Associations between the Ecology of Virulent Rhodococcus equi and the Epidemiology of R. equi Pneumonia on Australian Thoroughbred Farms

The ecology of virulent strains of Rhodococcus equi on horse farms is likely to influence the prevalence and severity of R. equi pneumonia in foals. This study examined the association between the ecology of virulent R. equi and the epidemiology of R. equi pneumonia by collecting air and soil samples over two breeding seasons (28 farm-year combinations) on Thoroughbred breeding farms with different reported prevalences of R. equi pneumonia. Colony blotting and DNA hybridization were used to detect and measure concentrations of virulent R. equi. The prevalence of R. equi pneumonia was associated with the airborne burden of virulent R. equi (both the concentration and the proportion of R. equi bacteria that were virulent) but was not associated with the burden of virulent R. equi in the soil. Univariable screening and multivariable model building were used to evaluate the effect of environmental and management factors on virulent R. equi burdens. Lower soil moisture concentrations and lower pasture heights were significantly associated with elevated airborne concentrations of virulent R. equi, as were the holding pens and lanes, which typically were sandy, dry, and devoid of pasture cover. Few variables appeared to influence concentrations of virulent R. equi in soil. Acidic soil conditions may have contributed to an elevated proportion of virulent strains within the R. equi population. Environmental management strategies that aim to reduce the level of exposure of susceptible foals to airborne virulent R. equi are most likely to reduce the impact of R. equi pneumonia on endemically affected farms.     

Diversity of isolates of Rhodococcus equi from Australian Thoroughbred horse farms

Pulsed field gel electrophoresis of restriction endonuclease digested genomic DNA from a collection of clinical isolates of Rhodococcus equi was used to compare strain diversity on different Thoroughbred horse farms over time. Restricted diversity was found among the isolates tested, as the same strains were detected on multiple farms and in multiple years. Marked variation occurred in strain prevalence with some strains being represented by single isolates, and the most prevalent by 26 isolates. There were dominant strains on some farms and the prevalence of some strains differed between farms. Infection with multiple strains was noted in some cases where multiple isolates from a single foal were examined.     

Epidemiology of Rhodococcus equi strains on Thoroughbred horse farms

Pulsed-field gel electrophoresis of restriction endonuclease-digested genomic DNA from a large collection of clinical isolates of Rhodococcus equi, an important pathogen of foals, was used to compare strain distribution between farms and over time. Forty-four strains were found among 209 isolates, with 5 of these accounting for over half the isolates and the 22 strains isolated more than once accounting for 90% of the isolates. The average genotypic diversity on each farm and in each year was found to be less than the genotypic diversity of the isolates taken as a whole, with 5.2% of the total diversity being due to differences between farms and 5.5% to differences between years. A small number of strains on each farm were found to have caused at least half the clinical cases of disease, and these varied between farms and, to a lesser extent, years. Most strains were found on more than one farm, and some very similar restriction patterns were found among isolates from different continents, indicating that strains can be very widespread. Multiple strains were isolated in five of the six cases in which more than one isolate from a single foal was examined, indicating that disease may commonly be caused by simultaneous infection with multiple strains. It was concluded that there are a number of different strains of R. equi which carry the vapA gene, and these strains tend to be widespread, but individual farms tend to have particular strains associated with them.     

Live virulent Rhodococcus equi, rather than killed or avirulent, elicits protective immunity to R. equi infection in mice

Mice inoculated intravenously with a sublethal dose of live virulent Rhodococcus equi ATCC 33701 that contained an 85-kb virulence plasmid were immune to a lethal intravenous challenge of ATCC 33701. This immunity depended upon the dose of immunization and developed rapidly: mice primed with 10(5) live ATCC 33701 eliminated the challenged bacteria more rapidly than mice primed with doses ranging from 10(2) to 10(4) bacteria, and mice given 10(5) live ATCC 33701 intravenously withstood the lethal challenge as early as 5 days after the initial inoculation. However, this protective immunity did not develop in mice immunized with doses of heat-killed ATCC 33701 ranging from 10(6) to 10(8), or in mice immunized with doses of live ATCC 33701P-, a plasmid-cured derivative (avirulent), in doses ranging from 10(5) to 10(7). These mice had positive antibody titers against R. equi at the challenge (14 days after priming). Adoptive transfer of resistance to virulent R equi was obtained with spleen cells from mice immunized with live ATCC 33701, but not monoclonal antibody to 15- to 17-kDa virulence-associated antigens. These results revealed that live ATCC 33701P-, a plasmid-cured derivative of virulent R equi, could not elicit protective immunity, and are consistent with previous observations that protective immunity was induced by live virulent, but not killed organisms.     

