Development of a model of natural infection with Mycobacterium bovis in white-tailed deer.

The objective of this study was to develop a suitable experimental model of natural Mycobacterium bovis infection in white-tailed deer (Odocoileus virginianus), describe the distribution and character of tuberculous lesions, and to examine possible routes of disease transmission. In October 1997, 10 mature female white-tailed deer were inoculated by intratonsilar instillation of 2 x 10(3) (low dose) or 2 x 10(5) (high dose) colony forming units (CFU) of M. bovis. In January 1998, deer were euthanatized, examined, and tissues were collected 84 to 87 days post inoculation. Possible routes of disease transmission were evaluated by culture of nasal, oral, tonsilar, and rectal swabs at various times during the study. Gross and microscopic lesions consistent with tuberculosis were most commonly seen in medial retropharyngeal lymph nodes and lung in both dosage groups. Other tissues containing tuberculous lesions included tonsil, trachea, liver, and kidney as well as lateral retropharyngeal, mandibular, parotid, tracheobronchial, mediastinal, hepatic, mesenteric, superficial cervical, and iliac lymph nodes. Mycobacterium bovis was isolated from tonsilar swabs from 8 of 9 deer from both dosage groups at least once 14 to 87 days after inoculation. Mycobacterium bovis was isolated from oral swabs 63 and 80 days after inoculation from one of three deer in the low dose group and none of four deer in the high dose group. Similarly, M. bovis was isolated from nasal swabs 80 and 85 days after inoculation in one of three deer from the low dose group and 63 and 80 days after inoculation from two of four deer in the high dose group. Intratonsilar inoculation with M. bovis results in lesions similar to those seen in naturally infected white-tailed deer; therefore, it represents a suitable model of natural infection. These results also indicate that M. bovis persists in tonsilar crypts for prolonged periods and can be shed in saliva and nasal secretions. These infected fluids represent a likely route of disease transmission to other animals or humans.     

Aerosol exposure of white-tailed deer (Odocoileus virginianus) to Mycobacterium bovis

Tuberculosis due to Mycobacterium bovis affects both captive and free-ranging Cervidae in the United States. Various animal models have been developed to study tuberculosis of both humans and animals. Generally, tuberculosis is transmitted by aerosol and oral routes. Models of aerosol exposure of large animals to M. bovis are uncommon. In order to develop a reliable method of aerosol exposure of white-tailed deer (Odocoileus virginianus) to M. bovis, 12 healthy white-tailed deer, aged 8-10 mo, were infected by aerosol exposure to 2 x 10(5) to 1 x 10(6) colony forming units (CFU) (high close, n = 4) of M. bovis or 6 x 10(2) to 1.6 x 10(3) CFU (low dose, n = 8) of M. bovis. Tuberculous lesions were more widely disseminated in (leer receiving the high dose, while lesions in deer receiving the low dose were more focused on the lungs and associated lymph nodes (tracheobronchial and mediastinal). Aerosol delivery of M. bovis to white-tailed deer results in a reliable manner of experimental infection that may be useful for studies of disease pathogenesis, immune response, mycobacterial shedding, and vaccine efficacy.     

Experimental inoculation of North American opossums (Didelphis virginiana) with Mycobacterium bovis

Eight North American opossums (Didelphis virginiana) were inoculated with 1 x 10(5) colony forming units of Mycobacterium bovis to investigate their potential as reservoir hosts for bovine tuberculosis in Michigan. Four animals received this dose orally and four were inoculated intramuscularly (i.m.). In each group, two animals were euthanized 1 mo postinoculation (PI) and two at 2 mo PI. Four control animals were housed separately and sacrificed in the same manner as those inoculated. One of four orally inoculated opossums and three of four i.m.-inoculated opossums were positive for M. bovis by culture of tissues obtained at necropsy. The oral recipient had positive cultures from intestine and pooled lymphoid samples. Pooled lymphoid samples were positive in three i.m.-inoculated animals and two of these also had positive liver and lung cultures. One animal with gross and histologic lesions compatible with tuberculosis had negative tissue cultures. The findings suggest that opossums are susceptible to M. bovis infection by multiple routes, although their relative susceptibility compared to true reservoir hosts appears to be low.     

