Cholesterol oxidase (ChoE) is not important in the virulence of Rhodococcus equi

To analyze further the role in virulence of the prominent cholesterol oxidase (ChoE) of Rhodococcus equi, an allelic exchange choE mutant from strain 103+ was constructed and assessed for virulence in macrophages, in mice, and in foals. There was no difference between the mutant and parent strain in cytotoxic activity for macrophages or in intra-macrophage multiplication. No evidence of attenuation was obtained in macrophages and in mice, but there was slight attenuation apparent in four intra-bronchially infected foals compared to infection of four foals with the virulent parent strain, based on a delayed rise in temperature of the choE-mutant infected foals. However, bacterial colony counts in the lung 2 weeks after infection were not significantly different, although there was a slight but non-significant (P=0.12) difference in lung:body weight ratio of the choE mutant versus virulent parent infected foals (mean 2.67+/-0.25% compared to 4.58+/-0.96%). We conclude that the cholesterol oxidase is not important for the virulence of R. equi.     

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.     

A physical map of the 85 kb virulence plasmid of Rhodococcus equi 103.

A physical map of the 85 kb virulence plasmid pOTS from Rhodococcus equi 103 was constructed. The restriction map contains 2 AsnI, 5 BglII, 9 EcoRI, 4 HindIII, and 3 XbaI sites. The positions of the EcoRI and HindIII of pOTS are identical to that of the 85 kb virulence plasmid of R. equi ATCC 33701 reported recently by others. EcoRI restriction fragment sizes were similar in the 85 kb plasmids isolated from 4 horse derived R. equi but, except apparently for the 28.3 and possibly 2.0 and 1.5 kb fragments, were different in an 80.1 kb plasmid isolated from a pig source R. equi.     

Two cases of equine abortion caused by Rhodococcus equi

Rhodococcus equi was isolated from lung, liver, spleen, and stomach content of two aborted equine fetuses of 7 and 8 months gestation from two different farms. Lesions included diffuse pyogranulomatous pneumonia with numerous Gram-positive coccobacilli within the cytoplasm of macrophages, multinucleated Langhans giant cells and neutrophils, and enhanced extramedullary hematopoiesis with megakaryocytosis within the liver and spleen. Detection of R. equi was made by bacteriology and immunohistochemistry for R. equi and VapA, the virulence factor of R. equi. R. equi and VapA were identified within the lungs of both fetuses, and its distribution correlated with lesions. Fetal lesions were similar to those observed in foals. We speculate that the fetuses contracted infection from the placenta by normal breathing movements or by swallowing of the amniotic fluid contaminated with R. equi.     

Characterization of virulence plasmid types in Rhodococcus equi isolates from foals,pigs, humans and soil in Hungary

Rhodococcus equi isolates (204) obtained from foals (lung abscesses, lymph nodes, nasal discharge, rectal swabs) bred in 15 studs located throughout Hungary, isolates from soil samples, lymph nodes of pigs and from lesions of human patients were examined to determine genotypic diversity of virulence-associated plasmids. Isolates were tested for the presence of 15-17 kDa virulence-associated protein antigen (VapA) and 20k Da (VapB) genes by polymerase chain reaction (PCR). Plasmid DNAs were isolated and analysed by digestion with restriction endonucleases for estimation of size and comparison of polymorphisms. Of 146 clinical isolates from foals in 15 studs, 129 (88.3%) gave positive results for the VapA gene, showing a 564 bp product of the expected size in the PCR amplification. Of the 129 clinical isolates from foals, 123 contained an 85 kb type I plasmid and the remaining six contained an 87 kb type I plasmid. Of 48 soil isolates from two horse studs, 26 (54.2%) were positive for VapA gene and contained an 85 kb type I plasmid. Of three pig isolates, one was positive for VapA gene and contained an 85 kb type I plasmid, and the remaining two were positive for the VapB gene, showing a 827 bp product of the expected size in the PCR amplification and were R. equi of intermediate virulence which contained a 95 kb type S5 plasmid. Of the seven human isolates, five were positive for VapB gene by PCR, these were R. equi of intermediate virulence, which contained a 95 kb type S5 plasmid. These results revealed that virulent R. equi strains harbouring a virulence plasmid of 85 kb type I or 87 kb type I, which have been found in clinical isolates from Europe and North and South America, are widespread in Hungary. Furthermore, same intermediately virulence plasmid type was found in both human and pig isolates.     

