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.     

In vitro phagocytosis and killing of Corynebacterium equi by alveolar macrophages of foals

Bronchoalveolar lavage was performed 5 times, sequentially, on 3 healthy foals while each foal was 6 to 63 days of age. Phagocytosis and bactericidal assays were performed on recovered alveolar macrophages. Corynebacterium equi and alveolar macrophages at a ratio of 10:1 were incubated for 1 hour in medium containing 1% heat-inactivated rabbit anti-C equi serum. After incubation, greater than 90% of the alveolar macrophages contained at least 1 ingested bacterium and each alveolar macrophage contained 9.4 +/- 1.0 bacteria (mean +/- SE). After alveolar macrophages and C equi were incubated for 1 hour in medium containing heat-inactivated pooled normal horse serum, approximately 24% of the alveolar macrophages contained at least 1 bacterium and each alveolar macrophage contained 0.8 +/- 0.7 bacteria. From 6 to 61 days of age, each foal had significantly (P less than 0.05) decreased phagocytic activity by alveolar macrophages, but a significant change in killing of C equi by alveolar macrophages was not found in the foals from 21 to 61 days of age. After incubating alveolar macrophages and C equi for 4 hours in vitro, approximately 75% of ingested C equi remained viable.     

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.     

Rhodococcus equi

Rhodococcus equi is an important cause of subacute or chronic abscessating bronchopneumonia of foals up to 3-5 months of age. It shares the lipid-rich cell wall envelope characteristic of the mycolata, including Mycobacterium tuberculosis, as well as the ability of pathogenic members of this group to survive within macrophages. The possession of a large virulence plasmid in isolates recovered from pneumonic foals is crucial for virulence. The plasmid contains an 27 kb pathogenicity island (PI) that encodes seven related virulence-associated proteins (Vaps), including the immunodominant surface-expressed protein, VapA. Only PI genes are differentially expressed when the organism is grown in macrophages in vitro. Ten of the PI genes, including six Vap genes, have signal sequences, suggesting that they are exported from the cell to interact with the macrophage. Different PI genes are regulated by temperature, pH, iron, oxidative stress and probably also by magnesium, all environmental changes encountered after environmental R. equi are inhaled in dust and are ingested into macrophages in the lung. The basis of pathogenicity of R. equi is its ability to multiply in and eventually to destroy alveolar macrophages. Infectivity is largely or exclusively limited to cells of the monocyte-macrophage lineage. Current evidence suggests that infection of foals with virulent R. equi results in some foals in subversion of cell-mediated immunity and development of an ineffective and sometimes lethal Th2-based immune response. Significant progress has been made recently in the development of R. equi-E. coli shuttle vectors, transformation and random and site specific mutagenesis procedures, all of which will be important in molecular dissection of the mechanisms by which R. equi subverts normal macrophage killing mechanisms and cell-mediated immunity.     

Rhodococcus equi: equine neutrophil chemiluminescent and bactericidal responses to opsonizing antibody

The opsonic capacity of serum containing R. equi-specific antibody was compared with antibody-deficient sera using luminol-dependent chemilumenscence (LDCL) and bactericidal assays. These assays incorporated peripheral blood polymorphonuclear neutrophilic leukocytes (PMNL) exposed to R. equi opsonized with neonatal equine pre-colostral serum (control) or serum from foals with R. equi infections (principal). All sera were complement inactivated at 56 degrees C for 30 min. Bacteria were obtained from the lung of a foal with R. equi pneumonia. Neutrophils were obtained from one adult horse for LDCL and another for bactericidal assays. Chemiluminescence of PMNL exposed to R. equi opsonized with control or principal sera was measured in a liquid scintillation counter. Mean peak LDCL within 1 h was significantly (P less than 0.01) higher with principal sera (2.4 X 10(5) cpm) than with control sera (0.018 X 10(5) cpm). A radioisotope bactericidal assay was used to determine the effect of control or principal sera on PMNL capacity to kill R. equi. Mean peak percent kill of R. equi by PMNL within 2 h was significantly (P less than 0.01) higher with principal sera (95.2%) than with control sera (54.6%). Enzyme-linked immunosorbent assay (ELISA) values for R. equi-specific antibody were determined on all sera. Mean ELISA values were significantly (P less than 0.01) higher for principal sera (71.8) than for controls (0.0). This investigation documents the presence and biological effectiveness of opsonic activity in complement-inactivated sera from foals with R. equi infections and R. equi-specific antibody.     

