Molecular epidemiology of virulent Rhodococcus equi from foals in Brazil: virulence plasmids of 85-kb type I, 87-kb type I, and a new variant, 87-kb type III

We investigated the prevalence of virulent Rhodococcus equi in clinical isolates from 41 foals (19 sporadic and seven endemic cases) in Brazil between 1991 and 2003. Of the 41 virulent isolates, six contained an 85-kb type I plasmid, 33 contained an 87-kb type I plasmid, both of which have been found in isolates from the Americas, and the remaining two contained a new variant, which did not display the EcoRI, EcoT22I and BamHI digestion patterns of the 11 representative plasmids already reported (85-kb types I-IV; 87-kb types I and II; 90-kb types I-V). We tentatively designated the new variant as the '87-kb type III' plasmid, because its BamHI digestion pattern is similar to that of the 87-kb type I plasmid. This is the first report of the molecular epidemiology surveillance of virulent R. equi in clinical isolates from Brazilian foals.     

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.     

Two new variants of the Rhodococcus equi virulence plasmid, 90 kb type III and type IV, recovered from a foal in Japan

Calves were vaccinated orally, subcutaneously or intraperitoneally with a smooth, plasmid-cured strain of Salmonella enterica serovar typhimurium, strain 81. Oral vaccination was not effective, as only 1/5 calves survived challenge with virulent S. typhimurium. Strain 81 was attenuated for calves, as only a slight rise in rectal temperatures was detected after vaccination. The organism was excreted by some calves in the faeces, but no signs of diarrhoea were observed after vaccination. After parenteral vaccination, strain 81 was able to reach the intestines, gastric associated lymphoid tissues and other internal lymphoid tissues and remained viable for up to 14 days in the bovine host. After oral challenge with a virulent strain, 9/10 vaccinated calves survived challenge as opposed to 4/10 control calves (p<0.5). Diarrhoea was present in all calves of the control groups, but in only 4/10 of the vaccinated calves. The clinical reactions of the vaccinated calves were milder than in the control calves, as the rises in rectal temperatures were lower, diarrhoea was less severe, and the challenge strain was present in fewer organs from vaccinated calves than control calves. This study showed that parenterally administered Salmonella vaccines can induce both mucosal and systemic immunity, and it is postulated that this capability of strain 81 is related to its colonisation of lymphoid tissues and other systemic and intestinal tissues. This study confirmed that plasmid-cured strains were attenuated in the bovine host and conferred significant protection after parenteral vaccination, but not oral vaccination.     

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)     

Rhodococcus equi: clinical manifestations, virulence, and immunity

Pneumonia is a major cause of disease and death in foals. Rhodococcus equi, a gram-positive facultative intracellular pathogen, is a common cause of pneumonia in foals. This article reviews the clinical manifestations of infection caused by R. equi in foals and summarizes current knowledge regarding mechanisms of virulence of, and immunity to, R. equi. A complementary consensus statement providing recommendations for the diagnosis, treatment, control, and prevention of infections caused by R. equi in foals can be found in the same issue of the Journal.     

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.     

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.     

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.     

Rhodococcus (Corynebacterium) equi: bactericidal capacity of neutrophils from neonatal and adult horses

The capacity of hematogenous polymorphonuclear neutrophilic leukocytes (PMNL) to kill Rhodococcus equi was compared in horses of various ages. A radioisotope bactericidal assay was used to determine the capacity of PMNL to kill R equi. Assays were conducted on PMNL from horses in 3 groups: group I, 13 foals with a mean age of 3.3 days; group II, 10 group-I foals at a mean age of 35.7 days; and group III, adult dams of group-I foals. Bacteria were obtained from the lungs of a foal with R equi pneumonia and opsonized with fresh adult equine serum that contained R equi specific antibody. The mean peak percentage of R equi killed by PMNL was 78.9 for group I, 90.1 for group II, and 87.9 for group III. There was no significant difference (P greater than 0.05) among groups; however, 15% of foals in group I (2 foals) had a mean peak percentage of 30.5 killed, which was significantly (P less than 0.05) lower than the percentage for other foals in group I. The results of our investigation indicated that the capacity of PMNL to kill opsonized R equi is similar in neonatal, young, and adult horses. However, some neonatal foals have a substantially lower capacity to kill R equi, which may be an important factor in the pathogenesis of R equi infections.     

