Virulent Rhodococcus Equi Research Articles

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.

Retraction for Liu et al., "Appearance of Multidrug-Resistant Virulent Rhodococcus equi Clinical Isolates Obtained in China".

Retraction for Liu et al., "Appearance of Multidrug-Resistant Virulent Rhodococcus equi Clinical Isolates Obtained in China".

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

BackgroundDiagnostic accuracy of real‐time, quantitative PCR (qPCR) assays to quantify virulent Rhodococcus equi using rectal swab samples has not been systematically evaluated.ObjectiveTo evaluate the accuracy of qPCR of rectal swab samples to differentiate foals with pneumonia from healthy foals of similar age from the same environment.AnimalsOne hundred privately owned foals born in 2021 from 2 farms in New York.MethodsAn 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.ResultsThe 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 ImportanceAlthough 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.

BackgroundIntragastric 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.HypothesisFecal concentration of virulent R. equi before IB infection with R. equi is positively associated with protection from pneumonia in foals.AnimalsTwenty‐one university‐owned foals.MethodsSamples 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).ResultsFecal 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 ImportanceGreater natural ingestion of LVRE might contribute to protection against pneumonia among foals.

Plasmid Profiles of Virulent Rhodococcus equi Strains Isolated from Infected Foals in Poland.

Rhodococcus equi is an important bacterial pathogen in foals up to 6 months old, widespread in horse farms all over the world. It was found that only virulent R. equi strains expressing 15–17 kDa virulence-associated protein (VapA) and having large virulence plasmid of 85–90 kb containing vapA gene are pathogenic for horses. To date, 12 plasmid types have been reported in VapA positive strains from horses. There are no data concerning plasmid types of Polish field R. equi strains isolated from horses and horse farm environment. The aim of the study is to determine plasmid profiles of virulent R. equi strains isolated in Poland from dead foals as well as from soil samples taken from horse breeding farms. Plasmid profiles of 10 clinical strains derived from 8 farms and 11 environmental strains from 3 farms, confirmed as virulent by PCR, were compared with 12 reference strains containing the known plasmid size and type. Plasmid DNAs were analysed by digestion with the restriction endonucleases BamHI, EcoRI, EcoT22I, and HindIII for detailed comparison and estimation of plasmid sizes. The results of RFLP analysis revealed that all except one isolates used in the study are classified as VapA 85 kb type I plasmid. One strain harboured VapA 87 kb type I plasmid. This is the first report of plasmid types of Polish field R. equi strains. The results of our preliminary investigations on horse farms located in central and eastern Poland indicate that the virulent R. equi strains thus far isolated from diseased foals and horse farms environment represent a highly uniform plasmid pattern.

Virulent Rhodococcus equi isolates from foals in Poland – pVapA characteristics and plasmid new variant, 85-kb type V

Virulent Rhodococcus equi isolates from foals in Poland – pVapA characteristics and plasmid new variant, 85-kb type V

Antimicrobial susceptibility of Australian virulent Rhodococcus equi isolates collected between 1991 and 2014

Antimicrobial susceptibility of Australian virulent Rhodococcus equi isolates collected between 1991 and 2014

Clinical Research Abstracts of the British Equine Veterinary Association Congress 2015.

