance. These are crucial mechanisms necessary for effective control of inflammatory reactions and adequate cell growth and proliferation early in life25. Therefore, it is no surprise that the finely regulated immune system of the foal differs from that of the adult horse in the way it responds to immune challenges. The overall competency of the foal’s innate immune system has been addressed through investigations of phagocyte function and expression of cytokines, activation markers, and cell receptors.

Phagocyte function

As the most abundant white blood cell in the peripheral blood, neutrophils are critical effectors cells of innate immunity26. In healthy foals up to 3 weeks of age, neutrophils have impaired in vitro yeast phagocytic activity in the presence of autologous serum. However, in the presence of pooled serum from adult horses or specific IgG, the same neutrophils are able to phagocytose yeast cells. This finding suggests that neutrophils are competent phagocytes at birth, and that the cause for early decreased phagocytosis is associated with the neonatal serum27. The fact that serum IgG concentrations remain low in foals during the first few months of life, implies that other opsonic factors, such as complement, may be of greater importance in neutrophil phagocytosis28. Indeed, it has been shown that the inhibition of complement-mediated opsonisation by heat inactivation reduces the phagocytic capacity of neutrophils29.

Studies evaluating early neutrophil killing capacity in foals have shown contradictory results. When chemiluminescence was used to quantify oxidative burst, Demmers et al. found that the killing capacity of neutrophils was significantly lower following birth but, from 3 months onwards, values were equal to or exceeded that of mature horses27. However, a previous study using a similar experimental design showed no significant changes in chemiluminescence response in

3 foals from birth to 6 months of age14. When flow cytometry was used to access oxidative burst activity in foal neutrophils, no age-dependent changes were identified30. The reasons for these conflicting results are not clear.

Peripheral blood phagocytic activity of leukocytes in the healthy foal has been evaluated via a simultaneous phagocytosis and oxidative burst activity assay31. No age-dependent maturation of phagocyte function was identified, and phagocytosis was optimized in the presence of opsonizing serum. Therefore, as shown with neutrophils, these cells were also deemed competent at birth.

Bronchoalveolar lavages obtained from foals of 1 to 21 days of age have shown a numerical deficiency in alveolar macrophages up to 2 weeks of age, when compared to older horses (2-3 years of age)32. Alveolar macrophages obtained from 2-3-day old foals also demonstrated significant impaired chemotactic function. These numerical and chemotactic deficiencies of the neonatal foal have been suggested to play a role in the foal’s susceptibility to respiratory disease.

Expression of cytokines, activation markers, and cell receptors

Non-phagocytic mechanisms may also play a role in protection against intracellular pathogens. The induction of protective resistance to infectious diseases is influenced by the pattern and magnitude of cytokine gene expression33. In other species, neutrophils produce a variety of cytokines and chemokines that modulate the immune response by recruiting and activating other effector cells of the immune system34. Neutrophil expression of interleukine (IL) 6 and IL-8 in healthy newborn foals (< 24 hours of birth) is significantly greater than expression at 2-, 4-, and 8- weeks of age35. The role of IL-6 in infection is controversial, but the majority of the evidence suggests it is required for protection against a number of bacterial infections36–39. IL-8 is a potent chemoattractant of neutrophils, monocytes and T-cells, and has been shown to enhance killing of

Mycobacterium tuberculosis by neutrophils40–42. Therefore, it is possible that in newborn foals enhanced expression of IL-6 and IL-8 is needed for pathogen protection.

Most recently, it has been demonstrated that cytosine-phosphate guanine

4 oligodeoxynucleotide (CpG-ODNs) can activate cytokine expression and reactive oxygen species

(ROS) production by neutrophils in foals older than 2 weeks of age43. Similar studies using foals younger than 2 weeks revealed that in vitro stimulation with CpG-ODN also significantly increased pro-inflammatory cytokine gene expression and degranulation of neutrophils collected from foals as young as 1 day old44. When CpG-ODN was subsequently administered intramuscularly to live foals at 1 and 7 days of age, there was a significant increase in the IFN-γ mRNA expression by the foal’s neutrophils, suggesting that the administration of this compound may improve the foal’s early immune responses45.

Cytokines released from innate immune cells play key roles in the regulation of the immune response33. Recent studies in neonatal humans, mice and other species have shown that neonates are deficient in the production of IFN-γ and IL-646–48. The impaired production of these pro- inflammatory cytokines has been proposed as the basis of the deficiency in neonatal innate immunity, which contributes to impaired neonatal host defenses in newborns26,49. Based on studies that evaluated PBMCs, foals have also being suggested as being IFN-γ deficient at birth50–52, and this immunodeficiency has been assumed to contribute to the vulnerability of neonatal foals to a wide variety of pathogens51,53.

IL-1 plays an important role in nonspecific immunological defense. It enhances activity of natural killer cells, promotes maturation and proliferation of B cells, chemotactically attracts neutrophils and macrophages, and initiates synthesis of acute phase proteins by the liver, along with IL-6 and TNF-α 54,55. While its innate functions are crucial to the immune system, the ability of IL-1 to evoke protective immunity is limited. IL-1α and IL-1β deficient mice have shown no unique susceptibility to common pathogens56. In fact, instances of IL-1 over-expression have lead to health complications. Sepsis, for example, is a common ailment of neonatal foals. During sepsis in foals, IL-1 is released early as part of an inflammatory response, resulting in tissue damage57.

TGF-β has potent immunomodulating properties that can be contradictory depending on the cytokine environment and surrounding cell type58. Although TGF-β is chemotactic for neutrophils and monocytes, it also limits inflammatory responses and promotes wound healing59.

Toll-like receptors (TLRs) are key components of the innate immune system, because they recognize conserved structural pathogen-associated molecular patterns (PAMPs) and initiate signaling cascades upon ligand biding to activate immune cells. TLR-8 recognizes single-stranded

RNAs and plays a role in the innate anti-viral immune response60. Foal neutrophils of all ages express TLR-8 mRNA constitutively, and stimulation of the TLR-8 signaling pathway induces mRNA expression of inflammatory cytokines IL-6 and IL-8 in neutrophils from foals at 1 and 14 days of age61. TLR-9 detects bacterial DNA, which is distinguished by unmethylated CpG motifs, and its signaling promotes functional activity of neutrophils against intracellular pathogens 60.

Neutrophils from newborns and two-month-old foals express levels of TLR-9 mRNA comparable to those of adult horse neutrophils, and stimulation of foal and adult neutrophils with CpG induces

IFN-γ, IL-8, and IL-12 mRNA expression, while TNF-α mRNA is downregulated43. Foal dendritic cells and macrophages express TLR-9 mRNA comparable to adult horse cells, however, CpG-ODN treatment did not induce specific maturation and cytokine expression in foal macrophages and dendritic cells62. Also, despite high levels of TLR expression, the in vitro activation of TLR-2 had no immunomodulatory effect over TLR-4-mediated inflammatory response in peripheral blood mononuclear cells (PBMCs) from neonatal foals, contrary to what has been reported in several in vivo studies in experimental animals63–65. The lower functional response observed after TLR activation in neonatal foals has been proposed as a strategy to limit the immune responses to the bacterial colonization of epithelial surfaces during the early period of life63.

Adaptive immunity of the healthy foal

Cell-mediated immunity

Although much of the lymphoid tissue develops during gestation, the foal’s adaptive immune system is naive at birth, thus lacking pathogen-specific B and T effector cells, as well as

B and T memory cells and preformed antibodies. Consequently, they are highly susceptible to intestinal and respiratory pathogens, including rotavirus, R. equi and others, many of which rarely occur in adults and only as opportunistic pathogens66.

The exposure to an abundant and diverse population of pathogens in early life induces a massive expansion of antigen-specific lymphocyte populations, reflected by a 2-3 times increase in the number of circulating lymphocytes and an increase in mass of secondary lymphoid tissues30.

In healthy foals, absolute circulating CD4+ and CD8+ T lymphocytes and B cells increase linearly up to 3 months of age, plateauing at a number greater than that of adult horses for a few months, before dropping toward adult horse reference values67. Peripheral leukocyte function has been assessed in the foal. While an age-dependent peripheral blood leukocyte proliferation in response to mitogen stimulation (Concanavalin A) was described during the first few weeks of life, general immune function (lymphokine activated killing activity, opsonized phagocytosis, and oxidative burst activity) of newborns was similar to the adult horse30. Neonatal antigen presenting cells

(APCs) also undergo age-dependent changes over the first few months of life. Foal macrophages and dendritic cells express relatively lower levels of major histocompatibility complex (MHC) class

II antigen in comparison to adult horses, suggesting a limited capacity to present processed extracellular antigens to T cells before exposure to environmental stimulation causes increased expression of immunoregulatory molecules62,68. In addition, fewer numbers of mature dendritic cells (CD14-CD1b+CD86+) are detected in foals younger than 3 months of age when compared to adult horses69.

Although the majority of the peripheral lymphoid organs develop throughout fetal life, only an insignificant number of T cells can be detected in the equine fetal lung, and the bronchus- associated lymphoid tissue (BALT) will only be detected in 12-week or older foals60,68.

Consequently, leukocyte populations in the bronchoalveolar lavage fluid (BALF) increase with age, with the numbers in foals aged 1 week being only 10% of those in adults68. Adult-like CD4+ and CD8+ BALF T cell concentrations are reached by 3 and 10 weeks of age, respectivelly70. Very few B lymphocytes or plasma cells are identified in the BALF before 8 weeks of life30. As observed with peripheral blood lymphocytes, lymphocytes from the BALF collected from foals younger than

6 weeks of age express lower MHC class 2 molecules than in adult horses70. Few alveolar macrophages are recovered in BALF up to two weeks of life but, by the third week, greater than

70% of the recovered cells are alveolar macrophages. While healthy foals have significantly higher percentages of macrophages in the BALF at one month compared with 12 months of age, the lymphocyte percentages are significantly higher at 12 months of age compared with 1 week of life69.

As for T cell immunity, T helper (Th) cell subsets play major roles in regulating adaptive immunity. Studies in mice have shown that a Th1 response, characterized by IFN-γ production, is sufficient to effectively promote pulmonary clearance of R. equi whereas a Th-2 response, characterized by IL-4 production, is detrimental72,73. Whether a CD4+ T helper lymphocyte differentiates into its Th1 or Th2 subset depends largely on the cytokine environment58. IL-12, for example, is produced by activated macrophages and dendritic cells and strongly stimulates the naïve T helper cell to differentiate into Th1 cells74,75. Once T cells have differentiated into a Th1 or

Th2 subset, the immune response tends to persist in that direction because Th1 cells produce cytokines that inhibit Th2 cell development and vice versa. Both human and murine neonatal T cells are impaired in producing Th1 cytokines IL-2 and IFN-γ under relatively neutral conditions76.

Thus, evidence from other species indicates that it is important to characterize cytokine expression and to evaluate cytokine expression as a function of age in foals. Characterizing cytokine expression and examining quantitative changes could help define the role of cytokines in the susceptibility of foals to infectious diseases.

Currently, Th-cell responses in neonatal and young foals are controversial. Several studies have indicated a weak Th1 immune response in healthy newborn and young foals. For example, neonatal PBMCs have been shown to express less IFN-γ, transforming growth factor (TGF)-β and

IL-1α mRNA transcripts in comparison with cells from adult horses, but their expression significantly increased with age reaching adult levels within the first year of life50,51. The neonate’s inability to express the IFN-γ gene and produce IFN-γ protein has also been reported in cells from bronchoalveolar lavage (BAL) obtained from foals at 1 month of age51. These findings suggest that a maturational development of a Th1 response occurs during the early life, and that the weaker Th1 response occurs during the neonatal period, which may contribute to the foal’s susceptibility to

8 intracellular pathogens. However, one study has shown that young foals can produce small amounts of IFN-γ via Th1 and CD8+ cytotoxic T cells and that regulatory IL-10 production by T cells is developmentally mature in foals77.

Humoral immunity

Although immunoglobulin production in foals begins in utero, serum IgM and IgG levels are low and not protective at birth. Regardless of colostrum intake, it is only by 2-3 months of life that significant concentrations of endogenous IgM and IgG in the serum are observed67. If passive transfer successfully occurs, the total serum IgG and IgM reach the lowest concentrations when the foal is 1-3 months old, due to the decay of colostrum-derived antibodies67,78. One important aspect of this dynamic phase of circulating colostrum-derived antibodies and endogenously-produced antibodies is the need for humoral protection, not only at birth but also in the initial few months of life. Therefore, what is considered to be standard adequate transfer of antibodies through colostrum at birth (e.g., at least 800 mg/dL), questionably, in theory, provides humoral protection in the first

3 months of life, when considering an IgG half-life of approximately 30 days, and a minimal circulating level of 500 mg/dL. Nonetheless, levels greater than 1000-1200 mg/dL would provide even greater protection in the first few months of life. Indeed, adequate transfer of immunoglobulins occurs naturally at levels much higher than 800 mg/dL, and varies among breeds30,60.

Only recently the full complement of the horse Ig heavy chain constant region genes has been described, increasing the known number of IgG subclasses to seven79. Earlier studies described five equine IgG subclasses named IgGa, IgGb, IgGc, IgG(T), and IgG(B). Following the identification of the seven horse heavy chain constant region genes, the IgG subclasses have been reassigned as IgG1 to IgG780. Of the originally described IgG subclasses, IgGa corresponds to

IgG1, IgGb to IgG4 and IgG7, IgGc to IgG6, and IgG(T) to both IgG3 and IgG579. In order to remain faithful to the findings of the referenced authors, the IgG subclass nomenclature here will adhere to those used in the original publication.

Several studies evaluating humoral immunity of foals have focused on IgG antibody because of its important opsonic activity81,82. Analysis of the different effector function capabilities of the seven equine IgG subclasses revealed that IgG1, IgG3, IgG4, IgG5 and IgG7, but not IgG2 and IgG6, elicit a strong respiratory burst from equine peripheral blood leukocytes. IgG1, IgG3,

IgG4 and IgG7, but not IgG2, IgG5 and IgG6, bind complement C1q and activate complement via the classical pathway 83.

IgGb is the most abundant IgG subclass in adult horse serum (followed by IgG(T) and

IgGa) and colostrum (followed by IgGa and IgG(T)), yet its endogenous synthesis could not be detected in healthy foals until 63 days after birth78. In contrast, IgGa, IgG(T) and IgA can be detected in foal serum within the first 5-8 weeks of life. IgGa increases by 2-3 months to concentrations 3-fold higher in foal than in adult serum. IgGb concentration in foal serum was lower than that of IgGa or IgG7 between 12 weeks and 8 months and had not attained adult concentrations by one year of age84. Since the effector function of these IgG subclasses overlap widely, the delay in IgGb production may be functionally compensated by the higher circulating

IgGa and adult-like IgG(T). It should be emphasized that endogenous antibody production in foals was measured in the presence of residual maternal antibodies and therefore, the true onset of antibody production in foals may have been underestimated. Foal Ig production starts before birth for IgGa, shortly after birth for IgG(T) and IgA, and likely begins at around 5 weeks of age for

IgGb25.

Concentrations of immunoglobulins have been determined in nose wash samples obtained from foals immediately after parturition and on days 1, 7, 14, 28, 42 and 63 of life. The major isotype reported in nasal secretions of foals older than 28 days was IgA, while IgGa and IgGb were the predominant immunoglobulins in foals younger than 14 days of age. The combination of few lymphocytes in the lungs with the lack of IgA in the upper airway of young foals, has been suggested as a predisposing factor to respiratory disease85.

Foal’s innate immune response to R. equi

The importance of neutrophils in controlling early R. equi tissue infection and decreasing disease severity in mice was demonstrated by Martens et al., 200586. Anti-neutrophil monoclonal antibody-induced neutrophil deficiency during the first week after experimental infection with R. equi resulted in more severe disease and significantly increased bacterial tissue concentrations. In the foal, the protective role of neutrophils against R. equi has been controversial. The in vitro bactericidal capacity of neutrophils against opsonized R. equi was found to be statistically similar among neonates, 1-month old foals, and adult horses87. However, some neonatal foals (15%) showed a substantially reduced capacity to kill R. equi, which was suggested as a potential important factor in the pathogenesis of R. equi infections. Further, relatively lower circulating neutrophil concentrations in foals at 2 and 4 weeks of age were associated with a significant higher likelihood of subsequent development of R. equi pneumonia compared to foals with higher concentrations88. On the other hand, several authors have suggested that foal neutrophils are functional cells and are not a contributing factor in the pathogenesis of R. equi pneumonia. Yager et al. have described an intact in vitro bactericidal capacity of neutrophils in neonatal foals89. Age- related changes in basal and R. equi-stimulated cytokine gene expression by foal neutrophils have shown that they are able to increase mRNA expression of many pro-inflammatory cytokines, including IFN-γ, in response to in vitro stimulation with virulent R. equi, and the magnitude of this increase in expression is influenced by age starting at birth35,43.

Dendritic cells (DCs) are vitally important in the initial stages of infection for the production of a successful and efficient immune response. Maturational differences have been found between DCs of foals and adults, including a reduced ability of DCs of foals to produce TNF-

α in response to LPS stimulation69. While R. equi challenged foal DCs produce equivalent amounts of IL-12, a cytokine which promotes a Th1 response and IFN-γ production, they failed to upregulate

MHCII expression when compared to adult DCs90,. This could lead to impaired ability to interact with and stimulate naïve T-cells. Differential expression of additional 19 immunologically relevant genes has been identified in foal and adult monocyte-derived DCs in response to R. equi infection,

11 providing promising targets for further research into the host response to R. equi, and the susceptibility of foals to this disease91.

Given the structural similarity between Mycobacterium tuberculosis and R. equi, the role of MHC-unrestricted cytotoxic T lymphocytes, and the expression of CD-1 molecules in antigen- presenting cells of foals and adult horses has been investigated92. Protective immune responses to

M. tuberculosis involve classical major histocompatibility complex MHC-restricted T cells that recognize peptide antigen, as well as MHC-independent T cells that recognize mycobacterial lipid antigen presented by CD-1 molecules. The CD-1b isoform is evolutionarily conserved and is present on equine monocyte-derived macrophages. However, both CD-1b and MHCII expression are significantly lower in foal monocyte-derived macrophages compared with adult horses, which could partially explain the unique susceptibility of foals to R. equi infection. Most interestingly, infection of monocyte-derived macrophages induced down-regulation of CD-1b on the cell surface, which may represent a novel mechanism by R. equi to avoid detection and killing of infected cells by the immune system.

Whether macrophage age and lineage affect intracellular survival of R. equi and modulate the production of cytokines after infection has been investigated in vitro93. Intracellular survival of virulent R. equi in both monocyte-derived and bronchoalveolar macrophages from foals showed significantly greater bacterial replication in 3-month old foals than in 3-day old foals, 2-week old foals, 1-month old foals, and adult horses. Regardless of age, intracellular replication of R. equi was significantly greater in bronchoalveolar than in monocyte-derived macrophages. Expression of IL-4 mRNA was significantly higher in monocyte-derived macrophages whereas expression of

IL-6, IL-18 and TNF-α was significantly higher in bronchoalveolar macrophages. It is possible that the decreased killing capacity of macrophages in foals of 3 months of age may play a role in the timing of disease presentation. Furthermore, preferential intracellular survival of R. equi in bronchoalveolar macrophages may explain the pulmonary tropism of the pathogen.

Foal’s adaptive immune response to R. equi

Cell-mediated immunity

Since immunocompetent adult horses are immune to rhodococcal pneumonia, a better understanding of the mechanisms of immune clearance in these animals could help define the requirements for a protective immune response in foals. Upon intrabronchial challenge of adult horses with virulent R. equi, and subsequent follow up of local and peripheral blood responses, clearance of bacteria was associated with increased mononuclear cells (primarily lymphocytes) in the bronchoalveolar lavage fluid, and inversion of the normal macrophage:lymphocyte ratio94.

Furthermore, flow cytometric analysis of the bronchoalveolar lavage fluid demonstrated that clearance correlated with significant increases in pulmonary T-lymphocytes, including both CD4+ and CD8+ cells. These results are compatible with previous work performed in mice, which also showed that both CD4+ and CD8+ T-cells played a role in clearance of R. equi95. When adult horses were challenged with either virulent or avirulent R. equi (plasmid-cured derivative), clearance of the virulent strain was associated with increased numbers of pulmonary CD4+ and CD8+ lymphocytes producing IFN-γ, while the plasmid-cured strain was cleared in horses without a significant increase in IFN-γ producing T lymphocytes in the bronchoalveolar lavage fluid96.

Interestingly, there was no change in the IFN-γ-positive cells in the peripheral blood of horses that cleared virulent R. equi from the lung, suggesting that a local, type 1 recall response was sufficient to confer protection. Furthermore, the cytotoxic T lymphocytes (CTLs) via non-classical MHC molecules (CD-1b), have been demonstrated to contribute to the immune control of R. equi in adult horses 97.

