Pyogranulomatous skin disease and cellulitis in a cat caused by Rhodococcus equi

This report describes a case of Rhodococcus equi infection causing pyogranulomatous skin disease and cellulitis in a two-year-old female domestic shorthaired cat. The case differed from previously reported cases in cats in its clinical presentation and in the locations of the lesions, which were similar to those seen in horses. The presence of an intracellular organism was confirmed by cytology and on histopathology. The aetiological diagnosis was confirmed by routine biochemical tests specific for R. equi on a pure isolate obtained from a biopsy specimen. The report also reviews the literature of the documented feline cases and discusses the common pitfalls in the diagnosis of such infections.     

The effect of immunosuppression on resistance to Rhodococcus equi in mice

Rhodococcus equi, a natural pathogen of horses, produces lesions in mice following experimental infection. The effect of various immunosuppressing agents on the sequential development of these lesions has been assessed by measuring the growth of R. equi following intravenous or intranasal challenge and by histological examination. Cyclophosphamide treatment of mice, challenged intranasally, resulted in the development of lesions not unlike that seen in experimental and natural infection in foals. Cortisone acetate also impaired bacterial clearance from the lungs and affected the accumulation of mononuclear cells at infective foci. Most of the agents chosen to impair macrophage function failed to affect the resistance of mice to R. equi. Carbon, carrageenan and silica failed to alter significantly the growth kinetics of R. equi. Dextran sulphate depressed the rate of pulmonary clearance of organisms and affected the ability of animals to eliminate R. equi following rechallenge. Overall, these results support other evidence that cell mediated immunity is involved in host resistance to R. equi and that activated macrophages play a role in acquired immunity to this organism.     

Disseminated Rhodococcus equi infection in dromedary camels (Camelus dromedarius)

Rhodococcus (R). equi, a recognized pathogen in horses, is emerging as a human opportunistic pathogen, especially in immunocompromized people. It affects also New World camelids, but there are no reports of R. equi infection in Old World camelids yet. Four cases of disseminated R. equi infection in adult breeding dromedaries occurred at one camel farm near Dubai within 16 months of each other. At necropsy the lungs were diffusely consolidated with large caseous areas. Histology revealed severe suppurative to necrotising pneumonia with multiple encapsulated abscesses. Immunohistochemistry enabled the detection of 15- to 17-kDa antigens (VapA) of R. equi in the lung sections. High numbers of R. equi were isolated from the lung lesions as well as from liver, spleen and mediastinal lymph nodes, indicative of septicaemia. The isolated strains were PCR-positive for the specific virulence plasmid (VapA-Gen) of R. equi, indicating virulent strains and containing an 85-kb type I plasmid. This is the first report of disseminated R. equi infection in Old World camelids. Since adult camels in general do not suffer from bacterial caused pneumonia (except tuberculosis), this is a new emerging disease for camels.     

Experimental infection of piglets by aerosols of Rhodococcus equi.

The purpose of this study was to investigate experimental infection of the piglet as a model of Rhodococcus equi pneumonia in the foal. Three litters of eight piglets each were exposed to an aerosol of 3.4 X 10(7) R. equi per piglet per day for seven consecutive days. Over the next 23 days the piglets were observed for clinical signs of disease. Periodically after infection one piglet from each litter was killed, the lungs were cultured quantitatively for R. equi and the gross and microscopic pulmonary lesions were assessed. The only clinical evidence of disease was the occurrence of elevated temperatures in the infected piglets. Rhodococcus equi was slowly cleared from the piglets' lungs during the 23 days following aerosolization. Piglets sacrificed seven to ten days after aerosolization had the most extensive pulmonary lesions, consisting of severe consolidation of the cranioventral lobes. Microscopic examination revealed thickened interalveolar septa and alveoli containing many neutrophils and macrophages with intracytoplasmic Gram-positive coccobacilli. The pulmonary lesions in these piglets differed from those of naturally infected foals in that they were not characterized by macrophage-rich abscesses and the infection gradually resolved.     