The interaction of Rhodococcus equi and foal neutrophils in vitro

Polymorphonuclear neutrophil leukocytes (PMNL) from 8 healthy foals (2-14 weeks of age) and 2 foals with bacterial pneumonia were separated from whole blood using a 2 step Percoll gradient. Purified PMNL were tested for bactericidal function against Rhodococcus equi and Staphylococcus aureus in the presence of normal horse serum. The percentage uptake after a 15-min pre-incubation of PMNL and bacteria was also calculated. Ultrastructural examination of the interaction of R. equi and normal foal PMNL was performed after 15 min incubation. Results indicated that foal PMNL effectively phagocytose and destroy R. equi and S. aureus in the presence of normal horse serum. The mean percent uptake for R. equi was 99.3 +/- 0.4% and for S. aureus 99.9 +/- 0.1%. Further, 97.8 +/- 0.1% ingested R. equi and 98.4 +/- 0.1% ingested S. aureus were destroyed in the 15-min incubation period. Over the 3-h incubation, 91.9% of remaining R. equi were killed, but only 49.2 +/- 31.9% of S. aureus (P less than 0.01). Total bactericidal effect of foal PMNL, however, was 99.3 +/- 0.4% against R. equi and 99.9 +/- 0.1% against S. aureus. The percentage uptake and total bactericidal efficacy of neutrophils from sick foals was greater than 95%. Ultrastructural examination of the PMNL-R. equi interaction after 15 min incubation revealed phagocytosis of the bacteria and morphologic changes consistent with neutrophil degranulation. This study suggests that a defect in PMNL bactericidal capability is not likely to be a contributing factor in the pathogenesis of R. equi pneumonia in foals.     

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.     

Rhodococcus equi pneumonia in the foal--part 1: pathogenesis and epidemiology

Rhodococcus equi pneumonia is a worldwide infectious disease of major concern to the equine breeding industry. The disease typically manifests in foals as pyogranulomatous bronchopneumonia, resulting in significant morbidity and mortality. Inhalation of aerosolised virulent R. equi from the environment and intracellular replication within alveolar macrophages are essential components of the pathogenesis of R. equi pneumonia in the foal. Recently documented evidence of airborne transmission between foals indicates the potential for an alternative contagious route of disease transmission. In the first of this two-part review, the complexity of the host, pathogen and environmental interactions that underpin R. equi pneumonia will be discussed through an exploration of current understanding of the epidemiology and pathogenesis of R. equi pneumonia in the foal.     

Immunohistochemical observations on pneumonic lesions caused by Rhodococcus equi in foals.

An immunohistochemical analysis of Rhodococcus equi-induced pneumonia in 10 foals was performed by biotin-streptavidin system. The detection of R. equi was more sensitive in immuno-stain using anti-R. equi serum than in Gram's stain. This bacteria also reacted to anti-BCG serum. Lysozyme and alpha 1-antitrypsin were detectable in macrophages. A particularly intense staining was observed in association with intracellular bacteria. Though a degree of reaction for alpha 1-antichymotrypsin was very low in comparison with lysozyme and alpha 1-antitrypsin, it was also demonstrated in macrophages ingesting R. equi. These bacteria were almost intact under an electron microscope. Therefore, the surface components of R. equi may play important roles of protection from intracellular enzymes of macrophages. The cells containing intracytoplasmic IgM, IgG or IgA were a few in number and scattered predominantly around the pneumonic lesion. It is considered that the bactericidal activity by immunoglobulins may be weak in comparison with phagocytosis by macrophages.     

Evaluation of fecal samples from mares as a source of Rhodococcus equi for their foals by use of quantitative bacteriologic culture and colony immunoblot analyses

OBJECTIVE: To determine whether mares are a clinically important source of Rhodococcus equi for their foals. SAMPLE POPULATION: 171 mares and 171 foals from a farm in Kentucky (evaluated during 2004 and 2005). PROCEDURES: At 4 time points (2 before and 2 after parturition), the total concentration of R equi and concentration of virulent R equi were determined in fecal specimens from mares by use of quantitative bacteriologic culture and a colony immunoblot technique, respectively. These concentrations for mares of foals that developed R equi-associated pneumonia and for mares with unaffected foals were compared. Data for each year were analyzed separately. RESULTS: R equi-associated pneumonia developed in 53 of 171 (31%) foals. Fecal shedding of virulent R equi was detected in at least 1 time point for every mare; bacteriologic culture results were positive for 62 of 171 (36%) mares at all time points. However, compared with dams of unaffected foals, fecal concentrations of total or virulent R equi in dams of foals with R equi-associated pneumonia were not significantly different. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that dams of foals with R equi-associated pneumonia did not shed more R equi in feces than dams of unaffected foals; therefore, R equi infection in foals was not associated with comparatively greater fecal shedding by their dams. However, detection of virulent R equi in the feces of all mares during at least 1 time point suggests that mares can be an important source of R equi for the surrounding environment.     

