Development and validation of the Axiom-MaruPri SNP chip for genetic analyses of domesticated old world camelids.

Camels play a crucial socio-economic role in sustaining the livelihoods of millions in arid and semi-arid regions. They possess remarkable physiological attributes which enable them to thrive in extreme environments, and provide a source of meat, milk and transportation. With their unique traits, camels embody an irreplaceable source of untapped genomic knowledge. This study introduces Axiom-MaruPri, a medium-density SNP chip meticulously designed and validated for both Camelus bactrianus and Camelus dromedarius. Comprising of 182,122 SNP markers, derived from the re-sequenced data of nine Indian dromedary breeds and the double-humped Bactrian camel, this SNP chip offers 34,894 markers that display polymorphism in both species. It achieves an estimated inter-marker distance of 14 Kb, significantly enhancing the coverage of the camel genome. The medium-density chip has been successfully genotyped using 480 camel samples, achieving an impressive 99 % call rate, with 96 % of the 182,122 SNPs being highly reliable for genotyping. Phylogenetic analysis and Discriminant Analysis of Principal Components yield clear distinctions between Bactrian camels and dromedaries. Moreover, the discriminant functions substantially enhance the classification of dromedary camels into different breeds. The clustering of various camel breeds reveals an apparent correlation between geographical and genetic distances. The results affirm the efficacy of this SNP array, demonstrating high genotyping precision and clear differentiation between Bactrian and dromedary camels. With an enhanced genome coverage, accuracy and economic efficiency the Axiom_MaruPri SNP chip is poised to advance genomic breeding research in camels. It holds the potential to serve as an invaluable genetic resource for investigating population structure, genome-wide association studies and implementing genomic selection in domesticated camelid species.

Design and validation of high-density SNP array of goats and population stratification of Indian goat breeds.

Goats are the supporting pillars of rural economy contributing significantly to meat and milk production in India. It is a species targeted for fulfilling the interdependent goals of poverty reduction and creation of employment for supporting the rural income. The increased demand for goat products necessitates their genetic characterization and improvement to augment the production of native breeds. Bi-allelic, genome wide, densely placed single nucleotide polymorphism (SNP) markers are most suitable for this purpose. This paper describes the design and validation of an Affymetrix Axiom-based high-density (HD) SNP chip for goats. The array was designed using a panel of 225 samples from 15 diverse goat breeds of India. In total, more than 38 million high quality SNPs were subjected to stringent filtering and 626,975 SNPs were finally tiled on the array. The average coverage of SNPs in our chip is one SNP per four kilobase (kb), providing a denser coverage of the goat genome than previously available arrays. The HD chip (Axiom_Cahi) was validated by genotyping 443 samples from 26 indigenous goat breeds/populations. The results revealed 95.83% markers to be highly informative and polymorphic in Indian goats. Multivariate analysis indicated population structuring, as 15 breeds could be segregated using the designed array. Phylogenetic analysis suggested stratification of breeds by geographic proximity. This HD SNP chip for goats is a valuable resource for genomic selection, genome wide association as well as population genetic studies in goats.

Distinct and shared B cell responses of tuberculosis patients and their household contacts.

This study was aimed at identifying the B cell responses which could distinguish between 'latent tuberculosis infection (LTBI)' and active TB disease. Study subjects were smear-positive TB patients (n = 54) and their disease-free household contacts (HHCs, n = 120). The sera were used for determination of antibody levels (ΔOD values) against Mycobacterium tuberculosis membrane (MtM) antigens by ELISA and for visualisation of seroreactive MtM antigens by immunoblotting. B cell subsets in whole blood samples were determined by flow cytometry. In TB sera, levels of IgG antibodies were significantly higher than IgM and IgA whereas IgM and IgA antibody levels were comparable. Conversely, HHC sera had significantly higher IgM antibody levels than IgG and IgA. The ratio of IgM to IgG antibodies in HHCs were also significantly higher than in patients. Immunoblotting revealed that some of the MtM antigens (<10, ~12 and ~25 kDa) reacted with TB as well as HHC sera whereas some other antigens (~16, ~36, ~45 and ~60 kDa) reacted with most of TB and a subset of HHC sera. Frequencies of classical memory B cells (cMBCs, CD19+CD27+) were significantly higher, and of IgG+ cMBCs were significantly lower in HHCs than in patients. Frequencies of IgA+ cMBCs in HHCs and patients were comparable but both were significantly higher than the corresponding frequencies of IgG+ cMBCs. Frequencies of IgA+ atypical MBCs (aMBCs, CD19+CD27-) in HHCs and patients were also comparable and significantly higher than the IgG+ aMBCs. The plasmablast (CD19+CD27++CD38++) frequencies in HHCs and patients were comparable. These results suggest that the IgM/IgG antibody ratio, antibody binding to selected MtM antigens and relative frequencies of MBC subsets could indicate protective or pathogenic immune responses following the primary infection with Mtb. Responses that orchestrate protection leading to a 'quiescent' LTBI may provide clues to an effective vaccination strategy against TB.

