Evaluation of vaccine candidates against Rhodococcus equi in BALB/c mice infection model: cellular and humoral immune responses.

Rhodococcus equi (R. equi) is a zoonotic opportunistic pathogen that mainly causes fatal lung and extrapulmonary abscesses in foals and immunocompromised individuals. To date, no commercial vaccine against R. equi exists. We previously screened all potential vaccine candidates from the complete genome of R. equi using a reverse vaccinology approach. Five of these candidates, namely ABC transporter substrate-binding protein (ABC transporter), penicillin-binding protein 2 (PBD2), NlpC/P60 family protein (NlpC/P60), esterase family protein (Esterase), and M23 family metallopeptidase (M23) were selected for the evaluation of immunogenicity and immunoprotective effects in BALB/c mice model challenged with R. equi. The results showed that all five vaccine candidate-immunized mice experienced a significant increase in spleen antigen-specific IFN-γ- and TNF-α-positive CD4 + and CD8 + T lymphocytes and generated robust Th1- and Th2-type immune responses and antibody responses. Two weeks after the R. equi challenge, immunization with the five vaccine candidates reduced the bacterial load in the lungs and improved the pathological damage to the lungs and livers compared with those in the control group. NlpC/P60, Esterase, and M23 were more effective than the ABC transporter and PBD2 in inducing protective immunity against R. equi challenge in mice. In addition, these vaccine candidates have the potential to induce T lymphocyte memory immune responses in mice. In summary, these antigens are effective candidates for the development of protective vaccines against R. equi. The R. equi antigen library has been expanded and provides new ideas for the development of multivalent vaccines.

First isolation and whole-genome sequencing of Trueperella abortisuis from a goat in Canada.

Objective: The present study reports the first isolation and whole-genome sequencing of a Trueperella abortisuis bacterium from a goat. Animals and sample: The T. abortisuis was isolated from the uterus of a goat following an abortion. Procedure: The T. abortisuis was identified by pure culture phenotype and MALDI-TOF analysis and further characterized by whole-genome sequencing. Results: This isolate was reliably identified as T. abortisuis and showed similar properties to type strain T. abortisuis DSM 19515T, which was recovered from a sow following an abortion. The assembled genome of this isolate was 2 564 866 bp long with a GC content of 63.9%. A total of 30 virulence-related genes were determined, suggesting the pathogenic potential of this organism. Conclusion and clinical relevance: This study details the first isolation of T. abortisuis from goats. The genotypic findings of this isolate will serve as a baseline description for any similar future studies.

Premier isolement et séquençage du génome entier de Trueperella abortisuis provenant d'une chèvre au Canada. Objectif: La présente étude rapporte le premier isolement et séquençage du génome entier d'un isolat de Trueperella abortisuis provenant d'une chèvre. Animaux et échantillon: Le T. abortisuis a été isolé de l'utérus d'une chèvre à la suite d'un avortement. Procédure: Le T. abortisuis a été identifié par un phénotype de culture pure et analyse par MALDI-TOF, puis caractérisé par séquençage du génome entier. Résultats: Cet isolat a été identifié de manière fiable comme étant T. abortisuis et a montré des propriétés similaires à la souche type T. abortisuis DSM 19515T, qui a été récupérée chez une truie après un avortement. Le génome assemblé de cet isolat mesurait 2 564 866 pb avec une teneur en GC de 63,9 %. Au total, 30 gènes liés à la virulence ont été déterminés, suggérant le potentiel pathogène de cet organisme. Conclusion et pertinence clinique: Cette étude détaille le premier isolement de T. abortisuis chez la chèvre. Les résultats génotypiques de cet isolat serviront de description de base pour toute étude future similaire.(Traduit par Dr Serge Messier).

Case Series: Microbacterium aurum Bacteremia in Immunosuppressed Patients-An Emerging Threat.

