Analysis of porcine body size variation using re-sequencing data of miniature and large pigs.

BACKGROUND: Domestication has led to substantial phenotypic and genetic variation in domestic animals. In pigs, the size of so called minipigs differs by one order of magnitude compared to breeds of large body size. We used biallelic SNPs identified from re-sequencing data to compare various publicly available wild and domestic populations against two minipig breeds to gain better understanding of the genetic background of the extensive body size variation. We combined two complementary measures, expected heterozygosity and the composite likelihood ratio test implemented in "SweepFinder", to identify signatures of selection in Minipigs. We intersected these sweep regions with a measure of differentiation, namely FST, to remove regions of low variation across pigs. An extraordinary large sweep between 52 and 61 Mb on chromosome X was separately analyzed based on SNP-array data of F2 individuals from a cross of Goettingen Minipigs and large pigs. RESULTS: Selective sweep analysis identified putative sweep regions for growth and subsequent gene annotation provided a comprehensive set of putative candidate genes. A long swept haplotype on chromosome X, descending from the Goettingen Minipig founders was associated with a reduction of adult body length by 3% in F2 cross-breds. CONCLUSION: The resulting set of genes in putative sweep regions implies that the genetic background of body size variation in pigs is polygenic rather than mono- or oligogenic. Identified genes suggest alterations in metabolic functions and a possible insulin resistance to contribute to miniaturization. A size QTL located within the sweep on chromosome X, with an estimated effect of 3% on body length, is comparable to the largest known in pigs or other species. The androgen receptor AR, previously known to influence pig performance and carcass traits, is the most obvious potential candidate gene within this region.

Short communication: Uncovering quantitative trait loci associated with resistance to Mycobacterium avium ssp. paratuberculosis infection in Holstein cattle using a high-density single nucleotide polymorphism panel.

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiological agent of Johne's disease in cattle. Johne's disease is a disease of significant economic, animal welfare, and public health concern around the globe. Therefore, understanding the genetic architecture of resistance to MAP infection has great relevance to advance genetic selection methods to breed more resistant animals. The objectives of this study were to perform a genome-wide association study of previously analyzed 50K genotypes now imputed to a high-density single nucleotide polymorphism panel (777K), aiming to validate previously reported associations and potentially identify additional single nucleotide polymorphisms associated with antibody response to MAP infection. A principal component regression-based genome-wide association study revealed 15 putative quantitative trait loci (QTL) associated with the MAP infection phenotype (serum or milk ELISA tests) on 9 different chromosomes (Bos taurus autosomes 5, 6, 7, 10, 14, 15, 16, 20, and 21). These results validated previous findings and identified new QTL on Bos taurus autosomes 15, 16, 20, and 21. The positional candidate genes NLRP3, IFi47, TRIM41, TNFRSF18, and TNFRSF4 lying within these QTL were identified. Further functional validation of these genes is now warranted to investigate their roles in regulating the immune response and, consequently, cattle resistance to MAP infection.

Association of TLR4 polymorphisms with Mycobacterium avium subspecies paratuberculosis infection status in Canadian Holsteins.

Mycobacterium avium ssp. paratuberculosis (MAP) causes chronic enteritis in cattle that results in substantial financial losses to the cattle industry worldwide. Given that susceptibility to MAP infection is determined in part by genetics, marker-assisted selection may help in the breeding of animals that are more resistant to MAP infection. The toll-like receptor 4 gene (TLR4) was selected as a potential candidate gene because of its role in innate immunity and its involvement in MAP recognition and infection. The objective of this study, therefore, was to identify associations between TLR4 polymorphisms and susceptibility to MAP infection in Canadian Holstein cows. Two biologically relevant SNPs, including c.-226G>C in the 5'-untranslated region and the non-synonymous SNP c.2021C>T in the potential TIR domain, were selected for an association analysis with MAP infection status in 409 Canadian Holsteins. The haplotype C-T from these combined SNPs yielded significant association with susceptibility to MAP infection, supporting the involvement of TLR4 in susceptibility to MAP infection.

