Pneumonic Foals Research Articles

Differential Effects of Rhodococcus equi Virulence-Associated Proteins on Macrophages and Artificial Lipid Membranes.

Virulence-associated protein A (VapA) of Rhodococcus equi is a pathogenicity factor required for the multiplication of virulent R. equi strains within spacious macrophage vacuoles. The production of VapA is characteristic for R. equi isolates from pneumonic foals. VapB and VapN proteins in R. equi isolates from infected pig (VapB) and cattle (VapN) have amino acid sequences very similar to VapA and consequently have been assumed to be its functional correlates. Using model membrane experiments, phagosome pH acidification analysis, lysosome size measurements, protein partitioning, and degradation assays, we provide support for the view that VapA and VapN promote intracellular multiplication of R. equi by neutralizing the pH of the R. equi-containing vacuole. VapB does not neutralize vacuole pH, is not as membrane active as VapA, and does not support intracellular multiplication. This study also shows that the size of the sometimes enormous R. equi-containing vacuoles or the partitioning of purified Vaps into organic phases are not features that have predictive value for virulence of R. equi, whereas the ability of Vaps to increase phagosome pH is coupled to virulence. IMPORTANCE Rhodococcus equi is a major cause of life-threatening pneumonia in foals and occasionally in immunocompromised persons. Virulence-associated protein A (VapA) promotes R. equi multiplication in lung macrophages, which are the major host cells during foal infection. In this study, we compare cellular, biochemical, and biophysical phenotypes associated with VapA to those of VapB (typically produced by isolates from pigs) or VapN (isolates from cattle). Our data support the hypothesis that only some Vaps support multiplication in macrophages by pH neutralization of the phagosomes that R. equi inhabit.

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.

A Common Practice of Widespread Antimicrobial Use in Horse Production Promotes Multi-Drug Resistance

The practice of prophylactic administration of a macrolide antimicrobial with rifampin (MaR) to apparently healthy foals with pulmonary lesions identified by thoracic ultrasonography (i.e., subclinically pneumonic foals) is common in the United States. The practice has been associated epidemiologically with emergence of R. equi resistant to MaR. Here, we report direct evidence of multi-drug resistance among foals treated with MaR. In silico and in vitro analysis of the fecal microbiome and resistome of 38 subclinically pneumonic foals treated with either MaR (n = 19) or gallium maltolate (GaM; n = 19) and 19 untreated controls was performed. Treatment with MaR, but not GaM, significantly decreased fecal microbiota abundance and diversity, and expanded the abundance and diversity of antimicrobial resistance genes in feces. Soil plots experimentally infected with Rhodococcus equi (R. equi) and treated with MaR selected for MaR-resistant R. equi, whereas MaR-susceptible R. equi out-competed resistant isolates in GaM-treated or untreated plots. Our results indicate that MaR use promotes multi-drug resistance in R. equi and commensals that are shed into their environment where they can persist and potentially infect or colonize horses and other animals.

Rhodococcus equi-specific hyperimmune plasma administration decreases faecal shedding of pathogenic R. equi in foals.

Rhodococcus equi is the most common cause of pneumonia in young foals. Pneumonic foals are an important source of environmental contamination as they shed higher amounts of R. equi in their faeces than unaffected foals. As R. equi-specific hyperimmune plasma (HIP) lessens clinical pneumonia, we hypothesise that its use would result in decreased faecal shedding of R. equi by foals. Neonatal foals were either given HIP (n=12) or nothing (n=9, control) shortly after birth and were then experimentally infected with R. equi Faeces were collected before and on weeks 2, 3, 5 and 7 after infection. Presence of virulent R. equi was tested using qPCR. There was strong evidence of an association between HIP administration and a decrease in faecal shedding of virulent R. equi (P=0.031 by Pearson chi-squared test). Foals in the control shed significantly more R. equi (colony-forming units/ml) than foals that received HIP (P=0.008 by Mann-Whitney rank-sum test). While our study is the first to report this additional benefit of HIP administration, future studies are needed to evaluate the implications of its use under field conditions.