Bronchopneumonia caused by Rhodococcus equi is an important disease of young horses throughout the world. Although early diagnosis and treatment improves the prognosis, this also increases the amount of antimicrobial usage and therefore increases the likelihood of resistance developing. The aim of this study was to determine the level of resistance to commonly prescribed antimicrobial agents of 97 virulent Rhodococcus equi isolates. Analysis of archived samples. Virulent Rhodococcus equi isolates were collected between 1991 and 2014 from clinically affected horses and from air samples collected in the breathing zone of foals. Antimicrobial susceptibility of these isolates was assessed using a disc diffusion assay with a panel of agents. The minimum inhibitory concentration (MIC) was determined for rifampicin, erythromycin, tetracycline, and neomycin using a novel resazurin-based microtitre assay. Resistance to rifampicin was detected in 3 of the isolates (2 collected in 2013 and one in 2014) by both methods. The MIC for these isolates was 64 μg/ml (n = 2) and 16 μg/ml (n = 1). All isolates collected prior to 2013 had MICs<0.125 μg/ml, which was the limit of detection in this assay. Although no isolates were resistant to tetracycline, there was a general increase in MIC in isolates collected in recent years. No isolates were resistant to either neomycin or erythromycin, with MIC values ranging between 0.25 and 2 μg/ml for neomycin and 0.125-1 μg/ml for erythromycin. The success of the macrolide-rifampicin combination relies on the synergistic action of these 2 agents. Resistance to rifampicin will reduce the therapeutic efficacy of this treatment. It is of serious concern that the resistant isolates were all recently collected. Hopefully, recent research will lead to fewer asymptomatic foals receiving antimicrobials which will in turn reduce the likelihood of ongoing development of resistance. Ethical animal research: All organisms in this study were received by the laboratory from diagnostic accessions. Sources of funding: Funding for the study was provided by Rural Industries Research and Development Corporation and the University of Melbourne. G. Herbert was the recipient of a RN McCarthy scholarship from the Faculty of Veterinary Science. Competing interests: None declared.

Association of perinatal exposure to airborne Rhodococcus equi with risk of pneumonia caused by R equi in foals

To determine whether the concentrations of airborne virulent Rhodococcus equi in stalls housing foals during the first 2 weeks after birth are associated with subsequent development of R equi pneumonia in those foals. Air samples collected from foaling stalls and holding pens in which foals were housed during the first 2 weeks after birth. At a breeding farm in Texas, air samples (500 L each) were collected (January through May 2011) from stalls and pens in which 121 foals were housed on day 1 and on days 4, 7, and 14 after birth. For each sample, the concentration of airborne virulent R equi was determined with an immunoblot technique. The association between development of pneumonia and airborne R equi concentration was evaluated via random-effects Poisson regression analysis. Some air samples were not available for analysis. Of the 471 air samples collected from stalls that housed 121 foals, 90 (19%) contained virulent R equi. Twenty-four of 121 (20%) foals developed R equi pneumonia. Concentrations of virulent R equi in air samples from stalls housing foals that developed R equi pneumonia were significantly higher than those in samples from stalls housing foals that did not develop pneumonia. Accounting for disease effects, air sample concentrations of virulent R equi did not differ significantly by day after birth or by month of birth. Exposure of foals to airborne virulent R equi during the first 2 weeks after birth was significantly (and likely causally) associated with development of R equi pneumonia.

Association of airborne concentration of virulent Rhodococcus equi with location (stall versus paddock) and month (January through June) on 30 horse breeding farms in central Kentucky

To determine whether the concentration of airborne virulent Rhodococcus equi varied by location (stall vs paddock) and month on horse farms. Air samples from stalls and paddocks used to house mares and foals on 30 horse breeding farms in central Kentucky. Air samples from 1 stall and 1 paddock were obtained monthly from each farm from January through June 2009. Concentrations of airborne virulent R equi were determined via a modified colony immunoblot assay. Random-effects logistic regression was used to determine the association of the presence of airborne virulent R equi with location from which air samples were obtained and month during which samples were collected. Of 180 air samples, virulent R equi was identified in 49 (27%) and 13 (7%) obtained from stalls and paddocks, respectively. The OR of detecting virulent R equi in air samples from stalls versus paddocks was 5.2 (95% confidence interval, 2.1 to 13.1). Of 60 air samples, virulent R equi was identified in 25 (42%), 18 (30%), and 6 (10%) obtained from stalls during January and February, March and April, and May and June, respectively. The OR of detecting virulent R equi from stall air samples collected during May and June versus January and February was 0.22 (95% confidence interval, 0.08 to 0.63). Foals were more likely to be exposed to airborne virulent R equi when housed in stalls versus paddocks and earlier (January and February) versus later (May and June) during the foaling season.

