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Master of Science / Department of Animal Sciences and Industry / Joann M. Kouba / The objectives of these studies were to (1) characterize mare milk and fecal bacteria, and foal fecal microbiota from birth to 4 mo and (2) determine the effect of weaning and weaning method on foal fecal bacterial composition. Next generation sequencing of the V4 region of the bacterial 16S rRNA gene was performed using the Illumina Miseq according to Earth Microbiome Project protocols and sequencing data was analyzed using QIIME. In experiment 1, mare milk, mare fecal, and foal fecal samples were collected from 9 mare and foal pairs at birth (d 0), d 2, 7, and 1, 2, 3 and 4 mo. In experiment 2, 9 foals were separated into 2 treatments: abrupt (n = 5) and gradual (n = 4) weaning methods. Fecal samples were collected the day before weaning (d-1), the day of weaning (d 0) and post-weaning on d 1, 2, 3, 4, and 7. Blood was collected for analysis of cortisol concentration at 0800 h on d -1, 1, 2, and at 0800 h and 1100 h on d 0 and 4. Heart rate was recorded in 10 min intervals on the day of weaning starting 1 h before weaning to 2 h post-weaning, and again for 1 h starting 24 h after weaning. Results from experiment 1 showed newborn foal meconium and mare milk were similar in species diversity and composition; however, large shifts in composition and increases in foal fecal bacterial diversity occurred within the first week. By 1 mo, foal fecal bacterial composition did not differ in composition from mare feces at the phylum level (P = 1.0). Firmicutes, Bacteriodetes, Verrucomicrobia, and Spirochaetes were the dominant phyla found in feces of foals 1 mo and older and adult mare feces. For experiment 2, there were no differences in species diversity (P > 0.05) or separations in bacterial community structure between weaning methods or before and after weaning. There were minor shifts in relative abundance of specific phyla and genera in response to weaning. Foals in the abrupt treatment group had increased cortisol concentrations on d 1 (P < 0.05) and increased heart rate for 50 min after weaning on d 0 (P < 0.05). The foal is born with fecal microbial communities similar to milk that rapidly change during the first week of life, reaching the same composition of its dam by the first month. The foal fecal microbiota matures prior to weaning, therefore weaning did not cause drastic changes in bacterial composition. Although acute stress was increased in abruptly weaned foals, stress associated with different weaning methods did not influence the fecal microbiota within the first week post-weaning.

The purpose of this study was to determine the safety and pharmacokinetic profile of firocoxib in healthy neonatal foals. Foals are more sensitive to the side effects of nonsteroidal anti-inflammatory drugs, (NSAIDs), particularly due to immature renal clearance mechanisms and ulcerogenic effects on gastric mucosa. Firocoxib, a novel second generation NSAID, is reported to have reduced side effects due to its COX-2 selectivity. The pharmacokinetic profile of firocoxib in neonates has not been established, making reliable dosing difficult. We hypothesized that firocoxib given per os at the labeled dose to neonatal foals would be absorbed and not be associated with clinically significant adverse events. Seven healthy American Quarter Horse foals of mixed gender were administered 0.1mg/kg firocoxib orally q24h for nine consecutive days, commencing at 36h of age. Blood samples were collected for firocoxib analysis using high pressure liquid chromatography with fluorescence detection at 0 (dose #1 only), 0.25, 0.5, 1, 2, 4, 8, 16 and 24 hours after doses #1, 5 and 9. For all other doses (2, 3, 4, 6, 7 and 8) blood was collected immediately prior to the next dose (24 hour trough). Elimination samples (36, 48, 72, 96, 120 and 144 hours) were collected after dose #9. Safety was assessed via physical examinations, changes in body weight, gastroscopy, complete blood count, serum biochemistry and urinalysis. Firocoxib was rapidly absorbed following oral administration with minimal accumulation after repeat dosing. After the initial dose, an average peak serum concentration (Cmax) of 89.50 ° 53.36 ng/mL (mean ° SD) was achieved (Tmax) in 0.54 ° 0.65 hours. Steady state was obtained after approximately 4 doses and the average maximum concentration (Cavg) in serum was 39.1 ° 8.4 ng/mL. After the final dose, the mean terminal half-life (T½?») was 10.46 ° 4.97 hours. Firocoxib was not detected in plasma 72 hours after the final dose (<2ng/mL). Bioavailability could not be determined as currently, there is no accompanying intravenous dose of firocoxib for this age group to permit the calculation. No significant abnormalities were noted on blood work, urinalysis or gastroscopy. This study demonstrated that firocoxib is absorbed after oral administration in neonatal foals with no observable adverse effects after multiple doses. / Master of Science