Molecular characterization of Rhodococcus equi isolates from horses in Poland: pVapA characteristics and plasmid new variant, 85-kb type V.

Background Rhodococcus equi is one of the most significant bacterial pathogens affecting foals up to 6 months of age worldwide. Rhodococcosis is present in Poland however information about molecular characterization of R. equi isolates is scarce.This study describes molecular characterization of Rhodococcus equi infection on 13 horse breeding farms in Poland between 2001 and 2012. Samples were collected by tracheobronchial aspiration from pneumonic foals or during necropsy. The R. equi isolates were genotyped by plasmid profiling and pulsed-field gel electrophoresis.ResultsTotally, 58 R. equi isolates were investigated. One isolate lost its plasmid. Among the 57 VapA-positive isolates, 48 contained 85-kb type I plasmid (82.8%), 8 contained 87-kb type I plasmid (13.8%). One isolate (1.7%) had a unique restriction cleavage pattern and the 2nd fragment of EcoRI digests of this plasmid DNA was about 2600 bases smaller than that of the 85 kb type I. This new plasmid variant was designated as the “85-kb type V”.Among the 58 isolates typeable with VspI-PFGE, ten PFGE clusters were detected. The majority of foals were infected mostly with isolates of low genetic diversity.ConclusionsMost of clinical isolates of R. equi from foals in Poland contain pVapA 85-kb type I and 87-kb type I similarly to the other European countries and the United States. However, the new variant of pVapA 85-kb type V was identified.The chromosomal variability was detected among some of the investigated isolates and the presence of farm-specific isolates might be possible.

Clinical Assessment of a Point-of-Care Serum Amyloid A Assay in Foals with Bronchopneumonia.

BackgroundDespite the paucity of data available, stall‐side serum amyloid (SAA) assays are commonly used to make diagnostic and treatment decisions in foals with bronchopneumonia.HypothesisMeasurement of SAA concentrations can accurately differentiate pneumonic from healthy foals.AnimalsFifty‐four pneumonic foals between 3 weeks and 5 months of age were compared to 44 healthy controls. In addition, 47 foals on a farm endemic for R. equi infections were studied.MethodsSerum samples were collected from pneumonic foals at hospital admission. Foals were categorized as having pneumonia caused by R. equi or by other microorganisms based on culture of a tracheobronchial aspirate. In addition, serum samples were obtained at 2‐week intervals from foals born at a farm endemic for R. equi. SAA concentrations were measured by a point‐of‐care assay. Diagnostic performance of SAA was assessed by use of receiver operating characteristic curves.ResultsConcentrations of SAA in foals with bronchopneumonia were significantly (P < 0.001) higher than those of healthy foals, but 15 of 54 pneumonic foals (28%) had SAA concentrations <5 μg/mL. There was no correlation between SAA concentrations and radiographic score in foals with R. equi pneumonia. The ability of SAA to predict development of R. equi pneumonia at the endemic farm was limited with a sensitivity of 64% and a specificity of 77%.Conclusion and clinical importanceOverall, SAA concentrations are significantly higher in pneumonic than in healthy foals. However, performance of SAA in detecting pneumonic foals is limited by the high proportion of false‐positive and false‐negative results.

Estimating the Sensitivity and Specificity of Real-Time Quantitative PCR of Fecal Samples for Diagnosis of Rhodococcus equi Pneumonia in Foals.

