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PubMed1996

Influence of soyabean meal supplementation on the resistance of Scottish blackface lambs to haemonchosis.

Protein supplementation improves the resistance of sheep to haemonchosis. This experiment investigated the Scottish blackface breed to establish whether dietary protein supplementation is still beneficial in a genetically resistant breed. Lambs were given either a basal diet or a diet supplemented with soyabean meal to give an additional 80 g crude protein kg dry matter-1. The lambs were given an initial loading dose of Haemonchus contortus, followed by a trickle infection for 10 weeks. The weight gains of the lambs given the supplemented diet were greater and their carcases were leaner, irrespective of infection status. Infected animals on the basal diet were more anaemic and hypoalbuminaemic than animals receiving the supplemented diet, although there were no statistically significant differences in mean worm burdens or faecal egg counts.

Analysis of Variance↗
PubMed1996

Response of Dorper and red Maasai lambs to trickle Haemonchus contortus infections.

Six-month-old red Maasai lambs were more resistant than Dorper lambs to repeated infections at one to two week intervals with 1000 Haemonchus contortus infective larvae. Resistance after infection was assessed by means of faecal egg counts, packed cell volumes, eosinophil counts, total serum protein concentrations and mortality rates. The weight gains of the infected animals were only marginally lower than those of their uninfected controls, most probably because of their significantly higher feed consumption, and evidently the infected lambs were not utilising all of the extra feed for growth. This absence of anorexia in spite of the infection was probably due to the palatability of the high protein diet fed to the lambs.

Animals↗
PubMed1996

Immunisation of sheep with an integral membrane glycoprotein complex of Haemonchus contortus and with its major polypeptide components.

Sheep were immunised against Haemonchus contortus with an integral membrane glycoprotein complex isolated from the intestines of the parasite as antigen. This antigen has been termed Haemonchus galactose-containing glycoprotein complex. Sodium dodecyl sulphate (SDS) polyacrylamide gel analysis has shown that it is composed of several polypeptides but so far these have proved refractory to separation when in the native state. However when dissociated by SDS, it was found to be as efficacious as in its native state, although it was less consistently protective when tested after being dissociated and reduced. An attempt was therefore made to identify the protective ingredient(s) of the dissociated complex by testing its major polypeptides individually after they had been separated on SDS polyacrylamide gels under non-reducing conditions. Partial protection was induced by protein bands with molecular weights of about 200 kD and less than 50 kD, but none of the individual fractions tested was as efficacious as the unseparated complex, suggesting that either more than one component was essential for a consistent effect or that the separation technique had damaged crucial protective epitopes.

Animals↗
PubMedDate not supplied

Duration of protective immunity against ovine haemonchosis following vaccination with the nematode gut membrane antigen H11.

To establish for how long protective antibody levels may be maintained, lambs were vaccinated with the gut membrane antigen H11 and challenged with Haemonchus contortus 14, 84, 126 or 168 days later. Compared to controls, mean faecal egg counts of vaccinated lambs were reduced by 97 per cent, 99 per cent, 92 per cent and 86 per cent respectively. Total worm burdens at postmortem five weeks after infection were reduced by 87 per cent, 94 per cent, 92 per cent and 62 per cent respectively. In vaccinated lambs, antibody levels to H11 peaked at about 60 days after the first vaccination and were maintained for the duration of the experiment. There was evidence of secondary antibody responses to H11 following challenge.

Animals↗
PubMed1999

Progress and expectations for helminth vaccines.

The large amount of scientific progress made in the last 5 years has allowed a more rational approach to the design of nematode vaccines to develop. Successful experimental trials have been published using two different approaches, one aiming to boost acquired host immunity through vaccination with natural immunogens, the other affecting parasite viability by targeting parasite molecules crucial for nutrition or survival in the host. The individual or combined action of these two vaccination procedures will need to be evaluated with respect to their potential effects on animal health and productivity in the field. To this effect, more data are required concerning the level and duration of immunity of the vaccine-induced protection using acceptable adjuvant systems. In addition, the age at which vaccination is effective and the effect of vaccination on highly susceptible or temporarily immunosuppressed individuals will need to be considered. In the case of gastrointestinal nematodes, the level of pasture contamination with infective larvae is dependent on the worm burdens in the host animal and, in turn, affects the buildup of natural resistance in the host. An appreciation of these complex interactive factors is best achieved through computer simulation models using the powerful simulation software that has recently become available. Further animal trials will need to be performed to establish the necessary data to incorporate into the models and to adapt the model outcomes to the trial results. These epidemiologic and simulation studies should be pursued in parallel with vaccine development so that a better appreciation is gained of the requirements of a successful commercial vaccine.

