Bacterial dissemination in Mycobacterium tuberculosis by CD+ T-cells & proinflammatory cytokines

Background & objectives: As CD4+ and CD8+ T lymphocyte numbers decline, the conventional, localized forms of tuberculosis shift to the atypical, disseminated forms. Variations in lymphocyte and immune cell expression levels affect how tuberculosis manifests in disseminated forms. Understanding the relationship between lymphocyte counts (CD4+ and CD8+) and pro-inflammatory cytokines such as tumour necrosis factor-alpha, interleukin-12 and interferon, we may therefore be able to shed light on how infections spread and suggest potential biomarkers for these immune factors. Methods: In this study, 15 guinea pigs were infected with Mycobacterium tuberculosis (M.tb) H37Rv strain and grouped into three groups of five each for further investigation. Serum samples and bronchoalveolar lavage (BAL) fluid were examined for the expression of pro-inflammatory cytokines and T-cell subsets in guinea pigs infected with pulmonary tuberculosis and disseminated tuberculosis. Results: We found that M.tb escapes macrophages due to pro-inflammatory cytokine dysregulation. Despite the protective immunity created by T-cells and cytokines, M.tb bacilli may spread to other organs due to inflammation induced by these immune components. A high number of T-cells and stimulated cytokine production are involved in triggering inflammation after necrotic tissue develops and tuberculosis spreads. Interpretation & conclusions: Our findings imply that increased bacilli in the spleen at the 8th wk of infection may be caused by the overexpression of CD4+ T-cell lymphocyte subsets and cytokines that generated inflammation during the 4th wk of infection. This is a pilot study with a small sample size and less assertive inference. Larger studies would be helpful to validate the results of the present investigation.

Mycobacterium bovis induced human tuberculosis in India: Current status, challenges & opportunities

Tuberculosis (TB) caused by Mycobacterium tuberculosis is a leading cause of human deaths due to any infectious disease worldwide. However, infection of Mycobacterium bovis, primarily an animal pathogen, also leads to the development of ‘human tuberculosis’. Infected animals have been considered the major source of M. bovis infection and humans get exposed to M. bovis through close contact with infected animals or consumption of contaminated milk, unpasteurized dairy products and improperly cooked contaminated meat. The information on the global distribution of bovine TB (bTB) is limited, but the disease has been reported from all the livestock-producing middle- and low-income countries of the world. In recent years, there is a renewed interest for the control of bTB to minimize human infection worldwide. In India, while the sporadic presence of M. bovis has been reported in domestic animals, animal-derived food products and human beings from different geographical regions of the country, the information on the national prevalence of bTB and transmission dynamics of zoonotic TB is, however, not available. The present article reviewed published information on the status of M. bovis-induced zoonotic TB to highlight the key challenges and opportunities for intervention to minimize the risk of M. bovis infection in humans and secure optimum animal productivity in India.

Single-nucleotide polymorphisms in CLEC7A, CD209 and TLR4 gene and their association with susceptibility to paratuberculosis in Indian cattle

The aim of this study was to identify the single-nucleotide polymorphisms (SNPs) in bovine candidate genes CLEC7A, CD209 and TLR4, and explore the association between these SNPs with the occurrence of bovine paratuberculosis (PTB) disease. For this purpose, 549 animals were screened by a panel of four diagnostic tests, namely Johnin PPD test, ELISA test, faecal microscopy and IS900 blood PCR against Mycobacterium avium ssp. paratuberculosis (MAP) to develop case–control populations. SNPs were genotyped by polymerase chain reaction-restriction fragment length polymorphism method. Genotypic–phenotypic associations were assessed by the PROCLOGISTIC procedure of SAS 9.3. Of the seven SNPs; rs110353594 in CLEC7A gene and rs8193046 in TLR4 gene were found to be associated with PTB. For SNP rs110353594, odds of CC and CT genotypes vs TT genotype was 1.543 (0.420–5.667; 95% CI) and 0.284 (0.104–0.774; 95% CI), respectively which means that CT genotype was more resistant than TT and CC genotypes against bovine PTB. For SNP rs8193046, odds of AA and AG genotypes versus GG genotype was 0.947 (0.296–3.034; 95% CI) and 3.947 (1.555–10.022; 95% CI), respectively, i.e. probability for getting an infection in animals with AG genotype was 3.94 times more as compared to GG genotype. Hence, a selection programme favouring CT genotype for rs110353594 and against AG genotype for rs8193046 may be beneficial for conferring resistance against bovine PTB

