Rhodococcus equi pneumonia in the foal – Part 1: Pathogenesis and epidemiology

The Veterinary Journal 192 (2012) 20–26

Contents lists available at SciVerse ScienceDirect

The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl

Review

Rhodococcus equi pneumonia in the foal – Part 1: Pathogenesis and epidemiology Gary Muscatello ⇑ Faculty of Veterinary Science, University of Sydney, NSW 2006, Australia

a r t i c l e

i n f o

Article history: Accepted 12 August 2011

Keywords: Rhodococcus equi Foals Pathogenesis Epidemiology

a b s t r a c t 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 twopart 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. Ó 2011 Elsevier Ltd. All rights reserved.

Introduction

Virulence

The Gram positive soil saprophytic coccobacillus Rhodococcus equi has been a recognised cause of pneumonia in the foal for close to 90 years (Magnusson, 1923). R. equi pneumonia is an important respiratory disease of the foal worldwide (Takai et al., 1995a; Cohen et al., 2003; Muscatello et al., 2006a; Venner et al., 2009). The disease is a major cause of wastage in foals and costs the equine industry millions of dollars to treat (Muscatello et al., 2006b). The incidence of R. equi pneumonia appears to be increasing, possibly reflecting intensified management of equine breeding farms and climate change (Muscatello et al., 2007; Knight and Muscatello, 2008). The disease continues to present clinicians and equine breeding managers with major challenges with respect to epidemiological and therapeutic control of the disease due to the complex and not yet fully understood host, pathogen and environmental interactions that contribute to the disease process. In the first part of this two-part review, the focus will be on the pathogenesis of the disease and our current understanding of the epidemiology of R. equi pneumonia. In part two of this review, an overview of diagnostics, therapeutics and management issues that need to be addressed to effectively control R. equi pneumonia on farms will be provided and discussed, based on our current understanding of the pathogenesis and epidemiology of R. equi pneumonia (Muscatello, 2011).

Role of Rhodococcus equi as an intracellular pathogen

⇑ Tel.: +61 2 9114 0790. E-mail address: [email protected] 1090-0233/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2011.08.014

The pathogenicity of R. equi relies on the ability of the bacterium to survive and replicate intracellularly, mostly within mononuclear phagocytes, such as alveolar macrophages in the lungs of infected foals (Hondalus and Mosser, 1994). The organism does so by interfering with endosomal maturation following phagocytosis and suppressing acidification of the phagosome in which it resides (Fernandez-Mora et al., 2005; Toyooka et al., 2005). Intracellular proliferation ultimately leads to necrosis of the phagocyte, usually accompanied by a pyogranulomatous inflammatory response that damages the surrounding tissue (Luhrmann et al., 2004).

Plasmids and virulence genes Only certain strains of R. equi appear to be capable of producing disease in the foal. These strains are known as virulent R. equi and possess one or more copies of a virulence plasmid of approximately 80–90 kilobases (Takai et al., 2000, 2001a; Rodriguez-Lazaro et al., 2006). To date, 12 virulence plasmid types have been recognised on the basis of their endonuclease restriction patterns, some of which appear to have geographical specificity (Takai et al., 2001b, 2003; Ribeiro et al., 2005). The virulence plasmid harbours a pathogenicity island that contains genes required for intracellular survival and replication and, hence, virulence. The most important family of virulence proteins encoded by this island are the virulence-associated proteins (Vaps). Of the nine vap genes (vap A, vapC–vapI, pseudo-vapE), six (vapA, vapC, vapD, vapE, vapG and vapH) appear to encode full

G. Muscatello / The Veterinary Journal 192 (2012) 20–26

length functional Vap proteins (Takai et al., 2000; Russell et al., 2004; Letek et al., 2008). Expression of vap genes encoding functional proteins is upregulated when the organism is within macrophages. The majority of vaps are regulated by temperature, pH and nutrients, with optimal expression occurring under mildly acidic conditions, at temperatures of approximately 37 °C and under conditions of restricted iron availability, highlighting their importance in host adaptation and R. equi pathogenesis (Takai et al., 1996a; Byrne et al., 2001; Ren and Prescott, 2003). The function of Vaps is not fully understood. However, there appears to be one Vap that is essential for virulence in the foal, a 15– 17 kDa cell surface lipoprotein known as virulence associated protein A (VapA). Strains of R. equi possessing a vapA deletion mutant plasmid exhibit attenuated virulence (Jain et al., 2003). Both VapA and the virulence plasmid are required for virulence in the foal, suggesting that there is an interaction between VapA and other non-Vap plasmid encoded proteins in the pathogenesis of R. equi pneumonia (Giguere et al., 1999a). A number of chromosomally encoded genes have been implicated in virulence. These include the isocitrate lyase gene aceA, the nitrate reductase gene narG, the gene encoding high temperature requirement A protein (HtrA) and genes encoding a two component signal transduction system, the phoPR operon. Mutations in these genes lead either to full attenuation or, in the case of the phoPR operon, hypervirulence (Ren and Prescott, 2004; Wall et al., 2005; Pei et al., 2007). It is likely that a variety of chromosomal and plasmid encoded proteins and their regulators interact to facilitate pathogenesis. Microarray expression studies and gene network modelling illustrate the extent of plasmid-chromosomal ‘cross-talking’ in virulent R. equi (Letek et al., 2010). This study showed that vaps make multiple connections with various chromosomal genes; in particular, co-regulation of genes encoding key metabolic enzymes with vaps enabled optimal intracellular fitness. This process of modifying gene expression and function through gene interactions is known as ‘cooption’. The evolution of virulent R. equi from a harmless saprophyte and gut commensal to a pathogen probably resulted from the acquisition of vaps and other host colonisation genes coupled with the subsequent cooption of bacterial chromosomal genes (Letek et al., 2010). An ‘intermediately virulent’ plasmid encoding VapB, a 20 kDa cell surface lipoprotein analogous to VapA, is often associated with R. equi isolates recovered from cervical lymphatic tissues of pigs and from humans (mostly immunocompromised patients) with cavitary pneumonia (Makrai et al., 2002; Ocampo-Sosa et al., 2007). These strains are classified as intermediately virulent on the basis of their relative pathogenicity in a murine model, with lethality observed after administration of 107 colony forming units (cfu) of intermediately virulent R. equi, which is 10-fold greater than that needed for virulent strains carrying the plasmid encoding the VapA protein (Takai et al., 1995b). Intermediately virulent strains of R. equi are not found in foals or their environment (Letek et al., 2008). Most human patients are infected with intermediately virulent strains, suggesting a porcine, rather than equine, zoonotic link (Takai et al., 1996b; Ocampo-Sosa et al., 2007).

