Antimicrobial Residue Accumulation Contributes to Higher Levels of Rhodococcus equi Carrying Resistance Genes in the Environment of Horse-Breeding Farms.

Antimicrobial residues excreted in the environment following antimicrobial treatment enhance resistant microbial communities in the environment and have long-term effects on the selection and maintenance of antimicrobial resistance genes (AMRGs). In this study, we focused on understanding the impact of antimicrobial use on antimicrobial residue pollution and antimicrobial resistance (AMR) in the environment of horse-breeding farms. Rhodococcus equi is an ideal microbe to study these associations because it lives naturally in the soil, exchanges AMRGs with other bacteria in the environment, and can cause disease in animals and humans. The environment is the main source of R. equi infections in foals; therefore, higher levels of multidrug-resistant (MDR) R. equi in the environment contribute to clinical infections with MDR R. equi. We found that macrolide residues in the environment of horse-breeding farms and the use of thoracic ultrasonographic screening (TUS) for early detection of subclinically affected foals with R. equi infections were strongly associated with the presence of R. equi carrying AMRGs in the soil. Our findings indicate that the use of TUS contributed to historically higher antimicrobial use in foals, leading to the accumulation of antimicrobial residues in the environment and enhancing MDR R. equi.

Serum activities of complement 1q and antibodies to the virulence-associated protein A are lower in foals that develop rhodococcal pneumonia.

OBJECTIVE: To determine the effects of transfusion of Rhodococcus equi hyperimmune plasma (REHIP) on serum concentrations of complement component 1q (C1q) and to examine the association of serum C1q and anti-rhodococcal antibodies of newborn foals with subsequent development of rhodococcal pneumonia. ANIMALS/SAMPLES: Foals (n = 205) from 2 Thoroughbred breeding farms in New York transfused with REHIP between January 1, 2022, and December 1, 2022. PROCEDURES: Blood was collected immediately before transfusion with REHIP and again from the contralateral vein immediately after transfusion. Foals were followed through weaning for clinical and ultrasonographic evidence of rhodococcal pneumonia. Serum samples were tested by ELISA for concentrations of C1q and for activity of IgG1 and IgG4/7 recognizing the virulence-associated protein A (VapA) of R equi. Logistic regression analysis was used to determine the association between rhodococcal pneumonia and levels of C1q and anti-VapA IgG1 and IgG4/7. RESULTS: REHIP significantly decreased C1q concentrations immediately after transfusion. Accounting for effects of farm and birth month, estimated odds of pneumonia were 2.1-fold (P = .0330) higher for foals with pretransfusion C1q concentrations less than or equal to the population median and 3.3-fold (P = .0051) higher for foals with posttransfusion IgG1 activity in the lower quartile. CLINICAL RELEVANCE: Both C1q and IgG appear to contribute to protection against R equi, and IgG1 appears to be especially important. Increasing IgG1 concentrations targeting rhodococcal proteins in REHIP or serum of foals might improve protection against R equi foal pneumonia.

Intramuscular but not nebulized administration of a mRNA vaccine against Rhodococcus equi stimulated humoral immune responses in neonatal foals.

OBJECTIVE: Design and evaluate immune responses of neonatal foals to a mRNA vaccine expressing the virulence-associated protein A (VapA) of Rhodococcus equi. ANIMALS: Cultured primary equine respiratory tract cells; Serum, bronchoalveolar lavage fluid (BALF), and peripheral blood mononuclear cells (PBMCs) from 30 healthy Quarter Horse foals. METHODS: VapA expression was evaluated by western immunoblot in cultured equine bronchial cells transfected with 4 mRNA constructs encoding VapA. The mRNA construct with greatest expression was used to immunize foals at ages 2 and 21 days in 5 groups: (1) 300 µg nebulized mRNA (n = 6); (2) 600 µg nebulized mRNA (n = 4); (3) 300 µg mRNA administered intramuscularly (IM) (n = 5); (4) 300 µg VapA IM (positive controls; n = 6); or (5) nebulized water (negative controls; n = 6). Serum, BALF, and PBMCs were collected at ages 3, 22, and 35 days and tested for relative anti-VapA IgG1, IgG4/7, and IgA activities using ELISA and cell-mediated immunity by ELISpot. RESULTS: As formulated, nebulized mRNA was not immunogenic. However, a significant increase in anti-VapA IgG4/7 activity (P < .05) was noted exclusively in foals immunized IM with VapA mRNA by age 35 days. The proportion of foals with anti-VapA IgG1 activity > 30% of positive control differed significantly (P = .0441) between negative controls (50%; 3/6), IM mRNA foals (100%; 5/5), and IM VapA (100%; 6/6) groups. Natural exposure to virulent R equi was immunogenic in some negative control foals. CLINICAL RELEVANCE: Further evaluation of the immunogenicity and efficacy of IM mRNA encoding VapA in foals is warranted.

