Serotypes of Rhodococcus equi isolated from horses, immunocompromised human patients and soil in Hungary

Two hundred and twelve Rhodococcus equi strains were isolated from soil, nasal and rectal swabs of horses and immunocompromised human patients in Hungary and serotyped using Prescott's serotyping system. One hundred and forty-seven strains (69.3%) belonged to serotype 1, 22 strains (10.4%) to serotype 2, 6 strains (2.8%) to serotype 3 and 1 strain (0.5%) to serotype 4. Serotypes 5, 6 and 7 were not found and 36 strains (17%) could not be typed. Serotype 1 (72%) was the type most commonly isolated from clinical samples of foals or from the soil of horse facilities. Six out of 8 R. equi strains from humans belonged to serotype 2, and two human strains were untypable. The data show that the prevalence of R. equi serotypes varies in different geographic areas of the country.     

Rhodococcus equi pneumonia in foals

Rhodococcus (Corynebacterium) equi pneumonia is diagnosed by thoracic auscultation, radiographic and hematologic examination, and transtracheal aspiration. Treatment may be unsuccessful because of the organism's tendency to cause pulmonary abscesses. A 2-month-old, depressed, anorectic, febrile Quarter Horse colt, previously unresponsive to penicillin therapy, had loud, moist breath sounds in the ventral lung fields. Chest radiographs revealed pneumonia. Based on culture and sensitivity tests on organisms isolated from transtracheal washes, chloramphenicol and erythromycin, and then oral trimethoprim-sulfadiazine, were given, in addition to supportive therapy. The animal was fully recovered within 2 months.     

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.     

Studies of the pathogenesis of Rhodococcus equi infection in foals

Pyogranulomatous pneumonia was induced in Thoroughbred foals by intranasal challenge with freeze-dried cultures of Rhodococcus equi (previously Corynebacterium equi). The incubation period was about 18 days and clinical signs were not seen for a further week. There were marked seasonal and individual foal differences in responses to infection. Elevations in serum caeruloplasmin oxidase activity and copper concentrations appeared to be sensitive indicators of infection. Serum zinc concentrations and serum alpha-mannosidase and alkaline phosphatase activities fell in the more severely infected foals. Use of trace elements and trace element-related parameters along with faecal culture for R. equi could prove useful for early diagnosis of field cases.     

Rhodococcus equi infection in foals: the science of 'rattles'

Infection with Rhodococcus (Corynebacterium) equi is a well-recognised condition in foals that represents a consistent and serious risk worldwide. The condition manifests itself primarily as one of pulmonary abscessation and bronchitis, hence the terminology of 'rattles' derived from its most obvious clinical sign, frequently terminal when first identified. This review addresses the clinical manifestation, bacteriology and pathogenesis of the condition together with recent developments providing knowledge of the organism in terms of virulence, epidemiology, transmission and immune responses. Enhanced understanding of R. equi virulence mechanisms and biology derived from the recently available genome sequence may facilitate the rational development of a vaccine and the improvement of farm management practices used to control R. equi on stud farms in the future. Reliance on vaccines alone, in the absence of management strategies to control the on-farm challenge is likely to be disappointing.     

Interaction of Rhodococcus equi with phagocytic cells from R. equi-exposed and non-exposed foals

The interaction of Rhodococcus equi with alveolar macrophages from adult horses, foals experimentally exposed to R. equi (sensitized foals) and non-exposed foals was studied using in vitro bactericidal assays, cytochemical staining and transmission electron microscopy. It was demonstrated that R. equi is a facultative intracellular parasite, able to survive and multiply within the alveolar macrophages of the host by interfering with phagosome-lysosome fusion. Opsonization of R. equi with antibody against capsular components was associated with increased phagosome-lysosome fusion and significantly enhanced (P less than 0.05) killing of the organism by alveolar macrophages from non-exposed foals. Macrophages from non-exposed foals were able to ingest the non-opsonized organism, but unable to kill greater than 65% of the infective dose by 6 h post-exposure. Alveolar macrophages from sensitized foals behaved as adult macrophages, able to kill greater than 95% of the infective dose by 6 h. Lymphocyte factors, derived by in vitro incubation of sensitized peripheral blood lymphocytes with R. equi surface antigens, enhanced macrophage bactericidal activity. Macrophages from non-exposed foals incubated in the presence of the lymphocyte factors had a 50% increase in killing of R. equi, while sensitized macrophages incubated with lymphocyte factors had a greater than 100% increase in killing capacity.     

Humoral immune response of foals to experimental infection with Rhodococcus equi

Humoral immune response to Rhodococcus equi in experimentally infected foals was studied with the enzyme-linked immunosorbent assay (ELISA) method. Class-specific antibodies were measured by ELISA in the sera of foals after intratracheal or oral inoculation with R. equi ATCC 6939 or T 48 and in the lung washings of a foal after intratracheal inoculation or of normal horses. After intratracheal or oral inoculation with R. equi, serum antibodies were first detected in immunoglobulin G (IgG) followed by IgM and IgA classes, but significant levels of IgM and IgA developed only in the foal infected intratracheally with R. equi T 48. Only the foal infected intratracheally with T 48 developed pneumonia. Anti-R. equi IgG and IgA antibodies appeared in lung washings of the intratracheally infected foal. There were differences in the antibody response to R. equi among the intratracheally infected foals, the orally infected foal and the naturally infected foal. These results suggest that the humoral immune response to R. equi may be affected by the type of R. equi strain and the route and extent of R. equi exposure.     

