R.J. Shaw

Discovery of quantitative trait loci for resistance to parasitic nematode infection in sheep: I. Analysis of outcross pedigrees

BackgroundCurrently most pastoral farmers rely on anthelmintic drenches to control gastrointestinal parasitic nematodes in sheep. Resistance to anthelmintics is rapidly increasing in nematode populations such that on some farms none of the drench families are now completely effective. It is well established that host resistance to nematode infection is a moderately heritable trait. This study was undertaken to identify regions of the genome, quantitative trait loci (QTL) that contain genes affecting resistance to parasitic nematodes.ResultsRams obtained from crossing nematode parasite resistant and susceptible selection lines were used to derive five large half-sib families comprising between 348 and 101 offspring per sire. Total offspring comprised 940 lambs. Extensive measurements for a range of parasite burden and immune function traits in all offspring allowed each lamb in each pedigree to be ranked for relative resistance to nematode parasites.Initially the 22 most resistant and 22 most susceptible progeny from each pedigree were used in a genome scan that used 203 microsatellite markers spread across all sheep autosomes. This study identified 9 chromosomes with regions showing sufficient linkage to warrant the genotyping of all offspring. After genotyping all offspring with markers covering Chromosomes 1, 3, 4, 5, 8, 12, 13, 22 and 23, the telomeric end of chromosome 8 was identified as having a significant QTL for parasite resistance as measured by the number of Trichostrongylus spp. adults in the abomasum and small intestine at the end of the second parasite challenge. Two further QTL for associated immune function traits of total serum IgE and T. colubiformis specific serum IgG, at the end of the second parasite challenge, were identified on chromosome 23.ConclusionDespite parasite resistance being a moderately heritable trait, this large study was able to identify only a single significant QTL associated with it. The QTL concerned adult parasite burdens at the end of the second parasite challenge when the lambs were approximately 6 months old. Our failure to discover more QTL suggests that most of the genes controlling this trait are of relatively small effect. The large number of suggestive QTL discovered (more than one per family per trait than would be expected by chance) also supports this conclusion.

Serum IgE responses during primary and challenge infections of sheep with Trichostrongylus colubriformis.

Serum IgE responses during primary or challenge infections with Trichostrongylus colubriformis were examined by measuring total IgE and parasite-specific IgE by ELISA. Three- to four-month-old lambs reared nematode-free received a single primary infection of 30,000 T. colubriformis-infective larvae (TcL3). Infections were terminated after 100 days by anthelmintic treatment, at which time faecal egg counts had fallen to low levels. Total serum IgE increased slowly from 20 days p.i. until anthelmintic treatment. The specific IgE response to T. colubriformis adult antigen peaked at 20-27 days p.i. before returning to near baseline levels. A further slight increase occurred between 56 and 100 days p.i. The animals were then divided equally into two groups. A first series of challenge infections consisting of either weekly 10,000 TcL3 infections (Group A) or a single 30,000 TcL3 infection (Group B) produced low faecal egg counts. Total and specific IgE to adult and L3-ES antigens increased rapidly over a 21-day period, then remained elevated irrespective of challenge regime. Termination of primary and the first challenge infections with anthelmintic resulted in a rapid decline in serum IgE responses but not other T. colubriformis-specific Ig responses. A second series of challenge infections consisting of repeated 10,000 (Group A) or 20,000 TcL3 infections (Group B) resulted in all IgE responses peaking within 7-8 days p.i. Marked softening of faeces coincided with peaks of specific and total IgE responses during challenge infections. The results of this study showed that infection of sheep with T. colubriformis leads to elevated levels of total and parasite-specific IgE in serum. IgE responses following challenge suggest involvement of this antibody isotype in protection from T. colubriformis infections.