Identification of 3 neutralizing linear epitopes on the VP8 outer capsid protein of group A equine rotavirus.

OBJECTIVE: To identify protective equine rotavirus group A (ERVA) VP8 epitopes and demonstrate that immunizing hens with synthetic peptides based on these epitopes would yield high-titered, neutralizing egg yolk antibodies for potential application in foals. ANIMALS: 26 rotavirus-positive, client-owned foals were included in the study. Five white leghorn hens were used for antibody production. METHODS: Chicken antibodies were raised against 3 synthetic epitope peptides from the VP8 protein of the common ERVA P-type, P4[12] using CD40-targeted streptavidin-peptide complexes. Antipeptide serum- and egg yolk antibodies were subject to ELISA and in vitro virus neutralization assays to evaluate binding and neutralization activities. Lyophilized anti-VP8 egg yolk antibodies were orally administered (30 g; q 24 h for 5 days) to foals with rotaviral diarrhea. Physical examinations were performed daily. The duration of diarrhea and any adverse effects were recorded. RESULTS: CD40-targeted vaccination of hens generated high titers of anti-VP8 serum and egg yolk antibodies after just 3 immunizations. These antibodies prevented in vitro infection of ERVA with titers of 128 in the serum and 94.5 in the yolk. Oral administration (30 g; q 24 h for 5 days) of lyophilized hyperimmune egg yolk to foals with rotaviral diarrhea did not reveal any adverse effects of the treatment. CLINICAL RELEVANCE: This study demonstrated that antibodies raised against neutralizing epitopes of the ERVA VP8 protein could prevent ERVA infection in vitro. Based on these results and previous work in other animals, in vivo evaluation of the therapeutic efficacy of anti-VP8 egg yolk antibodies is warranted.

Impact of surface receptors TLR2, CR3, and FcγRIII on Rhodococcus equi phagocytosis and intracellular survival in macrophages.

The virulence-associated protein A (VapA) produced by virulent Rhodococcus equi allows it to replicate in macrophages and cause pneumonia in foals. It is unknown how VapA interacts with mammalian cell receptors, but intracellular replication of avirulent R. equi lacking vapA can be restored by supplementation with recombinant VapA (rVapA). Our objectives were to determine whether the absence of the surface receptors Toll-like receptor 2 (TLR2), complement receptor 3 (CR3), or Fc gamma receptor III (FcγRIII) impacts R. equi phagocytosis and intracellular replication in macrophages, and whether rVapA restoration of virulence in R. equi is dependent upon these receptors. Wild-type (WT) murine macrophages with TLR2, CR3, or FcγRIII blocked or knocked out (KO) were infected with virulent or avirulent R. equi, with or without rVapA supplementation. Quantitative bacterial culture and immunofluorescence imaging were performed. Phagocytosis of R. equi was not affected by blockade or KO of TLR2 or CR3. Intracellular replication of virulent R. equi was not affected by TLR2, CR3, or FcγRIII blockade or KO; however, avirulent R. equi replicated in TLR2-/- and CR3-/- macrophages but not in WT and FcγRIII-/-. rVapA supplementation did not affect avirulent R. equi phagocytosis but promoted intracellular replication in WT and all KO cells. By demonstrating that TLR2 and CR3 limit replication of avirulent but not virulent R. equi and that VapA-mediated virulence is independent of TLR2, CR3, or FcγRIII, our study provides novel insights into the role of these specific surface receptors in determining the entry and intracellular fate of R. equi.

Equine bronchial epithelial cells are susceptible to cell entry with a SARS-CoV-2 pseudovirus but reveal low replication efficiency.