The immunological response of foals to Rhodococcus equi: a review

Normal horses of all ages regularly show evidence of having responded immunologically to R. equi, thus adding serological support to epidemiological evidence that this organism is a normal intestinal inhabitant. More animals from "diseased" farms show a stronger antibody response when compared with foals from "healthy" farms. Various serological tests have been used to detect evidence of infection and to relate antibody level to severity of disease. Anti-R. equi IgG antibody levels, as measured by ELISA, are raised significantly during natural infection. Clinical severity of pneumonia can be correlated with lower specific antibody responses. Following experimental infection, immunological responses can be detected by complement fixation, indirect immunofluorescence, ELISA, lymphocyte blastogenesis and skin testing. Very little work has been carried out to evaluate vaccines against R. equi infection and results have not been encouraging. Success in treatment has been reported following passive immunisation. Administration of immune leucocyte extracts has had no effect on morbidity or mortality rates. The widespread distribution of this organism, together with the relative infrequency of disease caused by it, suggest that R. equi may initiate infection only in such circumstances as a very high infectious challenge, immunological immaturity or deficiency in the host and genetic predisposition.     

Controversies in therapy of infections caused by Rhodococcus equi in foals

Pneumonia caused by Rhodococcus equi is one of the most important causes of disease and death in foals. R. equi can also be cultured from a large variety of extrapulmonary sites of infection. In the absence of an effective vaccine, ultrasonographic screening for early detection of pulmonary lesions has become routine practice at many farms endemic for pneumonia caused by R. equi. Consequently, the most frequently recognised form of R. equi infection at such farms is a subclinical form in which foals develop sonographic evidence of peripheral pulmonary consolidation or abscessation without necessarily manifesting clinical signs. Evidence exists that not all foals with ultrasonographic lesions will progress to develop clinical signs, and treating a large proportion of foals based on subclinical ultrasonographic findings has been linked to emergence of macrolide‐ and rifampin‐resistant R. equi at a horse farm. Selectively treating only those foals with larger lesion scores and monitoring foals with daily physical inspections and weekly thoracic ultrasonography offers an approach that could decrease antimicrobial drug use without significantly increasing mortality. Current evidence continues to support the combination of rifampin with a macrolide (azithromycin, clarithromycin or erythromycin) for treating clinical infections caused by R. equi despite recently described pharmacological interactions between these drugs. When infection with a macrolide‐resistant isolate is confirmed, limited effective alternatives exist.     

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

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

Rhodococcus equi pneumonia in 48 foals: response to antimicrobial therapy

Case records of 48 foals with pneumonia due to Rhodococcus equi were reviewed. Twenty of the 48 foals survived and 28 died or were euthanized. There was no significant difference between the survivors and non-survivors in the age of onset of illness, duration of illness prior to admission, the mean white blood cell (WBC) count, or the mean plasma fibrinogen content. All foals had R. equi isolated from a tracheobronchial aspirate or lung specimens obtained at necropsy. All organisms were susceptible in vitro (Kirby-Bauer) to erythromycin and gentamicin. Susceptibilities to other drugs were: trimethoprim-sulfamethoxazole (88%), tetracycline (87%), chloramphenicol (83%); 97% were resistant to cephalothin and 83% to penicillin. Thirteen of the 20 surviving foals were treated with erythromycin and/or rifampin. A decline in mortality rate was observed with the introduction of the combination of erythromycin and rifampin. None of the 17 foals treated with penicillin and gentamicin survived. Chronic, active, non-septic synovitis was confirmed in 17 foals. These foals had joint distension with mild or no apparent lameness.     

Rapid immunohistochemical detection of Rhodococcus equi in impression smears from affected foals on postmortem examination

The first objective of this study was to develop an immunohistochemical procedure for rapid detection of Rhodococcus equi in impression smears from affected organs of foals on postmortem examination. The second aim was to demonstrate whether R. equi can be detected in smears of tracheal exudates collected from the same foals using an immunohistochemical method. Impression smears and cryostat and paraffin-embedded sections were made from the lungs and mediastinal lymph nodes of three foals (A, B and C) that had died of respiratory disease caused by R. equi, and also from the caudal mesenteric lymph node of foal A. Impression smears were made from the tracheal exudates of all foals. An affinity purified rabbit IgG was used for the immunohistochemical demonstration of R. equi. This antibody reacted with serotype 1 of R. equi in Ouchterlony's immunodiffusion and in the passive haemagglutination test, but not with other serotypes or with Streptococcus equi ssp. equi or Staphylococcus aureus, and failed to give an immunohistochemical reaction with Mycobacterium bovis or M. paratuberculosis. The immunohistochemical method proved to be of identical sensitivity to bacterial culture; moreover, from the lungs and mediastinal lymph nodes of one foal, R. equi could only be detected by this method. R. equi was demonstrated in smears of the tracheal exudates of all three foals. The results of this study indicate that the immunohistochemical method may be used for the rapid detection of R. equi in impression smears from the affected organs, especially abscesses, obtained postmortem, and possibly as a tool for diagnosing R. equi pneumonia in live foals by examining smears of tracheal aspirates.     