In contrast to adult horses, infection of foals with virulent R. equi resulted in decreased local IFN-γ mRNA expression in lung CD4+ cells98. The inability of foals to respond to infection with adult-like T cell responses and with comparable IFN-γ production has been deemed responsible for the increased susceptibility of foals to intracellular pathogens such as R. equi51,52,99,100. On the other hand, foal innate immune cells, such as monocyte-derived dendritic cells, respond to virulent R. equi infection with increased IL-12 mRNA expression compared to

13 cells from adult horses.90 Foal PBMCs also showed a clear upregulation of IFN-γ mRNA, decreased

IL-4 gene expression and greater IFN-γ/IL-4 transcript ratios in response to R. equi infection compared with adult horses101. This showed that Th-cells of young foals can generally produce

IFN-γ but have a substantially reduced capacity to develop into IL-4 producing Th2-cells.

The effect of inoculum size on cell-mediated immune responses of foals challenged with

R. equi has been studied in foals challenged intrabronchially between 7-12 days of age, with either a 106 or 108 cfu inoculum. Proliferative response assay as well as quantification of mRNA cytokine expression (IL-2, IL-4, IL-10, and IFN-γ) after in vitro stimulation with soluble R. equi antigen were performed on mononuclear cells harvested from bronchial lymph nodes 15 days after infection. Concanavalin A and C. pseudotuberculosis were positive and negative controls.

Proliferative responses to positive and negative controls and R. equi antigen, as well as expression of mRNA for IL-2, IL-4, IL-10, and IFN-γ were not significantly different between the two groups.

However, there was a tendency towards a higher IFN-γ/IL-4 ratio in the lower inoculum group.

Therefore, a potential influence of the inoculum size over cell-mediated responses of R. equi infected foals should not be completely discarded.

CTL activity has been investigated in immunocompetent foals102. As suspected, CTL activity was shown to be deficient in foals younger than 3 weeks. This observation coincides with the period of time when foals are first exposed to a contaminated environment. At 6 weeks of age, some foals had developed CTL activity, although not as efficient as in adult horses. At 8 weeks of age, all foals showed significant R. equi-specific CTL activity. In addition, as in adults, killing of

R. equi targets was not MHCII restricted. These findings suggest that acquisition of CTL activity in association with development of protective immune responses may occur due to natural exposure and immunological maturation.

Humoral immunity

Since adult horses are immune and remain clinically normal upon challenge, understanding their humoral response to R. equi may help elucidate a protective humoral response. Lopez et al.

(2002), challenged 12 adult horses with virulent R. equi and subsequently evaluated serum levels of IgGa, IgGb, IgG(T), IgA and IgM against both whole R. equi and recombinant VapA protein.

While all horses remained clinically sound, levels of R. equi and VapA specific IgGa and IgGb were dramatically enhanced at 14 days post-challenge. In another instance, 6 healthy adult horses were challenged with R. equi and their humoral response evaluated81. An IgGa-dominant response was observed, as well as consistently higher IgGa-to-IgGb and IgGa-to-IgG(T) ratios when compared to pneumonic foals.

VapA specific IgG subisotype responses in healthy exposed foals, and foals with clinical

R. equi pneumonia have been compared81. Pneumonic foals produced an IgGb-dominant response to VapA, which was significantly greater than in healthy, exposed foals. Whereas the IgG subisotype response of pneumonic foals trended toward higher IgG(T) compared to clinically normal foals, IgGa was significantly higher in the clinically normal foals. Also, both IgGa-to-IgGb and IgGa-to-IgG(T) ratios tended to be higher in healthy, exposed foals. Since in mice and humans, the antibody isotype reflects the Th1- Th2 bias of the immune response, the authors concluded that a similar bias may occur in horses, and that IgGb and IgG(T) are products of a Th2 response, while

IgGa is a product of a Th1 response. Furthermore, it was suggested that foals which develop R. equi pneumonia have a Th2-biased, ineffective immune response whereas foals which become immune develop a Th1-biased immune response.

Changes over time in VapA specific IgG and IgG subisotypes have been further characterized in challenged, naturally infected and not infected foals103. Challenged foals were intratracheally infected with 103 cfu/foal of R. equi (206+ UKVDL) at 1, 2 or 3 weeks of age.

Another group of foals that was not challenged, but was housed in the same pasture as the challenged foals, and developed subclinical lung lesions with positive R. equi tissue culture were considered naturally infected. Foals that were not challenged, remained disease-free, and had negative lung tissue culture for R. equi were considered not infected. All foals were bled at birth and weekly thereafter for 8 weeks. Following birth, VapA-specific IgGs significantly decreased over time in all foals as a result of normal decay of passively transferred antibodies. Both VapA-

15 specific IgGa and IgG(T) significantly increased after experimental challenge, however, the rise in

IgG(T) occurred earlier. Only a significant increase in VapA-specific IgG(T) over time was seen after natural infection. However, due to the terminal nature of this challenge study, the IgG profile of foals that developed clinical pneumonia either after challenge or natural infection could not be determined. Furthermore, the small sample size of naturally infected foals (n = 6) precluded the determination of whether VapA-specific IgG(T) could be used to differentiate rhodococcal from other pneumonias under field conditions.

Most recently, the performance of VapA-specific ELISA for IgG and its subclasses IgGa,

IgGb and IgG(T) in the early diagnosis of pneumonia caused by R. equi was evaluated104. When used to identify infected foals, VapA-specific IgG, IgGa and IgGb had no diagnostic value. In contrast, IgG(T) had high sensitivity (90%) and specificity (96%) at a cut-off of 2.7 Elisa Units. A major limitation of this study was the lack of definitive diagnosis of naturally infected foals; hence, further investigation of this test in field conditions is needed.

The effect of inoculum size on humoral immune response of foals challenged with R. equi has also been studied105. Foals were challenged intrabronchially, between 7-12 days of age, with either 106 or 108 cfu inoculum. R. equi-specific IgM, IgGa, IgGb, IgGc, and IgG(T) concentrations in serum were determined before infection and on day 15 post-infection. The mean ratios of post- infection to pre-infection IgG(T) and IgM concentrations were significantly higher in foals infected with the greater inoculum, indicating that the inoculum size modulates the humoral response of infected foals.

Disease prevention strategies

Currently used strategies

Since rhodococcal pneumonia is an insidious disease, clinical signs may not be apparent until pathological changes are well progressed1,2. Therapy of all foals affected with subclinical disease is not warranted as a recent controlled clinical trial on an endemic farm demonstrated that many foals with subclinical pulmonary lesions recover without therapy106. Mass treatment of foals

16 with antibiotics could generate negative consequences for both equine and human medicine due to the development of anti-microbial resistance. Consequently, the rationale for screening is that detection foals in the early stages of disease will improve therapeutic outcomes. Currently, the most widely used methods for prevention of rhodococcal pneumonia rely on highly sensitive but poorly specific screening techniques for early detection of pneumonia, and on administration of HIP. The latter confers only partial protection, is expensive and may not readily available. Although screening for early identification of disease on farms with recurrent history of foals affected by R. equi is recommended by the American College of Veterinary Internal Medicine107 , evidence for the recommendation is relatively weak, with no supporting controlled studies.

A variety of screening techniques performed serially have been described, including visual inspection of foals, monitoring rectal temperatures, clinical signs of pneumonia or extrapulmonary disorders, hematological parameters, serology, and thoracic imaging using either radiography or ultrasonography, with empiric recommendation that screening begin around 3 weeks of age108.

Some tests appear to be ineffective for screening, such as serum concentrations of R. equi specific

IgG, serum amyloid A, or plasma fibrinogen109–112. White blood cell (WBC) counts performed at monthly intervals appeared to have clinically acceptable sensitivity and specificity for early detection of R. equi pneumonia at 1 farm112. Limitations of using WBC for screening are the time and effort required to collect blood samples, possible lag from time of submission, and the potential for false-positive results attributable to other infectious or inflammatory conditions that may be common among foals, or to stress-associated leukocytosis. Use of thoracic ultrasonography as a screening tool offers a number of advantages: it can be quickly learnt and performed, provides immediate results, and has a greater specificity for detection of pulmonary disease compared to

WBC. However, the main disadvantage of a highly sensitive screening program is an increase in apparent incidence of disease, leading to an increased number of foals treated for presumptive R. equi pneumonia. As the proportion of which foals might recover spontaneously remains unkown, and because R. equi infections can cause severe disease, many breeding farms elect to treat all foals with positive results of screening tests. This approach has had more than a significant negative

17 financial impact in the equine industry worldwide. Overtreatment of foals over the past decade has led to an alarming emergence of multi-drug resistant R. equi on different continents113,114, indicating immediate need for efficient disease prevention rather than reliance on treatment.

Administration of commercially available hyperimmune plasma (HIP) products containing antibodies against R. equi is heavily relied upon as a disease preventive measure. Despite the historical controversy regarding the efficacy of HIP, its use has generally proved effective in significantly reducing the severity of R. equi pneumonia in foals following experimental challenge115–119. However, studies evaluating the efficacy of various HIP preparations under field conditions have shown equivocal results120–122. Many factors may account for these disparate results such as differences in hyperimmunization protocol, study design, clinical outcome assessment, and timing of administration. Most recently, a lack of product uniformity among the different commercially available R. equi HIP products in the US was reported123. VapA specific IgGs were significantly different between products and varied between lots of the same product, with coefficients of variation ranging from 17 to 123%. These results may also explain previously reported disparities in HIP efficacy.

Neither the optimal amount, the number of doses, or the foal’s age at HIP administration time has been determined. Administration of HIP at 7 and 9 days after aerosol infection of foals with R. equi did not confer protection, suggesting that its administration prior to infection may be of importance124. Because of evidence that many foals become infected early in life, it is commonly recommended that foals receive transfusion of at least 1 liter of HIP within the first 24-48 hours after birth21,24,107. The rationale for administering a second dose of HIP at 2-4 weeks of age is based on the possibility that early administration of HIP may result in the decline of passively transferred antibodies to a non-protective level at a time foals are still susceptible to R. equi.

Vaccine

For decades, numerous attempts have been made to develop an effective vaccine for use in foals that is safe, immunogenic and efficacious. However, R. equi vaccine candidates based upon

18 the traditional vaccine platforms such as live125, killed126,127 and attenuated128 have not been successful. Modern molecular based vaccines, such as DNA129,130, subunit131 and genetically attenuated132,133R. equi have shown some potential, however, these vaccines have not conferred protection in mice. More recently, bacterial vector vaccines have shown positive results in murine models134–136, but are yet to be further developed and shown to be protective in foals.

The most difficult challenge yet to be overcome is the need to vaccinate foals at a very young age, with a vaccine that will benignly modulate their naïve, immunodeviant immune system generating an effective immunity within the first week of life.

Research Hypotheses

Despite all the efforts that have been directed towards a better understanding of the foal’s immune response to R. equi, much remains unknown. In an attempt to make a positive contribution not only to the existing research body, but also to impact the routine of those who deal with R. equi in the field, my research has focused on seeking improved diagnostic and preventive tools.

Therefore, the following hypotheses are proposed:

H1 – Specific anti-VapA IgG(T) is a marker of clinical disease in infected foals. (Chapter 2)

H2 – Cytokine expression profile in whole blood of progressor and regressor foals is significantly different. (Chapter 3)

H3 – Higher levels of passively acquired VapA antibodies delivered by HIP are associated with disease regression in naturally exposed foals. (Chapter 4)

H4 – Specific Rhodococcus equi pili antibodies passively acquired by foals via colostrum ingestion from mares previously immunized with a pili-based candidate vaccine confers protection against induced rhodococcal pneumonia.

CHAPTER 2

VAPA SPECIFIC IGG(T) WAS NOT A RELIABLE PREDICTOR OF CLINICAL

PNEUMONIA IN FOALS AFTER CHALLENGE OR NATURAL INFECTION WITH R. EQUI

Introduction

Rhodococcus equi, a gram-positive facultative intracellular pathogen, has worldwide importance as a cause of pneumonia in young foals107. There is no commercially available vaccine against rhodococcal pneumonia, and although controversial, intravenous administration of R. equi hyperimmune plasma (HIP) shortly after birth is commonly practiced in an attempt to prevent disease. Control of R. equi pulmonary infections currently relies on early detection of disease using thoracic ultrasonography and initiation of treatment with antimicrobial agents prior to the development of clinical signs. This approach is highly sensitive; however, it lacks specificity. In particular, it fails to differentiate infected foals in which disease will progress and become symptomatic (progressors) from the majority of infected foals in which lung lesions will regress and spontaneously resolve, as they remain free of symptoms of pneumonia (regressors)106. Use of a low ultrasonographic score threshold to guide therapeutic decision has led to overtreatment of foals, and most importantly, to multi-drug bacterial resistance worldwide affecting not only horses but also immune-compromised humans 137–139 .

While little is known about the factors that determine susceptibility of foals for development of rhodococcal disease, it is likely that protection involves opsonizing antibodies.

Specifically, immunoglobulin G (IgG) antibodies are thought to be important since entry of R. equi into macrophages mediated by Fcγ receptors leads to enhanced bacterial killing12,14,81,82.

Furthermore, an increase in serum IgG against R. equi is observed after natural exposure and oral or intra-tracheal inoculation103,140,141. However, not all IgG subisotypes are equally effective.

Although IgG(T) has been shown to be a potent complement activator in horses, a VapA specific

IgG(T) response was not seen after natural exposure of adult horses or healthy foals, and it has been

20 typically associated with a non-protective T helper 2 (Th2) immune responses in R. equi-infected foals81,83.

Recently, it has been demonstrated that an early response to experimental infection of neonatal foals using a low dose (103 cfu/foal), as well as to natural infection, is characterized by a significant increase in specific IgG(T) at 5-8 weeks of life103. Furthermore, when compared to IgG,

IgGa, and IgGb, IgG(T) was shown to be the best predictor of R. equi infection in either experimentally challenged or naturally infected foals104. Despite the low number of naturally infected foals in the first study, and the lack of confirmed diagnosis of the presumptively naturally infected foals included in the second study, these findings suggest that IgG(T) may be useful to differentiate rhodococcal from other causes of pneumonia affecting young foals. However, further research is needed to evaluate VapA specific IgG subisotypes as potential markers for the development of clinical disease.

In light of the challenge that identification of progressor foals currently presents, the main objective of this study was to investigate the use of IgG(T) to identify progressor foals among the

R. equi-infected population. Therefore, our hypotheses were three-fold: (1) serum concentrations of IgG(T) are significantly higher in progressor foals than in regressor foals, after challenge or natural infection; (2) IgG(T) is a reliable predictor of disease outcome in naturally infected foals; and (3) IgG(T) outperforms other IgG subisotypes in predicting clinical pneumonia in naturally infected foals.

Materials and Methods

R. equi challenge

Two low-dose challenge studies were performed in subsequent years (2016 and 2017), using modifications of the previously described infection model24. R. equi 103+ was provided by

Bioniche Animal Health. The bacteria were stored as a frozen stock in 20% glycerol and streaked onto a tryptic soy agar yeast extract (TSAYE) plates and incubated for 48 hours at 37°C. A single typical mucoid, creamy colony was selected from the plate, inoculated into tryptic soy yeast broth

(TSBYE) and incubated for another 48 hours at 37°C. Bacterial concentration was initially estimated based on optical density (OD) of the culture media using a spectrophotometer at 600nm wavelength and confirmed by serial dilution plating. All isolates were tested for the VapA-carrying plasmid using PCR at the UKVDL. The agar plates were held at room temperature for an additional

72 hours when colony morphology was evaluated. Culture medium was serially diluted using sterile

PBS to obtain a total concentration of bacteria of 104 cfu/ml. One ml of 104 cfu/ml was used to inoculate each foal. Inocula were kept refrigerated during transportation to the farm, and until challenge was performed. An additional inoculum sample that had been taken to the farm, was plated onto TSAYE plates once back in the laboratory, in order to confirm bacterial viability and concentration.

Foals were challenged at 3-7 days of age following confirmation of optimal passive transfer. Foals were deemed clinically healthy based on a normal complete blood cell (CBC) count, fibrinogen and thoracic ultrasonography. Prior to challenge, mare and foals were brought into stalls and the foals were sedated using a combination of valium (0.2 mg/kg) and butorphanol (0.05 mg/kg). A flexible tube was used to deliver bacteria to the mid trachea (intratracheal instillation).

Confirmation of R. equi pneumonia was obtained either by collecting post-mortem samples from lung lesions, or by performing in vivo trans-tracheal wash 4 weeks after challenge. Subsequent aerobic bacterial culture was performed at the UKVDL. Sample collection was discontinued if treatment for pneumonia was perceived as necessary based on observation of significant clinical signs of pneumonia.

Sera and HIP samples

Sera samples from challenged foals were obtained immediately before, and 2, 4, 6, and 8 weeks after challenge. None of the challenged foals received HIP. All sera were archived at -20°C for batch analysis.

Potentially naturally infected foals were located on R. equi-endemic farms in the central

Kentucky area. Foals were included in the study if they had adequate passive transfer and were deemed clinically healthy at 1 week of age based on history and physical examination. Systemic

22 disease or treatment for respiratory disease was used as exclusion criteria. All foals were born during the 2017 foaling season. Since all enrolled farms had a history of rhodococcal disease, routine administration of 1L of HIPa within 24 hours of birth was practiced. HIP samples were collected at administration time. Sera samples were also collected from each foal’s dam at the time of HIP administration. Sera samples from the foals were collected by the farm’s veterinarian at 7 days (timepoint 1), and at 1, 2 and 3 months of age (timepoints 2-4). Serum was also collected when a foal developed specific signs of pneumonia, and a diagnosis of R. equi pneumonia was made based on the presence of one or more specific clinical signs of pneumonia and detection of lesions by ultrasonography (See “Foal evaluation and classification”). All sera were archived at -

20C for batch analysis.

Foal evaluation and classification

After challenge, mares and foals returned to pasture and were evaluated for 8 weeks. Foals were observed daily for increased respiratory rate or effort, mucoid or purulent nasal discharge, lethargy and anorexia. Rectal temperature was recorded twice a day. Complete physical examination was performed twice a week in order to detect specific signs of pneumonia, including increased respiratory rate and effort, abnormal lung sounds on thoracic auscultation, presence of purulent nasal discharge, presence of spontaneous cough and presence of submandibular and/or retropharyngeal lymph node enlargement. In order to objectively assess respiratory disease severity, a scoring system was designed (Table 2.1). Bilateral lung ultrasonography was performed prior to, and biweekly after challenge using a 7.5MHz linear probe (CTS-7700V-SIUI, Universal

Medical System Inc., Bedford Hills, NY). In order to monitor disease progression, each lung was scored based on the sum of the maximum diameters of each lesion, and a total score was obtained by adding both lung scores. All physical and ultrasound examinations of challenged foals were performed by the author.

a MgBiologics, Ames, IO, USA.

Table 2. 1 Physical examination scoring system. F = Fahrenheit degrees; bpm = breaths per minute;

LNs = lymph nodes; *to be scored according to the severity of the abnormal lung sounds.

Potentially naturally infected foals were physically examined at 7 days of age. Thereafter, physical and lung ultrasonographic examinations were performed at 1, 2 and 3 months of age, or when specific clinical signs of pneumonia developed. While the same physical examination and lung ultrasonographic parameters and guidelines were used to evaluate challenged and naturally infected foals, four different veterinarians performed the examinations and assigned the respective scores to the naturally infected foals.

Experimentally and naturally infected foals were categorized as regressors based on the presence of ultrasonographic lesions compatible with rhodococcal pneumonia142, that spontaneously resolved over time, in the abscence of any specific clinical signs of pneumonia or medical treatment. Based on the presence of one or more specific clinical sign(s) of lower respiratory infection in addition to ultrasonographic lesions compatible with rhodococcal pneumonia, foals were categorized as progressors. For potentially naturally infected foals, if neither ultrasonographic lesions compatible with rhodococcal pneumonia or specific clinical sign(s) of lower respiratory infection were detected over the first 3 months of life, they were categorized as no respiratory abnormalities or NRA.

VapA Purification

Recombinant VapA protein was produced and purified using an Escherichia coli strain that contained a VapA plasmid fused to glutathione-S-transferase, as previously described82. The purity of the protein was assessed by sulfate-polyacrylamide gel electrophoresis (SDS-page)b.

Source of reference control sera

Positive and negative control sera were included on each ELISA plate. Serum from a mare previously vaccinated with killed VapA+ R. equi was used as positive control. Negative control serum included fetal equine serum (FES) and serum obtained from a 4-month old foal whose necropsy and lung tissue culture results were negative for R. equi. Commercially available R. equi specific hyperimmune plasmas were used to construct standard curves. The standard curves for

IgG, IgGa, and IgGb were constructed using one product (ReSolution)c while a different one

(EquiplasRea)d was used for IgG(T). All reference sera were divided into 100uL aliquots and stored at -20C until used.

VapA-specific enzyme-linked immunoabsorbent assay

The ELISA for IgG was based on previously described methods 81,104,140. Briefly, 96-well microplatese were coated with VapA (0.5ug/well) in carbonate bufferf and allowed to incubate overnight at 4C. Afterwards, a mixture of blocking buffer (polyvinyl alcohol [Mowiol 6-98] 1%

[w/v] in distilled water) and phosphate-buffered saline (PBS, to a final concentration of 1:1 [v/v]) was added. Serum was diluted (1:100) in PBS with 0.05 Tween-202 (PBST), added to duplicate wells and incubated at 37C for 1 hour. Either goat anti-horse IgG conjugated with horseradish peroxidase (HRPg; 1:10,000 dilution) or murine anti-IgGa (1:2 dilution, CVS48), anti-IgGb (1:5 dilution, CVS39), or anti-IgG(T) (1:2 dilution, CVS40; all hybridomas provided by P. Lunn, North

Carolina State University) were then added and incubated for 1 hour at 37C. The IgG subisotypes

b Bio-Rad, Philadelphia, PA, USA. c MgBiologics, Ames, IO, USA. d Plasvacc USA Inc, Templeton, CA, USA. e Thermo Scientific, Rochester, NY, USA. f Sigma Aldrich, St. Louis, MO, USA. g Jackson ImmunoResearch, West Grove, PA, USA.