Seroepidemiological survey of Rhodococcus equi infection in asymptomatic horses and donkeys from southeast Turkey

In order to assess the level of Rhodococcus equi infection in southeast Turkey, 679 sera from healthy foals and adult horses and 78 sera from donkeys were tested by indirect ELISA using a R. equi reference strain (ATCC 33701) as antigen. Eighty (11.7%) sera from horses and 9 (11.5%) sera from donkeys with titres >0.85 were positive. The prevalence of seropositive horses in Sanliurfa Province was higher than in Diyarbakir Province; 56 (13.9%) horses in Sanliurfa Province and 24 (8.7%) horses in Diyarbakir Province were defined as seropositive. In Sanliurfa Province 14.5% of female (n=343) and 10.1% of male (n = 59) horses tested were defined as seropositive, while in Diyarbakir Province more males (11.4%, n=114) were seropositive than females (6.7%, n=163). Horses 1 to 5 years of age were found to have the highest seropositivity rate in both provinces. A total of 78 sera from donkeys were investigated in Sanliurfa Province, of which 9 (11.5%) were positive by ELISA. Among the 9 positive sera, 6 (12.8%) were from donkeys 1-5 years old and 3 (13.6%) were from donkeys >5 years of age. No positive sera were found in donkeys less than 1 year old. Five (12.5%) sera of females and 4 (10.5%) sera of males tested were positive. These results indicate the existence of R. equi in the horse populations in Sanliurfa and Diyarbakir Provinces. Similar infection rates were found for donkeys in Sanliurfa. This suggests the importance of serological surveys to diagnose R. equi infection in the region and to prevent the zoonotic risk.     

Seroprevalence of Neospora,Toxoplasma gondii and Sarcocystis neurona antibodies in horses from Jeju island,South Korea

Parasite-specific antibody responses to Neospora spp. and Toxoplasma gondii, antigens were detected using the indirect fluorescent antibody test (IFAT) and immunoblot analysis in a korean equine population located on Jeju island, South Korea (126 degrees 12' E and 33 degrees 34' N). For comparison, a naturally infected Neospora hughesi horse and an experimentally inoculated T. gondii equid (pony) were used. In addition, all samples were tested for antibodies to Sarcocystis neurona by immunoblot analysis. A total of 191 serum samples from clinically normal horses were evaluated. Only 2% (4 out of 191) and 2.6% (5 out of 191) of the samples had showed reactivity at 1:100 using the IFAT for Neospora spp. and T. gondii, respectively. For T. gondii, two samples matched the antigen banding pattern of the positive control by immunoblot analysis. No sample was positive for N. hughesi by immunoblot analysis in this study. Overall, there was a 1% seroprevalence for T. gondii antibodies in the horses tested based on immunoblot analysis. The seroprevalence for S. neurona and N. hughesi antibodies was 0%. We concluded that these horses are either not routinely exposed to these parasites or antibody titers are not sufficiently elevated to be detectable. It is most likely the former explanation since Jeju island equine farms are isolated from the main land, and the horses were all less than 3 years of age. This na?ve population of horses could be useful when evaluating S. neurona serodiagnostic tests or evaluating potential S. neurona vaccines since exposure risks to S. neurona and closely related parasites are negligible.     

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.     

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.     

Genotypic characterization of VapA positive Rhodococcus equi in foals with pulmonary affection and their soil environment on a warmblood horse breeding farm in Germany

Pulsotypes of VapA positive Rhodococcus equi isolated from foals and soil on a farm in Germany were characterized on the basis of nasal and tracheal samples simultaneously collected in 2003 from 217 foals with sonographic evidence of pneumonia or pulmonary abscesses. Of the 217 double samples, R. equi was isolated in 118 (54%) of the tracheal samples and in 52 of the nasal swab samples (24%) (P<0.001). Furthermore, 37 and 55 isolates were also randomly selected from nasal swabs and the tracheal samples, respectively, and further processed to determine the presence of VapA by colony blot enzyme-linked immunosorbent assay. This method showed that 26 (68%) of the nasal swabs and 40 (73%) of the tracheal samples were VapA-positive. R. equi was isolated from 56 (87%) of the 64 soil samples taken from the paddocks and stables in March and from 17 (68%) of the 25 samples taken in July of 2003. Three (21%) of these randomly selected 14 isolates from March and 13 (81%) of the 16 from July were VapA-positive. The VapA positive isolates from foals and soil were genotyped by plasmid profiling, restriction fragment length polymorphism analysis and pulsed-field gel electrophoresis. Of the 83 isolates, 80 contained an 85-kb type I plasmid and three contained an 87-kb type I plasmid. Pulsed-field gel electrophoresis yielded four distinct VspI profiles dividing 83 isolates into three major (A1, 51; D, 14; and 11 isolates) and three minor (C, 4; A3, 2; and A2, 1 isolates) groups. These results suggest that the majority of foals were exposed to and infected with three pulsotypes of VapA positive R. equi containing an 85-kb type I plasmid.     

