Mycobacterium bovis

Successful use of liposomes as immunological adjuvants in vaccines requires simple, easy to scale up technology capable of high-yield antigen entrapment. Recent work from this laboratory has led to the development of techniques that can generate liposomes of various sizes containing soluble antigens such as proteins or particulate antigens such as whole, live, or attenuated bacteria or viruses. Entrapment of proteins is carried out by the dehydration-rehydration procedure, which entails freeze-drying of a mixture of "empty" small unilamellar vesicles and free antigens. Upon rehydration, the large multilamellar vesicles that are formed incorporate up to 80% of the antigen used. When such liposomes are microfluidized in the presence of nonentrapped material, their size is reduced to about 100 nm in diameter, with much of the originally entrapped antigen still associated with the vesicles. A similar technique applied to the entrapment of particulate antigens (e.g., Bacillus s...

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only vaccine available against tuberculosis, and the strains used worldwide represent a family of daughter strains with distinct genotypic characteristics. Here we report the complete genome sequence of M. bovis BCG Moreau, the strain in continuous use in Brazil for vaccine production since the 1920s.

The n-hexane extract of Lovage root was found to significantly inhibit the growth of both Mycobacterium smegmatis mc 2 155 and Mycobacterium bovis BCG, and therefore a bioassay-guided isolation strategy was undertaken. (Z)-Ligustilide, (Z)-3-butylidenephthalide, (E)-3-butylidenephthalide, 3-butylphthalide, a-prethapsenol, falcarindiol, levistolide A, psoralen and bergapten were isolated by chromatographic techniques, characterized by NMR spectroscopy and MS, and evaluated for their growth inhibition activity against Mycobacterium tuberculosis H 37 Rv using the whole-cell phenotypic spot culture growth inhibition assay (SPOTi). Cytotoxicity against RAW 264.7 murine macrophage cells was employed for assessing their degree of selectivity. Falcarindiol was the most potent compound with a minimum inhibitory concentration (MIC) value of 20 mg/L against the virulent H 37 Rv strain; however, it was found to be cytotoxic with a half-growth inhibitory concentration (GIC 50) in the same order of magnitude (SI < 1). Interestingly the sesquiterpene alcohol a-prethapsenol was found to inhibit the growth of the pathogenic mycobacteria with an MIC value of 60 mg/L, being more specific towards mycobacteria than mammalian cells (SI~2). Colony forming unit analysis at different concentrations of this phytochemical showed mycobacteriostatic mode of action.

Some of the authors of this publication are also working on these related projects: Development of MALDI-TOF methods for detection and classification of Brucella spp., Mycobacterium spp., Salmonella e Escherichia coli in bovines View project Flábio Araújo Brazilian Agricultural Research Corporation (… 129 PUBLICATIONS 1,023 CITATIONS

Normal and Bacillus Calmette-Guerin (BCG) vaccine-induced rabbit alveolar macrophage homogenates were fractionated by isopycnic density gradient centrifugation. Superoxide dismutase-inhibitable NAD(P)H-dependent nitro-blue tetrazolium reductase was found localised to endoplasmic reticulum and mitochondria. The normal macrophages tended to contain more of this activity than the BCG-induced macrophages. Two superoxide dismutases were found: cyanide-sensitive superoxide dismutase was predominantly present in the cytosol, with a small proportion in mitochondria; cyanide-resistant superoxide dismutase was found confined to mitochondria. Neither differed in specific activity betw-en the normal and BCG-induced macrophages.

The only currently available vaccine against tuberculosis (TB) is Mycobacterium bovis Bacille Calmette-Guerin (BCG), which has inconsistent efficacy to protect against the disease in adults. M. tuberculosis (MTB) cell wall components have been implicated in the pathogenicity of TB and therefore have been a prime target for the identification and characterization of cell wall proteins with potential application in vaccine development. In this regard, proteoliposomes (PLs) derived from mycobacteria containing lipids and cell wall proteins could be potential vaccine candidates against TB. In the present study PLs derived from BCG were prepared. These homogeneous population of spherical microparticles was then immunized into Balb/c mice. Sera of immunized animals showed high IgG response and strong cross-reactivity against different MTB antigens.These results showed that BCG PLs could be potential vaccine candidates against TB.

PE and PPE proteins appear to be important for virulence and immunopathogenicity in mycobacteria, yet the functions of the PE/PPE domains remain an enigma. To decipher the role of these domains, we have characterized the triacylglycerol (TAG) hydrolase LipY from Mycobacterium tuberculosis, which is the only known PE protein expressing an enzymatic activity. The overproduction of LipY in mycobacteria resulted in a significant reduction in the pool of TAGs, consistent with the lipase activity of this enzyme.