The objectives of these two studies were to characterize normal growth and resultant changes in conformation and walk kinematics of warmblood foals. The first study quantified linear and angular conformation changes of 13 warmblood foals during the first 9 mo of growth. An objective photographic method of evaluating conformation was used to obtain all data. All linear measurements increased significantly over the investigated ages and growth rates were highest in the first 2 mo of growth. Total percentage of growth during the study was greatest for neck and back length. Distal limb growth was minimal over the investigated ages. Metacarpal growth slowed earlier than many other traits. Length of the metatarsus increased minimally during the studied ages with significant growth occurring only between 23 wk and post-weaning measurements. Increasing wither heights were positively associated with increases in scapula, humerus, radius, ilium, femur, tibia and metatarsal and metacarpal lengths. Angular conformation also changed significantly during growth. Trends in angular changes were generally less clear than those for linear variables. Scapula, femur and hock angles significantly increased and humerus angle decreased with age. Utilizing a plumb line from the hock upward, the distance of the hindlimb behind the body was quantified. The distance out behind decreased significantly between 1 and 15 wk. Distance out behind was positively correlated with tibia angle at all ages. The second study quantified linear and temporal kinematics of the walk in growing foals. Nine warmblood foals from the first study were filmed as they walked over a uniform concrete surface covered in 13mm thick rubber matting. Speed was controlled through the use of a uniform handler with a metronome. Trait means at ages 3, 11, 21 wk and post-weaning were compared. Length variables were standardized to percent of total stride length. Temporal variables were standardized to percent of stride duration. Stride length and duration increased significantly with age. Step lengths, stance duration and protraction and retraction durations did not change across ages. Over-stride decreased significantly with age, potentially due to increased back length in older foals. Linear distance between diagonal hooves during stance increased with age, and was negatively correlated to the decrease in over-stride. While older foals appeared to display a more regular, 4-beat walk rhythm, timing between lateral and diagonal footfalls remained significantly different at all ages. Both conformation and kinematics changed during growth. Characterizing conformational changes due to growth can allow a better understanding of how foal conformation and gait change during growth and may predict these traits in adults, thus allowing selection of top performance prospects at a younger age. / Master of Science

Master of Science / Department of Clinical Sciences / Elizabeth Davis / Differences in pharmacokinetics and drug disposition exist between young and adult animals which become especially important for drugs with a narrow therapeutic index. While the pharmacokinetics and plasma concentrations of intravenous lidocaine have been studied in adult horses, determination of the disposition in foals is necessary before appropriate clinical use can be determined. This study examined the disposition of intravenous lidocaine in healthy (phase I) and hospitalized (phase II) foals. Phase I consisted of 6 healthy 4-10 week old foals administered a 6-hour intravenous lidocaine infusion. Phase II consisted of 8 hospitalized foals (2-136 days old) administered intravenous lidocaine. A bolus (1.3 mg/kg) of lidocaine was administered intravenously to all foals followed by a 50 µg/kg/min infusion. Plasma lidocaine and monoethylglycinexylidide (MEGX) concentrations were determined. In phase I, plasma lidocaine concentrations remained below the suggested adult target range of 1-2 µg/mL with MEGX concentrations approximately half that of the parent drug. Total body clearance of lidocaine was 72.2 ± 7.8 mL/min/kg, elimination half-life (t₁/₂) was 26.3 ± 3.7 min, peak concentration (C[subscript]m[subscript]a[subscript]x) was 0.79 ± 0.07 µg/mL, and the volume of distribution (V[subscript]d) was 1.8 ± 0.4 L/kg. The C[subscript]m[subscript]a[subscript]x for MEGX was 0.36 ± 0.11 µg/mL, t₁/₂ was 60 ± 6 min and time to peak concentration (T[subscript]m[subscript]a[subscript]x) was 279.6 ± 90.3 min. In phase II, the severely compromised foals that were eventually euthanized had the largest fluctuations in plasma lidocaine and MEGX concentrations; foals that were discharged from the hospital had plasma concentrations below the target adult range similar to foals in phase I. In conclusion, despite low plasma lidocaine concentrations, the clinical benefits observed in foals may be due to the presence of metabolites. Further research in a larger population of unhealthy foals is required before comprehensive dosing recommendations can be made.

