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.     

Quantitative fecal culture for early diagnosis of Corynebacterium (Rhodococcus) equi enteritis in foals.

Quantitative culture of Corynebacterium (Rhodococcus) equi from feces of 17 foals on a farm (A) with an endemic C. equi infection problem and 26 foals on a farm (B) without the disease in the past decade was done with a selective medium at weekly or monthly intervals from April to August of 1984. Corynebacterium equi was observed in the feces of 16 of 17 foals on farm A, and 19 of 26 foals on farm B. The mean viable count of C. equi in one gram of feces was 4.1 +/- 3.7 (log10) on farm A, and 3.9 +/- 3.4 (log10) on farm B. Corynebacterium equi was recovered from feces of foals as young as two weeks old. Almost all foals at an age between two to four weeks shed the bacteria in the feces. During the observation period two foals showed clinical signs: fever, diarrhea, and cough, at four or five weeks old. At the same time the bacterial count per gram of feces increased from 4 to 7 or 8 (log10). They shed large number of bacteria in the feces and continued to show the clinical signs until death at 10 or 11 weeks old. One of the foals was diagnosed as having had C. equi enteritis and pneumonia by the postmortem recognition of lesions with bacteriological confirmation. The quantitative culture of the feces of foals at weekly intervals after birth on farm A was found to be very useful as an aid in early diagnosis of C. equi enteritis in foals.     

Review of Corynebacterium (Rhodococcus) equi lung abscesses in foals: pathogenesis, diagnosis and treatment

Corynebacterium (Rhodococcus) equi is becoming increasingly significant as a cause of bronchopneumonia and lung abscessation in foals. The organism can survive within macrophages and may thus escape normal pulmonary defence mechanisms, particularly in immunocompromised animals. The disease has hitherto been associated with mortality rates as high as 80 per cent, partly as a result of inappropriate therapy. The selection of lipid-soluble antibiotics capable of intracellular penetration is critical for the successful treatment of C equi lung abscesses. A combination of two such antibiotics, erythromycin (25 mg/kg three times daily) and rifampicin (5 mg/kg twice daily) has been used on foals since 1981. Most of these animals had radiographic evidence of extensive lung abscessation, and in all cases the presence of C equi was confirmed on culture of tracheal aspirates. The duration of therapy ranged from four to nine weeks. Mild gastritis and diarrhoea were occasionally noted, but never such as to require termination of the therapy. No other adverse side effects were encountered. The success rate, as judged by a return to normal of chest radiographs and plasma fibrinogen concentrations, has exceeded 80 per cent.     

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.     

Antibody to equi factor(s) in the diagnosis of Corynebacterium equi pneumonia of foals.

Antibody to equi factor(s) in cases of Corynebacterium equi pneumonia in foals was detected using C. pseudotuberculosis exotoxin sensitized calf red blood cells. The test was standardized using antitoxin produced in rabbits by injection of equi factor(s). All sera from ten foals with culture-diagnosed C. equi pneumonia had antibodies to equi factor(s) (titre range 8-256, mean 74.0) and nine sera from 11 foals with suspected C. equi pneumonia also showed antibodies (titre range 4-512, mean 136.4). Two of five pneumonia foals with transtracheal aspirate cultures not yielding C. equi had such antibodies. Fifty-eight of 59 control horse sera had no antibodies; the one positive serum came from a foal on a farm where C. equi pneumonia was endemic. By contrast only five of 15 foals with experimentally-induced C. equi pneumonia had antibodies to equi factor(s), probably because the acute nature of the disease produced did not mimic the chronic course of the natural disease. Antibody to equi factor(s) can be used in the diagnosis of naturally-occurring corynebacterial pneumonia in foals.     

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.     