Pneumonia and the susceptible foal Clinical manifestations In foals, the most typical or classical manifestation of R. equi disease is chronic pyogranulomatous bronchopneumonia characterised by abscesses, which may be scattered extensively throughout the lung (Zink et al., 1986; Giguere and Prescott, 1997). In Australia, the disease has assumed the colloquial term

21

‘rattles’ due to the rattling and wheezing heard on thoracic examination. Early clinical signs of R. equi pneumonia in foals may include a sporadic or intermittent cough, often most apparent on mustering, yarding or when stressed during handling. Pyrexia, lethargy, respiratory distress, decreased appetite, a dry coat and illthrift are clinical features that may be apparent in foals suffering from the classical chronic form of R. equi pneumonia (Giguere and Prescott, 1997). Occasionally, foals will present with acute respiratory distress, often dying within a few hours or days. This acute form is characterised by an interstitial pneumonia with diffuse miliary pyogranulomatous lesions in the lung on postmortem examination (Martens et al., 1982). A variety of disease sequelae can accompany R. equi pneumonia in the foal, including ulcerative enterocolitis and typhlitis associated with the ingestion of sputum containing high concentrations of the bacterium (Johnson et al., 1983). The enteric form of disease is characterised by inflammation of lymphatic tissue, including the Peyer’s patches, with granulomatous or suppurative inflammation of the mesenteric and/or colonic lymph nodes (Zink et al., 1986; Reuss et al., 2009). Less common sequelae associated with bacteraemia include septic arthritis, osteomyelitis and hepatic and renal abscesses (Chaffin et al., 1995; Paradis, 1997). Non-septic polysynovitis, particularly of the tibiotarsal and stifle joints, is a common sequela to R. equi pneumonia. This immunemediated condition has been reported in approximately one third of R. equi pneumonia cases. The joint effusion usually resolves, without any apparent consequence, as the pneumonia resolves (Sweeney et al., 1987). Other immune-mediated conditions, such as uveitis, haemolytic anaemia and facial pemphigus, may also accompany R. equi pneumonia (Reuss et al., 2009). Immunology of Rhodococcus equi pneumonia Typically, R. equi pneumonia is seen in foals between 1 and 6 months of age. The immunological factors that predispose foals in this age group to develop R. equi pneumonia are unclear but are thought to be related to the immaturity of the immune system of the foal. The innate, humoral and cell mediated immune responses all appear to play roles in susceptibility to R. equi pneumonia (Giguere et al., 1999b; Darrah et al., 2004; Cauchard et al., 2004; Dawson et al., 2010). Since virulent R. equi are intracellular within macrophages, cellmediated immune responses and activation of these cells by interferon (IFN)-c are required to eliminate infection (Nordmann et al., 1993). In humans, R. equi pneumonia occurs predominantly in patients with acquired immunodeficiency syndrome (AIDS), predisposed by the suppression of cell-mediated immunity resulting from selective CD4+ helper T cell depletion caused by human immunodeficiency virus. Viral suppression of cell-mediated immunity in foals infected with equine herpesvirus 2 (EHV-2) may explain the apparent association between EHV-2 infection and R. equi pneumonia in Northern Europe (Nordengrahn et al., 1996). A T-helper 1 (Th1) cell-mediated response is considered to be more beneficial in controlling virulent R. equi infection because these cells are a potent source of IFN-c (Giguere et al., 1999b). Conversely, a T-helper 2 (Th2) cell-mediated response is considered to be detrimental and is often associated with the development of clinical pneumonia (Giguere et al., 1999b). The lack of IFN-c production in newborn foals and an age-dependent increase in IFN-c production to adult levels by the age of 3 months initially suggested that foals have an inherent Th2 bias and are unable to mount an effective Th1 response (Breathnach et al., 2006). However, although the foal has inherently low levels of IFN-c, similar to many other neonates, they are able to increase IFN-c production and mount a Th1 response when presented with the appropriate antigenic stimulus (Jacks et al., 2007).

22

G. Muscatello / The Veterinary Journal 192 (2012) 20–26

A dose-dependent Th1/Th2 response has been observed in foals experimentally infected with virulent R. equi via the respiratory route (Jacks and Giguere, 2010). An intrabronchial inoculum of 1  108 cfu of virulent R. equi led to a Th2 response bias and clinical pneumonia within 12 days of exposure, with severe pulmonary lesions at postmortem examination. In contrast, an inoculum of 1  106 cfu resulted in a stronger Th1 response, mild lung pathology and subclinical disease. These results indicate that the number of virulent R. equi inhaled by the foal will influence the immune response and the clinical outcome. Antigen specific immunoglobulin (Ig) G plays an important role in opsonisation, promoting phagocytosis of virulent R. equi by alveolar macrophages and down-regulation of intracellular growth through enhanced bacterial killing capacity (Hietala and Ardans, 1987; Cauchard et al., 2004). Low antibody titres in foals may contribute to their age-related susceptibility to R. equi pneumonia (Prescott, 1991; Hines and Hietala, 1996). The lowest circulating antibody titres in the foal are usually between 1 and 3 months of age through the combined effects of waning maternally derived antibodies and low endogenous antibody production (Hietala et al., 1985). This period corresponds to the age at which the majority of R. equi pneumonia cases are diagnosed (Zink et al., 1986; Horowitz et al., 2001; Muscatello et al., 2006a). In foals infected intratracheally with R. equi, there are substantial increases in mucosal IgA; a specific VapA B cell epitope is highly reactive with mucosal IgA from infected foals (Taouji et al., 2002). The apparent absence of IgA from the nasal mucosa during the first 4 weeks of life may increase the susceptibility of foals to R. equi pneumonia (Sheoran et al., 2000). Protection against R. equi pneumonia following oral immunisation with live virulent R. equi reflects the necessity of appropriate cell-mediated as well as local and systemic antibody responses in protective immunity (Chirino-Trejo et al., 1987; Hooper-McGrevy et al., 2005). If an effective live-attenuated R. equi vaccine were to be developed, oral immunisation may provide a valid means of protection against respiratory disease due to this organism. Immunogenetics It is unclear whether there is a genetic basis for susceptibility to R. equi pneumonia in foals. Allelic variants in a number of immunity related genes of foals with R. equi pneumonia have been observed. Specific genotypes relating to the cytokine receptor for interleukin 7 (IL7R), transferrin (Tf) and a lysosomal transporter protein (NRAMP1) influence susceptibility to R. equi pneumonia (Mousel et al., 2003; Halbert et al., 2006; Horin et al., 2010). The completion of sequencing of the equine genome and the increased affordability of tailored immunogene chips will no doubt result in the identification and exploration of more immune related genes in an attempt to identify foal genotypes susceptible or resistant to R. equi pneumonia. Ecology of virulent Rhodococcus equi