Immunity to Rhodococcus equi: antigen-specific recall responses in the lungs of adult horses

Rhodococcal pneumonia is an important disease of young horses that is not seen in immunocompetent adults. Since all foals are normally exposed to Rhodococcus equi in their environment, we hypothesized that most develop protective immune responses. Furthermore, these antigen-specific responses were hypothesized to operate throughout adult life to prevent rhodococcal pneumonia. A better understanding of the mechanisms of immune clearance in adult horses would help define the requirements for an effective vaccine in foals. Adult horses were challenged with virulent R. equi by intrabronchial inoculation into the right lung, and pulmonary immune responses were followed for 2 weeks by bronchoalveolar lavage. Local responses in the inoculated right lung were compared to the uninfected left lung and peripheral blood. Challenged horses rapidly cleared R. equi infection without significant clinical signs. Clearance of bacteria was associated with increased mononuclear cells in bronchoalveolar lavage fluid (primarily lymphocytes) and inversion of the normal macrophage:lymphocyte ratio. There was no significant increase in neutrophils at 7 days post-challenge. Flow cytometric analysis of bronchoalveolar lavage fluid demonstrated that clearance correlated with significant increases in pulmonary T-lymphocytes, both CD4+ and CD8+. Prior to challenge, most adult horses demonstrated low proliferative responses when pulmonary lymphocytes were stimulated with soluble R. equi ex vivo. However, clearance was associated with marked increases in lymphoproliferative responses to soluble R. equi antigen and recombinant VapA, a virulence associated protein of R. equi and candidate immunogen. These results are compatible with previous work in mice which showed that both CD4+ and CD8+ T-cells play a role in immune clearance of R. equi. Recognition of VapA in association with clearance lends further support to its testing as an immunogen. Importantly, the cellular responses to R. equi challenge were relatively compartmentalized. Responses were more marked and the sensitivity to antigen dose was increased at the site of challenge. The blood, including peripheral blood mononuclear cells, was an insensitive indicator of local pulmonary responses.     

Intracranial abscess caused by Rhodococcus equi infection in a foal

CASE DESCRIPTION: A 4-month-old Missouri Fox Trotter colt was examined for a 5-week history of head tilt after treatment for suspected pulmonary Rhodococcus equi infection. CLINICAL FINDINGS: Computed tomography revealed osteolysis of the occipital, temporal, and caudal portion of the parietal bones of the left side of the cranium. A soft tissue mass compressing the occipital region of the cerebral cortex and cerebellum was associated with the osteolytic bone. TREATMENT AND OUTCOME: A rostrotentorial-suboccipital craniectomy approach was performed to remove fragmented occipital bone, debulk the intracranial mass, and obtain tissue samples for histologic examination and bacterial culture. All neurologic deficits improved substantially within 3 days after surgery. Bacterial culture of the resected soft tissue and bone fragments yielded R equi. CLINICAL RELEVANCE: Intracranial surgery in veterinary medicine has been limited to dogs and cats; however, in select cases, extrapolation of surgical techniques used in humans and small animals can assist with intracranial procedures in horses.     

Evaluation of equine immunoglobulin specific for Rhodococcus equi virulence-associated proteins A and C for use in protecting foals against Rhodococcus equi-induced pneumonia

OBJECTIVE: To determine whether purified equine immunoglobulin specific for Rhodococcus equi virulence-associated proteins A and C (VapA and VapC) can confer passive protection against R. equi-induced pneumonia in foals. ANIMALS: Twenty-eight 3-week-old mixed-breed pony foals. PROCEDURE: 7 foals received IV injections of equine hyperimmune plasma (HIP) against whole-cell R. equi, and 7 received purified equine immunoglobulin specific for VapA and VapC 1 day prior to intrabronchial infection with R. equi strain 103+. Eleven foals were not treated prior to infection, and 3 control foals were neither treated nor infected. Heart rate, respiratory rate, and rectal temperature were recorded twice daily, and serum fibrinogen concentration and WBC count were determined every other day following infection. Foals were euthanatized 14 days following infection, and lung lesions and concentration of R. equi in lungs were assessed. RESULTS: The onset of clinical signs of pneumonia was significantly delayed in the HIP- and immunoglobulin-treated groups, compared with the untreated infected group. Moreover, pulmonary lesions were less severe in the treated groups, and significantly fewer R. equi organisms were cultured from the lungs of treated foals. CONCLUSIONS AND CLINICAL RELEVANCE: Degree of protection against R. equi-induced pneumonia provided by purified immunoglobulin specific for VapA and VapC was similar to that provided by commercially available HIP. Results not only suggest that immunoglobulin is the primary component of HIP that confers protection against R. equi-induced pneumonia in foals but also indicate that antibodies against R. equi VapA and VapC are protective.     