Associations between the Exposure to Airborne Virulent Rhodococcus equi and the Incidence of R equi Pneumonia among Individual Foals

Rhodococcus equi is a significant cause of pneumonia, resulting in disease and sometimes death of foals. It is believed that infection occurs by inhalation of dust contaminated with virulent R equi. Although association between the airborne concentration of virulent R equi and the incidence of foal pneumonia at breeding farms has been documented, studies at the level of individual foals have not been reported. Thus, the objective of this study was to determine whether the magnitude of airborne virulent R equi was significantly associated with risk of R equi pneumonia for individual foals. The concentration of virulent R equi was significantly ( P < .001) greater in stalls than paddocks among samples collected from 47 foals at a breeding farm in central Kentucky. The presence of airborne virulent R equi in stalls was significantly ( P = .045) more likely at 7 days of age for foals subsequently found to be affected by rhodococcal pneumonia. Additionally, airborne concentrations of virulent R equi in stalls were significantly greater at 7 and 14 days of age than at birth. Presence of the mare and foal at the time of sampling was significantly ( P < .001) associated with increased airborne concentrations of virulent R equi in stalls. These findings suggest that environments containing airborne virulent R equi during the first week of life may influence the risk of subsequent disease for a foal.

Investigation of the antagonistic activity of spore probiotics against Rhodococcus equi in experiments on mice

A method is developed for studying the antagonistic activity of the spore probiotics Sakhabactisubtil and Irilis in vivo against a virulent Rhodococcus equi. Intraperitoneal injection of sterile filtrates of these spore probiotics into mice that received the antibiotic Ampiox and were infected with a lethal dose of the virulent R. equi strain promotes the survival of 70–100% of the animals and reduced the number of rhodococci in internal organs by 6–9 times.

Effects of location for collection of air samples on a farm and time of day of sample collection on airborne concentrations of virulent Rhodococcus equi at two horse breeding farms

To determine whether airborne concentrations of virulent Rhodococcus equi at 2 horse breeding farms varied on the basis of location, time of day, and month. 2 farms in central Kentucky with recurrent R equi-induced pneumonia in foals. From February through July 2008, air samples were collected hourly for a 24-hour period each month from stalls and paddocks used to house mares and their foals. Concentrations of airborne virulent R equi were determined via a modified colony immunoblot technique. Differences were compared by use of zero-inflated negative binomial methods to determine effects of location, time, and month. Whether mares and foals were housed predominantly in stalls or paddocks significantly affected results for location of sample collection (stall vs paddock) by increasing airborne concentrations of virulent R equi at the site where horses were predominantly housed. Airborne concentrations of virulent R equi were significantly higher from 6:00 pm through 11:59 pm than for the period from midnight through 5:59 am. Airborne concentrations of virulent R equi did not differ significantly between farms or among months. Airborne concentrations of virulent R equi were significantly increased when horses were predominantly housed at the site for collection of air samples (ie, higher in stalls when horses were predominantly housed in stalls and higher in paddocks when horses were predominantly housed in paddocks). Concentrations of virulent R equi among air samples collected between the hours of 6:00 am and midnight appeared similar.

In vitro antimicrobial activity of gallium maltolate against virulent Rhodococcus equi

The objective of this study was to determine the in vitro antimicrobial activity of gallium maltolate (GaM) against Rhodococcus equi. A total of 98 virulent bacterial isolates from equine clinical cases were examined, of which 19 isolates were known to be resistant to macrolides and rifampin. Isolates were cultured with various concentrations of GaM and minimal inhibitory concentration (MIC) values were determined after 24 and 48h. Both the MIC50 and the MIC90 after 24h of growth were 3,560ng/mL (8μM) and after 48h of growth were 14,241ng/mL (32μM). There were no apparent differences between MICs of macrolide-resistant and macrolide-susceptible isolates.