-a-producing CD4 + cells were low among control subjects (median, 0.33% and 0.78%, respectively). By contrast, among patients with TB, strong IFN-g and TNF-a responses were demonstrated (median, 24.0% and 32.4%, respectively), regardless of whether the TB was pulmonary or nonpulmonary. Measurement of type 1 cytokine production by CD4 + T lymphocytes in response to PPD in BAL fluid is a promising new diagnostic test for active TB in immunocompetent individuals.

The pentameric form of the cholera toxin B subunit (CTB) is known to be a strong mucosal adjuvant and stimulates antigen-specific secretory immunoglobulin A (IgA) and systemic antibody responses to antigens when given by mucosal routes. To deliver CTB for prolonged periods of time to the respiratory mucosa, we constructed a Mycobacterium bovis bacillus Calmette-Guérin (BCG) strain that produces and secretes assembled pentameric CTB. Mice immunized intranasally (i.n.) with recombinant BCG (rBCG) developed a stronger anti-BCG IgA response in bronchoalveolar lavage fluids (BALF) than mice immunized with nonrecombinant BCG. The total IgA response in the BALF of mice immunized with rBCG was also stronger than that in BALF of mice immunized with the nonrecombinant strain. The induction of IgA was well correlated with an increased production of transforming growth factor ␤1. Simultaneous administration of intraperitoneally delivered ovalbumin and of i.n. delivered CTB-producing BCG induced a long-lasting ovalbumin-specific mucosal IgA response as well as a systemic IgG response, both of which were significantly higher than those in mice immunized with nonrecombinant BCG together with ovalbumin. These results suggest that the CTB-producing BCG may be a powerful adjuvant to be considered for future mucosal vaccine development.

I.R. GRANT, H.J. BALL AND M.T. ROWE. 1996. T h e thermal inactivation of Mycobacterium avium, Myco. bovas, Myco. fortuitum, Myco. intracellulare and Myco. kansaii in milk at 63.5"C was investigated. Survivors were enumerated after heating for 0, 5, 10, 15, 20 and 30 min and thermal death curves were constructed for each species. Mycobacterium bovis and Myco. fortuitum were found to exhibit linear thermal death curves and neither species demonstrated any survival after heating at 63-5"C for 30 min (equivalent to holder pasteurization). In contrast, Myco. avium, Myco. intracellulare and Myco. tansaszi yielded thermal death curves which exhibited significant 'tailing' and all three strains survived holder pasteurization.

Bovine tuberculosis (TB) is an important neglected zoonosis in Cameroon, where many communities depend on their livestock for livelihood and the incidence of human TB and TB-HIV/AIDS co-infection are high and increasing annually. The aim of this study was to estimate the prevalence of bovine TB in cattle in the highlands of Cameroon. The magnitude and trend of detecting TB lesions in slaughtered cattle (1994 to 2010) and tuberculin skin tests (TST) in 2853 cattle (84 herds) of 39 livestock rearing communities were analysed. Of 129 165 slaughtered cattle inspected, 599 (0.46%; 95% CI: 0.43%-0.50%) showed suspected TB lesions among a total of 983 (0.76%; 95% CI: 0.71%-0.81%) identified pathologies. The monthly TB detection rates ranged from 0.30% (95% CI: 0.20%-0.40%) to 0.81% (95% CI: 0.64%-0.98%) and annual rates from 0.04% (95% CI: 0%-0.11%) to 1.46% (95% CI: 1.22%-1.69%). The rates were not affected (P < 0.05) by season and fluctuating peaks were also recorded. The comparative TST revealed that bovine TB was widely distributed in live cattle (4.67%; 95% CI: 3.89%-5.44%) and was higher (χ 2 = 17.50, P ≤ 0.001) in the Western highlands than Adamawa plateaux. Comparative TST bovine TB reactors were higher (P < 0.05) in cattle managed in semi-intensive and beef production systems compared to the others. Animals in small herds showed higher (χ 2 = 4.283, P = 0.038) rates than those in large herds. Bovine TB prevalence in exotic/upgraded cattle was comparable to that of the Red Bororo zebu but higher than the rates in Guadali (χ 2 = 4.971, P = 0.026) and White Fulani (χ 2 = 5.6, P = 0.018) zebus. Among the indigenous zebus, the rate was higher in Red Bororo than the Guadali (χ 2 = 6.244, P = 0.012) and White Fulani (χ 2 = 6.568, P = 0.010). Sex did not influence (χ 2 = 0.410, P = 0.522) bovine TB prevalence in this study but diagnosis of the disease was higher (χ 2 = 5.787; P = 0.016) among adult/older cattle than in younger animals. Further analysis of the TST responses revealed that atypical mycobacterial infections was widespread and 6.83% of tested animals showed positive reactions at both bovine and avian tuberculin injection sites and a strong association (χ 2 = 2.512; P = 0.113) between skin responses to both tuberculins. The study confirms that bovine TB is prevalent in live cattle and meat production abattoirs in Cameroon and we recommend strict a interpretation of TST results for maximum diagnosis of the disease in the local environment. A need for comprehensive investigation of the molecular epidemiology, zoonotic risks and the public health importance of bovine TB in Cameroon cannot be overemphasised.

