Noah D. Cohen

Objective: Because of lefamulin’s antimicrobial spectrum and intrapulmonary disposition, the objective of this study was to estimate MICs for macrolide- and rifampin-susceptible (MRS) and macrolide- and rifampin-resistant (MRR) isolates of Rhodococcus equi to lefamulin either alone or in combination with rifampin. Methods: Gradient diffusion and broth microdilution were performed to determine MICs for lefamulin alone using MRS (n = 43) and MRR isolates (61) of R equi . Twenty MRR and 20 MRS isolates were tested for synergistic effects using a checkerboard assay and the fractional inhibitory concentration index as an outcome measure. Results: For both MRR and MRS isolates, MIC values were significantly higher using broth microdilution (median MIC, 32 µg/mL; range, 8 to 64 µg/mL) than gradient diffusion (median MIC, 12 µg/mL; range, 5 to 28 µg/mL) testing. The fractional inhibitory concentration values of MRR isolates (median, 0.562; range, 0.250 to 1.000) were significantly higher than those of MRS isolates (median, 0.280; range, 0.039 to 1.038). In addition, the proportion of isolates exhibiting synergistic interaction was significantly greater among MRS isolates (16 of 20 [80%]) than among MRR isolates (6 of 20 [30%]). Conclusions: Overall, the MIC values for lefamulin against R equi were high relative to concentrations considered therapeutic in humans. When combined with rifampin, lefamulin exhibited synergistic effects for MRS but not MRR isolates. Clinical Relevance: Need exists for alternative antimicrobials to treat rhodococcal foal pneumonia. Results indicate that studies in foals of the pharmacokinetics and pulmonary disposition of lefamulin in combination with rifampin are warranted.

Objective: To develop an accurate serological test for detecting horses infected with Streptococcus equi subsp equi (SEE). Methods: The study was designed as a prospective experiment. First, 89 proteins either expressed on the surface or secreted by SEE were cloned, expressed in a cell-free culture system, and printed onto a microarray. Microarrays were probed with serum from 30 horses with strangles, 30 horses with no history of infection or immunization against SEE, and 18 horses infected with Streptococcus equi subsp zooepidemicus (SEZ); serum samples were collected between 2020 and 2021 from Texas horses. Informatic analysis identified a novel protein we identified as protein 92 (P92). Serum samples from 102 horses with strangles, 104 control horses, and 24 horses infected with SEZ were tested with indirect ELISA for either P92 or the M protein of SEE (SeM); horses were from various US states collected during 2022 and 2023. Data were analyzed by comparing areas under the receiver operator characteristic curves and by generalized linear modeling to compare antibody activities among groups with the Tukey method for pairwise comparisons. Results: The microarray identified a new protein (P92) for strangles testing. The area under the receiver operator characteristic curve for the P92 ELISA (95%; 95% CI, 92% to 98%) was significantly greater than that of the SeM ELISA (88%; 95% CI, 83% to 92%). Conclusions: The P92 ELISA improves the accuracy of diagnostic testing for strangles. Clinical Relevance: A newly identified protein offers a target for improving strangles diagnostic testing.