25 plates were washed, and goat anti-mouse IgG-HRPh (1:2000 dilution) added for an additional hour at 37C. Plates were washed between each step with PBST using an ELISA plate washer (MW

96/384)i. The substrate (3,3’,5,5’-tetra-methylbenzidine, peroxidase substrate)j was then added for

5 minutes. The reaction was stopped using stop solutionk. Absorbance was read at 450 nm using an

ELISA plate readerl. Results were converted to ELISA units (EU) using a logarithmic trend line from the standard curve generated for IgG and each subisotype143. A coefficient of determination

(r2) of ≥ 0.90 for the standard curve was required for the results to be considered valid144.

Statistical Analysis

Data collected from the challenge studies was analyzed using commercial software

(SigmaPlot, SPSS, Chicago, IL, USA). Average physical and lung ultrasonography scores were compared between progressor and regressor groups at each timepoint using Student’s t-test.

Physical examination and lung ultrasonography scores from regressor and progressor foals were compared with respect to their IgG profiles expressed as ELISA Units (EU) using Pearson correlation coefficient. IgG profiles were compared overtime, and between progressor and regressor groups using two-way repeated measures ANOVA.

Data collected from naturally infected foals were analyzed using commercial softwares

(SigmaPlot, SPSS, Chicago, IL, USA). Maternal serum and HIP IgG profiles were compared among the three groups of foals using ANOVA on ranks. Average physical and lung ultrasonography scores were compared between progressor, regressor and NRA groups at each time-point using one-way ANOVA. Physical examination and lung ultrasonography scores from regressor, progressor, and NRA foals were compared to their IgG profile expressed as ELISA Units

(EU) using Pearson correlation coefficient. VapA IgG profile was compared overtime, and between regressor, progressor, and NRA groups using two-way repeated measures ANOVA.

h Bethyl Laboratories, Inc, Montgomery, TX 77356 USA i Beckman Coulter, Brea, CA, USA. j KPL, Gaithersburg, MD, USA. k KPL, Gaithersburg, MD, USA. l Bio-Rad, Philadelphia, PA, USA.

In order to evaluate the potential use of IgG(T) as a field diagnostic test, only samples collected under field conditions were used to evaluate the diagnostic performance of the different

IgG subisotypes and total IgG. Logistic regression and receiver operating characteristic (ROC) curves were used to assess the overall diagnostic performance of each IgG subisotype and total IgG at each timepoint, and as the sum of each IgG subisotype and total IgG produced over the first 3 months of life (time-points 2-4). In order to evaluate the diagnostic performance of each IgG subisotype and total IgG to identify progressor foals among the infected population, ROC curves were constructed using progressors and regressor foals at each timepoint, and also using the paired results from the first 3 months of life.

Significance was set at P<0.05.

Results

Challenged foals

Twenty-two and 14 foals challenged in the years of 2016 and 2017, respectively, were included. The median age at challenge was 4.6 days, and the inoculum per foal ranged from 12,600-

25,200 cfu. Bacterial isolates used to challenge the foals were VapA-positive and developed typical colonies on TSAYE plates, when plated either before or shortly after challenge. Ultrasonographic lesions consistent with R. equi pneumonia were observed in all foals after challenge. Time of ultrasonographic lung lesion appearance varied from 3 to 4 weeks after challenge. During the first challenge study 6 randomly selected foals were euthanized and necropsied (4 progressor and 2 regressor foals). Time of euthanasia varied from 4 to 7 weeks after challenge. R. equi pneumonia was confirmed in all 6 foals. During the second challenge study 2 foals were treated for rhodococcal pneumonia. Transtracheal wash was performed in all foals included in this study, and R. equi culture of the obtained fluid yielded positive growth in 26 (72%) foals. Only in one foal R. equi culture was accompanied of Streptococcus equi zooepidemicus. Non-pathogenic bacteria were isolated from the remaining 10 foals. Overall, 12 foals were classified as progressors, and 24 foals as regressors.

Average physical examination and lung ultrasonographic scores were significantly different between progressor and regressor foals (Figs. 2.1 and 2.2).

Figure 2. 1 Average physical examination scores of progressor and regressor foals before and after

R. equi challenge at physical examination times 1-16. * indicates statistical difference between groups.

Figure 2. 2 Average lung ultrasonographic scores of progressor and regressor foals before and after

R. equi challenge at physical examination times 1-16. * indicates statistical difference between groups.

Significant correlations were found between IgGa and physical examination (p = 0.0431) and lung ultrasonography (p = 0.0185) at four weeks after challenge, and also between total IgG and lung ultrasonography scores (p = 0.0121), and IgGb and lung ultrasonography scores (p =

0.0052) at six weeks after challenge (Table 2.2).

Table 2. 2 Pearson correlation coefficient between the IgG profile of progressor and regressor foals and their physical examination (PE score) and thoracic ultrasound (US score) scores. * indicates significant correlation between variables.

Progressor foals had significantly higher total IgG than before challenge (p = 0.007), and higher than regressor foals 8 weeks after challenge (p = 0.013) (Fig. 2.3). Progressor foals had significantly higher IgGa than before challenge at 6 (p = 0.004) and 8 (p = 0.003) weeks after challenge, and than regressor foals also at 6 (p = 0.003) and 8 (p = 0.004) weeks after challenge

(Fig. 2.4). Progressor foals had significantly higher IgGb than before challenge at 6 (p = 0.02) and

8 (p = 0.002) weeks, and to regressor foals also at 6 (p = 0.04) and 8 (p = 0.018) weeks after challenge (Fig. 2.5). Although progressor foals had increased IgG(T) at 6 weeks after challenge, no significant differences were detected between groups at any timepoint, or overtime within each group (p = 0.101) (Fig. 2.6). The IgG profile means and standard deviations for progressor and regressor foals at each timepoint are shown in Table 2.3.

Figure 2. 3 Total VapA specific IgG of progressor and regressor foals before (PC = pre-challenge), and 2 (2w), 4 (4w), 6 (6w), and 8 (8w) weeks after challenge. Bars represent sample standard deviation. *indicates statistical difference between groups; letter “a” indicates statistical difference between pre-challenge values within progressor group.

Figure 2. 4 VapA specific IgGa of progressor and regressor foals before (PC = pre-challenge), and

2 (2w), 4 (4w), 6 (6w), and 8 (8w) weeks after challenge. Bars represent sample standard deviation.

*indicates statistical difference between groups; letter “a” indicates statistical difference between pre-challenge values within progressor group.

Figure 2. 5 VapA Specific IgGb of progressor and regressor foals before (PC = pre-challenge), and

2 (2w), 4 (4w), 6 (6w), and 8 (8w) weeks after challenge. Bars represent sample standard deviation.

*indicates statistical difference between groups; letter “a” indicates statistical difference between pre-challenge values within progressor group.

Figure 2. 6 VapA specific IgG(T) of progressor and regressor foals before (PC = pre-challenge), and 2 (2w), 4 (4w), 6 (6w), and 8 (8w) weeks after challenge. Bars represent sample standard deviation.

Progressor Total IgG SD IgGa SD IgGb SD IgG(T) SD PC 1.07 0.71 1.45 0.52 2.37 0.99 1.31 0.11 2w 0.72 0.25 1.38 0.30 2.11 0.57 1.41 0.12 4w 2.19 2.29 20.44 28.84 3.73 3.16 7.26 7.61 6w 5.74 7.25 70.49 157.30 11.45 14.09 14087.45 44399.59 8w 8.46 8.33 87.12 154.59 15.67 14.02 711.19 1380.53 Regressor Total IgG SD IgGa SD IgGb SD IgG(T) SD PC 3.99 9.78 4.03 6.39 6.86 14.22 1.37 0.13 2w 1.74 2.42 2.28 2.14 3.55 3.83 1.38 0.10 4w 1.94 1.24 9.62 11.08 3.98 2.17 1726.51 5839.61 6w 2.65 1.86 11.15 10.01 5.54 3.83 321.86 905.07 8w 2.99 2.29 11.78 10.74 7.21 6.17 546.38 1740.01

Table 2. 3 Mean (± SD) VapA specific IgG profile of progressor and regressor foals before (PC) and 2 (2w), 4 (4w), 6 (6w) and 8 (8w) weeks after challenge.

Naturally infected foals

Ninety-eight sample sets consisting of foal serial sera, dam’s sera and HIP samples, were studied. Fourteen foals were classified as progressors, 6 as regressors, and 78 as NRAs.

No significant statistical differences were detected between IgG profiles from the dams of progressor, regressor and NRA foals. However, HIP received by progressor (p = 0.011) and regressor (p = 0.021) foals had significantly higher amounts of IgGa than the HIP administered to the NRA foals (Fig. 2.7). Similarly, the HIP received by progressor (p = 0.036) and regressor (p =

0.049) foals also had significantly higher amounts of IgGb than the HIP administered to the NRA foals (Fig. 2.8).

Figure 2. 7 VapA specific IgGa in HIP received by naturally exposed foals (progressor, regressor,

NRA). Bars represent sample standard deviation *indicates statistical difference in comparison to the NRA group.

Figure 2. 8 VapA specific IgGb in HIP received by naturally exposed foals (progressor, regressor,

NRA). Bars represent sample standard deviation *indicates statistical difference in comparison to the NRA group.

Average physical examination and thoracic ultrasonographic scores were significantly higher for progressor than NRA foals at timepoints 3 (p = 0.027) and 4 (p < 0.001). No significant correlations were found between the IgG profile of progressor and regressor foals and their physical examination and thoracic ultrasonographic scores (Table 2.4).

TP2 Total IgG IgGa IgGb IgG(T) All P and R PE Score -0.1315 -0.1737 0.0422 -0.0506 US Score -0.2618 -0.3163 0.3283 0.0911 Progressors PE Score -0.2691 -0.3730 -0.0872 -0.0832 US Score -0.4300 -0.3699 -0.3513 -0.0142 Regressors PE Score N/A N/A N/A N/A US Score -0.3429 -0.1985 -0.0692 -0.3028 TP3 Total IgG IgGa IgGb IgG(T) All P and R PE Score -0.1565 -0.0941 -0.2468 0.7061 US Score -0.0600 -0.0109 -0.1859 0.5484 Progressors PE Score -0.4005 -0.2760 -0.5853 0.7462 US Score -0.2045 -0.1542 -0.4406 0.5609 Regressors PE Score N/A N/A N/A N/A US Score -0.5349 0.1464 -0.4572 0.3316 TP4 Total IgG IgGa IgGb IgG(T) All P and R PE Score 0.6907 0.6031 0.7196 0.0538 US Score 0.1629 0.3201 0.3684 0.5926 Progressors PE Score 0.5498 0.5083 0.5210 0.0159 US Score -0.0447 0.2666 0.2468 0.6585 Regressors PE Score 0.0709 0.0857 -0.2409 -0.5610 US Score 0.9077 0.0818 0.4352 0.4509

Table 2. 4 Pearson correlation coefficient between VapA specific IgG profile of progressor and regressor foals and their physical examination (PE score) and thoracic ultrasound (US score) scores.

There were no significant total IgG differences between groups (Fig. 2.9). Progressor foals had significantly higher titers of IgGa in comparison to regressor foals at timepoint 2 (p = 0.015)

(Fig. 2.10). Progressor foals also had significantly higher titers of IgGb than regressor foals at timepoints 1 (p = 0.036), and significantly higher than regressor (p = 0.04) and NRA (p = 0.045) foals at timepoint 2. NRA foals had significantly higher titers of IgGb than regressor foals at timepoint 2 (p = 0.036) (Fig. 2.11). Finally, progressor foals had significantly higher titers of

IgG(T) than NRA foals at timepoint 3 (p <0.001) (Fig. 2.12). Differences in IgG profiles within each group of foals overtime are indicated on the figures (Figs. 2.9-12). The IgG profile means and

37 standard deviations for progressor, regressor and NRA foals at each timepoint are shown in Table

2.5.

Figure 2. 9 Total VapA specific IgG of progressor, regressor, and NRA foals over time (1,2,3 and

4 sample collection time points). Bars represent sample standard deviation. Letter “a” indicates statistical difference between pre-challenge values within NRA group.

Figure 2. 10 VapA specific IgGa of progressor, regressor, and NRA foals over time (1,2,3 and 4 sample collection time points). Bars represent sample standard deviation. *indicates statistical difference between progressor and regressor groups; letters “a” and “b” indicate statistical difference between pre-challenge values within NRA and progressor group, respectively.

Figure 2. 11 VapA specific IgGb of progressor, regressor, and NRA foals over time (1,2,3 and 4 sample collection time points). Bars represent sample standard deviation. *indicates statistical difference between progressor and regressor groups; letters “a” and “b” indicate statistical difference between pre-challenge values within NRA and progressor groups, respectively.

Figure 2. 12 VapA specific IgG(T) of progressor, regressor, and NRA foals over time (1,2,3 and 4 sample collection time points). Bars represent sample standard deviation. # indicates statistical

40 difference between progressor and NRA groups; letter “b” indicates statistical difference between pre-challenge values within progressor group.

Progressor Total IgG SD IgGa SD IgGb SD IgG(T) SD Time point 1 99.45 40.81 111.42 93.45 268.16 210.90 4.47 3.33 Time point 2 91.21 49.20 98.22 57.67 253.01 159.37 3.81 2.07 Time point 3 63.44 33.91 61.41 38.86 117.25 58.98 20.14 56.30 Time point 4 54.99 40.57 37.70 27.24 89.16 59.85 3.16 2.28 Regressor Total IgG SD IgGa SD IgGb SD IgG(T) SD Time point 1 65.92 58.66 66.05 42.00 132.80 81.29 5.11 3.50 Time point 2 98.75 150.12 28.78 22.56 78.82 54.50 3.70 3.64 Time point 3 34.94 30.14 33.42 14.98 51.06 32.79 12.28 19.62 Time point 4 22.78 15.14 22.12 16.82 29.64 16.10 2.48 1.28 NRA Total IgG SD IgGa SD IgGb SD IgG(T) SD Time point 1 96.34 51.84 74.66 41.37 200.43 107.09 4.25 3.05 Time point 2 80.17 56.03 77.92 59.50 191.28 146.55 4.41 4.79 Time point 3 60.54 47.77 49.30 62.03 101.97 80.06 4.96 9.44 Time point 4 38.75 41.32 28.47 27.59 69.51 75.49 4.31 6.74

Table 2. 5 Mean and standard deviation (SD) VapA specific IgG profile of progressor, regressor and NRA foals at timepoints 1-4.

Logistic regression indicated that IgGa and IgGb at timepoint 2, and also the sum of IgGa and IgGb produced over the first 3 months, were predictors of outcome (progressor versus regressor foals) (Table 2.6), as illustrated by the respective ROC curves (Figs. 2.13-15). Both total IgG and

IgG(T) were poor predictors of outcome at each timepoint, and as the sum of total IgG and IgG(T) produced over the first 3 months (Table 2.6).

Logistic regression of progressor vs regressor foals predicted by listed parameters Parameter Time point Estimate** P value AUC for ROC curve model Total IgG 1 0.00941 0.1786 0.7262 2 -0.00054 0.8751 0.3214 3 0.0175 0.1284 0.7455 4 0.0441 0.1011 0.1011 Paired (2-4) 0.00313 0.4278 0.69 IgGa 1 0.00714 0.2646 0.5952 2 0.0416 *0.0239 0.8929 3 0.027 0.1237 0.7636 4 0.0224 0.2376 0.7 Paired (2-4) 0.0159 *0.0496 0.82 IgGb 1 0.00594 0.1591 0.6905 2 0.0119 *0.0338 0.8929 3 0.0177 0.0532 0.8364 4 0.0539 0.0576 0.9 Paired (2-4) 0.00761 *0.043 0.89 IgG(T) 1 -0.0369 0.6834 0.5595 2 0.9359 0.9359 0.625 3 0.00286 0.7235 0.5273 4 0.1428 0.492 0.492 Paired (2-4) 0.00308 0.8156 0.45 **Analysis of maximum likelihood estimates

Table 2. 6 Logistic regression of progressor versus regressor foals predicted by each single and paired parameter. * indicates variable as significant outcome predictor.

ROC Curve for Model Area Under the Curve = 0.8929 13 1.00 18

0.75

i t

i 0.50

e S

0.25

0.00 Points labeled by observation number

0.00 0.25 0.50 0.75 1.00 1 - Specificity

Figure 2. 13 ROC curve analysis of VapA specific IgGa at timepoint 2 as a predictor of clinical disease (progressor) among R. equi infected foals (progressors and regressors). The area under the curve in indicated on the graph.

ROC Curve for Model Area Under the Curve = 0.8929 13 1.00 18

0.75

i t

i 0.50

e S

0.25

0.00 Points labeled by observation number

0.00 0.25 0.50 0.75 1.00 1 - Specificity

Figure 2. 14 ROC curve analysis of VapA specific IgGb at timepoint 2 as a predictor of clinical disease (progressor) among R. equi infected foals (progressors and regressors). The area under the curve in indicated on the graph.

Section 2 ROC Curves

1.0

0.8

0.6

Sensitivity 0.4

C-Igg tot, A = 0.69 0.2 C-IgGa, A = 0.82 C-Iggb, A = 0.89 C-Iggt, A = 0.45

0.0

0.0 0.2 0.4 0.6 0.8 1.0 1 - Specificity

Figure 2. 15 ROC curve analysis total VapA specific IgG and IgG subisotypes serially measured over the first 3 months of life as predictors of clinical disease (progressor) among R. equi infected foals (progressors and regressors). The areas under the curve (A) are indicated on the graph.

Discussion

Consistent with previous reports81,103, our results demonstrate that infected foals, either after challenge or natural infection, had an increased production of IgG(T) (Tables 2.3 and 2.5).

The peak of IgG(T) production in either group occurred within the same age range (6-8 weeks of age), suggesting that the experimental model was successful in mimicking the timing of natural infection. Although challenged and naturally infected progressor foals had higher IgG(T) titers compared to the respective regressor and NRA groups, these differences reached statistical significance only among the naturally infected foals, during the second month. Clinically, this result has no value since thoracic ultrasonography remains the most practical and sensitive tool to detect early infection. The decreasing IgG(T) titer observed after it peaked in challenged and naturally

45 infected progressor foals did not indicate a decrease in IgG(T) production, but was due to the loss of samples from euthanized and treated foals (Fig. 2.6).

When IgG(T) was further evaluated as a possible marker of progressor foals among the naturally infected population, the results indicated that it failed to predict the development of clinical disease (Table 2.6). Overlap in individual titers between progressor and regressor groups indicated the limited value of this assay for diagnostic purpose (Table 2.5). Therefore, IgG(T) was not a reliable predictor of clinical pneumonia in the naturally infected foals on any of the sampling times. In addition, diagnostic accuracy was not increased by testing serial serum samples collected over the first 3 months of life in the studied populations.

Furthermore, IgG(T) did not outperform the other IgG subisotypes in predicting clinical disease among infected foals (Fig. 2.15). Instead, IgGa and IgGb measured either during the first month of life or as serial serum samples, were the most reliable indicators of progressor foals in the studied population (Table 2.6 and Fig. 2.15). However, this finding may have been confounded by the passive immunity acquired via HIP, as it was not possible to eliminate its contribution to the titers. Despite encouraging results from the challenge study indicating that IgGa and IgGb may serve as clinical disease markers among infected foals, confirmatory field studies including foals that did not receive HIP are necessary before its use as a diagnostic tool is recommended.

Previous studies have shown lack of accuracy of serology in differentiating R. equi-affected from unaffected foals at time of clinical diagnosis109,110. The poor diagnostic performance of serological assays has been attributed to the widespread exposure of foals to R. equi at young ages leading to seroconversion without clinical disease. Most recently, IgG(T) has been suggested as a potential marker of infection by R. equi either after challenge or natural infection 103. Since unchallenged foals were not available, IgG(T) could not be evaluated as marker of infection after challenge. Nonetheless, if the challenged progressor and regressor foals were grouped together and treated as one group of infected foals, a number of differences in the humoral response after challenge were observed when compared to the previous report. Unlike our results, previously challenged foals showed a significant decrease in all IgGs at 2-4 weeks after birth that was

46 attributed to decay of passively acquired colostral antibodies. This discrepancy is likely a result of varying amounts of VapA specific antibodies carried by the dams in the different studies. A wide range of R. equi antibody titers in healthy adult horses has been described140. In contrast to our results, Sanz et al. showed that both IgGa and IgG(T) significantly increased after challenge, and only IgG(T) significantly increased after natural infection. In our challenged group of foals, there were no significant differences in the IgG profile over time in comparison to pre-challenge values, despite the increase of IgG titers after challenge (data not shown). The R. equi strain and dose used in the present study were different than previously reported145 which might explain the differences in the humoral response from that in the Sanz et al. study. Furthermore, a significant increase in

IgG(T) was not detected overtime in the naturally infected foals (data not shown) in contrast to what was reported by Sanz et al.. Although a definitive diagnosis was not obtained for naturally infected foals, the present study included a much larger number of sampled subjects under field conditions than previously reported.