Growth of R. equi is inhibited in extremely alkaline (pH > 9) and acidic (pH < 5) conditions and the organism is susceptible to heat treatment >45 °C (Hughes and Sulaiman, 1987; Hebert et al., 2010). Virulent R. equi has been shown to be acid tolerant, with vap expression being regulated by pH and nutrient stress. However, as yet, no selective growth or inhibitory factors have been identified that affect concentrations of virulent R. equi in soil (Benoit et al., 2001; Ren and Prescott, 2003). Strategies to reduce virulent R. equi burdens in soil by modifying the soil environment through the application of soil disinfectants, such as chlorine, iodine and lime, are currently being explored. Faecal shedding of Rhodococcus equi Faecal excretion of virulent R. equi is an important contributor to environmental contamination on equine breeding farms (Takai, 1997). Most adult horses shed <2000 R. equi cfu/g faeces, with virulent strains comprising <10–15% of the adult R. equi faecal population (Grimm et al., 2007). In contrast, foals shed 1  103 to 1  104 R. equi cfu/g faeces, with excretion concentrations peaking between 3 and 12 weeks of age and virulent strains compromising 10–40% of the faecal R. equi population (Takai et al., 1994; Muscatello et al., 2007). In foals with R. equi pneumonia, the faecal concentration of the organism can range from 106 to 108/g, with virulent strains representing 80% or more of the faecal R. equi population (Takai et al., 1986a, 1994; Muscatello, 2005). On the basis of these findings, it is hypothesised that the faeces of foals, particularly those with R. equi pneumonia, are the most potent sources of virulent R. equi contamination on farms. Thus, many authors have recommended removal of faeces to reduce environmental burdens and disease prevalence (Prescott, 1987; Barton, 1991; Prescott and Yager, 1991). Gastrointestinal ecology and physiology Gut colonisation with R. equi commences early in the foal’s life, with the organism being detected in the faeces of foals within the first week of life (Takai et al., 1986b). This reflects the ubiquitous nature of the organism in the environment of the newborn foal and the high likelihood of exposure to the pathogen early in life. The apparent protective immunity against R. equi pneumonia conferred to foals experimentally challenged by oral immunisation with live virulent R. equi suggests that early oral ingestion and colonisation is important in immune priming (Chirino-Trejo et al., 1987; Hooper-McGrevy et al., 2005). Investigations into the ecology of R. equi in the gastrointestinal tract of foals demonstrate that the organism primarily replicates in the caecum, with substantial increases in the concentrations of total R. equi (both avirulent and virulent strains) in the caecum, colon and rectum (Chicken et al., 2008). There is no evidence of preferential proliferation of virulent R. equi in the intestine of foals. Further investigation into the caecal micro-environment and the significance of symbiotic relationships with hindgut protozoa are warranted.

Coprophilic-soil ecology Disease ecology and epidemiology R. equi is a soil saprophyte with a coprophilic-soil life cycle. It persists and replicates in soil, from where it is ingested by grazing herbivores, survives passage through the gastrointestinal tract and is excreted in faeces back into the soil habitat (Barton and Hughes, 1984; Barton, 1991). Growth of R. equi in soil is enhanced by the presence of horse faeces, with neutral to mildly alkaline pH at 30 °C; the organism thrives on volatile fatty acids found in horse faeces and is considered to be ubiquitous on equine breeding farms (Hughes and Sulaiman, 1987).

Prevalence and severity of Rhodococcus equi pneumonia R. equi pneumonia occurs endemically on some farms, but sporadically, or not at all, on many other farms. The prevalence and severity of disease on endemic farms can vary from season to season (Chaffin et al., 2003a; Muscatello et al., 2006a). In Australia, 1– 10% of Thoroughbred foals are diagnosed with R. equi pneumonia annually, with mortalities 61% commonly observed. Early diagno-

G. Muscatello / The Veterinary Journal 192 (2012) 20–26

23

sis and prompt and improved antimicrobial therapy have all contributed to minimising foal mortalities (Muscatello et al., 2006b). Many endemically infected farms experience morbidity of P20% and case fatalities ranging from 5% to 100% for R. equi pneumonia (Chaffin et al., 2003b; Muscatello et al., 2006a; Venner et al., 2009). Increased airborne concentrations of virulent R. equi in the farm environment is associated with an increased prevalence of R. equi pneumonia in foals (Muscatello et al., 2006a).

given farm developing R. equi pneumonia (Martens et al., 2000; Muscatello et al., 2006a; Cohen et al., 2008). The coprophilic-soil life cycle of R. equi in the horse means that contamination of the soil on an equine breeding farm is progressive and probably irrelevant as long as virulent strains remain soil bound and unable to be inhaled into the lungs.

Incubation period

Aerosol infection through dust is considered to be the major route of pulmonary infection with virulent R. equi in the foal (Giguere and Prescott, 1997; Muscatello et al., 2006a). Epidemiological studies in Japan showed that the airborne R. equi concentration in the air of stables increased when weather conditions became warmer, drier and windier and that this coincided with an increased incidence of disease (Falcon et al., 1985; Takai et al., 1987). An epidemiological study based on culture and differentiation of airborne R. equi highlighted the relationship between airborne virulent R. equi and the prevalence of R. equi pneumonia on Australian Thoroughbred farms (Muscatello et al., 2006a). The prevalence and incidence of R. equi pneumonia on farms was associated with the airborne virulent R. equi burden and the age of the foals on the farm. Farms with high airborne virulent R. equi burdens experienced a high prevalence of R. equi pneumonia (>9%). Environmental factors on these farms, such as low soil moisture, poor grass cover and high ambient temperatures, promoted the aerosolisation of virulent R. equi from the soil. In the same study, a relationship between airborne virulent R. equi, the age of foals and the incidence of R. equi pneumonia was observed (Muscatello et al., 2006a). The majority of R. equi pneumonia cases (65%, n = 269) were diagnosed in foals aged 1– 2 months and the incidence of R. equi pneumonia peaked during periods when high airborne virulent R. equi burdens corresponded with high numbers of foals 1–3 months of age in residence on farms. The tight regulation of Thoroughbred breeding results in high concentrations of foals <3 months of age during the spring and early summer when airborne virulent R. equi burdens are likely to be increasing as conditions become warm and dusty (Muscatello et al., 2006a). This may explain the high prevalence of R. equi pneumonia in Thoroughbred foals compared to other breeds, such as Standardbreds, whose breeding season is less tightly regulated, with a greater temporal spread of foaling (Hutchins et al., 1980; Muscatello et al., 2006a).