The bacterium Rhodococcus equi is a common cause of severe pneumonia in foals worldwide. Foals are exposed to R. equi from birth and are most susceptible in the first weeks of life. In North America, control measures are limited to transfusion to newborn foals of hyperimmune plasma whose potency is assessed by surpassing a threshold of IgG against the virulence- associated protein A (VapA) of R. equi . Here we report results of vaccination of pregnant mares with mRNA encoding a GPI-anchored virulence-associated protein A (VapA) of R. equi induced VapA-specific IgG subisotypes transferred to their foals during the 63-day period after birth. Pregnant mares were randomly assigned to receive either VapA mRNA vaccine (n=10) at 8 and 10 months of gestation or no vaccine (n=10). Serum was collected from mares at 8 months of gestation (before vaccination for immunized mares) and ≈24 hours after foaling and from foals at ages 1, 35, and 63 days. Mean relative concentrations of were low before immunization in all mares and did not change over time in unvaccinated mares; however, mean relative concentrations of anti-VapA IgG ₁ , IgG _4/7 , and IgG _3/5 of mares were significantly increased after VapA mRNA immunization and strongly correlated with the corresponding concentrations in their foals’ sera at age ≈24 hours. Concentrations of anti-VapA IgGs remained increased in foals of vaccinated mares compared with control foals for at least 35 days. IMPORTANCE: Results of our study indicate that maternal vaccination of pregnant mares with a GPI-anchored VapA mRNA vaccine provides humoral immunity to foals when they are most susceptible to infection. These findings indicate that further evaluation of the efficacy of this vaccine to prevent pneumonia in foals infected with R. equi is warranted.

Objective: To develop a multiplex real-time PCR assay for simultaneous detection of Rhodococcus equi ( choE ), virulence ( vapA ), and macrolide-resistance determinants ( erm [46], erm [51]), and evaluate their clinical and temporal trends. Methods: Clinical R equi isolates (n = 229) obtained from the Hagyard Equine Medical Institute laboratory were collected sequentially over a 3-year surveillance period (2021 to 2023) from 14 states and underwent broth microdilution susceptibility testing. A cohort of 214 isolates with high-quality DNA was characterized using quantitative PCR. Geographic distribution, seasonal trends, and genotypic-phenotypic concordance were evaluated. Results: The multiplex PCR assay demonstrated 100% analytical specificity. The virulence gene vapA was present in 94.4% (202 of 214) of isolates. The macrolide resistance determinant erm (46) was highly prevalent at 62.6% (134 of 214), showing a significant seasonal increase during July to December periods at 76.6% (vs 58.2% in January to June). Crucially, erm (51) was detected in a single isolate (0.5%), representing its first recorded clinical identification in R equi . Unstratified genotypic-phenotypic agreement for macrolides was fair (κ = 0.22 to 0.25). However, stratification by genomic copy number revealed near-perfect concordance (κ ≥ 0.97) exclusively among isolates harboring > 50,000 target copies. Alternative agents (gentamicin, ceftiofur, minocycline, doxycycline) maintained 97% to 100% susceptibility, regardless of erm (46) status. Conclusions: While erm (46) is widely distributed across R equi –endemic farms, actual phenotypic resistance expression is strongly copy-number dependent. The clinical emergence of erm (51) expands the known resistome of this pathogen. Clinical Relevance: Incorporating quantitative molecular screening into routine R equi diagnostics provides crucial real-time therapeutic guidance, supporting precision medicine and targeted antimicrobial stewardship on endemic equine farms.

Rhodococcus equi is a facultative intracellular pathogen and a major cause of pneumonia in foals worldwide. The standard treatment for affected foals is a combination of a macrolide with rifampicin. The widespread use of this combination has led to the emergence of multidrug‐resistant (MDR) strains of R. equi . Consequently, antimicrobial susceptibility testing is essential for guiding treatment of rhodococcal pneumonia. Unfortunately, clinical breakpoints for macrolides and other antimicrobials against R. equi for foals are lacking, thereby complicating clinical interpretation of in vitro results. This review summarises current knowledge of antimicrobial resistance in R. equi , the methods used for susceptibility testing and the challenges of interpreting susceptibility testing for guiding treatment of this intracellular infection. We discuss the limitations of extrapolating breakpoints from other species of bacteria and animals and emphasise the importance of integrating minimum inhibitory concentration data, knowledge of intracellular antimicrobial activity and principles of pharmacokinetics and pharmacodynamics to inform therapeutic decisions. In the absence of standardised interpretive criteria of results of susceptibility testing, we propose evidence‐based guidelines to support veterinarians and diagnostic laboratories in managing R. equi infections of foals.