Blood stream infection with Microbacterium species in humans is rare and frequently linked to the presence of immunosuppressed conditions such as patients on chemotherapy or corticosteroids. Presence of indwelling catheters is also a potential risk factor for M. aurum infection. No case report has been documented in the literature regarding the pathogenic potential of M. aurum in causing bacteremia. This is the first case series reporting bacteremia by M. aurum describing the risk factors and sensitivity pattern of this pathogen. In this case series, we have described bacteremia caused by M. aurum. The risk factors and sensitivity pattern of this pathogen have also been evaluated. Here, we describe the clinical course and presentation of three patients whose blood culture showed growth of M. aurum. Indwelling venous catheter for hemodialysis or for chemotherapy for the treatment of acute lymphoblastic leukemia was found to be a risk factor in two patients. Rheumatoid arthritis was the underlying condition in the second patient and was started on immunosuppressants. Blood samples were collected during the febrile period. The blood culture samples of all these patients had pure isolates of M. aurum, identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. All three patients were managed according to the sensitivity reports and were discharged in stable condition.

Experimental infection of goats with pVAPN-harboring Rhodococcus equi causes latent infection in the lymph nodes.

Rhodococcus equi has recently been identified in various animals, including ruminants. Several studies have highlighted the emergence of pVAPN-harboring strains, isolated from multiple abscesses, in the liver and lungs of ruminants. Epidemiological evidence strongly suggests that pVAPN-harboring strains are pathogenic in ruminants. This study aims to replicate the disease in goats through experimental infection. Intravenous administration of the pVAPN-harboring strain (Yokkaichi), pVAPA-harboring strain (ATCC33701), and pVAPN-cured strain (Yokkaichi_P-), each at 1.0 × 107 CFU/head, was conducted in 24-month-old goats (n = 1 per group). During the observation period, goats treated with Yokkaichi or ATCC33701 exhibited transient increases in body temperature and white blood cell count, alongside a decrease in body weight from the administration day. Conversely, goats treated with Yokkaichi_P- displayed no significant changes in these values. The Yokkaichi-treated goat demonstrated a >10-fold increase in anti-VapN antibody titers from 11 to 14 days postadministration, whereas the other two goats exhibited no variation in anti-VapA and VapN antibody titers. Pathological autopsy analysis of organs harvested 28 days postadministration revealed no characteristic lesions on gross examination. However, the inoculated strain (vapN-positive R. equi) was exclusively recovered from the tracheobronchial lymph node in the Yokkaichi-treated goat. Immunohistochemistry detected a VapN-positive reaction in the tracheobronchial lymph node, confirming latent infection despite the absence of dramatic suppurative lesions seen in ruminants. Overall, this study highlights the latent infection in lymph nodes induced by the pVAPN-harboring strain, despite the absence of overt pathological manifestations.

Mycolyltransferase is important for biofilm formation and pathogenesis of Tsukamurella keratitis.

Tsukamurella, a group of multi-drug resistant, Gram-positive, aerobic, and partially acid-fast bacteria, are emerging causes of bacterial conjunctivitis and keratitis. However, the pathogenesis of Tsukamurella keratitis is largely unknown. To address this, we used New Zealand White rabbits to develop the first eye infection model and conducted in vitro tests to study the pathogenesis mechanisms of Tsukamurella. There is increasing evidence that biofilms play a significant role in ocular infections, leading us to hypothesize that biofilm formation is crucial for effective Tsukamurella infection. In order to look for potential candidate genes which are important in biofilm formation and Tsukamurella keratitis. We performed genome sequencing of two ocular isolates, T. pulmonis-PW1004 and T. tyrosinosolvens-PW899, to identify potential virulence factors. Through in vitro and in vivo studies, we characterized their biological roles in mediating Tsukamurella keratitis. Our findings confirmed that Tsukamurella is an ocular pathogen by fulfilling Koch's postulates, and using genome sequence data, we identified tmytC, encoding a mycolyltransferase, as a crucial gene in biofilm formation and causing Tsukamurella keratitis in the rabbit model. This is the first report demonstrating the novel role of mycolyltransferase in causing ocular infections. Overall, our findings contribute to a better understanding of Tsukamurella pathogenesis and provide a potential target for treatment. Specific inhibitors targeting TmytC could serve as an effective treatment option for Tsukamurella infections.