Immune responses to Mycobacterium tuberculosis membrane-associated antigens including alpha crystallin can potentially discriminate between latent infection and active tuberculosis disease.

Changes in expression of membrane antigens may accompany the transition of Mycobacterium tuberculosis (Mtb) from 'dormant' to 'active' states. We have determined whether antibody and T cell responses to Mtb membrane (MtM)-associated antigens, especially the latency-induced protein alpha crystallin (Acr), can discriminate between latent tuberculosis infection (LTBI) and active TB (ATB) disease. Study subjects comprised a previously described cohort of healthcare workers (HCWs, n = 43) and smear-positive ATB patients (n = 10). HCWs were further categorized as occupational contacts (OC, n = 30), household contacts of TB (HC, n = 8) and cured TB (CTB, n = 5). Levels (ΔOD) of serum antibody isotypes (IgG, IgA and IgM) were determined by ELISA and blood T cell proliferative responses were determined by flow cytometry using Ki67 protein as marker for DNA synthesis. Antibodies to MtM and Acr were predominantly IgG and their levels in HCWs and ATB did not differ significantly. However, HCWs showed a significantly higher level of anti-MtM IgM and a significantly lower level of anti-Acr IgA antibodies than the ATB patients. Also, a larger proportion of HCWs showed a high (>1) ΔODAcr/ΔODMtM ratio for IgG. HCWs also showed a higher, though not significantly different from ATB, avidity of anti-MtM (IgG) antibodies. A higher proportion of HCWs (35% of OC, 62.5% of HC and 20% of CTB), compared with ATB (10%) showed a positive T cell response to Acr along with significant difference (P <0.05) between HC and ATB. A significant correlation (r = 0.60, P <0.0001) was noted between T cell responses of HCWs towards Acr and MtM (reported earlier by us) and both responses tended to decline with rising exposure to the infection. Even so, positive responses to Acr (38.5%) were significantly lower than to MtM (92%). Neither antibody nor T cell responses to either antigen appeared affected by BCG vaccination or reactivity to tuberculin. Results of the study suggest that the levels of IgM antibodies to MtM, IgA antibodies to Acr and proliferative T cell responses to both the antigens can potentially discriminate between LTBI and active TB disease. They also underscore the necessity of SOPs for antibody assays.

Buffalo milk transcriptome: A comparative analysis of early, mid and late lactation.

The expression of genes and their regulation during lactation in buffaloes remains less understood. To understand the interplay of various genes and pathways, the milk transcriptome from three lactation stages of Murrah buffalo was analyzed by RNA sequencing. The filtered reads were mapped to the Bubalus bubalis as well as Bos taurus reference assemblies. The average mapping rate to water buffalo and Btau 4.6 reference sequence, was 75.5% and 75.7% respectively. Highly expressed genes (RPKM > 3000), throughout lactation included CSN2, CSN1S1, CSN3, LALBA, SPP1 and TPT1. A total of 12833 transcripts were common across all the stages, while 271, 205 and 418 were unique to early, mid and late lactation respectively. Majority of the genes throughout lactation were linked to biological functions like protein metabolism, transport and immune response. A discernible shift from metabolism in early stage to metabolism and immune response in mid stage, and an increase in immune response functions in late lactation was observed. The results provide information of candidate genes and pathways involved in the different stages of lactation in buffalo. The study also identified 14 differentially expressed and highly connected genes across the three lactation stages, which can be used as candidates for future research.

Synergy between tuberculin skin test and proliferative T cell responses to PPD or cell-membrane antigens of Mycobacterium tuberculosis for detection of latent TB infection in a high disease-burden setting.

