Identification of Corynebacterium pseudotuberculosis from sheep by PCR-restriction analysis using the RNA polymerase β-subunit gene (rpoB)

Caseous lymphadenitis, caused by Corynebacterium pseudotuberculosis, has a high prevalence in many regions of the world, including Argentina and Brazil. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for the identification of this microorganism was designed based on the hypervariable region of the polymorphic RNA polymerase β-subunit gene (rpoB). All available CorynebacteriumrpoB sequences were analyzed by computer-assisted restriction analysis. The rpoB PCR-RFLP pattern predicted by using endonucleases MseI and StuI clearly differentiated C. pseudotuberculosis from sixty-one other Corynebacterium species. This method was successfully applied to identify twelve wild C. pseudotuberculosis ovine isolates and one caprine isolate. It was also used to differentiate C. pseudotuberculosis from Arcanobacterium pyogenes, an ovine pathogen with similar clinical characteristics. These results indicate that this new molecular method can be used for the reliable identification of the pathogen, essential for the timely detection of infected animals and for epidemiological studies.     

Double-antibody sandwich enzyme-linked immunosorbent assay and immunoblot analysis used for control of caseous lymphadenitis in goats and sheep.

A double-antibody sandwich ELISA for detection of antibodies directed against the exotoxin of Corynebacterium pseudotuberculosis, the cause of caseous lymphadenitis (CL) in small ruminants, was developed. A concentrated exotoxin was used. For interpretation of ELISA results, these sera were tested: sequentially obtained sera of C pseudotuberculosis-inoculated goats and sheep that were monitored for 68 weeks; sequentially obtained sera from 80 goats of 3 flocks with CL; sera from 652 goats of 7 flocks without CL; sera from 160 sheep of 4 flocks without CL; and 2,265 caprine and 208 ovine sera submitted for diagnostic testing. Data regarding the infection status and history of 10,454 of the 23,302 animals were collected after testing; most of these were goats that had been part of a CL control program. Specificity and sensitivity of the ELISA were nearly 100%. Subsequently, 31,978 animals from which no data on infection status of flocks had been collected were then tested. It was concluded that the ELISA is a useful diagnostic test for CL eradication programs. Sera with doubtful or inconclusive ELISA results were examined by use of immunoblot analysis. Proteins from C pseudotuberculosis culture supernatant were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose. Six proteins with molecular mass of 68, 65, 39, 38, 31, and 29 kDa reacted with sera from goats and sheep with experimentally induced or naturally acquired infection. Immunoblot analysis was valuable in classifying sera with doubtful or inconclusive results by ELISA.     

Comparative molecular characterization of Corynebacterium pseudotuberculosis of different origin

Ribotyping and susceptibility to 17 antimicrobial agents were used to compare 37 isolates of Corynebacterium pseudotuberculosis (28 from horses, 1 from cattle, 3 from sheep and 5 from goats) derived from various types of lesions, and different geographic locations. According to the presence of nitrate reductase, all but one isolate from horses reduced nitrate (nitrate-positive), whereas all isolates from sheep and goats were unable to reduce nitrate (nitrate-negative). The ribotype of the nitrate-negative isolate from a horse with ulcerative lymphangitis was identical to all the other isolates from horses, and different than the ribotype of nitrate-negative isolates from sheep and goats. Ribotyping with one of the restriction endonucleases, Apa I, revealed differences between, but not within, the two biotypes. However, ribotyping with Pst I endonuclease revealed one variant within the equine biotype and one variant within the ovine biotype. The minimum inhibitory concentration (MIC; μg/ml) of antimicrobial agents against isolates from nitrate-negative and nitrate-positive groups was very similar, with the exception of isolates from sheep and goats which had a higher MIC for amikacin than isolates from horses and cattle.     

