Molecular Typing of Mycobacterium bovis Isolates in Argentina: First Description of a Person‐to‐Person Transmission Case

Bovine tuberculosis is caused by Mycobacterium bovis, a mycobacterium highly similar to M. tuberculosis that belongs to the M. tuberculosis complex. The main host of M. bovis is cattle but it also affects many other mammalians including humans. Tuberculosis in humans caused by either M. bovis or M. tuberculosis is clinically hard to distinguish. During 2004–2005, samples from 448 patients with diagnosis of TB were collected from different regions of Argentina. The PRA technique identified 400 isolates with representative patterns of mycobacterium. The predominant ones were the M. tuberculosis complex, the M. avium–M. intracellulare complex and M. gordonae. Samples with M. tuberculosis complex PRA restriction profiles were analyzed with a multiplex PCR to differentiate between M. tuberculosis and M. bovis. Multiplex PCR identified nine M. bovis. The results allowed the possibility to establish that 2% of pulmonary tuberculosis was due to M. bovis. Isolates of M. bovis from humans were examined using spoligotyping. These isolates presented five different spoligotypes. The main spoligotype was also the most frequently one found in cattle. The remaining human spoligotypes (grouped in clusters) are occasionally found in cattle. Variable number tandem repeat (VNTR) analysis identified five different patterns. By combining the results of spoligotyping and VNTR analysis, we were able to differentiate seven M. bovis isolates. The remaining two M. bovis samples showed the same spoligotype and VNTR profile and belonged to household contacts. An MDR‐M. bovis was isolated from the samples of these household contacts. The identification of two epidemiologically linked cases of human M. bovis infection suggests person‐to‐person transmission of an MDR‐M. bovis.     

Direct Detection of Mycobacterium bovis in Bovine Lymph Nodes by PCR

Thirty‐five lymph node samples were taken from animals with macroscopic lesions consistent with Mycobacterium bovis infection. The animals were identified by postmortem examination in an abattoir in the northwestern region of state of Paraná, Brazil. Twenty‐two of the animals had previously been found to be tuberculin skin test positive. Tissue samples were decontaminated by Petroff’s method and processed for acid‐fast bacilli staining, culture in Stonebrink and Lowenstein‐Jensen media and DNA extraction. Lymph node DNA samples were amplified by PCR in the absence and presence (inhibitor controls) of DNA extracted from M. bovis culture. Mycobacterium bovis was identified in 14 (42.4%) lymph node samples by both PCR and by culture. The frequency of PCR‐positive results (54.5%) was similar to that of culture‐positive results (51.5%, P > 0.05). The percentage of PCR‐positive lymph nodes increased from 39.4% (13/33) to 54.5% (18/33) when samples that were initially PCR‐negative were reanalysed using 2.5 μl DNA (two samples) and 1 : 2 diluted DNA (three samples). PCR sensitivity was affected by inhibitors and by the amount of DNA in the clinical samples. Our results indicate that direct detection of M. bovis in lymph nodes by PCR may be a fast and useful tool for bovine tuberculosis epidemic management in the region.     

The prevalence and distribution of Mycobacterium bovis infection in European badgers (Meles meles) as determined by enhanced post mortem examination and bacteriological culture

The accurate diagnosis of Mycobacterium bovis infection in badgers is key to understanding the epidemiology of tuberculosis in this species and has significant implications for devising strategies to limit spread of the disease. In this study, badgers (n = 215) in the Republic of Ireland were examined at post mortem and tissues were collected from a range of anatomical locations and pooled into groups for bacterial culture of M. bovis. By assessing confirmed gross visible lesions (VL) alone, infection was detected in 12.1% of badgers. However, by including the results of all culture positive pooled samples, the overall infection prevalence increased significantly to 36.3%. Two-thirds (66.7%) of infected animals had no visible lesions (NVL). While the thoracic cavity (lungs and pulmonary lymph nodes) was found to be the most common site of infection, in a proportion of animals infection was absent from the lungs and draining lymph nodes and was confined to the lymph nodes of the carcase or the head. This may indicate an early extrapulmonary dissemination of infection or alternatively, in the case of the head lymph nodes, a secondary pathogenic pathway involving the lymphoid tissues of the upper respiratory tract (URT).     

