Interferon gamma and IP 10 mRNA quantitative real time PCR in whole blood culture of guinea pig and cattle to multi-antigen recombinant protein cocktail and PPD of Mycobacterium bovis (3/86Rv)

In vivo tuberculin skin test and in vitro IFNγ assays are the most explored diagnostics for Mycobacterium bovis infection (Bovine Tuberculosis) in animals. However, there are other potential biomarkers like IP10 which may allow diagnosis of tuberculosis. In this study, IFNγ and IP10 mRNA responses in guinea pig and cattle were determined by quantitative RT-PCR in whole blood samples stimulated PPDs and recombinant protein cocktail containing eight purified rCFP2, rESAT6, rCFP10, rMTC28, rMPB53, rMPB63, rMPB64 and rMPB83 proteins. Blood samples were obtained from M. bovis sensitised guinea pigs and tuberculin test positive cattle. Both guinea pigs and cattle blood cultures produced a significant increase in mRNA level of IFNγ and IP10 when stimulated with protein cocktail as compared to that of bovine PPD. Upon different antigenic stimulations, IP10 mRNA responses were followed the similar kinetics to those of IFNγ with high correlation (r = 0.97 for guinea pig and 0.96 for cattle)

PCR-SSCCP analysis in detecting point mutations targeting rpoB, katG and inhA genes for determining multi-drug resistance in Mycobacterium bovis and Mycobacterium tuberculosis strains

The usefulness of polymerase chain reaction-single stranded confirmation polymorphism (PCR-SSCP) for determination of rifampicin and isoniazid resistance in Mycobacterium tuberculosis and M. bovis cultures from human and animal origin was investigated. Mycobacteria (81) in the study included, viz. 12 MDR-TB samples, 35 sputum samples, 3 lung and lymphnode tissues from bovines and 11 M. tuberculosis and 18 M. bovis culture, M. tuberculosis H37Rv and M. bovis BCG strain). All the mycobacterial cultures were characterized on growth characteristics, biochemical test pattern, MTB complex specific IS6110 PCR and species specific 12.7 kb multiplex PCR. PCR-SSCP was used to determine resistance against rifiampin by targeting rpoB gene (305 bp) and isoniazid by targeting katG (237 bp) and inhA (261 bp). Rifampicin resistance was detected by PCR-SSCP in 1 out of 12 MDR-TB samples (8.3%), while isoniazid resistance was detected in 66.7% of MDR-TB samples using PCRSSCP of katG and 75% of MDR-TB samples using inhA SSCP analysis

A preliminary assessment of new bovine and avian tuberculin in cattle by intra-dermal tests and whole blood interferon gamma assay

The present study reports ante-mortem diagnosis of bovine tuberculosis employing tuberculin test using a new tuberculin PPDB (M. bovis, 3/86) and interferon gamma assay in cattle whole blood culture stimulated using a commercial bovine IFN- γ assay kit. Dairy cattle (87) categorized in groups A, B and C were tested using tuberculin test and gamma interferon (IFN-γ). Fresh batches of bovine PPD (from M. bovis 3/86) and avian PPD (M. avium N–17) were prepared and were standardized by conforming to sterility, safety and potency. For tuberculin testing cervical single intradermal test (SIT) and caudal fold test (CFT) were performed using tuberculins at concentration of 1mg/ml and at the concentration of 0.3mg/ ml as a stimulating antigens in IFN-γ test. Out of 80 cattle (group A + B), 44 (55%) were reactor to tuberculin, 21 (26.25%) non reactor and 15 (18.75%) were inconclusive animals and by IFN-γ test 36 (45%) were M. bovis positive, 10 (12.5%) negative, 13 (16.25%) inconclusive and 21 (26.25%) were M. avium positive animals. In group C (not tuberculin tested) only 4 (57.14%) out of 7 cattle were positive. Relative sensitivity of IFN-γ test (63.88%) was higher than the tuberculin test (52.27%). Our study indicated that skin tests (CFT and SIT) and IFN-γ test can be used for successful control and eradication of bovine tuberculosis as IFN-γ test had higher sensitivity

Comparative evaluation of pncA gene, IS6110 and 12.7-Kb fragment based PCR assays for simultaneous detection of Mycobacterium tuberculosis complex (M. tuberculosis and M. bovis) in cultured strains and clinical specimens