Serodiagnosis of inapparent caseous lymphadenitis in goats and sheep, using the synergistic hemolysis-inhibition test

The synergistic hemolysis-inhibition (SHI) test, a serologic test for the detection of infection with Corynebacterium pseudotuberculosis, was applied to serum samples from 196 goats and 76 sheep, including animals both with and without C pseudotuberculosis abscesses. Fifty-one of 52 (98%) goats and 27 of 28 (96%) sheep with abscesses caused by C pseudotuberculosis had seropositive titers. Seropositivity continued on subsequent samplings, even after superficial lesions were completely healed. The SHI test may detect subclinically infected animals, as well as animals with clinically recognizable lesions. Of the animals without abscesses, 53 of 186 (28%) goats and 4 of 41 (10%) sheep were seropositive. Either the SHI test is lacking in specificity or these titers are a reflection of a past or a current infection without any grossly visible abscesses.     

Molecular characterization of Corynebacterium pseudotuberculosis isolates using ERIC-PCR

Caseous lymphadenitis is an infectious sheep and goats disease caused by Corynebacterium pseudotuberculosis and characterized by abscesses in superficial and visceral lymph nodes. C. pseudotuberculosis strains isolated from these hosts have been shown to be very difficult to type by the existing methods. The aim of this study is evaluating the potential of the Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) as a tool for molecular typing of C. pseudotuberculosis strains isolated in sheep. One hundred and twenty seven isolates of C. pseudotuberculosis were isolated from lesions suspected to have had caseous lymphadenitis collected from sheep at the slaughterhouse. Animals were from 24 flocks in 13 municipalities of the Minas Gerais State, Brazil. Species identification of the isolates was performed by routine biochemical tests and mPCR. Fingerprint was performed by RAPD using ERIC-1R, ERIC-2 and ERIC-1R+ERIC-2 primers. Seventeen different genotypes were generated by ERIC 1-PCR, 21 genotypes by ERIC 2-PCR and 21 genotypes by ERIC 1+2-PCR. Hunter-Gaston Discrimination Index (HGDI) found for ERIC 1, ERIC 2, ERIC 1+2 PCR were 0.69, 0.87, and 0.84, respectively. For most herds evaluated observed at most three different genotypes among isolates from animals of these property, in all ERIC-PCR assays. However a few flocks observed between four and nine genotypes per flock. The W Kendall value found for correlation among the three techniques of ERIC-PCR was 0.91 (P<5.0 x 10(-6)). The results show that ERIC-PCR has good discriminatory power and advantages over other DNA-based typing methods, making it a useful tool to discriminate C. pseudotuberculosis isolates.     

Synergistic hemolysis-inhibition titers associated with caseous lymphadenitis in a slaughterhouse survey of goats and sheep in Northeastern Brazil

A survey of caseous lymphadenitis was conducted at a goat and sheep slaughterhouse in Northeastern Brazil One hundred and fifty-eight goats and 43 sheep were examined for the presence of abscesses, with bacterial culturing of purulent material to define the etiological agent. Blood was collected simultaneously for determination of serological titer via the synergistic hemolysis-inhibition test which measures antibodies to an exotoxin of Corynebacterium pseudotuberculosis. Thirteen and nine-tenths percent of the goats had abscesses, with a high proportion having mediastinal or pulmonary lesions (9.5%). Two sheep had abscesses, both with internal organ involvement. Corynebacterium pseudotuberculosis was the most frequently isolated organism. Of 22 goats with abscesses, 20 were positive via the synergistic hemolysis-inhibition test. Both of the sheep with abscesses had positive synergistic hemolysis-inhibition titers. The proportion of serological reactors was greater than the proportion of animals with abscesses. The synergistic hemolysis-inhibition test may be detecting subclinically infected animals.     

