Effects of intravenous administration of ascorbic acid (vitamin C) on oxidative status in healthy adult horses.

BACKGROUND: Ascorbic acid (AA) is an antioxidant that might be beneficial for adjunctive treatment of sepsis in horses. The optimal dose and effects on oxidative status are unknown. HYPOTHESIS: Ascorbic acid administration will increase plasma AA concentrations and decrease determinants of reactive oxygen metabolites (dROM), basal and stimulant-induced intraerythrocytic reactive oxygen species (ROS) concentrations, and stimulant-induced neutrophil ROS production, and increase plasma antioxidant capacity (PAC) in a dose-dependent manner. ANIMALS: Eight healthy horses. METHODS: Randomized placebo-controlled crossover study. Each horse received 4 single-dose IV treatments including AA at 25, 50, and 100 mg/kg and saline (placebo) with each treatment separated by ≥1 week. Blood was collected at baseline, 2 and 6 hours for assessment of plasma dROM and PAC via photometer, intraerythrocytic ROS by flow cytometry, and stimulant-induced neutrophil ROS by a fluorometric assay. Plasma AA concentrations were measured by high-performance liquid chromatography/electrochemical detection. RESULTS: Ascorbic acid at 100 mg/kg resulted in decreased dROM 2 hours after treatment (P = .03, 95% CI 5.51-121.2, point estimate 63.3). There was no effect of AA on basal or stimulant-induced intraerythrocytic ROS (P = .88, 95% CI -0.156 to 0.081, point estimate -0.037; P = .93, 95% CI -0.123 to 0.112, point estimate -0.006, respectively), basal or stimulant-induced neutrophil ROS (P ≥ .12, 95% CI -644.9 to 56.2, point estimate -294.4), or PAC (P ≥ .64, 95% CI -1567 to 463.4, point estimate -552.0) at any dose or timepoint. Plasma AA concentrations increased in a dose-dependent manner. CONCLUSIONS AND CLINICAL IMPORTANCE: High-dose administration of AA might provide antioxidant benefits in horses.

Validation of a commercially available photometric analytical system for assessment of plasma oxidative status in healthy horses.

Reactive oxygen species (ROS) are the end-products of physiologic functions in health. Oxidative stress occurs when endogenous antioxidants are insufficient to neutralize ROS in the system. As a result, ROS can damage DNA, RNA, proteins, lipids, and cell organelles. To obtain accurate measurements of plasma oxidative stress, levels of both oxidants and antioxidants must be measured. This study validates a commercially available, semi-quantitative, photometric analytical system that measures systemic determinants of reactive oxygen metabolites (dROM) and plasma antioxidant capacity (PAC) in stored equine plasma. The objectives of this work were: 1) to validate a photometric analytical system to quantify dROM and PAC in equine plasma; and 2) to determine expected results for these tests in healthy adult horses. We hypothesized that this system would reliably and reproducibly assess dROM and PAC in equine plasma. We observed expected, dose-dependent increases in dROM generated by adding increasing concentrations of H2O2 or ascorbic acid to equine plasma to provide samples containing a known quantity of oxidants or antioxidants respectively. Mean dROM value in healthy horses was 103.3 ±20.7 U. Carr and mean PAC was 2881.0 ± 313.9 U. Cor. This system reliably and reproducibly quantified dROM and PAC in equine plasma samples.

Equine blood cultures: Can we do better?