192-199PCR based molecular techniques help in discrimination of two closely related Mycobacterium tuberculosis and M.bovis. Here, we analyzed 24 M. bovis, 39 M. tuberculosis, 21 fresh acid-fast positive sputum samples and standardmycobacterial strains with pncA, 12.7 Kb and IS6100 based PCR assays. DNA from cultures and sputum yielded a positiveamplification of 185 bp with M. tuberculosis specific reverse primer pncAMT-2 but not with M. bovis specific reverseprimer pncAMB-2 and all M. bovis strains showed a positive amplification of 185 bp with M. bovis specific reverse primerpncAMB-2 but not with M. tuberculosis specific reverse primer pncAMT-2. The 12.7 Kb fragment based PCR performed onDNA extracted from cultures of M. tuberculosis and sputum yielded product of 168 bp while M. bovis showed 262 bpproducts. M. tuberculosis complex specific IS6110 PCR assay performed on DNA extracted from M. tuberculosis, M. boviscultures and sputum samples yielded M. tuberculosis complex specific 123-bp amplified products. The sequence analysis ofrepresentative PCR products of IS6110 and 12.7 Kb fragment showed 99-100% and 100% identity in amplicon products,respectively. To test reliability of primers, M. tuberculosis and M. bovis cultures were mixed and subjected to IS6110, pncAand 12.7 Kb PCR assay. pncA primers could not successfully and reliably discriminate the mixed culture, however, 12.7 Kbfragment primers successfully discriminated the mixed culture of M. tuberculosis and M. bovis

Random amplified polymorphic DNA (RAPD) analysis of Mycobacterium tuberculosis strains in India

The usefulness of random amplification of polymorphic DNA (RAPD) analysis for typing Indian strains of M. tuberculosis was investigated. M. tuberculosis H37Rv, M. tuberculosis DT and 42 clinical isolates of M. tuberculosis were subjected to RAPD-PCR using 7 random decamer primers. All 7 primers were found to be differentiated and produced specific RAPD profiles. The polymorphic amplicons served as RAPD markers for M. tuberculosis. The dendrograms, obtained by different primers, showed the discriminatory ability of the primers. RAPD analysis provided a rapid and easy means of identifying polymorphism in M. tuberculosis isolates, and it was found to be a valuable alternative epidemiological tool. In addition, the results of the present study showed heterogeneity in the M. tuberculosis strains in the population studied

Evaluation and comparison of native and recombinant LipL21 protein-based ELISAs for diagnosis of bovine leptospirosis

A 21-kDa leptospiral lipoprotein (LipL21) was evaluated for its diagnostic potential to detect bovine leptospirosis by ELISA. Both native LipL21 (nLipL21) and recombinant LipL21 (rLipL21) proteins were tested and compared regarding diagnostic efficiency, and no statistically significant difference was observed. The sensitivity of rLipL21 ELISA for 62 microscopic agglutination test (MAT) positive sera was 100% and the specificity with 378 MAT negative sera was 97.09%. Thus, rLipL21 protein-based ELISA could be used as an alternative to MAT for the diagnosis of bovine leptospirosis

Eliminating Tuberculosis

47-49For a successful TB elimination programme, we need to take lessons from the Polio Campaign which was a huge success because the major stakeholders such as hospitals, government, NGOs, social workers and many more came together to make India Polio-free

Comparative evaluation of pncA gene, IS6110 and 12.7-Kb fragment based PCR assays for simultaneous detection of Mycobacterium tuberculosis complex (M. tuberculosis and M. bovis) in cultured strains and clinical specimens

PCR based molecular techniques help in discrimination of two closely related Mycobacterium tuberculosis and M.bovis. Here, we analyzed 24 M. bovis, 39 M. tuberculosis, 21 fresh acid-fast positive sputum samples and standardmycobacterial strains with pncA, 12.7 Kb and IS6100 based PCR assays. DNA from cultures and sputum yielded a positiveamplification of 185 bp with M. tuberculosis specific reverse primer pncAMT-2 but not with M. bovis specific reverseprimer pncAMB-2 and all M. bovis strains showed a positive amplification of 185 bp with M. bovis specific reverse primerpncAMB-2 but not with M. tuberculosis specific reverse primer pncAMT-2. The 12.7 Kb fragment based PCR performed onDNA extracted from cultures of M. tuberculosis and sputum yielded product of 168 bp while M. bovis showed 262 bpproducts. M. tuberculosis complex specific IS6110 PCR assay performed on DNA extracted from M. tuberculosis, M. boviscultures and sputum samples yielded M. tuberculosis complex specific 123-bp amplified products. The sequence analysis ofrepresentative PCR products of IS6110 and 12.7 Kb fragment showed 99-100% and 100% identity in amplicon products,respectively. To test reliability of primers, M. tuberculosis and M. bovis cultures were mixed and subjected to IS6110, pncAand 12.7 Kb PCR assay. pncA primers could not successfully and reliably discriminate the mixed culture, however, 12.7 Kbfragment primers successfully discriminated the mixed culture of M. tuberculosis and M. bovis

