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).     

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.     

Ecology of Rhodococcus equi

A selective broth enrichment technique was used to study the distribution of Rhodococcus equi in soil and grazing animals. Rhodococcus equi was isolated from 54% of soils examined and from the gut contents, rectal faeces and dung of all grazing herbivorous species examined. Rhodococcus equi was not isolated from the faeces or dung of penned animals which did not have access to grazing. The isolation rate from dung was much higher than from other samples and this was found to be due to the ability of R. equi to multiply more readily in dung. Delayed hypersensitivity tests were carried out on horses, sheep and cattle, but only horses reacted significantly. The physiological characteristics of R. equi and the nature of its distribution in the environment suggested that R. equi is a soil organism.     

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.     

Influence of Rhodococcus equi on the respiratory burst of resident alveolar macrophages from adult horses

Opsonized Rhodococcus equi activated the respiratory burst of resident alveolar macrophages (AM) from adult horses in a logarithmic-linear, mass-related manner. The effect of R equi was not significantly different from that of equal masses of opsonized zymosan A. Therefore, R equi does not appear to attenuate the respiratory burst of equine AM. The stimulatory effect of R equi was not reflected by increased production of superoxide anion (O2-), but increased activity of the hexose monophosphate shunt was observed. These results suggest a similarity between the respiratory burst of AM from horses and that of AM from rabbits. We concluded that resident AM from adult horses do not produce O2- concurrently with an increase in activity of the hexose monophosphate shunt when stimulated with either opsonized zymosan A or opsonized R equi. This suggests that O2- is not an important component of the antibacterial defenses of equine AM. Whether equine AM are incapable of producing O2- or require different stimuli to produce it was not determined.     

Disseminated Rhodococcus equi infection in two goats.

Rhodococcus equi infection was diagnosed in two goats from the same herd. At necropsy, numerous caseating granulomas were disseminated throughout the liver, lungs, abdominal lymph nodes, medulla of right humerus, and the right fifth rib of goat No. 1, and the liver of goat No. 2. Histopathologic examination confirmed the presence of multiple caseating granulomas in these organs. Numerous gram-positive and Giemsa-positive coccobacilli were identified within the cytoplasm of macrophages. Aerobic bacterial cultures of the liver and lung from both goats yielded a pure growth of R. equi. R. equi antigens were immunohistochemically identified in caseating granulomas from both goats. However, the 15- to 17-kd virulence antigens of R. equi were not detected, suggesting possible infection by an avirulent strain of this organism.     

Attempts to find phenotypic markers of the virulence plasmid of Rhodococcus equi.

Four isolates of Rhodococcus equi, from pneumonic foals, and containing the 85 kb virulence plasmid, a porcine isolate containing an 80 kb plasmid, and their plasmid cured derivatives, were examined for 239 phenotypic properties in an attempt to find characters other than the virulence-associated protein (VapA) which might be encoded by the virulence plasmid in organisms grown at 37 degrees C. Tests chosen included those which have previously given variable results for R. equi isolates, since such variability might be attributed to plasmid curing, and characteristics which have been described as properties of plasmids of Rhodococcus species other than R. equi. Tests included cadmium resistance, Congo red binding, resistance to 26 antibiotics, conventional clinical microbiological tests, utilization of 95 different carbon sources, enzymatic activities in API ZYM, fluorogenic assays for exo- and endopeptidase, glycosidase activities, and testosterone degradation. Apart from production of VapA by foal isolates, no phenotypic property was identified in the plasmid-positive isolates. Phenotypic characteristics of R. equi that have not been described before, and might be useful in identification were: metabolism of N-acetyl-beta D-glucopyranoside, alpha- and beta-hydroxybutyric, alpha-ketobutyric and N-acetyl-glutamic acids, of methylpyruvate, heptanoate, nonanoate and stearate esters; exopeptidase activity against alanine-alanine-tyrosine, alanine-phenylalanine-lysine, glycine-arginine, lysine-alanine, and valine-glycine-alanine; endopeptidase activity against arginine and methionine; and hydrolysis of bis-phosphate ester.     