OBJECTIVE: To examine the susceptibility of cultured primary equine bronchial epithelial cells (EBECs) to a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus relative to human bronchial epithelial cells (HBECs). SAMPLE: Primary EBEC cultures established from healthy adult horses and commercially sourced human bronchial epithelial cells (HBECs) were used as a positive control. METHODS: Angiotensin-converting enzyme 2 (ACE2) expression by EBECs was demonstrated using immunofluorescence, western immunoblot, and flow cytometry. EBECs were transduced with a lentivirus pseudotyped with the SARS-CoV-2 spike protein that binds to ACE2 and expresses the enhanced green fluorescent protein (eGFP) as a reporter. Cells were transduced with the pseudovirus at a multiplicity of infection of 0.1 for 6 hours, washed, and maintained in media for 96 hours. After 96 hours, eGFP expression in EBECs was assessed by fluorescence microscopy of cell cultures and quantitative PCR. RESULTS: ACE2 expression in EBECs detected by immunofluorescence, western immunoblotting, and flow cytometry was lower in EBECs than in HBECs. After 96 hours, eGFP expression in EBECs was demonstrated by fluorescence microscopy, and mean ΔCt values from quantitative PCR were significantly (P < .0001) higher in EBECs (8.78) than HBECs (3.24) indicating lower infectivity in EBECs. CLINICAL RELEVANCE: Equine respiratory tract cells were susceptible to cell entry with a SARS-CoV-2 pseudovirus. Lower replication efficiency in EBECs suggests that horses are unlikely to be an important zoonotic host of SARS-CoV-2, but viral mutations could render some strains more infective to horses. Serological and virological monitoring of horses in contact with persons shedding SARS-CoV-2 is warranted.

Development of a novel real-time PCR multiplex assay for detection of Streptococcus equi subspecies equi and Streptococcus equi subspecies zooepidemicus.

Strangles is a contagious bacterial disease of horses caused by Streptococcus equi subspecies equi (SEE) that occurs globally. Rapid and accurate identification of infected horses is essential for controlling strangles. Because of limitations of existing PCR assays for SEE, we sought to identify novel primers and probes that enable simultaneous detection and differentiation of infection with SEE and S. equi subsp. zooepidemicus (SEZ). Comparative genomics of U.S. strains of SEE and SEZ (n = 50 each) identified SE00768 from SEE and comB from SEZ as target genes. Primers and probes for real-time PCR (rtPCR) were designed for these genes and then aligned in silico with the genomes of strains of SEE (n = 725) and SEZ (n = 343). Additionally, the sensitivity and specificity relative to microbiologic culture were compared between 85 samples submitted to an accredited veterinary medical diagnostic laboratory. The respective primer and probe sets aligned with 99.7 % (723/725) isolates of SEE and 97.1 % (333/343) of SEZ. Of 85 diagnostic samples, 20 of 21 (95.2 %) SEE and 22 of 23 SEZ (95.6 %) culture-positive samples were positive by rtPCR for SEE and SEZ, respectively. Both SEE (n = 2) and SEZ (n = 3) were identified by rtPCR among 32 culture-negative samples. Results were rtPCR-positive for both SEE and SEZ in 21 of 44 (47.7 %) samples that were culture-positive for SEE or SEZ. The primers and probe sets reported here reliably detect SEE and SEZ from Europe and the U.S., and permit detection of concurrent infection with both subspecies.

A Randomized, Self-Controlled Case Series Evaluating Core Osteostixis of Osseous Cyst-Like Lesions of the Navicular Bone to Improve Lameness in Horses with Podotrochlear Syndrome.