A system for the tetracycline-inducible regulation of gene expression in mycobacteria has been developed. We have sub-cloned the tetRO region from the Corynebacterium glutamicum TetZ locus into a mycobacterial shuttle plasmid, making expression of genes cloned downstream of tetRO responsive to tetracycline. Using the luxAB-encoded luciferase from Vibrio harveyi as a reporter (pMind-Lx), we observed a 40-fold increase in light output from Mycobacterium smegmatis cultures 2 h after adding 20 ng ml−1 of tetracycline. Similarly, exposure to the drug resulted in up to 20-fold increase in relative light units from M.bovis BCG carrying the reporter construct, and a 10-fold increase for M.tuberculosis. Tetracycline induction was demonstrated in log and stationary phase cultures. To evaluate whether this system is amenable to use in vivo, J774 macrophages were infected with M.bovis BCG[pMind-Lx], treated with amikacin to kill extracellular bacteria, and then incubated with tetracycline. A 10-fold increase in light output was measured after 24 h, indicating that intracellular bacteria are accessible and responsive to exogenously added tetracycline. To test the use of the tetracycline-inducible system for conditional gene silencing, mycobacteria were transformed with a pMind construct with tetRO driving expression of antisense RNA for the ftsZ gene. Bacterial cells containing the antisense construct formed filaments after 24 h exposure to tetracycline. These results demonstrate the potential of this tetracycline-regulated system for the manipulation of mycobacterial gene expression inside and outside cells.

The original Mycobacterium bovis Bacillus Calmette Guérin vaccine strain has developed into several different substrains which have been used for production of BCG vaccines throughout the world since 1921. Based on the latest genetic and antigenic knowledge, as well as the early literature reports on BCG vaccination, we are able to fit the different pieces of the BCG puzzle together and outline the origin of the different substrains of M. bovis BCG. The BCG vaccine substrains analysed demonstrate two distinct patterns, with an abrupt change consisting of a loss of several genes and altered biochemical characteristics in strains originating from Institut Pasteur after 1927. Further evidence from the literature is provided that a change occurred in virulence of the BCG parent strain at Institut Pasteur in the late 1920s. Based on this information a genealogical tree is proposed and discussed.

The word Influenza comes from mid–18th century Italian and literally means ‘influence’. Similar to how the coronavirus was once best known to cause the common cold, in 1918 Influenza was felt to be so benign a disease that it was not reportable in the United States. And it was not until 1933 that Influenza was “discovered”. So just what was causing the deaths in 1918? And why did the corona “virus” turn so deadly so quickly? Before we can be certain of anything, these questions need to be answered. But to this point, they have not been answered. In the meantime, during both events there was a deadly pandemic of tuberculosis going on, a pandemic which even in 2020 kills one person every 21 seconds for a total of at least 1.5 to 1.8 million dead in 2018 alone. Yet Robert Koch, TB’s discoverer, was repeatedly forced to call it a virus, to assuage other authorities of his time. And, as we shall soon see, the concept that only a new and virulent strain of a virus can be so infectious and kill so quickly is not only fallacious, but dangerous -and completely at odds with what used to be called the acute form of “galloping consumption” [tuberculosis] which did and still can kill in a matter of hours, days or weeks.
“Viral” influenza was informally first mentioned as being behind America’s 1918 influenza pandemic not by a direct study of the disease in humans, but rather from studies on animal diseases. In 1918, J.S. Koen, a veterinarian, observed a disease killing thousands of pigs which he believed to be the same disease as the now infamous influenza Pandemic of 1918. He felt that it was a virus. It was solely his belief. Yet new evidence and older historical findings bring up the possibility that influenza doesn’t originate from a virus –despite the indefatigable efforts, up to the present, of flu enthusiasts to viralize the 1918-19 pandemic. (Burnet F, Clark E. 1942) (Morens DM, Taubenberger JK. 2009). As we shall see, such efforts on the part of viral devotees are nothing new, and began in force with scant evidence in Great Britain during the 1st World War, also known as the Great War.