Blood culture is considered the gold standard test for documenting bacteraemia in patients with suspected bacterial sepsis in veterinary and human medicine. However, blood culture often fails to yield bacterial growth even though the clinical picture is strongly suggestive of bacterial sepsis, or contaminating organisms can overgrow the true pathogen, making accurate diagnosis and appropriate management of this life-threatening condition very challenging. Methodology for collecting blood cultures in equine medicine, and even in human hospitals, is not standardised, and many variables can affect the yield and type of microorganisms cultured. Microbiological culture techniques used in the laboratory and specific sample collection techniques, including volume of blood collected, aseptic technique utilised, and the site, timing and frequency of sample collection, all have substantial impact on the accuracy of blood culture results. In addition, patient-specific factors such as husbandry factors, the anatomical site of the primary infection, and changing microflora in different geographic locations, also can impact blood cultures. Thus, blood cultures obtained in practice may not always accurately define the presence or absence of, or specific organisms causing, bacteraemia in horses and foals with suspected sepsis. Erroneous blood culture results can lead to inappropriate antimicrobial use, which can result in poor outcomes for individual patients and contribute to the development of antimicrobial resistance in the patient's microflora and the environmental microcosm. This review summarises current indications and methodology, and specific factors that may be optimised, for equine blood culture, with particular focus on available literature from neonatal foals with suspected bacterial sepsis. To standardise and optimise blood culture techniques in horses and foals, future research in this area should be aimed at determining the optimal volume of blood that should be collected for culture, and the ideal site, timing, and frequency of sample collection.

Pharmacokinetic properties of pergolide mesylate following single and multiple-dose administration in donkeys (Equus asinus).

BACKGROUND: Donkeys with clinical signs of pituitary pars intermedia dysfunction are treated with oral pergolide mesylate despite the lack of species-specific pharmacokinetic data. OBJECTIVE: To evaluate the pharmacokinetics of intragastric and oral pergolide mesylate in healthy donkeys (Equus asinus). STUDY DESIGN: Pharmacokinetic study. METHODS: Six healthy donkeys were administered pergolide mesylate (Prascend®) at 2 µg/kg bodyweight (bwt) intragastrically once, then once daily per os (PO) for 5 days. Blood samples were collected at 0, 10, 20, 30 and 45 min and 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36 and 48 h after the single intragastric dose, once daily immediately before the PO dose, and then again at the above times after Day 5 of once daily oral dosing. Plasma pergolide concentration was quantified via ultra-performance liquid chromatography-tandem mass spectrometry. Pergolide concentration versus time data after the first and last doses were analysed based on noncompartmental pharmacokinetics using commercial software. Paired t-tests were used to compare single and multiple doses (p < 0.05). In a follow-up study, a single oral dose was then administered to two donkeys followed by concurrent blood sampling from the jugular and cephalic veins to evaluate the effect of route of administration on pergolide pharmacokinetics. RESULTS: Cmax , Tmax AUC, and t½λz differed significantly (p ≤ 0.03) after single and multiple doses, with significantly lower Cmax (0.16 ± 0.16 ng/ml) and t½λz (9.74 ± 1.35 h) after intragastric dosing on Day 1 than after 5 days of oral dosing (3.74 ± 2.26 ng/ml and 16.35 ± 5.21 h, respectively). Pergolide plasma concentrations were higher in jugular vein samples compared to cephalic vein samples after a single oral dose. MAIN LIMITATIONS: Small sample size; varied administration routes. CONCLUSIONS: Pergolide mesylate (dosed at 2 µg/kg bwt) is bioavailable in donkeys after intragastric and PO administration. Differences in pharmacokinetics were noted after multiple doses, related to different routes of administration and sublingual absorption of pergolide.

Effects of intravenous administration of peripheral blood-derived mesenchymal stromal cells after infusion of lipopolysaccharide in horses.

BACKGROUND: A systemic and dysregulated immune response to infection contributes to morbidity and mortality associated with sepsis. Peripheral blood-derived mesenchymal stromal cells (PB-MSC) mitigate inflammation in animal models of sepsis. Allogeneic PB-MSC administered IV to horses is well-tolerated but therapeutic benefits are unknown. HYPOTHESIS: After IV lipopolysaccharide (LPS) infusion, horses treated with PB-MSC would have less severe clinical signs, clinicopathological abnormalities, inflammatory cytokine gene expression, and oxidative stress compared to controls administered a placebo. ANIMALS: Sixteen horses were included in this study. METHODS: A randomized placebo-controlled experimental trial was performed. Sixteen healthy horses were assigned to 1 of 2 treatment groups (1 × 109 PB-MSC or saline placebo). Treatments were administered 30 minutes after completion of LPS infusion of approximately 30 ng/kg. Clinical signs, clinicopathological variables, inflammatory cytokine gene expression, and oxidative stress markers were assessed at various time points over a 24-hour period. RESULTS: A predictable response to IV LPS infusion was observed in all horses. At the dose administered, there was no significant effect of PB-MSC on clinical signs, clinicopathological variables, or inflammatory cytokine gene expression at any time point. Antioxidant potential was not different between treatment groups, but intracellular ROS increased over time in the placebo group. Other variables that changed over time were likely due to effects of IV LPS infusion. CONCLUSIONS AND CLINICAL IMPORTANCE: Administration of allogeneic PB-MSC did not cause clinically detectable adverse effects in healthy horses. The dose of PB-MSC used here is unlikely to exert a beneficial effect in endotoxemic horses.