Introduction: Podotrochlear syndrome is a common cause of lameness in Quarter Horses involving both soft tissue and bony structures within the heel region. Current surgical treatment of podotrochlear syndrome addresses pathological changes affecting the soft tissue structures of the navicular region but does not address either edema or cyst-like lesions of the navicular bone. Objective: The objective of this randomized, self-controlled case series was to determine whether core osteostixis improved lameness in Quarter Horses with podotrochlear syndrome characterized by bilateral magnetic resonance imaging (MRI) findings of osseous cyst-like lesions of the navicular bone. Methods: Seven Quarter Horses that had not responded to standard medical management were included. Each horse had an affected forefoot randomly assigned to surgical treatment with navicular bursoscopy and core osteostixis; the contralateral limb was assigned to navicular bursoscopy only. Video recordings were used to assign lameness scores and make comparisons of each limb at baseline and 24 weeks post-operatively by an observer blinded to the surgical treatment. A second MRI was performed 24 weeks after surgery to reevaluate navicular bone edema, osseous cyst-like lesions of the navicular bone, and tears of the deep digital flexor tendon (DDFT). Results: Reduction of lameness score from baseline was significantly (P = 0.0254) greater for the limbs treated with core osteostixis than limbs treated with bursoscopy. New DDFT tears were noted in 3 of 7 limbs treated with core osteostixis and in 1 of 7 bursoscopy limbs. Conclusion: Results of this study suggest that core osteostixis of the navicular bone combined with navicular bursoscopy can improve lameness in horses with osseous cyst-like lesions. Further evaluation of this technique is warranted.

Feasibility of a point-of-care ultrasound protocol for cardiorespiratory evaluation of horses in different clinical settings.

BACKGROUND: A point-of-care ultrasound (POCUS) protocol for evaluation of the cardiac and respiratory systems in horses does not exist. OBJECTIVES: (a) Describe the windows of a POCUS protocol for cardiorespiratory assessment of horses (CRASH); (b) Estimate the number of acoustic windows that can be acquired by a sonographer-in-training; (c) Estimate the time required to complete the protocol for specific groups of horses; (d) Describe the sonographic abnormalities detected in horses presented with cardiovascular, respiratory, or systemic disease. ANIMALS: Twenty-seven healthy horses, 14 horses competing in athletic events, and 120 horses with clinical disease. METHOD: A pocket-sized ultrasound device was used to acquire 7 sonographic cardiorespiratory windows in various clinical scenarios. The duration of the examination was timed, and images were evaluated for diagnostic quality. Abnormalities in horses with clinical disease were determined by an expert sonographer. RESULTS: The CRASH protocol could be performed in healthy and diseased horses in hospital, barn, and competition settings between 5.5 ± 0.9 (athletic horses) and 6.9 ± 1.9 min (horses with clinical disease). Thoracic windows were obtained most consistently, followed by right parasternal long-axis echocardiographic windows. Frequently detected abnormalities were pleural fluid, lung consolidation, B-lines, and moderate-to-severe left-sided heart disease. CONCLUSIONS: The CRASH protocol was feasible using a pocket-sized ultrasound device in various groups of horses, could be completed rapidly in a variety of settings, and frequently identified sonographic abnormalities when evaluated by an expert sonographer. The diagnostic accuracy, observer agreement, and utility of the CRASH protocol merit further evaluation.

Agreement of Temperatures Measured Using a Non-Contact Infrared Thermometer With a Rectal Digital Thermometer in Horses.

Evaluating the body temperature of horses is an essential tool for monitoring horse health and biosecurity in groups of horses. Temperatures of horses and foals are determined most often using rectal thermometry. Rectal thermometry has limitations that include safety considerations for horses and humans. Thus, we investigated the agreement between a noncontact infrared thermometer (NCIT) and a rectal digital thermometer in 142 horses and 34 foals. For each horse and foal, measurements using the NCIT were collected from the forehead (n = 2) or neck (n = 1) and with a rectal digital thermometer (n = 1). Although the NCIT demonstrated good reliability (i.e. repeatability of measurements), a large negative bias (nearly 2°F (-16.7°C) in adult horses and >3°F (-16.1°C) in foals) was observed between readings from the NCIT and the rectal thermometer in healthy horses. Although horses with febrile illness were not included in the study, our results indicate that the large and inconsistent bias observed with the NCIT indicates that these devices will not be a suitable substitute for rectal thermometry for obtaining valid estimates of core body temperature in horses.

Conservation of vaccine antigen sequences encoded by sequenced strains of Streptococcus equi subsp. equi.