Vaccination with Mycobacterium bovis BCG Strains Danish and Pasteur in White‐tailed Deer (Odocoileus virginianus) Experimentally Challenged with Mycobacterium bovis

Wildlife reservoirs of Mycobacterium bovis represent serious obstacles to the eradication of tuberculosis in domestic livestock and the cause for many faltering bovine tuberculosis eradication programmes. One approach in dealing with wildlife reservoirs of disease is to interrupt inter‐species and intraspecies transmission through vaccination of deer or cattle. To evaluate the efficacy of BCG vaccination in white‐tailed deer, 35 deer were assigned to one of three groups; one s.c. dose of 10⁷ CFU of M. bovis BCG Pasteur (n = 12); 1 s.c. dose of 10⁷ CFU of M. bovis BCG Danish (n = 11); or unvaccinated deer (n = 12). After vaccination, deer were inoculated intratonsilarly with virulent M. bovis. Lesion severity scores of the medial retropharyngeal lymph node, as well as all lymph nodes combined, were reduced in vaccinated deer compared to unvaccinated deer. BCG Danish vaccinated deer had no late stage granulomas characterized by coalescent caseonecrotic granulomas containing numerous acid‐fast bacilli compared to BCG Pasteur vaccinated or unvaccinated deer where such lesions were present. Both BCG strains were isolated as late as 250 days after vaccination from deer that were vaccinated but not challenged. In white‐tailed deer, BCG provides protection against challenge with virulent M. bovis. Issues related to vaccine persistence, safety and shedding remain to be further investigated.     

Detection of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> in tissue samples of cattle and buffaloes

Tissue samples were collected at random from cattle (Bos taurus) and buffalo (Bubalus bubalis) from an abattoir of the district of Lahore and were analyzed for the presence of Mycobacterium avium subsp. paratuberculosis and Mycobacterium bovis through acid-fast staining and polymerase chain reaction (PCR). Body condition of animals and diarrhea were recorded. Most of the animals were emaciated. Diarrhea was noticed in 15.6% of buffaloes and 19.2% of cattle. Intestinal pathology was observed in 29% of buffaloes and 32.8% of cattle. Number of mesenteric lymph node (MLN) showing gross lesions was a bit higher (35.6%) in cattle than buffalo (31.2%). Acid-fast staining of tissue scraping smears revealed the presence of acid-fast bacilli (AFB) in 17.4% intestinal and 16.4% MLN tissue samples in buffalo, while in cattle 19.2% intestinal and 17.8% MLN were found positive for AFB. In buffaloes, PCR confirmed 12.8% intestinal and 12.4% MLN positive samples for M. avium subsp. paratuberculosis. However, in cattle, PCR analysis demonstrated 14.2% positive results for M. avium subsp. paratuberculosis in both MLN and intestinal tissue samples. PCR also confirmed M. bovis in 5.8% of cattle and 5% of buffalo MLN and intestinal tissues. PCR positive tissue samples for M. avium subsp. paratuberculosis were from those animals which were emaciated, having diarrhea, and severe gross lesions. AFB were also detected in tissue scraping smears of these animals. It is concluded that infection by various mycobacterium species can be differentiated by PCR, which is not possible by acid-fast staining technique.     

Experimental Mycobacterium bovis infection of cattle: Effect of dose of M. bovis and pregnancy on immune responses and distribution of lesions

Groups of l8-month-old cattle were inoculated intratracheally with 5 X 10⁵ colony forming units (high dose) or 500 colony forming units (low dose) of Mycobacterium bovis to determine an appropriate dose to induce lesions similar to those seen in the natural disease. An additional group of 21–28 weeks pregnant cattle were inoculated with the high dose of M. bovis to determine if pregnancy increased the susceptibility of cattle to M. bovis infection. By 23–24 weeks after challenge, the high dose of M. bovis had induced extensive lung lesions, and tuberculous lesions were observed in the lymph nodes of the head, neck, and thoracic and abdominal cavities. In contrast, the low dose of M. bovis induced predominantly small lesions (< 1 cm diameter) which were localised to the lungs and pulmonary lymph nodes. The lesions induced by the low dose were similar to those seen in the natural disease in cattle. The majority of the high dose group cattle produced strong antibody responses to M. bovis culture filtrate, while only one low dose animal produced a detectable response. All of the M. bovis-inoculated cattle produced strong cellular immune responses to bovine PPD (skin test and interferon-γ responses). Pregnancy did not appear to affect the susceptibility to M. bovis infection, and immune responses of the cattle in this group at the end of the study were similar to those in the high dose non-pregnant group. However, from the first test after calving, the interferon-y responses of peripheral blood cultures to bovine PPD were low compared with the responses prior to calving.     