Diagnostic contribution of thoracic ultrasonography in 17 foals with Rhodococcus equi pneumonia

The aim of this retrospective study was to determine the clinical usefulness of thoracic ultrasonography compared to thoracic radiography in evaluation of Rhodococcus equi pneumonia. Criteria for patient inclusion in this study were: (1) isolation of R. equi from transtracheal aspirate, (2) radiographic evaluation of the pulmonary parenchyma, and (3) sonographic evaluation of the pulmonary parenchyma. Seventeen foals met this criteria and their medical records were reviewed. Pyogranulomatous pneumonia was identified radiographically in 13 foals. Severe consolidative pneumonia with no detectable abscessation was identified radiographically in three others. Both consolidation and abscessation were identified radiographically in one. In this foal only consolidation was ultrasonographically identified. Ultrasonographically, pulmonary abscessation was identified in 12 foals and pulmonary consolidation with no detectable abscessation was identified in three others. Sonographic examination allowed detection of only pleural irregularities in one foal, which was subsequently found to have pyogranulomatous pneumonia radiographically. Results indicate that ultrasonography may be an accurate alternative imaging modality for detection of pulmonary pathology attributed to R. equi pneumonia in foals when thoracic radiography is not available.     

Protection of foals against experimental Rhodococcus equi pneumonia by oral immunization.

Two groups of three one to three week old foals were immunized orally on four occasions over five weeks with two strains of Rhodococcus equi, a clinical isolate from a pneumonic foal and a laboratory passaged Congo red negative variant of this strain. Three nonimmunized foals of similar age acted as controls. Three weeks after the last immunization, all foals were challenged on five occasions over seven days by aerosol infection with about 10(10) of the pneumonic foal isolate on each occasion. Control foals became seriously ill and were euthanized. Immunization with either strain protected foals equally against the challenge, and resulted in rapid lung clearance. Oral immunization can thus protect foals against severe challenge with R. equi. The proteins associated with Congo red colony staining appear not to be involved in protective immunity.     

Evaluation of 5 serologic assays to detect Rhodococcus equi pneumonia in foals

OBJECTIVE: To determine the sensitivity and specificity of 5 serologic assays used to diagnose Rhodococcus equi pneumonia in foals and to determine whether any of the assays could be used to identify affected foals prior to the onset of clinical signs or to differentiate between affected and unaffected foals when clinical signs first become apparent. DESIGN: Nested case-control study. ANIMALS: 26 foals. PROCEDURE: Serum samples were obtained from all foals at 2, 4, and 6 or 7 weeks of age. Additional samples were obtained from affected foals at the time of diagnosis of R equi pneumonia and from age-matched unaffected foals. Samples were tested with 3 ELISA, an agar gel immunodiffusion assay, and a synergistic hemolysis inhibition assay. RESULTS: Sensitivity and specificity data indicated that none of the assays could be used to reliably differentiate affected from unaffected foals at any testing period. Proportions of foals that had an increase in test values between paired samples collected at 4 and 6 or 7 weeks of age were not significantly different between affected and unaffected foals. For all assays, result values increased significantly over time; however, the rate of increase was not significantly different between affected and unaffected foals. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that serologic assays, whether performed on single or paired samples, cannot be used to reliably establish, confirm, or exclude a diagnosis of R equi pneumonia in foals.     

Virulence-associated protein characterisation of Rhodococcus equi isolated from bovine lymph nodes

Rhodococcus equi has a low pathogenicity in cattle, but it occasionally causes lymph node granulomas, which are detected at abattoir post mortem inspection, and must be distinguished from tuberculous granulomas. Lymph node lesions were detected in 6719 cattle, from a total of 3,263,622 cattle examined post mortem in abattoirs, in the Republic of Ireland, during 1997 and 1998. Histological examination was performed on all lesions, principally for the purpose of identifying animals with tuberculosis. A total of 1122 of the lesions were cultured on blood agar and on Stonebrinks and Lowenstein-Jensen medium containing pyruvate, because the histological findings were difficult to interpret or were suggestive of R. equi infection. R. equi was isolated from 264 lesions. Almost all of the R. equi granulomas were confined to a single lymph node, and were present predominantly in the retropharyngeal, bronchial and mediastinal lymph nodes. R. equi granulomas were present in a significantly higher proportion of the lesions detected in steers and heifers compared to cows. The prevalence in the total population of 3.3 million cattle examined post mortem was 0.008%. The 15-17kDa antigens, associated with virulence in this organism, and the 20kDa antigen, associated with intermediate virulence, were not detected in isolates from 146 cattle, analysed by immunoblot assays. A PCR assay to detect the plasmid gene encoding the 15-17kDa antigens was also negative for isolates from these 146 animals. Plasmids were not detected in 30 isolates which were examined.