Lawsonia intracellularis and Virulent Rhodococcus equi Infection in a Thoroughbred Colt

A 26-month-old thoroughbred colt with a 4-month history of continuous diarrhoea and weight loss was subject to necropsy examination. The small intestinal mucosa was thickened and this change particularly affected the terminal ileum. Microscopical examination revealed multifocal epithelial hyperplasia, with multifocal granulomas and marked lymphocytic infiltration of the lamina propria. Numerous gram-negative argyrophilic curved bacilli were observed within the cytoplasm of affected enterocytes. Macrophages and epithelioid cells forming the granulomas had abundant, lightly eosinophilic, foamy cytoplasm, with occasional large, clear vacuoles containing gram-positive coccobacilli. Immunohistochemical studies suggested that the argyrophilic bacilli were Lawsonia intracellularis and the gram-positive coccobacilli were Rhodococcus equi. L. intracellularis-specific DNA fragments were amplified from the affected ileocaecal mucosa by polymerase chain reaction. Virulent R.equi (VapA positive) was isolated in pure culture from the liver and mesenteric lymph nodes. These results suggested that the two intracytoplasmic organisms had induced multifocal proliferative and granulomatous enteritis accompanied by severe and extensive lymphocytic infiltration.

Effects of inoculum size on cell-mediated and humoral immune responses of foals experimentally infected with Rhodococcus equi: A pilot study

The objective of this pilot study was to compare the cytokine profile as well as cell-mediated and antibody responses of foals infected with a low inoculum of virulent Rhodococcus equi resulting in subclinical pneumonia to that of foals infected with a high inoculum resulting in severe clinical pneumonia. The mean (±SD) ratio of post-infection to pre-infection anti- R. equi IgG(T) concentration was significantly ( P = 0.002) higher in foals infected with the high inoculum (195 ± 145; range 62–328) compared to foals infected with the low inoculum (3.9 ± 4.5; range 0.5–11). Similarly, mean (±SD) ratio of post-infection to pre-infection IgM concentration was significantly ( P = 0.002) higher in foals infected with the high inoculum (12 ± 4.0; range 7.4–14) compared to foals infected with the low inoculum (2.5 ± 1.5; range 1.2–4.7). Proliferative responses to R. equi antigens as well as expression of mRNA for IL-2, IL-4, IL-10, and IFN-γ in BLN were not significantly different between the two groups. There was a tendency ( P = 0.073) towards a higher IFN-γ/IL-4 ratio in the low inoculum group. This study demonstrates that the size of inoculum modulates the IgG subisotype response and possibly the cytokine profile of foals.

Intermediately virulent Rhodococcus equi isolates from pigs in Slovenia: discovery of new plasmid types and assessment of genetic diversity by pulsed-field gel electrophoresis

The presence of large plasmids in 30 &lt;I&gt;Rhodococcus equi&lt;/I&gt; strains isolated from pig lymph nodes with granulomatous changes was investigated. Plasmid DNAs were isolated and digested with the restriction endonucleases &lt;I&gt;Bam&lt;/I&gt;HI, &lt;I&gt;Eco&lt;/I&gt;RI, &lt;I&gt;Eco&lt;/I&gt;T22I and &lt;I&gt;Hind&lt;/I&gt;III for detailed comparison and estimation of plasmid sizes. A total of nine isolates were identified as intermediately virulent (VapB-positive), harbouring large plasmids of type 5 (&lt;I&gt;n&lt;/I&gt; = 5) and four new variants that we tentatively designated as type 19 (&lt;I&gt;n&lt;/I&gt; = 1), 20 (&lt;I&gt;n&lt;/I&gt; = 1), 21 (&lt;I&gt;n&lt;/I&gt; = 1) and 24 (&lt;I&gt;n&lt;/I&gt; = 1). All isolates were subjected to genotyping with pulsed-field gel electrophoresis (PFGE). High genetic diversity was observed: 21 distinct genotypes were detected; five were found in multiple isolates and the others were unique. Isolates of the same plasmid type exhibited different PFGE profiles and vice versa. In a few cases, multiple strains from certain farms were analysed, the majority of which exhibited diverse PFGE profiles. Our findings demonstrate the presence of a wide variety of &lt;I&gt;R. equi&lt;/I&gt; strains even in small confined environments such as farms. This is the first molecular epidemiology study of intermediately virulent &lt;I&gt;R. equi&lt;/I&gt; isolates from Slovenian pigs.