Identification and characterisation of an aspartyl protease inhibitor homologue as a major allergen of Trichostrongylus colubriformis

Allergens were identified from the gastrointestinal nematode of sheep, Trichostrongylus colubriformis, by probing Western blots of infective larvae (third stage) somatic antigen with IgE purified from the serum of sheep grazed on worm contaminated pasture. A 31 kDa allergen was frequently recognised by sera from immune sheep, particularly those deriving from a line that has been genetically selected over 23 years for parasite resistance. Using a proteomic approach, the 31 kDa allergen was identified as an aspartyl protease inhibitor homologue. The entire coding sequence of T. colubriformis aspartyl protease inhibitor (Tco-api-1) was obtained and the mature protein expressed in Escherichia coli. Anti-Tco-API-1 antibodies revealed that a commonly observed 21 kDa T. colubriformis allergen species is a truncated form of Tco-API-1. Specific IgE responses to T. colubriformis aspartyl protease inhibitor were significantly correlated with the degree of resistance to nematode infection as measured by faecal egg count in sheep. Surprisingly, IgE responses to Tco-API-1 were not correlated with breech soiling (dag score), which is thought to be caused, in part, by allergic hypersensitivity to worms. Therefore, a specific IgE response to this allergen may be a suitable marker for identifying lambs at an early age that will develop strong immunity to gastrointestinal nematodes.

Sequential cellular and humoral responses in the abomasal mucosa and blood of Romney sheep dosed with Trichostrongylus axei

Abomasal cannulae were surgically placed in 7 2-year-old New Zealand Romney sheep which had been maintained parasite-free from birth. Four of these sheep were randomly selected and dosed orally with 10,000 infective Trichostrongylus axei larvae per week for 8 weeks, while the remaining 3 sheep served as uninfected controls. Abomasal biopsy, blood and faecal samples were obtained from all sheep at regular intervals from 5 days before and until 58 days after the first infection. The sheep were then killed, worm burdens assessed and abomasal and small intestinal samples collected Faecal egg counts of all 4 dosed sheep were low and only one (No. 701) had a substantial worm burden (8400) post mortem. Overall, levels of mucosal mast cells/globule leukocytes, eosinophils, T19+ cells and larval migration inhibitory activity increased significantly in the abomasal mucosa of the dosed sheep compared to the controls. The CD4+:CD8+ cell ratio in the abomasal mucosa of the dosed sheep also increased compared to that of the controls (P = 0.06). In blood, T. axei-specific antibody (total and IgG1) and eosinophil numbers increased significantly in the dosed sheep. Mucosal cells staining for IgE (IgE+), and blood and mucosal eosinophils showed the earliest substantive increases in number followed by increases in specific serum antibody levels, numbers of mucosal cells fluorescing under UV light (UVf) and T19+ cells. The difference in the IgE+ and UVf cell responses indicated that expansion of globule leukocyte numbers lagged behind that of mucosal mast cells. The results supported the concept of CD4+ T cell help in the abomasal mucosa and defined the sequential expression of components of the immunological responses potentially mediating resistance to T. axei. In sheep No. 701, persistence of adult worms was associated with lower mucosal IgE+ cell and eosinophil responses compared with the other dosed sheep.

Genetic and phenotypic relationships among Trichostrongylus colubriformis-specific immunoglobulin E, anti-Trichostrongylus colubriformis antibody, immunoglobulin G1, faecal egg count and body weight traits in grazing Romney lambs

Variation in total and Trichostrongylus colubriformis-specific immunoglobulin E (IgE) was investigated in four- and six-month old Romney lambs reared on pasture, where they were exposed to natural challenge with nematode parasites. The lambs, which were from an experimental progeny test flock (n=64 sires), were born in spring over three years and weaned at an average age of three months (December). Data from the flock were analysed to obtain heritability estimates for IgE and genetic correlations between IgE and other immunological, parasitological and production traits. In addition, correlated responses of IgE to long-term genetic selection for high, control or low faecal nematode egg count (FEC) were investigated in a set of experimental breeding lines. Repeatabilities and heritabilities of IgE traits in the progeny-test flock were similar to those calculated previously for anti-T. colubriformis antibody (Ab), immunoglobulin G1 (IgG1) and FEC (generally >0.30). Genetic correlations of the loge transformed IgE traits (January and March samples) with loge (FEC+100) on three sampling occasions were all negative (−0.22 to −0.37). However, positive correlations (0.17 and 0.43) were found between loge IgE and dag score, indicating a tendency for there to be more severe breech soiling in lambs with elevated serum IgE. In the 1995 and 1996 selection-line lamb crops (after 17 and 18 years of selective breeding for high or low FEC), both total and T. colubriformis-specific IgE levels were higher (by between 59 and 103%) in the low than in the high line (P<0.001). IgE levels in the controls were intermediate between those of the high and low lines but closer to those of the low line. The results are in line with other evidence which suggests that greater genetic resistance to gastrointestinal nematode infection in Romney lambs is associated with an increase in activity of the TH2 arm of the immune system which mediates inflammatory responses against multicellular parasites.