Tuberculosis continues to be an important disease both in humans and animals. It causes morbidity, mortality and economic loss worldwide. The occurrence of Mycobacterium bovis disease in humans, domesticated and wild animals confirms the relevance of this zoonosis. M. bovis in humans continues to be reported in industrialised countries and in immigrants from regions of the world where tuberculosis in cattle is endemic. The real incidence of M. bovis in humans in developing countries continues to be roughly under-estimated due to the scarcity of appropriate laboratory facilities to isolate and to differentiate M. bovis strains. In Latin America, less than 1% of tuberculosis cases are reported as being due to M. bovis. However, the economic relevance that meat and dairy industries play in these countries stimulates the promotion of bovine tuberculosis eradication programmes. Human-to-human airborne transmission of M. bovis does occur and it may be important where human immunodeficiency virus (HIV) infection in humans is prevalent, M. bovis infection in cattle is enzootic and pasteurisation of dairy products is not routinely practised. Eradication of M. bovis in cattle and pasteurisation of dairy products are the cornerstones of prevention of human disease. Measures should be developed to identify and control M. bovis infection in wild animals as these may be important reservoirs of infection for domesticated food-producing animals. There is a need for medical and veterinary professionals to cooperate on disease outbreaks. The information presented herein strongly supports the ‘One World/One Health/One Medicine’ concept.

The immunostimulatory activity of lipids associated with the mycobacterial cell wall has been recognized for several decades and exploited in a large variety of different adjuvant preparations. Previously, we have shown that a mycobacterial lipid extract from Mycobacterium bovis bacillus Calmette-Guérin delivered in cationic liposomes was a particular efficient Th1-inducing adjuvant formulation effective against tuberculosis. Herein, we have dissected the adjuvant activity of the bacillus Calmette-Guérin lipid extract showing that the majority of the activity was attributable to the apolar lipids and more specifically to a single lipid, monomycoloyl glycerol (MMG), previously also shown to stimulate human dendritic cells. Delivered in cationic liposomes, MMG induced the most prominent Th1-biased immune response that provided significant protection against tuberculosis. Importantly, a simple synthetic analog of MMG, based on a 32 carbon mycolic acid, was found to give rise to comparable high Th1-biased responses with a major representation of polyfunctional CD4 T cells coexpressing IFN-␥, TNF-␣, and IL-2. Furthermore, comparable activity was shown by an even simpler monoacyl glycerol analog, based on octadecanoic acid. The use of these synthetic analogs of MMG represents a promising new strategy for exploiting the immunostimulatory activity and adjuvant potential of components from the mycobacterial cell wall without the associated toxicity issues observed with complex mycobacterial preparations.

To our knowledge, this is the first sequential study of cytokines in tissue sections of developing and healing tuberculous (BCG) lesions. In situ hybridization, immunohistochemical, and RT-PCR techniques were used. Cytokine mRNAs showed a biphasic pattern. The percentage of mononuclear cells (MN) containing IL-1␤, TNF-␣, MCP-1, and IL-8 mRNAs was highest in 1-to 3-day lesions, apparently because of the nonspecific inflammatory response caused by the tubercle bacilli in the BCG vaccine. At 5 days, this percentage was significantly reduced. With IFN-␥, the peak and trough were delayed by 2 days. By 9 days, the percentage of MN containing the mRNAs of all five cytokines had again increased and the rabbits had become tuberculin-positive. In general, MCP-1 and TNF-␣ proteins and the vascular adhesion molecules, ICAM, VCAM, and perhaps ELAM, peaked at about 3 days. Many mononuclear cells surrounding the central areas of solid and liquefied caseous necrosis contained chemokine IL-8 mRNA. IL-8 is known to attract PMN, and PMN were present nearby. In contrast, MN containing chemokine MCP-1 mRNA were present more peripherally in areas rich in macrophages and lymphocytes. The early nonspecific cytokine response seems to be an adjuvant effect of the mycobacteria in BCG vaccine in that it causes a rapid entry of macrophages, lymphocytes, granulocytes, and probably dendritic cells into local sites of antigen deposition. This effect should be considered in developing improved vaccines for the prevention of tuberculosis, because BCG vaccines producing a strong early cytokine response should be more immunogenic than BCG vaccines with similar antigens producing a weak response.