Experimental aerosol inoculation of Mycobacterium bovis in North American opossums (Didelphis virginiana)

The goal of this study was to evaluate the susceptibility of North American opossums (Didelphis virginiana) to aerosol inoculation of Mycobacterium bovis at two dose levels in order to gain information on disease pathogenesis, fecal shedding of the organism, and the potential role that opossums play in the spread of this disease in nature. Six opossums received high dose (1 x 10(7) colony forming units (cfu) by aerosol inoculation, six opossums received low dose (1 x 10(3) cfu inoculation, and six opossums were sham-inoculated with sterile water and served as controls. Lungs were the most frequently infected tissues, with nine of 12 inoculated opossums positive for M. bovis on culture. Gross lesions consisted of multifocal pneumonia and enlarged lymph nodes. Microscopically, granulomatous pneumonia and granulomatous lymphadenitis associated with acid-fast bacilli were present in eight of 12 inoculated opossums. Fecal shedding of M. bovis was uncommon at both inoculation doses. While opossums were highly susceptible to aerosol inoculation of M. bovis, they did not become emaciated or develop widely disseminated lesions. From this study, opossums may transmit tuberculosis by aerosol infection to other opossums in close contact and serve as a source of infection to carnivores that feed upon them, however, transmission of the disease to large herbivores by fecal shedding or direct contact may be less likely.     

Experimental inoculation of meadow voles (Microtus pennsylvanicus), house mice (Mus musculus), and Norway rats (Rattus norvegicus) with Mycobacterium bovis

Mycobacterium bovis has a wide host range that includes several wildlife species, and this can hamper attempts to eradicate bovine tuberculosis from livestock. The purpose of this study was to determine if common rodent species, namely meadow voles (Microtus pennsylvanicus), house mice (Mus musculus), and Norway rats (Rattus norvegicus), that inhabit the bovine tuberculosis endemic area of Michigan, can be experimentally infected with M. bovis. The objectives of the study were: 1) to determine if these rodent species can be infected, and if so, to document attendant pathologic processes/pathogenesis; 2) to detect any fecal shedding of M. bovis; and 3) to evaluate the relative susceptibility of the three species to M. bovis infection. For each species (n=36) there were two treatment (n=12/group) and one or two control groups depending on species (n=6-12/group); the maximum study duration was 60 days. The meadow vole treatments consisted of high dose inocula that were given by oral or intranasal routes, whereas the house mice and Norway rats were given only oral inocula at either a high or low dose. Of the three species, meadow voles were most susceptible to M. bovis infection. Upon intranasal inoculation, all 12 voles were infected as determined by gross and microscopic lesions and culture of M. bovis from tissue and feces. Seven of the 12 meadow voles inoculated orally were infected. House mice also were susceptible; M. bovis was isolated from 14 of 24 animals. Only one Norway rat in the high dose treatment group was positive by culture and this was the only animal from which minimal attendant lesions were observed. Results of this study indicate that meadow voles and house mice can be infected with M. bovis and might serve as spillover hosts. Concerted efforts should, therefore, be made to reduce or eliminate these rodents on premises where M. bovis-infected livestock are present.     

Molecular detection of Mycobacterium bovis and Mycobacterium bovis BCG (Pasteur) in soil

PCR primers specific for the Mycobacterium tuberculosis complex were used to detect the presence of Mycobacterium bovis BCG (Pasteur) in soil microcosms and Mycobacterium bovis in environmental samples taken from a farm in Ireland with a history of bovine tuberculosis. M. bovis genes were detected in soil at 4 and 21 months after possible contamination. Gene levels were found in the range of 1 x 10(3) to 3.6 x 10(3) gene copies g of soil(-1), depending on the sampling area. Areas around badger setts had the highest levels of detectable genes and were shown to have the highest levels of gene persistence. M. bovis-specific 16S rRNA sequences were detected, providing evidence of the presence of viable cells in Irish soils. Studies of DNA turnover in soil microcosms proved that dead cells of M. bovis BCG did not persist beyond 10 days. Further microcosm experiments revealed that M. bovis BCG survival was optimal at 37 degrees C with moist soil (-20 kPa; 30% [vol/wt]). This study provides clear evidence that M. bovis can persist in the farm environment outside of its hosts and that climatic factors influence survival rates.     