Rhodococcus equi infection of cats

Six cases of Rhodococcus equi infection in cats are described. One cat had pneumonia and died. The remaining five cats had cutaneous lesions affecting the feet in four of the cats and the metacarpus in one cat, and all these cats recovered with the aid of antibiotics. All the Rhodococcus equi isolates were sensitive to amoxycillin/clavulanic acid and, where used, at least 14–16 days of treatment was needed to help eliminate the infection. Histologically and cytologically the reaction was pyogranulomatous and many macrophages in the lesions contained large numbers of Gram-positive bacteria.     

The pathogenesis of Rhodococcus equi pneumonia in foals

The pathogenesis of Rhodococcus equi pneumonia in foals is reviewed. The main routes of infection are respiratory and alimentary. The latter is probably the chief route of exposure in all foals and probably leads to development of specific immunity. Susceptible foals, those whose maternal immunity wanes before generation of their own immune response, readily develop disease if exposed aerogenously to sufficient numbers of R. equi. Management and environmental circumstances have a major role to play in determining the magnitude of this challenge and, therefore, in the prevalence of the disease. Infection of a naive foal leads to severe, suppurative bronchopneumonia with suppurative lymphadenitis of regional nodes and, in approximately 50% of animals, to necrotizing enterocolitis. The foal is uniquely susceptible to R. equi pneumonia; comparable experimental infections do not produce progressive destructive pulmonary lesions in other animal species. In the naive foal lung, R. equi behaves as a facultative intracellular pathogen, avoiding destruction within the alveolar macrophage by inhibiting phagolysosome fusion and possibly by causing lysosomal degranulation. The role of putative virulence factors, such as equi factor, remains to be elucidated.     

Role of CD4+, CD8+ and double negative T-cells in the protection of SCID/beige mice against respiratory challenge with Rhodococcus equi.

To evaluate the contributions of T-lymphocyte subsets in pulmonary immunity against Rhodococcus equi, C.B-17 SCID/beige mice were adoptively transferred with splenic lymphocytes from congenic BALB/c mice previously infected with R. equi. Spleen cells were enriched for either CD4+ or CD8+ populations before inoculation, Flow cytometry showed that each enriched population contained less than 0.5% cross contamination. Groups of adoptively transferred SCID/beige mice were sacrificed 6 and 13 d after intranasal infection with R. equi. Bacterial clearance was measured in the lungs, liver and spleen. Lesion development was assessed by gross and histopathological score and the fate of transferred cells assessed by flow cytometry and by immunohistochemistry. SCID/beige mice receiving either CD4+ or CD8+ T-cells were able to clear the infection better than control mice. On d 6 post-infection, bacterial numbers were significantly lower in the lungs of CD4+ transferred mice as compared to CD8+ mice. By d 13, both groups had cleared R. equi from all organs. CD4+ cells were however identified in the lung and spleen of CD8+ recipients at d 13 making conclusions about the role of CD8+ cells in R. equi clearance impossible. By contrast, no significant increases in CD8+ lymphocytes were observed in the organs of CD4+ recipients. All mice developed suppurative bronchopneumonia but lesions were most severe in the CD4+ group. Immunohistochemistry and flow cytometry confirmed that CD4+ and CD8+ cells had migrated to the lungs of adoptively transferred mice. Serum antibody against R, equi was not detected by ELISA in the recipients. SCID/beige mice receiving CD4-CD8- cells were unable to clear R. equi. The study supports the suggestion that CD4+ cells have a central role in R. equi clearance in mice.     

Effect of prophylactic administration of hyperimmune plasma to prevent Rhodococcus equi infection on foals from endemically affected farms

The effect on foals of prophylactic administration of hyperimmune plasma to prevent R. equi infection was investigated on three farms at which R. equi infection was endemic. Sixteen foals between 10 and 39 days of age were intravenously given 1-21 of hyperimmune plasma. ELISA antibody titres against R. equi were significantly increased and maintained at high levels for over 30 days in most of the recipient foals. The prevalence of R. equi infection was 6.3% (1/16) in the foals that received the immune plasma, and 26.3% (5/19) in the control foals not given the immune plasma on the three farms. For 2 years before and after this field trial on the three farms, 18 of 64 foals (28.1%) showed clinical signs of respiratory tract infection and four of them died of R. equi pneumonia. Heavy contamination of horses and their environment with virulent R. equi was detected by colony blotting, and plasmid profiles also suggested that foals on the three farms were constantly exposed to virulent R. equi. The results of this field trial support previous observations by some researchers that the administration of hyperimmune plasma to foals in the early days of life promotes prevention of R. equi infection on endemic farms; however, the mechanism of hyperimmune plasma protection remains unclear.     