The Mycobacterium bovis BCG vaccine for commercial use is classically produced as surface pellicles by culture on synthetic medium. Under these conditions, reproducibility of the cultures and quality assessment are hampered by slow growth of the bacilli, the formation of bacterial aggregates and a high proportion of dead bacilli after processing and final formulation of the vaccine. Here, we established dispersed cultures of M. bovis BCG in synthetic media in small-scale bioreactors. These cultures allow recording and adjusting of culture parameters and give rise to single bacilli with a high degree of live bacteria. In the murine model, bioreactor-grown M. bovis BCG exhibited slightly stronger replication and persistence than the vaccine produced under the classical conditions. The protective efficacy against challenge with M. tuberculosis was identical for both vaccine preparations.

From 2005 to 2007, Mycobacterium tuberculosis complex (MTC) strains were isolated from cattle, goats and pigs samples collected at the Bodija abattoir and from human samples from tuberculosis patients and livestock traders at the Akinyele cattle market in Ibadan, Southwestern Nigeria. Seventy four isolates obtained from humans (24) and livestock (50) were identified as MTC strains. Thirty two isolates were spoligotyped. Nineteen of these 32 isolates were identified as M. tuberculosis whilst 13 were identified as Mycobacterium bovis. M. bovis was isolated from two humans, whereas M. tuberculosis was isolated from a bovine, a pig and a goat. All the M. bovis isolates identified in this study belonged to the Africa 1 clonal complex. Multiple locus VNTR [variable number of tandem repeats] analysis (MLVA) was carried out on the 74 isolates. Three major clusters were defined. Group A consisted of 24 M. tuberculosis isolates (MLVA genotypes 1-18). One strain was isolated from a bovine and one from a pig. Group B consisted of 49 M. bovis strains (MLVA genotypes 19-48), mainly of cattle origin but also included four goat, nine pig and two human isolates. Group C consisted of a single M. tuberculosis isolate (MLVA genotype 49) obtained from a goat. Spoligotyping and MLVA confirmed it as clustering with the East Africa Indian clade found in humans in Sudan and the Republic of Djibouti. The isolation of three M. tuberculosis strains from livestock raises the question of their epidemiological importance as a source of infection for humans.

Background: Bovine Tuberculosis (BTB) is a widespread and endemic disease of cattle in Ethiopia posing a significant threat to public health. Regular surveillance by skin test, bacteriology and molecular methods is not feasible due to lack of resource. Thus, routine abattoir (RA) inspection will continue to play a key role for national surveillance. We evaluated efficiency of RA inspection for diagnosis of Mycobacterium bovis infection and discussed its public health implications in light of a high risk of human exposure. Methods: The study was conducted in five abattoirs: Addis Ababa, Adama, Hawassa, Yabello and Melge-Wondo abattoirs. The efficiency of routine abattoir (RA) inspection was validated in comparison to detailed abattoir (DA) inspection, followed by culture and microscopy (CM) and region of difference (RD) deletion analysis. Diagnostic accuracies (with corresponding measures of statistical uncertainty) were determined by computing test property statistics (sensitivity and specificity) and likelihood estimations using web-based SISA diagnostic statistics software. Post-test probability of detecting TB infected carcasses was estimated using nomograms. Agreement between RA and DA inspections was measured using kappa statistics. The study was conducted and reported in accordance with standards for reporting of diagnostic accuracy (STARD) requirements. Both routine and detailed meat inspection protocols were performed on a subpopulation of 3322 cattle selected randomly from among 78,269 cattle slaughtered during the study period. Three hundred thirty seven carcasses identified through detailed meat inspection protocols were subjected to culture and microscopy; of the 337, a subset of 105 specimens for culture and microscopy were subjected to further molecular testing. Results: There was a substantial agreement between RA and DA inspections in Addis Ababa (Kappa = 0.7) and Melge-Wondo abattoirs (Kappa = 0.67). In Adama, Hawassa and Yabello abattoirs, the agreement was however poor (Kappa ≤ 0.2). RA inspection was able to detect only 117 of the total 3322 carcasses inspected (3.5%). The sensitivity (Sn) and specificity of RA inspection were 28.2% (95/337) [95%CI: 23.4-33.0] and 99.3% (2963/2985) [95%CI: 99.0-99.6], respectively, when DA inspection was considered as reference test. When culture and microscopy (CM) was considered as reference test, the Sn and Sp of RA were 55.2% (58/105) [95%CI: 45.7-64.7] and 84.1% (195/232) [95%CI: 79.3-88.8]. RA inspection failed to detect 71.8% (242/337) and 44.8% (47/105) of TB infected carcasses as judged by DA inspection and CM, respectively. On the other hand, a much higher sensitivity of DA was obtained when CM and RD deletion analysis were considered as reference tests (96.3% (105/109) and 100.0% (24/24), respectively).