Associations between the presence of virulence determinants and the epidemiology and ecology of zoonotic Escherichia coli

The severity of human infection with pathogenic Escherichia coli depends on two major virulence determinants (eae and stx) that, respectively, produce intimin and Shiga toxin. In cattle, both may enhance colonization, but whether this increases fitness by enhancing cattle-to-cattle transmission in the field is unknown. In E. coli O157, the almost uniform presence of the virulence determinants in cattle isolates prevents comparative analysis. The availability to this study of extensive non-O157 E. coli data, with much greater diversity in carriage of virulence determinants, provides the opportunity to gain insight into their potential impact on transmission. Dynamic models were used to simulate expected prevalence distributions for serogroups O26 and O103. Transmission parameters were estimated by fitting model outputs to prevalence data from Scottish cattle using a Bayesian Markov chain Monte Carlo (MCMC) approach. Despite similar prevalence distributions for O26 and O103, their transmission dynamics were distinct. Serogroup O26 strains appear well adapted to the cattle host. The dynamics are characterized by a basic reproduction ratio (R(0)) of >1 (allowing sustained cattle-to-cattle transmission), a relatively low transmission rate from environmental reservoirs, and substantial association with eae on transmission. The presence of stx(2) was associated with reduced transmission. In contrast, serogroup O103 appears better adapted to the noncattle environment, characterized by an R(0) value of <1 for plausible test sensitivities, a significantly higher transmission rate from noncattle sources than serogroup O26, and an absence of fitness benefits associated with the carriage of eae. Thus, the association of eae with enhanced transmission depends on the E. coli serogroup. Our results suggest that the capacity of E. coli strains to derive fitness benefits from virulence determinants influences the prevalence in the cattle population and the ecology and epidemiology of the host organism.     

Interaction of virulent and non-virulent Rhodococcus equi human isolates with phagocytes, fibroblast- and epithelial-derived cells

Abstract Rhodococcus equi is a facultative, intracellular, Gram-positive coccobacillus, increasingly reported in pneumonia of AIDS-infected patients. We investigated killing resistance properties of human R. equi virulent and avirulent human strains. Avirulent β-lactam-susceptible strains had lower intracellular colony forming units after 45 min incubation in murine macrophages J774 and human monocyte-macrophage TPH-1 than those of virulent strains. Only virulent β-lactam-resistant strains persisted within macrophages for at least 18 min only. A β-lactam-resistant mutant was obtained from a β-lactam-susceptible strain after selection in a penicillin G-containing culture medium. This mutant strain, like the natural virulent strains, persisted within macrophages, harboured cell-associated appendages, produced phage-like particles and induced, after its intravenous inoculation, a chronic infection in BALB/c nude mice. Supernatant culture of virulent strains transferred partial macrophage-killing resistance properties to avirulent strains. The same supernatant was toxic for L-929, HeLa and Vero cell cultures. These supernatant effects were heat-inactivated, trypsin-inactivated and did not seem to be linked to phage-like particle presence. These data argue that virulence, β-lactam-resistance, and macrophage-killing resistance are associated in human R. equi isolates. Moreover, only virulent strains produced uncharacterized toxic factors.     

Oral Administration of Electron-Beam Inactivated Rhodococcus equi Failed to Protect Foals against Intrabronchial Infection with Live,Virulent R. equi

There is currently no licensed vaccine that protects foals against Rhodococcus equi–induced pneumonia. Oral administration of live, virulent R. equi to neonatal foals has been demonstrated to protect against subsequent intrabronchial challenge with virulent R. equi. Electron beam (eBeam)-inactivated R. equi are structurally intact and have been demonstrated to be immunogenic when administered orally to neonatal foals. Thus, we investigated whether eBeam inactivated R. equi could protect foals against developing pneumonia after experimental infection with live, virulent R. equi. Foals (n = 8) were vaccinated by gavaging with eBeam-inactivated R. equi at ages 2, 7, and 14 days, or gavaged with equal volume of saline solution (n = 4), and subsequently infected intrabronchially with live, virulent R. equi at age 21 days. The proportion of vaccinated foals that developed pneumonia following challenge was similar among the vaccinated (7/8; 88%) and unvaccinated foals (3/4; 75%). This vaccination regimen did not appear to be strongly immunogenic in foals. Alternative dosing regimens or routes of administration need further investigation and may prove to be immunogenic and protective.     

Influence of inoculation route on virulence of Rhodococcus equi in mice.

Virulence of R. equi ATCC 33701 was compared by the intraperitoneal (i.p.) and intravenous (i.v.) routes in mice. Strain ATCC 33701 was more virulent by the i.v. than the i.p. route. The LD50 of strain ATCC 33701 by either route correlated with the initial number of bacteria in the liver and spleen at day 0.     