BACKGROUND: Streptococcus equi subspecies equi (S equi) is the cause of Strangles, one of the most prevalent diseases of horses worldwide. Variation within the immunodominant SeM protein has been documented, but a new eight-component fusion protein vaccine, Strangvac, does not contain live S equi or SeM and conservation of the antigens it contains have not been reported. OBJECTIVE: To define the diversity of the eight Strangvac antigens across a diverse S equi population. STUDY DESIGN: Genomic description. METHODS: Antigen sequences from the genomes of 759 S equi isolates from 19 countries, recovered between 1955 and 2018, were analysed. Predicted amino acid sequences in the antigen fragments of SEQ0256(Eq5), SEQ0402(Eq8), SEQ0721(EAG), SEQ0855(SclF), SEQ0935(CNE), SEQ0999(IdeE), SEQ1817(SclI) and SEQ2101(SclC) in Strangvac and SeM were extracted from the 759 assembled genomes and compared. RESULTS: The predicted amino acid sequences of SclC, SclI and IdeE were identical across all 759 genomes. CNE was truncated in the genome of five (0.7%) isolates. SclF was absent from one genome and another encoded a single amino acid substitution. EAG was truncated in two genomes. Eq5 was truncated in four genomes and 123 genomes encoded a single amino acid substitution. Eq8 was truncated in three genomes, one genome encoded four amino acid substitutions and 398 genomes encoded a single amino acid substitution at the final amino acid of the Eq8 antigen fragment. Therefore, at least 1579 (99.9%) of 1580 amino acids in Strangvac were identical in 743 (97.9%) genomes, and all genomes encoded identical amino acid sequences for at least six of the eight Strangvac antigens. MAIN LIMITATIONS: Three hundred and seven (40.4%) isolates in this study were recovered from horses in the UK. CONCLUSIONS: The predicted amino acid sequences of antigens in Strangvac were highly conserved across this collection of S equi.

Transfusion of hyperimmune plasma for protecting foals against Rhodococcus equi pneumonia.

The bacterium Rhodococcus equi causes pneumonia in foals that is prevalent at breeding farms worldwide. In the absence of an effective vaccine, transfusion of commercial plasma from donor horses hyperimmunised against R. equi is used by many farms to reduce the incidence of pneumonia among foals at farms where the disease is endemic. The effectiveness of hyperimmune plasma for controlling R. equi pneumonia in foals has varied considerably among reports. The purposes of this narrative review are: (1) to review early studies that provided a foundational basis for the practice of transfusion of hyperimmune plasma that is widespread in the United States and in many other countries; (2) to summarise current knowledge of hyperimmune plasma for preventing R. equi pneumonia; (3) to provide an interpretive summary of probable explanations for the variable results among studies evaluating the effectiveness of transfusion of hyperimmune plasma for reducing the incidence of R. equi pneumonia; (4) to review mechanisms by which hyperimmune plasma might mediate protection; and (5) to consider risks of transfusing foals with hyperimmune plasma. Although the weight of evidence supports the practice of transfusing foals with hyperimmune plasma to prevent R. equi pneumonia, many important gaps in our knowledge of this topic remain including the volume/dose of hyperimmune plasma to be transfused, the timing(s) of transfusion, and the mechanism(s) by which hyperimmune plasma mediates protection. Transfusing foals with hyperimmune plasma is expensive, labour-intensive, and carries risks for foals; therefore, alternative approaches for passive and active immunisation to prevent R. equi pneumonia are greatly needed.

Wound swabs versus biopsies to detect methicillin resistant Staphylococcus aureus in experimental equine wounds.

OBJECTIVE: To compare: (1) the load and diversity of cultivatable bacterial species isolated from tissue biopsies with cultures from surface swabs, and (2) the ability of each technique to detect methicillin-resistant Staphylococcus aureus (MRSA) in a model of MRSA-infected equine wounds. STUDY DESIGN: Experimental in vivo study. ANIMALS: Three light-breed adult horses. METHODS: Four 2.5 × 2.5 cm full-thickness skin wounds were created on the dorsolateral aspect of each forelimb. Five days later, each wound was inoculated with a pure culture of MRSA (ATCC 43300). One hundred microlitres of 0, 5 × 108 , 5 × 109 or 5 × 1010 colony forming units (CFU)/ml was used to inoculate each wound. Surface swabs (Levine technique) and tissue biopsy samples (3 mm punch biopsy) were obtained at 2, 7, 14, and 21 days after inoculation. Quantitative aerobic culture was performed using routine clinical techniques. RESULTS: A similar bacterial profile was identified from the culture of each wound-sampling technique and there was moderate correlation (R = 0.49, P < .001) between the bacterial bioburdens. Agreement was fair (κ = 0.31; 95% CI, 0.129-0.505) between the sampling techniques in identification of MRSA. Methicillin-resistant Staphylococcus aureus was isolated more frequently (P = .016) from cultures of tissue biopsies (79%; 76/96) than from surface swabs (62%; 60/96). CONCLUSION: Bacterial load and diversity did not differ between sampling techniques but MRSA was detected more often from the cultures of tissue biopsies. CLINICAL SIGNIFICANCE: Tissue biopsy should be preferred to culture swab in wounds where MRSA is suspected.