BackgroundReal‐time, quantitative PCR (qPCR) methods for detecting Rhodococcus equi in feces have been developed as a noninvasive, rapid diagnostic test for R. equi pneumonia, but have not been evaluated in a large population of foals.ObjectiveThe objective of this study was to evaluate the clinical utility of fecal PCR as a diagnostic test for R. equi pneumonia in foals using receiver operating characteristic (ROC) methods.Animals186 foals born in 2011 at an R. equi‐endemic ranch in Texas.MethodsFecal samples were collected at the time of onset of clinical signs for pneumonic foals (n = 31). Foals with pneumonia were matched by age and birth date to healthy (n = 31) and subclinical (n = 124) control foals; fecal samples were collected from these controls. DNA was extracted from feces using commercial kits and concentration of virulent R. equi in feces was determined by qPCR.ResultsConcentration of R. equi in feces differed significantly (P < .05) among groups. The area under the ROC curve for fecal qPCR for diagnosis of R. equi pneumonia was 89% (95% CI, 83–99), with a sensitivity of 94% and specificity of 72%.Conclusions and Clinical Importance qPCR of feces can be useful as an alternative to tracheobronchial aspiration for the diagnosis of R. equi in foals with clinical signs of pneumonia. Caution should be used in extrapolating results of this study to other populations because fecal concentration of R. equi might vary by geographic location or management practices.

Validation and evaluation of VapA‐specific IgG and IgG subclass enzyme‐linked immunosorbent assays (ELISAs) to identify foals with Rhodococcus equi pneumonia

Rhodococcus equi (Rhodococcus hoagii/Prescottella equi) is a common cause of foal pneumonia, but its diagnosis remains a challenge for equine veterinarians. While the VapA-specific (virulence-associated protein A) immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) has low sensitivity and specificity for detecting pneumonic foals, little is known about VapA-specific IgG subclasses. To evaluate the performance of VapA-specific ELISA for IgG and its subclasses IgGa, IgGb and IgG(T) in the early diagnosis of pneumonia caused by R. equi. Assay validation followed by assessment of diagnostic performance using archived samples from animals of known status. Serum samples from exposed (n = 125) and nonexposed adult horses (n = 10) and from experimentally challenged and naturally infected foals were used for ELISA validation. Post mortem and tissue culture records of the last 24 years from the Institute for Experimental Pathology at the University of Iceland in Keldur, Iceland laboratory were evaluated to confirm the absence of R. equi cases in Iceland. The diagnostic performance of VapA-specific IgG and its subclasses was evaluated using banked serum samples from pneumonic (n = 21) and healthy foals (n = 80). To evaluate each IgG assay, a cut-off value was selected based on receiver operating characteristic curve analysis and used to calculate sensitivity and specificity. The intra- and interassay coefficients of variation were calculated for each ELISA. Using sera from Iceland, where R. equi infection has not been reported, the VapA-specific IgG ELISA differentiated exposed from nonexposed horses. When used to identify infected foals, VapA-specific IgG, IgGa and IgGb had no diagnostic value. In contrast, IgG(T) had high sensitivity and specificity. Horses from Iceland are not exposed to VapA(+) R. equi and can serve as negative controls. VapA-specific IgG subclasses, with the exception of IgG(T), are poor predictors of disease. Further investigation on the use of IgG(T) as a diagnostic tool in field conditions is needed.

Chimeric vapA/groEL2 DNA vaccines enhance clearance of Rhodococcus equi in aerosol challenged C3H/He mice

Rhodococcus equi remains a significant bacterial pathogen, causing severe pyogranulomatous pneumonia in foals aged 1-3 months. There is no effective vaccine currently available for the prevention of R. equi pneumonia. DNA vaccines are known to offer specific advantages over conventional vaccines. The aim of this study was to demonstrate efficacy of our recombinant DNA vaccine candidates, namely pcDNA3-Re1, pcDNA3-Re3 and pcDNA3-Re5 by combining a heat shock protein GroEL2 to a virulence-associated protein A (VapA) from R. equi to protect C3H/He mice against the R. equi infection. VapA was shown to be strongly recognised by sera from pneumonic foals. All vaccines elicited at least a doubling of the IgG2a/IgG1 ratio in comparison to the controls, indicating a bias to the Th1 response, which is postulated to be crucial for bacterial clearance and protective immunity against intracellular pathogens including R. equi. In addition, the immunised mice showed a significant reduction in R. equi in their lungs at 7 days after the aerosol challenge in comparison to PBS treated mice. However, examination of lung pathology 14 days after the challenge showed no gross differences in pathological changes between the unvaccinated and vaccinated animals. The lack of significant pathological changes suggests that the precise level of protection against R. equi pneumonia in the murine model of infection may not represent a true effectiveness of the potential vaccine candidates, indicating the mouse may not be the ideal non-equine model for vaccine studies and (or) the incomplete immunogenic antigen of vapA-based DNA vaccine constructs that mount an inadequate cell-mediated immune response against the R. equi infection.