Age-related changes in cytokine expression by neutrophils of foals stimulated with virulent Rhodococcus equi in vitro

Although evidence exists that neutrophils play a vital role in resistance to infection with Rhodococcus equi, the means by which neutrophils exert their effects have not been clearly defined. In the present study we evaluated differences in cytokine expression by unstimulated and R. equi-stimulated neutrophils obtained from newborn foals and subsequently at 2-, 4-, and 8-weeks of age. Stimulation with virulent R. equi induced significantly ( P < 0.05) greater expression of IFNγ, TNFα, IL-6, IL-8, IL-12p40, IL-12p35, and IL-23p19 mRNA relative to expression by unstimulated neutrophils, and there were significant effects of age on expression of IL-6, IL-8, IL-12p40 and IL-23p19. Neutrophil expression of IL-6 and IL-8 in newborn foals was significantly greater than expression at 2-, 4-, and 8-weeks of age. Expression of IL-12p40 by R. equi-stimulated neutrophils from newborn and 2-week-old foals did not differ from that of unstimulated neutrophils; however, expression of IL-12p40 by neutrophils from 4- and 8-week-old foals was significantly greater when stimulated by R. equi than without stimulation. These results demonstrate that foal neutrophils increase mRNA expression of many pro-inflammatory cytokines, including IFNγ, in response to in vitro stimulation with R. equi, and that the magnitude of this expression with respect to IL-6, IL-8, IL-12p40 and IL-23p19 is influenced by age. The clinical importance of the age-related difference in R. equi-induced expression of IL-12p40 to susceptibility to R. equi pneumonia remains to be determined.

Association of soil concentrations of Rhodococcus equi and incidence of pneumonia attributable to Rhodococcus equi in foals on farms in central Kentucky

To determine whether soil concentrations of total or virulent Rhodococcus equi differed among breeding farms with and without foals with pneumonia caused by R equi. 37 farms in central Kentucky. Procedures-During January, March, and July 2006, the total concentration of R equi and concentration of virulent R equi were determined by use of quantitative bacteriologic culture and a colony immunoblot technique, respectively, in soil specimens obtained from farms. Differences in concentrations and proportion of virulent isolates within and among time points were compared among farms. Soil concentrations of total or virulent R equi did not vary among farms at any time point. Virulent R equi were identified in soil samples from all farms. Greater density of mares and foals was significantly associated with farms having foals with pneumonia attributable to R equi. Among farms with affected foals, there was a significant association of increased incidence of pneumonia attributable to R equi with an increase in the proportion of virulent bacteria between samples collected in March and July. Results indicated that virulent R equi were commonly recovered from soil of horse breeding farms in central Kentucky, regardless of the status of foals with pneumonia attributable to R equi on each farm. The incidence of foals with pneumonia attributable to R equi can be expected to be higher at farms with a greater density of mares and foals.

Virulence-associated protein-specific serum immunoglobulin G-isotype expression in young foals protected against Rhodococcus equi pneumonia by oral immunization with virulent R. equi

The purpose of this study was to determine whether foals immunized orally from 2 days of age with virulent Rhodococcus equi developed a protective pulmonary immune response and to characterise the antibody response of the immunized foals to the virulence-associated proteins (Vaps) of the bacterium. Two groups of foals were used. One ( n = 4) was given live R. equi ATCC 33701 orally at 2, 7, and 14 days of age. The second group comprised three non-immunized foals age-matched to the vaccinates. At 3 weeks of age, 1 week after the final immunization, both groups were challenged intrabronchially with virulent R. equi ATCC 33701 and observed for 2 weeks post-challenge. Unvaccinated foals became clinically pneumonic and had high fever with increased heart and respiratory rates and severe pneumonia evident at necropsy. Foals of the immunized group remained healthy and lung lesions were not found post-mortem. Thus, it is possible to immunize young foals orally to protect them by 3 weeks of age against lung challenge with R. equi, even in the presence of maternal antibodies. The antibody response of the immunized foals confirmed that VapA and VapC are highly immunogenic. The immunoglobulin G isotype-related serum antibody response of immunized compared to non-immunized foals had an IgGT bias and a relatively low IgGa:IgGb ratio, both features different from what has been previously observed in immune adults and immune foals. This suggests that the serum IgG isotype profile of antibody cannot be used as a measure of evidence of protection against R. equi pneumonia.