Gary Muscatello

Associations between the Ecology of Virulent Rhodococcus equi and the Epidemiology of R. equi Pneumonia on Australian Thoroughbred Farms

ABSTRACT The ecology of virulent strains of Rhodococcus equi on horse farms is likely to influence the prevalence and severity of R. equi pneumonia in foals. This study examined the association between the ecology of virulent R. equi and the epidemiology of R. equi pneumonia by collecting air and soil samples over two breeding seasons (28 farm-year combinations) on Thoroughbred breeding farms with different reported prevalences of R. equi pneumonia. Colony blotting and DNA hybridization were used to detect and measure concentrations of virulent R. equi. The prevalence of R. equi pneumonia was associated with the airborne burden of virulent R. equi (both the concentration and the proportion of R. equi bacteria that were virulent) but was not associated with the burden of virulent R. equi in the soil. Univariable screening and multivariable model building were used to evaluate the effect of environmental and management factors on virulent R. equi burdens. Lower soil moisture concentrations and lower pasture heights were significantly associated with elevated airborne concentrations of virulent R. equi, as were the holding pens and lanes, which typically were sandy, dry, and devoid of pasture cover. Few variables appeared to influence concentrations of virulent R. equi in soil. Acidic soil conditions may have contributed to an elevated proportion of virulent strains within the R. equi population. Environmental management strategies that aim to reduce the level of exposure of susceptible foals to airborne virulent R. equi are most likely to reduce the impact of R. equi pneumonia on endemically affected farms.

Current understanding of the equine immune response to Rhodococcus equi. An immunological review of R. equi pneumonia.

Rhodococcus equi is recognised to cause chronic purulent bronchopneumonia in foals of less than 6 months of age. Virulent strains of the bacteria possess a large 80-90 kb plasmid encoding several virulence-associated proteins, including virulence-associated protein A (VapA), which is associated with disease. R. equi pneumonia can represent significant costs and wastage to the equine breeding industry, especially on stud farms where the disease is endemic. This article reviews knowledge of the equine immune response, both in the immune adult and susceptible neonate, with respect to this pathogen. Humoral immune responses are addressed, with a discussion on the use of hyperimmune and normal adult equine plasma as prophylactic tools. The role that innate immune mechanisms play in the susceptibility of some foals to R. equi infection is also highlighted. Likewise, cell-mediated immune components are reviewed, with particular attention directed towards research undertaken to develop an effective vaccine for foals. It is possible that the implementation of a single immunoprophylaxis strategy to prevent R. equi infection on farms will yield disappointing results. Combined prophylactic protocols that address husbandry practices, environmental and aerosol contamination levels, enhancement of innate immunity, good quality hyperimmune plasma for the neonate, and vaccinal efficacy in the developing foal may be required.

Detection of Virulent Rhodococcus equi in Exhaled Air Samples from Naturally Infected Foals

Virulent Rhodococcus equi causes pyogranulomatous bronchopneumonia in foals. The route of infection of foals has been considered to be inhalation of aerosolized bacteria from soil that is contaminated with equine feces. Thus, disease caused by R. equi has been regarded as an opportunistic infection of environmental origin and not a contagious disease. In this study, we report the exhalation of virulent R. equi from the respiratory tract of naturally infected foals. A handheld air-monitoring system was used to recover virulent R. equi from the exhaled breath of foals, and the concentration of virulent R. equi organisms in exhaled air was compared to the concentration in environmental air samples taken from the holding pens and lane areas on farms. R. equi strains carrying the vapA gene of the virulence plasmid were detected by using colony blotting and DNA hybridization techniques in cultures of exhaled air from 67% (37/55) of foals tested. The concentration of virulent R. equi organisms in exhaled air from foals was significantly higher than that in environmental air (P < 0.001). There were no significant differences in the median concentrations of virulent R. equi bacteria exhaled by clinically healthy or diseased foals. The high concentrations of virulent R. equi bacteria in exhaled air suggested that aerosol transmission between foals is possible and may have a significant impact on the prevalence of R. equi pneumonia on farms. The air sampling technique described is potentially useful as a noninvasive method for the detection and quantification of virulent R. equi in the respiratory tract of foals.