Prevalence of Mycobacterium bovis infection in feral pigs in the Northern Territory

SUMMARY The prevalence of Mycobacterium bovis infection in populations of feral pigs from five areas in the Northern Territory was examined. In total 790 pigs were necropsied and positive cultures of M bovis were obtained from two pigs (0.25%) and a mycobacterial granuloma was found in one pig. The observed prevalence of M bovis infection in feral pigs is significantly less (x²= 139.8, df = 1, P < 0.001) than the results of a comparable survey conducted during the early 1970s before the implementation of the Brucellosis and Tuberculosis Eradication Campaign. The prevalence of all types of macroscopic lesions resembling tuberculosis was significantly (x²= 338.7, df = 1, P < 0.001) less than the earlier survey. The results are further support for the hypothesis that in the Northern Territory feral pigs are an end-host for M bovis infection, and that the previous high prevalence of M bovis recorded in feral pigs in the 1970s was caused by the close association between these animals and large populations of M bovis-infected buffalo and cattle.     

Single-nucleotide polymorphism-based epidemiological analysis of Korean Mycobacterium bovis isolates

BackgroundBovine tuberculosis (TB) is caused by Mycobacterium bovis, a well-known cause of zoonotic tuberculosis in cattle and deer, and has been investigated in many physiological and molecular studies. However, detailed genome-level studies of M. bovis have not been performed in Korea.ObjectivesTo survey whole genome-wide single-nucleotide polymorphism (SNP) variants in Korean M. bovis field isolates and to define M. bovis groups in Korea by comparing SNP typing with spoligotyping and variable number tandem repeat typing.MethodsA total of 46 M. bovis field isolates, isolated from laryngopharyngeal lymph nodes and lungs of Korean cattle, wild boar, and Korean water deer, were used to identify SNPs by performing whole-genome sequencing. SNP sites were confirmed via polymerase chain reaction using 87 primer pairs.ResultsWe identified 34 SNP sites with different frequencies across M. bovis isolates, and performed SNP typing and epidemiological analysis, which divided the 46 field isolates into 16 subtypes.ConclusionsThrough SNP analysis, detailed differences in samples with identical spoligotypes could be detected. SNP analysis is, therefore, a useful epidemiological tracing tool that could enable better management of bovine TB, thus preventing further outbreaks and reducing the impact of this disease.     

A study of cattle-to-cattle transmission of Mycobacterium bovis infection

Twenty steers, positive to the single intradermal comparative tuberculin test (SICTT), were selected fromherds with a recent history of Mycobacterium bovis infection. Ten steers, negative to SICTT, were selected from herds with no history of M. bovis infection and served as in-contact animals. The animals were divided into 10 groups, each consisting of two SICTT-positive (reactor) animals and one in-contact animal. Each group was housed in an individual loose-box for a period of 1 year. Five of the groups were fed a restricted diet for part of the experiment. All cattle were slaughtered at the end of the study period and examined at post mortem. Transmission of infection to an in-contact animal occurred in four of the 10 groups. One of the four in-contact animals, which became infected, had a retropharyngeal lymph node tubercle and M. bovis was isolated from lymph nodes without visible lesions from the other three. Two of the infected in-contact animals without visible lesions did not show any detectable cell-mediated immune response. There was no evidence that dietary, restriction had any effect on transmission of disease.     

Isolation of Mycobacterium bovis from brushtail possums with non-visible lesions

AIM: To determine the prevalence of Mycobacterium bovis infection in brushtail possums (Trichosurus vulpecula) that did not have macroscopic lesions of bovine tuberculosis, and to evaluate culture of pooled tissues from multiple possums as a method for determining the M. bovis-infection status of wildlife populations in New Zealand.

METHODS: Pools of selected tissues were collected from possums from four different populations known to be infected with M. bovis. Tissue pools from individual animals, and combined pools from multiple animals, were cultured for M. bovis.

RESULTS: In the four populations investigated, the prevalence of possums with macroscopic lesions confirmed by culture to be infected with M. bovis ranged from 1 to 19 (mean 31/283; 10.9)%. The prevalence of possums with non-visible lesions that were culture positive for M. bovis in the same populations ranged from 4 to 10 (mean 24/283; 8.5)%. The mean of the log10 cfu of M. bovis of the macroscopic lesions and of the culture-positive samples that did not have visible lesions was 3.85 (SE 0.26) and 1.46 (SE 0.26) log₁₀ cfu, respectively (p<0.01). Mycobacterium bovis was cultured from pools of 30–50 animals in the four populations studied.

