Multiplex PCR Detection Of Alleles Responsible For BZ-R And Cloning, Expression And Characters Of New Gene HC38 Of Haemonchus Contortus

Haemonchus contortus is an important gastrointestinal nematode parasite of ruminants in many parts of the world, which belongs to Haemonchus genus of Trichostrongylidae family. It is a blood-feeding nematode parasite that infects the abomasums (fourth or true stomach) and small intestine of ruminants. Infection can lead to anaemia, loss of condition and the death of the host (especially lambs) in severe cases, which cause significant economic loss of husbandry industry. Benzimidazole (BZ) has been an important anthelmintics for the control of this parasite for many years. Large-scale use and improper dose rates of the drug have been identified as the major cause for the development of resistance to this drench in many countries. With the delay in the commercial development of an effective vaccine against this parasite and the increasing awareness of consumers to drug residues, the detection resistance will be important for sustainable animal production and very little is known about the BZ-R situation in China, we commenced this scientific research as follows: 1. Cloning, analysis genomic DNA fragment of β-tubulin from H. contortusAustralia benzimidazole-resistant strain and Chinese Xinjiang Shihezi strainand egg hatch assayA DNA fragment of 798bp was produced from Australia benzimidazole resistant-strain (Au-BZ-R) and Chinese Xinjiang Shihezi strain of H. contortus (XJ) by PCR with a pair of primers designed on |3 -tubulin genomic DNA sequence of H. contortus from GenBank; The sequencing of the cloned target fragment showed: The identity of 4 sequences with the one in GenBank was 90.6-95.1%. The code of residue 200 of the gene was TAC (Tyr) and TTC (Phe) from Au-BZ-R and XJ respectively. The fifth exon containing the code of residue 200 of the gene from XJ was amplified and analysed. The results showed: nucleotide acid of two sequences from Australia differs slightly from the one on GenBank (Identities 98.5%), but the amino acidsequence was similar. Three sequences obtained from XJ were also the same, except the amino acid was different at the second base of the residue 200. Furthermore, there was a TCC genotype in XJ. The ED50 of the H. contortus from Australian BZ-R strain to Albendazole as determined by EHA was 0.54ug/ml, while that of XJ was 0.0023ug/ml.2. Establishment of mutiplex PCR to detect allele responsible for benzimidazole-resistance or susceptibility of H, contortusA multiplex PCR was developed to detect benzimidazole-resistance (BZ-R) in H. contortus using two pairs of primers based on β -tubulin genomic DNA sequence of the parasite on GenBank. This was achieved by optimizing the reaction conditions including primers concentration and proportion, appropriate Taq DNA polymerase, and the determination of template quantity from worms with two recombinant plasmids pMD18-T-AuFl and pMD18-T-XJF1-3. Au-BZ-R was used as the resistant control, while the benzimidazole-susceptibility (BZ-S) strain from Shanghai was the susceptibility control. The results of the mutiplex PCR were as follows: The Au-BZ-R strain produced fragments F1 and F3, indicting a resistance pattern, while the BZ-S from Shanghai showed fragments F2 and F3, a susceptibility pattern. Sensitivity experiments showed that multiplex PCR could detect small quantities of template DNA up to 50ng. The PCR patterns of the assayed heterozygote mixture showed that Fl, F2, and F3 were present. The method was able to detect the BZ-R when pMD18-T-AuFl consisted of only 4x10~12ng in the template mixture and its ratio in total template was 1/ ( 7. 5 x 10~15). The ED50 of Albendazole of the Australian BZ-R strain was 0.54 ug/ml,as compared to the Shanghai BZ-S strain of 0.0023ug/ml using EHA.3. Prevalence of benzimidazole resistant of H. contortus in sheep and goats in 5 regions of ChinaThe benzimidazole resistances of H. contortus strains (473 worms) from Shihezi and Yining of Xinjiang region, Wuhe of Anhui province, Nanjing and Xuzhou of Jiangsu province were detected using the multiplex PCR and EHA with Au-BZ-R and Shanghai strains (192 w...