Serologic response and lesions in goats experimentally infected with Corynebacterium pseudotuberculosis of caprine and equine origins

Fifteen goat kids were experimentally inoculated with Corynebacterium pseudotuberculosis. Five were given a strain of caprine origin (nitrate-negative biotype) intradermally, 5 were given a strain of equine origin (nitrate-positive biotype) intradermally, and 5 were inoculated intranasally with the caprine-origin strain. Animals were monitored for 127 days. The goats given the inocula intradermally developed abscesses; those given caprine-origin strain had multiple lesions both peripherally and in visceral locations (primarily endothoracic abscesses), whereas those given the equine-origin strain had abscesses only at injection sites and draining nodes. The difference in extent of lesions could be due to biotypic bacterial differences or to the individual strains used. Intranasally inoculated goats did not develop abscesses and were essentially no different from controls. The cranial part of the respiratory tract may not be an important portal of entry for C pseudotuberculosis. Serum samples obtained monthly from all animals were subjected to the synergistic hemolysis-inhibition test, which measures antibodies to the exotoxin of C pseudotuberculosis. Animals with abscesses developed titers within 1 month of inoculation. Animals without abscesses remained seronegative. The synergistic hemolysis-inhibition test may be a reliable diagnostic assay for caseous lymphadenitis in goats.     

Characterization of strains of corynebacterium pseudotuberculosis.

Twenty-five strains of Corynebacterium pseudotuberculosis isolated from lesions of caseous lymphadenitis in goats were examined for their biochemical characteristics, antimicrobial susceptibility, and phospholipase D activity. The strains were uniform in biochemical reactions, cultural characteristics, and susceptibility to antimicrobial agents. Presence of urease and phospholipase D and absence of pyrazinamidase were valuable criteria in the identification of C. pseudotuberculosis.     

Enteritis in sheep, goats and pigs due to Yersinia pseudotuberculosis infection

The features of naturally occurring Yersinia pseudotuberculosis serotype III infections in 16 sheep, one goat and 3 pigs, and Y. pseudotuberculosis serotype I infections in 3 goats, are described. Affected animals usually had diarrhoea and were in poor condition or emaciated. A number were moribund or dead when submitted for necropsy. Thickening of the caecal and colonic mucosa was the only gross lesion attributable to Y. pseudotuberculosis infection, with liver or other visceral abscesses not being seen. Characteristic microabscesses were demonstrated in the intestinal mucosa of 10 sheep, one goat and one pig infected with Y. pseudotuberculosis serotype III and one goat infected with Y. pseudotuberculosis serotype I. Sheep, goats and pigs dosed orally with Y. pseudotuberculosis serotype III, the serotype isolated most commonly from these species, developed intestinal infection. In sheep and pigs, infection was accompanied by diarrhoea. Haematological changes and specific antibodies were elicited in all 3 species in response to infection. Microabscesses were seen in the intestinal mucosa of all experimentally exposed animals. The occurrence of field cases and the results of experimental exposure confirm that Y. pseudotuberculosis serotype III is an enteropathogen of sheep, goats and pigs. The association of Y. pseudotuberculosis serotype I with lesions in a goat, indicates that this bacterium may also be a pathogen of this species. It is concluded that Y. pseudotuberculosis serotype III is an enteric pathogen of a wide range of ungulate species including cattle, buffalo, deer, antelopes, sheep, goats and pigs. Serotypes I and II, while having a more restricted host range, are probably also pathogens of ungulates and, in particular, deer, antelopes and goats.     

Corynebacterium ulcerans from diseased wild boars

Two Corynebacterium strains were isolated from lymph nodes of wild boars showing severe alterations caused by caseous lymphadenitis. The wild boars came from different districts in southern Germany; one was found dead, the other had been shot. The two Corynebacterium strains obtained were both positive for phospholipase D. Further analysis of biochemical profiles did not allow unambiguous differentiation between Corynebacterium ulcerans and Corynebacterium pseudotuberculosis. Fourier-transformed infrared spectroscopy as well as partial sequencing of the genes for 16S rRNA and RNA polymerase beta subunit (rpoB) clearly identified both strains as Corynebacterium ulcerans. The tox gene for diphtheria toxin (DT) could be detected in both porcine isolates by PCR. Partial DNA sequencing of this tox gene showed significant differences from sequences described for other Corynebacterium ulcerans strains and a higher degree of similarity to that of Corynebacterium diphtheria. Production of diphtheria toxin could not be detected. These results indicate that wild game could be a reservoir for zoonotic Corynebacterium ulcerans.     