Circulating Hypothalamic-Pituitary-Adrenal Axis Hormones and Insulin Concentrations in Horses and Ponies.

Mechanisms resulting in breed predispositions to insulin dysregulation (ID) are poorly characterized. Cortisol antagonizes insulin, and free, biologically active cortisol can be increased in ID. Breed-related differences in serum free cortisol fraction (FCF) could contribute to ID, but FCF has not been quantified in equidae predisposed to ID, such as ponies. To compare FCF and other hypothalamic-pituitary-adrenal (HPA) axis hormones between horses and ponies during health and ID. We hypothesized: (1) FCF is higher in ponies than horses in health, and is higher still in ponies with ID and obesity; and (2) FCF is positively correlated with insulin in horses and ponies during health and ID. Thirty-three horses and 24 ponies were sampled before morning feeding in their normal routine. Plasma ACTH and insulin and serum total cortisol concentrations and FCF were measured. ID was defined as evidence of hyperinsulinemia at rest or after oral sugar administration. Data were compared with Mann-Whitney tests and Spearman correlation analysis (P < 0.05). Total cortisol, free cortisol, insulin concentrations, and FCF were comparable in healthy horses (n = 24) and ponies (n = 12), but ACTH concentrations were 29% higher in ponies than in horses (P = 0.016). In animals with ID, total cortisol, free cortisol, and insulin concentrations were similar between horses and ponies, but FCF was increased 40% in ponies (n = 12) compared to horses (n = 9). These data demonstrate differences in insulin, ACTH, and free cortisol during health and ID between ponies and horses.

Congenital Cataracts and Microphakia with Retinal Dysplasia and Optic Nerve Hypoplasia in a Calf.

Case Description. A two-month-old, female, Aberdeen-Angus calf was presented for congenital cataracts and blindness in both eyes (OU). The dam had a reported history of visual defects (not specified) and had produced other affected calves (per owner history). Ophthalmic examination revealed mature bilateral cataracts, attenuation of the iridic granules, persistent pupillary membranes, and dyscoric pupils. Additionally, the calf had a poor body condition, prognathism, dome-shaped head, excessive nasal drainage, limb contracture, and fever. Histopathology of both eyes revealed lenticular degeneration (congenital cataracts), retinal dysplasia, and optic nerve hypoplasia. BVDV IHC detected antigen within only the left eye (OS), consisting of intrahistiocytic and endothelial immunoreactivity within the ciliary body, iris, and choroid. No BVDV immunoreactivity could be detected in the right eye (OD). This case highlights the unique ocular changes present in in utero BVDV infection of cattle with a different immunohistochemical staining profile than previously described.

Epidemiology and Molecular Basis of Multidrug Resistance in Rhodococcus equi.