The emergence of wildlife reservoirs of Mycobacterium bovis infection in cattle as well as increased interregional trade with associated spread of M. bovis has led to renewed interest in the use of vaccines for the control of bovine tuberculosis (TB). Field efficacy trials performed in the early 20th century demonstrated the partial effectiveness of bacilli Calmette-Guerin (BCG) for the control of bovine TB. Recent experimental trials with cattle have demonstrated that: (1) subunit vaccines may boost immunity elicited by BCG in cattle, (2) T cell central memory immune responses evoked by protective vaccines correlate with protection upon subsequent M. bovis challenge, (3) BCG is particularly protective when administered to neonates, and (4) differentiation of infected from vaccinated animals (DIVA) is feasible in cattle using in vitro or in vivo methods. In regards to wildlife reservoirs, the efficacy of BCG delivered orally has been demonstrated for brushtail possums (in field trials) as well as Eurasian badgers, wild boar, and white-tailed deer (each in experimental challenge studies). Vaccine delivery to wildlife reservoirs will primarily be oral, although a parenteral route is being deployed for badgers in England. Vaccine efficacy trials, both experimental challenge and field studies, with cattle and their wildlife reservoirs represent a primary example of the one health approach, with outcomes relevant for both veterinary and medical applications.

Tuberculosis (TB) is a leading cause of death worldwide despite the availability of effective chemotherapy for over 60 years. Although Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccination protects against active TB disease in some populations, its efficacy is suboptimal. Development of an effective TB vaccine is a top global priority that has been hampered by an incomplete understanding of protective immunity to TB. Thus far, preventing TB disease, rather than infection, has been the primary target for vaccine development. Several areas of research highlight the importance of including preinfection vaccines in the development pipeline. First, epidemiology and mathematical modeling studies indicate that a preinfection vaccine would have a high population-level impact for control of TB disease. Second, immunology studies support the rationale for targeting prevention of infection, with evidence that host responses may be more effective during acute infection than during chro...

Bovine tuberculosis (TB), mainly caused by Mycobacterium bovis, is a zoonotic disease with implications for Public Health and having an economic impact due to decreased production and limitations to the trade. Bovine TB is subjected to official eradication campaigns mainly based on a test and slaughter policy using diagnostic assays based on the cell-mediated immune response as the intradermal tuberculin test and the gamma-interferon (IFN-γ) assay. Moreover, several diagnostic assays based on the detection of specific antibodies (Abs) have been developed in the last few years with the aim of complementing the current diagnostic techniques in the near future. This review provides an overview of the current ante-mortem diagnostic tools for diagnosis of bovine TB regarding historical background, methodologies and sensitivity (Se) and specificity (Sp) obtained in previous studies under different epidemiological situations.

We have devised a novel approach to active immunotherapy based on modification of autologous cancer cells with the hapten, dinitrophenyl (DNP). The treatment program consists of multiple intradermal injections of DNP-modified autologous tumor cells mixed with BCG. Administration of DNP-vaccine to patients with metastatic melanoma induces a unique reaction - the development of inflammation in metastatic masses. Histologically, this consists of infiltration of T lymphocytes, most of which are CD8+. These T cells usually produce gamma interferon in situ. Moreover, they represent expansion of T cell clones with novel T cell receptor structures. Occasionally, administration of DNP-vaccine results in partial or complete regression of measurable metastases. The most common site of regression has been small lung metastases. Administration of DNP-vaccine to patients in the post-surgical adjuvant setting produces a more striking clinical effect. We have treated 214 patients with clinically ev...

This study shows a representative stratified cluster sample survey of the prevalence of comparative intradermal tuberculin test in cattle from four regions in Ethiopia. Using a cut-off for positivity of 2 mm, it assesses possible risk factors for tuberculin-positive reaction in cattle. Seventy-three villages in 24 kebeles (administrative units) were randomly selected, from which 2216 cattle from 780 owners were tested. In addition, 450 of these cattle owners were interviewed for risk factor assessment. Ninety-nine percent of the tested cattle in this rural livestock production system were traditional zebus. The individual overall prevalence of cattle bovine tuberculosis (BTB)e was 3%, with the highest found in Meskan Mareko, in Central Ethiopia (7.9%) and the lowest in Woldia, in the North East edge of the Rift Valley (1.2%). Generalised Linear Mixed Models (GLMM) with random effect on kebeles was used to analyse risk factors of cattle reactors and human tuberculosis (TB) infection....

