No abstract

A 615 bp full length cDNA encoding a Teladorsagia circumcincta glutathione 2 transferase (TcGST) was cloned, expressed in Escherichia coli and the recombinant 2 protein purified and its kinetic properties determined. The predicted protein consisted 3 of 205 amino acids and was present as a single band of about 24 kDa on SDS-PAGE. 3 Multiple alignments of the protein sequence of TcGST with homologues from other 3 helminths showed that the highest identity of 53-68% with haem-binding nematode 3 proteins designated as members of the nu class of GSTs. Substrate binding sites and 3 conserved regions were identified and were generally conserved. The predicted 3-3 dimensional structures of TcGST and HcGST revealed highly open binding cavities 3 typical of this class of GST, considered to allow greater accessibility to diverse 3 ligands compared with other classes of GST. At 25 o C, the optimum pH for TcGST 3 activity was pH 7, the V max was 1535 ± 33 nmoles.min -1 .mg -1 protein and the apparent 3 K m for the substrate 1-chloro-2,4-dinitrobenzene (CDNB) was 0.22 ± 0.01 mM (mean 4 ± SD, n = 2). Antibodies in both serum and saliva from field-immune, but not 4 nematode-naïve, sheep, recognised recombinant TcGST in enzyme-linked 4 immunosorbent assays. The recognition of the recombinant protein by antibodies 4 generated by exposure of sheep to the native enzyme indicates similar antigenicity of 4 the two proteins. These findings could aid in the design of novel drugs and vaccine 4 antigens for economically important parasites of livestock.

Internal parasitism, a significant cause of production losses in sheep, is routinely controlled by anthelmintic drenches. A better understanding and alternative control strategies are needed to combat the increasing resistance. This study investigated the presence of resident bacteria in the field strain of Haemonchus contortus. Adult female worms were collected from the abomasa of sheep. DNA was extracted from adult female worms and parasite eggs laid in vitro using long or shorter enzymatic incubation methods. Polymerase Chain Reaction (PCR) was performed using universal bacterial and phylum Firmicutes-specific primers; PCR products were cloned and sequenced. The analysis of the sequences shows a majority of the sequences belong to rumen bacteria, mainly Ruminococcus. Streptococcus was detected in four eggs, and adult worm samples and the sequences had a very high homology to the Streptococcus sequences in the database. Clostridium was detected only in the adult samples, whereas Nevskia and Pseudomonas were detected only in the egg samples. Three antibiotics, Ampicillin (Amp), Gentamycin (Gen) and Tetracycline (Tet), individually or combination, were tested to establish proof of concept that abomasal nematode parasites can be controlled by killing the resident bacteria. A larval migration inhibition assay was used to test the hypothesis. Tet (10 and 20 mM) resulted in around 30% mortality in larvae. Amp and Gen did not result in significant levels of larval mortality but, when given in combination, resulted in significant mortality of the larvae, suggesting the role of antibiotics in controlling the parasites by targeting the resident bacteria.

A 1332 bp full length cDNA encoding Teladorsagia circumcincta isocitrate lyase (TciICL) and a 1575 bp full length cDNA encoding T. circumcincta malate synthase (TciMS) were cloned, expressed in Escherichia coli and the recombinant proteins purified. The predicted TciICL protein of 444 amino acids was present as a single band of about 52 kDa on SDS-PAGE and the recombinant TciMS of 525 amino acids formed a single band about 62 kDa. Multiple alignments of the combined bifunctional TciICL-MS protein sequence with homologues from other nematodes showed that the greatest similarity (89-92%) to the homologues of Ancylostoma ceylanicum, Haemonchus contortus and Haemonchus placei and 71-87% similarity to the other nematode sequences. The 3-dimensional structures, binding and catalytic sites were determined for TciICL and TciMS and shown to be highly conserved. Substrate and metal ion binding sites were identified and were completely conserved in other homologues.TciICL was confirmed as a functional enzyme. At 30 o C, the optimum pH was pH 7.5, the Vmax was 275 ± 23 nmoles.min -1 .mg -1 protein and the apparent Km for the substrate isocitrate was 0.7 ± 0.01µM (mean ± SEM, n = 3). Addition of 10 mM metal ions (except Mg 2+ ) or 1 mM inhibitors reduced the recombinant TciICL activity by 60-90%.Antibodies in both serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant TciICL in ELISA, supporting similar antigenicity to that of the native enzyme.

No abstract