Bovine CLEC7A genetic variants and their association with seropositivity in Johne's disease ELISA.

Mycobacterium avium ssp. paratuberculosis (MAP) infection in cattle causes significant economic losses to the dairy and beef industries resulting from reduced productivity, premature culling and mortality. Bovine Dectin-1, an important pattern recognition molecule that is able to generate a proinflammatory response by acting alongside Toll like receptor (TLR) signaling, is known to co-operate with TLR2 to specifically activate a macrophage proinflammatory response against mycobacterial infections. Therefore, the goal of this study was to identify single nucleotide polymorphisms (SNPs) in the gene encoding bovine Dectin-1 (CLEC7A) and to assess their association with susceptibility to MAP infection in dairy cattle. Blood and milk samples, collected from commercial dairy operations, were tested for MAP infection using blood and milk ELISAs and a resource population consisting of 197 infected and 242 healthy cattle was constructed. Pooled DNA was used for sequencing and eight single nucleotide polymorphisms (SNPs) were identified. Identified SNPs were genotyped on the resource population using the iPLEX MassARRAY system and statistical analysis was performed using logistic regression fitting the additive and dominance effects of each SNP in the model. Out of a total of eight identified SNPs, five were successfully genotyped, and three out of these five SNPs were found to be in complete linkage. Statistical analysis revealed a strong association between a non-synonymous SNP c.589A>G (p=0.008), and MAP infection status of the resource population inferred by seropositivity in MAP antibody specific ELISAs. This SNP c.589A>G was located in the geneic region that encodes the carbohydrate recognition domain of bovine Dectin-1. Therefore, further investigation of its functional relevance is warranted.

Comparative study of sensitivity of rapid diagnostic (hexagon) test with calculated malarial parasitic density in peripheral blood.

BACKGROUND: Different diagnostic test kits are used for rapid diagnosis of malaria. Most are based on antigen detection (pLDH, Pan Aldolase, HRP-2). In context of Nepal the diagnostic reliability and sensitivity of these tests is unknown. Hexagon Malaria Combi™ is one of the most commonly used test kit in Nepal for rapid diagnosis of malaria. The aim of the present study is to evaluate the sensitivity of the Hexagon malaria Combi test in comparison with parasitic density by microscopy technique. METHODS: A Cross sectional prospective study was conducted in three districts of Nepal from September to November 2009. Blood samples were collected from the suspected cases of malaria. Thick and thin smear were prepared from all the samples and Giemsa stain was done. Simultaneously RDT (hexagon) for malaria was done. When RDT was found to be positive, blood was serially diluted in 6 tubes as 1:2, 1:4, 1:8, 1:16, 1:32 and 1:64. RDT was done on diluted blood till RDT test gave negative result. Parasitic density was calculated for undiluted and diluted blood samples and sensitivity of RDT in various parasitic densities was calculated. RESULTS: Hexagon malaria combi test is sensitive (86%) when malarial parasitic density is >500/µl. Sensitivity was found to be directly related to parasitic density. Its sensitivity is very low (2.9%) when parasitic density is less than 500/ µl. CONCLUSIONS: The sensitivity of rapid diagnostic test (hexagon Combi test detecting malarial pLDH antigen) is high only if the parasitic density is more than 500/µl.

Bovine PGLYRP1 polymorphisms and their association with resistance to Mycobacterium avium ssp. paratuberculosis.