Fitness cost conferred by the novel erm(51) and rpoB mutation on environmental multidrug resistant-Rhodococcus equi.

Rhodococcus equi is a common cause of severe pneumonia in foals. Emergence of macrolide-resistant R. equi isolated from foals and their environment has been reported in the United States. A novel erm(51) gene was recently identified in R. equi in soil from horse farms in Kentucky. Our objective was to determine the effect of the erm(51) gene and associated rpoB mutation on the fitness of multidrug resistant-R. equi (MDR-R. equierm(51)+, rpoB+) under different nutrient conditions. Bacterial growth curves were generated for 3 MDR-R. equierm(51)+, rpoB+ isolates and 3 wild-type (WTN) R. equi isolates recovered from environmental samples of farms in central Kentucky. Growth was measured over 30.5 h in brain-heart infusion broth (BHI), minimal medium (MM), and minimal medium without iron (MM-I). All isolates had significantly (P < 0.05) higher growth in BHI compared to either MM or MM-I. MDR-R. equierm(51)+, rpoB+ exhibited significantly lower growth compared to WTN isolates in BHI (nutrient-rich condition), but not in either MM or MM-I (nutrient-restricted conditions). This study indicates that under nutrient-rich conditions fitness of MDR-R. equierm(51)+, rpoB+ is reduced relative to susceptible isolates; however, under nutrient-restricted conditions MDR-R. equierm(51)+, rpoB+ isolates grow similarly to susceptible isolates. These findings indicate that MDR-R. equierm(51)+, rpoB+ might be outcompeted by susceptible isolates in nature when practices to reduce antimicrobial pressure, such as reducing antimicrobial use in foals, are implemented. But it also raises the concern that these resistant genotypes might persist in the environment of horse-breeding farms in the face of selective pressures such as antimicrobials or nutrient restriction.

Fecal concentration of Rhodococcus equi determined by quantitative polymerase chain reaction of rectal swab samples to differentiate foals with pneumonia from healthy foals.

BACKGROUND: Diagnostic accuracy of real-time, quantitative PCR (qPCR) assays to quantify virulent Rhodococcus equi using rectal swab samples has not been systematically evaluated. OBJECTIVE: To evaluate the accuracy of qPCR of rectal swab samples to differentiate foals with pneumonia from healthy foals of similar age from the same environment. ANIMALS: One hundred privately owned foals born in 2021 from 2 farms in New York. METHODS: An incident case-control study design was used. Rectal swabs were collected from all foals diagnosed with R. equi pneumonia at 2 horse-breeding farms (n = 47). Eligible pneumonia cases (n = 39) were matched by age to up to 2 healthy (n = 53) control foals; rectal swabs were collected from control foals on the day of diagnosis of the index case. DNA was extracted from fecal swabs and the concentration of virulent R. equi (ie, copy numbers of the virulence-associated protein A gene [vapA] per 100 ng fecal DNA) was estimated by qPCR. RESULTS: The area under the ROC curve for qPCR of fecal swabs was 83.7% (95% CI, 74.9-92.6). At a threshold of 14 883 copies of vapA per 100 ng fecal DNA, specificity of the assay was 83.0% (95% CI, 71.7-92.4) and sensitivity was 79.5% (95% CI, 66.7-92.3). CONCLUSIONS AND CLINICAL IMPORTANCE: Although fecal concentrations of virulent R. equi are significantly higher in pneumonic foals than healthy foals of similar age in the same environment, qPCR of rectal swabs as reported here lacks adequate diagnostic accuracy for clinical use.

Association of pneumonia with concentrations of virulent Rhodococcus equi in fecal swabs of foals before and after intrabronchial infection with virulent R. equi.