Immune rejection of Trichostrongylus colubriformis in sheep; a possible role for intestinal mucus antibody against an L3‐specific surface antigen

Sheep that have been immunized by multiple truncated infections with the parasitic nematode Trichostrongylus colubriformis contain anti‐larval activity in their intestinal mucus and high‐speed mucus supernatants. This activity induces T. colubriformis L3 to clump in vitro and causes a significant reduction in larval establishment in naive sheep after infusion of larvae and mucus into the intestinal lumen via a duodenal cannula. In this report, we provide evidence that one factor contributing to the anti‐larval activity of immune mucus is antibody against a 35‐kDa L3‐specific cuticular antigen. The anti‐larval activity in mucus is > 100 kDa by membrane filtration, is heat labile and sensitive to either protease digestion or reduction with DTT. Immunoblotting showed that mucus and supernatants of ultracentrifuged mucus from immune sheep contained IgG1 and IgA antibodies that recognized predominantly a larval antigen with an estimated molecular weight of 35 kDa on SDS‐PAGE. Antibodies eluted from the surface of washed larvae that had been incubated in immune mucus also reacted specifically with the 35 kDa antigen on blots of larval homogenate. Immunofluorescence and immunogold electron microscopy showed that the 35 kDa antigen is present on the epicuticle of L3 and is shed during the moult to L4. The antigen is not present in eggs, L1, L2, L4 or adult worms and is found only in extracts of sheaths and L3 before infection and up to 4 days after infection. We hypothesize that the binding of antibody to the larval surface prevents larvae from establishing at their preferred site, causing them to be eliminated from the intestine. Monoclonal antibody PAB‐1 recognizes the 35 kDa T. colubriformis larval antigen and also cross‐reacts with antigens of similar molecular weight on blots of L3 extracts of the parasitic nematodes Haemonchus contortus and Ostertagia circumcincta; and with a 22‐kDa antigen on blots of L3 extracts from Cooperia curticei and Nematodirus spathiger. This indicates that an antigenically related surface antigen with immunizing potential is present on several nematode species and can be identified by mAb PAB‐1. The 35 kDa T. colubriformis larval antigen and related molecules in other nematodes are potential novel targets for stimulating host‐protective immunity against nematode infections.

Studies on the role of mucus and mucosal hypersensitivity reactions during rejection of Trichostrongylus colubriformis from the intestine of immune sheep using an experimental challenge model

Nematode-naive sheep and sheep immunised by truncated infections with Trichostrongylus colubriformis were fitted with intestinal cannulae to allow administration of challenge infection and collection of intestinal fluids. Sheep were slaughtered at various times after challenge and the distribution of larvae along the small intestine was determined. Results showed that immune sheep had significantly fewer larvae in their intestines and that some sheep could expel the challenge infection within 2 h. Mucus samples from immune sheep contained increased parasite-specific antibody, histamine and anti-parasite activity as measured by larval migration inhibition assay. Higher levels of antibody and histamine were seen in intestinal fluids of immune sheep after challenge. Immunisation of sheep by truncated infections stimulated serum IgE and resulted in significantly higher numbers of IgE-positive cells in gut tissue sections before challenge and at 2 h and 24 h after challenge. Immune sheep also had greater numbers of mucosal mast cells and globule leucocytes after challenge, compared with naive sheep. When challenge larvae were mixed with mucus from immune sheep and infused back into naive recipient sheep, there was a distinct displacement of the larval population towards the distal part of the intestine, compared with the profile of larval establishment after infusion with mucus from naive sheep. These results are further evidence for an immediate hypersensitivity reaction in the intestine of immune sheep, where challenge larvae are expelled within 2 h and confirm the direct anti-larval properties of mucus. The cannulated-sheep challenge model described here will be a useful tool to unravel the mechanism of larval rejection from immune sheep and could lead to novel therapies.

The production costs of anthelmintic resistance in sheep managed within a monthly preventive drench program.