Recombinant secretory proteins based new ‘Cocktail ELISA’ as a marker assay to differentiate infected and vaccinated cows for Mycobacterium avium subspecies paratuberculosis infection

Johne's disease is endemic in the domestic livestock population of India. Recently, we developed highly effective 'Indigenous vaccine' to control Johne’s disease in animals. In order to gain disease free status as per World Organization for Animal Health, it is essential to have a marker assay to differentiate between infected and vaccinated animals before vaccine can be used in the field. We have developed a new marker assay ‘Cocktail ELISA’ using six ‘recombinant secretary proteins’ (MAP 1693c, MAP 2168c, MAP Mod D, MAP 85c, MAP Pep AN and MAP Pep AC) and evaluated for diagnosis of Johne’s disease along with 'Indigenous ELISA kit'. This ‘Cocktail ELISA’ successfully differentiated the infected, vaccinated and healthy (non-infected) cows and will facilitate the use of Johne’s disease vaccine to control the disease in cows at national levels.

‘Indigenous’ and ‘Ethanol Vortex’ ELISA kits for diagnosis of Mycobacterium avium subsp. paratuberculosis infection in cattle: Is there a ‘globally relevant kit’ in the ‘Reverse Ice-burg’ environment?

Highly versatile and robust 'Indigenous ELISA kit' for the diagnosis of Mycobacterium avium subsp. paratuberculosis infection in cattle herds was compared with 'Ethanol Vortex (EV) ELISA kit' of USA. Of 160 (118 vaccinated and 42 non-vaccinated) cattle screened, 129 and 35 were positive in 'Indigenous' and 'Ethanol Vortex' ELISA kits, respectively. 'I-ELISA', using 'semi-purified protoplasmic antigen' from native highly prevalent biotype ('Indian Bison type') of MAP of goat origin, was highly sensitivitive (91.4%) as compared to the 'EV-ELISA. 'I-ELISA kit using whole cell sonicate from native 'S 5' (‘Indian Bison type’) strain of MAP as 'antigen source' was significantly superior than EV-ELISA kit using surface antigens from 'Linda' strain ('cattle type') of cattle origin in USA. Therefore, 'i-ELISA kit' may be recommended for the screening of domestic cattle herds against MAP infection in India. The present study has demonstrated that in the diagnosis of chronic infections and diseases, such as Johne's disease, 'Indigenous kits' are significantly superior to kits made in other countries, EV-ELISA, in the present case, particularly screening of native cattle herds endemically infected with MAP.

Evaluation of goat based ‘Indigenous vaccine’ against Bovine Johne’s Disease in endemically infected native cattle herds.