Rhodococcus equi pneumonia in the foal--part 1: pathogenesis and epidemiology.

Rhodococcus equi pneumonia is a worldwide infectious disease of major concern to the equine breeding industry. The disease typically manifests in foals as pyogranulomatous bronchopneumonia, resulting in significant morbidity and mortality. Inhalation of aerosolised virulent R. equi from the environment and intracellular replication within alveolar macrophages are essential components of the pathogenesis of R. equi pneumonia in the foal. Recently documented evidence of airborne transmission between foals indicates the potential for an alternative contagious route of disease transmission. In the first of this two-part review, the complexity of the host, pathogen and environmental interactions that underpin R. equi pneumonia will be discussed through an exploration of current understanding of the epidemiology and pathogenesis of R. equi pneumonia in the foal.

Comparison of concentrations of Rhodococcus equi and virulent R. equi in air of stables and paddocks on horse breeding farms in a temperate climate.

REASONS FOR PERFORMING STUDY Rhodococcoccus equi is a significant cause of bronchopneumonia in foals worldwide. Infection of the lungs is believed to result from inhalation of virulent R. equi in dust from contaminated environments. A measure of infectious risk in an environment is the level of airborne contamination. OBJECTIVES To assess and compare the level of airborne virulent R. equi in paddocks and stables. METHODS Air samples were collected sequentially over the 2003 foaling season from the paddocks and stables on 3 Irish horse breeding farms affected by R. equi pneumonia. Colony blotting and DNA hybridisation techniques allowed quantitation of virulent R. equi. RESULTS The odds of detecting airborne virulent R. equi in stables were 173 times greater than in paddocks. The median airborne concentration of virulent R. equi was significantly higher (P < 0.001) in stables than in paddocks on all farms. These observations suggested that stables were high-risk areas for infection. CONCLUSIONS AND POTENTIAL RELEVANCE Our results indicate that contaminated stables are a significant risk factor in the epidemiology of R. equi pneumonia on horse-breeding farms in a temperate climate, such as in Ireland. Management strategies that improve the air hygiene of stables, through better ventilation, use of less fragile bedding material and the use of fogging agents to reduce the airborne concentration of virulent R. equi, may reduce the incidence and severity of R. equi pneumonia on farms.

Assessment of oxidative stress biomarkers in exhaled breath condensate and blood of Thoroughbred foals.

This study reports preliminary data on systemic and local biomarkers of oxidative stress (OS) in Thoroughbred foals. Blood and exhaled breath condensate (EBC) were sampled from 13 foals on two farms. Values of reactive oxygen metabolites (d-ROMs), biological antioxidant potential (BAP), antioxidant barrier (OXY-ads), thiol antioxidant barrier (SHp) and advanced oxidation protein products (AOPPs) were determined in blood, while EBC samples were assayed for hydrogen peroxide (H(2)O(2)) levels. Systemic and local OS biomarkers did not differ between farms and gender. Increased oxidative stress index (OSI) values in a foal recovering from pneumonia and elevated H(2)O(2) in EBC coupled with low SHp and elevated AOPPs in the blood of a foal with overt upper respiratory tract disease suggested that the OS markers measured in this study may relate to the respiratory health status of foals.

Rhodococcus equi pneumonia in the foal--part 2: diagnostics, treatment and disease management.

Various challenges face clinicians and farm managers in diagnosing, treating and preventing Rhodococcus equi pneumonia. The use of ultrasound imaging has aided in the early diagnosis of the disease, reducing treatment duration and improving therapeutic outcomes. Antimicrobial resistance in R. equi is an emerging issue that necessitates prudent antimicrobial therapy of diseased foals. Alternative methods of disease transmission, such as contagious foal-to-foal aerosol transmission, may need to be addressed to complement dust reduction environmental strategies and to minimise the overall risk of exposure of foals to highly concentrated inhaled doses of the organism. Effective management of foals and land aimed at reducing aerosol exposure to virulent R. equi is likely to yield significant reductions in the prevalence and severity of R. equi pneumonia.