Increased pulmonary activation of nuclear factor-kappaB (NF-kappaB) in foals inoculated with Rhodococcus equi is associated with increased expression of inflammatory cytokines

Previous studies revealed that foals inoculated with virulent Rhodococcus equi had significantly higher pulmonary levels of interleukin-1beta, interleukin-12 p40, interferon-gamma, and tumor necrosis factor-alpha mRNA compared to foals inoculated with an avirulent plasmid-cured derivative. The purpose of this study was to determine if the increases in cytokine expression were associated with increased pulmonary activation of nuclear factor-kappaB (NF-kappaB). Electrophoretic mobility shift assays were performed on pulmonary nuclear protein extracted from foals treated with phosphate-buffered saline, or inoculated with either virulent or avirulent R. equi. NF-kappaB activation was increased in the nuclear extracts from foals inoculated with virulent R. equi at 14 days after inoculation when increased cytokine expression was also observed. Southwestern histochemistry revealed activated NF-kappaB in multinucleated giant cells that often contained bacteria. These results indicate that the cytokine response to R. equi is at least partially mediated by NF-kappaB activation.     

Electron microscopic investigation of intracellular events after ingestion of Rhodococcus equi by foal alveolar macrophages

It has been suggested that R. equi causes pulmonary disease in foals by persisting within the lung as a facultative intracellular parasite of alveolar macrophages. This paper describes an ultrastructural study of the intracellular events after ingestion of R. equi by foal alveolar macrophages, in an attempt to determine the mechanism of intracellular survival of R. equi. Secondary lysosomes of alveolar macrophages recovered from foals by bronchoalveolar lavage were labelled with electron-dense ferritin, and the cells were challenged with either viable or formalin-killed R. equi. After 0-, 3-, 8- or 24-h incubation, the cells were fixed and processed for electron microscopy. There was no evidence of phagosome-lysosome fusion after ingestion of either viable or non-viable R. equi by foal alveolar macrophages. Rhodococcus equi persisted and multiplied within dilated phagosomes, which were often lined by elongate microvillous structures. After 24-h incubation, 75% of the ingested bacteria were still structurally intact. Macrophages with ingested viable R. equi were irreversibly damaged and released intracellular bacteria into the surrounding medium. These data confirm that R. equi is a facultative intracellular parasite of foal alveolar macrophages and is able to persist and multiply within the phagosome, apparently inhibiting phagosome-lysosome fusion by some as yet unknown mechanism.     

Equine humoral immune response to Rhodococcus (Corynebacterium) equi

An enzyme-linked immunosorbent assay was developed to test equine serum for the presence of antibodies to Rhodococcus (Corynebacterium) equi. Experimental ponies had no detectable antibody to R equi before exposure to the bacterium. After experimental inoculation, animals in groups that received live R equi subcutaneously or intranasally/intratracheally developed high titers to R equi. Noninoculated controls remained seronegative. Serum was also collected from horses of various ages that were naturally exposed to R equi. There was a wide range of anti-R equi titers in these horses. Because experimentally infected horses seroconverted when some naturally infected foals did not seroconvert, the function of antibody in resistance to R equi infection remains unknown.     

Rhodococcus equi: clinical manifestations, virulence, and immunity

Pneumonia is a major cause of disease and death in foals. Rhodococcus equi, a gram-positive facultative intracellular pathogen, is a common cause of pneumonia in foals. This article reviews the clinical manifestations of infection caused by R. equi in foals and summarizes current knowledge regarding mechanisms of virulence of, and immunity to, R. equi. A complementary consensus statement providing recommendations for the diagnosis, treatment, control, and prevention of infections caused by R. equi in foals can be found in the same issue of the Journal.     

Effects of iron modulation on growth and viability of Rhodococcus equi and expression of virulence-associated protein A

