Ecology of Rhodococcus equi in horses and their environment on horse-breeding farms

Quantitative culture of R. equi in the feces of dams and foals, in the air of the stalls and in the soil of the paddocks was carried out on three horse-breeding farms during the foaling season. The isolation rates of R. equi from the feces of dams from the 3 farms suddenly increased to approximately 80% at the end of March, when the snow in the paddocks finished melting, and remained at that level during April and May. The mean number of R. equi and the isolation rate of R. equi from the feces of dams on the farms were investigated for 5 weeks before and 5 weeks after delivery. During the 10 weeks, there were no differences in the isolation rate or in the mean number of R. equi from the feces of dams. R. equi was first isolated from the feces of the foals born in February and the middle of March at 3-4 weeks of age, on the other hand, it was first isolated from the feces of foals born in the end of March and April at 1-2 weeks of age. The number of R. equi in the soil collected from the paddocks used by dams during the winter was approximately 10(2)-10(4) g-1 of soil during the experiment. R. equi was isolated from the air in the stalls at the end of March and the number of R. equi in the air increased particularly on dry and windy days.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Ecology of Rhodococcus (Corynebacterium) equi in soil on a horse-breeding farm

The ecology of Rhodococcus (Corynebacterium) equi in soil was studied on a horse-breeding farm. R. equi was cultured from soil at a depth of 0, 10, and 20 cm on the six sites of the farm at monthly intervals for 10 months from March to December of 1983. The highest numbers of R. equi were found in the surface soil. The mean number of bacteria in soil samples at every depth increased remarkably from 0 or 10(2) to 10(4) colony-forming units (CFU) g-1 of soil in the middle of April, and later decreased gradually. R. equi inoculated into six soil exudate broths prepared from surface soils at separate sites yielded suspensions with different optical densities, indicating differences in growth. The distribution of serotypes in the soil was similar to that in the horses on the farm. These findings indicated that R. equi could multiply in the soil and flourish in the cycle existing between horses and their soil environment.     

广东省部分马场土壤马红球菌(R.equi)的分布调查

采集广东省3个城市、4个马场的马厩、训练场、打圈场及过道中的土壤、沙粒样本共计355份,采用优化改良的菌落印记方法对广东省部分马场土壤马红球菌(Rhodococcus equi,R.equi)进行分布调查。结果显示,马场土壤中普遍存在R.equi,其平均菌量达10~(6 ) CFU/g;训练场和打圈场的R.equi菌量和分离率都相对较高,而马场和季度之间的R.equi菌量和分离率均无明显差异,表明R.equi在马场土壤中的分布与马匹活动有关,与季节、地理位置等其他因素无明显相关性;因此,马场应注意马匹活动区域的定期清洁和消毒。本研究通过系统的流行病学方法进行调查与分析,为R.equi防控及治疗提供理论基础和依据,并为我国R.equi种质资源库的建立提供基本资源,具有重要的公共卫生学意义。     

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.     

Isolation of virulent Rhodococcus equi from native Japanese horses

R. equi was isolated from soil samples obtained from the environment of seven native Japanese horse breeds (Hokkaido, Kiso, Noma, Misaki, Tokara, Miyako and Yonaguni) and from fecal samples collected from three native horse breeds (Hokkaido, Kiso and Misaki). Virulent R. equi at various levels (ranging from 0.5 to 12.9%) was isolated from the feces or soil environment of Hokkaido, Kiso and Misaki horses. Isolates were investigated both for the presence of 15- to 17-kDa antigens (virulence-associated protein antigens; VapA) by colony blotting, using the monoclonal antibody 10G5, and the gene of VapA by PCR. Plasmid DNAs extracted from positive isolates were digested with restriction endonucleases, and the digestion patterns of the plasmids of virulent isolates were divided into three types. Two of the three types (87-kb type II and 90-kb type I) had already been reported in Japanese isolates, and a new type (tentatively designated as 90-kb type II) had been found in isolates from Kiso horses. Six virulent R. equi isolates from the Hokkaido horses contained an 87-kb type II plasmid. Eight of 24 isolates from the Kiso horses contained an 87-kb type II plasmid, and the remaining 16 contained a 90-kb type II (a new type) plasmid. Two isolates from the Misaki horses contained a 90-kb type I plasmid. These results demonstrate the geographic difference in the distribution of virulence plasmids in R. equi isolates among native Japanese horses.     