A field trial to evaluate a whole cell vaccine for the prevention of caseous lymphadenitis in sheep and goat flocks.

A field trial to evaluate a whole cell vaccine for the prevention of caseous lymphadenitis (CLA) in sheep and goats was performed in one goat herd and one sheep flock over a period of three years. In goats, there was a nonstatistically significant trend for fewer cases of CLA in the vaccinated animals compared to the controls. In sheep, from six months to 36 months postinitial vaccination, the proportion of vaccinated sheep that developed CLA was significantly less (p less than 0.05) than in the control sheep. The antibody titers to Corynebacterium pseudotuberculosis as detected by microagglutination assay were significantly different (p less than 0.0001) at all times except at the initial vaccination. Swellings occurred at the vaccination site at an incidence level of 29.6% in goats and 34.1% in sheep. The vaccine appeared to be efficacious in reducing the proportion of sheep that developed CLA when challenged naturally in a field situation.     

Molecular epidemiologic features of Corynebacterium pseudotuberculosis isolated from horses

OBJECTIVE: To characterize isolates of Corynebacterium pseudotuberculosis from horses, cattle, and sheep in Colorado, Kentucky, Utah, and California in samples collected during perceived epidemics of infection (increased numbers of cases identified) in 2002 and 2003, and determine how closely isolates were related and their possible source. SAMPLE POPULATION: 54 isolates of C pseudotuberculosis from 49 horses, 4 cattle, and 1 sheep. PROCEDURES: Random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) assay, PCR assay for the gene encoding the phospholipase D (PLD) toxin, biochemical analyses, and tests for susceptibility to 17 antimicrobial drugs were performed. RESULTS: All isolates reduced nitrate to nitrite, most yielded positive results for the PLD toxin gene, and all were susceptible to antimicrobial drugs. Ten genetic types were detected by use of RAPD PCR assay; types III to X were isolated from horses, cattle, or both in 1 or more states. Types III and IX were isolated from both horses and cattle. Types VII and VIII were isolated in only 1 state, but the number of isolates in these groups was small. In contrast, all other types were isolated in 2 or more states. All isolates from Utah were type III, but the other 3 states had isolates from more than 1 type. CONCLUSIONS AND CLINICAL RELEVANCE: These data are consistent with a clonally expanding epidemic of infection in Utah and an increase in number of infections caused by multiple strains of C pseudotuberculosis not derived from a single source in the other states. The increase in number of infections could be the result of reporting bias, environmental factors facilitating infection, or host factors such as greater herd susceptibility.     

Microorganisms associated with abscesses of sheep and goats in the south of Iran

A total of 86 abscesses (45 and 41) abscesses of sheep and goats, respectively) were examined for their causal agents; 44 of these abscesses were located subcutaneously, and the remaining 42 were in lungs, livers, intestines, and udders. A total of 23 different types of microorganisms were isolated from 78 abscesses; bacteria were not detected in the remaining eight abscesses. Microorganisms isolated were: species of the genera Corynebacterium, Staphylococcus, and Streptococcus and Pasteurella, Escherichia coli, and other gram-negative rods. Peptostreptococcus anaerobius and Eubacterium tortuosum were isolated in pure culture from one abscess each in goats. Two to four different microorganisms were associated with 25 of the 86 abscesses. It was determined that 11 isolates of Corynebacterium pyogenes, 2 isolates of Pasteurella haemolytica, 1 of P multocida, and 1 of C pseudotuberculosis were lethal to mice. Two of the C pyogenes isolates from subcutaneous spreading abscesses of goats were pathogenic for rabbits; these two isolates produced similar suppurative inflammation in goats experimentally, but did not cause death. As determined by experimental inoculation, goats were more susceptible than sheep to the two isolates. In nature, subcutaneous abscesses of goats associated with C pyogenes were of a more diffusive type which resulted in generalization of the infection and death of the animals. Postmortem examination of ten goats dying of field infection showed the presence of larvae of the warble-fly Przhevalskiana silenus at the site of infection.     