Animals↗
PubMed2000

Cellular profiles in the abomasal mucosa and lymph node during primary infection with Haemonchus contortus in sheep.

Cellular changes in the abomasal tissue and draining abomasal lymph nodes were examined after primary infection of lambs with Haemonchus contortus for 3, 5 or 27-36 days. Infection with H. contortus larvae resulted in a rapid and selective increase in the percentage of CD4(+) T-cells in the abomasal lymph node at 3 days post-infection (PI). By 5 days PI, the lymph node weight had increased two-fold; however, the percentage of lymphocyte populations in the abomasal lymph node resembled that seen in uninfected sheep. Lymph node weights remained at increased levels in the adult nematode infected sheep and down-regulation of B-cell surface markers (sIg and MHC Class II) was apparent in this group. Significant increases in the percentage of CD4(+) T-cells co-expressing MHC Class II, but not CD25, were observed in the larval infected groups except in adult nematode infected sheep. Increased numbers of eosinophils, CD4(+), gamma delta(+) T-cells and B-cells were found in the abomasal tissue by 5 days PI, but no further increases in these cell populations were observed in the adult nematode infected group. In contrast, the level of both lamina propria and intraepithelial mast cells observed in the abomasal mucosa was highest in the sheep carrying an adult nematode burden. These findings indicate that sheep are able to generate an early immune response to infection with H. contortus larvae, characterised by the activation of CD4 T-cells and B-cells in the draining lymph nodes and recruitment of eosinophils, CD4(+) and gamma delta-TCR,WC1(+) T-cells and B-cells in larval infected tissues. However, these changes do not seem to be maintained during infection with the adult parasite where increases in mast cell numbers dominate the local response, indicating that different parasite stages may induce distinct and possibly counteractive immune responses.

Abomasum↗
PubMed2001

A single-chain variable region immunoglobulin library from the abomasal lymph node of sheep infected with the gastrointestinal nematode parasite Haemonchus contortus.

Sheep immunoglobulin (Ig) heavy-chain (V(H)DJ(H)) and lambda light-chain variable region (V(lambda)J(lambda)) nucleotide coding sequence was isolated by reverse transcriptase-polymerase chain reaction (RT-PCR) from abomasal lymph node (ALN) B cells of immune sheep challenged with the gastrointestinal nematode parasite Haemonchus contortus. Single-chain antibodies (scFv) were then constructed with the purified V(H)DJ(H) and V(lambda)J(lambda) Ig gene region DNA using oligonucleotides to PCR and join the variable regions to a central [Gly(4)Ser](3)-linker. In a similar fashion 5'-SfiI and 3'-NotI restriction endonuclease sites were added for cloning into a phagemid expression vector. Expression of sheep scFv from pHFA phagemid in an amber-suppresser strain of Escherichia coli, after infection with filamentous phage, resulted in 10(9) sheep scFv antibodies displayed as a library on phagemid particles. Western blot analysis demonstrated sheep scFv gene expression in E. coli cell lysate and on purified library phage. In addition, four rounds of scFv-library selection against H. contortus surface antigen resulted in a 300-fold increase in the elution titre of phage recovered from parasite surface antigen. Nearly 1000 of the selected and eluted scFvs were expressed in an attempt to identify monoclonal sheep scFv against parasite antigen. Only low affinity clones were isolated during screening of this sheep scFv-library, suggesting different strategies will be needed for isolation of specific high affinity recombinant antibody in future studies.

Amino Acid Sequence↗
PubMed2001

Molecular analysis and nematode resistance association of a polymorphism at the 5' end of the sheep IgE gene.