Equine cell-mediated immune response to Rhodococcus (Corynebacterium) equi

A lymphocyte blastogenic assay was developed to serve as an in vitro correlate of cell-mediated immunity to Rhodococcus (Corynebacterium) equi (R equi) in the equine species. Lymphocytes obtained from a group of experimental ponies showed no response in cell culture to R equi heat extract or lysozyme extract antigens. Ponies were assigned to groups for experimental inoculation. Three ponies were inoculated subcutaneously with live R equi, 3 were given live R equi by intranasal and intratracheal routes, and 4 ponies were left untreated. Lymphocytes from all inoculated ponies had a mitogenic response to R equi antigens in lymphocyte blastogenic assays performed between the 7th and 40th days after inoculation. Lymphocytes from noninoculated control ponies remained unresponsive to R equi antigens. Delayed-type hypersensitivity reactions developed in all experimentally exposed ponies after intradermal administration of the R equi antigen preparations. In a 2nd phase of experimentation, blastogenesis assays were performed on lymphocytes from horses in herds with endemic R equi infections. Results indicated that many of the animals had significant (stimulation index greater than 2) cell-mediated responses to the bacterium, but there was no distinct correlation between the immune response and clinical history. These data indicated that cell-mediated immunity is involved in the interaction of the equine immune system with R equi.     

Enzyme-linked immunosorbent assay for diagnosis of equine infectious anemia

An enzyme-linked immunosorbent assay (ELISA) was elaborated for the detection of specific antibody to equine infectious anemia (EIA) antigen. Sera from horses experimentally infected with EIA virus were assayed by ELISA, complement fixation (CF) and immunodiffusion (ID) tests for antibody to EIA antigen. The ELISA technique was found to be much more sensitive than CF and ID tests. In addition, EIA specific antibody could be detected by ELISA at an earlier stage of infection than by CF or ID techniques. The applicability of the technique to diagnosis of EIA is discussed.     

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.     

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.     

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.     

The pathology of experimental Corynebacterium equi infection in foals following intragastric challenge

The intragastric inoculation of a suspension of Corynebacterium equi on five consecutive days induced severe ulcerative colitis, typhlitis, and lymphadenitis of colonic and cecal nodes in two ponies necropsied three weeks after infection. No gross lesions were observed in two ponies necropsied ten days after infection. A single inoculum of equivalent size failed to induce gross lesions in four ponies killed at ten or 20 days after infection. Microscopic lesions consistent with early C. equi infection of Peyer's patches were seen in two of the ponies killed ten days after infection. Only one small pulmonary abscess occurred in one foal, suggesting that intestinal lesions are not likely the usual precursor of pulmonary disease in naturally infected foals. The gross and microscopic lesions in the experimentally infected ponies were typical of the intestinal form of naturally occurring C. equi associated disease in foals.     

Changes in structure of lymphatic organs in foals died due to Rhodococcus equi infection

The aim of this study was to investigate the structure of lymphatic organs in foals which died due to Rhodococcus equi infection. The material for this study consisted of 9 foals, of both genders, various breeds, 6-14 weeks old. The routine histopathological examination and immunocytochemical reaction for cytokeratine-3 receptors revealed the disturbances in the thymus epithelial cells development. This was manifested by a complete lack of cells expressing cytokeratine-3 receptors, as well as absence of normal thymocytes differentiation. The supposed thymus epithelial cells were very few, expressing faint reaction of cytokeratine-3 receptors. The relative deficiencies in the lymphatic tissue development in peripheral lymphatic organs were observed. It was concluded, that the observed pathological changes may indicate the inherited, not described yet gene failure, which was responsible for the thymus epithelial cells developmental disturbances, and was expressed with various intensity in examined foals. The causes of the fatal course of rhodococcosis in foals were primary disturbances in the immune competence.     

Ecology of Rhodococcus (Corynebacterium) equi in soil on a horse-breeding farm

The ecology of Rhodococcus (Corynebacterium) equi in soil was studied on a horse-breeding farm. R. equi was cultured from soil at a depth of 0, 10, and 20 cm on the six sites of the farm at monthly intervals for 10 months from March to December of 1983. The highest numbers of R. equi were found in the surface soil. The mean number of bacteria in soil samples at every depth increased remarkably from 0 or 10(2) to 10(4) colony-forming units (CFU) g-1 of soil in the middle of April, and later decreased gradually. R. equi inoculated into six soil exudate broths prepared from surface soils at separate sites yielded suspensions with different optical densities, indicating differences in growth. The distribution of serotypes in the soil was similar to that in the horses on the farm. These findings indicated that R. equi could multiply in the soil and flourish in the cycle existing between horses and their soil environment.