Rudolf Pichler

Candidate gene approach for parasite resistance in sheep--variation in immune pathway genes and association with fecal egg count.

Sheep chromosome 3 (Oar3) has the largest number of QTLs reported to be significantly associated with resistance to gastro-intestinal nematodes. This study aimed to identify single nucleotide polymorphisms (SNPs) within candidate genes located in sheep chromosome 3 as well as genes involved in major immune pathways. A total of 41 SNPs were identified across 38 candidate genes in a panel of unrelated sheep and genotyped in 713 animals belonging to 22 breeds across Asia, Europe and South America. The variations and evolution of immune pathway genes were assessed in sheep populations across these macro-environmental regions that significantly differ in the diversity and load of pathogens. The mean minor allele frequency (MAF) did not vary between Asian and European sheep reflecting the absence of ascertainment bias. Phylogenetic analysis revealed two major clusters with most of South Asian, South East Asian and South West Asian breeds clustering together while European and South American sheep breeds clustered together distinctly. Analysis of molecular variance revealed strong phylogeographic structure at loci located in immune pathway genes, unlike microsatellite and genome wide SNP markers. To understand the influence of natural selection processes, SNP loci located in chromosome 3 were utilized to reconstruct haplotypes, the diversity of which showed significant deviations from selective neutrality. Reduced Median network of reconstructed haplotypes showed balancing selection in force at these loci. Preliminary association of SNP genotypes with phenotypes recorded 42 days post challenge revealed significant differences (P<0.05) in fecal egg count, body weight change and packed cell volume at two, four and six SNP loci respectively. In conclusion, the present study reports strong phylogeographic structure and balancing selection operating at SNP loci located within immune pathway genes. Further, SNP loci identified in the study were found to have potential for future large scale association studies in naturally exposed sheep populations.

Bovine monocyte derived dendritic cell based assay for measuring vaccine immunogenicity in vitro

During both human and animal vaccine development phases, animal testing is necessary to demonstrate vaccine efficacy. Since the number of antigen candidates for testing is usually large when developing a potential vaccine, it is too costly, time consuming and would involve higher risks to carry out selection using in vivo models. The currently available in vitro assays that measure immunogenicity do not adequately reproduce the in vivo state and this is especially true for vaccine research in livestock species. With this in mind, we have developed a bovine monocyte derived dendritic cell (MODC)s based assay to prime CD4 and CD8 lymphocytes in order to investigate vaccine immunogenicity in vitro. MODCs were generated, pulsed with diphtheria toxoid (DT) and co-cultured with lymphocytes for priming. Immunogenicity was measured through antigen recall when antigen pulsed MODC were re-introduced to the co-culture and proliferation of CD4 and CD8 positive lymphocytes were quantified using expressed Ki-67. Having developed the protocol for the assay, we then employed two licenced vaccines against blue tongue virus and rabies virus to validate the assay. Our results show the ability of the assay to satisfactorily measure immunogenicity in cattle. The assay could be used to identify antigens that induce CD4 and CD8 T cell responses prior to embarking on in vivo experiments and can also be used for the quality control of established vaccines in vaccine production facilities as a supplement for in vivo experiments.

Construction of two whole genome radiation hybrid panels for dromedary (Camelus dromedarius): 5000RAD and 15000RAD

The availability of genomic resources including linkage information for camelids has been very limited. Here, we describe the construction of a set of two radiation hybrid (RH) panels (5000RAD and 15000RAD) for the dromedary (Camelus dromedarius) as a permanent genetic resource for camel genome researchers worldwide. For the 5000RAD panel, a total of 245 female camel-hamster radiation hybrid clones were collected, of which 186 were screened with 44 custom designed marker loci distributed throughout camel genome. The overall mean retention frequency (RF) of the final set of 93 hybrids was 47.7%. For the 15000RAD panel, 238 male dromedary-hamster radiation hybrid clones were collected, of which 93 were tested using 44 PCR markers. The final set of 90 clones had a mean RF of 39.9%. This 15000RAD panel is an important high-resolution complement to the main 5000RAD panel and an indispensable tool for resolving complex genomic regions. This valuable genetic resource of dromedary RH panels is expected to be instrumental for constructing a high resolution camel genome map. Construction of the set of RH panels is essential step toward chromosome level reference quality genome assembly that is critical for advancing camelid genomics and the development of custom genomic tools.

A novel snapback primer probe assay for the detection and discrimination of sympatric Haemonchus species using DNA melting analysis

Different sympatric species of Haemonchus parasites infecting ruminants and camels can be distinguished morphologically, but involves tedious microscopic examinations, measurements and several other limitations. Information on internal transcribed spacer-2 (ITS-2) sequence provides confirmatory differentiation of sympatric Haemonchus species. The present study introduces a novel, snapback primer probe based, real time PCR assay for the differentiation of three sympatric Haemonchus species, H. contortus (Hco), H. placei (Hpl) and H. longistipes (Hlo). The assay was designed to amplify a region of 130bp within the ITS-2 gene that included three diagnostic mutational sites capable of discriminating Hco, Hpl and Hlo. Following melt curve analysis, species-specific diagnostic melt peaks were obtained for Hco, Hpl and Hlo with a mean melting temperature of 56.6±0.3°C, 64.4±0.1°C and 54.4±0.1°C respectively. The test for analytical sensitivity revealed the ability of the assay to detect up to 5 copies per reaction. To evaluate the discriminating power of the assay, 174 samples from adult worms and 3rd stage larvae belonging to different Haemonchus species and various other nematode species including Cooperia curticei, Trichostrongylus axei, Trichostrongylus colubriformis, and Teladorsagia circumcincta were tested. Additionally, DNA extracted from 25 fecal egg samples was also tested and the specificity of the assay was verified by sequencing the ITS-2 gene of all the Haemonchus positive and non-Haemonchus samples. The assay worked accurately with 100% specificity in at least three real time PCR platforms. The assay is an effective alternative to the sequencing approach and is expected to be helpful for the screening of individual adult and larval Haemonchus parasites. However, caution needs to be applied while interpreting the results from fecal egg samples due to varying levels of sympatric co-infections from different Haemonchus species. The present study is the first report on the application of snapback primer probe methodology for the differentiation of nematode parasites.