We performed spoligotyping and 12-mycobacterial interspersed repetitive unit-variable number tandem repeats (MIRU-VNTRs) typing to characterise Mycobacterium bovis isolates collected from tissue samples of bovines with lesions suggestive for tuberculosis during slaughter inspection procedures in abattoirs in Brazil. High-quality genotypes were obtained with both procedures for 61 isolates that were obtained from 185 bovine tissue samples and all of these isolates were identified as M. bovis by conventional identification procedures. On the basis of the spoligotyping, 53 isolates were grouped into nine clusters and the remaining eight isolates were unique types, resulting in 17 spoligotypes. The majority of the Brazilian M. bovis isolates displayed spoligotype patterns that have been previously observed in strains isolated from cattle in other countries. MIRU-VNTR typing produced 16 distinct genotypes, with 53 isolates forming eight of the groups, and individual isolates with unique VNTR profiles forming the remaining eight groups. The allelic diversity of each VNTR locus was calculated and only two of the 12-MIRU-VNTR loci presented scores with either a moderate (0.4, MIRU16) or high (0.6, MIRU26) discriminatory index (h). Both typing methods produced similar discriminatory indexes (spoligotyping h = 0.85; MIRU-VNTR h = 0.86) and the combination of the two methods increased the h value to 0.94, resulting in 29 distinct patterns. These results confirm that spoligotyping and VNTR analysis are valuable tools for studying the molecular epidemiology of M. bovis infections in Brazil. Romano MI 1999. Usefulness of spoligotyping in molecular epidemiology of Mycobacterium bovis -related infections in South America. J Clin Microbiol 37: 296-303.

A study to determine the secretion of Mycobacterium spp. in milk from indigenous cattle was carried out in pastoral cattle reared in the Southern Highlands to Tanzania. The study was aimed at elucidating the dangers associated with milk-borne zoonoses in a society where milk is normally consumed raw. Out of 805 milk samples, 31 (3.9%) were positive for mycobacteria. There was a preponderance of atypical mycobacteria (87%) whereas only two isolates (6.5%) were confirmed as M. bovis. Atypical mycobacteria included: M. terrae (n = 7), M. fortuitum (n = 2), M. flavescens (n = 13), M. gordonae (n = 1) and M. smegmatis (n = 4). Although the number of M. bovis positive samples was low, the habit of pooling milk may still pose great public health dangers to milk consumers in this part of the world. Moreover, isolation of atypical mycobacteria should also be considered to be a danger to human health in countries such as Tanzania, where the number of people with lowered immunity due to HIV in...

A cross sectional study was conducted on 906 apparently healthy camels slaughtered at Akaki and Metehara abattoirs to investigate the pathology of camel tuberculosis (TB) and characterize its causative agents using postmortem examination, mycobacteriological culturing, and multiplex polymerase chain reaction (PCR), region of difference-4 (RD4)-based PCR and spoligotyping. The prevalence of camel TB was 10.04% (91/906) on the basis of pathology and it was significantly higher in females (x 2 = 4.789; P = 0.029). The tropism of TB lesions was significantly different among the lymph nodes (x 2 = 22.697; P = 0.002) and lung lobes (x 2 = 17.901; P = 0.006). Mycobacterial growth was observed in 34% (31/91) of camels with grossly suspicious TB lesions. Upon further molecular characterization using multiplex PCR, 68% (21/31) of the colonies showed a positive signal for the genus Mycobacterium, of which two were confirmed Mycobacterium bovis (M. bovis) by RD4 deletion typing. Further characterization of the two M. bovis at strains level revealed that one of the strains was SB0133 while the other strain was new and had not been reported to the M. bovis database prior to this study. Hence, it has now been reported to the database, and designated as SB1953. In conclusion, the results of the present study have shown that the majority of camel TB lesions are caused by mycobacteria other than Mycobacterium tuberculosis complex. And hence further identification and characterization of these species would be useful towards the efforts made to control TB in camels.