The development and spread of antimicrobial resistance are major concerns for human and animal health. The effects of the overuse of antimicrobials in domestic animals on the dissemination of resistant microbes to humans and the environment are of concern worldwide. Rhodococcus equi is an ideal model to illustrate the spread of antimicrobial resistance at the animal-human-environment interface because it is a natural soil saprophyte that is an intracellular zoonotic pathogen that produces severe bronchopneumonia in many animal species and humans. Globally, R. equi is most often recognized as causing severe pneumonia in foals that results in animal suffering and increased production costs for the many horse-breeding farms where the disease occurs. Because highly effective preventive measures for R. equi are lacking, thoracic ultrasonographic screening and antimicrobial chemotherapy of subclinically affected foals have been used for controlling this disease during the last 20 years. The resultant increase in antimicrobial use attributable to this "screen-and-treat" approach at farms where the disease is endemic has likely driven the emergence of multidrug-resistant (MDR) R. equi in foals and their environment. This review summarizes the factors that contributed to the development and spread of MDR R. equi, the molecular epidemiology of the emergence of MDR R. equi, the repercussions of MDR R. equi for veterinary and human medicine, and measures that might mitigate antimicrobial resistance at horse-breeding farms, such as alternative treatments to traditional antibiotics. Knowledge of the emergence and spread of MDR R. equi is of broad importance for understanding how antimicrobial use in domestic animals can impact the health of animals, their environment, and human beings.

Association between antimicrobial treatment of subclinical pneumonia in foals and selection of macrolide- and rifampicin-resistant Rhodococcus equi strains at horse-breeding farms in central Kentucky.

OBJECTIVE: To compare soil concentrations of macrolide- and rifampicin-resistant Rhodococcus equi strains (MRRE) on horse-breeding farms that used thoracic ultrasonographic screening (TUS) to identify foals with subclinical pneumonia combined with subsequent administration of macrolides and rifampin to affected foals (TUS farms) versus soil concentrations on farms that did not (non-TUS farms), determine whether the combined use of TUS and antimicrobial treatment of subclinically affected foals was associated with soil concentration of MRRE, and assess whether there were temporal effects on soil concentrations of MRRE during the foaling season. SAMPLES: 720 soil samples and 20 completed questionnaires from 20 horse-breeding farms (10 TUS farms and 10 non-TUS farms) in central Kentucky. PROCEDURES: A questionnaire was used to gather information from participating farms about their 2019 foaling season. Soil samples were collected during January, March, May, and July 2019 for bacterial culture and antimicrobial susceptibility testing to identify any isolates of MRRE. Results were compared for TUS farms versus non-TUS farms. Linear mixed-effects modeling was used to evaluate for potential associations between the soil concentration of MRRE and the use of TUS. RESULTS: Overall, the sum of the mean soil concentrations of MRRE was significantly higher for TUS farms (8.85 log10-transformed CFUs/g) versus non-TUS farms (7.37 log10-transformed CFUs/g). CONCLUSIONS AND CLINICAL RELEVANCE: Our findings indicated that farms that use TUS to identify foals with subclinical pneumonia for antimicrobial treatment select for antimicrobial-resistant R equi strains. Because prognosis is worse for foals infected with resistant versus nonresistant strains of R equi, prudent use of antimicrobials to treat foals with subclinical pulmonary lesions attributed to R equi is recommended.

Evidence for anti-inflammatory effects of firocoxib administered to mares with experimentally induced placentitis.

PROBLEM: Minimal evidence exists supporting therapeutic selections for equine placentitis. The goal of this study was to characterize the anti-inflammatory effects of firocoxib when administered to mares with placentitis. METHODS: Mares (gestation D270-300) were assigned to: INFECT (n = 6; placentitis, no treatment), FIRO (n = 6; placentitis, firocoxib, 0.1 mg/kg, PO, daily), and NORM (n = 6; no infection/treatment). Allantoic fluid (8 hours, 24 hours, birth) and amniotic fluid (birth) were collected from mares after infection. Concentrations of IL-1ß, IL-6, TNF-α, IL-10, PGF2α , and PGE2 in fluids were measured by ELISA. mRNA expression of IL-1ß, IL-6, TNF-α, IL-8, IL-10, matrix metalloproteinases (MMPs) -1, 3, and 9 in fetal membranes/fetuses was quantified using real-time PCR. RESULTS: Allantoic TNF-α concentrations were lowest in FIRO at 8 hours and 24 hours post-infection; IL-6 concentrations were lower in FIRO than NORM at 8 hours, lower in FIRO than INFECT at 24 hours post-inoculation, and lower in NORM than FIRO or INFECT at birth. Marginal mean allantoic IL-ß and IL-10 concentrations were lower in FIRO and NORM than INFECT. Amniotic fluid cytokines were lowest in NORM with all measurements in that group being below the limit of detection. Allantoic PGF2α concentrations were lower in FIRO and INFECT than NORM at 8 hours post-inoculation, and lower in FIRO than INFECT or NORM at 24 hours post-inoculation. Allantoic PGE2 concentrations were lower in FIRO than INFECT. Amniotic PGF2α and PGE2 concentrations were lower in NORM than INFECT. In fetal membranes, group differences with respect to IL-1ß, IL-6, IL-8, and MMP1 were dependent on tissue type. CONCLUSIONS: Data suggest a suppressive effect of firocoxib administration on cytokine and prostaglandin production in mares with placentitis.

