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Immunogenicity of synthetic Rhodococcus equi virulence-associated protein peptides in neonate foals.

Rhodococcus equi infection is considered the most common cause of pneumonia in foals less than 6 months of age. Immunization of foals and/or mares may become a procedure of choice for prevention. The present work documents the antibody response of neonate foals to R. equi virulence-associated protein (Vap) vaccine candidate peptides. A mixture of 4 R. equi (ATCC 33701) Vap peptides was selected based on their hydrophilicity and recognition by naturally acquired IgG antibodies from 13 adult horses and 33 neonate foals from France and Japan. They were combined with a water-based nanoparticular adjuvant to promote a protective immune response including both Th1 cytokine pattern and antibody response. A single intramuscular injection resulted in an IgG antibody response 30 days later, although inconsistently. In responding animals, no bias in IgG subclass distribution was observed, and antibody response was associated with enhanced serum opsonic activity. In conclusion, data indicate that synthetic Vap peptides combined with nanoparticular adjuvant were immunogenic and resulted in a significant increase in IgG antibodies against the corresponding virulent R. equi strain in a majority of foals.

Actinomycetales Infections↗

Rhodococcus equi peritonitis in continuous ambulatory peritoneal dialysis.

The human Rhodococcus equi (R. equi) infection is now emerging, although extrapulmonary manifestation and isolation from patients without human immunodeficiency virus (HIV) infection remains unusual. Considerable effort is required to correctly identify and diagnose this facultative pathogen in patients with peritonitis in end-stage renal failure (ESRF) on continuous ambulatory peritoneal dialysis (CAPD). In the six cases of R. equi CAPD peritonitis reported in this series, diagnoses were made, on average, after 15 days and prolonged antibiotic therapy with morbidity in two patients. A diagnosis of R. equi should be considered in patients with suspected diphtheroid or Nocardia CAPD peritonitis, even with no history of animal contact. This study is the largest series on R. equi CAPD peritonitis and highlights the impact of this disease.

Actinomycetales Infections↗

Clinical application of a polymerase chain reaction assay in the diagnosis of pneumonia caused by Rhodococcus equi in a horse.

Diagnosis of pneumonia caused by Rhodococcus equi can be made more rapidly by use of a polymerase chain reaction (PCR) assay than by use of conventional bacteriologic culture techniques. Use of a PCR assay aids in the differentiation between virulent and avirulent strains of R equi, and the assay may be used to identify R equi in feces and soil of breeding farms.

Actinomycetales Infections↗

Identification and mutagenesis by allelic exchange of choE, encoding a cholesterol oxidase from the intracellular pathogen Rhodococcus equi.

The virulence mechanisms of the facultative intracellular parasite Rhodococcus equi remain largely unknown. Among the candidate virulence factors of this pathogenic actinomycete is a secreted cholesterol oxidase, a putative membrane-damaging toxin. We identified and characterized the gene encoding this enzyme, the choE monocistron. Its protein product, ChoE, is homologous to other secreted cholesterol oxidases identified in Brevibacterium sterolicum and Streptomyces spp. ChoE also exhibits significant similarities to putative cholesterol oxidases encoded by Mycobacterium tuberculosis and Mycobacterium leprae. Genetic tools for use with R. equi are poorly developed. Here we describe the first targeted mutagenesis system available for this bacterium. It is based on a suicide plasmid, a selectable marker (the aacC4 apramycin resistance gene from Salmonella), and homologous recombination. The choE allele was disrupted by insertion of the aacC4 gene, cloned in pUC19 and introduced by electroporation in R. equi. choE recombinants were isolated at frequencies between 10(-2) and 10(-3). Twelve percent of the recombinants were double-crossover choE mutants. The choE mutation was associated with loss of cooperative (CAMP-like) hemolysis with sphingomyelinase-producing bacteria (Listeria ivanovii). Functional complementation was achieved by expression of choE from pVK173-T, a pAL5000 derivative conferring hygromycin resistance. Our data demonstrate that ChoE is an important cytolytic factor for R. equi. The highly efficient targeted mutagenesis procedure that we used to generate choE isogenic mutants will be a valuable tool for the molecular analysis of R. equi virulence.

Alleles↗

The intracellular bacterium Rhodococcus equi requires Mac-1 to bind to mammalian cells.