Tuberculin skin test (TST) is used most widely for the detection of latent tuberculosis infection (LTBI), even though evidences suggest that it could be underreporting the prevalence of LTBI particularly in high disease-burden settings. We have explored whether in vivo (TST) and in vitro (cell-proliferative) T cell responses to PPD can serve as complementary measures. In addition, we also probed whether in vitro T cell response to cell-membrane antigens (Mem) of Mycobacterium tuberculosis (MTB) can serve as a biomarker for LTBI. Study subjects comprised 43 healthcare workers (HCWs), and 9 smear-positive TB patients served as 'disease control'. To measure proliferative T cell responses, 0.1 ml blood (diluted 1:10) was incubated (5 days) with test or control antigen. Cells were stained with fluorescent antibodies to T cell (CD3+/CD4+/CD8+) surface markers and, after fixation and permeabilization, to nuclear proliferation marker Ki67. Data was acquired on a flow cytometer. HCWs who had an intimate exposure to MTB showed significantly higher TST positivity (85%) than the rest (43%), notwithstanding their BCG vaccination status. The proliferative responses of CD4+ and CD8+ subsets of T cells were comparable. Sixty seven and 100% TST-negative HCWs, respectively, were positive for proliferative T cell response to PPD and MTBMem. Cumulative positivity (TST or in vitro) was 86% with PPD and 100% with MTBMem indicating complementarity of the two responses. As standalone in vitro assay, MTBMem provided a significantly higher positivity (95%) than PPD (67%). T cell responses of TB patients were 'generally' depressed, having implications for the development of immunological assays for 'progressive' LTBI. Altogether, these results demonstrate that in vivo and in vitro T cell responses to PPD are complementary and in vitro response to MTBMem can be developed as a highly sensitive biomarker for LTBI.

Antibody and inflammatory response-mediated severity of pandemic 2009 (pH1N1) influenza virus.

Influenza A virus causes significant morbidity and mortality each year worldwide due to antigenic drift, punctuated by infrequent pandemics following antigenic shift. H1N1 subtype of pandemic 2009 (pH1N1) influenza virus lineages has continued to circulate in humans and raised severe concerns about its pandemic developments. The pathogenesis of the disease and its progression as post-infectious sequelae is not well understood. Moderate inflammatory response protects against the ill effects and hyper-inflammatory response promotes the pathogenesis in disease progression. Samples were screened by RT-PCR and classified in pandemic 2009 (pH1N1), Influenza A virus infected patient. Further antibody titer was analyzed by hemagglutination inhibition assay and cytokine/chemokine response by Cytometric bead array assy. Screening of 216 patients shows 63 were belongs to pH1N1 influenza virus infection and 47 were Influenza A virus infected and 106 samples were negative for these viruses, were used as a disease control. Apart from that 100 samples were taken for healthy control. Lower antibody titer was found in patient infected with pH1N1/Influenza A virus and expression of cytokines (IL-6, IL-8, and IL-10) and chemokine MCP-1 was higher in patient infected with pH1N1 compare to healthy/disease control however there was no significant difference observed in the expression of pro-inflammatory cytokines TNF-α and antiviral cytokine IFN-γ in pH1N1 influenza virus infected patients.

N-acetyltransferase and cytochrome P450 2E1 gene polymorphisms and susceptibility to antituberculosis drug hepatotoxicity in an Indian population.

Background. Antituberculosis drug hepatotoxicity (ATDH) is common in India. Isoniazid, a constituent of most anti- tuberculosis drug regimens, is metabolized by N-acetyltransferase (NAT2) and cytochrome P450 2E1 (CYP2E1) enzymes. We therefore studied the association of some single-nucleotide polymorphisms (SNPs) in these enzyme genes with ATDH. Methods. Allelic and genotypic frequencies at three SNP loci in the NAT2 gene (rs1799929, rs1799930 and rs1799931) and one locus (rs2031920) in the CYP2E1 gene were studied using restriction fragment length polymorphism in 33 patients who developed ATDH following an isoniazid- containing antituberculosis drug regimen and 173 healthy blood donors. After confirming adherence of the control data to the Hardy-Weinberg equilibrium model, genotype and allele frequencies in the two groups were compared. Results. For SNP rs1799930 in the NAT2 gene, 7 (21%), 21 (64%) and 5 (15%) patients, and 93 (54%), 62 (36%) and 18 (10%) controls had GG, GA and AA genotypes, respectively (p=0.003; odds ratio [OR] for GA v. GG=4.50 [95% CI 1.80-11.22] and for AA v. GG=3.69 [1.05-12.93]). Allele frequency for G nucleotides for this SNP was 0.53 among patients and 0.72 among controls (OR 2.24 [1.31-3.84], p=0.007). The allele and genotype frequencies of the other NAT2 SNPs and the CYP2E1 SNP showed no significant difference between cases and controls. All the 33 patients and 151 (87%) of 173 controls had mutant allele at one or more of the three NAT2 SNP loci (p=0.03). The presence of two or more mutant alleles, a marker of slow acetylator status, was more frequent in patients (23/33 [70%]) than in controls (73/173 [42%]; OR 3.23 [95% CI 1.45-7.19], p=0.004). Conclusion. In India, the risk of ATDH is increased in persons with 'A' allele at SNP rs1799930 in the NAT2 gene, but is not associated with rs2031920 polymorphism in the CYP2E1 gene.