Monitoring of inflammatory blood biomarkers in foals with Rhodococcus Equi pneumonia during antimicrobial treatment.

Rhodococcus equi (R. equi), a gram-positive facultative intracellular pathogen, is a common cause of pneumonia in foals and represents a major cause of disease and death. The aim of the present study was to investigate the time-depended changes in White Blood Cells (WBC), basophils (Baso), neutrophils (Neu), lymphocytes (Lymf), monocytes (Mon), eosinophils (Eos), platelet (PLT) counts, fibrinogen (Fbg) concentration, interferon (IFN-α, IFN-γ) and interleukins (IL-2 and IL-10) in foals with clinical R. equi pneumonia. The main treatment was with azithromycin-rifampicin for 14 days. Blood was sampled prior to, 7 and 14 days after starting therapy. Treatment was associated with significantly decreased counts of WBC, (25.6 ± 6.7 and 14.2 ± 2,7 × 103/ml), Neu (18.6 ±6.2 and 10.7 ± 3.1 × 103/ml), Mon (1.5 ± 0.5 and 0.9 ± 0.2 × 103/ml) and Fbg (539 ± 124 and 287 ± 26 g/dl) between day 0 and day 14. IL-2 and IL-10 concentrations were significantly increased (P = 0.028, P = 0.013, respectively) after treatment, whereas IFN-α and IFN-γ concentrations were not. The diagnostic potentials of INF-α, INF-γ, IL-2 and IL-10 per se seems not very high, however, the study suggests that the activity change of selected interleukins in the course of the disease may be associated with amelioration. We concluded that patterns of serum concentration changes of INF-α, INF-γ, IL-2 and IL-10 may help in the study of the innate immune response in foals during infection and treatment of R. equi pneumonia.

Tsukamurella Bacteremia in a Surgical Patient: Case Report and Review of the Literature.

Background: Tsukamurella species were first isolated in 1941. Since then, 48 cases of Tsukamurella bacteremia have been reported, a majority of which were immunosuppressed patients with central venous catheters.A case is described and previous cases of Tsukamurella bacteremia are reviewed. Patients and Methods: A 70-year-old total parenteral nutrition (TPN)-dependent female with recurrent enterocutaneous fistula (ECF), developed leukocytosis one week after a challenging ECF takedown. After starting broad-spectrum antibiotic agents, undergoing percutaneous drainage of intra-abdominal abscess, and subsequent repositioning of the drain, her leukocytosis resolved. Blood and peripherally inserted central catheter (PICC) cultures grew Tsukamurella spp. The patient was discharged to home with 14 days of daily 2 g ceftriaxone, with resolution of bacteremia. Conclusions: Tsukamurella spp. are a rare opportunistic pathogen predominantly affecting immunocompromised patients, with central venous catheters present in most cases. However, there have been few reported cases in immunocompetent individuals with predisposing conditions such as end-stage renal disease and uncontrolled diabetes mellitus.

Whole genome-based antimicrobial resistance, virulence, and phylogenetic characteristics of Trueperella pyogenes clinical isolates from humans and animals.

Trueperella pyogenes is an opportunistic zoonotic bacterial pathogen, whose antimicrobial resistance, virulence, and genetic relatedness between strains from animals and humans are barely studied. These characteristics were therefore analyzed for clinical T. pyogenes strains from 31 animals of 11 different species and 8 humans determining their complete circular genome sequence and antimicrobial susceptibility. The MICs of 19 antimicrobials including 3 antiseptics correlated to the resistance genes identified in silico within the genomes revealing a predominance of resistance to streptomycin (aadA9), sulfamethoxazole (sul1), and tetracycline (tet(33), tet(W/N/W)) among strains from humans and cattle. Additional resistance genes (erm(X), erm(56), cmx, drfA1, aadA1, aph(3'')-Ib (strA), aph(6)-Id (strB), aac(3)-IVa, aph(4)-Ia) were found only sporadically. The resistance genes were localized on genetic elements integrated into the chromosome. A cgMLST-based phylogenetic analysis revealed two major clusters each containing genetically diverse strains. The human strains showed the closest relatedness to strains from cattle. Virulence genes coding for fimbriae (fimA, fimC), neuroamidase (nanP, nanH), pyolysin (plo), and collagen binding protein (cbpA) were identified in strains from different hosts, but no correlation was observed between virulence factors and strain origin. The existence of resistance genes typically found in Gram-negative bacteria within the Gram-positive T. pyogenes indicates a wider capacity to adapt to antimicrobial selective pressure. Moreover, the presence of similar antimicrobial resistance profiles found in cattle and human strains as well as their closest relatedness suggests common zoonotic features and cattle as the potential source for human infections.