BACKGROUND: Intragastric administration of virulent Rhodococcus equi protects foals against subsequent experimental intrabronchial (IB) infection, but it is unknown whether R. equi naturally ingested by foals contributes to their susceptibility to pneumonia. HYPOTHESIS: Fecal concentration of virulent R. equi before IB infection with R. equi is positively associated with protection from pneumonia in foals. ANIMALS: Twenty-one university-owned foals. METHODS: Samples were collected from experimental studies. Five foals were gavaged with live, virulent R. equi (LVRE) at age 2 and 4 days; the remaining 16 foals were not gavaged with LVRE (controls). Fecal swabs were collected from foals at ages 28 days, immediately before IB infection. Foals were monitored for clinical signs of pneumonia, and fecal swabs were collected approximately 2 weeks after IB infection. Swabs were tested by quantitative PCR for concentration of virulent R. equi (ie, copy numbers of the virulence-associated protein A gene [vapA] per 100 ng fecal DNA). RESULTS: Fecal concentrations of virulent R. equi (vapA) before IB infection were significantly (P < .05) lower in control foals (25 copies/100 ng DNA [95% CI, 5 to 118 copies/100 ng DNA) that developed pneumonia (n = 8) than in healthy control foals (n = 8; 280 copies/100 ng DNA; 95% CI, 30 to 2552 copies/100 ng DNA) or those gavaged with LVRE (707 copies/100 ng DNA, 95% CI, 54 to 9207 copies/100 ng DNA). CONCLUSIONS AND CLINICAL IMPORTANCE: Greater natural ingestion of LVRE might contribute to protection against pneumonia among foals.

Rhodococcus equi foal pneumonia: Update on epidemiology, immunity, treatment and prevention.

Pneumonia in foals caused by the bacterium Rhodococcus equi has a worldwide distribution and is a common cause of disease and death for foals. The purpose of this narrative review was to summarise recent developments pertaining to the epidemiology, immune responses, treatment, and prevention of rhodococcal pneumonia of foals. Screening tests have been used to implement earlier detection and treatment of foals with presumed subclinical R. equi pneumonia to reduce mortality and severity of disease. Unfortunately, this practice has been linked to the emergence of antimicrobial-resistant R. equi in North America. Correlates of protective immunity for R. equi infections of foals remain elusive, but recent evidence indicates that innate immune responses are important both for mediating killing and orchestrating adaptive immune responses. A macrolide antimicrobial in combination with rifampin remains the recommended treatment for foals with R. equi pneumonia. Great need exists to identify which antimicrobial combination is most effective for treating foals with R. equi pneumonia and to limit emergence of antimicrobial-resistant strains. In the absence of an effective vaccine against R. equi, passive immunisation remains the only commercially available method for effectively reducing the incidence of R. equi pneumonia. Because passive immunisation is expensive, labour-intensive and carries risks for foals, great need exists to develop alternative approaches for passive and active immunisation.

Differences in the Accessory Genomes and Methylomes of Strains of Streptococcus equi subsp. equi and of Streptococcus equi subsp. zooepidemicus Obtained from the Respiratory Tract of Horses from Texas.

Streptococcus equi subsp. equi (SEE) is a host-restricted equine pathogen considered to have evolved from Streptococcus equi subsp. zooepidemicus (SEZ). SEZ is promiscuous in host range and is commonly recovered from horses as a commensal. Comparison of a single strain each of SEE and SEZ using whole-genome sequencing, supplemented by PCR of selected genes in additional SEE and SEZ strains, was used to characterize the evolution of SEE. But the known genetic variability of SEZ warrants comparison of the whole genomes of multiple SEE and SEZ strains. To fill this knowledge gap, we utilized whole-genome sequencing to characterize the accessory genome elements (AGEs; i.e., elements present in some SEE strains but absent in SEZ or vice versa) and methylomes of 50 SEE and 50 SEZ isolates from Texas. Consistent with previous findings, AGEs consistently found in all SEE isolates were primarily from mobile genetic elements that might contribute to host restriction or pathogenesis of SEE. Fewer AGEs were identified in SEZ because of the greater genomic variability among these isolates. The global methylation patterns of SEE isolates were more consistent than those of the SEZ isolates. Among homologous genes of SEE and SEZ, differential methylation was identified only in genes of SEE encoding proteins with functions of quorum sensing, exopeptidase activity, and transitional metal ion binding. Our results indicate that effects of genetic mobile elements in SEE and differential methylation of genes shared by SEE and SEZ might contribute to the host specificity of SEE. IMPORTANCE Strangles, caused by the host-specific bacterium Streptococcus equi subsp. equi (SEE), is the most commonly diagnosed infectious disease of horses worldwide. Its ancestor, Streptococcus equi subsp. zooepidemicus (SEZ), is frequently isolated from a wide array of hosts, including horses and humans. A comparison of the genomes of a single strain of SEE and SEZ has been reported, but sequencing of further isolates has revealed variability among SEZ strains. Thus, the importance of this study is that it characterizes genomic and methylomic differences of multiple SEE and SEZ isolates from a common geographic region (viz., Texas). Our results affirm many of the previously described differences between the genomes of SEE and SEZ, including the role of mobile genetic elements in contributing to host restriction. We also provide the first characterization of the global methylome of Streptococcus equi and evidence that differential methylation might contribute to the host restriction of SEE.