Objective: To characterize age-related trajectories of the relative concentrations of anti–virulence-associated protein A (VapA) immunoglobulin (Ig)-G ₁ , IgG _3/5 , IgG _4/7 , and IgA in serum and bronchoalveolar lavage fluid (BALF) as indicators of systemic and intrapulmonary immune responses in foals naturally exposed to Rhodococcus equi . Methods: Blood and BALF samples were collected at ages 4, 14, 28, and 56 days from healthy Quarter Horse foals (n = 19) naturally exposed to R equi . Samples were tested via ELISA for relative concentration of IgG ₁ , IgG _3/5 , IgG _4/7 , and IgA recognizing the VapA of R equi . Data were analyzed using linear mixed-effects modeling to compare VapA ELISA results by age for IgG subisotypes and IgA. Results: Serum relative concentrations of VapA-specific IgG ₁ , IgG _3/5 , IgG _4/7 , and IgA of 19 foals did not differ significantly during the first 28 days of age but increased nearly 10-fold by age 56 days. Similarly, BALF relative concentrations of VapA-specific IgG ₁ , IgG _3/5 , and IgG _4/7 in these foals did not differ significantly during the first 28 days of age but increased by about 10-fold by age 56 days. In contrast, BALF relative concentrations of VapA-specific IgA increased progressively with age. Conclusions: Natural exposure results in progressive increase in BALF concentration of IgA but not IgGs, indicating intrapulmonary IgA response to virulent R equi . Clinical Relevance: Foals do not mount strong antibody responses to R equi when they are most vulnerable to infection. Further investigation is warranted to determine if increasing intrapulmonary antirhodococcal IgA is a correlate of protective immunity or merely a marker of exposure.

Background: Despite the availability of a killed whole-virus (KV) vaccine, diarrhea caused by equine rotavirus group A (ERVA) remains a significant health concern for foals in the United States. The vaccine is administered to pregnant mares, with foals protected by passive transfer of colostral antibodies. However, KV-induced immunity is only partially protective and maternal antibody levels in foals are often low and wane rapidly. To address these limitations, we developed a mRNA-based ERVA vaccine encoding the highly conserved VP8* protein to evaluate whether it can provide improved immune protection. Methods: Pregnant mares (n = 12 per group) were immunized either at months 8 and 10 of gestation with the VP8* mRNA or at months 8, 9, and 10 of gestation with the KV. Serum samples were collected from mares before and after immunization and from their foals at ages 1, 35, and 49 days. Serum samples were tested by indirect ELISA for VP8*-specific relative antibody concentrations and relative concentrations were compared for effects of study group and sample-time using linear mixed-effects regression. To detect functional antibodies against ERVA, a virus neutralization titer assay was performed to compare titers between mares vaccinated with the mRNA vaccine (and their foals) and unvaccinated control mares (and their foals). Results: Mares vaccinated with VP8* mRNA had significantly (p < 0.05) higher antibody concentrations after foaling than mares in the KV group, and foals of VP8* mRNA-vaccinated mares had significantly (p < 0.05) higher concentrations through age 49 days than foals in the KV group. In addition, the VP8* mRNA vaccine elicited higher titers of ERVA-neutralizing antibodies against both G3 and G14 strains. Conclusions: Longer-lasting, higher concentrations of virus-neutralizing antibodies might provide superior duration of immunity to ERVA in foals from mares vaccinated with VP8* mRNA.