CONCLUSIONS: The finding of M. bovis infection in possums with non-visible lesions identified a potential deficiency of declaring possum populations free of M. bovis on the basis of absence of macroscopic lesions. The culturing of pools of selected tissues from multiple animals without visible lesions can be used to reduce laboratory costs of possum surveys without a major reduction in the ability to detect M. bovis infection.     

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.     

Prevalence of Mycobacterium bovis infection in traditionally managed cattle at the wildlife-livestock interface in South Africa in the absence of control measures

Cattle are the domestic animal reservoir for Mycobacterium bovis (M. bovis) which also affects other domestic animals, several wildlife species and humans leading to tuberculosis. The study area is in a resource-poor community that is surrounded by several game parks, where M. bovis infection has been previously diagnosed in wildlife. A cross-sectional study was carried out to determine the prevalence of M. bovis infection in 659 cattle from a total of 192 traditionally managed herds using the BOVIGAM® interferon gamma assay (IFN-γ). Infection was confirmed by post mortem examination and M. bovis isolation from three test-positive cattle. Genotyping of the M. bovis isolates was done using spoligotyping and VNTR (variable number of tandem repeats typing). The apparent M. bovis prevalence rate in cattle at animal level was 12% with a true population prevalence of 6% (95% Confidence interval (C.I) 3.8 to 8.1) and a herd prevalence of 28%. Spoligotyping analysis revealed that the M. bovis isolates belonged to spoligotype SB0130 and were shared with wildlife. Three VNTR profiles were identified among the SB0130 isolates from cattle, two of which had previously been detected in buffalo in a game reserve adjacent to the study area. The apparent widespread presence of M. bovis in the cattle population raises a serious public health concern and justifies further investigation into the risk factors for M. bovis transmission to cattle and humans. Moreover, there is an urgent need for effective bTB control measures to reduce infection in the communal cattle and prevent its spread to uninfected herds.

     

Experimental tuberculosis in the European badger (Meles meles) after endobronchial inoculation with Mycobacterium bovis: II. Progression of infection

The aim of the study was to describe, over a period of 24 weeks, the pathological and bacteriological changes in badgers experimentally infected with Mycobacterium bovis. The badgers were infected by endobronchial instillation of 2.5 × 10⁴ colony forming units (cfu) M. bovis. After infection, the badgers were examined at 3 weekly intervals when blood and tracheal aspirates were collected. At 6, 12, 18 and 24 weeks post-infection (pi) three animals were euthanized and a detailed pathological and bacteriological examination was performed to assess the nature of the experimental disease. During the course of the study only one badger developed clinical signs of disease: a subcutaneous swelling on its head, first observed at 18 weeks pi. At post-mortem examination gross and histological lesions of tuberculosis were observed and M. bovis was recovered from all, except one badger. In the majority of badgers the endobronchial route of inoculation resulted in the establishment of infection that over 24 weeks was non-progressive with limited dissemination of infection from the thoracic cavity, mainly to the hepatic and mesenteric lymph nodes. However, in one of the badgers examined at 18 weeks pi and one at 24 weeks pi, infection was widely disseminated. The disease induced by the endobronchial inoculation displayed the characteristics of disease observed in naturally infected badgers.     

Tuberculosis Infection in Animal and Human Populations in Three Districts of Western Gojam,Ethiopia

Tuberculosis concurrent infection in cattle and their respective owners in North‐western Ethiopia had been investigated. Two hundred and ten cattle owners and 1220 heads of their cattle were included in the study to determine degree of tuberculosis infection in cattle owned by tuberculosis patients and tuberculosis patients. Comparative intradermal tuberculin test, bacteria culturing, acid fast staining and biochemical tests were used to conduct the study. The prevalence of tuberculosis was significantly (P < 0.001) higher in cattle owned by tuberculosis patients than in cattle owned by non‐tuberculosis owners, and infection with tuberculosis was threefold greater in cattle owned by tuberculosis‐positive owners. Further more, cattle owners who consumed raw milk were at higher risk (P < 0.001, OR = 3.23) for tuberculosis infection than those who consumed boiled milk. Mycobacterium tuberculosis (15.4%), Mycobacterium bovis (44.1%) and atypical mycobacteria (38.5%) were identified from milk collected from tuberculin‐positive cows using biochemical tests. Similarly M. tuberculosis (74.5%), M. bovis (14.9%) and atypical mycobacteria (8.5%) were identified from sputum and fine needle aspiration specimens of tuberculosis patient cattle owners. Mutual transmission of mycobacterium from animals to humans and vice versa has been signified.     