Prevalence of virulent Rhodococcus equi in isolates from soil collected from two horse farms in South Africa and restriction fragment length polymorphisms of virulence plasmids in the isolates from infected foals, a dog and a monkey

The prevalence of virulent Rhodococcus equi in soil isolates from two horse farms in South Africa and nine clinical isolates from six foals, a foal foetus, a dog, and a monkey was investigated. The isolates were tested for the presence of virulence plasmid DNA and 15- to 17-kDa antigens by immunoblotting. Rhodococcus equi was isolated from almost all of the soil samples obtained from the two farms with 5.0 x 10(1) to 3.3 x 10(4) colony forming units per gram of soil. Virulent R. equi was isolated from three soil samples from one of the farms and appeared in 3.8% (three of 80 isolates), but not in any of the 182 isolates from the other farm. Of the three virulent R. equi isolates, one contained an 85-kb type I plasmid and two an 87-kb type I plasmid. Of nine clinical isolates from the foals, foal foetus, dog and monkey, five from the foals were virulent R. equi which expressed the virulence-associated antigens and contained a virulence plasmid 85-kb type I, and were all isolated from cases of pneumonia typical of that induced by R. equi in young foals living in widely separated areas in South Africa. The isolates from the other four foals, the dog and the monkey were avirulent R. equi.     

Analysis of virulence plasmid gene expression of intra-macrophage and in vitro grown Rhodococcus equi ATCC 33701

Rhodococcus equi is a soil organism that infects macrophages of foals and immunocompromised humans. Virulence in foal isolates is tightly associated with an 80kb plasmid, which includes a pathogenicity island (PI) with a virulence-associated gene family, vap. A DNA microarray containing 66 of 69 putative open reading frames (ORFs) of the virulence plasmid was developed. Virulence plasmid gene expression of R. equi grown in macrophages or under different conditions in vitro was compared against in vitro growth at 30 degrees C, pH=7. When grown in macrophages, all seven vap family genes as well as six ORFs within, but not outside, the PI were induced. Cluster analysis of the gene expression matrix assembled from different growth conditions suggested that those genes that actively responded to environmental changes divided broadly into two groups. One group, orf1, 2, 5, 6-8, 12-15, 19, and 20 (which includes all the vap genes), was induced at 37 degrees C, mostly by low iron, and to a lesser extent by the synergy of low calcium and pH=5. The second group, orf3, 9, and 10, was induced at 37 degrees C by magnesium depletion (produced by EDTA treatment of growth medium). Temperature (37 degrees C) was the most important factor inducing gene expression for the both groups. Iron restriction led to down-regulation of Group II genes and magnesium restriction led to down-regulation of Group I genes. A putative consensus IdeR binding site was identified upstream of vapA, suggesting that vapA is a member of an IdeR regulon in R. equi. Expression of genes inside macrophages was most closely but not completely mimicked by growth of bacteria at 37 degrees C, pH=5, under conditions of restricted iron, calcium and magnesium; that is, similar to environmental factors found inside macrophages.     

The effect of experimental infection with Rhodococcus equi on immunodeficient mice

To investigate the pathogenesis of respiratory lesions caused by the facultative intracellular pathogen, Rhodococcus equi, pulmonary clearance was compared in four groups of genetically defined mice, chosen for their specific deficits in immune and inflammatory responses. Complement-deficient A/J, immunodeficient nu/nu (nude), scid/scid.bg/bg (SCID/beige), C57BI/6J.bg/bg (beige) and normal Swiss mice (SW) received approximately 10(7) R. equi intranasally on day 0. Bacterial clearance was assessed in lung, liver and spleen on days 1, 4, 7 and 14. Pulmonary clearance was not significantly different between SW and A/J mice. Beige mice cleared R. equi more rapidly and completely than A/J and SW, indicating that deficits in phagocytic and NK cell function associated with the bg/bg gene did not compromise clearance. Pulmonary clearance in immunodeficient SCID/beige mice paralleled that of the SW and A/J mice initially but bacterial proliferation produced significant differences from SW mice at day 14. Nude mice were unable to clear R. equi from day 1, resulting in the death of two nude mice at day 11. Both SCID/beige and nude mice developed severe pyogranulomatous bronchopneumonia, whereas A/J and SW mice developed transient pulmonary lesions. Beige mice developed minimal lung lesions. Significant systemic bacterial proliferation occurred only in nude and SCID/beige mice. We conclude that deficiencies in complement components, phagocytic and NK cells do not impair the pulmonary clearance of R. equi but that a competent cellular immune system is required to prevent pneumonia and death. The difference in early phase pulmonary clearance in nude and SCID/beige mice indicates two phases are important for clearance. An acapsular mutant of R. equi was completely cleared from the lungs of SCID/beige mice suggesting an important role for the capsule in virulence for mice.     