The effect of foal or adult horse plasma on equine monocyte-derived dendritic cell phenotype and function.

Immunological and endocrine immaturity in foals increases foal morbidity and mortality from bacterial sepsis. Dendritic cells (DC) are critical in activating the adaptive immune response, but foal DC are phenotypically and functionally different than those of adult horses. Age-related variations in availability of some soluble plasma factors, such as hormones, might govern some age-related differences in DC function. Effects of exposure to plasma factors on equine DC phenotype and function have not been described. We hypothesized that exposure to plasma from foals or adult horses would differentially impact monocyte-derived DC (MoDC) phenotype and function. Eight healthy adult horses and 8 healthy foals were divided into pairs of one adult horse and one foal. Blood was collected from each pair for MoDC generation when foals were 1 and 30 days of age. MoDC from horses and foals were then exposed to killed whole-cell bacteria in the presence of their own age-matched plasma, plasma from the opposite-aged animal in the pair, and serum-free medium alone (control). Expression of DC-relevant surface markers (MHC class-II, CD86, and CD14) and endocytosis capability were measured by flow cytometry. Supernatant cytokine concentrations (IL-4, IL-17, IFN-γ, and IL-10) were quantified with a validated bead-based immunoassay. Data were analyzed using linear mixed-effects and Tobit regression models (P < 0.05). The percentage of MoDC expressing surface markers MHC class-II and CD86 was reduced in MoDC derived from 1-day-old foals in comparison to adult horse MoDC when cultured in medium alone or with either source of plasma (P = 0.0001). Foal and adult horse MoDC cultured in either source of plasma expressed more CD86 and less CD14 than cells cultured in serum-free medium alone (P ≤ 0.02). Adult horse and foal MoDC exposed to bacterial antigen in the presence of 1-day-old foal plasma secreted less IL-10 (P ≤ 0.0008) compared to those cultured in adult horse plasma. Endogenous production of IL-17 by MoDC from foals at day 1 of age cultured in adult plasma was increased compared to foal MoDC cultured in serum-free medium (P = 0.004). Phagocytosis of killed, labeled Staphylococcus aureus was reduced when MoDC generated from foals or adult horses were exposed to plasma from foals at day 1 or 30 of age (P ≤ 0.03). Age-related variation in soluble plasma factors appear to regulate equine MoDC function, but specific plasma factors capable of regulating MoDC phenotype or function were not defined in this study.

Variability in peripheral blood enrichment techniques can alter equine leukocyte cellularity, viability and function.

Peripheral blood is commonly sampled to assess the health status of human and veterinary patients. Venous blood collection is a minimally invasive procedure, and in the horse, the common collection site is the jugular vein. Post blood collection, sample processing for leukocyte enrichment can vary by research laboratory with the potential to yield different effects on the enriched cells and their function. The focus of the present study was to compare a common blood dilution-leukocyte enrichment technique using a Histopaque gradient medium (His) to a modified leukocyte buffy coat syringe-lymphocyte separation medium technique (Syr- LSM) with peripheral blood from 12 healthy horses. The endpoints examined included cell recovery/mL of blood, cell viability, leukocyte enrichment purity, leukocyte cell marker subset phenotype, leukocyte spontaneous and mitogen-induced proliferation and secretory TNFα concentrations. Leukocyte cell recovery/mL of whole blood and cell viability was significantly increased in enriched leukocytes from the Syr-LSM technique. Interestingly, the percentage of CD8+ and CD21+ were significantly increased with the His technique as was Con A-induced proliferation. Still, leukocyte cell purity and TNFα concentrations from the 72 h cell culture supernatants were comparable across the two enrichment techniques. To summarize, the type of whole blood leukocyte enrichment technique employed can affect the results of immunologic assay endpoints possibly altering data interpretation.