A correlation between disease severity and IgG(T) production may still exist when advanced clinical cases are selected, explaining the previous association of IgG(T) with non- protective responses82. For purposes of this study, administration of treatment for pneumonia was a criterion for termination of sample collection, therefore collection of samples from affected foals at later stages in the disease process was not performed.

Although all naturally exposed foals received HIP of the same commercial brand, previous reports have documented a lack of homogeneity of commercial HIP146, which might have resulted in progressor and regressor foals receiving more IgGa and IgGb than NRA foals. This possibility reinforces a need for standardization of HIP products, as well as the importance of cell-mediated immune responses to achieve protective immunity.

As expected, both physical examination and lung ultrasonography scores were significantly higher for progressor than regressor foals. In addition, among the challenged foals, the significant positive correlations between total IgG, IgGa and IgGb with either score indicated that the paired variables increased together. This association is most likely due to the fact that foals

47 with clinical disease (progressor) produced the highest titers of total IgG, IgGa and IgGb at weeks

6 and 8 after challenge. The lack of significant correlations between IgG(T) and either scores emphasizes the dissociation of clinical parameters and IgG(T).

One marked difference between IgG profiles of challenged and potentially naturally infected foals was the overall trend of IgG antibody concentrations. While both populations were allowed to nurse ad libitum from their dams, only naturally infected foals received HIP, which explains the decreasing antibody titers over time as the exogenous antibodies decayed. Since the concentration of circulating IgGs was low in the dam’s sera (data not shown), increasing antibody titers over time observed in challenged foals was likely due to seroconversion in response to the inoculated R. equi.

This study had several important limitations, including the limited numbers of naturally infected foals that could be classified as progressor or regressor. Pressure to treat foals with small ultrasonographic lung lesions limited the number of field samples. Studies including larger sample populations of progressor and regressor foals under field conditions where there is less pressure to treat subclinically affected foals could yield data more likely to be statistically significant. Another limitation of this study was that a definitive diagnosis could not be obtained from naturally infected foals. Therefore, our results should be interpreted based on the high positive predictive value of ultrasonographically detecting characteristic lung lesions in foals from endemic farms107. Finally, although additional equine IgG subisotypes have been described79, we elected to use those subisotypes described in prior studies to allow for comparison. Additional work will be needed to assess all IgG subisotypes, once specific reagents become available.

In conclusion, titers of IgG(T) from progressor and regressor foals greatly overlap, regardless of infection mode. Consequently, IgG(T) was not a reliable predictor of disease outcome as a single sample measurement, or as a serial sample test over the first 3 months of life. These conclusions should be interpreted with caution taking all the study’s limitations into account.

CHAPTER 3

WHOLE BLOOD EXPRESSION OF T CELL MARKERS AND CYTOKINES OF

PROGRESSOR AND REGRESSOR FOALS CHALLENGED WITH RHODOCOCCUS EQUI

Introduction

While the majority of foals infected with Rhodococcus equi develop a protective immune response and are able to spontaneously recover without showing clinical signs of pneumonia

(regressors), others exhibit a unique susceptibility to infection which culminates in progressive, clinical disease (progressors)106,147. The determining reasons for either outcome are not completely understood which hinders development of a highly desirable diagnostic tool capable of differentiating between these two populations of infected foals.

After activation, T cell subsets express different cytokines and mediate distinct effector functions during immune responses. Th1 cells are characterized by interferon-gamma (IFN-γ) production and are required in defending against infection with many intracellular pathogens148.

Th2 cells produce interleukin-4 (IL-4) and interleukin-5 (IL-5), and promote development of antibody-mediated immune responses against extracellular bacteria and parasites149. Tregs express

IL-10, and have major functions in maintaining peripheral tolerance, immune regulation, and in limiting inflammation during autoimmune diseases150. In addition to distinctive cytokine secretion profiles, differential expression of T cell markers provides valuable insight about their diversity.

It is widely recognized that neonates of different species generally mount poor immune responses, particularly a helper T cell type 1 response (Th1) that is crucial for cell-mediated immunity76. Neonates often demonstrate an inappropriate bias to a helper T cell type 2 (Th2) response151. Without a proper Th1 cell-mediated immune response, neonates have an impaired ability to overcome viral and bacterial infections, putting them at risk for infectious diseases26. In neonatal and young foals, however, Th-cell responses remain debatable. Although several studies

49 have suggested that the Th-1 immune response of healthy newborn and young foals is deficient51,99,152, there is also evidence of the ability of CD4+ and CD8+ T cells to produce adult levels of interferon-gamma (IFN-γ) by day 5 after birth in response to in vitro stimulation with phorbol myristate acetate (PMA)77. Furthermore, in response to intra-bronchial challenge with virulent R. equi, newborn foals were able to mount an INF-γ response with decreased IL-4 expression similar to that of adult horses101,105,153.

Regulatory T cells (Tregs) also play a key role in balancing immune responses as they regulate immunity after birth and maintain immune tolerance150. Previous reports have demonstrated that Tregs are not only prevalent and functional in foals77 but are also able to express greater amounts of IL-10 than adult horses following stimulation with lipopolysaccharide (LPS)154.

Most recently, circulating T regs (CD4+CD25highFoxP3+) have been identified at significantly higher concentrations in young foals, and displayed higher suppressive capability than in yearlings and adult horses155. These findings support the notion that T-cell responses of healthy foals may be biased towards regulatory immune responses.

To date, neither the whole blood expression of INF-γ, IL-4, IL-5 and IL-10 or of the T cell markers for CD4+ (CD4 and MHCII), CD8+ (CD8 and Granzyme B), and Tregs (CD25 and

FoxP3) of progressor and regressor foals after challenge with R. equi has been evaluated.

Establishing a better understanding of the immune system of foals is crucial for elucidating the susceptibility of young foals to infectious diseases. Here, we characterize the whole blood expression of T cell markers and cytokines in foals after experimental infection and search for differences between progressor and regressor foals.

Materials and Methods

R. equi challenge

Two low-dose infection studies were performed (2016 and 2017), with modifications from the previously described infection model24. R. equi 103+ was provided by Bioniche Animal Health.

The bacteria were stored as a frozen stock in 20% glycerol and streaked onto a tryptic soy agar

50 yeast extract (TSAYE) plate and incubated for 48 hours at 37°C. To prepare the inoculum, a single typical mucoid, creamy colony was selected from the plate, inoculated into tryptic soy yeast broth

(TSBYE) and incubated for another 48 hours at 37°C. Bacterial concentrations were initially estimated based on optical density (OD) of the culture media using a spectrophotometer at 600nm wavelength and confirmed by serial dilution plating. All the final isolates were tested for the presence of VapA-carrying plasmid using PCR at the UKVDL. The agar plates were held at room temperature for additional 72 hours at which point colony morphology was evaluated. Culture medium was serially diluted using sterile PBS to obtain the total concentration of bacteria of 104 cfu/ml. One ml of 104 cfu/ml was used to inoculate each challenged foal. The bacterial suspensions were kept refrigerated during transportation to the farm, and until challenge was performed. An additional aliquot taken to the farm, was later plated onto TSAYE plates in the laboratory, in order to confirm bacterial viability and concentration.

Foals were challenged at 3-7 days of age if adequate passive transfer had been documented, were deemed clinically healthy based on a normal complete blood cell (CBC) count, fibrinogen and thoracic ultrasonography. Prior to challenge, mare and foals were brought into stalls and the foals were sedated using a combination of valium (0.2 mg/kg) and butorphanol (0.05 mg/kg). A flexible tube was used to deliver the bacteria to the mid trachea (intratracheal instillation).

Confirmation of R. equi infection was obtained from challenged foals either by collecting post-mortem samples from lung lesions, or attempted by performing an in vivo trans-tracheal wash

4 weeks after challenge. Subsequent aerobic bacterial culture was performed at the UKVDL.

Foal evaluation and classification

After challenge, mare and foal returned to pasture and were evaluated for the following 8 weeks. Foals were observed daily for increased respiratory rate or effort, mucoid or purulent nasal discharge, lethargy and anorexia. Rectal temperature was recorded twice a day. Complete physical examination was performed twice a week, in order to detect specific signs of pneumonia including: increased respiratory rate and effort, presence of purulent nasal discharge, presence of spontaneous cough and presence of submandibular and/or retropharyngeal lymph node enlargement. Bilateral

51 lung ultrasonography was performed on all challenged foals prior to, and biweekly after challenge.

A 7.5MHz linear probe (CTS-7700V-SIUI, Universal Medical System Inc., Bedford Hills, NY) was used for all examinations. All the physical and ultrasound examinations of challenged foals were performed by the same veterinarian (F. Cesar).

Challenged foals were categorized as regressors based on the presence of ultrasonographic lesions compatible with rhodococcal pneumonia142, that spontaneously resolved over time, without the detection of any specific clinical signs of pneumonia or medical intervention. Based on the presence of one or more specific clinical sign(s) of lower respiratory infection in addition to ultrasonographic lesions compatible with rhodococcal pneumonia, foals were categorized as progressors.

Sample collection

Venous blood samples were collected from foals at 2 and 6 weeks after R. equi challenge.

Approximately 3ml of blood were collected into Tempus Blood RNA Tubes (Applied Biosystems,

Foster City, CA – Catalog # 4342792) after which tubes were vigorously shaken for 10-20 seconds to ensure adequate cell lysis. All tubes were stored frozen at -20o C for batch analysis.

Gene expression analysis

Total RNA was extracted using the iPrep Total RNA Kit (Invitrogen, Carlsbad, CA-

Catalog # 59270) and the iPrep Purification Instrument (Invitrogen), per the manufacturer’s directions, with the exception that pelleted RNA was resuspended in 600-mL viral lysis buffer

(Invitrogen - Catalog # 12282500). Gene expression was determined using the relative quantitation method156 where averaged results from regressor foals at 2 weeks post-challenge were used as the calibrator for each gene. β-Glucuronidase (β-Gus) was used as the housekeeping gene for all samples51, and samples were assayed using commercially available primers and probes

(ThermoFisher Scientific, Waltham, MA): β-Gus (Ec03470630_m1), INF-γ (Ec03468606_m1),

IL-4 (Ec03468790_m1), IL-5 (Ec03468691_m1), IL-10 (Ec03468647_m1), CD4 (AI89L0G),

MHCII(Ec03468998_m1), CD8 (AIQJCBQ), CD25 (Ec03469221_m1), and FoxP3

(Ec04319948_m1). Relative quantity data were logarithmically transformed before performing the

52 statistical analysis. Amplification data from real time PCR with efficiency outside 90-110% were not included.

Statistical Analysis

All statistical analyses were carried out using SigmaPlot 13.0 (SPSS Inc., Chicago, IL).

Two-way analysis of variance (ANOVA) was used to detect significant differences in differential gene expression between timepoints and groups. Results were considered statistically significant if

P≤ .05.

Results

Challenged foals

Twenty-two and 13 foals were challenged in 2016 and 2017, respectively. The median age at challenge was 4.6 days, and the inoculum received per foal ranged from 12,600-25,200 cfu. All bacterial isolates were VapA-positive and produced typical colonies on TSAYE plates.

Ultrasonographic lesions consistent with R. equi pneumonia were observed in all foals after challenge. Ultrasonographic lung lesion appeared between 3 to 4 weeks after inoculation. During the first challenge experiment 6 randomly selected foals were euthanized and necropsied. Time of euthanasia varied from 4 to 7 weeks after challenge. R. equi pneumonia was confirmed in all 6 foals. Transtracheal washes were performed on all foals, of which 26 (72%) were positive for R. equi following culture. Streptococcus equi zooepidemicus was also isolated from one of these foals.

Non-pathogenic bacteria were isolated from the remaining 10 foals. Overall, 13 foals were classified as progressors, and 22 foals as regressors.

Gene Expression

While expression of INF-γ (p = 0.019) significantly increased, expression of IL-4 (p =

0.024), CD4 (p = 0.004), MHCII (p = 0.010), FoxP3 (p < 0.001), and CD25 (p < 0.001) significantly decreased over time in challenged foals (Fig. 3.1).

Figure 3. 1 Gene amplification measured by RT-qPCR denoted as logarithmic transformed relative measure of mRNA expressions (RQ) of IFN-γ , IL-4, CD4, MHCII, FoxP3, and CD25 at 2 (2W) and 6 weeks (6W) in all foals after challenge. Bar with asterisk denotes significant difference between timepoints.

When foals were grouped into progressors and regressors, INF-γ expression significantly increased overtime in regressor foals (p = 0.015) (Fig. 2). Expression of CD4 significantly decreased overtime in progressor foals (p = 0.01) (Fig. 2). Expression of MHCII significantly decreased over time in regressor foals (p = 0.012) (Fig. 2). Expression of FoxP3 and CD25 significantly decreased over time in progressor (p < 0.001) and regressor (p < 0.001) foals (Fig.

3.2). No significant differences in expression of any cytokine or cell marker were detected between progressor and regressor at any time.

Figure 3. 2 Gene amplification measured by RT-qPCR denoted as logarithmic transformed relative measure of mRNA expressions (RQ) of IFN-γ , CD4, MHCII, FoxP3, and CD25 at 2 (2W) and 6 weeks (6W) in regressor (R) and progressor (P) foals after challenge. Letters “a” and “b” denote significant difference between timepoints within regressor and progressor groups, respectively.

Discussion

Our results support previous reports demonstrating a predominant induction of INF-γ expression and simultaneous downregulation of IL-4 following R. equi challenge of young foals101 or in vitro stimulation of their PBMCs with PMA77. When challenged foals were grouped as progressors and regressors, a significant increase in differential expression of IFN-γ was observed only among regressor foals. Since Th1 cells are characterized by IFN-γ production and are required in defending against infection with many intracellular pathogens148, this response may indicate that regressor foals produced a stronger Th1 response after challenge in comparison to progressor foals, and successfully controlled the infection. However, since the samples consisted of a pool of mRNA from whole blood and not from a specific cell population, the source of the

INF-γ is unknown. Nonetheless, a significant decrease in differential expression of CD4 was observed among progressor foals, reinforcing the idea that CD4+, Th1 cells played a role in the production of INF-γ and disease resolution in regressor foals.

The overall increase in INF- γ expression with concomitant CD4 and MHCII downregulation over time, may indicate that CD8+ T lymphocytes also played a role in producing

INF-γ in infected foals. By using a flow cytometric method for intracytoplasmic detection of INF-

γ in cell of bronchoalveolar lavage fluid (BALF) from adult horses after challenge, clearance of virulent R. equi was shown to be associated with increased numbers of pulmonary CD8+ T lymphocytes producing IFN-γ 96. However, no change was observed in IFN-γ-positive cells in peripheral blood, implicating a local type 1 recall response in adult horses 96. An accelerated development of R. equi specific cytotoxic T Lymphocytes (CTL) has been reported after oral inoculation of 3-week old foals with virulent R. equi157. Further, adult-like PBMC expression of

IFN-γ mRNA by foals stimulated with R. equi lipid antigen or live bacteria has also been reported

157. At this time, additional sources of IFN-γ mRNA expression by cells of the innate immune system cannot be discounted, including γδ T-lymphocytes, NKT cells, or NK cells49.

Downregulation of the genes (CD4, MHCII, FoxP3 and CD25) in whole blood over time

57 can be explained by either absolute or relative decrease in their expression. Decreased relative expression of CD4 and MHCII can be reasoned based on previous reports of decreased CD4+ and increased CD8+ cell proportions in peripheral blood of naturally infected foals88. Similarly, a relative decrease in FoxP3 and CD25 expression would be expected with aging of the foals as the proportion of Tregs in circulation decreases155. The fact that the expression of both FoxP3 and

CD25 were significantly lower over time among progressor and regressor foals further supports the idea that age drove this response.

Ultimately, there were no significant differences in the cytokine and cell marker expression profiles of regressor and progressor foals, precluding their use as diagnostic markers for disease outcome. However, many possibilities remain to be explored in an attempt to identify potential mRNA expression patterns that differ among progressor and regressor foals. Cytokine expression profiles of specific cell populations, such as peripheral blood mononuclear cells, should be evaluated and compared between these 2 populations of foals.

CHAPTER 4

THE IMPACT OF VAPA IGGS FROM RHODOCOCCUS EQUI HYPERIMMUNE PLASMA

ON DISEASE OUTCOME AFTER NATURAL EXPOSURE

Introduction

Rhodococcus equi, a gram-positive facultative intracellular pathogen, is the most common cause of pneumonia in foals between 3 weeks and 5 months of age147. It causes considerable economic losses at horse-breeding farms at which infection is endemic. Rhodococcal pneumonia in foals is an insidious disease and early diagnosis is not easy. Moreover, despite numerous attempts over the past decades to develop a vaccine for use in foals, no vaccine candidate has been shown to be effective. In addition, mass treatment of foals with antibiotics may incur serious long-term effects such as the induction of multi-drug bacterial resistance, a concern not only for the equine industry but also for human medicine, and therefore113,114, is strongly discouraged. Thus the importance of disease preventive measures aimed to minimize the financial and welfare impacts of

R. equi on the equine industry cannot be overstated.

Many farms with enzootic rhodococcal pneumonia rely on the use of R. equi specific hyperimmune plasma (HIP) as a prophylactic measure. Although the efficacy, protective component, or minimum effective dose of R. equi HIP are yet to be established82,121,122, a great number of in vitro, field and challenge study results have supported the protective role of antibodies and HIP against R. equi infection14,115–121,146,158–160. HIP administration to foals is usually performed within 24-48 hours after birth, as it has been shown that foals that received HIP later in life were not protected124. It may also involve the administration of a second dose 4-8 weeks after the first dose160, at a time when the foal’s systemic antibodies reach a nadir25.

Although R. equi is an ubiquitous soil saprophytic bacterium, virulence requires the presence of plasmid that expresseses virulence-associated protein A (VapA)161. VapA antibodies are thought to increase opsonization of R. equi and its killing by phagocytes14,82. Indeed, in vitro opsonization of R. equi with immune serum enhanced phagolysossomal fusion and bacterial killing

59 by macrophages14. Likewise, opsonization of R. equi with specific antibodies from commercially available HIP promoted phagocytic function of macrophages and neutrophils, increased their oxidative burst activity and TNF-alpha production158. Therefore, opsonizing antibodies could be important in conferring early protection to young foals, while their cell-mediated immunity matures.

Marked variation in the VapA IgG profile of 4 commercially available R. equi HIP in the

USA has been reported146. The VapA IgG profile was significantly different between products and within three different HIP lots of each product, with coefficients of variation ranging from 17 to

123%. The wide HIP variation in IgG profile has raised concern among veterinarians, farm owners and managers. Further evaluation of the influence of VapA specific IgG profile of HIP profile on incidence of rhodococcal pneumonia of naturally exposed foals is warranted.

The purposes of this study were (1) to determine variation in the IgG profile between different units and lots of on HIP product commercially available in the USA, and (2) to identify associations between IgG titers in HIP and subsequent development of rhodococcal pneumonia in foals on an endemic farm located in central KY, USA.

Material and Methods

Sera and HIP samples

Samples were collected in 2015 and 2016 from all foals born on a Rhodococcus-endemic breeding farm in central Kentucky. All foals born received 1L of hyperimmune plasma m intravenously within the first 24 hours of life. Foals were excluded from the study if failure of passive transfer or systemic illness was detected. Sera were collected at 36-48 hours after administration of HIP from all foals and also from their dams at the time of HIP administration. A

3ml sample of HIP was also collected and kept refrigerated. All samples were stored at -20o Celsius for batch analysis.

m MgBiologics, Ames, IO, USA.

VapA Purification

Source of reference control sera

Positive and negative control sera were included in each ELISA plate. Serum from a mare previously vaccinated with killed VapA+ R. equi was used as the positive control. Negative controls included fetal equine serum (FES) and serum obtained from a 4-month old foal whose necropsy and lung culture results were negative for R. equi. Commercially available R. equi specific hyperimmune plasmas were used to construct standard curves. The standard curves for IgG, IgGa, and IgGb were constructed using one product (ReSolution)o while a different one (EquiplasRea)p was used for IgG(T). All reference sera were divided into 100uL aliquots and stored at -20o Celsius until used.

VapA-specific enzyme-linked immunoabsorbent assay

The ELISA for VapA-specific IgG was based on previously described methods 81,104,140.

Briefly, 96-well microplatesq were coated with VapA (0.5ug/well) in carbonate bufferr and allowed to incubate overnight at 4C. Afterwards, a mixture of blocking buffer (polyvinyl alcohol [Mowiol

6-98] 1% [w/v] in distilled water) and phosphate-buffered saline (PBS, to a final concentration of

1:1 [v/v]) was added. Serum was diluted (1:100) in PBS with 0.05 Tween-202 (PBST), added to duplicate wells and incubated at 37C for 1 hour. Goat anti-horse IgG conjugated with horseradish peroxidase (HRPs; 1:10,000 dilution) or murine anti-IgGa (1:2 dilution, CVS48), anti-IgGb (1:5 dilution, CVS39), or anti-IgG(T) (1:2 dilution, CVS40; all hybridomas provided by P. Lunn, North

Carolina State University) were then added and incubated for 1 hour at 37C. The plates were then

n Bio-Rad, Philadelphia, PA, USA. o MgBiologics, Ames, IO, USA. p Plasvacc USA Inc, Templeton, CA, USA. q Thermo Scientific, Rochester, NY, USA. r Sigma Aldrich, St. Louis, MO, USA. s Jackson ImmunoResearch, West Grove, PA, USA.