The pathogenic equine streptococci

Streptococci pathogenic for the horse include S. equi (S. equi subsp. equi), S. zooepidemicus (S. equi subsp. zooepidemicus), S. dysgalactiae subsp. equisimilis and S. pneumoniae capsule Type III. S. equi is a clonal descendent or biovar of an ancestral S. zooepidemicus strain with which it shares greater than 98% DNA homology and therefore expresses many of the same proteins and virulence factors. Rapid progress has been made in identification of virulence factors and proteins uniquely expressed by S. equi. Most of these are expressed either on the bacterial surface or are secreted. Notable examples include the antiphagocytic SeM and the secreted pyrogenic superantigens SePE-I and H. The genomic DNA sequence of S. equi will greatly accelerate identification and characterization of additional virulence factors and vaccine targets. Although it is the most frequently isolated opportunist pyogen of the horse, S. zooepidemicus has been the subject of few contemporary research studies. Variation in the protectively immunogenic SzP proteins has, however, been well characterized. Given its opportunist behavior, studies are urgently needed on regulation of virulence factors such as capsule and proteases. Likewise, information is also very limited on virulence factors and associated gene regulation of S. dysgalactiae subspecies equisimilis. It has recently been shown that equine isolates of Streptococcus pneumoniae are clonal, a feature shared with S. equi. All equine isolates express capsule Type III, are genetically similar, and have deletions in the genes for autolysin and pneumolysin. In summary, the evolving picture of the interaction of the equine pathogenic streptococci and their host is that of multiple virulence factors active at different stages of pathogenesis. The inherent complexity of this interaction suggests that discovery of effective combinations of immunogens from potential targets identified in genomic sequence will be laborious.     

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.     

Mutation and virulence assessment of chromosomal genes of Rhodococcus equi 103

Rhodococcus equi can cause severe or fatal pneumonia in foals as well as in immunocompromised animals and humans. Its ability to persist in macrophages is fundamental to how it causes disease, but the basis of this is poorly understood. To examine further the general application of a recently developed system of targeted gene mutation and to assess the importance of different genes in resistance to innate immune defenses, we disrupted the genes encoding high-temperature requirement A (htrA), nitrate reductase (narG), peptidase D (pepD), phosphoribosylaminoimidazole-succinocarboxamide synthase (purC), and superoxide dismutase (sodC) in strain 103 of R. equi using a double-crossover homologous recombination approach. Virulence testing by clearance after intravenous injection in mice showed that the htrA and narG mutants were fully attenuated, the purC and sodC mutants were unchanged, and the pepD mutant was slightly attenuated. Complementation with the pREM shuttle plasmid restored the virulence of the htrA and pepD mutants but not that of the narG mutant. A single-crossover mutation approach was simpler and faster than the double-crossover homologous recombination technique and was used to obtain mutations in 6 other genes potentially involved in virulence (clpB, fadD8, fbpB, glnA1, regX3, and sigF). These mutants were not attenuated in the mouse clearance assay. We were not able to obtain mutants for genesfurA, galE, and sigE using the single-crossover mutation approach. In summary, the targeted-mutation system had general applicability but was not always completely successful, perhaps because some genes are essential under the growth conditions used or because the success of mutation depends on the target genes.     

In vivo pathogenicity and resistance to phagocytosis of Streptococcus equi strains with different levels of capsule expression.

The glossy non-encapsulated strain of Steptococcus equi, NCTC 9682, was compared with the matt strain Hidaka/95/2 which expresses a medium sized capsule and with the mucoid CF32 which expresses a large sized capsule in phagocytosis assays and for virulence in inoculated horses. The three strains, NCTC 9682, Hidaka /95/2 and CF32 produced 2.0, 3.1, and 5.3 mg/g wet cells respectively after 3 h incubation, but similar amounts of M-like proteins, cytotoxin and mitogen. NCTC 9682 showed no resistance to phagocytosis by equine neutrophils regardless of the presence of opsonin while strains Hidaka /95/2 and CF32 showed almost complete resistance to phagocytosis. Furthermore, NCTC 9682 produced no clinical disease although it infected the guttural pouch and caused seroconversion. Typical strangles with guttural pouch invasion was observed in all horses infected with encapsulated strains.     

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.     

Experimental infection of mice with Rhodococcus equi: differences in virulence between strains

The growth kinetics in outbred mice of clinical and environmental isolates of Rhodococcus equi were followed by serial bacterial enumeration of organ homogenates. Clinical isolates multiplied until Day 4 before being progressively cleared, but could still be recovered from the liver at 3-4 weeks post-infection. Intravenous inoculation of clinical strains was associated with histopathological responses very similar to those elicited by intravenous infection with various facultative intracellular parasites. Whereas lesions in mice and foals at 7-9 days following respiratory infection are those of severe bronchopneumonia with massive consolidation, a week later the patterns of host response have diverged as the murine lesions resolve. The type strain, NCTC 1621 and 4-6 environmental isolates were eliminated without prior multiplication and these strains caused negligible lesions. The two environmental strains which behaved as the clinical strains were recovered from a stud with an R. equi problem. No association of colonial morphology of R. equi with virulence was apparent.     