Previous work using Southern analysis of genomic DNA detected a polymorphism at the 5' end of the sheep and cattle IgE gene. Identical length differences found between fragments following digestion with restriction enzymes indicated that the basis for the polymorphism was an insertion/deletion event. To characterise the polymorphism, the entire cattle and sheep Cvarepsilon genes were sequenced including 668bp of 5' untranslated DNA. Sequence comparison revealed a high degree of similarity between the ovine and bovine genes at both the nucleotide and amino acid level. A feature of the 5' untranslated DNA was the presence of an 87bp repeat starting at -365 upstream of the Cvarepsilon start site. PCR primers were designed to span most of the 5' untranslated sequence, including the repeat unit, and used to amplify genomic DNA from a panel of 40 sheep. Three alleles were found with frequencies of 0.7, 0.29, 0.01 which were identical to the Southern analysis results. Sequencing of the two commonest alleles revealed the basis for the polymorphism was a 36bp deletion from the 87bp repeat. Association studies in a sheep selection flock phenotypically assessed for parasite resistance found a highly significant association between one of the IgE alleles and resistance to the intestinal nematode parasite Trichostrongylus colubriformis (P=0.005). Attempts to confirm this finding in two other flocks using linkage analysis and genotype association failed to find any significant associations between the IgE polymorphism and resistance to either T. colubriformis or Haemonchus contortus.

Alleles↗
PubMed2001

Isolation, characterisation and expression of mRNAs encoding the ovine CC chemokines, monocyte chemoattractant protein (MCP)-1alpha and -2.

CC chemokines are important mediators of immune responses, orchestrating the differential recruitment of various leukocyte populations. Despite the large number of known CC chemokines in other species, no cDNA encoding ovine CC chemokines have been isolated. A homology cloning strategy was utilised to isolate the cDNA of ovine CC chemokines. Full-length monocyte chemoattractant protein (MCP)-1alpha and -2 cDNA have been isolated. The predicted ovine MCP-1alpha amino acid sequence shares 87 and 75% identity with bovine MCP-1alpha and porcine MCP-1, respectively. The predicted ovine MCP-2 amino acid sequence shares 92 and 85% identity with bovine and porcine MCP-2, respectively. Northern blot analysis of MCP-1alpha revealed that it is strongly expressed in cells isolated from mammary lavage fluid (MAL) of ewes given intramammary infusions of Haemonchus contortus. Weak signals were detected in mammary and abomasal tissue. Southern blot analysis of reverse transcriptase-polymerase chain reaction (RT-PCR) products indicates that MCP-1alpha mRNA levels increase in abomasum after challenge with H. contortus. MCP-1alpha mRNA levels were also increased in bronchoalveolar lavage (BAL) cells and lung tissue after house dust mite extract (HDME) challenge. Similarly, MCP-2 mRNA was detected by Northern blot analysis at high levels in MAL cells after H. contortus intramammary infusion, and increased in BAL cells and lung tissue in HDME-challenged sheep. MCP-2 mRNA was not detected by Northern blots in whole mammary or abomasal tissue, but Southern blot analysis of RT-PCR products also indicates that MCP-2 mRNA increases in abomasal tissue after challenge with H. contortus. Hence, two ovine CC chemokine mRNA have been isolated that are up-regulated in response to parasite infection and allergen challenge. Ultimately the isolation of these and other ovine CC chemokines will help elucidate a wide variety of immune responses in sheep.

Amino Acid Sequence↗
PubMed1996

Genetic control of resistance to helminths in sheep.

Uncertainties over the continued effectiveness of currently available anthelmintics and the massive costs associated with development of new drugs have provided an impetus to search for alternative measures to control gastrointestinal nematodes in sheep. One option is to exploit the genetically determined variability in resistance existing within host populations. A number of selection experiments, comprising divergent and control lines, have been initiated to investigate the nature of this genetic regulation. It was found that the heritability of worm-egg counts in faeces after infection ranges from 0.2 to 0.4, indicating that worthwhile genetic gains can be achieved in commercial breeding programmes. Immune responses directed against parasites are under genetic control and appear to be the major factor responsible for the interline differences. Consequently, selection for increased resistance to gastrointestinal nematodes has resulted in an enhanced reactivity across a broad range of immunological functions (humoral, cellular and effector responses). These mechanistic studies have relevance to the development of vaccines and vaccination strategies, as well as for the application of phenotypic and genetic markers to measure resistance more accurately or to identify genetically resistant animals independently of infection.

Animals↗
PubMed2000

EST sequencing of the parasitic nematode Haemonchus contortus suggests a shift in gene expression during transition to the parasitic stages.