Isolation of vapB-positive Rhodococcus equi from submaxillary lymph nodes with or without granulomatous lesions in growing-finishing pigs in Japan.

To investigate the etiological role of vapB-positive Rhodococcus equi in pigs, R. equi was isolated from the submaxillary lymph nodes with or without macroscopically detectable lesions of apparently healthy growing-finishing pigs at a slaughterhouse in Toyama Prefecture, Japan. R. equi was isolated from 57 (24.6%) of 232 pigs with macroscopically detectable lymph node lesions, and 56 (98.2%) of the 57 isolates were vapB-positive. R. equi was isolated from 10 (2.4%) of 420 pigs without lymph node lesions, and six (60%) of the 10 isolates were vapB-positive. Plasmid DNA was isolated from the 62 vapB-positive isolates and digested with EcoRI and NsiI to obtain the plasmid profile. Fifty-two (83.9%), three (4.8%), and four (6.5%) isolates contained pVAPB subtypes 1, 2, and 3, respectively, while the remaining three isolates were of pVAPB subtypes 9, 13, and 14, respectively. Twelve specimens from lymph nodes with macroscopically detectable lesions were randomly selected for histopathological staining. Granulomatous lesions resembling tuberculosis were found in 11 of the 12 specimens, and the remaining specimen showed typical foci of malakoplakia in the lymph node. The isolation rates of R. equi and vapB-positive R. equi from lymph nodes with macroscopically detectable lesions were significantly higher (P<0.05) than those of lymph nodes without lesions, suggesting an etiologic association between vapB-positive R. equi and macroscopically detectable granulomatous lesions in porcine submaxillary lymph nodes. Previous reports on the prevalence of vapB-positive R. equi in pigs are reviewed and discussed.

Antimicrobial efficiency of bromhexine hydrochloride against endometritis-causing Escherichia coli and Trueperella pyogenes in bovines.

In this study, the main agents associated with endometritis in cows in the state of Santa Catarina, Brazil, were identified and the resistance profile and virulence mechanisms of the bacterial isolates were evaluated. Isolates of Escherichia coli and Trueperella pyogenes were tested for their biofilm forming ability and the antimicrobial action of bromhexine hydrochloride in combination with other antimicrobials. A total of 37 uterine lavage samples were collected from cows with endometritis. Of the 55 bacteria isolated, 25.4% were identified as T. pyogenes and 16.3% as E. coli. The bacterial isolates showed greater resistance to sulfamethoxazole + trimethoprim (58.2%) and tetracycline (56.3%). Among the species, E. coli showed the highest resistance rates, with 100% of isolates showing resistance to amoxicillin, streptomycin, and gentamicin. The results of the minimum inhibitory concentration for the T. pyogenes isolates showed that 91.6% of the isolates were resistant to enrofloxacin and tetracycline, and 75% were resistant to ceftiofur and sulfamethoxazole + trimethoprim. All E. coli and T. pyogenes isolates showed biofilm forming ability. The plo, fimA, and nanH genes were identified in 100% of T. pyogenes isolates. In parallel, 100% of E. coli isolates had the fimH gene, and 11.1% had the csgD gene. Bromhexine hydrochloride showed antimicrobial activity against 100% of E. coli isolates and 66.6% of T. pyogenes isolates. Furthermore, when associated with antimicrobials, bromhexine hydrochloride has a synergistic and additive effect, proving to be an option in the treatment of endometritis in cows and an alternative for reducing the use of antimicrobials.