Pneumonia due to natural infection with virulent R. equi in foals is typically chronic, with an ill-defined incubation period. Incubation periods range from 6 to 18 days in foals experimentally dosed with 104 cfu virulent R. equi (Wada et al., 1997; Barton and Embury, 1987). In foals from Argentina and Japan, Horowitz et al. (2001) calculated a theoretical incubation period of 49 days on the basis of an observed log-normal distribution of age of onset of clinical signs of R. equi pneumonia and age at death. The observed distribution fitted the Sartwell epidemiological model, which states that the incubation period of an infectious disease originating from a point-source follows a log-normal distribution (Sartwell, 1950). Given the ubiquitous nature of the pathogen in the foals’ environment, the period immediately after birth was suggested as the time at which the point-source exposure to virulent R. equi occurred (Horowitz et al., 2001). However, given that experimental observations suggest a shorter incubation period, an alternative hypothesis to explain the log-normal distribution observed in natural infection is that of a point-source exposure to the protective effects of colostrum and maternally derived antibodies soon after birth instead of a point-source exposure to virulent R. equi. Farm practices and characteristics For many years, the application of good preventive health practices was believed to reduce the impact and risk of R. equi pneumonia in the foal. This theory was largely based on observed decreases in prevalence and mortality in single farm studies associated with the implementation of improved preventive health practices (Bain, 1963; Debey and Bailie, 1987; Clarke, 1989). However, more recent epidemiological studies have not demonstrated an association between good preventive health practices, such as vaccination, anthelminthic usage and the administration of hyperimmune plasma, and reduced prevalence or risk of R. equi pneumonia on equine breeding farms (Chaffin et al., 2003c; Muscatello et al., 2006b). Farm characteristics and environmental conditions are thought to play a role in determining the prevalence and severity of R. equi pneumonia (Debey and Bailie, 1987; Giguere and Prescott, 1997). Large acreage, high foal density and population size and a high proportion of transient mares during the breeding season are some of the key farm characteristics associated with high prevalence and risk of R. equi pneumonia (Chaffin et al., 2003b; Muscatello et al., 2006b). Soil environment A commonly held view is that the most significant factor associated with risk of R. equi pneumonia is the level of virulent R. equi in the foals’ environment, specifically the concentration in soil and aerosolised dust (Prescott and Yager, 1991; Takai, 1997; Cohen et al., 2005). Recent studies comparing soil R. equi populations with disease prevalence on farms have indicated that the burden (both concentration and proportion) of virulent R. equi in soil is not an accurate reflection of the prevalence of R. equi pneumonia on a given farm, nor does it accurately reflect the risk of foals raised on a

Contaminated air

Infectious ‘hot spots’ Specific sites or ‘hot spots’ on farms are thought to represent a high infectious risk for R. equi pneumonia (Prescott et al., 1984; Chaffin et al., 2003a; Cohen et al., 2005; Muscatello et al., 2006a,c). Early studies from Canada showed that R. equi concentrations in yards adjacent to stables on farms with endemic R. equi pneumonia were higher than on other farms where the disease was not endemic (Prescott et al., 1984). Other studies have shown associations between dusty environments and length of stabling and increased risk of R. equi pneumonia (Chaffin et al., 2003a; Cohen et al., 2005). On Australian Thoroughbred farms, high traffic areas, such as laneways (paths along which horses are moved to and from pens and paddocks) and holding pens (outdoor fenced areas used to confine horses for veterinary and farriery procedures), had approximately twice the concentration of airborne virulent R. equi compared to grass covered paddocks used for grazing mares and foals (Muscatello et al., 2006a). A similar study was performed in Ireland, where dense pasture cover and high soil moisture meant the environmental conditions differed markedly from Australia.

24

G. Muscatello / The Veterinary Journal 192 (2012) 20–26

When stabled, a foal was 17.3 times more likely to encounter airborne virulent R. equi than when in an outdoor paddock environment (Muscatello et al., 2006c). Contagious disease theorem Subclinically and clinically affected foals have been shown to exhale substantially higher concentrations of virulent R. equi in their breath than dust derived aerosols, suggesting the potential for an alternative contagious method of transmission of infection (Muscatello et al., 2009). The contagious spread theory means that an infectious herd mate may represent a significant infectious risk to a susceptible foal from both respiratory and faecal sources. These findings support the theory that inhalation of virulent R. equi is the main route of pulmonary infection by R. equi in foals. Furthermore, the size of the aerosolised virulent R. equi challenge derived from the foal’s environment, either in soil from faecal contamination or possibly in the exhaled breath of infectious herd mates, will influence the likelihood of R. equi pneumonia developing in a susceptible foal (Prescott et al., 2010). Conclusions The interactions between virulent R. equi, the developing immune system of the foal and the level of respiratory exposure and environment contamination with the organism on equine breeding farms all play critical roles in determining the severity and prevalence of R. equi pneumonia. The pathogenesis of this disease relies on the capacity for the virulent organism to survive and replicate within alveolar macrophages following inhalation, dependent on the presence of a plasmid encoding VapA. Airborne virulent R. equi burdens appear to be the best measure of disease risk on farms, but must be evaluated in relation to age and susceptibility of foals in those environments. When young susceptible foals (<2 months of age) encounter high airborne virulent R. equi burdens, the disease risk is likely to be high. The possibility of foal-to-foal aerosol transmission of concentrated virulent organisms may provide an alternative to environmental derived aerosols in disease transmission. Despite advances in our understanding of the organism and its ecology, there are still important gaps in our understanding of natural disease due to R. equi and the nature of immunity in the foal. There is a need to identify immunogenetic factors predisposing foals to the disease. The discovery of specific innate host factors related to disease risk may help clarify the pathogenesis and complex epidemiology of this disease. Conflict of interest statement The author of this paper has no financial or personal relationships with people or organisations that could inappropriately influence or bias the content of this paper. References Bain, A.M., 1963. Corynebacterium equi infection in the equine. Australian Veterinary Journal 39, 116–121. Barton, M.D., 1991. The ecology and epidemiology of Rhodococcus equi. In: Proceedings of the 6th International Conference on Equine Infectious Diseases, Cambridge, UK, pp. 77–81. Barton, M.D., Embury, D.H., 1987. Studies of the pathogenesis of Rhodococcus equi infections in foals. Australian Veterinary Journal 64, 332–339. Barton, M.D., Hughes, K.L., 1984. Ecology of Rhodococcus equi. Veterinary Microbiology 9, 65–76. Benoit, S., Benachour, S., Taouji, Y., Auffray, Y., Hartke, A., 2001. Induction of vap genes encoded by the virulence plasmid of Rhodococcus equi during acid tolerance. Research in Microbiology 152, 439–449. Breathnach, C.C., Sturgill-Wright, T., Stiltner, J.L., Adams, A.A., Lunn, D.P., Horohov, D.W., 2006. Foals are interferon gamma-deficient at birth. Veterinary Immunology and Immunopathology 112, 199–209.