Expressed sequence tags from the parasitic nematode Haemonchus contortus were generated in order to identify anchor loci for comparative mapping between nematode genomes and candidate targets for future control measures. In total, 370 SL1 trans-spliced cDNAs from different developmental stages representing 195 different genes were partially sequenced. From these expressed sequence tags 50% were similar to genes with a known or predicted function and 19% were similar to nematode sequences with no ascribed function. From the first, free-living L1 and L3 stages relatively many cDNAs matched to housekeeping genes, and 11% (L1) or 23% (L3) of the encoded proteins were predicted to contain signal peptides. In contrast, no function could be ascribed to most of the cDNAs from the early L5 and adult parasitic stages, but for 30% (L5) or 55% (adult) of the encoded proteins a signal sequence was predicted. This limited analysis suggests that during the transition from the free-living to parasitic stages gene expression shifts towards the synthesis of less conserved extracellular proteins. These proteins offer the best perspectives for vaccine development and the development of anthelmintic drugs. In contrast, cDNAs from the first larval stages may be most suitable for comparative mapping with the free-living nematode Caenorhabditis elegans.

Animals↗
PubMed2001

Expression of Haemonchus contortus pepsinogen in Caenorhabditis elegans.

A genomic copy of a gut-expressed Haemonchus contortus candidate vaccine antigen, pepsinogen, was isolated using the polymerase chain reaction (PCR). The isolated sequence was 4 kb in length and contained eight introns ranging in size from 54 to 1475 base pairs. This sequence, together with its 3' non-coding DNA region containing a polyadenylation signal sequence, was cloned into the Bluescript SK(+) vector immediately downstream of the Caenorhabditis elegans cpr-5 gene promoter. This promoter has been shown previously to direct protein expression to the gut of C. elegans. The construct was micro-injected into DR96 unc-76(e911) mutant C. elegans together with a rescue plasmid and transgenic worms identified by reversion back to wild-type phenotype. Two transgenic lines of C. elegans were established. The presence of the injected construct and of the Haemonchus pepsinogen transcript in transgenic worms was confirmed by PCR analysis. Correct splicing of intronic sequences was observed. Immunohistochemistry showed expression of the Haemonchus pepsinogen protein in the gut of transgenic C. elegans, with reactivity evident in the larval and adult stages. Expression of the Haemonchus pepsinogen in C. elegans affirms the role of C. elegans as a model for parasitic nematodes and demonstrates its potential as a vector for expression of candidate vaccine antigens from parasitic nematodes.

Animals↗
PubMed2003

Selection at a gamma-aminobutyric acid receptor gene in Haemonchus contortus resistant to avermectins/milbemycins.

Gamma-aminobutyric acid (GABA) Type A receptors are inhibitory chloride channels in membranes of vertebrate and invertebrate neuromuscular cells. Gating of the channels by GABA leads to an influx of chloride ions into, and hyperpolarisation of, the cell. GABA receptors are believed to form channels by the association of five protein molecules of varying subunit types, with the second transmembrane (M2) domain of each protein molecule forming a central pore through which chloride ions can pass. We have analysed by single-strand conformation polymorphism the genetic variation of a GABA-receptor gene, HG1, from two sets of unselected and anthelmintic-selected strains of the parasitic nematode Haemonchus contortus. Significant differences in allele frequencies were detected between one unselected strain and its derived ivermectin-selected strain and between the other unselected strain and its derived ivermectin- and moxidectin-selected strains. In each set of strains, one allele increased substantially in frequency in the drug-selected strains relative to their respective unselected strains. The selected allele, however, differed between the two sets of strains. Similar analyses were performed on a phosphoenolpyruvate carboxykinase gene and a nicotinic acetylcholine receptor subunit gene. No significant differences were found in allele frequencies between the unselected and their derived anthelmintic-selected strains. These results indicate the GABA receptor as a possible site of action for avermectins and milbemycins, and suggest its involvement in resistance to these anthelmintics.

Animals↗
PubMed1997

Molecular cloning and characterisation of a developmentally regulated putative metallopeptidase present in a host protective extract of Haemonchus contortus.

