| Dissertation note |
Recently, DNA-based techniques have been applied for specific identification and characterization of parasites and also to evaluate the level of infections. The most commonly used group of anthelmintics is Benzimidazoles (BZ). BZ includes Thiabendazole, Mebendazole, Albendazole, Oxfendazole, Oxibendazole, Febantel and Netobimin. It binds to β- tubulin and inhibits polymerization of tubulin dimers to microtubules H. contortus involves mutations in β-tubulin genes at specific amino acid sites determine the BZ resistance. This study involves the Haemnochus contortus (barber pole worm) present in abomasums of small ruminants. n= 300 total samples were randomly be collected from the different sampling locations. Out of 300 samples of worms, 30 worms had been subjected to DNA extraction and PCR reaction. PCR recipe was prepared with following composition with total volume of 20μl, Master mix (enzynomics) 10μl, Distilled water 4μl, Forward primer 2μl, Reverse primer 2μl and genomic DNA 2μl. After PCR product has been obtained, gel extraction was done by PureLink Quick Gel Extraction Kit of INVITROGEN for purifying the DNA to allow it for gene sequencing. Only selected positive amplification products had been subjected to DNA gene sequencing and were sent to Philco company for ABI type sequencing. The polymorphic sites had been identified by using the standard set of ambiguity nucleotides through BLAST searches at NCBI/primers used to generate the β- tubulin gene sequence from H. contortus. DNA nucleotide sequences obtained are aligned on BIO EDIT SEQUENCE ALIGNMENT EDITOR software to understand the mutation, deletion, replacement of nucleotides. Genetic variation exists in all the samples sent for gene sequencing. These findings show that genetic diversity and genetic variation existing in β-tubulin isotype 1 of the H. contortus population of small ruminants in Jhang and Toba Tek Singh districts of Central Punjab, Pakistan. Samples exhibits different gene sequence and are from Jhang and Toba Tek Singh district. Genetic sequences are retrieved with the sequence and research papers of previously work done on GENBANK NCBI shows 100% query cover and ident with the isolates and clone of China, UK, USA, Australia, Brazil and somewhat similarity of 94% query cover and 100% ident with isolates of India. Some of those H. contortus worms which have been identified as BZ resistant strains were subjected in vitro anthelmintic activity of the herbal extract. Herbal formulation manifests the anthelmintic activity in dose-dependent manner in AMA and EHT. Mortality of worms was comparable with the reference drug used Levamisole at 200mg/ml concentration just from the start of exposure in AMA. Conclusion drawn from the experiment is that the herbal extract efficacy is comparable with the efficacy of the synthetic anthelmintics and can be marketed as these are field friendly, environment safely, economically and socially efficient. However before starts commercialization and marketing of such products at large scale, these materials must be subjected to standardized protocols and procedures of parasitology to be accomplished.
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