Rhodococcus equi.

Rhodococcus equi is an important cause of subacute or chronic abscessating bronchopneumonia of foals up to 3-5 months of age. It shares the lipid-rich cell wall envelope characteristic of the mycolata, including Mycobacterium tuberculosis, as well as the ability of pathogenic members of this group to survive within macrophages. The possession of a large virulence plasmid in isolates recovered from pneumonic foals is crucial for virulence. The plasmid contains an 27 kb pathogenicity island (PI) that encodes seven related virulence-associated proteins (Vaps), including the immunodominant surface-expressed protein, VapA. Only PI genes are differentially expressed when the organism is grown in macrophages in vitro. Ten of the PI genes, including six Vap genes, have signal sequences, suggesting that they are exported from the cell to interact with the macrophage. Different PI genes are regulated by temperature, pH, iron, oxidative stress and probably also by magnesium, all environmental changes encountered after environmental R. equi are inhaled in dust and are ingested into macrophages in the lung. The basis of pathogenicity of R. equi is its ability to multiply in and eventually to destroy alveolar macrophages. Infectivity is largely or exclusively limited to cells of the monocyte-macrophage lineage. Current evidence suggests that infection of foals with virulent R. equi results in some foals in subversion of cell-mediated immunity and development of an ineffective and sometimes lethal Th2-based immune response. Significant progress has been made recently in the development of R. equi-E. coli shuttle vectors, transformation and random and site specific mutagenesis procedures, all of which will be important in molecular dissection of the mechanisms by which R. equi subverts normal macrophage killing mechanisms and cell-mediated immunity.

Immunoglobulin G Subisotype Responses of Pneumonic and Healthy, Exposed Foals and Adult Horses toRhodococcus equiVirulence-Associated Proteins

Rhodococcus equi causes severe pyogranulomatous pneumonia in foals and in immunocompromised humans. Replication of virulent isolates within macrophages correlates with the presence of a large plasmid which encodes a family of seven virulence-associated proteins (VapA and VapC to VapH), whose functions are unknown. Although cell-mediated immunity is thought to be crucial in eliminating R. equi infection, antibody partially protects foals. The antibody response to both VapA and VapC was similar in six adult horses and six naturally exposed but healthy foals, as well as in eight foals with R. equi pneumonia. The immunoglobulin G (IgG) subisotype response of pneumonic foals to Vap proteins was significantly IgGb biased and also had a trend toward higher IgGT association compared to the isotype association of antibody in adult horses and healthy exposed foals. This suggests that in horses, IgGb and IgGT are Th2 isotypes and IgGa is a Th1 isotype. Furthermore, it suggests that foals which develop R. equi pneumonia have a Th2-biased, ineffective immune response whereas foals which become immune develop a Th1-biased immune response. Pneumonic foals had significantly more antibody to VapD and VapE than did healthy exposed foals. This may indicate a difference in the expression of these two Vap proteins during persistent infection. Alternatively, in pneumonic foals the deviation of the immune response toward VapD and VapE may reflect a bias unfavorable to R. equi resistance. These data indicate possible age-related differences in the equine immune response affecting Th1-Th2 bias as well as antibody specificity bias, which together favor the susceptibility of foals to R. equi pneumonia.

Abstracts

Ultrasonographic determination of gestational age in pregnant bitches, Luvoni, G. C. & Grioni, A. (2000) Determination of gestational age in medium and small size bitches using ultrasonographic fetal measurements. Journal...