Differential antigen recognition by serum antibodies from three bovid hosts of Mycobacterium bovis infection

Cattle, bison and buffaloes are susceptible to Mycobacterium bovis, the causative agent for bovine tuberculosis. Accurate and timely identification of infected animals is critical for improved management and control of disease in these species. Bovids develop humoral immune responses to M. bovis infection making serological tests attractive for tuberculosis screening. However, optimization and validation of antibody assays designed for various animal species require understanding of antigen recognition patterns in each target host. The objective of this study was to characterize serological reactivity profiles generated by cattle, American bison, and African buffaloes in tuberculosis. Serum samples from M. bovis-infected animals were tested for the presence of IgM and IgG antibodies to MPB70/MPB83 and CFP10/ESAT6 chimeric proteins using Dual-Path Platform technology. All three host species showed IgG responses of higher magnitude and frequency than IgM responses; further, IgM seroreactivity was limited to MPB70/MPB83, whereas IgG antibodies recognized both test antigens. In cattle, the IgM and IgG responses were elicited mainly by MPB70/MPB83, whereas bison and buffaloes showed similar IgG seroreactivity rates for MPB70/MPB83 and CFP10/ESAT6 antigens. The findings demonstrate distinct patterns of predominant antigen recognition by different bovid species in M. bovis infection.     

Bacteriological, serological, pathological and immunohistochemical studies of Mycoplasma bovis respiratory infection in veal calves and adult cattle at slaughter

Mycoplasma bovis is an important cause of calf pneumonia worldwide. In this study, we examined 140 cattle at slaughter comprising 70 veal calves and 70 beef cattle; 115 animals with pneumonic lesions and 25 without. Lung samples were submitted for bacteriological, histological, and M. bovis-immunohistochemical analyses. Serology for M. bovis was positive in 76% of beef cattle and 100% of veal calves. M. bovis was isolated only from veal calves in 16 out of 64 pneumonic cases. M. bovis was detected by immunohistochemistry in seven bacteriologically positive cases. M. bovis antigen was associated with bronchogenic necrosuppurative or fibrinonecrotizing lesions. Bacteriologically positive and immunohistochemical negative cases were associated with catarrhal bronchointerstitial pneumonia. Results suggest that M. bovis infection may develop into a severe necrosuppurative bronchopneumonia or fibrinonecrotizing pneumonia when associated with a high number of intralesional organisms or, conversely, into a mild catarrhal bronchointerstitial pneumonia when associated with a low number of organisms.     

Mycobacteria isolated from deer in New Zealand from 1970–1983

Mycobacterium bovis was isolated from 504 deer from 1970 to 1983. It was first isolated from feral red deer (Cervus elaphus) in New Zealand in 1970, and from farmed deer in 1978. Cervine tuberculosis has emerged as a significant problem in farmed deer and in 1983 M. bovis was found on 40 different farms. Thirty-five isolates of Mycobacterium avium-intracellulare have been cultured from deer but were associated with clinical disease in only four cases. Mycobacterium nonchromogenicum, Mycobacterium diernhoferi, Mycobacterium gastri, Mycobacterium chelonei, Mycobacterium smegmatis and Mycobacterium vaccae were isolated from deer but were not considered to be pathogenic.     

THE DISTRIBUTION OF MYCOBACTERIUM BOVIS IN QUEENSLAND CATTLE HERDS WITH OBSERVATIONS ON THE LABORATORY DIAGNOSIS OF TUBERCULOSIS

SUMMARY Of 1,791 tissues submitted from 1975 to 1978 for histological examination for tuberculosis, 78.5% were classified as tuberculosis, 8.1% as club-forming granulomas, 3.3% as hydatid cysts, 3.4% as other granulomas and 2.4% as squamous cell carcinomas. Cultural examination of 2,682 tissues for mycobacteria yielded Mycobacterium bovis from 42.88%, other mycobacteria from 7.64% and 0.89%Rhodococcus (Corynebacterium) equi. Of 1,478 tissues on which both examinations were performed, culture and histology results were in agreement on 94.1%. M. bovis was isolated on one or more occasions from 564 herds with the highest herd prevalence being recorded in western areas of the state. Culture of 1,024 tissues from no visible lesion (NVL) reactors yielded 0.59%M. bovis, 15.04% other mycobacteria and 0.19%R. equi. Of 120 isolates of other mycobacteria from tissues of NVL reactors, 39 were Runyon Group III (20 M. avium complex) and 61 Runyon Group IV. Approximately half of the herds from which NVL tissues were collected were confirmed M. bovis infected properties.     

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.