Optimization of single step multiplex PCR for detection of Eimeria tenella and Eimeria necatrix from commercial broilers

Multiplex polymerase chain reaction (PCR) was optimized for detection of 2 important species of Eimeria (Eimeria tenella and E. necatrix). This optimized protocol was used to screen 100 fecal samples collected from broiler birds in Lahore periphery. Out of 100 samples we found 6 tenella and only 1 E. necatrix positive by this method. The detection limit of oocysts by this method was as leastas 16 oocysts. This optimized multiplex PCR method can be used as routine diagnostic tool for detection of E. tenella and E. necatrix and can be extended up to the detection of 7 Eimeria species in future

Assessment of nematodes in Punjab Urial (Ovis vignei punjabiensis) population in Kalabagh game reserve: development of a DNA barcode approach

Punjab urial (Ovis vignei punjabiensis) is a wild sheep of Pakistan, considered a vulnerable species by IUCN. Major threats to urial populations include habitat loss and poaching, causing severe declines in its population. Nematode infections may also compromise urial survival, but little is known about Punjab urial gastrointestinal nematodes. In this study, a novel DNA barcoding approach was developed using ITS-I as a target region, with a primer pair designed to amplify frequently reported nematode species for small ruminants. The novel primer pair was validated in silico and in vitro and subsequently used to determine the presence of nematodes in Punjab urial samples from Kala Bagh Game Reserve, District Mianwali (Pakistan). DNA barcoding revealed a higher prevalence of Haemonchus contortus (73.91%), Trichuris ovis (16.30%) and Trichostrongylus axei (3.26%) in Punjab urial. This study demonstrates that the novel DNA barcoding approach is a robust tool to detect nematode parasites from faecal samples of Punjab urial. This method can be used to detect nematode infections in wild and domestic hosts for surveillance and population conservation

DNA Amplification Techniques for the Detection of Toxoplasma gondii Tissue Cysts in Meat Producing Animals: A Narrative Review Article

Background: Toxoplasma gondii is an intracellular parasite, which infects one-third population of world. Humans and animals acquire infection by ingesting oocytes from feces of cats or by meat of other animals having cysts that may lead to congenital, ocular or cephalic toxoplasmosis. Either it is important to detect T. gondii from meat of food animals from retail shops or directly at slaughterhouses, which is meant for export. Methods: The current research was done without time limitation using such terms as follows: “Toxoplasma gondii”, “Meat”, “Tissue cyst”, “PCR”, “LAMP”, “Screening” and “Immunological assay” alone or in combination, in English language. The used electronic databases for searching included as follows: PubMed, Scopus, Google Scholar, Web of Science and Science Direct. The searches were limited to the published papers to English language. Results: Sensitivity of different molecular techniques for diagnosis of Toxoplasma is real-time PCR > LAMP > conventional PCR. In addition to these DNA analysis tools, bioassay in mice and cats is considered as “gold standard” to detect T. gondii. Conclusion: This review article will help the readers for grasping advantages and limitations of different diagnostic tools for screening meat samples for T. gondii. This review also makes bibliography about the type of meat sample to be processed for diagnosis and different primers or sequences to be targeted for T. gondii by number of researches for its detection from meat or tissue sample using DNA amplification techniques

Demographic history of the Punjab urial and implications for its management

The Punjab urial (Ovis vignei punjabiensis) is endemic to Northern Punjab, Pakistan, and is categorized as vulnerable by the International Union for Conservation of Nature Red List of Threatened Species. The urial population has declined by 30% over the last 3 generations. We used non-invasive fecal samples to identify individuals and estimate population size of Punjab urial in the Kalabagh Game Reserve, Pakistan. We genotyped samples using 12 microsatellite markers to assess genetic variation, population structure, and demographic changes. Microsatellite analysis revealed high levels of genetic variation in urials in terms of expected and observed heterozygosity and allelic diversity. The population structure of the Punjab urial in the Kalabagh Game Reserve, based solely on microsatellite variation using Bayesian clustering, indicated 3 different clusters in the reserve. Results revealed that the urial population may be facing inbreeding pressure because its ancestral effective population size has declined from between 20,000 and 50,000 to ≤1,000 animals today. This reduction has partly occurred because of a bottleneck that occurred about 10,000 years ago. Results also indicate that 1 urial population cluster has the signature of a bottleneck, which may be due to population isolation. The 3 urial clusters are small and broadly dispersed in a large territory, meaning they could be extirpated without any opportunity for natural re-population through dispersion. The results of our study support a management strategy that encourages maintaining connectivity between urial localities within the Kalabagh Game Reserve, increased diversity so the effective population size may recover from the historical decline, and the use of data generated here as a baseline of urial genetic diversity in the reserve for monitoring diversity over the long term

Population demographic history and population structure for Pakistani Nili-Ravi breeding bulls based on SNP genotyping to identify genomic regions associated with male effects for milk yield and body weight.

The domestic Nili-Ravi water buffalo (Bubalus bubalis) is the best dairy animal contributing 68% to total milk production in Pakistan. In this study, we identified genome-wide single nucleotide polymorphisms (SNPs) to estimate various population genetic parameters such as diversity, pairwise population differentiation, linkage disequilibrium (LD) distribution and for genome-wide association study for milk yield and body weight traits in the Nili-Ravi dairy bulls that they may pass on to their daughters who are retained for milking purposes. The genotyping by sequencing approach revealed 13,039 reference genome-anchored SNPs with minor allele frequency of 0.05 among 167 buffalos. Population structure analysis revealed that the bulls were grouped into two clusters (K = 2), which indicates the presence of two different lineages in the Pakistani Nili-Ravi water buffalo population, and we showed the extent of admixture of these two lineages in our bull collection. LD analysis revealed 4169 significant SNP associations, with an average LD decay of 90 kb for these buffalo genome. Genome-wide association study involved a multi-locus mixed linear model for milk yield and body weight to identify genome-wide male effects. Our study further illustrates the utility of the genotyping by sequencing approach for identifying genomic regions to uncover additional demographic complexity and to improve the complex dairy traits of the Pakistani Nili-Ravi water buffalo population that would provide the lot of economic benefits to dairy industry