61 washed, and goat anti-mouse IgG-HRPi (1:2000 dilution) added for an additional hour at 37oC.

Plates were washed between each step with PBST using an ELISA plate washer (MW 96/384)t.

Substrate (3,3’,5,5’-tetra-methylbenzidine, peroxidase substrate)u was then added for 5 minutes.

The reaction was stopped using stop solutionv. Absorbance was read at 450 nm using an ELISA plate reader w . Results from the sera samples were converted to ELISA units (EU) using a logarithmic trend line from the standard curve generated for total IgG and each subisotype143. A coefficient of determination (r2) of ≥ 0.90 for the standard curve was required for the results to be considered valid144.

Foal evaluation and classification

Foals were observed daily by trained farm staff for increased respiratory rate or effort, mucoid or purulent nasal discharge, lethargy and anorexia. The farm veterinarian performed a complete physical examination at the time of sample collection, and at lung ultrasonography examination. All foals were screened with lung ultrasonography by the farm veterinarian at 30. In cases where ultrasonographic lung lesions compatible with R. equi142 were identified, serial lung ultrasonographic examinations (once a week) were performed for as long as the lung lesions were visualized. Foals negative for lung lesions at 30 days, and which remained clinically normal thereafter, were not subsequently subjected to additional ultrasonography. All physical and ultrasonographic findings were recorded and stored at the farm as medical records.

Each year, upon completion of sample collection, medical records were reviewed for the presence of specific signs of pneumonia including increased respiratory rate and effort, abnormal lung auscultation, purulent nasal discharge, spontaneous cough, tracheal rattle, submandibular and/or retropharyngeal lymph node enlargement, and lung lesions detected by ultrasonography.

Foals were categorized as regressors based on the presence of ultrasonographic lesions indicative of rhodococcal pneumonia, which spontaneously resolved over time, without detection of clinical

t Bethyl Laboratories, Inc, Montgomery, TX 77356 USA u Beckman Coulter, Brea, CA, USA. v KPL, Gaithersburg, MD, USA. w Bio-Rad, Philadelphia, PA, USA.

62 signs of pneumonia or medical treatment. Based on the presence of one or more clinical sign(s) of lower respiratory infection in addition to ultrasonographic detection of lung lesions indicative of rhodococcal pneumonia, foals were categorized as progressors. Foals lacking ultrasonographic lesions indicative of rhodococcal pneumonia or specific clinical sign(s) of lower respiratory infection over the first 4 months of life, were categorized as no respiratory abnormalities (NRA).

Statistical Analysis

Data were analyzed using a commercial software (SigmaPlot, SPSS Inc., Chicago, IL,

USA). Variation of IgG titers, expressed as ELISA units (EU), among all HIP units and lots were assessed using coefficients of variation (CV%). Differences in IgG titers between HIP lots were assessed using Kruskal-Wallis one-way analysis of variance on ranks. Differences in IgG titers of the dam’s serum, the foal’s serum and the HIP samples grouped as progressors, regressors and

NRAs were assessed using Kruskal-Wallis one-way analysis of variance on ranks. Multiple comparisons were performed using Dunn’s method. Shapiro-Wilk test was used to assess for data normality. Significance was set at p<0.05.

Results

Fifty-five and 79 foals were initially enrolled in 2015 and 2016, respectively. However, only 122 foals had complete records and were further considered for data analysis. Overall, 30 foals were categorized as regressors, 13 as progressors, and 79 as NRAs. Twenty different HIP lots of the same product were used in the study.

Coefficients of variation determined for each IgG subisotype among all HIP samples and different lots are shown in Table 4.1.

Coefficient of Variation (%) Lot# IGG total IgGa IgGb IgG(T) 1 49.39 51.74 54.50 32.79 2 45.02 57.73 32.89 28.15 3 58.52 63.38 55.71 39.15 4 57.81 65.87 59.07 58.03 5 N/A N/A N/A N/A 6 39.18 55.54 46.33 47.53 7 60.43 61.40 59.22 58.09 8 70.83 91.88 79.37 31.83 9 51.96 56.79 71.16 52.33 10 N/A N/A N/A N/A 11 N/A N/A N/A N/A 12 0.44 0.24 0.35 0.34 13 0.52 0.47 0.40 2.48 14 0.38 0.25 0.27 0.26 15 0.56 0.62 0.33 0.09 16 0.51 0.46 0.44 0.71 17 0.28 0.22 0.30 0.51 18 0.21 0.46 0.56 0.10 19 0.48 0.27 0.29 0.23 20 0.38 0.41 0.30 0.51 Overall 0.53 0.85 0.75 3.84

Table 4. 1 Coefficients of variation (%) determined for VapA specific IgG subisotypes for all HIP samples (Overall) and different lots. N/A = coefficient of variation could not be calculated because there was only a single sample within the lot #.

There were significant differences in the median values of the total IgG (p = 0.025), IgGa

(p <0.001), IgGb (p <0.001), and IgG(T) (p <0.001), between HIP lot numbers (Figs. 4.1-4).

Figure 4. 1 Total VapA specific IgG concentration in 20 lots of HIP. The overall difference was significant (p=0.025).

Figure 4. 2 VapA specific IgGa concentration in 20 lots of HIP. The overall difference was significant (p<0.001).

Figure 4. 3 VapA specific IgGb concentration in 20 lots of HIP HIP. The overall difference was significant (p<0.001).

Figure 4. 4 VapA specific IgG(T) concentration in 20 lots of HIP. The overall difference was significant (p<0.001).

There were no significant differences in the dam’s, HIP, or the foal’s VapA specific IgGa,

IgGb, or IgG(T) between progressor, regressor and NRA foals. Total IgG was significantly different only in the HIP received by progressor, regressor and NRA foals. Regressor foals received

HIP with significantly higher concentrations of total IgG than progressor (p=0.018), and NRA

(p=0.026) foals (Fig. 4.5). When only progressor and regressor foals were compared, regressor foals received HIP with significantly higher concentrations of total IgG than progressor foals

(p=0.012). (Fig. 4.6)

Figure 4. 5 Total VapA specific IgG concentration in the HIP received by NRA, regressor and progressor foals. Asterisk denotes significant difference between the regressor and the other groups.

Figure 4. 6 Total VapA specific IgG concentration in the HIP received by regressor and progressor foals. Asterisk denotes significant difference between groups.

Discussion

Consistent with previous reports146, we demonstrated that IgG varies greatly within and between different lots of the same HIP product. Indeed, our results show an even greater inter-lot variation (53 to 384%) than what was previously reported for the same product (17 to 123%).

Although the amount of total IgG received by NRA and infected foals, i.e., progressor and regressor foals together, in HIP was not significantly different (data not shown), regressor foals received HIP with significantly greater amounts of total IgG than progressor foals (Fig. 4.6). This result suggests that marked variation in total IgG content may have had an impact on the clinical outcome of naturally exposed foals.

Our results support a role of IgGs in alleviating severity of rhodococcal pneumonia since foals receiving more IgG were less severely affected. As demonstrated in vitro, opsonizing IgGs promote R. equi phagocytosis and killing162,163. VapA antibodies, in particular, have been shown to

68 be important in protecting adult horses164, foals165 and mice82 against rhodococcal disease. In 2016, a low-dose challenge study designed to closely mimic natural exposure, evaluated HIP efficacy and showed that treated foals were less severely affected with rhodococcal pneumonia118.

Although regressor foals received significantly higher amounts of IgG than progressor foals, the present observational study does not prove a cause-effect relationship between these two variables. The possibility that R. equi-specific antibodies are not solely responsible for the observed results cannot be discounted. Other protective constituents of plasma might include fibronectin, interferon, cytokines, complement factors, and other proteins, several of which might aid in immunologic defenses166. Nevertheless, a recent study showed that foals given HIP or purified equine immunoglobulin specific for VapA and VapC had less severe pulmonary lesions and fewer

R. equi in lungs than controls given saline82. Furthermore, disease outcomes observed in this study were likely influenced by individual cell-mediated mechanisms.

The present observational study had inherent limitations that need to be taken into account when evaluating the results. All foals born at the farm received HIP, therefore no untreated foals were available to serve as controls. Consequently, the HIP efficacy in conferring protection against disease could not be determined. Also, the invasiveness, risks, costs and time associated with sterile trans-tracheal washes, precluded the use of this technique for definitive diagnosis of rhodococcal disease. Therefore, results should be interpreted based on the high positive predictive value of ultrasonographic detection of the characteristic R. equi lung lesions in foals from endemic farms107.

Finally, some foals ultimately classified as NRAs may have had subclinical disease with undetected lung lesions, since only one lung ultrasonographic examination was performed at 30 days of age.

In conclusion, a great lack of uniformity in IgG content within and between different lots of a HIP product was detected. Furthermore, it is probable that this variation might have had an impact on disease outcome, as regressor foals received significantly greater amounts of IgG.

CHAPTER 5

R. EQUI PILI-BASED VACCINE CANDIDATE FAILED TO CONFER PROTECTION TO

FOALS AGAINST CHALLENGE

Introduction

Rhodococcus equi, a facultative intracellular and soil saprophytic bacteria, has worldwide distribution, and is considered one of the most important pathogens of early life in horses1. Alveolar macrophages are the primary survival and replication niche for R. equi18. In infected foals, a life- threatening pyogranulomatous pneumonia develops within the first months of life2. The reasons for the unique susceptibility of young foals to this infection are not completely understood. In addition to the absence of immunological memory, neonates of most species have diminished innate immune responses, decreased antigen presenting cell function, and are less able to mount type-1 immune responses51,167. Indeed, epidemiological evidence from field and challenge studies indicates that foals are most susceptible to rhodococcal infections shortly after birth21–24. Many endemically infected farms experience morbidity of 70% or greater, and case fatalities ranging from

13% to 40% from R. equi pneumonia3–6. This disease has a major negative financial impact in the equine industry due to the ubiquitous pathogen distribution, high cumulative disease incidence in affected farms, and costly preventive and treatment measures.

Current methods for control and prevention rely on highly sensitive but poorly specific screening techniques for early detection of pneumonia, and on administration of R. equi hyper immune plasma, which may only confer partial protection, and is cost-prohibitive in many occasions. Standard-of-care treatment of affected foals with antimicrobials consists of a combination of macrolide and rifampin, which may last for months, has important potential adverse effects, and is not consistently successful107. Furthermore, overtreatment of foals over the past decade has led to an alarming emergence of multi-drug resistant R. equi on different continents113,114, urging for efficient disease prevention rather than reliance on treatment.

Numerous attempts have been made to develop an effective vaccine for use in foals that is safe, immunogenic and efficacious. However, candidates based upon the traditional vaccine platforms such as live125, killed126,127 and attenuated128 have not been successful. Molecular based vaccines, such as DNA129,130, subunit131 and genetically attenuated132,133R. equi with theoretical potential have not conferred protection in mice. More recently, bacterial vector vaccines have shown positive results in murine models134–136, but not yet in foals.

Notwithstanding failed attempts to actively immunize foals, evidence of a role of antibody in partial protection against R. equi infection14,82,123,165 suggests that vaccination of mares and subsequent passive transfer of immunity to foals via colostrum could confer some degree of protection. Initial unsuccessful attempts to prevent pneumonia by immunization of dams with inactivated or killed R. equi 120,168,169, may be explained by failure to include important virulence factors. Later research has established the role of the virulence-associated plasmid (pVAP) and its encoded factors as a critical determinant of R. equi pathogenicity 170–173. Although the mode of action of the Vap proteins in the host-pathogen interaction remains unkown, vaccination strategies involving passively acquired Vap-A immunoglobulins by foals via colostrum have been reported as encouragingly effective160,163.

Sequencing of the R. equi 103+ genome has revealed potential virulence determinants, including the horizontal gene transfer island encoding cytoadhesive Flp type IVb subfamily pili16.

The R. equi pili (Rpl) form long, rigid appendages, usually 2-5 per bacterial cell16, and mediate bacterial attachment to alveolar macrophages, a feature essential for lung colonization in mice9.

Vaccination of mice with a synthetic Rpl peptide elicited full protection against experimental challenge (Jose Vazquez-Boland, personal communication). Unpublished in vitro studies have indicated that Rpl antibodies enhance alveolar macrophage bacterial uptake and killing (Jose

Vazquez-Boland, personal communication). Furthermore, Rpl has been identified as immunogenic

R. equi antigen, based on the detection of Rpl antibodies in foals with clinical rhodoccocal disease

(Jose Vazquez-Boland, personal communication).

In this context, development of an effective vaccine would have immediate welfare and financial benefits worldwide. With this purpose, we hypothesized that Rpl antibodies passively acquired via colostrum of dams immunized with the Rpl antigen would protect foals against experimental infection of foals with Rhodococcus equi 103+.

Materials and Methods

Study Design

Twenty-seven healthy, pregnant mares were included in this study. Approximately 2 months prior to predicted foaling date, individual baseline serum Rpl antibody titers were determined by Rpl ELISA. Mares were initially grouped based on their predicted foaling week.

Within each group, mares with the lowest Rpl antibody titers were assigned into the non-vaccinated or control group, while mares with the highest Rpl antibody titers were assigned into the vaccinated or treated group.

Based on preliminary data (not shown), each mare was vaccinated with two consecutive doses, given 6 and 4 weeks prior to predicted foaling date. Sterile saline (1 mL) was administered to the non-vaccinated group at the same time. Injection sites were monitored daily for local reactions for duration of the study. Serum samples were collected at 4 and 6 weeks post- vaccination.

Starting 10 days prior to predicted foaling date, each mare was checked twice daily for udder development and presence of colostrum. When colostrum was detected, 3-5 ml sample were collected and stored at -20oC. Only colostrum collected nearest to the approximate parturition time were assayed for Rpl antibody.

All foals were born unattended and had free-access to colostrum. Within 48 hours post- partum, a physical examination, lung ultrasonography, complete blood work and an IgG SNAP

Test (IDEXX, Pharmaceuticals Inc.) were performed. Foals were included in the study if deemed clinically healthy, and without any significant clinical or ultrasonographic abnormalities, failure of passive transfer (IgG < 800 mg/dL) or blood work abnormalities. All foals were challenged at 3-7

72 days of age, and evaluated for the presence and severity of pneumonia for the following 8 weeks.

Sera were collected immediately prior to challenge, as well as a sample of their bronchoalveolar lavage fluid (BALF) to determine its Rpl antibody concentrations.

Vaccine (provided by Jose Vazquez-Boland’s group)

A 19-mer peptide, corresponding to the C-terminus of the Rpl protein, fused to GST protein was expressed in Escherichia coli (Fig. 1). Recombinant protein was purified by affinity

chromatography. Antigen quality control included LPS quantification and culture to confirm sterility. Each vaccine dose included 300 μg of purified Rpl, as well as 120 μl of aqueous polymeric adjuvant with a profile similar to aluminum salt (MontanideTM Pet Gel, SEPPIC, France), in a final volume

of 1 ml. Vaccine injections were administered intramuscularly, on alternating sides of the neck.

Figure 5. 1 Overview of Rpl subunit recombinant vaccine production.

Rpl ELISA – serum, colostrum and BALF samples (performed by Jose Vazquez-Boland’s group)

An indirect enzyme-linked immunoassay (ELISA) was performed using streptavidin- coated plates and a synthetic biotin-labeled pili peptide. Standard curves, positive and negative control serum samples were included on each plate. Standard curves were constructed with serial dilutions of a positive control serum. Sera from adult horses vaccinated with Rpl were used as

73 positive controls. Sera from the same horses collected prior to vaccination were used as negative controls. Rabbit anti-horse HRP IgG (ab6921, ABCAM, USA) was used as the conjugated secondary antibody. After addition of the substrate, absorbance was read using an ELISA plate reader. Results from the test sera were converted to ELISA units (EU) utilizing a logarithmic trend line from the standard curve143. A minimum coefficient of determination (r2) of ≥ 0.90 for the standard curve was determined for the results to be considered valid.

Serum, colostrum and BALF samples were assayed at a 1:100, 1:2000, and 1:1 dilution, respectively. Colostrum from vaccinated mares, containing the lowest amounts of Rpl antibodies, were used as negative controls. Colostrum from vaccinated mares, containing the highest amounts of Rpl antibodies, were used as positive controls. Likewise, the BALF samples containing the lowest and highest amounts of Rpl antibodies were used as negative and positive controls, respectively.

R. equi challenge

A low-dose challenge study was performed with modifications of the previously described infection model24. R. equi 103+ was provided by Bioniche Animal Health. Bacteria were stored as a frozen stock in 20% glycerol and streaked onto a tryptic soy agar yeast extract (TSAYE) plate and incubated for 48 hours at 37°C. A single typical mucoid, creamy colony was selected from the plate, inoculated into tryptic soy yeast broth (TSBYE) and incubated for another 48 hours at 37°C.

Bacterial concentrations were initially estimated based on optical density (OD) at 600nm wavelength and confirmed by serial dilution plating. All isolates were tested for the presence of

VapA-carrying plasmid using PCR at the UKVDL. Agar plates were held at room temperature for an additional 72 hours at which time colony morphology was evaluated. Culture media were serially diluted using sterile PBS to obtain a total concentration of bacteria of 104 cfu/ml. One ml of 104 cfu/ml was used to inoculate each foal. The inocula were refrigerated during transportation to the farm, and until challenge was performed. An additional sample of inoculum taken to the farm, was later plated on TSAYE in the laboratory, to confirm bacterial viability and concentration.

Foal assessment

After challenge, mare and foal returned to pasture and were evaluated for the following 8 weeks. Foals were observed daily for increased respiratory rate or effort, mucoid or purulent nasal discharge, lethargy and anorexia. Rectal temperature was recorded twice a day. A complete physical examination was performed twice a week to detect signs of pneumonia including increased respiratory rate and effort, abnormal lung auscultation, purulent nasal discharge, spontaneous cough, and submandibular and/or retropharyngeal lymph node enlargement. In order to objectively assess respiratory disease severity, a scoring system was designed (Table 2.1).

Bilateral lung ultrasonography was performed on all foals prior to, and biweekly after challenge using a 7.5MHz linear probe (CTS-7700V-SIUI, Universal Medical System Inc.,

Bedford Hills, NY). Lungs were scored based on the sum of the maximum diameters of each lesion, and a total score was obtained by adding both lung scores. All physical and ultrasound examinations were performed by the author.

Total white blood cell counts and fibrinogen were obtained once a week. Sample collection was discontinued if treatment for rhodococcal pneumonia was perceived as necessary based on the development of significant clinical signs of pneumonia, accompanied or not by fever.

Confirmation of R. equi pneumonia was obtained either by performing an in vivo transtracheal wash 4 weeks after challenge, or by post-mortem evaluation. Five randomly selected vaccinated foals and five age-matched non-vaccinated control foals were euthanized. All necropsies were performed by a board certified pathologist at the UKVDL, blinded to treatment.

The lungs of each animal were separated, and each lung lobe weighed. The percentage of lung

75 tissue showing pleural and parenchymal lesions consistent with disease due to R. equi (% of affected lung parenchyma) was determined as previously described175. Gross lung lesion scores were generated based on the percentage of pneumonic lung on the pleural surface (abscess scores) using digital images with a grid. Sterile samples of approximately 100 mg collected from 3 pre- determined sites within each lung (Acr, Dorso-medial, and DDB), right and left lung lymph nodes, as well as from a tracheal lymph node, were homogenized and serially diluted in phosphate buffer before plated on TSYEA (R. equi growth form lung homogenates). Lung tissue and tracheobronchial lymph nodes were also collected for bacteriological culture and histopathology.

Carcasses were evaluated for signs of extrapulmonary lesions.

Outcome evaluation

Vaccine efficacy was evaluated comparing disease incidence and disease severity between vaccinated and control groups. Disease incidence was determined based on the presence of characteristic lung lesions detected on ultrasonography. Disease severity was assessed based physical examination scores, lung ultrasonography scores, clinical pathology data, and post- mortem evaluations of the affected lungs.

Statistical Analysis

Concentrations of Rpl immunoglobulins in sera from vaccinated and control mares were compared overtime using two-way repeated measures analysis of variance (ANOVA). Colostrum,

BALF and sera, were evaluated using independent measures, t-test for differences in their concentrations of Rpl immunoglobulins between groups. Pearson’s correlation was used to test for association between concentration of Rpl IgG in sera from mares and foals, mare’s colostrum and foal’s BALF.

Differences in physical examination, ultrasonographic and abscess scores between foals of vaccinated and non-vaccinated mares were evaluated using the Holm-Sidak method. Differences in white blood cell count and fibrinogen concentrations over time and between groups were

76 evaluated using two-way repeated measures ANOVA. Percentages of affected lung parenchyma and R. equi posititve culture from lung homogenates (cfu counts) were compared between groups using independent measures, t-test.

For all analyses, data normality and equal variance were tested using Shapiro-Wilk and

Brown-Forsythe tests, respectively. Significance was set at P<0.05.