Rhodococcus equi is a facultative intracellular bacterium of macrophages that causes disease in immunocompromised individuals, particularly those with AIDS. In this report, we demonstrate that R. equi binding to mammalian cells requires complement and is mediated primarily by the leukocyte complement receptor, Mac-1. Bacteria bind to macrophages poorly unless exogenous complement is added to the incubation medium. The addition of fresh nonimmune serum, which contains no detectable antibodies to R. equi, greatly enhances bacterial binding to macrophages, whereas heat inactivation of this serum or immunological depletion of C3 from the serum reduces binding to levels only slightly higher than those of binding under serum-free conditions. Human serum depleted of C2 or C4 is fully opsonic, indicating that complement activation and fixation occur by the alternative pathway. The serum-dependent binding of rhodococci to macrophages is mediated primarily by the macrophage complement receptor type 3, Mac-1 (CD11b/CD18). Bacteria do not bind to fibroblastoid or epithelial cells that lack this receptor. Most of the bacterial binding to macrophages is inhibited by a monoclonal antibody to Mac-1 but is unaffected by a monoclonal antibody to complement receptor type 1. Furthermore, opsonized, but not unopsonized, bacteria bind to purified Mac-1 immobilized on plastic. In addition, in the presence of opsonic complement, rhodococci bind efficiently to fibroblastoid cells transfected with cloned Mac-1 but relatively poorly to cells transfected with the complement receptor type 1. Hence, R. equi fixes complement by activating the alternative complement pathway, and this fixation is a requirement for bacterial adhesion and invasion. Furthermore, complement fixation defines rhodococcal host cell tropism, since R. equi binds specifically and exclusively to cells expressing Mac-1.

Animals↗

Rhodococcus equi causing human pulmonary infection: review of 29 cases.

Rhodococcus equi is a gram-positive pleomorphic bacillus that has been identified as a life-threatening pulmonary pathogen in the immunocompromised host. Infection with R equi may go unrecognized by physicians unacquainted with its presentation and unaware of the organism's ability to mimic diphtheroids and to stain weakly positive with an acid-fast stain.

Actinomycetales Infections↗

Successful treatment of Rhodococcus equi pulmonary infection in a renal transplant recipient.

The rhodococcus is a mycobacterium-like organism which is normally a pathogen in foals. It usually spreads by direct contact or by aerosol from horse faeces and causes pyogranulomatous pulmonary infections. Occasionally, it acts opportunistically to infect immuno-compromised human hosts, most commonly those with the acquired immune deficiency syndrome (AIDS). Here we report a pulmonary infection by Rhodococcus equi in a renal transplant recipient who was successfully treated. The literature on this infection in transplant recipients is also reviewed with respect to manifestations and treatment.

Actinomycetales Infections↗

The immunogenicity of Rhodococcus equi GroEL2-based vaccines in a murine model.

Rhodococcus equi is a significant intracellular bacterial pathogen in foals. However, at present there is no commercially available vaccine for the prevention of R. equi-induced disease in these animals. Studies have shown that GroEL based vaccines can afford protection against some intracellular pathogens. In this study, the R. equi gene encoding the heat shock protein GroEL2 was cloned and sequenced, with a view to using it as a vaccine candidate. The promoter region of the gene contained two copies of controlling inverted repeat of chaperone expression (CIRCE) motifs, which are well-recognised transcriptional regulators of bacterial heat shock proteins. The R. equi GroEL2 was expressed in E. coli BL21 DE3 with a C-terminal His-tag and sequenced to confirm its identity. The R. equi purified His-tagged GroEL2 protein and a groEL2-based DNA vaccine were used in separate experiments to immunise BALB/c mice. The recombinant protein-based vaccine elicited a mixed Th1/Th2 response whereas the DNA vaccine was found to elicit a predominantly Th1 biased immune response. However, when vaccinated mice were challenged intravenously with 1.5 x 10(7) R. equi neither vaccine elicited enhanced bacterial clearance from the spleen or liver in this model. The reasons for this apparent lack of success are discussed.

Actinomycetales Infections↗

Rhodococcus equi: an emerging pathogen.

More than 100 cases of Rhodococcus equi infection have been reported since the first description of human disease caused by this organism. The vast majority of patients infected with R. equi are immunocompromised, and two-thirds have human immunodeficiency virus infection. The clinical manifestations of R. equi infection are diverse, although 80% of patients have some pulmonary involvement. The organism is easily cultured from specimens of infected tissue or body fluid, but it may be misdiagnosed as a contaminant. Treatment is often prolonged, and relapses at distant sites are common. This article summarizes the history, diagnosis, clinical features, and treatment of infection with this emerging pathogen.

Actinomycetales Infections↗

Random insertion mutagenesis of the intracellular pathogen Rhodococcus equi using transposomes.