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.     

Serotypes of Rhodococcus equi isolated from horses, immunocompromised human patients and soil in Hungary

Two hundred and twelve Rhodococcus equi strains were isolated from soil, nasal and rectal swabs of horses and immunocompromised human patients in Hungary and serotyped using Prescott's serotyping system. One hundred and forty-seven strains (69.3%) belonged to serotype 1, 22 strains (10.4%) to serotype 2, 6 strains (2.8%) to serotype 3 and 1 strain (0.5%) to serotype 4. Serotypes 5, 6 and 7 were not found and 36 strains (17%) could not be typed. Serotype 1 (72%) was the type most commonly isolated from clinical samples of foals or from the soil of horse facilities. Six out of 8 R. equi strains from humans belonged to serotype 2, and two human strains were untypable. The data show that the prevalence of R. equi serotypes varies in different geographic areas of the country.     

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.     

Rhodococcus equi in fecal and environmental samples from Kansas horse farms

Horse farms in Kansas were surveyed for the incidence of Rhodococcus equi. Fecal specimens and soil or cobweb samples were collected from each farm and cultured on selective media. One control farm (with no history of R. equi infection), one farm which had an outbreak 3 and 4 years previously and 2 farms which had R. equi-infected foals that season were surveyed. In addition, fecal samples from 21 horses hospitalized in the Kansas State University Veterinary Hospital were cultured. There was no significant difference in the incidence of R. equi in fecal samples from the 2 farms with recent disease problems. The farm with a history of disease had a significantly higher percentage of positive fecal cultures than the 2 farms with a more recent history of disease. Neither ration composition nor sex of the horses appeared to affect the fecal culture results. Fecal samples from 2 birds on 1 farm were positive for R. equi.     

Comparison of concentrations of Rhodococcus equi and virulent R. equi in air of stables and paddocks on horse breeding farms in a temperate climate

REASONS FOR PERFORMING STUDY: Rhodococcoccus equi is a significant cause of bronchopneumonia in foals worldwide. Infection of the lungs is believed to result from inhalation of virulent R. equi in dust from contaminated environments. A measure of infectious risk in an environment is the level of airborne contamination. OBJECTIVES: To assess and compare the level of airborne virulent R. equi in paddocks and stables. METHODS: Air samples were collected sequentially over the 2003 foaling season from the paddocks and stables on 3 Irish horse breeding farms affected by R. equi pneumonia. Colony blotting and DNA hybridisation techniques allowed quantitation of virulent R. equi. RESULTS: The odds of detecting airborne virulent R. equi in stables were 173 times greater than in paddocks. The median airborne concentration of virulent R. equi was significantly higher (P < 0.001) in stables than in paddocks on all farms. These observations suggested that stables were high-risk areas for infection. CONCLUSIONS AND POTENTIAL RELEVANCE: Our results indicate that contaminated stables are a significant risk factor in the epidemiology of R. equi pneumonia on horse-breeding farms in a temperate climate, such as in Ireland. Management strategies that improve the air hygiene of stables, through better ventilation, use of less fragile bedding material and the use of fogging agents to reduce the airborne concentration of virulent R. equi, may reduce the incidence and severity of R. equi pneumonia on farms.     