Antisera from lambs immunised with the Haemonchus contortus integral membrane protein complex, Haemonchus galactose-containing glycoprotein (H-gal-GP), the lambs being refractory to subsequent challenge, were used to identify several clones from an adult H. contortus lambda gt11 cDNA library. Using gene-specific oligonucleotide primers in conjunction with primers directed to a conserved nematode Spliced Leader (SL) sequence and to the polyA+ tail of mRNA, the remaining 5' and 3' sequences of one of these clones, metallopeptidase-1 (MEP1), were amplified. The 2.4 kb full-length coding sequences was subsequently amplified in a single reaction. Sequence analysis identified MEP1 as encoding a putative zinc metallopeptidase, which shared limited homology with the mammalian type II integral membrane protein neutral endopeptidase (NEP). Southern blotting indicated that MEP1 belonged to a multigene family. MEP1 was expressed in bacteria as a glutathione-S-transferase (GST) fusion protein, and a specific antiserum raised in sheep. This antiserum recognised several polypeptide components of H-gal-GP. Immunolocalisation studies showed that MEP1 encoded a protein located on the luminal surface of the nematode gut. Both MEP1 mRNA and protein are developmentally regulated with expression being limited to the blood-feeding stages of H. contortus.

Amino Acid Sequence↗
PubMed1997

Molecular characterization and expression of two putative protective excretory secretory proteins of Haemonchus contortus.

It has been shown that vaccination with two low molecular mass excretory secretory (ES) antigens of 15 and 24 kDa, respectively, afforded a substantial degree of protection against Haemonchus contortus to sheep. In vitro cultivation of the parasite usually yields a limited amount of these proteins and therefore, recombinant DNA technology was employed to clone the cDNAs encoding the ES proteins of interest and to express them in a convenient vector system. The N-terminal amino acid sequences of the two ES products were determined. Specific 5' primers were used in combination with an oligo (dT) 3' primer to amplify the appropriate cDNAs by polymerase chain reaction (PCR). A lambda ZAPII cDNA library was constructed from mRNA of L5 larvae and subsequently screened with the PCR products. The full length clone of the 15 kDa ES protein coded for a 17.2 kDa precursor molecule of 148 amino acids with a signal peptide of 30 amino acids. The full length clone of the 24 kDa ES protein coded for a 24.6 kDa precursor protein of 222 amino acids with a leader sequence of 19 residues. The expression of both ES products appeared to be developmentally regulated; mRNA encoding occurs only in the parasitic life stages. A cDNA of each ES protein was sub-cloned, without the leader sequence, into a pQE9 expression vector. Both purified recombinant proteins were recognized by sera from H. contortus hyperimmunised sheep as judged by immunoblot analysis, suggesting that antigenic determinants were also present on the recombinant proteins.

Amino Acid Sequence↗
PubMed1998

Ivermectin resistance in nematodes may be caused by alteration of P-glycoprotein homolog.

Resistance to ivermectin and related drugs is an increasing problem for parasite control. The mechanism of ivermectin resistance in nematode parasites is currently unknown. Some P-glycoproteins and multidrug resistance proteins have been found to act as membrane transporters which pump drugs from the cell. A disruption of the mdrla gene, which encodes a P-glycoprotein in mice, results in hypersensitivity to ivermectin. Genes encoding members of the P-glycoprotein family are known to exist in nematodes but the involvement of P-glycoprotein in nematode ivermectin-resistance has not been described. Our data suggest that a P-glycoprotein may play a role in ivermectin resistance in the sheep nematode parasite Haemonchus contortus. A full length P-glycoprotein cDNA from H. contortus has been cloned and sequenced. Analysis of the sequence showed 61-65% homology to other P-glycoprotein/multidrug resistant protein sequences, such as mice, human and Caenorhabditis elegans. Expression of P-glycoprotein mRNA was higher in ivermectin-selected than unselected strains of H. contortus. An alteration in the restriction pattern was also found for the genomic locus of P-glycoprotein derived from ivermectin-selected strains of H. contortus compared with unselected strains. P-glycoprotein gene structure and/or its transcription are altered in ivermectin-selected H. contortus. The multidrug resistance reversing agent, verapamil, increased the efficacy of ivermectin and moxidectin against a moxidectin-selected strain of this nematode in jirds (Meriones unguiculatus). These data indicate that a P-glycoprotein may be involved in resistance to ivermectin and other macrocyclic lactones in H. contortus.

ATP Binding Cassette Transporter, Subfamily B, Mem↗