Genotypic characterization of VapA positive Rhodococcus equi in foals with pulmonary affection and their soil environment on a warmblood horse breeding farm in Germany

Pulsotypes of VapA positive Rhodococcus equi isolated from foals and soil on a farm in Germany were characterized on the basis of nasal and tracheal samples simultaneously collected in 2003 from 217 foals with sonographic evidence of pneumonia or pulmonary abscesses. Of the 217 double samples, R. equi was isolated in 118 (54%) of the tracheal samples and in 52 of the nasal swab samples (24%) (P < 0.001). Furthermore, 37 and 55 isolates were also randomly selected from nasal swabs and the tracheal samples, respectively, and further processed to determine the presence of VapA by colony blot enzyme-linked immunosorbent assay. This method showed that 26 (68%) of the nasal swabs and 40 (73%) of the tracheal samples were VapA-positive.R. equi was isolated from 56 (87%) of the 64 soil samples taken from the paddocks and stables in March and from 17 (68%) of the 25 samples taken in July of 2003. Three (21%) of these randomly selected 14 isolates from March and 13 (81%) of the 16 from July were VapA-positive.The VapA positive isolates from foals and soil were genotyped by plasmid profiling, restriction fragment length polymorphism analysis and pulsed-field gel electrophoresis. Of the 83 isolates, 80 contained an 85-kb type I plasmid and three contained an 87-kb type I plasmid. Pulsed-field gel electrophoresis yielded four distinct VspI profiles dividing 83 isolates into three major (A1, 51; D, 14; and 11 isolates) and three minor (C, 4; A3, 2; and A2, 1 isolates) groups. These results suggest that the majority of foals were exposed to and infected with three pulsotypes of VapA positive R. equi containing an 85-kb type I plasmid.     

Rhodococcus equi occipital bone osteomyelitis,septic arthritis and meningitis in a neurological foal

This case report describes a 3‐month‐old female Thoroughbred foal that presented following the acute onset of apparent respiratory distress, abnormal head carriage and severe neurological deficits referable to the brainstem or cranial cervical spinal cord. Ultrasonography revealed an abscessing pneumonia. Radiographs did not show evidence of bony pathology at the atlanto‐occipital region, an area consistent with the observed neurological deficits. With magnetic resonance imaging, atlantal and occipital osteomyelitis, atlanto‐occipital septic arthritis, and atlanto‐axial synovitis were diagnosed. Brainstem meningitis and extradural compression were also revealed. Culture of the right occipitoatlantal joint and cerebrospinal fluid yielded a pure culture of Rhodococcus equi. These findings and multifocal R. equi abscessation were confirmed at necropsy.     

Pasteurella multocida recovered from live turkeys: prevalence and virulence in turkeys

Swabs of the oropharynges of 801 live turkeys (621 meat birds and 180 breeders), collected from 15 flocks that had experienced an outbreak of fowl cholera and from 12 non-outbreak flocks, were screened for the presence of Pasteurella multocida. Turkeys from outbreak flocks were sampled within 2 to 9 weeks of the outbreak. Forty-nine isolates of P. multocida were recovered from turkeys in 11 of the outbreak flocks, and none were recovered from turkeys in non-outbreak flocks. Isolation rates varied from 0 to 72% of turkeys sampled in a flock. Nineteen isolates were tested for virulence by injecting them intravenously into turkeys, and 14 were lethal. Results demonstrated that for purposes of disease control, meat birds in fowl-cholera-outbreak flocks should be considered carriers of potentially virulent P. multocida for the life of the flock.     

Rhodococcus equi pneumonia in the foal--part 2: diagnostics, treatment and disease management

Various challenges face clinicians and farm managers in diagnosing, treating and preventing Rhodococcus equi pneumonia. The use of ultrasound imaging has aided in the early diagnosis of the disease, reducing treatment duration and improving therapeutic outcomes. Antimicrobial resistance in R. equi is an emerging issue that necessitates prudent antimicrobial therapy of diseased foals. Alternative methods of disease transmission, such as contagious foal-to-foal aerosol transmission, may need to be addressed to complement dust reduction environmental strategies and to minimise the overall risk of exposure of foals to highly concentrated inhaled doses of the organism. Effective management of foals and land aimed at reducing aerosol exposure to virulent R. equi is likely to yield significant reductions in the prevalence and severity of R. equi pneumonia.