Byrne, B.A., Prescott, J.F., Palmer, G.H., Takai, S., Nicholson, V.N., Alperin, D.C., Hines, S.A., 2001. Virulence plasmid of Rhodococcus equi contains inducible gene family encoding secreted proteins. Infection and Immunity 69, 650–656. Cauchard, J., Sevin, C., Ballet, J.J., Taouji, S., 2004. Foal IgG and opsonizing antiRhodococcus equi antibodies after immunization of pregnant mares with a protective VapA candidate vaccine. Veterinary Microbiology 104, 73–81. Chaffin, K.M., Honnas, C.M., Crabill, M.R., Schneiter, H.L., Brumbaugh, G.W., Briner, R.P., 1995. Cauda equina syndrome, diskospondylitis and a paravertebral abscess caused by Rhodococcus equi in a foal. Journal of the American Veterinary Medical Association 206, 215–220. Chaffin, K.M., Cohen, N.D., Martens, R.J., Edwards, R.F., Nevill, M., 2003a. Foal-related risk factors associated with development of Rhodococcus equi pneumonia on farms with endemic infection. Journal of the American Veterinary Medical Association 223, 1791–1799. Chaffin, K.M., Cohen, N.D., Martens, R.J., 2003b. Evaluation of equine breeding farm characteristics as risk factors for development of Rhodococcus equi pneumonia in foals. Journal of the American Veterinary Medical Association 222, 467–474. Chaffin, K.M., Cohen, N.D., Martens, R.J., 2003c. Evaluation of equine breeding farm management and preventative health practices as risk factors for development of Rhodococcus equi pneumonia in foals. Journal of the American Veterinary Medical Association 222, 476–485. Chicken, C., Gilkerson, J.R., Begg, A.P., Browning, G.F., Muscatello, G., 2008. Proliferation of virulent Rhodococcus equi in the gastrointestinal tract of foals. In: Proceedings of the 4th Havemeyer Workshop on Rhodococcus equi, Edinburgh, UK, p. 75. Chirino-Trejo, J.M., Prescott, J.F., Yager, J.A., 1987. Protection of foals against experimental Rhodococcus equi pneumonia by oral immunization. Canadian Journal of Veterinary Research 51, 444–447. Clarke, A.F., 1989. Management and housing practices in relation to Rhodococcus equi infection of foals. Equine Veterinary Education 1, 30–32. Cohen, N.D., Smith, K.E., Ficht, T.A., Takai, S., Libal, M.C., West, B.R., del Rosario, L.S., Becu, T., Leadon, D.P., Buckley, T., Chaffin, K.M., Martens, R.J., 2003. Epidemiologic study of results of pulsed-field gel electrophoresis of isolates of Rhodococcus equi obtained from horses and horse farms. American Journal of Veterinary Research 64, 153–161. Cohen, N.D., O’Conor, M.S., Chaffin, M.K., Martens, R.J., 2005. Farm characteristics and management practices associated with development of Rhodococcus equi pneumonia in foals. Journal of the American Veterinary Medical Association 226, 404–413. Cohen, N.D., Carter, C.N., Scott, M., Chaffin, M.K., Smith, J.L., Grimm, M.B., Kuskie, K.R., Takai, S., Martens, R.J., 2008. Association of soil concentrations of Rhodococcus equi and incidence of pneumonia attributable to Rhodococcus equi in foals on farms in central Kentucky. American Journal of Veterinary Research 69, 385–395. Darrah, P.A., Monaco, M.C.G., Jain, S., Hondalus, M.K., Golenbock, D.T., Mosser, D.M., 2004. Innate immune responses to Rhodococcus equi. Journal of Immunology 173, 1914–1924. Dawson, T.R., Horohov, D.W., Meijer, W.G., Muscatello, G., 2010. Current understanding of the equine immune response to Rhodococcus equi. An immunological review of R. equi pneumonia. Veterinary Immunology and Immunopathology 135, 1–11. Debey, M.C., Bailie, W.E., 1987. Rhodococcus equi in faecal and environmental samples from Kansas horse farms. Veterinary Microbiology 14, 251–257. Falcon, J., Smith, B.P., O’Brien, T.R., Carlson, G.P., Biberstein, E., 1985. Clinical and radiographic findings in Corynebacterium equi pneumonia in foals. Journal of the American Veterinary Medical Association 186, 593–599. Fernandez-Mora, E., Polidori, M., Luhrmann, A., Schaible, U.E., Haas, A., 2005. Maturation of Rhodococcus equi-containing vacuoles is arrested after completion of the early endosome stage. Traffic 6, 635–653. Giguere, S., Prescott, J.F., 1997. Clinical manifestation, diagnosis, treatment and prevention of Rhodococcus equi infections in foals. Veterinary Microbiology 56, 313–334. Giguere, S., Hondalus M, K., Yager, J.A., Darrah, P., Mosser, D.M., Prescott, J.F., 1999a. Role of the 85 kilobase plasmid and plasmid-encoded virulence-associated protein A in intracellular survival and virulence of Rhodococcus equi. Infection and Immunity 67, 3548–3557. Giguere, S., Wilkie, B.N., Prescott, J.F., 1999b. Modulation of cytokine response of pneumonic foals by virulent Rhodococcus equi. Infection and Immunity 67, 5041–5047. Grimm, M.B., Cohen, N.D., Slovis, N.M., Mundy, G.D., Harrington, J.R., Libal, M.C., Takai, S., Martens, R.J., 2007. Evaluation of fecal samples from mares as a source of Rhodococcus equi for their foals by use of quantitative bacteriologic culture and colony immunoblot analyses. American Journal of Veterinary Research 68, 63–71. Halbert, N.D., Cohen, N.D., Slovis, N.M., Faircloth, J., Martens, R.J., 2006. Variations in equid SLC11A1 (NRAMP1) genes and associations with Rhodococcus equi pneumonia in horses. Journal of Veterinary Internal Medicine 20, 974–979. Hebert, L., Cauchard, J., Doligez, P., Quitard, L., Laugier, C., Petry, S., 2010. Viability of Rhodococcus equi and Parascaris equorum eggs exposed to high temperatures. Current Microbiology 60, 38–41. Hietala, S.K., Ardans, A.A., 1987. Interaction of Rhodococcus equi with phagocytic cells from R. equi exposed and non-exposed foals. Veterinary Microbiology 14, 307–320. Hietala, S.K., Ardans, A.A., Sansome, A., 1985. Detection of Corynebacterium equispecific antibody in horses by enzyme-linked immunosorbent assay. American Journal of Veterinary Research 46, 13–15.