Equine strangles caused by Streptococcus equi subspecies equi ( S. equi ) remains a significant cause of morbidity and mortality in horses, and there is a need for improved diagnostic and vaccination strategies for addressing this pathogen. ORFeome phage display is a platform that allows for rapid screening for potential antigenic epitopes by construction of phage-displayed peptide libraries. In this study, an S. equi ORFeome library was used to screen serum from a panel of 17 horses with known exposure to S. equi to identify antigenic bacterial proteins. From this screen, three major S. equi proteins were identified: a novel proline-rich repeat domain protein, a serine peptidase, and the M-like protein SeM. These three proteins are predicted to be expressed on the surface of the bacterial cell by the presence of N- and C-terminal signals. The proline-rich repeat protein and serine peptidase were confirmed to be immunogenic by enzyme-linked immunoassay (ELISA) using the recombinant full-length proteins against sera from horses with strangles, horses infected with the related pathogen S. equi subsp. zooepidemicus , and healthy control horses. Due to the native IgG binding activity of SeM, ELISA against the full-length protein was not conducted, but the specificity of the antibody response against the recovered ORFeome clones was confirmed and an antigenic region identified. Both the proline-rich repeat protein and serine peptidase were found to be highly conserved in global S. equi genomes, indicating these proteins may be useful as vaccine candidates against S. equi or as diagnostic markers to specifically identify S. equi infections in horses. IMPORTANCE: This work utilized an ORFeome phage display platform to systematically identify antigenic epitopes produced by Streptococcus equi subspecies equi ( S. equi ), an important equine pathogen and the causative agent of horses strangles. Three major S. equi surface proteins were identified: a novel proline-rich repeat domain protein, a serine peptidase, and the M-like protein SeM. The proline-rich repeat protein and serine peptidase were confirmed to be immunogenic in horses with strangles, and their sequences were shown to be conserved in global S. equi genomes, in contrast to their diversity in S. equi subsp. zooepidemicus. With the well-characterized S. equi immunogenic protein SeM, this paper identified an immunogenic region outside of the reported critical IgG-binding region. This work provides novel insights to the understanding of the S. equi immunogenic proteins and provides peptide regions that could serve as vaccine candidates against S. equi or as diagnostic markers to specifically identify S. equi infections.

We developed a fluorescence resonance energy transfer (FRET)-based real-time PCR (qPCR) assay coupled with high-resolution melting (HRM) analysis to differentiate Streptococcus equi subsp. equi (SEE) from S. equi subsp. zooepidemicus (SEZ), two closely related equine pathogens with distinct clinical implications. The assay targets a region of the 23S rRNA gene containing single-nucleotide polymorphisms that differentiate the subspecies. By combining subspecies-specific probes with melting temperature ( T _m ) analysis, the assay accurately distinguished SEE, SEZ, and mixed infections without the need for post-PCR processing. The entire workflow—from DNA extraction to result interpretation—was completed in under 2 h. The assay demonstrated high sensitivity and specificity, detecting as few as one genome copy per reaction, and showed excellent reproducibility with 100% concordance to whole-genome sequencing (WGS) results. Notably, one nasal swab isolate biochemically identified as SEE (based on its fermentation profile) was reclassified as SEZ by the FRET-qPCR assay, a result confirmed by WGS. Among 127 equine clinical samples, SEE was detected in 18% (23/127) and SEZ in 82% (104/127). SEZ predominated in eye, sinus, transtracheal aspirate, uterine, and wound samples, whereas SEE was significantly associated with guttural pouch samples. This FRET-qPCR assay offers a rapid, accurate, and practical molecular diagnostic tool for distinguishing S. equi subspecies, supporting improved clinical management and outbreak control in equine populations. IMPORTANCE: Accurately identifying the cause of respiratory infections in horses is essential for proper treatment and preventing outbreaks. This study introduces a rapid and reliable test that distinguishes between Streptococcus equi subsp. equi (which causes the serious disease strangles) and its close relative S. equi subsp. zooepidemicus (which usually causes milder, less contagious infections). Traditional tests can be slow or give unclear results, especially for unusual strains. Our new one-step molecular test uses melting curve analysis to rapidly and accurately differentiate between these bacterial subspecies—even in mixed infections. The method, which matches the accuracy of whole-genome sequencing but is much faster and easier to use, can help veterinarians to make more accurate diagnoses and improve tracking and control of equine respiratory disease.