Molecular and infection biology of the horse pathogen Rhodococcus equi

The soil actinomycete Rhodococcus equi is a pulmonary pathogen of young horses and AIDS patients. As a facultative intracellular bacterium, R. equi survives and multiplies in macrophages and establishes its specific niche inside the host cell. Recent research into chromosomal virulence factors and into the role of virulence plasmids in infection and host tropism has presented novel aspects of R. equi infection biology and pathogenicity. This review will focus on new findings in R. equi biology, the trafficking of R. equi -containing vacuoles inside host cells, factors involved in virulence and host resistance and on host–pathogen interaction on organismal and cellular levels.     

A novel lipoarabinomannan from the equine pathogen Rhodococcus equi. Structure and effect on macrophage cytokine production

Rhodococcus equi is a major cause of foal morbidity and mortality. We have investigated the presence of lipoglycan in this organism as closely related bacteria, notably Mycobacterium tuberculosis, produce lipoarabinomannans (LAM) that may play multiple roles as virulence determinants. The lipoglycan was structurally characterized by gas chromatography-mass spectrometry following permethylation, capillary electrophoresis after chemical degradation, and (1)H and (31)P and two-dimensional heteronuclear nuclear magnetic resonance studies. Key structural features of the lipoglycan are a linear alpha-1,6-mannan with side chains containing one 2-linked alpha-d-Manp residue. This polysaccharidic backbone is linked to a phosphatidylinositol mannosyl anchor. In contrast to mycobacterial LAM, there are no extensive arabinan domains but single terminal alpha-d-Araf residue capping the 2-linked alpha-d-Manp. The lipoglycan binds concanavalin A and mannose-binding protein consistent with the presence of t-alpha-d-Manp residues. We studied the ability of the lipoglycans to induce cytokines from equine macrophages, in comparison to whole cells of R. equi. These data revealed patterns of cytokine mRNA induction that suggest that the lipoglycan is involved in much of the early macrophage cytokine response to R. equi infection. These studies identify a novel LAM variant that may contribute to the pathogenesis of disease caused by R. equi.     

Purification and characterization of the enantioselective nitrile hydratase from Rhodococcus equi A4

The nitrile hydratase from Rhodococcus equi A4 consisted of two kinds of subunits which slightly differed in molecular weight (both approximately 25 kDa) and showed a significant similarity in the N-terminal amino acid sequences to those of the nitrile hydratase from Rhodococcus sp. N-774. The enzyme preferentially hydrated the S-isomers of racemic 2-(2-, 4-methoxyphenyl)propionitrile, 2-(4-chlorophenyl)propionitrile and 2-(6-methoxynaphthyl)propionitrile (naproxennitrile) with E-values of 5-15. The enzyme functioned in the presence of 5-98% (v/v) of different hydrocarbons, alcohols or diisopropyl ether. The addition of 5% (v/v) of n-hexane, n-heptane, isooctane, n-hexadecane, pristane and methanol increased the E-value for the enzymatic hydration of 2-(6-methoxynaphthyl)propionitrile.     

Enantioselectivitiy of the nitrile hydratase from Rhodococcus equi A4 towards substituted (R,S)-2-arylpropionitriles

Rhodococcus equi A4 cells containing a nitrile hydratase and an amidase converted (R,S)-2-(4-methoxyphenyl)-propionitrile into the corresponding (S)-acid (e.e. 87%) and (R)-nitrile (e.e. > 95%) in 49% yield. The same reaction using (R,S)-2-(4-chlorophenyl)-propionitrile gave the (S)-acid (e.e. > 95%) and (R)-nitrile (e.e. 52%) in 20 and 34% yield, respectively.     

Reinvestigation of the virulence of Rhodococcus equi isolates from patients with and without AIDS

Rhodococcus equi emerged as a zoonotic pathogen of human immunodeficiency virus-infected patients over the last three decades. Two virulence plasmid types of R. equi, pVAPA and pVAPB associated with equine and porcine isolates, have been recognized, and more recently, pVAPN, a novel host-associated virulence plasmid in R. equi, was found in bovine and caprine isolates. We reinvestigated 39 previously reported isolates of R. equi from patients with and without acquired immunodeficiency syndrome (AIDS) by detecting vapA, vapB and vapN using PCR and plasmid profiling. After excluding one isolate that could not be cultured from frozen storage, eight isolates carried a virulence plasmid encoding vapA (pVAPA), 10 carried a virulence plasmid encoding vapB (pVAPB), seven carried a virulence plasmid encoding vapN (pVAPN) and 13 were negative for those genes. Of the 29 isolates from patients with AIDS, 7, 10 and 5 harboured pVAPA, pVAPB and pVAPN respectively. Among nine isolates from patients without AIDS, one and two harboured pVAPA and pVAPN respectively. This study demonstrated that pVAPN-positive R. equi existed in human isolates before 1994 and reaffirmed that equine-associated pVAPA-positive, porcine-associated pVAPB-positive and bovine- or caprine-associated pVAPN-positive R. equi are widely spread globally. Because domestic animals might be major sources of human infection, further research is needed to reveal the prevalence of pVAPN-positive R. equi infection in cattle and goats.     