G. Muscatello / The Veterinary Journal 192 (2012) 20–26 Hines, S.A., Hietala, S.K., 1996. Rhodococcal pneumonia: Humoral versus cellmediated immunity. Equine Veterinary Journal 28, 339–340. Hondalus, M.K., Mosser, D.M., 1994. Survival and replication of Rhodococcus equi in macrophages. Infection and Immunity 62, 4167–4175. Hooper-McGrevy, K.E., Wilkie, B.N., Prescott, J.F., 2005. Virulence-associated protein-specific serum immunoglobulin G isotype expression in young foals protected against Rhodococcus equi pneumonia by oral immunization with virulent R. equi. Vaccine 23, 5760–5767. Horin, P., Sabakova, K., Futas, J., Vychodilova, L., Necesankova, M., 2010. Immunityrelated gene single nucleotide polymorphisms associated with Rhodococcus equi infection in foals. International Journal of Immunogenetics 37, 67–71. Horowitz, M.L., Cohen, N.D., Takai, S., Becu, T., Chaffin, K.M., Chu, K.K., Magdesian, K.G., Martens, R.J., 2001. Application of Sartwell’s model (lognormal distribution of incubation periods) to age at onset and age at death of foals with Rhodococcus equi pneumonia as evidence of perinatal infection. Journal of Veterinary Internal Medicine 15, 171–175. Hughes, K.L., Sulaiman, I., 1987. The ecology of Rhodococcus equi and physicochemical influences on growth. Veterinary Microbiology 14, 241–250. Hutchins, D.R., Brownlow, M.A., Johnston, K.G., 1980. Corynebacterium equi infections in foals – Concepts and observations. Australian Veterinary Practitioner 10, 248–252. Jacks, S., Giguere, S., 2010. Effects of inoculum size on cell-mediated and humoral immune responses of foals experimentally infected with Rhodococcus equi: A pilot study. Veterinary Immunology and Immunopathology 133, 282–286. Jacks, S., Giguere, S., Crawford, P.C., Castleman, W.L., 2007. Experimental infection of neonatal foals with Rhodococcus equi triggers adult-like gamma interferon induction. Clinical and Vaccine Immunology 14, 669–677. Jain, S., Bloom, B.R., Hondalus, M.K., 2003. Deletion of vapA encoding virulence associated protein A attenuates the intracellular actinomycete Rhodococcus equi. Molecular Microbiology 50, 115–128. Johnson, J.A., Prescott, J.F., Markham, R.J., 1983. The pathology of experimental Corynebacterium equi infection in foals following intrabronchial challenge. Veterinary Pathology 20, 440–449. Knight, P.K., Muscatello, G., 2008. Climate change and the racing industry. In: Proceedings of the 17th International Conference of Racing Analysts and Veterinarians Antalya, Turkey, pp. 321–325. Letek, M., Ocampo-Sosa, A.A., Sanders, M., Fogarty, U., Buckley, T., Leadon, D.P., Gonzalez, P., Scortti, M., Meijer, W.G., Parkhill, J., Bentley, S., Vazquez-Boland, J.A., 2008. Evolution of the Rhodococcus equi vap pathogenicity island seen through comparison of host-associated vapA and vapB virulence plasmids. Journal of Bacteriology 190, 5797–5805. Letek, M., González, P., Macarthur, I., Rodríguez, H., Freeman, T.C., Valero-Rello, A., Blanco, M., Buckley, T., Cherevach, I., Fahey, R., Hapeshi, A., Holdstock, J., Leadon, D., Navas, J., Ocampo, A., Quail, M.A., Sanders, M., Scortti, M.M., Prescott, J.F., Fogarty, U., Meijer, W.G., Parkhill, J., Bentley, S.D., Vázquez-Boland, J.A., 2010. The genome of a pathogenic Rhodococcus: Cooptive virulence underpinned by key gene acquisitions. PLoS Genetics 6, e1001145. Luhrmann, A., Mauder, N., Sydor, T., Fernandez-Mora, E., Schulze-Luehrmann, J., Takai, S., Hass, A., 2004. Necrotic death of Rhodococcus equi infected macrophages is regulated by virulence associated plasmids. Infection and Immunity 72, 853–862. Magnusson, H., 1923. Spezifische infektiose pneumonie beim fohlen. Ein neuer eiterregar beim pferd. Archiv fur Wissenschaftliche und Praktische Tierheilkunde 50, 22–38. Makrai, L., Takai, S., Tamura, M., Tsukamoto, A., Sekimoto, R., Sasaki, Y., Kakuda, T., Tsubaki, S., Varga, J., Fodor, L., Solymosi, N., Major, A., 2002. Characterization of virulence plasmid types in Rhodococcus equi isolates from foals, pigs, humans and soil in Hungary. Veterinary Microbiology 88, 377–384. Martens, R.J., Fiske, R.A., Renshaw, H.W., 1982. Experimental subacute foal pneumonia induced by aerosol administration of Corynebacterium equi. Equine Veterinary Journal 14, 111–116. Martens, R.J., Takai, S., Cohen, N.D., Chaffin, K.M., Liu, H., Sakurai, K., Sugimoto, H., Lingsweiler, S.W., 2000. Association of disease with isolation and virulence of Rhodococcus equi from farm soil and foals with pneumonia. Journal of the American Veterinary Medical Association 217, 220–225. Mousel, M.R., Harrison, L., Donahue, J.M., Bailey, E., 2003. Rhodococcus equi and genetic susceptibility: Assessing transferrin genotypes from paraffin-embedded tissues. Journal of Veterinary Diagnostic Investigation 15, 470–472. Muscatello, G., 2005. Effects of Stud Management on Ecology of Virulent Rhodococcus equi. Dissertation. University of Melbourne, Melbourne, Australia. Muscatello, G., 2011. Rhodococcus equi pneumonia in the foal – Part 2: Diagnostics, treatment 4 and disease management. The Veterinary Journal 192, 27–33. Muscatello, G., Anderson, G.A., Gilkerson, J.R., Browning, G.F., 2006a. Associations between the ecology of virulent Rhodococcus equi and the epidemiology of R. equi pneumonia on Australian thoroughbred farms. Applied and Environmental Microbiology 72, 6152–6160. Muscatello, G., Gilkerson, J.R., Browning, G.F., 2006b. Rattles in Horses: Effects of Stud Management on Ecology of Virulent Rhodococcus equi. Australian Government Rural Industries Research and Development Corporation, ACT, Australia. Muscatello, G., Gerbaud, S., Kennedy, C., Gilkerson, J.R., Buckley, T., Klay, M., Leadon, D.P., Browning, G.F., 2006c. Comparison of concentrations of Rhodococcus equi and virulent R. equi in air of stables and paddocks on horse breeding farms in a temperate climate. Equine Veterinary Journal 38, 263–265. Muscatello, G., Leadon, D.P., Klay, M., Ocampo-Sosa, A., Lewis, D.A., Fogarty, U., Buckley, T., Gilkerson, J.R., Meijer, W.G., Vazquez-Boland, J.A., 2007. Rhodococcus