OBJECTIVE: To determine the importance of iron for in vitro growth of Rhodococcus equi, define potential iron sources in the environment and mechanisms by which R equi may obtain iron from the environment, and assess expression and immunogenicity of iron-regulated proteins. SAMPLE POPULATION: 10 virulent and 11 avirulent strains of R equi. PROCEDURE: In vitro growth rates and protein patterns of R equi propagated in media with normal, excess, or limited amounts of available iron were compared. Immunoblot analyses that used serum from foals naturally infected with R equi and monoclonal antibody against virulence-associated protein (Vap)A were conducted to determine immunogenicity and identity of expressed proteins. RESULTS: Excess iron did not alter growth of any R equi strains, whereas growth of all strains was significantly decreased in response to limited amounts of available iron. Virulent R equi were able to use iron from ferrated deferoxamine, bovine transferrin, and bovine lactoferrin. Only virulent R equi expressed an iron-regulated, immunogenic, surface-associated protein identified as VapA. CONCLUSIONS AND CLINICAL RELEVANCE: Iron is required for the growth and survival of R equi. Sources of iron for R equi, and mechanisms by which R equi acquire iron in vivo, may represent important virulence factors and novel targets for the development of therapeutic and immunoprophylactic strategies to control R equi infection in foals. Expression of VapA is substantially upregulated when there is a limited amount of available iron.     

Clinical evaluation of the serodiagnostic value of enzyme-linked immunosorbent assay for Rhodococcus equi infection in foals

An enzyme-linked immunosorbent assay (ELISA) for detection of serum IgG antibodies against Tween 20-extracted antigen of strain ATCC 6939 was applied in Hidaka, Japan to a total of 752 sick foals showing a variety of signs of infectious disease. An optical density (OD) value of more than 0.3 was tentatively fixed to be positive on the basis of readings made of healthy horse sera in previous studies. During a 2 year study, 138 of the 752 sick foals showed an OD value of 0.3 or higher and were designated as 'suspected of R. equi infection'. Age distribution during the initial medical examination of the 138 seropositive foals was significant in that most (64%) foals were age 31-60 days, with a sharp decrease in subjects beyond that age. Of the 138 foals suspected of having R. equi infection, 34 foals (25%) showed OD values of over 0.9 at the initial medical examination, in addition to high blood leucocyte counts and serum fibrinogen and alpha-globulin values. The infectious foals had been treated with antibiotics just before and after serodiagnosis and 126 foals (91%) recovered from the disease. However, no clinical improvement was observed in 12 foals (9%). At necropsy, these foals revealed suppurative pneumonia and lymphadenitis of gut associated lymph nodes accompanied by abdominal abscesses. All isolates from the pulmonary and abdominal abscesses revealed R. equi. These results suggest that OD readings in the high range are associated with severe disseminated infection with R. equi.     

Hematologic and immunophenotypic factors associated with development of Rhodococcus equi pneumonia of foals at equine breeding farms with endemic infection

Rhodococcus equi causes severe pyogranulomatous pneumonia in foals and in immunocompromised people. In mice, both CD4+ and CD8+ T lymphocytes contribute to host defense against R. equi, but CD4+ T lymphocytes are required for pulmonary clearance of the bacteria. In this prospective study of 208 foals at two equine breeding farms with endemic R. equi infections, we collected peripheral blood samples at 2 and 4 weeks of age and at the time of diagnosis of R. equi pneumonia. Samples were analyzed for concentrations of total and differential leukocytes, EqCD4+ and EqCD8+ T lymphocytes, and B lymphocytes. Thirty (14.4%) foals developed R. equi pneumonia. At the 2nd week of life, affected foals had significantly lower concentrations of white blood cells (WBC) and segmented neutrophils, significantly lower proportions of EqCD4+ T lymphocytes, and significantly higher proportions of EqCD8+ T lymphocytes. The EqCD4:EqCD8 ratio was significantly lower for affected foals. At the 4th week of life, affected foals had significantly lower concentrations of segmented neutrophils and EqCD4+ T lymphocytes than did unaffected foals. The ratio of EqCD4:EqCD8 was significantly lower for affected foals. Two- and 4-week-old foals with ratios of EqCD4:EqCD8<3 were significantly more likely to develop R. equi pneumonia. There was a significant farm effect which diluted our statistical power to detect differences; however; after adjusting for the farm effect, 2-week-old foals with ratios of EqCD4:EqCD8<3 remained significantly more likely to develop R. equi pneumonia. There were no significant differences in immunophenotypic variables between affected foals (at the time of diagnosis) and age-matched control foals. These data suggest that there are hematologic and immunophenotypic differences between affected and unaffected foals during the first 2-4 weeks of life, prior to onset of clinical signs of R. equi pneumonia. These differences may represent important immunologic mechanisms associated with increased susceptibility of individual foals to infection with R. equi. Because there was considerable overlap between values for affected and unaffected foals, we cannot yet recommend immunophenotyping of foals at endemically-infected farms as a clinically useful screening tool to identify foals at increased risk of developing R. equi pneumonia.     

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.