Risk of adverse effects in pneumonic foals treated with erythromycin versus other antibiotics: 143 cases (1986-1996).

To determine whether foals with pneumonia that were treated with erythromycin, alone or in combination with rifampin or gentamicin, had a higher risk of developing adverse effects, compared with foals treated with trimethoprim-sulfamethoxazole (TMS), penicillin G procaine (PGP), or a combination of TMS and PGP (control foals). Retrospective study. 143 foals < 240 days old. Information on age, sex, breed, primary drug treatment, total days of treatment with the primary drug, and whether the foal developed diarrhea, hyperthermia, or respiratory distress was obtained from the medical records. Relative risk (RR) and attributable risk (AR) were calculated to compare risk of adverse reactions between foals treated with erythromycin and control foals. Only 3 (4.3%) control foals developed diarrhea; none developed hyperthermia or respiratory distress. Foals treated with erythromycin had an 8-fold risk (RR, 8.3) of developing diarrhea, compared with control foals, and increased risks of hyperthermia (AR, 25%) and respiratory distress (AR, 15%). Results suggest that use of erythromycin to treat foals with pneumonia was associated with an increased risk of diarrhea, hyperthermia, and respiratory distress, compared with use of TMS or PGP.

Modulation of cytokine response of pneumonic foals by virulent Rhodococcus equi.

The ability of Rhodococcus equi to induce pneumonia in foals depends on the presence of an 85- to 90-kb plasmid. In this study, we evaluated whether plasmid-encoded products mediate virulence by modulating the cytokine response of foals. Foals infected intrabronchially with a virulence plasmid-containing strain of R. equi had similar gamma interferon (IFN-gamma) and interleukin-12 (IL-12) p35 but significantly higher IL-1beta, IL-10, IL-12 p40, and tumor necrosis factor alpha (TNF-alpha) mRNA expression in lung tissue compared to foals infected with the plasmid-cured derivative. IFN-gamma mRNA expression levels in CD4+ T lymphocytes isolated from bronchial lymph nodes (BLN) were similar for the two groups of R. equi-infected foals on day 3 postinfection. However, on day 14, in association with pneumonia and marked multiplication of virulent R. equi but with complete clearance of the plasmid-cured derivative, IFN-gamma mRNA expression in BLN CD4+ T lymphocytes was significantly (P < 0.001) higher in foals infected with the plasmid-cured derivative. These results suggests an immunomodulating role for R. equi virulence plasmid-encoded products in downregulating IFN-gamma mRNA expression by CD4+ T lymphocytes.

Role of the 85-kilobase plasmid and plasmid-encoded virulence-associated protein A in intracellular survival and virulence of Rhodococcus equi.

Rhodococcus equi is a facultative intracellular pathogen of macrophages and a cause of pneumonia in young horses (foals) and immunocompromised people. Isolates of R. equi from pneumonic foals typically contain large, 85- or 90-kb plasmids encoding a highly immunogenic virulence-associated protein (VapA). The objective of this study was to determine the role of the 85-kb plasmid and VapA in the intracellular survival and virulence of R. equi. Clinical isolates containing the plasmid and expressing VapA efficiently replicated within mouse macrophages in vitro, while plasmid-cured derivatives of these organisms did not multiply intracellularly. An isolate harboring the large plasmid also replicated in the tissues of experimentally infected mice, whereas its plasmid-cured derivative was rapidly cleared. All foals experimentally infected with a plasmid-containing clinical isolate developed severe bronchopneumonia, whereas the foals infected with its plasmid-cured derivative remained asymptomatic and free of visible lung lesions. By day 14 postinfection, lung bacterial burdens had increased considerably in foals challenged with the plasmid-containing clinical isolate. In contrast, bacteria could no longer be cultured from the lungs of foals challenged with the isogenic plasmid-cured derivative. A recombinant, plasmid-cured derivative expressing wild-type levels of VapA failed to replicate in macrophages and remained avirulent for both mice and foals. These results show that the 85-kb plasmid of R. equi is essential for intracellular replication within macrophages and for development of disease in the native host, the foal. However, expression of VapA alone is not sufficient to restore the virulence phenotype.