Isolation and characterisation of Rhodococcus equi from submaxillary lymph nodes of wild boars (Sus scrofa)

Rhodococcus equi has been isolated from the submaxillary lymph nodes of domesticated pigs, but little is known about the presence of R. equi in wild boars. The aim of the study was the evaluation of the incidence of R. equi in wild boars and the characterisation of them. Of 482 submaxillary lymph nodes of wild boars shot in 39 settlements throughout Hungary, R. equi was isolated from 60 specimens, and plasmid types of 82 isolates were examined. The isolates were tested for the presence of 15-17-kDa (VapA) and 20-kDa virulence-associated protein antigen (VapB) genes by polymerase chain reaction (PCR). Plasmid DNAs were isolated and analysed by digestion with restriction endonucleases to estimate size and compare their polymorphisms. None of the 82 isolates contained vapA gene but 21 isolates (25.6%) were positive for vapB gene showing 827bp product of the expected size in the PCR amplification. Sixty-one strains (74.4%) did not contain plasmid. The 21 isolates of intermediate virulence contained virulence plasmids that were identified as types 1 (1 isolate), 5 (16 isolates), 21 (1 isolate), and three new distinct plasmid variants (1-1-1 isolate), respectively. On the basis of restriction digestion patterns of plasmid DNAs, we tentatively designated the new variants as types 25-27, respectively. The prevalence of R. equi strains of intermediate virulence among the isolates originated from the submaxillary lymph nodes of wild boars (25.6%) is very similar to those of domestic pigs (26.8%) in Hungary, and plasmid type 5 is the predominating one in both groups. This is the first report of isolation of VapB-positive R. equi from wild boars in the world.     

The role of hyaluronic acid capsular material of Streptococcus equi subsp. zooepidemicus in mediating adherence to HeLa cells and in resisting phagocytosis.

Hyaluronic acid is thought to be one of the critical virulence factors of Streptococcus equi subsp. zooepidemicus. The present study was designed to study the role of hyaluronic acid capsular material in mediating adherence and to resist the phagocytosis of the host's immune defence. The studies were performed with two encapsulated S. equi subsp. zooepidemicus and two unencapsulated phase variants. The bacteria had been previously isolated from diseased pigs and monkeys in Indonesia. The presence of capsular material was determined using the hyaluronic acid decapsulation test and by electron microscopic studies. Both encapsulated bacteria showed mucoid colonies after cultivation on blood agar, grew with diffuse colonies in soft agar media and reacted negatively in the salt aggregation test. The unencapsulated bacteria grew with small colonies on blood agar, formed compact colonies in soft agar media and reacted positively in the salt aggregation test. Adherence and phagocytosis studies revealed that the encapsulated bacteria adhered significantly more to HeLa cells and were less phagocytosed by murine macrophages compared to unencapsulated bacteria. Pretreatment of the HeLa cells using hyaluronic acid or pretreatment of the bacteria by hyaluronidase decreased the adherence value of encapsulated bacteria. Pretreatment of bacteria with pronase had no effect. The presented results strongly indicate that the hyaluronic acid capsular material contributes to adherence properties of S. equi subsp. zooepidemicus and might help the bacteria to resist phagocytosis by macrophages.     

Development of a live, attenuated, potential vaccine strain of R. equi expressing vapA and the virR operon, and virulence assessment in the mouse

Pneumonia caused by Rhodococcus equi remains a significant problem in foals. The objective of this study was to develop a safe and efficacious attenuated strain of R. equi for eventual use in oral immunization of foals. The approach involved expression of vapA in a live, virulence plasmid-negative, strain of R. equi (strain 103-). PCR-amplified fragments of the vapA gene, with and without the upstream genes virR, orf5, vapH, orf7 and orf8 (orf4-8), were cloned into a shuttle vector pNBV1. These plasmids, named pAW48A and pAWVapA respectively, were electroporated into strain 103-. The presence of the recombinant vectors in the attenuated strain (103-) and the integrity of the inserted genes were confirmed, and both constructs expressed VapA. The virulence of the two strains was compared to that of wild type R. equi 103+ and negative controls by their intravenous inoculation into mice, followed by examination of liver clearance 4 days later. Mice inoculated with R. equi 103-, 103-/pAWVapA and 103-/pNBV1 completely cleared infection, whereas strain 103-/pAW48A persisted in 47% of mice.     

Cutaneous pyogranuloma in a cat caused by virulent Rhodococcus equi containing an 87 kb type I plasmid

A 2-year-old intact male domestic shorthaired cat presented with a chronic, nodular, ulcerated, cutaneous lesion on the right thoracic limb. Histological and cytological examination revealed a pyogranulomatous inflammation with basophilic organisms in the macrophages. A virulent form of Rhodococcus equi containing an 87 kb type I (VapA) virulence plasmid was identified from cultures of biopsy samples. This report describes the clinicopathological features, plasmid profile and virulence of this case of R equi infection.