Randomized, controlled trial comparing Rhodococcus equi and poly-N-acetyl glucosamine hyperimmune plasma to prevent R equi pneumonia in foals.

BACKGROUND: Hyperimmune plasma raised against ß-1→6-poly-N-acetyl glucosamine (PNAG HIP) mediates more opsonophagocytic killing of Rhodococcus equi (R equi) than does R equi hyperimmune plasma (RE HIP) in vitro. The relative efficacy of PNAG HIP and RE HIP to protect foals against R equi pneumonia, however, has not been evaluated. HYPOTHESIS: Transfusion with PNAG HIP will be superior to RE HIP in foals for protection against R equi pneumonia in a randomized, controlled, blinded clinical trial. ANIMALS: Four hundred sixty Quarter Horse and Thoroughbred foals at 5 large breeding farms in the United States. METHODS: A randomized, controlled, blinded clinical trial was conducted in which foals were transfused within 24 hours after birth with 2 L of either RE HIP or PNAG HIP. Study foals were monitored through weaning for clinical signs of pneumonia by farm veterinarians. The primary outcome was the proportion of foals that developed pneumonia after receiving each type of plasma. RESULTS: The proportion of foals that developed pneumonia was the same between foals transfused with RE HIP (14%; 32/228) and PNAG HIP (14%; 30/215). CONCLUSIONS AND CLINICAL IMPORTANCE: Results indicate that PNAG HIP was not superior to a commercially available, United States Department of Agriculture-licensed RE HIP product for protecting foals against R equi pneumonia under field conditions.

The opportunistic intracellular bacterial pathogen Rhodococcus equi elicits type I interferon by engaging cytosolic DNA sensing in macrophages.

Rhodococcus equi is a major cause of foal pneumonia and an opportunistic pathogen in immunocompromised humans. While alveolar macrophages constitute the primary replicative niche for R. equi, little is known about how intracellular R. equi is sensed by macrophages. Here, we discovered that in addition to previously characterized pro-inflammatory cytokines (e.g., Tnfa, Il6, Il1b), macrophages infected with R. equi induce a robust type I IFN response, including Ifnb and interferon-stimulated genes (ISGs), similar to the evolutionarily related pathogen, Mycobacterium tuberculosis. Follow up studies using a combination of mammalian and bacterial genetics demonstrated that induction of this type I IFN expression program is largely dependent on the cGAS/STING/TBK1 axis of the cytosolic DNA sensing pathway, suggesting that R. equi perturbs the phagosomal membrane and causes DNA release into the cytosol following phagocytosis. Consistent with this, we found that a population of ~12% of R. equi phagosomes recruits the galectin-3,-8 and -9 danger receptors. Interestingly, neither phagosomal damage nor induction of type I IFN require the R. equi's virulence-associated plasmid. Importantly, R. equi infection of both mice and foals stimulates ISG expression, in organs (mice) and circulating monocytes (foals). By demonstrating that R. equi activates cytosolic DNA sensing in macrophages and elicits type I IFN responses in animal models, our work provides novel insights into how R. equi engages the innate immune system and furthers our understanding how this zoonotic pathogen causes inflammation and disease.