Development of aRhodococcus equi–Escherichia coliPlasmid Shuttle Vector

Isolates ofRhodococcus equifrom pneumonic foals possess an 85- or 90-kb virulence-associated plasmid. A prominent, thermoregulated surface antigen, VapA, encoded by these plasmids is thought to be important in virulence. A 1.35-kb fragment containing the origin of replication ofR. equistrain 103 virulence-associated plasmid (pOTS) was identified, sequenced, and its location identified. A simpleR. equi–Escherichia colishuttle plasmid (pRE-1) derived from theE. coliplasmid pACYC177 and the pOTSoriwas developed. The plasmid transformed readily and was stable in either host and expressed kanamycin resistance but not β-lactamase inR. equi.An improved 5.9-kb vector, pRE-7, was developed from pRE-1 and pBluescript. Subcloning ofvapAinto the multiple cloning site of the β-galactosidase gene of pRE-7 resulted in weak expression of the gene both inE. coliandR. equi.The shuttle vector may be useful in examining regulation of virulence gene expression inR. equi.

Antigenic analysis of Rhodococcus equi preparations using different horse sera

An R. equi vaccine, prepared under conditions which induce the expression of many antigens, and which has given encouraging results in field trials, was analyzed by SDS-PAGE and immunoblots and compared with other R. equi preparations: a preparation made in with the same technique from a nonvirulent isolate (virulence associated protein negative, VapA-negative); a whole cell preparation of a VapA-positive R. equi, prepared as a standard bacterin; and a semipurified VapA preparation (APTX). The antigens in these preparations were analyzed using hyperimmune sera (from adult horses vaccinated with the R. equi vaccine), passively and actively immunized foals' sera, asymptomatic but serologically positive foals' sera, sera from R. equi pneumonic foals, an equine APTX antiserum, and a VapA monoclonal antibody (Mab). The vaccine under study had many proteins in high concentrations. Hyperimmune sera reacted strongly with vaccine antigens in the high molecular weight regions. In the low molecular weight range, it reacted in the 14 and less kDa zone. Sera from passively immunized foals reacted similarly but not so strongly. Actively immunized foals gave very weak reactions. With the APTX extract, the Mab reacted with bands at 15–17, 44 and 66 kDa; it reacted weakly with the whole cell and not with the VapA-negative preparations. The APTX antiserum and the Mab reacted strongly with the vaccine at the 14 and less kDa zone, and also with bands at 21, 44 and 66 kDa and very tenuously at 18 kDa, but not in the expected 15–17 kDa zone, suggesting that the native form of VapA is altered but without loss of antigenicity in the vaccine preparation. Our results suggest that other higher molecular weight antigens, in addition to VapA, may be important in inducing antibodies that protect young foals from R. equi pneumonia. These antigens are in high concentrations and in an immunogenic form in the vaccine.

A tracheoscopic technique for obtaining uncontaminated lower airway secretions for bacterial culture in the horse

Transtracheal aspirate (TTA) aerobic microbiology culture results from 30 ponies were compared to protected catheter brush (PCB) culture results obtained from the same ponies. The PCB procedure resulted in significantly fewer (P less than 0.05) isolates than those obtained by the TTA procedure and the PCB procedure resulted in significantly more samples with no bacteria growth than the TTA procedure. To address the possibility that the volume of tracheobronchial secretion obtained by PCB was insufficient as a microbiological sample, a sterile, double-sheathed, protected aspiration catheter (PAC) was developed. Comparisons were made between TTA and PAC aerobic microbiology culture results from 18 pneumonic foals and weanlings. There were no significant differences between the PAC and TTA procedures with respect to the number of bacteria isolates. It was concluded that the PAC technique may be used as an alternative to TTA for isolation of equine pulmonary bacteria and may be the preferred procedure because of the reduced risk of complications associated with the TTA.