Responses of testosterone hormone concentration,semen quality,and its related pro-inflammatory cytokines in bucks following Corynebacterium pseudotuberculosis and its mycolic acid infection

Tropical Animal Health and Production - Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis, a debilitating chronic disease of sheep and goats. Little is known about...     

陕南白山羊伪结核棒状杆菌分离鉴定及灭活铝胶疫苗研制

羊干酪性淋巴结炎又称羊伪结核病,是由伪结核棒状杆菌感染引起的一种人畜共患慢性接触性传染病,对山羊危害最为严重,目前尚无有效治疗措施。陕西省石泉县陕南白山羊饲养量较大,羊群也存在伪结核棒状杆菌的感染,造成较大的经济损失。为筛选对当地伪结核棒状杆菌敏感的药物和研制灭活疫苗,本研究从疑似伪结核病患羊采取多份新鲜的脓液,进行细菌分离鉴定;对获得的1株分离菌进行药物敏感性试验并用其研制灭活疫苗。结果显示,分离菌在固体培养基上形成白色、干燥、扁平、不透明、边缘整齐的中等大小菌落,革兰染色阳性,经16SrRNA检测和序列分析,证实分离到的菌株为羊伪结核棒状杆菌。药物敏感性试验结果显示,该分离菌对环丙沙星、头孢曲松、红霉素、卡那霉素、头孢噻肟、妥布霉素、强力霉素、庆大霉素、氯霉素高度敏感;对四环素、利福平、链霉素中度敏感。以该分离菌为种子菌制备的灭活铝胶疫苗无菌检验合格,试验接种山羊无不良反应,目前已经在采样羊场进行临床应用,进一步评价免疫效果。     

A comparison of four serological tests for the diagnosis of caseous lymphadenitis in sheep and goats

A double antibody sandwich ELISA (ELISA A) developed for the detection of Corynebacterium pseudotuberculosis infection in sheep and goats was modified to improve its sensitivity. To establish the sensitivity and specificity of this modified ELISA (ELISA B), sera from 183 sheep and 186 goats were tested using ELISAs A and B. Comparison was also made with two further ELISAs (C and D) developed in Australia that, respectively, detect antibodies to cell wall antigens or toxin.ELISA B had the best performance of the four tests. Its specificity was 98+/-1% for goats and 99+/-1% sheep. Its sensitivity was 94+/-3% for goats and 79+/-5% for sheep. ELISA B will now be tested for use in caseous lymphadenitis eradication and control programmes in The Netherlands. It will also be used in experimental studies of CL in Scotland.     

The use of a microagglutination assay for the detection of antibodies to Corynebacterium pseudotuberculosis in naturally infected sheep and goat flocks

Two goat flocks comprising 326 animals and four sheep flocks comprising 343 animals, all with a previously recognized problem of abscesses due to Corynebacterium pseudotuberculosis, were examined for the presence of abscesses and antibody titers to C. pseudotuberculosis as detected by direct microagglutination assay. In sheep there was a strong positive relationship between age and titer (p less than 0.0001). However, the relationship in goats between age and titer could not be determined due to a strong interaction between flock and age. When the relationship between abscesses and titer was examined, it was found that goats with abscesses had higher titers than those that did not (p less than 0.05), whereas there was no difference in titer between sheep with abscesses and those without (p = 0.5753). The sensitivity of the microagglutination test was poor to good for both species (52.3% for goats and 89.7% for sheep). The specificity of the test was fair to poor (64.9% for goats and 21.7% for sheep). Given a disease prevalence of 13.5% for goats and 8.5% for sheep the predictive value of the positive test was very poor (18.9% for goats and 9.6% for sheep) but the predictive value of the negative test was good to excellent (89.7% for goats and 95.8% for sheep). The poor specificity of the test and therefore the positive predictive value may be due in part to the criterion of classification of presence of disease, i.e. presence of an abscess at the time of sampling.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of Corynebacterium pseudotuberculosis isolates from sheep and goats by PCR