Tropheryma whipplei infection in the lung of a patient with long COVID: a case report.

BACKGROUND: Immune dysregulation in individuals with long COVID has been detected. Differential diagnosis of diffuse infiltration on chest CT in long COVID is challenging. CASE PRESENTATION: A 62-year-old man presented with a 10-month history of dyspnea after COVID-19 infection. Dyspnea became worse in the one month preceding presentation. The chest CT showed multifocal, subpleural, bilateral opacities due to long-COVID, and infiltration around the bronchovascular bundle in the bilateral lower lung field. The pathology for the transbronchial cryobiopsy (TBCB) first reported chronic inflammation (mainly interstitial pneumonia). The patient had positive results on tests for the antibody, RO-52+, EJ+. The presumptive diagnosis of connective tissue disease-interstitial lung disease was made. Prednisone and cyclophosphamide were given. At follow-up one month later, the chest CT showed new diffuse ground-glass infiltration. The previous TBCB specimen was re-evaluated. Foamy macrophages were found in the alveolar air space. Periodic acid-Schiff (PAS) staining was performed. Numerous intracytoplasmic organisms were detected, with morphologic features consistent with those of Tropheryma whipplei. The patient recovered after intravenous ceftriaxone and oral trimethoprim-sulfamethoxazole. The final diagnosis was lung T. whipplei infection and long COVID-19. CONCLUSION: This is the first case report of Tropheryma whipplei infection in the lung of a patient with long COVID-19. T. whipplei should be considered as a potential pathogen for diffuse lung infiltration in the post-COVID-19 era.

Recurrent sternal wound infection caused by Gordonia bronchialis after open heart surgery.

Gordonia bronchialis is an aerobic gram-positive bacilli and also weakly acid fast. It requires a long incubation time and extensive biochemical reactions for identification. Therefore, use of broad-range polymerase chain reaction (PCR) for amplification of genes such as 16S rRNA or hsp65 followed by sequencing or advanced techniques like MALDI-TOF MS is needed for identification. Here, we present a case of persistent sternal wound infection following open heart surgery, caused by G. bronchialis in a 58 years old male, identified using MALDI-TOF MS-based system. The patient improved with oral Cefpodoxime 200 mg BD for four weeks.

Update on emerging multidrug-resistant Rhodococcus equi.

Jose Vázquez-Boland, Jorge Val-Calvo and Mariela Scortti present a brief summary of the main aspects surrounding the recently identified multidrug-resistant Rhodococcus equi that emerged in the USA and the actions being taken to tackle the problem with support from the UK's Horserace Betting Levy Board.

First description of Rhodococcus equi infection in common bottlenose dolphin (Tursiops truncatus).

Rhodococcus equi is a terrestrial bacterium and a common pathogen in foals (Equus caballus), in which causes pneumonia. This report describes for the first time the infection caused by R. equi in a common bottlenose dolphin (Tursiops truncatus) stranded in the Calabrian coast, Italy. The post mortem examination of the animal revealed lesions in lung and colon. The animal was also positive to dolphin morbillivirus. The histological study showed lesions attributable to R. equi infection, such as pyogranulomatous bacterial pneumonia and chronic granulomatous colitis. Whole genome sequencing of the isolated strain confirmed its identification as R. equi.

Rhodococcus infection: a 10-year retrospective analysis of clinical experience and antimicrobial susceptibility profile.