Results

Fourteen mares were included in the vaccination group and 13 mares served as negative controls (Fig. 5.2).

Figure 5. 2 Baseline serum Rpl specific IgG titers of pregnant mares approximately 2 months prior to foaling. Each bar represents a control (blue) and a vaccinated (red) mare ranked from lowest to highest antibody titer.

No significant difference in pre-existing Rpl titers between the vaccinated and control mares (p = 0.689) was detected. However, a significant increase in Rpl specific IgG was observed in vaccinated mares at 4 (p <0.001) and 6 (p <0.001) weeks. Significant differences were observed

77 between groups at 4 (p<0.050) and 6 (p<0.050) weeks post-vaccination, when vaccinated mares had significantly higher concentrations of Rpl specific IgG (Fig. 5.3).

Figure 5. 3 Vaccinated and control mare’s Rpl specific IgG titers over time. Asterisk indicates significant difference from baseline. Letter “a” indicates significant difference between groups.

Colostrum of vaccinated mares had significantly higher concentrations of Rpl specific

IgG compared to controls (p<0.001) (Fig. 5.4).

Figure 5. 4 Titers of Rpl specific IgG in colostrum of control and vaccinated mares. Asterisk indicates significant difference between groups.

Significantly higher concentrations of Rpl specific IgG were found in the sera of foals of vaccinated mares at 3-7 days of age (p<0.001) (Fig. 5.5), and in their BALF (p= 0.006) (Fig. 5.6), compared to foals of control mares.

Figure 5. 5 Rpl specific IgG serum titers of foals of control and vaccinated mares 3-7 days after being born (before experimental infection). Asterisk indicates significant difference between groups.

Figure 5. 6 Rpl specific IgG BALF titers of foals of control and vaccinated mares 3-7 days after being born (before experimental infection). Asterisk indicates significant difference between groups.

There were positive significant correlations between the mare’s anti-Rpl titers at 4 weeks post-vaccination, the foal’s serum anti-Rpl titers and the foal’s BALF samples (Table 5.1).

Colostrum Foal's Serum Foal's BALF Mare's 4w post-vaccine 0.208 0.507 0.424 Pearson Correlation Coefficient 0.298 0.00699 0.0275 P value Colostrum 0.22 0.259 Pearson Correlation Coefficient 0.271 0.192 P value Foal's Serum 0.596 Pearson Correlation Coefficient 0.00103 P value

Table 5. 1 Pearson correlation coefficient and p values between the Rpl specific IgG titers of mares and foal’s serum, mare’s colostrum and foal’s BALF .

There was no difference in lung disease incidence among the foals born from vaccinated and control mares, as all foals in the vaccine group developed lung lesions detected

81 ultrasonographically while only one foal in the control group did not develop lung lesions that could be detected ultrasonographically (Table 5.2).

Ultrasound scores of foals born from vaccinated and control mares Foal Group Pre-challenge 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Q1 Control 0 0 0 0 0.4 0.4 1.1 1.4 0.7 0.2 0.3 0.2 0 0 0 0 0 Q7 Control 0 0 0 0 0 0 0 0 0 0 0 0 7.1 ε Q8 Control 0 0 0 0 0.3 0.6 11 35.1 38.1 ε Q12 Control 0 0 0 0 0 0 0 0 1.6 ε Q13 Control 0 0 0 0 0 0 1.3 4 5.8 6.1 1.3 0.4 0.6 0 0 ε Q15 Control 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Q17 Control 0 0 0 0 1.1 0 0 0 1.7 0.9 0 0 0 0 0 0 0 Q18 Control 0 0 0 0 0 0 0.2 3 3.2 0.8 0.5 0 0 0 0.3 0.3 0 Q19 Control 0 0 0 0 0 0 0 0 0 0 13.2 5.5 0.7 0.8 0.3 0 0.7 Q20 Control 0 0 0 0 0 1.9 4.3 4.2 0.2 0 0.8 3.4 3.7 3.4 0 0 0 Q21 Control 0 0 0 0 1.8 20.9 11.1 13 2.4 1 0.9 0.4 0.6 0 0 0 0 Q23 Control 0 0 0 0 0 0.6 3.2 8.2 7.7 4.2 3.7 0.4 1.8 ε Q29 Control 0 0 0 0 0 0 0 16.1 3.5 0.9 0 0 0 0 0 0 0 Q2 Vaccinated 0 0 0 0 0.5 1.3 3.3 3.6 13.9 21.1 10.3 3.9 2.3 2 1.8 ε Q4 Vaccinated 0 0 0 0 0.9 5.7 3.4 1.4 0.8 1.7 0.4 0 0.4 0.5 0.5 0 0 Q5 Vaccinated 0 0 0 0 0 0.7 0 0 0 0.3 0 0 0 ε Q6 Vaccinated 0 0 0 0 0 4.5 11.3 9.4 0.9 2.5 0.8 0 0.9 1.1 0.4 0 0 Q9 Vaccinated 0 0 0 0 5.3 13 5 3.6 6.5 4.7 2 2 1.3 0 0 0 0 Q10 Vaccinated 0 0 0 0 0.2 1 0 1.1 8 ε Q11 Vaccinated 0 0 0 0 0 1.6 1.6 4.2 4 3.7 1.1 0.8 0 0 0 0.4 0.3 Q14 Vaccinated 0 0 0 0 0.3 0.9 3.2 0.8 0 0 0 0 0 0 0 0 0 Q16 Vaccinated 0 0 0 0 0.4 0 0.8 2.9 15.7 ε Q22 Vaccinated 0 0 0 0 0 0 1 7.5 5.2 15.4 20.8 12.2 3.9 ε Q25 Vaccinated 0 0 0 0 0 0 0 0 0 0 0 3.8 4 4.2 3.2 2.2 0 Q26 Vaccinated 0 0 0 0 0 0 0 0.6 0.5 0.5 0 0.2 0 0 0 0 0 Q27 Vaccinated 0 0 0 0 0 0 1.4 4.8 12.3 7.5 2 1.9 1 0 0 0 0 Q28 Vaccinated 0 0 0 0 0.5 1 0 0 0 0 0 0 0 0 0 0 0

Table 5. 2 Ultrasound scores of all foals of control and vaccinated mares before and after challenge, at different examination times (1-16). ε indicates euthanasia.

There were no significant differences in physical examination scores (p=0.227) (Table 5.3), lung ultrasonography scores (p=0.986), abscess scores (p=0.310), % affected lung parenchyma

(p=0.548), R. equi growth from lung homogenate (p=0.690) (Table 5.4), white blood cells counts

(p=0.264) (Table 5.5), and fibrinogen (p=0.336) (Table 5.6) between foals born from vaccinated and control mares.

Physical examination scores of foals born from vaccinated and control mares Foal Group Pre-challenge 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Q1 Control 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 Q7 Control 0 0 0 0 0 0 0 0 0 0 0 0 0 ε Q8 Control 0 0 0 0 0 0 1 5 9 ε Q12 Control 0 0 0 0 0 0 0 0 0 ε Q13 Control 0 0 0 0 0 2 0 0 0 2 0 1 0 0 0 ε Q15 Control 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Q17 Control 0 0 0 0 2 0 0 0 0 1 2 2 2 0 2 1 0 Q18 Control 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Q19 Control 0 0 0 0 0 0 0 0 0 0 5 7 4 1 0 1 2 Q20 Control 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Q21 Control 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 Q23 Control 0 0 0 0 0 0 0 0 0 0 0 0 0 ε Q29 Control 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Q2 Vaccinated 0 0 0 0 0 1 0 0 1 2 2 0 0 0 0 ε Q4 Vaccinated 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 Q5 Vaccinated 0 0 0 2 0 0 0 0 0 0 0 0 0 ε Q6 Vaccinated 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 Q9 Vaccinated 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 Q10 Vaccinated 0 0 0 0 0 0 0 0 0 ε Q11 Vaccinated 0 0 0 0 0 0 0 2 0 2 0 0 0 0 0 0 0 Q14 Vaccinated 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 Q16 Vaccinated 0 0 0 0 0 0 0 0 2 ε Q22 Vaccinated 0 0 0 0 1 0 0 0 0 3 2 2 1 ε Q25 Vaccinated 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Q26 Vaccinated 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Q27 Vaccinated 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Q28 Vaccinated 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Table 5. 3 Physical examination scores of all foals of control and vaccinated mares before and after challenge, at different examination times (1-16). ε indicates euthanasia.

Post-mortem parameters of foals born from vaccinated and control mares Foal Group % total lung pareAnbcshceymssa S caoffreecteRd. equi culture (cfu/mg) Q07 Control 0.3 0 3.8 Q08 Control 26.57 26 10.4 Q12 Control 7.88 5 959.0 Q13 Control 0.83 1 6.6 Q23 Control 0.39 0 3.2 Q02 Vaccinated 4.09 4 0.0 Q05 Vaccinated 0.4 1 201.6 Q10 Vaccinated 8.15 6 11.4 Q16 Vaccinated 11.88 12 965.2 Q22 Vaccinated 5.44 7 4.2

Table 5. 4 Post-mortem parameters of foals of vaccinated and control mares.

White blood cell (WBC) count of foals born from vaccinated and control mares Foal Group Pre-challenge 1 2 3 4 5 6 7 8 Q1 Control 12.3 11.1 5.6 14.5 12 8.9 11.3 12.9 10.8 Q7 Control 8.1 6.8 7.5 8.3 12.3 11.2 12.8 ε Q8 Control 9.4 13.8 12.3 11.7 14.1 ε Q12 Control 9.2 10.1 13.9 12.7 11.4 ε Q13 Control 11.9 10.3 9.7 9 14.9 15 15.4 13.4 ε Q15 Control 7.2 6.5 7 7.3 7.8 6.7 12.6 10.3 12.9 Q17 Control 9.6 7.7 14 11.1 15.2 13.7 15 15.4 14.5 Q18 Control 10.5 14.8 9.7 9.6 11.5 15.6 10 11.7 7.4 Q19 Control 12.5 12 10.2 7.3 10.3 11.4 14.8 12 17.3 Q20 Control 8.6 12.2 9.6 8.9 9.2 10.2 13 9.9 9.2 Q21 Control 8.1 8.4 10.1 12.6 13.5 13.5 14.7 17.2 17.6 Q23 Control 9 17.8 6.5 11.1 16 19.2 12.3 ε Q29 Control 9 10.7 7.3 11.7 12.2 9.3 10.8 10.9 9.8 Q2 Vaccinated 9.7 8.9 20.9 14.4 18.8 8.3 9.2 18.8 ε Q4 Vaccinated 11.4 13.6 11.7 12.1 13.8 10.6 7.7 11.3 15.4 Q5 Vaccinated 7.8 8.3 11 9.2 11.4 9.6 11.3 ε Q6 Vaccinated 7.9 11.1 10.7 9.8 10.7 7.9 12.3 6.6 15.5 Q9 Vaccinated 10.8 10 8.8 12 14.1 10.4 9 10.7 11.2 Q10 Vaccinated 7 7.8 9.6 9 9 ε Q11 Vaccinated 9.4 8.7 16.9 6.4 7.6 6.9 9.9 10 9.9 Q14 Vaccinated 7.2 8.7 10.9 9.7 10.9 10.3 8.8 8 10.3 Q16 Vaccinated 10.4 11.6 17.1 12.9 11.3 ε Q22 Vaccinated 9.3 7.5 10.6 9.6 6.8 24.3 8.5 ε Q25 Vaccinated 9 20.9 35.8 18.6 14.9 11.6 16 16.5 15 Q26 Vaccinated 6.5 10.8 9.5 7.3 12 6.7 18.9 11.4 10.2 Q27 Vaccinated 7.3 9.1 9.8 6.9 8 12.5 13.1 11 11.3 Q28 Vaccinated 7.8 7.7 8.9 12.3 13.8 10.7 13.2 11.2 12

Table 5. 5 White blood cell count of all foals of control and vaccinated mares before and after challenge, at different examination times (1-8). ε indicates euthanasia.

Fibrinogen of foals born from vaccinated and control mares Foal Group Pre-challenge 1 2 3 4 5 6 7 8 Q1 Control 280 279 533 359 337 303 322 535 577 Q7 Control 268 312 345 400 422 394 337 ε Q8 Control 288 256 314 465 613 ε Q12 Control 314 596 636 404 357 ε Q13 Control 259 302 358 594 504 467 364 368 ε Q15 Control 323 374 336 329 321 462 451 380 344 Q17 Control 316 389 309 310 405 410 353 300 326 Q18 Control 319 389 309 310 405 410 353 300 326 Q19 Control 322 483 571 611 839 1053 982 889 607 Q20 Control 316 453 425 375 389 352 365 336 338 Q21 Control 296 359 409 367 654 631 546 545 500 Q23 Control 347 695 808 402 864 708 794 ε Q29 Control 270 360 365 362 382 337 314 375 9.8 Q2 Vaccinated 283 407 332 331 353 424 336 310 ε Q4 Vaccinated 403 358 461 464 423 391 369 375 419 Q5 Vaccinated 236 269 304 280 276 268 310 ε Q6 Vaccinated 227 280 319 368 410 509 343 554 594 Q9 Vaccinated 436 453 392 614 453 479 429 427 457 Q10 Vaccinated 412 472 668 413 396 ε Q11 Vaccinated 403 326 392 379 383 394 346 342 328 Q14 Vaccinated 267 219 396 371 275 296 319 290 285 Q16 Vaccinated 421 638 733 765 682 ε Q22 Vaccinated 264 377 639 556 718 720 726 ε Q25 Vaccinated 277 857 1070 769 546 532 564 396 402 Q26 Vaccinated 224 257 355 416 423 503 560 303 354 Q27 Vaccinated 263 344 340 350 509 474 345 379 320 Q28 Vaccinated 274 296 370 397 418 490 486 603 510

Table 5.6 Fibrinogen of all foals of control and vaccinated mares before and after challenge, at different examination times (1-8). ε indicates euthanasia.

Discussion

The results demonstrated that the Rpl vaccine candidate induced an anamnestic response in vaccinated pregnant mares, and the Rpl antibodies concentrated in the colostrum. Furthermore, foals that ingested colostrum from vaccinated mares had significantly higher serum titers of Rpl specific IgG and in BALF compared to foals of non-vaccinated mares. However, passively acquired

Rpl specific IgG of foals in the vaccine group did not confer protection against rhodococcal pneumonia, nor decrease disease severity after challenge compared to control foals.

Virulent R. equi survives and replicate within alveolar macrophages11,176. Vaccination against intracellular pathogens is usually focused on generation of cell-mediated immune responses in the host, where a pool of memory T-cells is induced and is able to mediate either killing of the infected cell or induce killing mechanisms in the infected cell177. In contrast, the Rpl vaccine composed of a synthetic peptide with an alum-like adjuvant was expected to mostly induce a humoral response that could be passively transferred via colostrum. Because the R. equi pilus has been shown to mediate bacterial attachment to host cells (JBV, personal communication), passive transfer of Rpl specific IgG was expected to opsonize and, therefore, prevent or decrease intracellular infection by R. equi. This would result in a lack of, or fewer lung lesions in the foals of vaccinated mares.

Mares were vaccinated at 4 and 6 weeks prior to their predicted foaling date to induce a peak antibody response during the last 15 days of gestation. It is during this time that the non- selective secretion of serum immunoglobulins into colostrum occurs178. Indeed, our results indicate that the antibody peak response in vaccinated mares likely occurred shortly before or at parturition

(Fig. 5.4). The results also indicate that induced Rpl specific IgG was present in the colostrum from vaccinated mares (Fig. 5.5), as well as transferred to their foal’s serum and BALF (Figs. 5.6 and

5.7). Interestingly, significant positive correlations were observed between the Rpl specific IgG titer in the mare and foal’s sera, and foal’s BALF, indicating specific immunoglobulins exit systemic circulation after oral uptake and become compartmentalized. A lack of association between titers in mare and foal’s sera, and foal’s BALF and colostral titers may be explained by

85 the suboptimal timing of colostrum collection. Since parturition was unattended, a colostrum sample collected immediately post-partum and before suckling was not possible. Marked decline in immunoglobulin concentrations in the equine colostrum after suckling has occurred has been described178 For that reason, we chose to analyze the colostrum collected up to 12 hours prior to parturition. It is possible that colostrum samples collected from immediate post-partum and prior to suckling would have shown different results. Nonetheless, systemic and local Rpl specific IgG present in sera and BALF of foals of vaccinated mares, did not protect against experimental challenge.

Previous failures of peptide vaccines have been attributed to use of single peptide epitopes as vaccine candidates, immune evasion mechanisms, failure to elicit a controlled and prolonged immune responses, and inappropriate design of clinical trials179. The Rpl vaccine was formulated to induce an immune response against a linear epitope, while the vast majority of antigen-antibody interactions involve binding to conformational epitopes180. An antigen epitope-antibody mismatch could explain the lack of vaccine efficacy. However, it is since the same antibody was fully protective against R. equi challenge in mice (JBV, personal communication).

Earlier studies that evaluated active immunization of mares with killed or inactivated R. equi with subsequent colostral transfer of immunity to foals failed in preventing foals from developing R. equi pneumonia120,169. These studies were performed before Takai et al., demonstrated the importance of live, virulent R. equi in eliciting a protective immune response in mice181. Most recently, promising results were observed after the immunization of a small number of pregnant mares with a water-based nanoparticle adjuvanted (IMS 3012) VapA protein163.

Vaccinated mares had higher serum IgG and opsonic activity, which was transferred to foals, compared to mares vaccinated with killed whole cell R. equi. Likewise, Becu et al., reported a significant reduction in R. equi related foal mortality when pregnant mares were vaccinated with soluble R. equi antigens, including VapA160. While protective responses were observed, a great variation in the humoral response produced by vaccinated mares was also reported160. Indeed, the

86 antibody production of vaccinated mares in the present study varied considerably, and cannot be excluded as a reason for vaccine failure.

Since vaccination of mares has not been consistently effective in preventing rhodococcal pneumonia in foals, attention has been redirected toward actively vaccinating foals. Vaccines centered on DNA technology have been shown to be effective in inducing desirable Th1 responses in mice129,130,182 However, most of these vaccines did not protect against R. equi infection in mice.

Therefore, additional work is needed to optimize the DNA technology for neonatal foals. Live, oral vaccines containing virulent R. equi have shown positive results when administered to foals117,127,134. Thus, the impact and significance of a live oral vaccine on the gastrointestinal tract of foals, fecal shedding and subsequent on-farm contamination need to be established to satisfy safety considerations. In agreement with the results of the present study, previous Vap-peptide vaccines have been shown to induce a Vap-specific immunoglobulin production, with a mixed Th1 and Th2 profile in mice, adult horses and neonatal foals81,153. Protection elicited by these Vap- peptide vaccine candidates has not been reported in foals.

Induction of effective immune responses through vaccination requires both humoral and cell-mediated immune responses. Dose, antigen type, route of administration, and adjuvant type are central considerations. Combination of a modified live attenuated oral vaccine with an adjuvant capable of stimulating a mixed Th1/Th2 response, with less potential to contaminate the environment, may be a promising vaccine strategy. Nevertheless, there is the difficult challenge of eliciting a protective response in immunologically immature foal exposed to R. equi within the first few days of life.

CHAPTER 6

CONCLUSIONS AND FUTURE DIRECTIONS

Although R. equi was first isolated from a horse almost 100 years ago, it remains a leading cause of mortality in foals. Sequence analysis of the R. equi 103+ genome, has yielded a better understanding of the pathogen’s biology yet much remains unknown to explain the foal’s unique susceptibility. In addition, lack of early diagnostic and prophylactic methods are a major frustration to the field veterinarian.

Development of a diagnostic tool to precisely identify foals at risk of becoming clinically affected by R. equi, and that would benefit from medical intervention would have an immediate positive impact on the horse industry. Early diagnosis of R. equi infection of the respiratory tract currently relies on screening techniques that are highly sensitive, although poorly specific resulting in widespread antimicrobial use. Based on the challenge model previously developed, IgG(T) was identified as a marker of infection. Unfortunately, when evaluated as a marker for clinical disease

IgG(T) was not a reliable predictor of disease outcome following experimental challenge or natural infection. However, the possibility of using IgG(T) as a disease marker has not been exhausted yet.

Only recently, the full complement of the horse immunoglobulin heavy chain constant regions genes was described. IgG(T) was then reassigned as IgG3 and IgG5. Additional research will be needed to assess these IgG(T) subisotypes as predictors of disease outcome once specific antibodies become commercially available.

Comparison of the whole blood cytokine expression profiles of progressor and regressor foals was performed to provide a reliable, easy-to-perfom R. equi diagnostic test. However, it did not reveal differences in cell markers or cytokine mRNA expression of value in differentitation progressor from regressor foals. Nonetheless, it was only a first step taken towards identifying potential mRNA expression patterns that may differ among foals that are susceptible to R. equi, and foals that mount an effective immune response. Cytokine expression profiles of specific cell populations, such as PBMCs or alveolar macrophages, should be evaluated.

This study further evaluated the variability of IgGs among units of a commercially available HIP, and showed that naturally infected foals remaining free of clinical disease had received greater concentrations of total IgG. Observational data from a large number of foals collected over a period of 2 years is likely to weigh in future decisions of horse owners, farm managers, and field veterinarians regarding the use of HIP.