Mycobacterium avium ssp. paratuberculosis (MAP) causes a chronic, granulomatous inflammatory condition of the intestines in ruminants and wild-type species. It causes significant economic losses to the dairy and beef industries owing to reduced productivity, premature culling and mortality. Bovine peptidoglycan recognition protein 1 is an important pattern recognition molecule that is capable of directly killing microorganisms. The goal of this study was to identify single nucleotide polymorphisms (SNPs) in the gene encoding bovine peptidoglycan recognition protein 1 and to assess their association with susceptibility to MAP infection in dairy cattle. Blood and milk samples were collected from Holsteins in Southwestern and Eastern Ontario and tested for MAP infection using blood and milk ELISAs. A resource population consisting of 197 infected (S/P > 0.25) and 242 healthy (S/P < 0.10) cattle was constructed. Sequencing of pooled DNA was used to identify three SNPs (c.102G>C, c.480G>A and c.625C>A) that were genotyped in the resource population. Statistical analysis was performed using a logistic regression model fitting the additive and dominance effects of each SNP in the model. SNP c.480G>A (P = 0.054) was found to be associated with susceptibility to MAP infection. Cows with a copy of the major allele 'G' at this locus had an odds ratio of 1.51 (95% CI: 0.99-2.31) for being infected with MAP.

Single nucleotide polymorphisms alter the promoter activity of bovine MIF.

Macrophage migration inhibitory factor (MIF) is a unique pro-inflammatory cytokine whose chief functions include modulating TLR4 expression, and suppressing the anti-inflammatory effects of glucocorticoids. Not surprisingly, MIF is involved in a number of inflammatory diseases and single nucleotide polymorphisms (SNPs) have been implicated in modulating disease severity. The objective of the present study was to determine if SNPs in 5' region of bovine MIF affects its promoter activity. Three SNPs were identified, -1078A>G, -395A>G, and -400G>C, all of which fall within predicted transcription factor binding regions. Reporter gene assays indicate that the identified SNPs have a significant effect of modulating MIF promoter activity. Finally, gene association analysis suggests a significant relationship of -395A>G with the susceptibility to Mycobacterium avium ssp. paratuberculosis infection, the causative agent of Johne's disease. Given the relationships revealed in the current study, it is clear that the role of MIF in bovine diseases such as Johne's disease merits further investigation.

Identification of polymorphisms in bovine TLR2 and CARD15,associations between CARD15 polymorphisms and milk somatic cell score in Canadian Holsteins, and functional relevance of SNP c.3020A>T.

Toll-like receptor-2 (TLR2) and caspase recruitmentdomain 15 (CARD15) are important pattern recognition receptors that play a role in the initiation of the inflammatory and subsequent immune response. They have been previously identified as susceptibility loci for inflammatory bowel diseases in humans and are, therefore, suitable candidate genes for inflammatory disease resistance in cattle. The objective of this study was to identify single nucleotide polymorphisms (SNPs) in the bovine TLR2 and CARD15 and evaluate the association of these SNPs with health and production traits in a population of Canadian Holstein bulls. A selective DNA pool was constructed based on the estimated breeding values (EBVs) for somatic cell score (SCS). Gene segments were amplified from this pool in PCR reactions and the amplicons sequenced to reveal polymorphisms. A total of four SNPs, including one in intron 10 (c.2886-14A>G) and three in exon 12 (c.3020A>T, c.4500A>C and c.4950C>T)were identified in CARD15; nonewere identified in TLR2. Canadian Holstein bulls (n=338) were genotyped and haplotypes were reconstructed. Two SNPs, c.3020A>T and c.4500A>C, were associated with EBVs for health and production traits. The SNP, c.3020A>T for example, was associated with SCS EBVs (p = 0.0097) with an allele substitution effect of 0.07 score. When compared to the most frequent haplotype Hap12(AC), Hap22(TC) was associated with increased milk (p < 0.0001) and protein (p = 0.0007) yield EBVs, and hap21(TA) was significantly associated with increased SCS EBV (p = 0.0120). All significant comparison-wise associations retained significance at 8% experimental-wise level by permutation test. The role of SNP c.3020A>T in MDP induced IL-1beta expression was investigated by real-time quantitative reverse transcription-PCR (real-time quantitative RT-PCR). The induction of IL-1beta by MDP was highly variable between individuals, and no association was observed between IL-1beta expression and SNP c.3020A>T genotypes. In summary, the association study indicates that SNP c.3020A>T might play a role in the host response against mastitis; however, it is not the sole determinant of MDP induced IL-1beta expression in blood leukocytes. Further detailed studies are needed to understand the functional implications of SNP c.3020A>T.