The extracytoplasmic function (ECF) sigma factors constitute a diverse group of alternative sigma factors that have been demonstrated to regulate gene expression in response to environmental conditions in several bacterial species. Genes encoding an ECF sigma factor of Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium smegmatis, designated sigE, were cloned and analyzed. Southern blot analysis demonstrated the presence of a single copy of this gene in these species and in Mycobacterium bovis BCG, Mycobacterium leprae, and Mycobacterium fortuitum. Sequence analysis showed the sigE gene to be highly conserved among M. tuberculosis, M. avium, M. smegmatis, and M. leprae. Recombinant M. tuberculosis SigE, when combined with core RNA polymerase from M. smegmatis, reconstituted specific RNA polymerase activity on sigE in vitro, demonstrating that this gene encodes a functional sigma factor. Two in vivo transcription start sites for sigE were also identified in M. smegmatis and M. bovis BCG. Comparison of wild-type M. smegmatis with a sigE mutant strain demonstrated decreased survival of the mutant under conditions of high-temperature heat shock, acidic pH, exposure to detergent, and oxidative stress. An inducible protective response to oxidative stress present in the wild type was absent in the mutant. The mycobacterial SigE protein, although nonessential for viability in vitro, appears to play a role in the ability of these organisms to withstand a variety of stresses.

We determined the prevalence of bovine tuberculosis in Jigawa State, northwestern Nigeria using the comparative intradermal tuberculin test in 22 herds consisting of 922 cattle with overall and individual prevalence of 1.08% (10/922) and 45.45% (10/22) obtained, respectively.

Although tuberculosis is endemic in cattle in Ethiopia, little information is available on tuberculosis in dromedaries. Thus, this study was designed to investigate the epidemiology of tuberculosis and its causative agents in dromedaries slaughtered at four representative abattoirs in eastern Ethiopia. A total of 293 dromedaries were examined by detailed postmortem examination and the prevalence of tuberculosis-compatible lesion (TCL) was 12.3% (36/293), and occurrence of lesion was significantly associated with female dromedaries (95% confidence interval (CI) [0.19–0.97]). Mycobacteria were isolated in 61% (22/36) of the dromedaries with gross lesions. Further characterization of the isolates using PCR showed that 68% (15/22) of the isolates were non-tuberculosis mycobacteria (NTM) while 13.6% (3/22) were Mycobacterium tuberculosis (M. tuberculosis) and 18% (4/22) were not members of the genus Mycobacterium. Spoligotyping of the three M. tuberculosis isolates revealed that one of the three isolates was SIT 21 while the remaining two isolates with octal values of 773357776763671 and 773357777763661 were not reported to the SITVIT database. The isolation of large proportion of NTM from tuberculosis-compatible lesions in dromedary is suggestive of these bacteria being pathogenic to the species, while the isolation of M. tuberculosis from dromedary carcasses highlights the zoonotic risk represented by consuming the meat.

Because of its immunodominancy, and because it is conserved in different geographical isolates of Plasmodiumfalciparum, the repetitive sequence of the circumsporozoite protein, (Asn-Ala-Asn-Pro). [(NANP).], has been envisaged for the development of an anti-falciparum malaria subunit vaccine. However, the murine immune response to (NANP),

The role of wildlife in tuberculosis epidemiology is being widely studied since it can affect the effectiveness of eradication campaigns in cattle. The health problem is enhanced when it concerns also wildlife welfare and biodiversity conservation. This study was performed to understand the epidemiology of Mycobacterium bovis population affecting livestock and wild animals in the Doñana National Park using bacteriology and molecular characterisation techniques. Tuberculosis research was performed on 1209 cattle and wild animals (artiodactyla and carnivore) collected over 6 years in the Park. One hundred and sixty-three animals were found to be infected with M. bovis, comprising 7.96% of the cattle and 20.53% of the wild animals tested. Spoligotyping revealed nine patterns, being SB1232 and SB1230 the most prevalent (77.30% and 15.34% of infected animals, respectively). MIRU-VNTR analysis of a selected panel of 92 isolates showed eight different profiles, including several spoligotypes within the same MIRU-VNTR profile. The discriminatory capacity of both techniques in this panel was similar. The results obtained by combination of both techniques corroborate that wildlife species are infected with the M. bovis strains which are more prevalent in cattle and reveal their persistence. Genotype variation between isolates strongly suggests micro-evolutionary events in the M. bovis population in the same area. This study in the Doñana National Park exposes the risk of introduction of domestic animals into wildlife areas when there is not a warranty of disease freedom, appropriate diagnostic techniques and control measures.

Please cite this article as: Santema, W., Overdijk, M., Barends, J., Krijgsveld, J., Rutten, V., Koets, A., Searching for proteins of Mycobacterium avium subspecies paratuberculosis with diagnostic potential by comparative qualitative proteomic analysis of mycobacterial tuberculins, Veterinary Microbiology (2008),