The identification of virulence factors in Rhodococcus equi has been severely hampered by the lack of a method for in vivo random insertion mutagenesis. This study reports the use of transposomes to generate random insertions of a gene conferring kanamycin resistance into the genome of R. equi ATCC 33701. Southern hybridisation using the kanamycin resistance gene as probe showed that insertion of transposome is random. This was confirmed following nucleotide sequence analysis of the junction between the transposome and chromosomal DNA. The presence of a 9 bp duplication of the target sequence showed that random integration of the transposome was due to a bona fide Tn5 transposition event.

Genome, Bacterial↗

Production of S-(+)-2-phenylpropionic acid from (R,S)-2-phenylpropionitrile by the combination of nitrile hydratase and stereoselective amidase in Rhodococcus equi TG328.

A new soil isolate, tentatively identified as Rhodococcus equi TG328, was found to be effective in the production of S-(+)-2-phenylpropionic acid from (R,S)-2-phenylpropionitrile. The conversion is catalysed by two enzymes. First, a nitrile hydratase converts the (R,S)-nitrile to (R,S)-2-phenylpropionamide. Second, a stereoselective amidase converts the S-(+)-amide to S-(+)-2-phenylpropionic acid. Conditions for optimal enzyme production and accumulation of S-(+)-2-phenylpropionic acid by resting cells were studied. The reaction of resting cells for 30 h at 10 degrees C with (R,S)-2-phenylpropionitrile resulted in the production of 100 g of S-(+)-2-phenylpropionic acid per litre of reaction mixture. The enantiometric excess of the purified S-(+)-2-phenylpropionic acid was 99.4%. The amount of S-(+)-2-phenylpropionic acid accumulated was enhanced by lower reaction temperatures. In addition, unreacted R-(-)-2-phenylpropionamide with 99.0% enantiometric excess was isolated.

Acetonitriles↗

The structure of the specific capsular polysaccharide of Rhodococcus equi serotype 4.

The specific capsular polysaccharide produced by Rhodococcus equi serotype 4 was found to be a high-molecular-weight acidic polymer composed of D-glucose, D-mannose, pyruvic acid and a previously unidentified 5-amino-3,5-dideoxynonulosonic (rhodaminic) acid in the proportions 2:1:1:1. Structural analysis, employing a combination of microanalytical methods, nuclear magnetic resonance spectroscopy, and mass spectrometric techniques, established that the polysaccharide consisted of linear repeating tetrasaccharide units having the sequence of residues shown below. In the native polysaccharide, the rhodaminic acid residues were present as their acetamido derivatives (RhoANAc) and carried 1-carboxyethylidene groups that bridged the O-7 and O-9 positions. Treatment of the capsular polysaccharide with dilute acetic acid and/or anhydrous hydrogen fluoride under hydrolytic/solvolytic conditions, resulted in the formation of four different oligosaccharide species. The 1H and 13C NMR resonances of these oligosaccharide fragments and of the native serotype 4 capsular polysaccharides were fully assigned by homo- and heteronuclear chemical shift correlation methods.

Animals↗

The LysR-type transcriptional regulator VirR is required for expression of the virulence gene vapA of Rhodococcus equi ATCC 33701.

The virulence of the intracellular pathogen Rhodococcus equi in foals is dependent on the presence of an 81-kb virulence plasmid encoding the virulence protein VapA. Expression of this protein is induced by exposure to oxidative stress, high temperatures, and low pHs, which reflect the conditions encountered by R. equi when it enters the host environment. The aim of this study was to determine whether the LysR-type transcriptional regulator VirR, which is encoded by the virulence plasmid, is required for the expression of vapA. It was shown that the virR gene is cotranscribed with four downstream genes, one of which encodes a two-component response regulator. The expression of VapA, as monitored by Western blotting, was completely dependent on the presence of virR. Maximal expression was observed when vapA was present together with the complete virR operon, suggesting that at least one of the virR operon genes, in addition to virR, is required for the expression of vapA to wild-type levels. The transcriptional start site of vapA was determined to be a cytidine located 226 bp upstream from the vapA initiation codon. His-tagged VirR protein was expressed in Escherichia coli and purified by nickel affinity chromatography. DNA binding studies showed that purified VirR binds to a DNA fragment containing the vapA promoter. We therefore conclude that VirR is required for the activation of vapA transcription.

Adaptation, Physiological↗

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.

Actinomycetales Infections↗

Comparison of tracheal aspiration with other tests for diagnosis of Rhodococcus equi pneumonia in foals.