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.     

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.     

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.     

Evaluation of nasotracheal aspiration as a diagnostic tool for Rhodococcus equi pneumonia in foals

The reliability of preparing bacteriological cultures from nasotracheal aspirates of foals routinely in order to diagnose R. equi pneumonia in foals was studied by isolating Rhodococcus equi from specimens obtained from 96 foals by nasotracheal aspiration with a silicon catheter. Results were compared with specimens obtained from 21 foals by transtracheal aspiration (percutaneous tracheal puncture). These 117 foals showed clinical signs of respiratory tract infection at sampling. R. equi was isolated from 14 of 21 (66.7%) specimens by transtracheal aspiration and from 59 of 96 (61.4%) specimens by nasotracheal aspiration, 649 of 655 isolates (99.1%) from the 73 positive specimens were virulent R. equi, and the culture-positive foals were diagnosed as having R. equi pneumonia. To assess the contamination of aspirates by organisms from the nasopharynx, the results of R. equi isolation from nasal swabs obtained from 56 of the 96 foals were compared to those obtained by nasotracheal aspiration from the same foals. R. equi was isolated from 2 of the 56 nasal swabs: one from a tracheal aspirate was positive, and the other was not. These results suggest that the nasotracheal aspiration technique, which is noninvasive and not associated with complications, could be used as an alternative to the transtracheal aspiration method, especially for the diagnosis of R. equi pneumonia in foals.     

Peripheral Blood Lymphocyte Subpopulations and Immunoglobulin Concentrations in Healthy Foals and Foals with Rhodococcus equi Pneumonia

Infectious diseases are common in foals aged 1-5 months. The objectives of this investigation were to evaluate immunologic parameters in foals from birth to weaning to establish reference values for the proportion of circulating lymphocytes that were helper (CD4+) or cytotoxic (CD8+) T cells, or B cells; to measure serum immunoglobulin (IgM and IgG) concentrations; and to compare these immunologic parameters to values in foals with naturally occurring Rhodococcus equi pneumonia and in adult horses. Peripheral blood lymphocyte subpopulations were determined by flow cytometric analysis, and serum IgG and IgM concentrations were determined by radial immunodiffusion. Flow cytometric analysis of lymphocyte subpopulations suggested age-related changes in the cell-mediated immune system in horses. Absolute circulating CD4+ and CD8+ T lymphocytes and B cells increased linearly up to 3 months of age. Circulating B cell concentrations from birth to 6 months of age were greater than values in adult horses and the lymphocyte differences among the age groups are mainly due to variation in B lymphocytes. Both absolute and proportional B cell concentrations were greater in foals with R equi pneumonia than in healthy foals at the same age. The increase in absolute cell counts of each subpopulation was dependent on the increase of absolute peripheral blood lymphocyte count. Serum IgG concentration increased linearly from 1 to 3 months of age, and serum IgM concentrations increased from 1 to 6 months of age. These data suggest age-dependent cell-mediated and humoral development in young 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.     

Phagocytic and bactericidal properties of bovine macrophages from non-lactating mammary glands

Macrophages were isolated from the mammary glands of non-lactating (dry) cows and their ability to phagocytose and kill staphylococci in vitro assessed. Normal bovine serum enhanced the uptake of staphylococci and was required for optimal killing in the bactericidal test. Dry gland secretion interfered with uptake. Secretions taken progressively into the dry period became more inhibitory. The phagocytic ability of macrophages was significantly less than that of neutrophils present in the same gland preparation when tested in the presence of dry gland secretion. A marked variation in the antibacterial activity of macrophages from different cows was noted.     

Diagnosis of Rhodococcus equi infection in foals by the agar gel diffusion test with protein antigen

A protein antigen that reacted in the agar gel diffusion (AGD) test and which had equi factor(s) activity, was partially purified from the culture supernatant of Rhodococcus equi by successive column chromatography on diethylaminoethyl cellulose and Sepharose 4B. Employing a standard foal serum, the concentration of this antigen was adjusted for the AGD test. Optimal dilutions of the antigen reacted in the AGD test with sera from foals naturally infected with serologically different R. equi. The antigen prepared was considered suitable for use in field surveys of R. equi infection. Accordingly, four groups of sera were tested: those from 18 foals diagnosed as being infected with R. equi, those from 54 control foals with culture-negative R. equi pneumonia, arthritis or cellulitis, those from 46 diseased foals suspected of having R. equi infection and those from 51 clinically normal foals. A positive precipitation reaction was observed with sera from 100% of the first group, 69.5% of the third group and 17.7% of the fourth group. A negative reaction was obtained with sera from 100% of the second group.