Rhodococcus equi in the soil environment of horses in Inner Mongolia, China

Little is known about the distribution of Rhodococcus equi in the soil environment of native horses in China. One hundred and eight soil samples were collected from native-horse farms in the Hulun Beier grasslands of eastern Mongolia, the Xilin Goler grasslands of southern Mongolia, and Tongliao City in Inner Mongolia, China. The isolation rates of R. equi from soil samples from the Hulun Beier and Xilin Goler grasslands ranged from 25.9% to 30.0%. In contrast, isolation rates from soil samples from Tongliao City were as high as 82.3% and the mean number of R. equi in soil samples from Tongliao City was 10 times more than those of samples from the grasslands. The 488 isolates were examined using PCR for the presence of genes that encode virulence-associated 15-17 kDa antigen protein (VapA) and the 20 kDa antigen protein (VapB). All isolates were negative for virulence-associated proteins. Plasmid profiles of these avirulent isolates showed that cryptic plasmids of various sizes were present with an incidence of 13.3% to 21.5%. The results of the present study contrast with those of our recent study (J. Vet. Med. Sci. 67:611-613, 2005), in which we reported that R. equi was absent from Mongolian horses in Ulaanbaatar, Mongolia. It is suggested that the difference between the results of these two studies is due to the mobile pasturing system in Mongolia and nonmobile pasturing system in Inner Mongolia.     

Epidemiologic study of results of pulsed-field gel electrophoresis of isolates of Rhodococcus equi obtained from horses and horse farms

OBJECTIVE: To compare isolates of Rhodococcus equi on the basis of geographic source and virulence status by use of pulsed-field gel electrophoresis (PFGE). SAMPLE POPULATION: 290 isolates of R equi (218 virulent isolates from foals and 72 avirulent isolates from feces, soil, and respiratory tract samples) obtained between 1985 and 2000 from horses and horse farms from 4 countries. PROCEDURE: DNA from isolates was digested with the restriction enzyme Asel and tested by use of PFGE. Products were analyzed for similarities in banding patterns by use of dendrograms. A similarity matrix was constructed for isolates, and the matrix was tested for nonrandom distributions of similarity values with respect to groupings of interest. RESULTS: There was little grouping of isolates on the basis of country, virulence status, or region within Texas. Isolates of R equi were generally < 80% similar, as determined by use of PFGE. Isolates from the same farm generally were rarely of the same strain. CONCLUSIONS AND CLINICAL RELEVANCE: Considerable chromosomal variability exists among isolates of R equiobtained from the same farm, sites withinTexas, or among countries from various continents. Only rarely will it be possible to link infections to a given site or region on the basis of analysis of isolates by use of PFGE of chromosomal DNA.     

Distribution of Rhodococcus equi in animals,birds and from the environment

Extract

Madam:— As well as causing sporadic infections in animals, Rhodococcus (Corynebacterium) equi has been isolated from the gastro-intestinal tract of healthy grazing animals and from the environment. ^{(1 )(5)(6)(8)(11)} R. equi has been isolated in this laboratory from cattle and pigs in association with tuberculosis- like lesions and from lungs and abscesses from horses and deer.     

Virulence genes and plasmid profiles in Rhodococcus equi isolates from domestic pigs and wild boars (Sus scrofa) in Brazil

The virulence genes and plasmid profiles of 23 Rhodococcus equi isolates from 258 lymph nodes from domestic pigs (129 nodes with lesions and 129 without lesions) and 120 lymph nodes from slaughtered wild boars (60 nodes with lesions and 60 without) were characterized. R. equi was obtained from 19 lymph nodes of domestic pigs, 17 with, and two without lesions, and from four lymph nodes with lesions, from wild boars. The 23 isolates were tested for the presence of vapA and vapB genes, responsible for the 15–17 and 20 kDa virulence-associated proteins, respectively, by PCR in order to characterize as virulent (VapA), intermediately virulent (VapB) and avirulent. Plasmid DNAs were isolated and analyzed by digestion with restriction endonucleases to estimate size and compare their polymorphisms. Of the 19 domestic pigs strains, seven (36.8%) were avirulent and 12 (63.2%) were intermediately virulent, with the intermediately virulent isolates being plasmid types 8 (8 isolates), 10 (2 isolates), 1 (1 isolate) and 29 (1 isolate). The plasmid type of four strains isolated from wild boars was also intermediately virulent type 8. None of the domestic pigs and wild boar isolates showed the vapA gene. These findings demonstrate a high occurrence of plasmid type 8 in isolates from pigs and wild boars, and the similarity of plasmid types in the domestic pigs, wild boars and human isolates in Brazil.     

