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.     

Cellular and humoral immune response of foals to vaccination with Corynebacterium equi.

Transformation of peripheral blood lymphocytes from pony foals vaccinated and subsequently infected with Corynebacterium equi was studied. Three foals were vaccinated on two occasions using a formalinized C. equi vaccine with aluminum hydroxide as an adjuvant. Three nonvaccinated foals served as controls. Foals were challenged intratracheally with 9 x 10(9) C. equi six weeks after the initial vaccination.Foals survived this infection for one to two weeks. Significant lymphocyte transformation in response to C. equi antigens was detected in two vaccinated foals at the third week after initial vaccination and in all vaccinated animals at the fifth week. No statistically significant transformation was seen in nonvaccinated foals before infection. Vaccinated and nonvaccinated foals showed responsive lymphocytes following challenge. Vaccination offered no obvious protection against experimental challenge but this failure was probably due to an excessive infective dose of organisms. Low levels of humoral antibodies were detected in some challenged foals. The pathological changes in the lungs of infected animals were comparable with, but more fulminating than, changes observed in the natural disease.     

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.     

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.     

Enzyme-linked immunosorbent assay for diagnosis of Corynebacterium (Rhodococcus) equi infection in foals

An enzyme-linked immunosorbent assay (ELISA) was used to diagnose Corynebacterium (Rhodococcus) equi infection in foals. In tests done with different antigen-extraction procedures (sodium dodecyl sulfate, sodium deoxycholate, polyoxy-ethylene [9] p-tert-octylphenol, polyoxy-ethylene [9-10] p-tert-octylphenol, sonification, homogenization, and heat treatment at 121 C), Tween 20 was a satisfactory reactive antigen. Using hyperimmune rabbit sera or infected foal sera, we investigated the specificity and the sensitivity of the ELISA with the Tween 20 antigen of the different serotypes or of the isolates. Corynebacterium equi strain ATCC 6939 antigen had the best activity for detecting antibodies to C equi in foals. Sera from 218 healthy horses, 11 healthy foals, 17 healthy newborn foals, a foal with suspected C equi infection, and 5 infected foals were evaluated for antibodies to C equi, using ELISA. The optical density values of 206 healthy horses, 17 healthy newborn foals, and 9 healthy foals were less than 0.1. Infected foal sera, except from foal 3, and serum from a foal with suspected C equi infection had higher optical density values. Using ELISA, specific antibodies against C equi were detected in a naturally infected 6-week-old foal after the foal had a rapid increase in the number of bacteria in the feces and after the initial development of clinical signs of illness at 5 weeks of age. Therefore, ELISA was useful for the early diagnosis of C equi infection in foals.     

Rhodococcus (Corynebacterium) equi: bactericidal capacity of neutrophils from neonatal and adult horses

The capacity of hematogenous polymorphonuclear neutrophilic leukocytes (PMNL) to kill Rhodococcus equi was compared in horses of various ages. A radioisotope bactericidal assay was used to determine the capacity of PMNL to kill R equi. Assays were conducted on PMNL from horses in 3 groups: group I, 13 foals with a mean age of 3.3 days; group II, 10 group-I foals at a mean age of 35.7 days; and group III, adult dams of group-I foals. Bacteria were obtained from the lungs of a foal with R equi pneumonia and opsonized with fresh adult equine serum that contained R equi specific antibody. The mean peak percentage of R equi killed by PMNL was 78.9 for group I, 90.1 for group II, and 87.9 for group III. There was no significant difference (P greater than 0.05) among groups; however, 15% of foals in group I (2 foals) had a mean peak percentage of 30.5 killed, which was significantly (P less than 0.05) lower than the percentage for other foals in group I. The results of our investigation indicated that the capacity of PMNL to kill opsonized R equi is similar in neonatal, young, and adult horses. However, some neonatal foals have a substantially lower capacity to kill R equi, which may be an important factor in the pathogenesis of R equi infections.     

Two techniques for detection of antibodies against Corynebacterium (Rhodococcus) equi in horse sera

Two techniques were developed to detect antibodies against the exosubstance of C. equi called equi-factor. In the first technique serum samples are tested against native equi-factor produced by the growth of C. equi on agar medium. A positive result is manifested by the development of precipitation lines. The second test is based on neutralization of prepurified equi-factor by antibody, resulting in the inhibition of its hemolytic synergism with staphylococcal beta toxin. Sera (125 samples) from horses of different ages, kept in localities with a history of C. equi infections, were examined. The first technique detected 65.6%, and the second 40% of positive cases.     

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.     

Antibody response of horses to Rhodococcus equi antigens.

The antigens extracted from strains belonging to seven capsular serotypes of Rhodococcus equi, as well as from two wild strains isolated from pneumonic foals, were examined. Whole-cell antigens and soluble products present in broth culture supernatants were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroblotted onto nitrocellulose, and stained with serum from hyperimmunized rabbits or foals. Foal sera used included sera from pneumonic animals with known titer to equi factors; from animals bled monthly on a farm with enzootic pneumonia, and from animals bled monthly on a farm with no history of R. equi pneumonia. The humoral response of foals to somatic antigen preparations was negligible, with few differences noted between sera from healthy, subclinically affected, and sick foals. The humoral response to R. equi broth culture supernatant products appeared more marked and was related to equi factor antibody titer. These findings suggest that the humoral response to R. equi whole-cell antigens is unimportant in protection against disease, which is consistent with the behavior of the organism as a facultative intracellular pathogen.     

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.     

Genetic Susceptibility to Rhodococcus equi

Rhodococcus equi pneumonia is a major cause of morbidity and mortality in neonatal foals. Much effort has been made to identify preventative measures and new treatments for R. equi with limited success. With a growing focus in the medical community on understanding the genetic basis of disease susceptibility, investigators have begun to evaluate the interaction of the genetics of the foal with R. equi. This review describes past efforts to understand the genetic basis underlying R. equi susceptibility and tolerance. It also highlights the genetic technology available to study horses and describes the use of this technology in investigating R. equi. This review provides readers with a foundational understanding of candidate gene approaches, single nucleotide polymorphism‐based, and copy number variant‐based genome‐wide association studies, and next generation sequencing (both DNA and RNA).     

Rhodococcus equi (Prescottella equi) vaccines; the future of vaccine development

For decades researchers have been targeting prevention of Rhodococcus equi (Rhodococcus hoagui/Prescottella equi) by vaccination and the horse breeding industry has supported the ongoing efforts by researchers to develop a safe and cost effective vaccine to prevent disease in foals. Traditional vaccines including live, killed and attenuated (physical and chemical) vaccines have proved to be ineffective and more modern molecular‐based vaccines including the DNA plasmid, genetically attenuated and subunit vaccines have provided inadequate protection of foals. Newer, bacterial vector vaccines have recently shown promise for R. equi in the mouse model. This article describes the findings of key research in R. equi vaccine development and looks at alternative methods that may potentially be utilised.     

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.     

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.