The novel and transferable erm(51) gene confers macrolides, lincosamides and streptogramins B (MLSB ) resistance to clonal Rhodococcus equi in the environment.

The use of mass antimicrobial treatment has been linked to the emergence of antimicrobial resistance in human and animal pathogens. Using whole-genome single-molecule real-time (SMRT) sequencing, we characterized genomic variability of multidrug-resistant Rhodococcus equi isolated from soil samples from 100 farms endemic for R. equi infections in Kentucky. We discovered the novel erm(51)-encoding resistance to MLSB in R. equi isolates from soil of horse-breeding farms. Erm(51) is inserted in a transposon (TnErm51) that is associated with a putative conjugative plasmid (pRErm51), a mobilizable plasmid (pMobErm51), or both enabling horizontal gene transfer to susceptible organisms and conferring high levels of resistance against MLSB in vitro. This new resistant genotype also carries a previously unidentified rpoB mutation conferring resistance to rifampicin. Isolates carrying both vapA and erm(51) were rarely found, indicating either a recent acquisition of erm(51) and/or impaired survival when isolates carry both genes. Isolates carrying erm(51) are closely related genetically and were likely selected by antimicrobial exposure in the environment.

Phenotypic characterization of equine monocyte-derived dendritic cells generated ex vivo utilizing commercially available serum-free medium.

The impact of culture conditions on equine monocyte-derived dendritic cells (MoDC) generation has not been fully characterized. We hypothesized that 1) MoDC could be cultured in a commercially available serum-free medium (AIM-V); and 2) that differential culture conditions would influence MoDC viability, yield and phenotype. MoDC generated from adult horses were cultured under variable conditions in a series of experiments. Viability was assessed using trypan blue and propidium iodide staining. Yield was determined by manual hemocytometer counting. Phenotype was assessed by flow cytometric analysis of surface markers (MHC class-II, CD86 and CD14). Data were analyzed using paired t-tests and repeated measures ANOVA. Two MoDC populations that differed in size and phenotype were identified: larger MoDC (LgMoDC) and smaller MoDC (SmMoDC). Medium type, plate chemistry, or length of monocyte adhesion time did not impact MoDC viability or yield. LgMoDC generated in serum-free medium expressed more MHC class-II and CD86 (P ≤ 0.03). A prolonged duration in culture reduced MoDC yield (P ≤ 0.04). MoDC can be consistently and reliably generated using AIM-V serum-free medium in standard tissue culture plates with a recommended culture duration of 3-4 days.

Clinical Pathology in the Foal.

The dynamic physiologic changes and unique diet during the neonatal period contribute to key differences in clinicopathologic test results of healthy foals relative to healthy adult horses. When reporting results, most diagnostic laboratories only provide reference intervals for mature horses. Thus, failure to recognize the unique differences that occur in foals relative to adult horses can lead to erroneous interpretation of neonatal clinical pathologic values. Thus, the main objective of this article was to review distinct features of common clinicopathologic tests in foals, relative to mature horses.

Prevalence and risk factors associated with emergence of Rhodococcus equi resistance to macrolides and rifampicin in horse-breeding farms in Kentucky, USA.