Molecular typing of VapA-positive Rhodococcus equi isolates from Jeju native horses, Korea

We recently demonstrated the presence of virulence-associated protein antigen (VapA)-positive Rhodococcus equi in Jeju Island, Korea. These bacteria contained one of two distinct plasmid types, a 90-kb type II plasmid, which has been found in isolates from the native Kiso horses of Japan, and a new variant, a 90-kb type V plasmid. However, the genotypic characters of the VapA-positive R. equi from Jeju native horses and their environments are poorly understood. Ninety-eight isolates from soil samples and 89 isolates from fecal samples were collected from five farms that breed or have bred Jeju native horses, and were tested for the presence of VapA by immunoblotting and PCR. Of the 98 soil isolates and 89 fecal isolates, seven and 13 were VapA-positive R. equi, respectively. In 2003, two Jeju foals died suddenly and were brought to the Faculty of Veterinary Medicine, Cheju National University, for postmortem examination. Pure cultures of R. equi were isolated from the lung lesions of both foals. Of the 16 clinical isolates, 14 were VapA-positive R. equi. Of the 34 VapA-positive clinical and environmental isolates, 16 contained the 90-kb type II plasmid and 18 contained a 90-kb type V plasmid. Pulsed-field gel electrophoresis (PFGE) patterns of the VapA-positive isolates from Jeju horses and Kiso horses, containing the 90-kb type II plasmid, were compared and formed two distinct groups. Furthermore, 18 virulent isolates containing the 90-kb type V plasmid formed two distinct PFGE groups (of 16 and two isolates). These results demonstrate that two virulence plasmid types are widespread in R. equi in Jeju native horses. However, there is little diversity in the PFGE patterns of virulent isolates, suggesting the clonal spread of virulent R. equi. The PFGE pattern of the virulent R. equi isolates from Jeju native horses in Korea is not identical to those of Kiso native horses in Japan.     

A mycolyl transferase mutant of Rhodococcus equi lacking capsule integrity is fully virulent

Rhodococcus equi is a mucoid Gram-positive facultative intracellular pathogen which can cause severe bronchopneumonia in foals and AIDS patients. A polysaccharide capsule which gives R. equi a mucoid appearance has long been suspected to be a virulence factor. Here, we describe a transposome mutant in the gene fbpA of strain R. equi 103 causing absence of a capsular structure. FbpA is a chromosomal gene homologous to antigen 85 (Ag85) mycolyl chain transferase gene of Mycobacterium tuberculosis. The mutant multiplied normally in isolated macrophages, was able to establish the unusual R. equi-containing vacuole in macrophages, was cytotoxic for macrophages, and was virulent in a mouse model. Colonies had a dry appearance on nutrient agar and defective capsule structure. Surprisingly, fbpA mutants cured of the virulence-associated plasmid were found in a phagosome that was more alkaline than that of the corresponding wild-type bacteria, were more cytotoxic and even multiplied to some extent. This study suggests that the capsule is not an important virulence factor of R. equi and that it may even counteract virulence traits.     

Effect of prophylactic administration of hyperimmune plasma to prevent Rhodococcus equi infection on foals from endemically affected farms

The effect on foals of prophylactic administration of hyperimmune plasma to prevent R. equi infection was investigated on three farms at which R. equi infection was endemic. Sixteen foals between 10 and 39 days of age were intravenously given 1-21 of hyperimmune plasma. ELISA antibody titres against R. equi were significantly increased and maintained at high levels for over 30 days in most of the recipient foals. The prevalence of R. equi infection was 6.3% (1/16) in the foals that received the immune plasma, and 26.3% (5/19) in the control foals not given the immune plasma on the three farms. For 2 years before and after this field trial on the three farms, 18 of 64 foals (28.1%) showed clinical signs of respiratory tract infection and four of them died of R. equi pneumonia. Heavy contamination of horses and their environment with virulent R. equi was detected by colony blotting, and plasmid profiles also suggested that foals on the three farms were constantly exposed to virulent R. equi. The results of this field trial support previous observations by some researchers that the administration of hyperimmune plasma to foals in the early days of life promotes prevention of R. equi infection on endemic farms; however, the mechanism of hyperimmune plasma protection remains unclear.