Rhodococcus equi is an opportunistic pathogen known to cause pulmonary and extrapulmonary disease among immunocompromised patients. Treatment is frequently challenging due to intrinsic resistance to multiple antibiotic classes. While non-equi Rhodococcus spp. are prevalent, their clinical significance is poorly defined. There is also limited data on antibiotic susceptibility testing (AST) of Rhodococcus infection in humans. We conducted a single-center, retrospective cohort study evaluating clinical characteristics, microbiologic profile, and AST of Rhodococcus infections between June 2012 and 2022 at our tertiary academic medical center. Identification of Rhodococcus spp. was performed by Sanger 16S rRNA gene sequencing and/or matrix-assisted laser desorption ionization-time of flight mass spectrometry, and AST was performed by agar dilution. Three hundred twenty-two isolates of Rhodococcus spp. were identified from blood (50%), pulmonary (26%), and bone/joint (12%) sources. R. equi/hoagii, R. corynebacterioides, and R. erythropolis were the most frequently isolated species, with 19% of isolates identified only to genus level. One hundred ninety-nine isolates evaluated for AST demonstrated high-level resistance to amoxicillin/clavulanate, cephalosporins, and aminoglycosides. More than 95% susceptibility to imipenem, vancomycin, linezolid, rifampin, and clarithromycin was observed. Non-equi species showed a significantly more favorable AST profile relative to R. equi. Clinically significant Rhodococcus infection was rare with 10 cases diagnosed (majority due to R. equi) and managed. The majority of patients received 2- or 3-drug combination therapy for 2-6 months, with favorable clinical response. Significant differences in AST were observed between R. equi and non-equi species. Despite high antimicrobial resistance to several antibiotic classes, imipenem and vancomycin remain appropriate empiric treatment options for R. equi. Future research evaluating mechanisms underlying antimicrobial resistance is warranted.

The impact of age on vitamin D receptor expression, vitamin D metabolism and cytokine production in ex vivo Rhodococcus equi infection of equine alveolar macrophages.

Rhodococcus equi (R. equi), a pneumonia-causing intracellular bacterium, results in significant morbidity and mortality in young foals, while healthy adult horses rarely develop disease. Survival and replication within alveolar macrophages (AMφ) are the hallmarks of R. equi's pathogenicity. The vitamin D receptor (VDR) and its ligand, the active vitamin D metabolite 1,25(OH)2D, are important in immune responses to intracellular bacteria. The vitamin D/VDR pathway regulates the downstream production of cytokines in infected human AMφ. The immunomodulatory role of the vitamin D/VDR pathway in equine leukocytes is unknown. The objective of the current study was to determine the impact of R. equi infection and age on synthesis of 1,25(OH)2D, VDR expression, and cytokine production in an ex vivo model of R. equi infection in equine AMφ. AMφ were collected from ten healthy foals at 2-, 4- and 8-weeks old and from nine healthy adult horses once via bronchoalveolar lavage. AMφ were mock infected (CONTROL) or infected with a virulent laboratory strain of R. equi for 7 days (INFECTED). VDR expression was determined via RT-qPCR from cell lysates. 1,25(OH)2D and cytokines were measured in cell supernatant by immunoassays. VDR expression was impacted by age (P = 0.001) with higher expression in AMφ from 8-week-old foals than from 2-week-old foals and adults. There was no significant effect of infection in foal AMφ, but in adults, relative VDR expression was significantly lower in INFECTED AMφ compared to CONTROL AMφ (P = 0.002). There was no effect of age or infection on 1,25(OH)2D concentration (P > 0.37). Mean TNFα production was significantly higher from INFECTED compared to CONTROL AMφ from 4- and 8-week-old foals and adults (P < 0.005). Mean IFNγ production was significantly higher from AMφ from foals at 8-weeks-old compared to 2-weeks-old (P = 0.013) and higher from INFECTED AMφ than from CONTROL AMφ in foals at 4-weeks-old and in adults (P < 0.027). The proportion of samples producing IL-1ß and IL-10 was also significantly higher from INFECTED compared to CONTROL AMφ isolated from 4-week-old foals (P < 0.008). Similarly, in adult samples, IL-17 was produced from a greater proportion of INFECTED compared to CONTROL samples (P = 0.031). These data document age-associated changes in VDR expression and cytokine production in equine AMφ in response to R. equi infection. This preliminary investigation supports the need for further research to fully elucidate if the vitamin D pathway has an immunomodulatory role in the horse.

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.

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.