‘Indigenous vaccine’ prepared from ‘Indian Bison Type’ a native bio-type of Mycobacterium avium subspecies paratuberculosis strain ‘S5’ of goat origin (goat based) was evaluated in indigenous cattle herds located in gaushalas (cow shelters), endemic for Bovine Johne’s disease. Cows (893) were randomly divided into vaccinated (702 = 626 adults + 76 calves) and control (191 = 173 adults + 18 calves) groups. Response to vaccination was evaluated on the basis of health (mortality, morbidity), productivity (growth rate, reproductive performance, total milk yield), immunological parameters (LTT, ELISA titer), survivability of animals naturally infected with MAP, bacterimia (by specific blood PCR), sero-conversion (by indigenous ELISA) and status of shedding of MAP in feces (by microscopy) in the two groups before and after vaccination. Reduction in MAP shedding [to the extent of 100% in Herd A; and from 82.1% (0 DPV) to 10.7% (270 DPV) in Herd C] was the major finding in vaccinated cows. Whereas, the control group cows have shown no improvement. As the first indicator of vaccine efficacy, MAP bacilli disappeared from the blood circulation as early as 15 days post vaccination, however, peak titers were achieved around 90 DPV. Peak titers initially declined slightly but were maintained later throughout the study period. Control animals did not show any pattern in antibody titers. Mortality was low in vaccinated as compared to the control groups. Vaccination of endemically infected native cattle herds with inactivated whole-cell bacterin of novel ‘Indian Bison Type’ bio-type of goat origin strain ‘S5’ effectively restored health and productivity and reduced clinical BJD. Application of goat based ‘indigenous vaccine’ for therapeutic management of BJD in native cattle herds (gaushalas) is the first of its kind.

Application of IS1311 locus 2 PCR-REA assay for the specific detection of ‘Bison type’ Mycobacterium avium subspecies paratuberculosis isolates of Indian origin.

Background & objectives: Of the three major genotypes of Mycobacterium avium subspecies paratuberculosis (MAP), ‘Bison type’ is most prevalent genotype in the domestic livestock species of the country, and has also been recovered from patients suffering from Crohn’s disease. Recently, a new assay based on IS1311 locus 2 PCR- restriction endonuclease analysis (REA) was designed to distinguish between ‘Indian Bison type’ and non-Indian genotypes. The present study investigated discriminatory potential of this new assay while screening of a panel of MAP isolates of diverse genotypes and from different geographical regions. Methods: A total of 53 mycobacterial isolates (41 MAP and 12 mycobacterium other than MAP), three MAP genomic DNA and 36 MAP positive faecal DNA samples from different livestock species (cattle, buffaloes, goat, sheep and bison) and geographical regions (India, Canada, USA, Spain and Portugal) were included in the study. The extracted DNA samples (n=92) were analyzed for the presence of MAP specific sequences (IS900, ISMav 2 and HspX) using PCR. DNA samples were further subjected to genotype differentiation using IS1311 PCR-REA and IS1311 L2 PCR-REA methods. Results: All the DNA samples (except DNA from non-MAP mycobacterial isolates) were positive for all the three MAP specific sequences based PCRs. IS1311 PCR-REA showed that MAP DNA samples of Indian origin belonged to ‘Bison type’. Whereas, of the total 19 non-Indian MAP DNA samples, 2, 15 and 2 were genotyped as ‘Bison type’, ‘Cattle type’ and ‘Sheep type’, respectively. IS1311 L2 PCR-REA method showed different restriction profiles of ‘Bison type’ genotype as compared to non-Indian DNA samples. Interpretation & conclusions: IS1311 L2 PCR-REA method successfully discriminated ‘Indian Bison type’ from other non-Indian genotypes and showed potential to be future epidemiological tool and for genotyping of MAP isolates.

Evaluation of ‘cattle’ and ‘Indian Bison’ type antigens of Mycobacterium avium subspecies paratuberculosis for diagnosis of Bovine Johne’s Disease using ‘indigenous ELISA’ and AGPT .

Two antigens (‘cattle’ type and ‘Indian Bison’ type) of Mycobacterium avium subspecies paratuberculosis were evaluated for diagnosis of Johne’s disease (JD) in a gaushala (cattle herd). Of the 160 cows of Sahiwal and Hariana breeds screened, 81 (50.6%) tested positive in ELISA and 66 (41.8%) in AGPT test. Using the two antigens, 33.5% tested positive in both the tests while 41.1% tested negative. Exclusively, only 8.2% tested positive in ELISA while 17.1% tested positive in AGPT. Two antigens together detected 58.9% prevalence of MAP in the gaushala. Individually, indigenous ELISA using antigen from native source of MAP proved superior to AGPT in the diagnosis of JD in cows.