Rhodococcus equi is a common cause of severe pneumonia in foals, and is an opportunistic pathogen in immunocompromised humans. In combination, erythromycin and rifampin are the most commonly used antimicrobials in treating R. equi in foals. To provide reliable treatment, it is imperative to determine the mean inhibitory concentrations (MICs) of other antimicrobial agents in the event that certain strains of R. equi develop resistance to the current treatment. Several strains of R. equi have developed resistance to various antibiotics. In this study, R. equi strain 288 was completely resistant to rifampin with a MIC > 256ug/ml. The MICs of ethambutol, clarithromycin, azithromycin, isoniazide, ethionamide, rifampin, erythromycin, and linezolid of ninety-five R. equi isolates were also determined in this study. These isolates were obtained from the lungs and transtracheal washes of foals. In addition to these strains, three National Committee for Laboratory Clinical Standards (NCCLS) quality control strains were also tested: R. equi ATCC 6939, R. equi ATCC 33701, and S. pneumoniae 49619. Each drug was tested in triplicate and the MIC 50’s and MIC 90’s were determined for each drug. Ethambutol, isoniazide, and ethionamide were completely ineffective against R. equi. with MICs > 250ug/ml. Rhodococcus equi strains were more susceptible to clarithromycin (MIC 90 = 0.23 ug/ml) than to azithromycin (MIC 90 = 2.33 ug/ml), rifampicin (MIC 90 = 0.67ug/ml), erythromycin (MIC 90 = 1.2ug/ml), and linezolid (MIC 90 = 4ug/ml).

Blood was obtained from 5 adult horses and 16 juvenile horses (foals) at the time of birth and subsequently at 2-, 4-, and 8-weeks of age. Neutrophils from adult horses were purified and incubated for 2 h and 4 h with media, avirulent R. equi, virulent R. equi, or recombinant-human granulocyte-macrophage colony stimulating factor (rhGM-CSF). Neutrophils from foals were purified and incubated for 2 h and 4 h with media or virulent R. equi. Total RNA was extracted from both adult and foal neutrophils immediately after purification to measure baseline expression levels (0 h), and immediately after each of the prescribed incubation times. For each sample, 1 µg of total RNA was reverse-transcribed and analyzed for differential gene expression using real-time PCR. After 2 h and 4 h incubation with virulent or avirulent R. equi, neutrophils from adult horses expressed significantly (P< 0.05) greater TNFα, IL-12p40, IL-6, IL-8, and IL-23p19 mRNA relative to expression by unstimulated neutrophils, but not IFNγ or IL-12p35 mRNA. Furthermore, virulent R. equi induced significantly greater IL-23p19 mRNA expression than avirulent R. equi. Stimulation with rhGM-CSF of adult equine neutrophils failed to induce significant changes in cytokine expression. In foal neutrophils, stimulation with virulent R. equi induced significantly greater expression of IFNγ, TNFα, IL-6, IL-8, IL-12p40, and IL-12p35 mRNA relative to expression by unstimulated neutrophils. Furthermore, there were significant effects of age on expression of IL-6, IL-8 and IL-12p40 mRNA. Neutrophil mRNA expression of IL-6 and IL-8 in newborn foals was significantly greater than expression at 2-, 4-, and 8-weeks of age. There was no significant difference between unstimulated and R. equi-stimulated neutrophils from newborn and 2-week-old foals in expression of IL-12p40; however, expression of IL-12p40 by R. equi-stimulated neutrophils from 4- and 8-week-old foals was significantly greater than expression by unstimulated neutrophils. These results demonstrate that R. equi-stimulated neutrophils are a source of many pro-inflammatory cytokines, and that the magnitude of this expression with respect to IL-6, IL-8, and IL-12p40 mRNA expression was influenced by age. Collectively, the data presented indicate a non-phagocytic role for neutrophils that may influence the type of adaptive immune response to R. equi.