25

equi infection in foals: The science of ‘rattles’. Equine Veterinary Journal 39, 470–478. Muscatello, G., Gilkerson, J.R., Browning, G.F., 2009. Detection of virulent Rhodococcus equi in exhaled air samples from naturally infected foals. Journal of Clinical Microbiology 47, 734–737. Nordengrahn, A., Rusvai, M., Merza, M., Ekstrom, J., Morein, B., Belak, S., 1996. Equine herpesvirus type 2 (EHV-2) as a predisposing factor for Rhodococcus equi pneumonia in foals: Prevention of the bifactorial disease with EHV-2 immunostimulating complexes. Veterinary Microbiology 51, 55–68. Nordmann, P., Ronco, E., Genounou, M., 1993. Involvement of interferon-c and tumour necrosis factor-a in host defence against Rhodococcus equi. Journal of Infectious Diseases 167, 1456–1459. Ocampo-Sosa, A.A., Lewis, D.A., Navas, J., Quigley, F., Callejo, R., Scortti, M., Leadon, D.P., Fogarty, U., Vazquez-Boland, J.A., 2007. Molecular epidemiology of Rhodococcus equi based on traA, vapA, and vapB virulence plasmid markers. Journal of Infectious Diseases 196, 763–769. Paradis, M.R., 1997. Cutaneous and musculoskeletal manifestations of Rhodococcus equi infection in foals. Equine Veterinary Education 9, 266–270. Pei, Y.L., Parreira, V., Nicholson, V.M., Prescott, J.F., 2007. Mutation and virulence assessment of chromosomal genes of Rhodococcus equi 103. The Canadian Journal of Veterinary Research 71, 1–7. Prescott, J.F., 1987. Epidemiology of Rhodococcus equi infections in horses. Veterinary Microbiology 14, 211–214. Prescott, J.F., 1991. Rhodococcus equi: An animal and human pathogen. Clinical Microbiology Review 4, 20–34. Prescott, J.F., Yager, J.A., 1991. The control of Rhodococcus equi pneumonia in foals. In: Proceedings of the 6th International Conference on Equine Infectious Diseases, pp. 21–25. Prescott, J.F., Travers, M., Yager-Johnson, J.A., 1984. Epidemiological survey of Corynebacterium equi infections on five Ontario horse farms. Canadian Journal of Comparative Medicine 48, 10–13. Prescott, J.F., Meijer, W.G., Vazquez-Boland, J.A., 2010. Rhodococcus. In: Gyles, C.L., Prescott, J.F., Songer, G., Thoen, C.O. (Eds.), Pathogenesis of Bacterial Infections in Animals, Fourth Ed. Blackwell, Iowa, pp. 149–165. Ren, J., Prescott, J.F., 2003. Analysis of virulence plasmid gene expression of intramacrophage and in vitro grown Rhodococcus equi ATCC 33701. Veterinary Microbiology 94, 167–182. Ren, J., Prescott, J.F., 2004. The effect of mutation on Rhodococcus equi virulence plasmid gene expression and mouse virulence. Veterinary Microbiology 103, 219–230. Reuss, S.M., Chaffin, M.K., Cohen, N.D., 2009. Extrapulmonary disorders associated with Rhodococcus equi infection in foals: 150 cases (1987–2007). Journal of the American Veterinary Medical Association 235, 855–863. Ribeiro, M.G., Seki, I., Yasuoka, K., Kakuda, T., Sasaki, Y., Tsubaki, S., Takai, S., 2005. Molecular epidemiology of virulent Rhodococcus equi from foals in Brazil: Virulence plasmids of 85-kb type I, 87-kb type I and a new variant, 87-kb type III. Comparative Immunology Microbiology and Infectious Diseases 28, 53–61. Rodriguez-Lazaro, D., Lewis, D.A., Ocampo-Sosa, A.A., Fogarty, U., Makrai, L., Navas, J., Scortti, M., Hernandez, M., Vazquez-Boland, J.A., 2006. Internally controlled real-time PCR method for quantitative species-specific detection and vapA genotyping of Rhodococcus equi. Applied and Environmental Microbiology 72, 4256–4263. Russell, D.A., Byrne, G.A., O’Connell, E.P., Boland, C.A., Meijer, W.G., 2004. The LysRtype transcriptional regulator VirR is required for expression of the virulence gene vapA of Rhodococcus equi ATCC 33701. Journal of Bacteriology 186, 5576– 5584. Sartwell, P., 1950. The distribution of incubation periods of infectious diseases. American Journal of Hygiene 51, 310–318. Sheoran, A.S., Timoney, J.F., Holmes, M.A., Karzenski, S.S., Crisman, M.V., 2000. Immunoglobulin isotypes in sera and nasal mucosal secretions and their neonatal transfer and distribution in horses. American Journal of Veterinary Research 61, 1099–1105. Sweeney, C.R., Sweeney, R.W., Divers, T.J., 1987. Rhodococcus equi pneumonia in 48 foals: Response to antimicrobial therapy. Veterinary Microbiology 14, 329–336. Takai, S., 1997. Epidemiology of Rhodococcus equi infections: Review. Veterinary Microbiology 56, 167–176. Takai, S., Iimori, S., Tsubaki, S., 1986a. Quantitative fecal culture for early diagnosis of Corynebacterium (Rhodococcus) equi enteritis in foals. Canadian Journal of Veterinary Research 50, 479–484. Takai, S., Ohkura, H., Watanabe, Y., Tsubaki, S., 1986b. Quantitative aspects of fecal Rhodococcus (Corynebacterium) equi in foals. Journal of Clinical Microbiology 23, 794–796. Takai, S., Fujimori, T., Katsuzuaki, K., Tsubaki, S., 1987. Ecology of Rhodococcus equi in horses and their environment on horse-breeding farms. Veterinary Microbiology 14, 233–239. Takai, S., Takahagi, J., Sato, Y., Yamaguchi, K., Kakizaki, S., Takehara, F., Matsukura, S., Tamada, Y., Tani, A., Sasaki, Y., Tsubaki, S., Kamada, M., 1994. Molecular epidemiology of virulent Rhodococcus equi in horses and their environment. In: Proceedings of the 7th International Conference on Equine Infectious Diseases Tokyo, Japan, pp. 183–187. Takai, S., Sasak, Y., Tsubaki, S., 1995a. Rhodococcus equi infection in foals – Current concepts and implications for future research. Journal of Equine Science 6, 105– 119. Takai, S., Imai, Y., Fukunaga, N., Uchida, Y., Kamisawa, K., Sasaki, Y., Tsubaki, S., Sekizaki, T., 1995b. Identification of virulence-associated antigens and plasmids