Bovine tuberculosis in free-ranging carnivores from Michigan

During a survey of carnivores and omnivores for bovine tuberculosis conducted in Michigan (USA) since 1996, Mycobacterium bovis was cultured from lymph nodes pooled from six coyotes (Canis latrans) (four adult female, two adult male), two adult male raccoons (Procyon lotor), one adult male red fox (Vulpes vulpes), and one 1.5-yr-old male black bear (Ursus americanus). One adult, male bobcat (Felis rufus) with histologic lesions suggestive of tuberculosis was negative on culture but positive for organisms belonging to the Mycobacterium tuberculosis complex when tested by polymerase chain reaction. All the tuberculous animals were taken from three adjoining counties where M. bovis is known to be endemic in the free-ranging white-tailed deer (Odocoileus virginianus) population. There were two coyotes, one raccoon, one red fox, and one bobcat infected in Alpena county. Montmorency County had two coyotes and one raccoon with M. bovis. Two coyotes and a bear were infected from Alcona County. These free-ranging carnivores/omnivores probably became infected with M. bovis through consumption of tuberculous deer. Other species included in the survey were opossum (Didelphis virginiana), gray fox (Urocyon cinereoargenteus), and badger (Taxidea taxus); these were negative for M. bovis.     

牛型纯化蛋白衍生物皮内变态反应试验阳性牛中分枝杆菌的分离与鉴定

为评价牛分枝杆菌纯化蛋白衍生物(purified protein derivative,PPD)单纯颈部皮内变态反应试验(single intradermal cervical tuberculin,SICT)在奶牛结核病检测中的特异性,采集54头SICT阳性牛的118份组织样品进行病原分离和鉴定。结果显示,14头SICT阳性牛样品中分离到抗酸阳性菌,占SICT阳性牛总数的25.9%(14/54)。其中,10头阳性牛样品中分离到分枝杆菌,占18.5%(10/54);1头阳性牛样品中分离到牛分枝杆菌,占1.9%(1/54)。从118份组织样品中分离到16株抗酸阳性菌,其中12株为分枝杆菌,分枝杆菌分离率为10.2%(12/118)。12株分枝杆菌中,1株为牛分枝杆菌,其余11株为禽分枝杆菌、土地分枝杆菌等非结核分枝杆菌。牛分枝杆菌与非结核分枝杆菌分别占分枝杆菌分离株的8.3%(1/12)和91.7%(11/12)。病原分离和鉴定结果表明,SICT阳性牛的牛分枝杆菌分离率较低,为确保检测结果的准确性,有必要采用其他检测方法进行验证。同时,可将分枝杆菌快速分离培养及菌种鉴定检测技术引入对皮内变态反应试验阳性牛的实验室诊断,进一步提高牛结核病检测的特异性。     

Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays

Whole-genome comparisons of the tubercle bacilli were undertaken using ordered bacterial artificial chromosome (BAC) libraries of Mycobacterium tuberculosis and the vaccine strain, Mycobacterium bovis BCG-Pasteur, together with the complete genome sequence of M. tuberculosis H37Rv. Restriction-digested BAC arrays of M. tuberculosis H37Rv were used in hybridization experiments with radiolabelled M. bovis BCG genomic DNA to reveal the presence of 10 deletions (RD1-RD10) relative to M. tuberculosis. Seven of these regions, RD4-RD10, were also found to be deleted from M. bovis, with the three M. bovis BCG-specific deletions being identical to the RD1-RD3 loci described previously. The distribution of RD4-RD10 in Mycobacterium africanum resembles that of M. tuberculosis more closely than that of M. bovis, whereas an intermediate arrangement was found in Mycobacterium microti, suggesting that the corresponding genes may affect host range and virulence of the various tubercle bacilli. Among the known products encoded by these loci are a copy of the proposed mycobacterial invasin Mce, three phospholipases, several PE, PPE and ESAT-6 proteins, epoxide hydrolase and an insertion sequence. In a complementary approach, direct comparison of BACs uncovered a third class of deletions consisting of two M. tuberculosis H37Rv loci, RvD1 and RvD2, deleted from the genome relative to M. bovis BCG and M. bovis. These deletions affect a further seven genes, including a fourth phospholipase, plcD. In summary, the insertions and deletions described here have important implications for our understanding of the evolution of the tubercle complex.     

Efficacy of oral and parenteral routes of Mycobacterium bovis bacille Calmette-Guerin vaccination against experimental bovine tuberculosis in white-tailed deer (Odocoileus virginianus): a feasibility study

We investigated the efficacy of oral and parenteral Mycobacterium bovis bacille Calmette-Guerin Danish strain 1331 (BCG) in its ability to protect white-tailed deer (Odocoileus virginianus) against disease caused by M. bovis infection. Twenty-two white-tailed deer were divided into four groups. One group (n=5) received 10(9) colony-forming units (cfu) BCG via a lipid-formulated oral bait; one group (n=5) received 10(9) cfu BCG in culture directly to the oropharynx, one group (n=6) was vaccinated with 10(6) cfu BCG subcutaneously, and one group served as a control and received culture media directly to the oropharynx (n=6). All animals were challenged 3 mo after vaccination. Five months postchallenge the animals were examined for lesions. Results indicate that both oral forms of BCG and parenterally administered BCG offered significant protection against M. bovis challenge as compared to controls. This study suggests that oral BCG vaccination may be a feasible means of controlling bovine tuberculosis in wild white-tailed deer populations.     

