Variable number tandem repeat analysis of Mycobacterium bovis isolates from Gyeonggi-do, Korea

Bovine tuberculosis (TB) is a major zoonosis that''s caused by Mycobacterium bovis (M. bovis). Being able to detect M. bovis is important to control bovine TB. We applied a molecular technique, the variable number tandem repeat (VNTR) typing method, to identify and distinguish the M. bovis isolates from Gyeonggi-do, Korea. From 2003 to 2004, 59 M. bovis clinical strains were isolated from dairy cattle in Gyeonggi-do, Korea, and these cattle had tuberculosis-like lesions. Twenty-four published MIRU-VNTR markers were applied to the M. bovis isolates and ten of them showed allelic diversity. The most discriminatory locus for the M. bovis isolates in Korea was QUB 3336 (h = 0.64). QUB 26 and MIRU 31 also showed high discriminative power (h = 0.35). The allelic diversity by the combination of all VNTR loci was 0.86. Six loci (MIRU 31, ETR-A and QUB-18, -26, -3232, -3336) displayed valuable allelic diversity. Twelve genotypes were identified from the 59 M. bovis isolates that originated from 20 cattle farms that were dispersed throughout the region of Gyenggi-do. Two genotypes [designation index (d.i.) = e, g] showed the highest prevalence (20% of the total farms). For the multiple outbreaks on three farms, two successive outbreaks were caused by the same genotype at two farms. Interestingly, the third outbreak at one farm was caused by both a new genotype and a previous genotype. In conclusion, this study suggests that MIRU-VNTR typing is useful to identify and distinguish the M. bovis isolates from Gyeonggi-do, Korea.     

Disseminated Mycobacterium avium complex infection in a cat: presumptive diagnosis by blood smear examination

Disseminated mycobacteriosis was diagnosed in a 4-year-old, castrated male Domestic Shorthair cat following the observation of one to three retractile, non-staining bacilli in neutrophils and monocytes on a Wright-Leishman-stained blood smear Organisms were bright red following acid-fast staining by Kinyoun's technique. The cat had a history of progressive weight loss, anemia, fever, and sporadic vomiting after eating. In addition to blood smears, mycobacteria also were observed in bone marrow aspirates. During necropsy, multiple small white nodules were observed in the spleen and liver. An enlarged sternal lymph node and ascites also were present. In histologic sections, mycobacteria were observed in granulomas within the lungs, liver, spleen, colon, mesenteric and sternal lymph nodes, omentum, and kidney. Mycobacterium avium complex was isolated from cultures of liver, spleen, lung, and kidney. Occult feline leukemia virus infection, detected by immunofluorescent testing of bone marrow aspirates, may have predisposed this cat to bacterial infection. The serum ELISA test for group-specific feline leukemia virus antigen was negative.     

副结核分枝杆菌MAP0862、MAP1345基因的串联表达及其在间接ELISA中的初步应用

为探索副结核特异性蛋白MAP0862与MAP1345在牛副结核病血清学诊断中的作用,将通过串联表达获得的融合蛋白MAP0862-1345纯化定量后包被酶标板,经过对反应条件的优化,初步建立了基于融合蛋白MAP0862-1345的间接ELISA诊断方法。使用建立的ELISA方法对牛副结核病阳性血清、牛结核病阳性血清、牛布病阳性血清、卡介苗免疫牛血清、健康牛血清检测的结果表明其具有较好的特异性;使用该方法与IDEXX副结核病抗体检测试剂盒共同对300份临床血清样本检测,总符合率为92.7%。     

Disseminated Mycobacterium avium infection in a dog with chronic diarrhoea

A 3-year-old Maltese-cross dog presented with a 4-month history of chronic diarrhoea and inappetence. Poorly regenerative anaemia, leukocytosis and hypoproteinaemia were evident on several occasions. Biopsies of stomach, duodenum and colon revealed marked infiltration of mucosae by macrophages containing many acid-fast bacilli. Similar organisms were numerous in a faecal smear. Melaena, hematochezia and severe abdominal pain developed and were unresponsive to therapy. Following euthanasia and necropsy, histiocytic cells containing acid-fast bacilli were found throughout the gastrointestinal tract, mesenteric and peripheral lymph nodes, spleen, liver, kidney and lungs. The organism was identified as Mycobacterium avium by bacterial culture and polymerase chain reaction testing.     

Use of the polymerase chain reaction assay for the detection of Mycobacterium avium subspecies paratuberculosis in blood and liver biopsies from experimentally infected sheep

OBJECTIVE: To evaluate a polymerase chain reaction-based assay for the detection of Mycobacterium avium subsp paratuberculosis in blood and liver biopsies from subclinically infected sheep. STUDY DESIGN: A direct PCR assay for the detection of M a paratuberculosis was applied to liver biopsy specimens and to samples of blood that were sequentially collected over 53 weeks from 14 sheep infected experimentally with the organism. RESULTS: Of 117 blood samples from the 14 experimentally infected sheep, two tested positive in the PCR assay. Both positive results were obtained in two subclinically infected sheep that had paratuberculosis later confirmed by histological examination at necropsy. However, the assay failed to detect the target DNA in samples of blood from five other sheep with histologically confirmed paratuberculosis. Similarly, the PCR assay on liver biopsy specimens collected 32 weeks after administration of M a paratuberculosis gave only two positive results, both of which were obtained in sheep with histological evidence of paratuberculosis. CONCLUSIONS: The PCR assay on blood and liver biopsies does not provide a useful method for the diagnosis of M a paratuberculosis infection in subclinically infected sheep.     