Optimization of an in vitro assay to detect Streptococcus equi subsp. equi

Streptococcus equi is the etiologic agent of a highly infectious upper respiratory disease of horses known as strangles. Bacterial culture methods and polymerase chain reaction (PCR) of nasopharyngeal washes and guttural pouch lavages are used routinely to test clinical and carrier animals for the presence of S. equi but no definitive or gold standard test method has been shown to be optimal. We hypothesized that (i) a flocked swab submerged in ten-fold serial dilution suspensions of S. equi prepared in 0.9% NaCl would detect more colony forming units (CFU) than a rayon swab when used to inoculate a blood agar plate, (ii) centrifugation of a 1ml aliquot of each suspension would improve the limit of detection (LOD) by bacterial culture and PCR compared to the culture or PCR of submerged swab samples, (iii) PCR of the centrifuged samples from each suspension would be more sensitive than aerobic culture alone, and (iv) PCR of a 1ml aliquot directly from a sample would be more sensitive than PCR of a sample following submersion of a flocked swab in 1ml saline. Using 7 ten-fold serial dilutions of S. equi in 0.9% NaCl, the LOD for 4 bacterial culture methods and 3 PCR methods were compared. The LOD of direct PCR and flocked swab culture was determined at 1cfu/ml. All PCR methods were equivalent to each other and were more sensitive than any of the culture methods at the lower dilutions. At higher cell densities (>100cfu/ml) flocked swab culture was not statistically better than rayon swab culture, but it was superior to all other methods tested.     

The taxonomic status of Rhodococcus equi

The species Corynebacterium equi was proposed for strains isolated from foals suffering from purulent pneumonia. The taxon has had a confused history and is currently listed under both Corynebacterium and Rhodococcus in the Approved Lists of Bacterial Names. Data from modern taxonomic studies indicate that Corynebacterium equi Magnusson 1923 should be reduced to a synonym of Rhodococcus equi (Magnusson) Goodfellow and Alderson 1980. Rhodococcus equi has repeatedly been shown to be a good species on the basis of chemical, molecular biological, numerical phenetic and serological data. Improved methods are needed to differentiate Rhodococcus equi from closely related species of Rhodococcus.     

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.     

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.     

Role of antibody to extracellular proteins of Rhodococcus equi in protection against R. equi pneumonia in foals

Rhodococcus equi produces two exoenzymes (REE), a cholesterol oxidase in large amounts and a phospholipase C, which cause lysis of sheep red blood cells (SRBC) sensitized with Staphylococcus aureus beta toxin. Two immunization studies were done in foals to determine the role of antibody to REE in protection against R. equi pneumonia. In the first study, three foals (mean age 10 days) were vaccinated four times at 2-week intervals with over 1 million units of partially purified exoenzymes (PREE). In the second study, three foals (mean age 19 days) were administered plasma from an adult horse vaccinated with PREE. Relatively low titres (16-32) of neutralizing antibody were detected in the foals of the former group, and passive transfer of neutralizing antibody (titres 32-64) occurred in the latter. Following immunization, principal foals and an equal number of similarly aged nonimmunized foals were challenged by aerosol with 1 x 10(10) live R. equi per day for 5 consecutive days. No severe clinical pneumonia developed in either group and, with one exception, only minor and resolving lung abscesses developed in these foals. These studies showed that antibody response of foals to immunization with PREE was poor, antibody to PREE did not prevent foals from developing lung abscesses following experimental infection, and that foals even as young as 3 weeks of age may be largely refractory to aerosol challenge with virulent R. equi.     

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.     

Polyacrylamide gel electrophoresis of whole-cell preparations of Rhodococcus equi.

The whole-cell proteins of ten strains of Rhodococcus equi isolated from horses, pigs, or humans, including the type strain ATCC 6939, were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The protein profiles of seven different capsular serotypes and the type strain were very similar when bacteria were cultured under the same conditions. Protein profiles were largely unaffected by incubation at two temperatures (30 degrees C, 37 degrees C) or times (12 h, 48 h). There were generally minor differences in protein profiles between strains grown in different media (brain heart infusion, nutrient, minca broths, tryptic soy-blood agar) with the marked exception of a prominent diffuse 17.5 kd protein which was expressed in nutrient broth. This protein was not produced by the type strain and was lost on repeated passage in vitro (50th, 100th passage) in two of three other strains examined.     

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.     

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.