Despite the lack of protection by the Rpl vaccine candidate reported here, the results emphasize the need of a more thorough approach when formulation vaccine antigens, as well as the importance of a cell-mediated immune response in addition to specific antibodies in order to obtain a protective immune response against R. equi.

Although several limitations were identified throughout this body of work, some significant contributions towards improving current R. equi diagnostic and preventive measures were still achieved. For the first time, the humoral immune response and the whole blood cytokine and cell marker expression profiles of progressor and regressor foals were characterized either after challenge or natural infection. Evidence favoring the use of HIP in the field was provided. Finally, suscessful collaborative research involving different academic institutes, local field veterinarians and horse farms was accomplished.

APPENDIX

Supplemental table A.1: VAPA ELISA results of challenged foals during the years 2016 and 2017 before and 2, 4, 6 and 8 weeks post-challenge.

Subject Group Time point Total IgG IgGa IgGb IgGt 2 Progressor Pre-challenge 0.50 1.13 1.49 1.19 2 Progressor 2w PC 0.48 1.14 1.65 1.44 2 Progressor 4w PC 0.53 2.59 1.76 2.13 2 Progressor 6w PC 1.62 7.27 3.28 147884.8 2 Progressor 8w PC 8 Progressor Pre-challenge 0.57 0.91 1.76 1.21 8 Progressor 2w PC 0.54 1.05 1.85 1.46 8 Progressor 4w PC 2.92 60.17 3.87 11.36 8 Progressor 6w PC 8 Progressor 8w PC 11 Progressor Pre-challenge 2.63 1.24 2.40 1.39 11 Progressor 2w PC 0.81 1.60 2.22 1.65 11 Progressor 4w PC 1.09 6.21 2.33 1.37 11 Progressor 6w PC 1.05 3.48 2.27 1.47 11 Progressor 8w PC 1.73 4.38 3.98 2.21 13 Progressor Pre-challenge 0.90 1.28 2.11 1.27 13 Progressor 2w PC 0.68 1.40 2.10 1.46 13 Progressor 4w PC 1.06 5.13 2.12 14.21 13 Progressor 6w PC 1.58 7.48 3.04 42.95 13 Progressor 8w PC 17 Progressor Pre-challenge 1.66 1.92 3.91 1.21 17 Progressor 2w PC 1.01 1.59 3.10 1.49 17 Progressor 4w PC 3.20 5.62 4.29 2.99 17 Progressor 6w PC 3.20 6.38 7.48 19.03 17 Progressor 8w PC 4.56 8.29 9.53 60.76 19 Progressor Pre-challenge 0.65 1.01 1.90 1.18 19 Progressor 2w PC 0.66 1.09 1.85 1.18 19 Progressor 4w PC 0.84 2.32 2.22 1.20 19 Progressor 6w PC 1.92 5.16 4.32 1.32 19 Progressor 8w PC 8.60 30.77 22.55 475.89 22 Progressor Pre-challenge 0.52 0.92 1.56 1.23 22 Progressor 2w PC 0.61 1.11 1.67 1.22 22 Progressor 4w PC 9.03 93.81 13.37 6.10 22 Progressor 6w PC 14.69 83.44 31.86 4879.66 22 Progressor 8w PC R8 Progressor Pre-challenge 0.48 1.15 1.60 1.42

R8 Progressor 2w PC 0.48 1.11 1.57 1.36 R8 Progressor 4w PC 1.40 5.73 2.81 1.60 R8 Progressor 6w PC 24.33 538.37 45.66 167.62 R8 Progressor 8w PC 26.77 434.89 44.67 66.03 R10 Progressor Pre-challenge 2.08 2.57 4.54 1.39 R10 Progressor 2w PC 1.24 1.97 3.22 1.41 R10 Progressor 4w PC 1.82 35.81 4.53 24.51 R10 Progressor 6w PC 4.79 48.25 11.33 443.74 R10 Progressor 8w PC 5.77 29.10 10.91 569.00 R13 Progressor Pre-challenge 1.42 2.10 3.16 1.45 R13 Progressor 2w PC 1.01 1.76 2.61 1.42 R13 Progressor 4w PC 1.26 7.21 2.77 2.82 R13 Progressor 6w PC 1.69 4.21 3.62 4.27 R13 Progressor 8w PC R14 Progressor Pre-challenge 0.60 1.67 1.78 1.45 R14 Progressor 2w PC 0.53 1.38 1.70 1.45 R14 Progressor 4w PC 1.83 15.52 2.47 16.33 R14 Progressor 6w PC 4.49 45.78 6.18 1511.63 R14 Progressor 8w PC 6.12 62.41 7.41 3797.02 R18 Progressor Pre-challenge 0.85 1.44 2.24 1.40 R18 Progressor 2w PC 0.59 1.38 1.82 1.44 R18 Progressor 4w PC 1.29 5.14 2.28 2.49 R18 Progressor 6w PC 3.77 25.58 6.94 5.40 R18 Progressor 8w PC 5.70 40.02 10.65 7.40 1 Regressor Pre-challenge 0.53 0.89 1.60 1.19 1 Regressor 2w PC 0.50 1.01 1.73 1.45 1 Regressor 4w PC 0.95 2.92 2.36 1.60 1 Regressor 6w PC 1.63 6.07 4.02 42.15 1 Regressor 8w PC 1.73 4.59 4.05 45.16 4 Regressor Pre-challenge 0.64 1.31 1.73 1.21 4 Regressor 2w PC 1.46 1.25 1.81 1.46 4 Regressor 4w PC 0.60 3.45 1.87 1.96 4 Regressor 6w PC 1.08 7.46 2.35 40.54 4 Regressor 8w PC 2.34 25.56 4.73 1605.36 5 Regressor Pre-challenge 1.26 6.79 2.25 1.23 5 Regressor 2w PC 1.45 2.84 2.07 1.45 5 Regressor 4w PC 0.92 4.07 2.75 3.23 5 Regressor 6w PC 5 Regressor 8w PC 6 Regressor Pre-challenge 0.55 1.04 1.59 1.17 6 Regressor 2w PC 1.49 1.55 1.73 1.49 6 Regressor 4w PC 2.30 17.45 3.96 24576.13 6 Regressor 6w PC 2.05 12.67 5.65 3411.15

6 Regressor 8w PC 1.98 4.91 3.47 67.25 9 Regressor Pre-challenge 1.51 2.01 2.93 1.18 9 Regressor 2w PC 0.87 1.50 2.47 1.46 9 Regressor 4w PC 2.26 11.62 4.42 3.24 9 Regressor 6w PC 8.62 41.48 14.13 13.70 9 Regressor 8w PC 6.25 39.85 19.31 22.95 14 Regressor Pre-challenge 6.48 5.57 6.91 1.23 14 Regressor 2w PC 2.47 3.65 6.31 1.52 14 Regressor 4w PC 1.79 3.08 3.54 1.80 14 Regressor 6w PC 1.14 2.35 2.74 2.00 14 Regressor 8w PC 1.05 1.85 2.63 2.19 18 Regressor Pre-challenge 2.17 3.36 4.57 1.19 18 Regressor 2w PC 1.55 2.18 3.69 1.46 18 Regressor 4w PC 0.96 2.14 2.78 1.38 18 Regressor 6w PC 0.96 1.84 2.22 2.34 18 Regressor 8w PC 0.78 1.65 2.27 2.61 20 Regressor Pre-challenge 1.21 1.27 2.39 1.22 20 Regressor 2w PC 0.86 1.21 2.23 1.18 20 Regressor 4w PC 1.34 4.96 3.20 2.61 20 Regressor 6w PC 3.38 18.48 11.17 237.63 20 Regressor 8w PC 8.60 21.40 21.62 7130.10 23 Regressor Pre-challenge 0.61 1.46 1.95 1.49 23 Regressor 2w PC 0.70 1.40 1.80 1.22 23 Regressor 4w PC 3.66 18.02 4.81 13330.95 23 Regressor 6w PC 3.92 12.05 5.69 2609.90 23 Regressor 8w PC 25 Regressor Pre-challenge 0.79 1.77 2.28 1.47 25 Regressor 2w PC 0.66 1.26 1.90 1.25 25 Regressor 4w PC 1.61 3.58 4.04 4.81 25 Regressor 6w PC 1.33 6.35 2.63 27.95 25 Regressor 8w PC 6.08 21.40 16.63 300.47 26 Regressor Pre-challenge 0.78 1.36 2.20 1.48 26 Regressor 2w PC 0.65 1.19 1.87 1.29 26 Regressor 4w PC 0.91 6.28 2.43 2.27 26 Regressor 6w PC 2.02 12.46 4.21 5.58 26 Regressor 8w PC 5.07 11.54 7.60 15.60 27 Regressor Pre-challenge 0.51 1.08 1.69 1.46 27 Regressor 2w PC 0.48 1.21 1.72 1.48 27 Regressor 4w PC 27 Regressor 6w PC 3.49 25.01 9.11 54.53 27 Regressor 8w PC 0.44 20.54 10.74 61.10 28 Regressor Pre-challenge 0.61 1.24 1.93 1.48 28 Regressor 2w PC 0.57 1.29 1.75 1.25

28 Regressor 4w PC 3.81 34.12 10.10 23.12 28 Regressor 6w PC 1.19 28.73 14.93 33.62 28 Regressor 8w PC 29 Regressor Pre-challenge 0.55 1.18 1.87 1.53 29 Regressor 2w PC 0.50 1.05 1.65 1.25 29 Regressor 4w PC 0.77 5.67 2.12 1.55 29 Regressor 6w PC 4.03 5.25 2.85 38.03 29 Regressor 8w PC R01 Regressor Pre-challenge 1.16 1.68 2.96 1.42 R01 Regressor 2w PC 0.82 1.42 2.44 1.44 R01 Regressor 4w PC 1.90 7.29 4.18 1.83 R01 Regressor 6w PC 2.55 11.12 3.61 9.52 R01 Regressor 8w PC 1.68 3.70 3.32 10.76 R02 Regressor Pre-challenge 7.38 10.48 11.70 1.46 R02 Regressor 2w PC 2.39 3.41 5.07 1.40 R02 Regressor 4w PC 1.87 4.89 3.78 3.65 R02 Regressor 6w PC 1.85 4.06 2.86 4.98 R02 Regressor 8w PC 1.30 3.29 2.76 5.73 R03 Regressor Pre-challenge 0.94 2.10 2.29 1.47 R03 Regressor 2w PC 0.72 1.73 2.00 1.37 R03 Regressor 4w PC 0.67 1.83 1.89 1.43 R03 Regressor 6w PC 1.01 1.95 2.02 2.34 R03 Regressor 8w PC 1.00 2.01 2.15 2.03 R04 Regressor Pre-challenge 47.17 30.66 68.25 1.45 R04 Regressor 2w PC 11.75 10.98 18.95 1.42 R04 Regressor 4w PC 5.62 5.40 9.53 1.44 R04 Regressor 6w PC 5.80 7.58 6.59 2.05 R04 Regressor 8w PC 3.84 9.30 4.48 3.02 R05 Regressor Pre-challenge 10.80 9.20 23.12 1.40 R05 Regressor 2w PC 5.00 4.69 8.91 1.35 R05 Regressor 4w PC 2.12 2.88 4.33 1.41 R05 Regressor 6w PC 1.88 3.11 3.79 1.60 R05 Regressor 8w PC 3.46 4.21 6.11 1.82 R07 Regressor Pre-challenge 0.51 1.17 1.64 1.46 R07 Regressor 2w PC 0.48 1.12 1.60 1.37 R07 Regressor 4w PC 0.93 5.09 2.30 4.86 R07 Regressor 6w PC 1.78 8.42 4.01 5.98 R07 Regressor 8w PC 2.22 9.95 4.34 8.54 R11 Regressor Pre-challenge 3.76 3.91 7.29 1.43 R11 Regressor 2w PC 2.71 3.25 5.02 1.38 R11 Regressor 4w PC 2.65 5.40 3.85 7.30 R11 Regressor 6w PC 2.49 4.79 3.99 210.46 R11 Regressor 8w PC

R12 Regressor Pre-challenge 1.22 1.84 2.85 1.48 R12 Regressor 2w PC 1.28 2.01 2.91 1.41 R12 Regressor 4w PC 2.01 15.70 3.27 4.88 R12 Regressor 6w PC R12 Regressor 8w PC R15 Regressor Pre-challenge 0.69 1.36 1.83 1.40 R15 Regressor 2w PC 0.69 1.29 1.97 1.45 R15 Regressor 4w PC 2.99 45.91 6.16 1.71 R15 Regressor 6w PC 3.46 12.83 7.78 2.99 R15 Regressor 8w PC 3.04 14.55 6.43 3.80

Supplemental table A.2: VAPA ELISA results of naturally exposed foals at time points 1-4

Foal Group Time Point Total IgG IgGa IgGb IgGt 2 Progressor 1 90.5 135.7 237.7 10.1 2 Progressor 2 43.3 60.5 243.2 5.2 2 Progressor 3 67.8 70.3 131.3 3.2 2 Progressor 4 2 Progressor 5 38 Progressor 1 176.2 370.1 838.9 1.8 38 Progressor 2 74.4 87.7 107.5 1.8 38 Progressor 3 96.5 80 213.4 2 38 Progressor 4 143.9 75.3 181.6 1.7 38 Progressor 5 84 Progressor 1 51.2 32.3 83 5.4 84 Progressor 2 44.2 45.6 89.3 3.6 84 Progressor 3 79.5 154.8 106.2 7.3 84 Progressor 4 76.1 82.4 181.6 6.5 84 Progressor 5 128 Progressor 1 55.5 67.3 107.2 6.6 128 Progressor 2 108 209.6 438.9 4.9 128 Progressor 3 116 71 205.2 3 128 Progressor 4 36.7 27.9 69.1 2.6 128 Progressor 5 40 Progressor 1 147.8 105.5 545.5 1.8 40 Progressor 2 118.1 50.2 340.5 3.5 40 Progressor 3 24.8 24.9 80.4 2 40 Progressor 4 45.4 39.8 82.8 2.1

Foal Group Time Point Total IgG IgGa IgGb IgGt 40 Progressor 5 28 Progressor 1 126.2 163.8 223.1 6.4 28 Progressor 2 154.1 195.6 220.1 3.3 28 Progressor 3 2.2 9.9 3.9 189.8 28 Progressor 4 28 Progressor 5 32 Progressor 1 136.8 238.1 458.3 3.6 32 Progressor 2 76.9 124.1 167.4 2.7 32 Progressor 3 44.6 43.5 115.1 2.1 32 Progressor 4 57.9 28.8 61.2 2.2 32 Progressor 5 75 Progressor 1 72.1 44 121.8 1.7 75 Progressor 2 203.9 123.9 299.8 7.4 75 Progressor 3 51.9 59.6 67.9 4.6 75 Progressor 4 40.7 26.2 72.5 7.1 75 Progressor 5 87 Progressor 1 93.9 43.1 133.8 4.6 87 Progressor 2 17.7 24.1 26.2 4.2 87 Progressor 3 87 Progressor 4 87 Progressor 5 66 Progressor 1 129.1 104.3 267.9 2 66 Progressor 2 63.7 69.1 184 1.6 66 Progressor 3 66 Progressor 4 66 Progressor 5 78 Progressor 1 49.1 43.9 120.8 1.8 78 Progressor 2 52.2 59.2 163.1 1.6 78 Progressor 3 78 Progressor 4 78 Progressor 5 3 Progressor 1 98.1 78.6 243.6 1.8 3 Progressor 2 102.2 70.8 414.2 2.6 3 Progressor 3 99.7 83.6 141.9 2.1 3 Progressor 4 3 Progressor 5 7 Progressor 1 116.3 60.2 209.1 12.1 7 Progressor 2 95.4 166.7 624.7 8.4 7 Progressor 3 46.3 44.1 121.6 3.9 7 Progressor 4 16 10.9 32.2 1.8 7 Progressor 5 12.4 7.5 21.6 1.6 37 Progressor 1 49.5 73 163.5 2.9 37 Progressor 2 122.8 88 223.3 2.5 37 Progressor 3 68.5 33.8 102.8 1.5

Foal Group Time Point Total IgG IgGa IgGb IgGt 37 Progressor 4 23.2 10.3 32.3 1.3 37 Progressor 5 4.8 3.5 8.7 1 127 Regressor 1 31.5 76.4 142.7 5.9 127 Regressor 2 79.3 18.5 64.4 3.6 127 Regressor 3 28.9 21.2 46.4 2.6 127 Regressor 4 127 Regressor 5 86 Regressor 1 169.4 74.9 248.6 8.8 86 Regressor 2 400.3 69.7 171.6 10.9 86 Regressor 3 87.5 56.1 107.6 8.5 86 Regressor 4 46.1 23.5 44.7 4 86 Regressor 5 57.7 36.7 109.5 4.3 70 Regressor 1 47.6 46.4 95.5 2.08 70 Regressor 2 27.8 29.7 83.4 1.4 70 Regressor 3 20.1 37.1 41.5 1.4 70 Regressor 4 24.7 24.7 22.7 1.2 70 Regressor 5 5.3 14.6 17.5 1.1 93 Regressor 1 90.6 129.5 158.9 9.5 93 Regressor 2 29.7 26.9 81.5 2.9 93 Regressor 3 26.6 18.6 36.8 1.9 93 Regressor 4 16.6 11.4 29.6 1.8 93 Regressor 5 8 6.2 21.9 1.4 34 Regressor 1 55.8 68 149.4 3.1 34 Regressor 2 54.8 26.6 70.2 2.2 34 Regressor 3 34 Regressor 4 4.6 3.3 6.5 3.7 34 Regressor 5 92 Regressor 1 0.6 1.1 1.7 1.3 92 Regressor 2 0.6 1.3 1.8 1.2 92 Regressor 3 11.6 34.1 23 47 92 Regressor 4 21.9 47.7 44.7 1.7 92 Regressor 5 69 NRA 1 46.9 41.7 168.3 10.5 69 NRA 2 54 64.1 165.1 4.1 69 NRA 3 70 143.2 194 8.1 69 NRA 4 78.2 29.7 81.6 6.5 69 NRA 5 102 NRA 1 114.6 67.8 167.7 9.2 102 NRA 2 36.5 32.4 47 3.9 102 NRA 3 18.5 12.9 34.5 5.9 102 NRA 4 5.5 5.1 12.7 2.1 102 NRA 5 6.6 3.7 17.6 2.1 98 NRA 1 139.2 257.9 257.9 2.1 98 NRA 2 19.9 168.7 168.7 1.4

Foal Group Time Point Total IgG IgGa IgGb IgGt 98 NRA 3 24.6 27.8 27.8 1.6 98 NRA 4 12.2 19.8 19.8 21.9 98 NRA 5 96 NRA 1 122.8 130.4 258.6 2 96 NRA 2 101 31.1 100.8 1.4 96 NRA 3 40.6 37.7 91.7 1.3 96 NRA 4 23.3 12.3 28.8 1.3 96 NRA 5 9 7.1 22.2 1 72 NRA 1 203.6 140.1 245.7 2.19 72 NRA 2 61.6 60.9 108.9 1.6 72 NRA 3 32.6 14.5 38.7 1.2 72 NRA 4 9.9 7.6 17.5 1.2 72 NRA 5 7.9 6 14.8 1 68 NRA 1 90.6 44.4 107.5 1.8 68 NRA 2 77.2 54.2 156.1 1.4 68 NRA 3 8.8 10.8 18.4 1.3 68 NRA 4 10.2 9.6 23.9 0.8 68 NRA 5 11.3 7.8 17.6 1.3 9 NRA 1 114.6 76.1 153.2 7.5 9 NRA 2 60.9 54.3 206.2 5.3 9 NRA 3 52 27.5 67.2 4.6 9 NRA 4 28.7 44.1 52.8 5.6 9 NRA 5 17.8 18.9 24.8 2.9 18 NRA 1 58.7 43.5 122.5 2.3 18 NRA 2 55.7 36.9 73.5 1.9 18 NRA 3 45.3 20.5 40.9 1.7 18 NRA 4 9.2 12.9 22.5 1.3 18 NRA 5 8.6 6.6 15.4 2.3 20 NRA 1 91.6 93.4 231.8 2.6 20 NRA 2 50 69 94.9 2.1 20 NRA 3 14 14.7 24.3 1.6 20 NRA 4 6.2 5.5 11 1.3 20 NRA 5 5.3 5.1 8.7 1.2 42 NRA 1 94.6 37.1 222.6 2.6 42 NRA 2 62.5 35.7 99.1 2.2 42 NRA 3 48.9 28.2 144 1.7 42 NRA 4 41.5 27 45.7 2 42 NRA 5 12 20.1 27.3 1.3 47 NRA 1 131.7 57.4 187.2 2.4 47 NRA 2 102.9 77.9 278 2.6 47 NRA 3 45.2 23.2 49.8 1.6 47 NRA 4 47 NRA 5 62 NRA 1 52.2 41.4 93.2 1.8