To determine the impact of anthelmintic resistance on the productivity of sheep grazed on pasture in a temperate climatic zone, 14 groups each of 20 lambs were grazed on pasture on which benzimidazole-resistant parasites had been detected previously, then treated every 28 days - seven groups with a benzimidazole anthelmintic (albendazole) and seven with monepantel, a member of a new anthelmintic action family which was assumed in advance to be completely effective in removing all established worms. Faecal egg counts and larval differentiation demonstrated the presence of albendazole resistance, predominantly in Teladorsagia circumcincta but also in Trichostrongylus spp. By days 84 and 112, egg counts were significantly higher in the albendazole-treated animals than in those treated with monepantel. The presence of anthelmintic resistance resulted in a reduction in live-weight of 2.8 kg, a significant increase in breech-soiling and a significant reduction in body condition score. Fourteen animals from each treatment were necropsied at a commercial abattoir and carcase weights and standard quality parameters recorded; there was a reduction in carcase weight of 2.8 kg in the albendazole-treated animals, and a difference in the carcase grades within each group. These measurements were used to calculate that the presence of anthelmintic resistance resulted in a 14% reduction in carcase value.

Quantification of total sheep IgE concentration using anti-ovine IgE monoclonal antibodies in an enzyme immunoassay

Monoclonal antibodies (mAbs) which recognize separate epitopes on ovine immunoglobulin E (IgE) have been used to develop a non-competitive antibody sandwich enzyme immunoassay (EIA) for quantitating ovine IgE. Purified anti-IgE mAb (YD3) coated onto polystyrene microtitre plates was used to capture IgE in serum samples. Biotinylated anti-IgE mAb (XB6) followed by streptavidin conjugated with horseradish peroxidase were used to detect captured IgE. Tetramethylbenzidine and H2O2 were used as enzyme substrate. A reference serum was prepared by pooling sheep sera containing elevated IgE levels. This reference serum was assigned a value of 100 units ml-1 and used to prepare standard curves for the EIA. The linear region of log-log transformed standard curve data covered a range of 0.05-0.8 units ml-1. The equation of a linear regression line fitted to this curve was used to determine sample concentrations. Using purified IgE, 1 unit of reference serum was equivalent to 0.86 micrograms ml-1 IgE. Maximum intra- and inter-assay coefficients of variation for the EIA were 4.6% and 9.7%, respectively. Subjecting serum samples to 15 freeze/thaw cycles, storage at room temperature for 16 days or incubation at 37 degrees C for 8 h resulted in minimal loss of IgE detection. Incubation of serum at 56 degrees C resulted in rapid reduction in detection of IgE by the EIA. The assay was used to determine IgE levels in adult sheep monospecifically infected with weekly doses of the nematode Trichostrongylus axei. Serum IgE levels increased from 9 to 16 days following first infection and reached maximum levels by days 35-58. Serum IgE responses closely followed IgE positive cell responses in the abomasal mucosa.

Production and characterisation of monoclonal antibodies recognising ovine IgE

Two monoclonal antibodies (mAbs), XB6 and YD3, which recognise ovine immunoglobulin E (IgE) were produced. Mast cells isolated from ovine intestinal mucosa were used as a source of IgE to immunize mice. Culture supernatants of hybridomas were screened by immunoassays on small-intestine tissue sections, isolated mucosal cells, and dot blots of lysed mast cell homogenate. Two mAbs were chosen for their specific binding to mast cells. Antigen bound by these mAbs was purified by immunoaffinity chromatography using XB6 mAb, and this produced two bands consistent with IgE heavy chain (86,000 Daltons) and immunoglobulin light chain (28,000 Daltons) when run under reducing conditions on SDS-PAGE gels. Purified IgE was shown on dot blots to react weakly with mAb to chimeric ovine IgE and strongly to polyclonal anti-sheep antibodies. The two mAbs induced an immediate hypersensitivity-like reaction when injected into the skin of sheep. The mAbs bound to mast cells and other mononuclear cells, presumably IgE-secreting B-cells in mesenteric lymph node sections. These mAbs proved useful for detecting IgE-bearing cells in various ovine tissues, for purifying mast cells from cell isolates by panning and immunomagnetic bead separation, for purifying serum IgE using immunoaffinity chromatography and for detecting IgE in an ELISA. Competitive binding assays showed that the two mAbs bind to different epitopes on IgE. These mAbs will be useful in research applications and in diagnostic assays.