Shared feed as a means of deer-to-deer transmission of Mycobacterium bovis

To determine the ability of experimentally inoculated white-tailed deer (Odocoileus virginianus) to transmit Mycobacterium bovis to na?ve deer through the sharing of feed, four deer were intratonsillarly inoculated with 4 x 10(5) colony-forming units of M. bovis. On a daily basis, feed not consumed by inoculated deer after approximately 8 hr was offered to four na?ve deer maintained in a separate pen, where direct contact, aerosol transmission, or transmission through personnel were prevented. After 150 days, na?ve deer were euthanized and examined. All na?ve deer had lesions consistent with tuberculosis and M. bovis was isolated from various tissues. The most commonly affected tissues were lung, tracheobronchial lymph nodes, and mediastinal lymph nodes. This study demonstrates the potential for indirect transmission of M. bovis through the sharing of feed. Intentional or unintentional feeding of deer by wildlife or agricultural interests in regions where M. bovis infection is endemic should be avoided because both direct and indirect transmission through sharing of feed are enhanced.     

牛分支杆菌与肺结核分支杆菌基因组的比较

通过比较基因组学的方法研究发现,牛分支杆菌与肺结核杆菌基因组的同源性为99．95％,但在牛分枝杆菌基因组中有11个缺失区,大小从1kb到12．7kb,遗传信息的缺失引起牛分枝杆菌的基因组减小;牛分枝杆菌与肺结核分枝杆菌H37Rv间存在着2437个单核苷酸多态性（SNPs）,与肺结核分枝杆菌CDC1551间存在着2423个单核苷酸多态性（SNPs）,牛分支杆菌与肺结核分枝杆菌在编码细胞壁和分泌蛋白上变异程度也是巨大的。研究结果揭示了牛分支杆菌与肺结核分枝杆菌的遗传关系,为研究分支杆菌疫苗和诊断试剂提供理论依据,对牛肺结核病的防治有着非常重要的意义。     

Characterization of a Mycobacterium bovis BCG insertion sequence related to the IS21 family

The structure and distribution of a Mycobacterium bovis BCG insertion element of the IS21 family were investigated. Several IS21-like elements found in mycobacterial genomes were separated in four types, following their nucleic acid similarities. The M. bovis BCG IS21 element is highly similar to IS1533 (class I), 70% similar to IS1534 (class II), 52% similar to IS1532 (class III) of Mycobacterium tuberculosis, and 54% similar to both an Mycobacterium avium serovar 2 and an M. avium silvaticum IS (class IV). The M. bovis BCG IS21 element of the class I appears to be present in a single copy in the genome of M. bovis BCG, M. bovis, M. tuberculosis and Mycobacterium africanum and to be absent from all other tested species of the Corynebacteria-Mycobacteria-Nocardia group.     

Methyl chloride utilising bacteria are ubiquitous in the natural environment

A 17-kDa protein (CadI) was induced by cadmium in Mycobacterium bovis and Mycobacterium tuberculosis. Comparison of the N-terminal sequence from M. bovis CadI with the annotated M. tuberculosis genome database identified Rv2641 as the encoding gene. Long and short promoter fragments from M. bovis cadI were fused to the lacZ reporter gene in pYUB76. Only the long fragment directed cadmium-inducible activity when electroporated into M. bovis. The cadI promoter has potential for both constitutive and inducible expression studies in M. bovis and M. tuberculosis.     

A combined DNA vaccine-prime, BCG-boost strategy results in better protection against Mycobacterium bovis challenge