The combination of a macrolide and rifampicin has been the mainstay of therapy in foals with Rhodococcus equi pneumonia for decades. Recent studies suggest that mass antimicrobial treatment of subclinically affected foals over time has selected for antimicrobial resistance. Our objective was to estimate the prevalence of R. equi strains resistant to macrolides and rifampicin at horse breeding farms in Kentucky. A hundred breeding farms in Kentucky were surveyed and R. equi were cultured from soil samples. Data were analyzed with logistic regression and generalized linear modeling (P < 0.05). Seventy-six percent (76%) of farms yielded resistant R. equi, and resistance to macrolides and rifampicin was associated with their use at farms. The present study is the first to report the prevalence and distribution of resistant isolates in the environment of farms in Kentucky, USA. Collectively, previous reports and the data presented herein provide irrefutable evidence of emerging antimicrobial resistance in R. equi with alarming prevalence. Widespread dissemination and maintenance of resistance genes in the environment where many other pathogenic bacteria exist is a concern for both animal and human health.

Effect of Macrolide and Rifampin Resistance on Fitness of Rhodococcus equi during Intramacrophage Replication and In Vivo.

The soil-dwelling, saprophytic actinomycete Rhodococcus equi is a facultative intracellular pathogen of macrophages and causes severe bronchopneumonia when inhaled by susceptible foals. Standard treatment for R. equi disease is dual-antimicrobial therapy with a macrolide and rifampin. Thoracic ultrasonography and early treatment with antimicrobials prior to the development of clinical signs are used as means of controlling endemic R. equi infection on many farms. Concurrently with the increased use of macrolides and rifampin for chemoprophylaxis and the treatment of subclinically affected foals, a significant increase in the incidence of macrolide- and rifampin-resistant R. equi isolates has been documented. Previously, our laboratory demonstrated decreased fitness of R. equi strains that were resistant to macrolides, rifampin, or both, resulting in impaired in vitro growth in iron-restricted media and in soil. The objective of this study was to examine the effect of macrolide and/or rifampin resistance on intracellular replication of R. equi in equine pulmonary macrophages and in an in vivo mouse infection model in the presence and absence of antibiotics. In equine macrophages, the macrolide-resistant strain did not increase in bacterial numbers over time and the dual macrolide- and rifampin-resistant strain exhibited decreased proliferation compared to the susceptible isolate. In the mouse model, in the absence of antibiotics, the susceptible R. equi isolate outcompeted the macrolide- or rifampin-resistant strains.

Multiple adrenocortical steroid response to administration of exogenous adrenocorticotropic hormone to hospitalized foals.

BACKGROUND: The hypothalamic-pituitary-adrenal axis regulates the response to sepsis-associated stress. Relative adrenal insufficiency or adrenocorticotropic hormone (ACTH):cortisol imbalance, defined as a poor cortisol response to administration of ACTH, is common and associated with death in hospitalized foals. However, information on other adrenal steroid response to ACTH stimulation in sick foals is minimal. OBJECTIVE: To investigate the response of multiple adrenocortical steroids to administration of ACTH in foals. ANIMALS: Hospitalized (n = 34) and healthy (n = 13) foals. METHODS: In this prospective study, hospitalized foals were categorized into 2 groups using cluster analysis based on adrenal steroids response to ACTH stimulation: Cluster 1 (n = 11) and Cluster 2 (n = 23). After baseline blood sample collection, foals received 10 µg of ACTH with additional samples collected at 30 and 90 minutes after ACTH. Steroid and ACTH concentrations were determined by immunoassays. The area under the curve (AUC) and Delta0-30 were calculated for each hormone. RESULTS: The AUC for cortisol, aldosterone, androstenedione, pregnenolone, 17α-OH-progesterone, and progesterone were higher in critically ill (Cluster 1) compared to healthy foals (P < .01). Delta0-30 for cortisol and 17α-OH-progesterone was lower in Cluster 1 (24%, 26.7%) and Cluster 2 (16%, 11.2%) compared to healthy foals (125%, 71%), respectively (P < .05). Foals that died had increased AUC for endogenous ACTH (269 versus 76.4 pg/mL/h, P < .05) accompanied by a low AUC for cortisol (5.5 versus 15.5 µg/dL/h, P < .05), suggesting adrenocortical dysfunction. CONCLUSION AND CLINICAL IMPORTANCE: The 17α-OH-progesterone response to administration of ACTH was a good predictor of disease severity and death in hospitalized foals.