Standardization and development of Pasteurella multocida inactivated adjuvanted vaccine against septic pasteurellosis in pigs

315-323In piggery, septic pasteurellosis caused by Pasteurella multocida (B:2) is an issue of concern, which needs an effective vaccine. Here, we prepared a double emulsified (DE) vaccine containing 2.5 mg inactivated antigenic mass of pig field strain (B:2) (named as soron) isolated from an outbreak of septicaemic death in pigs and P. multocida P52 cattle strain (B:2) and studied their efficiency in terms of immunity to direct challenge, duration of immunity and the role of humoral and cell-mediated immunity. Both of these strains showed presence of hgbB, pfhA, nanH, ptfA, and tbPA virulence genes. The sequence analysis of bands of 760 bp product using capsular primers were obtained for soron and P52 revealed 99.2% homology between these two strains, indicating differences at genetic level. nanH and pfhA genes of soron shared 99.2% and 92.7% homology with P52, respectively suggesting differences between these two strains at genetic level. SDS-PAGE analysis of cell wall of both strains showed presence of about 15 major protein bands whereas Western blot analysis with 21 day soron immunized pig serum showed 16, 33, 47, 63 and 83 kDa polypeptides in both strains. The duration of immune responses were monitored at 3, 6 and 9 months post immunization in pigs. By direct challenge, pigs showed that the vaccines were protective at 21 days and up to 270th day post immunization. Vaccines induced a serum ELISA IgG response that peaked on 60 DPI which declined gradually up to 270th DPI in both vaccines. Stimulation index measured by lymphocyte proliferation test (LTT) indicated that the vaccine, induced cell-mediated immune response and in general percent stimulation index (SI) was higher in pigs immunized with soron vaccine at 15 days post challenge infection. The results showed that pig strain (soron) would be a potential homologous strain of P. multocida for the vaccine against pasteurellosis in place of use of cattle P. multocida P52 strain

Tuberculin response in guinea pigs with recombinant proteins cocktail prepared from Indian strain of <em>Mycobacterium bovis</em> (3/86 Rv)

638-645The bovine tuberculosis caused by Mycobacterium bovis is a serious disease among cattle worldwide resulting in considerable economic loss. There is a need for a diagnostic test that can discriminate M. bovis infection from BCG vaccination and NTM sensitization in animals. In this study, we intended to find out the potential use of recombinant antigens from Indian strain of Mycobacterium bovis (3/86Rv) for the intradermal tuberculin test of cattle. Immunodominant proteins MPB64, MPB83 and ESAT6 from M. bovis (3/86 Rv) Indian strain were recombinantly overexpressed, purified and immunologically characterized (rMPB64, rMPB83 and rESAT6). Four different cocktail combinations viz., cocktail I of protein antigens contained rMPB64, rMPB83, rESAT6, rCFP10 with protein concentration of 0.5 µg each; cocktail II contained 0.5 µg of each of rMPB64, rMPB83, rESAT6; cocktail III with 1 µg of each rESAT6, rCFP10; and cocktail IV contained rMPB64 and rMPB83 with 1 µg concentration of each protein, were administered at a dose of 0.1 mL. The DTH response was measured in heat killed M. bovis and non-tuberculous mycobacteria (NTM) sensitized, bacille Calmette-Guerin (BCG) vaccinated and control guinea pigs.The first cocktail of rMPB64, rMPB83 and rESAT6 containing 1.5 µg showed almost similar to cocktails II and III but stronger DTH response even at lower individual protein concentrations (each 0.5 µg) than the rESAT6 and rCFP10 protein of third cocktail with higher individual protein concentration (each 1 µg). The fourth cocktail with rMPB64 and rMPB83 elicited less DTH response as compared to the all other formulated cocktails. Cocktail I of four protein antigens elicited highest response at 24 h. Guinea pig model sensitized with heat killed M. bovis was found to be an efficient model for evaluating DTH response elicited by recombinant proteins cocktails. None of the cocktails elicited positive erythematous reaction in NTM sensitized and BCG vaccinated guinea pigs. A diagnostic test based on above cocktails could discriminate M. bovis infection from BCG vaccinated  and NTM sensitizatized cattle