In this study, we demonstrated that calves vaccinated with a combined DNA vaccine encoding Ag85B, MPT- 64, and MPT-83 antigens from the Mycobacterium tuberculosis for the priming and subsequently boosting with BCG prior to experimental challenge with virulent Mycobacterium bovis (M. bovis) resulted in improved immune responses over immunizing. Vaccination with the combined DNA/BCG induced higher levels of antigen- specific gamma interferon (IFN-gamma) in whole-blood cultures 4 weeks after final vaccination and the level of antigen-specific IFN-gamma in response to Ag85, MPT-64, and MPT-83 were still higher 4 weeks after challenge when compared to the combined DNA group. There was a significant bias toward induction of CD4+ T cells rather than CD8+ T cells responses, and the mean percentage of CD4+ T cells was increased about 2.6-fold in peripheral blood mononuclear cells (PBMC) cultures in DNA prime-BCG boost vaccination when compared to the nonvaccinated group. In addition, DNA prime-BCG boost vaccination resulted in stronger humoral immune responses, and the levels of the specific antibodies to three antigens were increased two- to 32- fold when compared to the combined DNA group. Vaccination with the combined DNA/BCG induced a high level of protection against an intratracheal challenge with virulent M. bovis, based on a significant enhancement of six pathological and microbiological parameters of protection compared to the nonvaccinated group. Finally, the combined DNA/BCG increased the protective efficacy by more than 10-100-fold as measured by reduced CFU counts in the lungs from calves challenged with M. bovis compared to the combined DNA and BCG groups. These results suggest that use of the prime-boost strategy offers better protection against bovine tuberculosis than does the combined DNA vaccines and BCG.     

Antimycobacterial activities of 5-alkyl (or halo)-3'-substituted pyrimidine nucleoside analogs

Several 5-alkyl (or halo)-3'-azido (amino or halo) analogs of pyrimidine nucleosides have been synthesized and evaluated against Mycobacterium bovis, Mycobacterium tuberculosis and Mycobacterium avium. Among these compounds, 3'-azido-5-ethyl-2',3'-dideoxyuridine (3) was found to have significant antimycobacterial activities against M. bovis (MIC(50)=1μg/mL), M. tuberculosis (MIC(50)=10μg/mL) and M. avium (MIC(50)=10μg/mL).     

Evaluation of carriers used in the test methods of the Association of Official Analytical Chemists

Revision of the official test method for the determination of the tuberculocidal activity of disinfectants is being undertaken. The current procedure lacks precision and accuracy and is not quantitative. Variability associated with carriers and the lack of temperature control were evaluated in this paper. The use of porcelain versus stainless steel carriers was also evaluated. When carriers of either type were contaminated with Mycobacterium bovis BCG, the number of organisms on the carriers varied by as much as 1.0 on the log10 scale. The average number of organisms attached to each porcelain carried was 1.10 x 10(5) CFU (range, 2.7 x 10(4) to 2.7 x 10(5) CFU), whereas the average number of organisms attached to each stainless steel carrier was 1.38 x 10(5) CFU (range, 2.9 x 10(4) to 4.0 x 10(5) CFU). The average number of cells attached to the carrier was directly proportional to the number of cells in the contaminating cell suspension. Variations in drying time did not alter the number of cells attached to the carrier. When porcelain carriers were placed in a test solution, the average number of organisms washed from the carriers was 55% of the total, with a range of 19 to 80%, whereas for stainless steel carriers, the average number was 82% of the total, with a range of 52 to 96%. Data for B. subtilis spores were similar to those for M. bovis BCG, suggesting that there may be similar problems with the Association of Official Analytical Chemists sporicidal test, which uses carriers. It was also found that the lack of an exacting temperature control could influence the outcome of the test. Changes in temperature as little as 1 degree C could influence the rate of killing of M. bovis BCG.     

Analysis of the products of genes encompassed by the theoretically predicted pathogenicity islands of Mycobacterium tuberculosis and Mycobacterium bovis

Sequencing of the genomes of Mycobacterium tuberculosis and Mycobacterium bovis provides a unique opportunity to study the biology of these pathogens on the genomic level. The computational detection of anomalous gene clusters such as those encompassed by pathogenicity islands allows for a narrowing of the study into well-defined groups of genes. Pathogenicity islands of M. tuberculosis (strains H37Rv and CDC1551) as well as M. bovis genomes comprise a group of genes encoding proteins that have been shown to be of immunological importance. The cross-genomic comparison (M. tuberculosis vs M. bovis) resulted in the elucidation of unique proteins in M. tuberculosis. These proteins may play a significant role in the host recognition process.     

Infection of mice with aerosolized Mycobacterium tuberculosis: use of a nose-only apparatus for delivery of low doses of inocula and design of an ultrasafe facility

Aerosolized delivery of virulent or hypervirulent Mycobacterium tuberculosis requires careful consideration of methodology and safety. To maximize safety, we installed a nose-only aerosol apparatus that can reproducibly deliver a low dose (<100 CFU per mouse) of M. tuberculosis in a carefully designed biohazard facility.