Blood was obtained from 5 adult horses and 16 juvenile horses (foals) at the time of birth and subsequently at 2-, 4-, and 8-weeks of age. Neutrophils from adult horses were purified and incubated for 2 h and 4 h with media, avirulent R. equi, virulent R. equi, or recombinant-human granulocyte-macrophage colony stimulating factor (rhGM-CSF). Neutrophils from foals were purified and incubated for 2 h and 4 h with media or virulent R. equi. Total RNA was extracted from both adult and foal neutrophils immediately after purification to measure baseline expression levels (0 h), and immediately after each of the prescribed incubation times. For each sample, 1 µg of total RNA was reverse-transcribed and analyzed for differential gene expression using real-time PCR. After 2 h and 4 h incubation with virulent or avirulent R. equi, neutrophils from adult horses expressed significantly (P< 0.05) greater TNFα, IL-12p40, IL-6, IL-8, and IL-23p19 mRNA relative to expression by unstimulated neutrophils, but not IFNγ or IL-12p35 mRNA. Furthermore, virulent R. equi induced significantly greater IL-23p19 mRNA expression than avirulent R. equi. Stimulation with rhGM-CSF of adult equine neutrophils failed to induce significant changes in cytokine expression. In foal neutrophils, stimulation with virulent R. equi induced significantly greater expression of IFNγ, TNFα, IL-6, IL-8, IL-12p40, and IL-12p35 mRNA relative to expression by unstimulated neutrophils. Furthermore, there were significant effects of age on expression of IL-6, IL-8 and IL-12p40 mRNA. Neutrophil mRNA expression of IL-6 and IL-8 in newborn foals was significantly greater than expression at 2-, 4-, and 8-weeks of age. There was no significant difference between unstimulated and R. equi-stimulated neutrophils from newborn and 2-week-old foals in expression of IL-12p40; however, expression of IL-12p40 by R. equi-stimulated neutrophils from 4- and 8-week-old foals was significantly greater than expression by unstimulated neutrophils. These results demonstrate that R. equi-stimulated neutrophils are a source of many pro-inflammatory cytokines, and that the magnitude of this expression with respect to IL-6, IL-8, and IL-12p40 mRNA expression was influenced by age. Collectively, the data presented indicate a non-phagocytic role for neutrophils that may influence the type of adaptive immune response to R. equi.

Rhodococcus equi is a common cause of severe pneumonia in foals, and is an opportunistic pathogen in immunocompromised humans. In combination, erythromycin and rifampin are the most commonly used antimicrobials in treating R. equi in foals. To provide reliable treatment, it is imperative to determine the mean inhibitory concentrations (MICs) of other antimicrobial agents in the event that certain strains of R. equi develop resistance to the current treatment. Several strains of R. equi have developed resistance to various antibiotics. In this study, R. equi strain 288 was completely resistant to rifampin with a MIC > 256ug/ml. The MICs of ethambutol, clarithromycin, azithromycin, isoniazide, ethionamide, rifampin, erythromycin, and linezolid of ninety-five R. equi isolates were also determined in this study. These isolates were obtained from the lungs and transtracheal washes of foals. In addition to these strains, three National Committee for Laboratory Clinical Standards (NCCLS) quality control strains were also tested: R. equi ATCC 6939, R. equi ATCC 33701, and S. pneumoniae 49619. Each drug was tested in triplicate and the MIC 50’s and MIC 90’s were determined for each drug. Ethambutol, isoniazide, and ethionamide were completely ineffective against R. equi. with MICs > 250ug/ml. Rhodococcus equi strains were more susceptible to clarithromycin (MIC 90 = 0.23 ug/ml) than to azithromycin (MIC 90 = 2.33 ug/ml), rifampicin (MIC 90 = 0.67ug/ml), erythromycin (MIC 90 = 1.2ug/ml), and linezolid (MIC 90 = 4ug/ml).

While Rhodococcus equi (R. equi) remains the most common cause of subacute or chronic granulomatous bronchopneumonia in foals, development of a relevant model to study this bacterium has proven difficult. As a result, the reasons for the underlying foal’s susceptibility to this disease are not well understood. Furthermore, data regarding the immune response of foals to R. equi infection remains controversial. We hypothesized that foals are susceptible to R. equi early in life and that this susceptibility decreases with age. Also, we hypothesized that specific subclasses of IgG antibodies to the virulence-associated protein of R. equi, VapA, predict the outcome of exposure. The objectives of this study were: (1) to develop an R. equi challenge model that resulted in slow progressive disease in some foals as well as spontaneous regression of lesions in others, (2) using the developed model, to investigate the age-related susceptibility of young foals to R. equi, (3) to describe the humoral immune response of foals following experimental challenge and natural infection. The use of a low dose of R. equi to challenge neonatal foals resulted in slow, progressive disease characterized by pulmonary abscessation and spontaneous regression in approximately 50% of the foals. When this low dose was used in 1, 2 or 3-week-old foals, a marked decrease in disease susceptibility was observed as the foals aged. The immunological responses seen after experimental challenge reflect those observed after natural infection. While there was a significant increase of VapA-specific IgG and IgG subclasses over time in both pneumonic and healthy foals, use of VapA-specific IgG(T) showed good sensitivity and specificity when used as a diagnostic tool for R. equi pneumonia. In summary, this study shows that foal susceptibility to R. equi occurs early in life and decreases with age. Whereas all foals developed VapA-specific IgG antibodies post-exposure, IgG(T) appeared to be predictive of infection.