Genetic association of polymorphisms in bovine TLR2 and TLR4 genes with Mycobacterium avium subspecies paratuberculosis infection in Indian cattle population

Toll like receptors (TLRs) are pattern recognition molecules involved in cellular recognition of Mycobacterium avium subspecies paratuberculosis (MAP), the infectious agent causing Paratuberculosis (PTB), a notified disease of domestic and wild ruminants. The present study was undertaken to investigate the presence of single nucleotide polymorphisms (SNPs) in TLR2 and TLR4 gene and to evaluate association of these SNPs with occurrence of PTB in Indian cattle. A total of 213 cattle, were subjected to multiple diagnostic tests viz. Johnin PPD, ELISA test (Indigenous and Parachek kit method), fecal microscopy and fecal culture for detection of MAP infection. Based on screening results 51 animals each were assigned to case and control population. Two SNPs viz. rs55617172, rs41830058 in TLR2 gene and two SNPs viz. rs8193046, rs8193060 in TLR4 gene and were genotyped by PCR-RFLP method. All SNPs were found to be polymorphic except rs41830058 in the case-control population. Both SNPs in TLR4 gene but none in TLR2 genes were significantly associated with the occurrence of PTB in our population. The genotypes in SNP rs8193046 and SNP rs8193060 were significantly (P?<?0.01) different in case-control population. These findings suggest that SNPs rs8193046 and rs8193060 are likely a potential marker against MAP infection and a selection programme eliminating AG genotype for rs8193046 and CT genotype for rs8193060 might be beneficial in conferring resistance to MAP infection in Indian cattle population.

     

Effect of selected single nucleotide polymorphisms in SLC11A1, ANKRA2, IFNG and PGLYRP1 genes on host susceptibility to Mycobacterium avium subspecies paratuberculosis infection in Indian cattle

Paratuberculosis (PTB) is a chronic infectious enteritis of ruminants, caused by Mycobacterium avium subspecies paratuberculosis (MAP) that brings huge economic loss to the dairy farmers. The study was conducted to explore the association of selected SNPs in IFNG, SLC11A1, ANKRA2 and PGLYRP1 genes with resistance to PTB disease in Indian cattle population. A case-control resource population was established based on the results of diagnostic tests used for detection of MAP infection status viz. ELISA, Johnin PPD test, faecal microscopy and IS900 blood PCR. The PCR-RFLP method was used for genotyping of SNPs. SNPs rs109453173 in SLC11A1, rs110853455 in IFNG and rs41933863 in ANKRA2 genes were significantly (P<0.05) associated with resistance to MAP infection. For SNP rs109453173, GG genotype and G allele was found to be associated with resistance against MAP infection than CC and CG genotypes and C allele, respectively. For SNP rs110853455, AG genotype was found to be associated with susceptibility to MAP infection than AA and GG genotype. For SNP rs41933863, the AG genotype provided three and six times more resistance against MAP infection than GG and AA genotype. The results of this study are suggestive of SNPs rs109453173, rs110853455 and rs41933863 as potential markers for screening MAP resistant cattle and a breeding programme favouring GG genotype and G allele for rs109453173, AG genotype for rs41933863 and against AG genotype for rs110853455 might confer resistance against MAP infection in Indian cattle. However, investigation of these SNPs in an independent and larger population will warrant the strength of association for resistance against MAP infection in cattle.

     

‘Nano-immuno test’ for the detection of live <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subspecies <Emphasis Type="Italic">paratuberculosis</Emphasis> bacilli in the milk samples using magnetic nano-particles and chromogen

Early rapid detection of Mycobacterium avium subspecies paratuberculosis (MAP) bacilli in milk samples is the major challenge since traditional culture method is time consuming and laboratory dependent. We report a simple, sensitive and specific nano-technology based ‘Nano-immuno test’ capable of detecting viable MAP bacilli in the milk samples within 10 h. Viable MAP bacilli were captured by MAP specific antibody-conjugated magnetic nano-particles using resazurin dye as chromogen. Test was optimized using true culture positive (10-bovine and 12-goats) and true culture negative (16-bovine and 25-goats) raw milk samples. Domestic livestock species in India are endemically infected with MAP. After successful optimization, sensitivity and specificity of the ‘nano-immuno test’ in goats with respect to milk culture was 91.7% and 96.0%, respectively. Whereas, it was 90.0% (sensitivity) and 92.6% (specificity) with respect to IS900 PCR. In bovine milk samples, sensitivity and specificity of ‘nano-immuno test’ with respect to milk culture was 90.0% and 93.7%, respectively. However, with respect to IS900 PCR, the sensitivity and specificity was 88.9% and 94.1%, respectively. Test was validated with field raw milk samples (goats-258 and bovine-138) collected from domestic livestock species to detect live/viable MAP bacilli. Of 138 bovine raw milk samples screened by six diagnostic tests, 81 (58.7%) milk samples were positive for MAP infection in one or more than one diagnostic tests. Of 81 (58.7%) positive bovine raw milk samples, only 24 (17.4%) samples were detected positive for the presence of viable MAP bacilli. Of 258 goats raw milk samples screened by six diagnostic tests, 141 (54.6%) were positive for MAP infection in one or more than one test. Of 141 (54.6%) positive raw milk samples from goats, only 48 (34.0%) were detected positive for live MAP bacilli. Simplicity and efficiency of this novel ‘nano-immuno test’ makes it suitable for wide-scale screening of milk samples in the field. Standardization, validation and re-usability of functionalized nano-particles and the test was successfully achieved in field samples. Test was highly specific, simple to perform and easy to read by naked eyes and does not require laboratory support in the performance of test. Test has potential to be used as screening test to estimate bio-load of MAP in milk samples at National level.     

The use of native turf mixtures to approach sustainable lawn in urban landscapes

Native grasses are excellent candidate species for manipulation to produce dwarf and turf type varieties as well as for producing cultivars with higher attractiveness and requiring less maintenance to be used as a turf grass in arid and semi-arid regions of the world. This investigation was conducted to explore visual qualities of native grasses and their mixtures compared to commercial turf. The field experiment was set out in a split-plot in time based on a randomized experimental design with three replications. We used two native monoculture accessions, perennial ryegrass (Lolium perenne L. ‘Yarand’) and (Lolium perenne L. ‘Shadegan’), Native low-variety Mixture (NM1): consisting of 50% Lolium multiflorum ‘Shadegan’? 50% Festuca spp. ‘Shadegan’, Native high-variety Mixture (NM2): consisting of 55% Lolium perenne L. ‘Yarand’? 35% Lolium perenne L. ‘Shadegan’? 5% L. multiflorum ‘Shadegan’ and 5% Festuca spp. ‘Shadegan’ and compared this with one commercial turf mixture that is commonly used in landscaping. Results indicated the effects of seasons and turf grass types and their interaction had significant effects on most variables including quality, season color, leaf texture, density, quality after clipping (p < 0.01). The visual quality measurements indicated the superiority of L. perenne ‘Shadegan’ over other native monoculture and polyculture and its ablility to compete with the commercial turf. The native turf mixture of NM2 showed several good characteristics. L. perenne ‘Yarand’ had statistically the lowest score for visual appeal as compared with the other turf types. This research suggests that the use of native grass species of L. perenne ‘Shadegan’ is worth investigating for better performance of the native landscape.