Tuberculosis in deer: perceptions, problems and progress

Since the emergence of deer farming as an alternative farming enterprise over the past 30 years, there has been an increasing awareness of the potential threat posed by tuberculosis (TB) to domesticated deer. TB, caused by Mycobacterium bovis, has been found in deer in every country involved with deer farming. Different types of TB control policies, which vary from whole-herd depopulation to selective testing and slaughter of reactor animals, have been implemented. Extensive research has been carried out, incorporating modern microbiological and immunological concepts and advanced molecular methodologies, to find new solutions for the eradication of TB from domesticated deer. This work has resulted in valuable new insights into the aetiology, transmission, pathogenesis, diagnosis, prevention and heritability of resistance to M. bovis infection in ruminants. This knowledge has complemented the existing literature database on bovine and human TB and will provide new strategies for improved diagnosis, vaccination and selective breeding to control TB, which should be relevant for human, domestic livestock and wildlife populations.     

Molecular fingerprinting confirms extensive cow-to-cow intra-herd transmission of a single Mycobacterium bovis strain

In this study we have characterized M. bovis isolates from a herd of cattle in Uvalde, Texas in which 52 of the 193 animals selected at random in 1994 from a herd of 331 were caudal fold skin-test positive. Thirty-two of 52 skin-test positive cattle had gross lesions at slaughter, and isolations of M. bovis were made from 29 animals. The herd was comprised of Red Devon cattle purchased between 1978 and 1980 (n = 26) and breeding bulls (n = 3) introduced at later times, and all were tuberculosis test negative at the time of purchase. Other animals were natural additions (offspring) of these cattle. One additional animal, a Holstein present on the ranch at the time of purchase in 1976, was retained to nurse orphaned and weak calves. Using several molecular fingerprinting techniques we have verified a clonal relationship among the M. bovis isolates consistent with infection originating with a single strain. The molecular fingerprint patterns demonstrate the stability of the profiles despite persistence and spread of the organism within the herd for two decades and confirms their use in epidemiological tracing.     

Pooled faecal culture for the detection of Mycobacterium avium subsp paratuberculosis in goats

OBJECTIVE: To evaluate pooled faecal culture for herd diagnosis of caprine Johne's disease and relate these findings to faecal shedding rates of Mycobacterium avium subsp paratuberculosis (Map). DESIGN: Radiometric broth culture was applied to several pooling dilutions, and shedding rates were estimated from a regression equation based on bacterial growth rates and known processing losses during radiometric culture. PROCEDURE: Sixteen faecal samples from goats naturally infected with sheep (n = 3) or cattle (n = 13) strains of Map, were diluted in normal goat faeces from 1 in 5 to 1 in 50. Cultures were confirmed by IS900 polymerase chain reaction and restriction endonuclease analysis, and mycobactin dependency. The numbers of viable Map in the culture inocula were determined by endpoint titration (most probable number) of nine samples and related to a cumulative growth index. RESULTS: A pooling dilution of 1 in 25 with an incubation period of 10 weeks detected 13 of 16 culture positive goats, all shedding > or = 2 x 10(4) Map per gram of faeces. Two samples containing very low numbers of Map (< 2 x 10(3)/g) were only culture positive from undiluted faeces. Thirteen of 16 goats were considered to be shedding low to moderate concentrations of Map (< 2 x 10(5)/g faeces). CONCLUSIONS: These data support a pooling dilution of 1 in 25 for application of pooled faecal culture as a diagnostic tool in caprine Johne's disease control. A test based on this dilution would reduce laboratory costs of whole herd testing in goats by approximately 40% relative to serology and 75 to 90% relative to individual faecal culture.     

Experimental tuberculosis in the European badger (Meles meles) after endobronchial inoculation of Mycobacterium bovis: I. Pathology and bacteriology

The aim was to develop an endobronchial infection procedure for the study of Mycobacterium bovis infection in badgers. The badgers were anaesthetised and a cannula was passed per os to the tracheal bifurcation. When in place 1 ml of M. bovis suspension was inoculated. Three concentrations of M. bovis suspension were used; <10 colony forming units (cfu), approximately 10(2) cfu and approximately 3 x 10(3) cfu. The badgers were examined at three weekly intervals for clinical signs of disease and a tracheal aspirate was collected at each examination. The badgers were euthanased 17 weeks post infection (pi) and at the post mortem examination a wide range of tissues were examined for gross and histopathological lesions of tuberculosis and cultured for M. bovis. A sample of bronchial alveolar lavage (BAL) fluid was collected at post mortem for culture. At post mortem examination 17 weeks after infection, gross and histopathological lesions of tuberculosis were observed in all badgers inoculated with the high and medium dose and 1/3 inoculated with the low dose. M. bovis was recovered from all inoculated badgers. Infection in the high dose group was more widely disseminated than in the other groups. The number of sites with gross and histopathological lesions increased with increasing dose of M. bovis. All tracheal aspirates were negative on culture and only one BAL, collected from a badger of the high dose group, was positive on culture. No clinical signs due to the experimental infection were observed. The endobronchial route of inoculation is an effective route for establishing experimental infection, and could be used for studies of tuberculosis pathogenesis, immunology of M. bovis infection in badgers and for challenging badgers in vaccine protection studies. Badgers appeared to be very susceptible to infection by this procedure even with a dose of < 10 cfu but appear to control and limit the resulting infection.