The present study was carried out to estimate the prevalence of caseous lymphadenitis (CL) in sheep and goats slaughtered at the local abattoir in Elazig province located in the east of Turkey, between September and December 2000. A total of 2046 sheep and 2262 goat carcasses were examined during the study period and 118 abscessed lymph nodes, 89 from sheep and 29 from goats, were collected. Corynebacterium spp. strains were isolated from 81.4% of the abscesses, giving an overall prevalence of 2.2%. The prevalence was 3.5 and 1.1% in sheep and goats, respectively. PCR on DNA extracted from 96 suspicious isolates, using a pair of Corynebacterium pseudotuberculosis-specific primers, was positive for 93. Although cross-reaction with C. ulcerans, a human/bovine species, was observed, the PCR assay used in this study may successfully be applied for the diagnosis of CL in goats and sheep as an alternative to conventional methods, owing to its advantages of specificity and speed.     

A serologic method for the detection of Corynebacterium pseudotuberculosis infections in horses

A serologic technique useful for detecting antibodies formed in horses in response to infection with Corynebacterium pseudotuberculosis is described. The test relies on the ability of C. pseudotuberculosis toxin to produce a wide zone of hemolysis when applied to erythrocytes previously treated with a sterile filtrate of Corynebacterium equi broth culture. The synergistic hemolytic activity can be neutralized by anti-C. pseudotuberculosis serum. This test was used to analyze sera from 616 horses for the presence of C. pseudotuberculosis antitoxin. Of 177 animals (see Table 2) found positive, there were 34 horses with bacteriologically confirmed, active infections and 18 with active but unconfirmed infections. In addition, 13 animals had a history of having had the disease and 112 had no history or evidence of having had the infection. The other 439 horses had negative titers. Statistical treatments confirmed the value of the test as an epidemiological tool but precluded using only titers for the diagnosis of active clinical disease.     

Evaluation of clinical characteristics, diagnostic test results, and outcome in horses with internal infection caused by Corynebacterium pseudotuberculosis: 30 cases (1995-2003)

OBJECTIVE: To determine clinical signs, results of diagnostic testing, and outcome in horses with internal Corynebacterium pseudotuberculosis infection. DESIGN: Retrospective study. ANIMALS: 30 horses. PROCEDURE: Information pertaining to clinical data, results of diagnostic tests, and costs of hospitalization and treatment was extracted from medical records of affected horses. RESULTS: Internal C. pseudotuberculosis infection was diagnosed on the basis of clinical signs, diagnostic imaging, and clinicopathologic data, including results of serologic tests and bacterial culture. The most common clinical signs were concurrent external abscesses, anorexia, fever, lethargy, weight loss, and signs of respiratory tract disease or abdominal pain. Clinicopathologic abnormalities included a geometric mean reciprocal serum synergistic hemolysin inhibition titer > or = 512, leukocytosis with neutrophilia, hyperglobulinemia, hyperfibrinogenemia, and anemia. Specific organ involvement was diagnosed in 27 of 30 horses. Affected organs included the liver (18 horses), lungs (12), kidneys (7), and spleen (3); multiple organs were affected in 10 horses. Treatment with antimicrobials for a median of 36 days (range, 7 to 97 days) was usually successful, yielding an overall survival rate of 71%. CONCLUSIONS AND CLINICAL RELEVANCE: Early diagnosis and long-term antimicrobial treatment were important for a successful outcome in horses with internal C. pseudotuberculosis infection. Ultrasonographic imaging was an important technique for identifying specific organs affected, aiding in obtaining samples for a definitive diagnosis, and monitoring response to treatment. Pregnant mares with internal infections are at risk for fetal loss. Preexisting chronic organ disease may be associated with a poor prognosis.