Susceptibility of rapidly growing mycobacteria and Nocardia isolates from cats and dogs to pradofloxacin

Rapidly growing mycobacteria (RGM) and Nocardiae can cause severe or refractory infections in cats and dogs. Prolonged antibacterial therapy is required to cure these infections. As fluoroquinolones have been used in combination therapy for treating RGM infections, isolates from the Mycobacterium smegmatis cluster (n=64), Mycobacterium fortuitum cluster (n=17), and M. mageritense cluster (n=2), collected from feline and canine patients, underwent susceptibility testing to pradofloxacin. The MIC(50), MIC(90) and tentative epidemiological cut-off (ECOFF) values as determined by microbroth dilution susceptibility testing that inhibited growth of the M. smegmatis and M. fortuitum clusters were 0.063, 0.125 and ≤ 0.25; and 0.125, 0.250 and ≤ 1.0 μg/mL, respectively. E-Test results showed similar trends but MICs were lower than those for microbroth dilution. In summary, pradofloxacin demonstrated effective in vitro activity against RGM isolates. Additionally, veterinary isolates of Nocardia nova (n=18), Nocardia farcinica (n=3) and Nocardia cyriacigeorgica (n=1) underwent microbroth dilution testing to ciprofloxacin, enrofloxacin and pradofloxacin. The MIC(50) and MIC(90) of pradofloxacin, ciprofloxacin and enrofloxacin that inhibited growth of Nocardia nova isolates were 2 (4), 8 (16), 16 (32) μg/mL, respectively. The tentative ECOFF values for pradofloxacin and ciprofloxacin were 32 μg/mL and for enrofloxacin 64 μg/mL. The MIC or MIC range for the three N. farcinica isolates of pradofloxacin, ciprofloxacin and enrofloxacin were 0.25-0.5, 2 and 2 μg/mL and for the single N. cyriacigeorgica isolate were 1, 4 and 4 μg/mL, respectively. On the basis on these results, fluoroquinolones appear to have limited therapeutic potential for most Nocardia infections.

Systemic and respiratory oxidative stress in the pathogenesis and diagnosis of Rhodococcus equi pneumonia.

REASONS FOR PERFORMING STUDY Oxidative stress (OS) is most simply defined as an imbalance between oxidants and antioxidants. Oxidative stress has been suggested to play roles in various equine respiratory diseases and the significance of OS in the pathogenesis of Rhodococcus equi pneumonia is unknown. OBJECTIVES To measure and relate biomarkers of OS to lesions consistent with R. equi pneumonia. STUDY DESIGN Case-control study. METHODS Various OS biomarkers were measured from blood and exhaled breath condensate (EBC) samples collected from 26 foals between 1 and 2 months of age (n = 12 cases and n = 14 controls) on 2 Thoroughbred farms endemically affected by R. equi pneumonia. Foals were defined as cases (positive) or controls (negative) based on ultrasonographic evidence of pulmonary abscessation (>15 mm in diameter). Haematology and biochemistry testing was also performed on blood samples collected from the foals. Comparison of biomarkers and key haematological and biochemical markers of inflammation between the groups was performed using 2 sample t tests. RESULTS Derivatives of reactive oxygen metabolites (d-ROMs) were significantly greater in case foals than in control foals (P = 0.027) and the oxidative stress index (OSI) was higher in case foals (P = 0.014). Hydrogen peroxide (H2 O2 ) concentrations in EBC were significantly greater in case foals than in control foals (P = 0.002). Meanwhile, there were no significant differences in traditional measures of inflammation between the 2 groups. CONCLUSIONS Measuring OS in both blood and EBC provided useful information in the early diagnosis of R. equi pneumonia.

Equine amnionitis and fetal loss: The case definition for an unrecognised cause of abortion in mares

A series of abortions occurred in mares in New South Wales during 2004 that involved similar and unusual findings on post mortem examination of aborted fetuses and fetal membranes. The term Equine Amnionitis and Fetal Loss (EAFL) was developed to describe the condition. This form of abortion had not been previously recognised in Australia. The pathology alone is not specific for EAFL and diagnosis requires demonstration of a combination of certain pathological and bacteriological features. The purpose of this paper is to describe patterns considered consistent with EAFL cases as a working case definition for use by veterinarians and veterinary pathologists in identifying future cases of EAFL. More detailed papers are in preparation to fully describe the epidemiological, histopathological, and microbiological aspects of EAFL.