26

G. Muscatello / The Veterinary Journal 192 (2012) 20–26

in Rhodococcus equi from patients with AIDS. Journal of Infectious Diseases 172, 1306–1311. Takai, S., Fukunaga, N., Kamisawa, K., Imai, Y., Sasaki, Y., Tsubaki, S., 1996a. Expression of virulence-associated antigens of Rhodococcus equi is regulated by temperature and pH. Microbiology and Immunology 40, 591–594. Takai, S., Fukunaga, N., Ochiai, S., Imai, Y., Sasaki, Y., Tsubaki, S., Sekizaki, T., 1996b. Identification of intermediately virulent Rhodococcus equi isolates from pigs. Journal of Clinical Microbiology 34, 1034–1037. Takai, S., Hines, S.A., Sekizaki, T., Nicholson, V.N., Alperin, D.C., Osaki, M., Takamatus, D., Nakamura, M., Suzuki, K., Ogino, N., Kakuda, T., Dan, H., Prescott, J.F., 2000. DNA sequence and comparison of virulence plasmids from Rhodococcus equi ATCC 33701 and 103. Infection and Immunity 68, 6840–6847. Takai, S., Murata, N., Kudo, R., Narematsu, N., Kakuda, T., Sasaki, Y., Tsubaki, S., 2001a. Two new variants of the Rhodococcus equi virulence plasmid, 90 kb type III and type IV, recovered from a foal in Japan. Veterinary Microbiology 82, 373– 381. Takai, S., Chaffin, K.M., Cohen, N.D., Hara, M., Nakamura, M., Kakuda, T., Sasaki, Y., Tsubaki, S., Martens, R.J., 2001b. Prevalence of virulent Rhodococcus equi in soil from 5 R. equi – Endemic horse-breeding farms and restriction fragment length polymorphisms of virulence plasmids in isolates from soil and infected foals in Texas. Journal of Veterinary Diagnostic Investigation 13, 489–494.

Takai, S., Son, W., Lee, D., Madarame, H., Seki, I., Yamatoda, N., Kimura, A., Kakuda, T., Sasaki, Y., Tsubaki, S., Lim, Y., 2003. Rhodococcus equi virulence plasmids recovered from horses and their environment in Jeju, Korea: 90-kb type II and a new variant, 90-kb type V. Journal of Veterinary Medical Science 65, 1313–1317. Taouji, S., Breard, E., Peyret-Lacombe, A., Pronost, S., Fortier, G., Collobert-Laugier, C., 2002. Serum and mucosal antibodies of infected foals recognized two distinct epitopes of VapA of Rhodococcus equi. FEMS Immunology and Medical Microbiology 34, 299–306. Toyooka, K., Takai, S., Kirikae, T., 2005. Rhodococcus equi can survive a phagolysosomal environment in macrophages by suppressing acidification of the phagolysosome. Journal of Medical Microbiology 54, 1007–1015. Venner, M., Reinhold, B., Beyerbach, M., Feige, K., 2009. Efficacy of azithromycin in preventing pulmonary abscesses in foals. The Veterinary Journal 179, 301–303. Wada, R., Kamada, M., Anzai, T., Takai, S., 1997. Pathogenicity and virulence of Rhodococcus equi in foals. Veterinary Microbiology 56, 301–312. Wall, D.M., Duffy, P.S., DuPont, C., Prescott, J.F., Meijer, W.G., 2005. Isocitrate lyase activity is required for virulence of the intracellular pathogen Rhodococcus equi. Infection and Immunity 73, 6736–6741. Zink, M.C., Yager, J.A., Smart, N.L., 1986. Corynebacterium equi infections in horses, 1958–1984: A review of 131 cases. The Canadian Veterinary Journal 27, 213– 217.