Foal Group Time Point Total IgG IgGa IgGb IgGt 62 NRA 2 38.9 40.8 93.7 1.6 62 NRA 3 14.4 20.1 34.4 1.4 62 NRA 4 7 6.6 16.5 1 62 NRA 5 8.5 9.3 14.6 3 36 NRA 1 46.3 213.5 378.7 2 36 NRA 2 237 101.8 587 9.5 36 NRA 3 57.8 40.4 55.2 3.2 36 NRA 4 46.9 19.4 88.1 2.1 36 NRA 5 41 NRA 1 186.4 59.8 286.3 1.6 41 NRA 2 80.1 58.5 262.4 6 41 NRA 3 64.8 68 238.4 3.2 41 NRA 4 32.8 29.9 74 2.9 41 NRA 5 43.7 18.3 61.9 2.2 56 NRA 1 160.7 113.4 225.1 2.3 56 NRA 2 94 80.2 176.2 6.2 56 NRA 3 49.5 60.5 111.2 4.5 56 NRA 4 103.5 78.4 100.5 7.3 56 NRA 5 13.8 13.3 29.9 2.4 57 NRA 1 190.1 112.3 214.5 2.3 57 NRA 2 112.3 79.9 260.3 7.3 57 NRA 3 39.6 19.7 170.1 4.5 57 NRA 4 121.9 93 155.8 4 57 NRA 5 76.1 51.5 89 0 109 NRA 1 84.1 47.7 165.4 9.5 109 NRA 2 32.9 21.3 93.7 4.1 109 NRA 3 141.4 121.5 176.4 2.3 109 NRA 4 76.2 50.3 118.9 2.1 109 NRA 5 41.8 37.5 91.1 2.4 117 NRA 1 217.2 90.6 453.9 7.7 117 NRA 2 18.9 25 84.1 7.2 117 NRA 3 156.2 60.5 188.7 2.2 117 NRA 4 19.9 21.5 62.4 1.6 117 NRA 5 17.8 9.6 22.6 8.5 118 NRA 1 106.2 91.6 216.9 7.7 118 NRA 2 59.3 43.1 130.1 6.6 118 NRA 3 134.7 48.8 174.9 4.4 118 NRA 4 72 84 158.1 3 118 NRA 5 37.1 32.2 68.9 8.2 120 NRA 1 145.1 110.6 280.5 8.1 120 NRA 2 14.1 11.8 61.5 3.9 120 NRA 3 105.6 102.5 167.6 2.2 120 NRA 4 58.3 33.5 73.4 2.4 120 NRA 5 22.1 20.9 45.3 2

Foal Group Time Point Total IgG IgGa IgGb IgGt 125 NRA 1 78.4 55.6 187.2 6.2 125 NRA 2 54.3 217.5 221.2 5.9 125 NRA 3 125 NRA 4 125 NRA 5 121 NRA 1 85 86.6 136.4 6.5 121 NRA 2 94.7 72.4 245.1 6 121 NRA 3 101 40 149.1 3.7 121 NRA 4 24.8 15.1 53.4 1.5 121 NRA 5 131 NRA 1 200.8 54.7 138.4 8.2 131 NRA 2 46 53.4 119.3 5.4 131 NRA 3 118.6 94.7 220.9 3.4 131 NRA 4 103.3 61.1 181.5 3 131 NRA 5 46 29.9 81.3 2 132 NRA 1 128.8 62.8 116.7 11.1 132 NRA 2 73.2 63.7 158.9 8.5 132 NRA 3 123.8 84.5 233.9 4.2 132 NRA 4 148.9 104.6 304.9 3.2 132 NRA 5 56.1 30.2 110.6 2 129 NRA 1 259 59.2 130.2 9.6 129 NRA 2 149.1 124.7 305 5 129 NRA 3 64.2 69.5 259.7 3.2 129 NRA 4 191.4 79.6 345.5 3 129 NRA 5 27.3 25 72.7 3.4 125 NRA 1 78.4 55.6 187.2 6.2 125 NRA 2 54.3 217.5 221.2 5.9 125 NRA 3 125 NRA 4 125 NRA 5 65 NRA 1 68.2 67.6 142.4 10.4 65 NRA 2 184.7 254 592.3 36.3 65 NRA 3 66.3 79.6 233.5 11.4 65 NRA 4 55.3 55.6 177.7 6.1 65 NRA 5 74.8 33.9 104.2 3.9 24 NRA 1 68.3 65.5 113.3 4.4 24 NRA 2 237.8 227.9 332.6 8.6 24 NRA 3 93.6 69.4 184.6 5.2 24 NRA 4 49.2 31.9 94.1 2.5 24 NRA 5 31.7 21.8 45.2 2.4 30 NRA 1 34.3 187.3 528.9 2 30 NRA 2 41.5 32.2 86.6 1.4 30 NRA 3 210.4 101.3 210.5 3.9 30 NRA 4 52.9 31.3 94.5 2.5

Foal Group Time Point Total IgG IgGa IgGb IgGt 30 NRA 5 29.6 17 52.1 2 8 NRA 1 85.9 68 214.8 7.1 8 NRA 2 151.9 149.2 657.5 17.4 8 NRA 3 134.8 82.7 169.9 6.9 8 NRA 4 90.9 60.6 136.8 3.3 8 NRA 5 31.2 22.5 80.6 2.7 12 NRA 1 65.7 63.4 100.3 6.9 12 NRA 2 54 48.3 105.3 4.6 12 NRA 3 114.1 36.4 102.3 6.6 12 NRA 4 84.4 76.2 196 4.1 12 NRA 5 47.5 27.7 77.3 3 22 NRA 1 68.8 82.6 255.5 2 22 NRA 2 69.5 87.9 174.4 5.3 22 NRA 3 185.4 78.3 266.4 4.3 22 NRA 4 54.5 77.8 168.3 3.3 22 NRA 5 50.8 21.7 93.2 2.2 1 NRA 1 125.4 199.4 362.4 1.9 1 NRA 2 130.1 163.5 593.6 2.7 1 NRA 3 85.8 62.7 256.8 1.7 1 NRA 4 11.8 6.5 15.7 1.2 1 NRA 5 5 NRA 1 59.9 53.4 182.3 1.8 5 NRA 2 157.1 181.8 347.3 3 5 NRA 3 9.4 8.6 16 3.4 5 NRA 4 5 NRA 5 19 NRA 1 120.6 88.4 328.8 3 19 NRA 2 109.9 171.7 190.8 2.7 19 NRA 3 66.3 50.8 126.9 1.7 19 NRA 4 19.3 16.7 29.8 1.4 19 NRA 5 25 NRA 1 92.2 159 405.6 3.3 25 NRA 2 193.1 217.8 665.6 3.3 25 NRA 3 83.8 49.3 93.1 1.8 25 NRA 4 25 14.8 28.9 1.4 25 NRA 5 31 NRA 1 95.7 69.4 206.3 2.6 31 NRA 2 110.5 208 214.1 2.6 31 NRA 3 92.8 43.1 80 1.7 31 NRA 4 31 NRA 5 39 NRA 1 170.4 59.6 435.1 2.8 39 NRA 2 70 69.6 242.4 2.5 39 NRA 3 19.8 34 32.6 1.6

100

Foal Group Time Point Total IgG IgGa IgGb IgGt 39 NRA 5 44 NRA 1 55.2 65 398.3 2.6 44 NRA 2 44 NRA 3 20.4 28.4 37 19.5 44 NRA 4 11.1 12.7 18.1 15.6 44 NRA 5 58 NRA 1 93.4 68.2 154.6 2.1 58 NRA 2 73.3 80.2 187.2 5.4 58 NRA 3 45.1 31 60.1 4 58 NRA 4 12.6 9.2 26.6 2 58 NRA 5 76 NRA 1 84.2 59.2 177.9 2 76 NRA 2 115 119.4 235.1 8.3 76 NRA 3 105 471.9 64.1 3.2 76 NRA 4 76 NRA 5 103 NRA 1 53.6 49.7 147.7 1.9 103 NRA 2 81.2 101.6 188.8 15.4 103 NRA 3 103 NRA 4 103 NRA 5 107 NRA 1 115.9 104.6 234.6 2.1 107 NRA 2 105.5 153.6 278.8 1.8 107 NRA 3 107 NRA 4 107 NRA 5 114 NRA 1 70.5 74.9 152.5 9.3 114 NRA 2 23.1 39.7 92.6 2.3 114 NRA 3 114 NRA 4 114 NRA 5 115 NRA 1 66.2 56.7 131 1.8 115 NRA 2 101.2 46.8 102.5 2.2 115 NRA 3 115 NRA 4 115 NRA 5 119 NRA 1 154.6 99.8 268.6 9.7 119 NRA 2 302.1 99.9 230.3 8.5 119 NRA 3 119 NRA 4 119 NRA 5 123 NRA 1 123 NRA 2 106.3 60.1 164.8 1.2 123 NRA 3

101

Foal Group Time Point Total IgG IgGa IgGb IgGt 123 NRA 5 126 NRA 1 126 NRA 2 48.2 59.1 130.6 3 126 NRA 3 126 NRA 4 126 NRA 5 130 NRA 1 130 NRA 2 72.5 109.9 219.6 1.8 130 NRA 3 130 NRA 4 130 NRA 5 10 NRA 1 83.2 69 186.8 9.3 10 NRA 2 49.5 40.2 124.2 4.3 10 NRA 3 46.3 26.7 43.7 2.3 10 NRA 4 12 6.8 18.3 1.7 10 NRA 5 43 NRA 1 37.1 33 118.4 2.3 43 NRA 2 43 NRA 3 16.8 10.8 29.1 0.9 43 NRA 4 4.1 3.3 7 1.4 43 NRA 5 46 NRA 1 66.2 63.9 214 5.3 46 NRA 2 40.8 16.9 46.1 2.4 46 NRA 3 2.7 2.7 5 1.5 46 NRA 4 46 NRA 5 48 NRA 1 53.5 34.5 109.7 2.2 48 NRA 2 44.4 33.9 82.2 2.1 48 NRA 3 9.4 4.4 9.3 1.4 48 NRA 4 48 NRA 5 50 NRA 1 66.6 51.3 157.5 6.1 50 NRA 2 66.3 30.6 99 2.4 50 NRA 3 24.3 28.2 27.3 12.2 50 NRA 4 50 NRA 5 51 NRA 1 0.8 1.3 2.4 1.2 51 NRA 2 0.8 1.4 1.9 1.5 51 NRA 3 51 NRA 4 51 NRA 5 82 NRA 1 39.7 50.3 108.8 2.7 82 NRA 2 39.9 31.5 85.2 2.2 82 NRA 3 19.7 34.3 36.1 3.1

102

Foal Group Time Point Total IgG IgGa IgGb IgGt 82 NRA 5 85 NRA 1 70.3 38 89.5 2 85 NRA 2 12.6 18.4 25.6 1.5 85 NRA 3 10.1 5.8 15.7 1.4 85 NRA 4 3.4 3.4 8.8 1 85 NRA 5 104 NRA 1 102.7 84 154.9 1.8 104 NRA 2 35.2 30.1 51 1.4 104 NRA 3 22 30.6 73.1 1.4 104 NRA 4 6.8 6 15.3 1.1 104 NRA 5 11 NRA 1 76.8 47.6 124.4 1.7 11 NRA 2 89.1 122.2 426.6 2.4 11 NRA 3 109.7 62.8 262.4 1.8 11 NRA 4 11 NRA 5 55 NRA 1 120.9 76.7 282.9 1.7 55 NRA 2 109.7 84.9 206.5 6.4 55 NRA 3 91 46.3 99.4 2.9 55 NRA 4 17 16.4 35.1 2.3 55 NRA 5 90 NRA 1 113 56.3 157.8 1.8 90 NRA 2 83.8 99 182 2 90 NRA 3 13.9 33.8 34.4 36.7 90 NRA 4 90 NRA 5 108 NRA 1 113.5 83.4 220.1 7.5 108 NRA 2 110.8 153.6 337 3.6 108 NRA 3 57.5 29.1 79.8 2.2 108 NRA 4 108 NRA 5 110 NRA 1 110 NRA 2 25.2 63.6 127.5 2.3 110 NRA 3 40.3 57.3 157.3 2.5 110 NRA 4 20.5 18 68.5 1.3 110 NRA 5 13 NRA 1 94.4 156.3 365.2 3 13 NRA 2 109.8 76.7 292.5 2 13 NRA 3 2.7 2.9 5.3 1.4 13 NRA 4 5.4 6.6 7.4 14.5 13 NRA 5 27 NRA 1 1.3 1.8 3.5 1.3 27 NRA 2 1.5 1.8 3.2 1.5 27 NRA 3 5.2 14.1 9.3 66.6

103

Foal Group Time Point Total IgG IgGa IgGb IgGt 27 NRA 5 29 NRA 1 175.1 129.9 262.5 1.4 29 NRA 2 53.4 43 63.7 1.5 29 NRA 3 29 NRA 4 29 NRA 5 59 NRA 1 124.4 83.6 208.3 2.3 59 NRA 2 66.1 35.5 97.2 1.6 59 NRA 3 16 15.1 31.8 2 59 NRA 4 11.7 11 16.5 2.2 59 NRA 5 5 3.7 7.8 2.2 60 NRA 1 0.9 1.6 2.5 1.4 60 NRA 2 0.9 1.7 2.1 1.4 60 NRA 3 1.3 3.8 2.2 7.6 60 NRA 4 2.2 6.4 4.2 9.1 60 NRA 5 80 NRA 1 47.1 25.4 64.1 4.1 80 NRA 2 19 37.7 94.7 2.4 80 NRA 3 80 NRA 4 80 NRA 5 89 NRA 1 155.3 126.1 473.4 2.9 89 NRA 2 93.1 38.9 45.4 1.9 89 NRA 3 37.7 18.4 23.2 2 89 NRA 4 89 NRA 5 94 NRA 1 78.8 51.6 103.7 8 94 NRA 2 121.5 114.1 201 6.7 94 NRA 3 35.1 20.9 50.7 2.5 94 NRA 4 94 NRA 5 95 NRA 1 105.4 100.9 170.6 3 95 NRA 2 70.5 45.4 174.8 2.6 95 NRA 3 70.1 60.9 179.1 2 95 NRA 4 95 NRA 5 6 NRA 1 38.3 43 209.5 1.9 6 NRA 2 53.8 65.9 377 2.4 6 NRA 3 101.9 49.9 138.1 1.6 6 NRA 4 11.8 7.2 23.1 1.3 6 NRA 5 17 NRA 1 160.9 60.7 164.4 1.8 17 NRA 2 104 73.9 225.8 6 17 NRA 3 100.8 52.1 103.7 2.3

104

Foal Group Time Point Total IgG IgGa IgGb IgGt 17 NRA 5 35 NRA 1 43.5 107 207.1 2.6 35 NRA 2 154.3 72.9 185.1 2.5 35 NRA 3 28.3 54.2 76.5 1.6 35 NRA 4 33.9 16.8 47.7 1.4 35 NRA 5 111 NRA 1 76.6 72.8 151.4 10.1 111 NRA 2 50.4 55.9 117.9 3.2 111 NRA 3 46.3 37 86.1 2.4 111 NRA 4 16.9 15.9 43.3 2.2 111 NRA 5 112 NRA 1 84.7 52.5 198.3 8 112 NRA 2 26.9 35.4 119.8 3.1 112 NRA 3 112 NRA 4 112 NRA 5 134 NRA 1 21.3 18 45.6 1.3 134 NRA 2 134 NRA 3 134 NRA 4 134 NRA 5

105

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VITA

FERNANDA BICUDO CESAR

ACADEMIC EDUCATION

Auburn University, Auburn, AL M.S., Clinical Pharmacology, July 2014 Thesis Title: “Disposition of levetiracetam in healthy adult horses” Advisor: Allison J. Stewart, BVSc, MS, DACVIM, DACVECC

University of Brasília, Brasilia, DF (Brazil) DVM (Doctor of Veterinary Medicine), July 2006

BOARD CERTIFICATION

American College of Veterinary Internal Medicine (ACVIM) Board Certified in Large Animal Internal Medicine, August, 2014

PROFESSIONAL EXPERIENCE

Large Animal Teaching Hospital, Auburn University, Auburn, AL Equine Internal Medicine Resident 2011 - 2014

Equine Medical Associates, Lexington, KY Intern in Equine Ambulatory Practice 2010 Clinical Mentor: James P. Morehead, DVM

Woodford Equine Hospital, Lexington, KY Intern in Equine Internal Medicine 2008 - 2010 Clinical Mentor: Lucas G. Pantaleon, DVM, MS, DACVIM

Equi-Vet, L.L.C, New Orleans, LA and Lexington, KY Intern in Equine Sports Medicine 2008 Clinical Mentor: Jay Addison, DVM

Patricia Brossi, DVM, MS, PhD - Sao Paulo, Brazil Equine Veterinarian 2007 – 2008

Sao Paulo Jockey Club Equine Hospital, Sao Paulo, SP (Brazil) Intern in Equine Medicine and Surgery 2006 – 2007

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RESEARCH GRANTS

Grayson-Jockey Club Research Foundation, Inc. Cesar, F.B., Horohov, D.W. Do IgGt antibodies identify foals at risk for rhodococcal pneumonia? February, 2016. $62,407

Animal Health and Disease, College of Veterinary Medicine, Auburn University Cesar, F.B., Stewart, A., Boothe, D.M., Ravis, W.A., Duran, S.H., Wooldridge, A.A., Wilborn, R.R. Disposition of levetiracetam in healthy adult horses. 2012-2014. $30,000.

National Council of Technological and Scientific Development, College of Veterinary Medicine, University of Brasilia Cesar, F.B., Selmi, A.L. Comparison between the analgesic efficacy of carprofen, vedaprofen and ketoprofen for post-operatory pain control in female dogs submitted to ovariosalpingohysterectomy. 2001-2002

PER REVIEWED PUBLICATIONS

Cesar, F.B., Stewart, A., Boothe, D.M., Ravis, W.A., Duran, S.H., Wooldridge, A.A. and Wilborn, R.R. (2017) Disposition of levetiracetam in healthy adult horses. Journal of Veterinary Pharmacology and Therapeutics, May 15, 2017. DOI: 10.1111/jvp.12417.

Cesar, F.B., Paudel, S., Horohov, D. (2016) The use of IgGt as a diagnostic tool in foals with naturally acquired Rhodococcus equi pneumonia. In: 2016 CRWAD proceedings.

Adams, A.A., Siard, M.H., Reedy, S.E., Braker, D., Elzinga, S., Sanz, M., Cesar, F.B., Lawson, C., Tucker, C., Mulholland, M., Horohov, D., Urschel, C., Ireland, J. Evaluating seasonal influences on hormone responses to a diagnostic test (thyrotropin-releasing hormone stimulation) advocated for early diagnosis of pituitary pars intermedia dysfunction. In: 2016 AAEP proceedings 62; 504-505.

Cesar, F.B., Joiner, K.S., Albanese, V.A., Groover, E.S., Waguespack, R.W. (2016) Osteoblastic osteosarcoma of the paranasal sinuses of an one-year-old American Quarter Horse colt. Journal of the American Veterinary Medical Association - Pathology in Practice, 248(7).

Cesar, F.B., Stewart, A., Boothe, D.M., Ravis, W.A., Duran, S.H., Wooldridge, A.A. and Wilborn, R.R. (2013) Disposition of levetiracetam in healthy adult horses. Journal of Veterinary Internal Medicine. In: 2013 ACVIM Forum Research Abstracts Program 27 (3); 604-756.

Mastrorilli, C., Cesar, F.B., Joiner, K.S, Wooldridge, A.A., Christopherson, P.W. Disseminated lymphoma with large granular lymphocyte morphology diagnosed in a horse via abdominal fluid and transtracheal wash cytology. Veterinary Clinical Pathology, May 4, 2015. DOI: 10.1111/vcp.12262.

Schumacher, J., DeGraves, F., Cesar, F., Duran, S. (2013) Efficacy of ketamine hydrochloride administered as a basilar sesamoid nerve block in alleviating foot pain caused by natural disease. Equine Veterinary Journal, 46(5), 639-641.

Barba, M., Barret, L., Cesar, F.B., Caldwell, F., Schumacher, J. (2013) Management of enterocutaneous fistula associated with an umbilical hernia in a two-year-old horse. Veterinary Records Case Reports, online publication November, 2013. DOI: 10.1136/vetreccr-2013-000014.

Cesar, F.B., Johnson, C.R. and Pantaleon, L.G. (2010) Suspected idiopathic intestinal lymphangiectasia in two foals with chylous peritoneal effusion. Equine Veterinary Education 22 (4); 172-178.

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HONOURABLE MENTIONS

American College of Veterinary Internal Medicine Resident Research Award, June 2013 Cesar, F.B., Stewart, A., Boothe, D.M., Ravis, W.A., Duran, S.H., Wooldridge, A.A. and Wilborn, R.R. Disposition of levetiracetam in healthy adult horses.

Auburn University Certificate of Achievement for Academic Excellence as Outstanding Graduate Student, July 2013

Phi Zeta Honor Society induction, October 2013

III International Symposium of the Athlete Horse Best Published Paper, April 2007 Cesar, F.B. and Laguna-Legorreta, G.G. (2007) Retrospective study of the fatal and non-fatal catastrophic injuries during Thoroughbred races at Soao Paulo’s Jockey Club racetrack between l996 and 2006. In: Proceedings III International Symposium of the Athlete Horse. Belo Horizonte, MG.

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RHODOCOCCUS EQUI&lt;/Em&gt; in the FOAL – IMPROVING DIAGNOSTIC

RHODOCOCCUS EQUI</Em> in the FOAL – IMPROVING DIAGNOSTIC