The effect of age on foal monocyte-derived dendritic cell (MoDC) maturation and function after exposure to killed bacteria.

Neonatal foals are uniquely susceptible to certain infections early in life. Dendritic cells (DC) are vital in the transition between the innate and adaptive immune response to infection, but DC biology in foals is not fully characterized. Monocyte-derived DC represent a suitable in vitro model similar to DC that differentiate from monocytes recruited from circulation. We hypothesized that foal monocyte-derived DC (MoDC) would exhibit age-dependent phenotypic and functional differences compared to adult horse MoDC. MoDC generated from 9 horses (collected once) and from 8 foals (collected at 1, 7, and 30 days-of-age) were exposed to killed whole cell Escherichia coli or Staphylococcus aureus bacteria. MoDC expression of MHC class II (MHC class-II), CD86, and CD14 were measured by flow cytometry, and supernatant cytokine concentrations of IL-4, IL-17, IFN-γ, and IL-10 were quantified with a validated immunoassay. The percentage of MoDC expressing MHC class-II and CD86 was lower and CD14 was higher for cells generated from 1-day-old foals compared to cells generated from adult horses (P < 0.0001). Bacterial exposure increased the percentage of cells expressing CD86 at all ages (P < 0.0001). Bacteria-exposed MoDC from 1-day-old foals produced significantly less IL-4, IL-17, and IFN-γ than adult MoDC produced in response to bacterial exposure (P ≤ 0.04). Following bacterial exposure, foal MoDC phenotype and cytokine secretion were different than those of mature horses. These differences could reduce the ability of foals to generate a protective immune response against bacterial infection.

Effect of Macrolide and Rifampin Resistance on the Fitness of Rhodococcus equi.

Rhodococcus equi is a leading cause of severe pneumonia in foals. Standard treatment is dual antimicrobial therapy with a macrolide and rifampin, but the emergence of macrolide- and rifampin-resistant R. equi isolates is an increasing problem. The objective of this study was to determine the effect of macrolide and/or rifampin resistance on fitness of R. equi Three unique isogenic sets were created, each consisting of four R. equi strains, as follows: a susceptible parent isolate, strains resistant to macrolides or rifampin, and a dual macrolide- and rifampin-resistant strain. Each isogenic set's bacterial growth curve was generated in enriched medium, minimal medium (MM), and minimal medium without iron (MM-I). Bacterial survival in soil was analyzed over 12 months at -20°C, 4°C, 25°C, and 37°C, and the ability of these strains to retain antimicrobial resistance during sequential subculturing was determined. Insertion of the mobile element conferring macrolide resistance had minimal effect on in vitro growth. However, two of three rpoB mutations conferring rifampin resistance resulted in a decreased growth rate in MM. In soil, macrolide- or rifampin-resistant R. equi strains exhibited limited growth compared to that of the susceptible R. equi isolate at all temperatures except -20°C. During subculturing, macrolide resistance was lost over time, and two of three rpoB mutations reverted to the wild-type form. The growth of rifampin-resistant R. equi colonies is delayed under nutrient restriction. In soil, possession of rifampin or macrolide resistance results in decreased fitness. Both macrolide and rifampin resistance can be lost after repeated subculturing.IMPORTANCE This work advances our understanding of the opportunistic environmental pathogen Rhodococcus equi, a disease agent affecting horses and immunocompromised people. R. equi is one of the most common causes of severe pneumonia in young horses. For decades, the standard treatment for R. equi pneumonia in horses has been dual antimicrobial therapy with a macrolide and rifampin; effective alternatives to this combination are lacking. The World Health Organization classifies these antimicrobial agents as critically important for human medicine. Widespread macrolide and rifampin resistance in R. equi isolates is a major emerging problem for the horse-breeding industry and might also adversely impact human health if resistant strains infect people or transfer resistance mechanisms to other pathogens. This study details the impact of antimicrobial resistance on R. equi fitness, a vital step for understanding the ecology and epidemiology of resistant R. equi isolates, and will support development of novel strategies to combat antimicrobial resistance.