Isolation of rare opportunistic pathogens in Hungary: case report and short review of the literature. Rhodococcus equi

Rhodococcus equi is a well-established pathogen in foal pneumonia and is increasingly recognized as a pathogen in immunocompromised humans. We have isolated a Gram-positive coccobacillus from 8 blood samples and lung tissues of a renal transplant patient. Colony morphology, growth in Lowenstein-Jensen medium, 21 biochemical reactions, the characteristic morphological cycle (coccus-rod-coccus) and the CAMP test established the R. equi diagnosis. Histological studies of 2 lung biopsy specimens revealed numerous microabscesses with aggregates of polymorphonuclear leukocytes surrounded by abundant foamy macrophages. Our isolates proved to be sensitive to majority of antibacterial drugs. The appropriate therapy (amoxicillin-clavulanate) proved to be effective, however six months later a relapse was observed. Data show that in spite of its rare occurrence, R. equi infection represents a diagnostic and therapeutic challenge. The taxonomical, epidemiological, clinico-pathological, diagnostic and therapeutic data of R. equi are discussed.     

Physical Factors and Reuse Affect the Production of Cholesterol Oxidase in Reactors Containing Calcium Alginate-Immobilized Rhodococcus equi No. 23

Summary Some physical factors including initial pH of medium, cultivation temperature and shaking speed as well as reuse affecting the production of cholesterol oxidase (CholOx) in reactors containing calcium alginate-immobilized cells of Rhodococcus equi No. 23 were investigated. Results revealed that the free cells showed the maximum CholOx in the culture with an initial pH of 5.0, while culture inoculated with immobilized cells exhibited a broad pH range, 6.0–9.0, for maximum CholOx production. The immobilized and free cells produced the maximum CholOx in the culture incubated at 30 and 25°C, respectively. The CholOx production decreased upon increasing the cultivation temperature. Increasing CholOx activity was also noted for both immobilized and free cells of R. equi No. 23 in the culture with increasing shaking speed. Under the optimal culture conditions, that were established, a higher maximum CholOx production of 0.94 unit/ml was found for immobilized R. equi No. 23 compared to that of 0.84 unit/ml for free cells after 48 h of cultivation. Furthermore, no gel leakage was noted after re-use of the calcium alginate-immobilized R. equi No. 23 for seven consecutive 48 h batch culture. The CholOx production in the seventh cycle was about 60.4% of that obtained in the first cycle.     

Facile synthesis of arabinomannose penta- and decasaccharide fragments of the lipoarabinomannan of the equine pathogen, Rhodococcus equi

Pentasaccharide repeating unit 20 of the lipoarabinomannan from the equine pathogen, Rhodococcus equi, and its dimer 31, were synthesized. The pentasaccharide was obtained by assembling a benzoylated 2,6-branched mannosyl trisaccharide acceptor 13 with a free hydroxyl group at C-2' of the mannose residue attached to the core mannose residue by (1 --> 6)-linkage, followed by coupling with 2,3,5-tri-O-benzoyl-alpha-D-arabinofuranosyl-(1 --> 2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate (18), and by deacylation. Meanwhile, the decamer 31 was obtained by firstly preparing a benzoylated mannose (1 --> 6)-linked tetrasaccharide backbone 26 with 2-, 2"-O-ClAc, and 2'-, 2'-O-Ac groups, respectively, then by dechloroacetylation and subsequent condensation with perbenzoylated trichloroacetimidate, and then by deacetylation and subsequent coupling with 18, and finally, by deacylation.     

On the epidemiology and etiology of pneumonia in adults

In a group of 74 hospitalized patients with the diagnosis of acute infectious pneumonia, the etiological contribution of viral and bacterial agents is analyzed in cases of clarified etiology and an assessment is made of the relationship between the explained etiology and the overall epidemiological situation. Etiology was clarified in 36 patients (48.6%). Viral and bacterial etiology was confirmed in 13.3% and 39.8% of the entire group respectively. In three cases, mixed viral and bacterial infection was reported. Most prominent among the viral agents were herpes simplex, parainfluenza, respiratory syncytial and influenza type B viruses. As far as the bacterial agents were concerned, the species most frequently isolated were Staphylococcus aureus, Streptococcus pneumoniae and a variety of Enterobacteriaceae. The relationship between the overall epidemiological situation and pneumonia etiology is discussed as well as the relevance of the diagnostic methods employed.