The diagnostic value of tracheal aspiration was evaluated through comparison with other diagnostic methods using an experimental model of Rhodococcus equi (R. equi) pneumonia in foals. Pneumonia was induced by spraying of the virulent R. equi strain ATCC 33701 into the trachea of foals. All foals developed fever from 11 to 16 days after bacterial inoculation. One foal was euthanized on day 26 due to its poor prognosis, and other foals euthanized on day 43. During the experiment, some tests for diagnosis of Rhodococcus equi pneumonia such as tracheal aspiration, radiography, serodiagnosis and fecal culture were carried out. R. equi was continually isolated from tracheal aspirates collected via a silicone catheter inserted transnasally on day 8 to day 32 after bacterial inoculation. On the other hand, radiography, serodiagnosis and fecal culture were demonstrated to be valuable diagnostic methods, but to be limited compared with tracheal aspiration. Indirect fluorescent antibody technique (IFA) using a monoclonal antibody against the 15- to 17-kDa virulence-associated antigens (VapA) of R. equi and PCR targeting the structural gene of VapA detected bacteria in tracheal aspirates less sensitively than the isolation technique although they were more rapid. Therefore, we conclude that a combination of tracheal aspiration and bacterial isolation was the most valuable method for routine diagnosis of R. equi pneumonia in foals.

Actinomycetales Infections↗

Evaluation of equine breeding farm management and preventative health practices as risk factors for development of Rhodococcus equi pneumonia in foals.

OBJECTIVE: To determine whether foal management practices, environmental management, and preventative health practices are risk factors for development of Rhodococcus equi pneumonia in foals. DESIGN: Prospective matched case-control study. ANIMALS: 2,764 foals on 64 equine breeding farms with 9,991 horses. PROCEDURE: During 1997, participating veterinarians completed paired data collection forms for comparison; 1 for an affected farm (containing > or = 1 foal with pneumonia caused by R equi) and 1 for a control farm. Information collected pertained to stabling facilities, environmental management, foal husbandry, and preventative equine health practices. RESULTS: Matched farm data compared by use of conditional logistic regression indicated that personnel on affected farms were more likely to attend foal births, test foals for adequacy of passive immunity, administer plasma or other treatments to foals to supplement serum immunoglobulin concentrations, administer hyperimmune plasma prophylactically to foals, vaccinate mares and foals against Streptococcus equi infection, and use multiple anthelmintics in deworming programs. Affected farms were also more likely to have foals that developed other respiratory tract disorders and were approximately 4 times as likely to have dirt floors in stalls used for housing foals as were control farms. CONCLUSIONS AND CLINICAL RELEVANCE: Rhodococcus equi pneumonia does not appear to be associated with poor farm management or a lack of attention to preventative health practices. Housing foals in stalls with dirt floors may increase the risk for development of R equi pneumonia.

Actinomycetales Infections↗

Prevalence of virulent Rhodococcus equi in soil from five R. equi-endemic horse-breeding farms and restriction fragment length polymorphisms of virulence plasmids in isolates from soil and infected foals in Texas.

Rhodococcus equi isolates (462) obtained from 64 soil samples collected on 5 R. equi-endemic horse-breeding farms and isolates from 100 infected foals in Texas were examined to determine the prevalence and genotypic diversity of virulence-associated plasmids. Isolates were tested for the presence of 15-17-kDa virulence-associated protein antigens (VapA) by immunoblotting and virulence-associated plasmids by PCR. Plasmid DNAs were isolated and analyzed by digestion with restriction endonucleases for estimation of size and comparison of polymorphisims. Rhodococcus equi were isolated from soil of all 5 farms; however, virulent R. equi were only isolated from 3 of the 5 farms and represented 18.8% (87 of 462) of total isolates. Of the 87 virulent soil isolates, 56 (64.5%) contained an 85-kb type I plasmid, 23 (26.4%) an 87-kb type I plasmid, 7 (8%) a newly defined 85-kb type III plasmid (Tx 43), and 1 (1.1%) a newly defined 85-kb type IV plasmid (Tx 47). Of the 100 isolates from infected foals, 96 were virulent. Of the 96 virulent isolates, 51 (53.1%) contained an 85-kb type I plasmid, 39 (40.6%) an 87-kb type I plasmid, 4 (4.2%) an 85-kb type III plasmid (Tx 43), and 2 (2.1%) an 85-kb type IV plasmid (Tx 47). There are at least 4 different R. equi virulence-associated plasmids in Texas, 2 of which have not previously been described. Based upon virulence plasmid typing, there is geographic diversity among isolates of R. equi from clinical and environmental samples on horse-breeding farms in Texas. There is not a strong correlation between the presence of virulent R. equi in farm soils and the R. equi disease status of those farms.

Actinomycetales Infections↗