Response to helminth infection of sheep selected for resistance to Haemonchus contortus

Lines of Merino sheep selected for increased (IRH) and decreased (DRH) resistance to Haemonchus contortus were compared with an unselected (CH) line, after approximately four generations of selection. Measurements were recorded on 69 IRH, 47 DRH and 84 CH animals. Following artificial challenge with H. contortus, the IRH line had significantly (P < 0.001) lower faecal egg counts than the CH and DRH lines (2730,12,720 and 17,400 epg, respectively). Significant differences (P<0.05) were found between all lines in the minimum packed cell volumes during artificial infection (25.7, 22.0 and 20.3%) and in faecal egg counts after natural infection (140, 3590 and 8750 epg). Differences were also recorded (P<0.05) following artificial challenge with Trichostrongylus colubriformis (490, 840 and 1340 epg). On a percentage basis, faecal egg counts in the IRH line deviated less from the CH line following artificial infection with T. colubriformis (42%) than with H. contortus (79%). The reverse was true for the DRH line (60 and 37%, respectively). Differences in egg output of this magnitude should have a marked effect on requirements for anthelmintic treatment, rate of development of drug resistance and level of pasture contamination when the lines are grazed separately.     

Selection for anthelmintic resistance by macrocyclic lactones in Haemonchus contortus.

Two morphologically marked strains of Haemonchus contortus, CAVRS (smooth-macrocyclic lactone resistant) and McMaster (linguiform-macrocyclic lactone susceptible), were used to investigate the selection for anthelmintic resistance following exposure to ivermectin (IVM), a non-persistent anthelmintic. and a more persistent anthelmintic, oral moxidectin (MOX). Three types of selection were investigated: (1) selection of resident worms at the time of treatment (Head selection); (2) selection of incoming-larvae post-treatment (Tail selection); and (3) selection of both resident population and incoming larvae (Head + Tail selection). The experimental animals were adult sheep and lambs. In the controls where there was no anthelmintic selection, the proportion of CAVRS in the adult worm population was the same as the proportion in larvae given to both adults and lambs indicating that CAVRS and McMaster H. contortus were equally infective. There was a significant effect of anthelmintic on total worm numbers in adult sheep with MOX treated adults having less worms, but selection type was non-significant. Anthelmintic type had a significant effect on numbers of resistant worms in adult sheep with less resistant worms in the MOX treated groups, but selection type had no effect. Analysis of variance of arcsine-transformed proportions of resistant worms found that the type of anthelmintic had a highly significant effect, with MOX treated adults having a higher proportion of resistant worms, while type of selection was not significant. In the lambs, nil treated controls and IVM Head + Tail and Tail selected groups had similar geometric mean total worm burdens while Head selected had less total worms. In the MOX treated lamb groups the worm burdens were similar within selection type but less than the IVM treated groups. In the lambs, the types of selection that resulted in more resistant worms were IVM Tail, MOX Head + Tail and MOX Tail. Resistant worm numbers were similar in both adult and lamb groups with Head selection by either MOX or IVM. Moxidectin selected out higher proportions of resistant worms than did IVM in the lambs, with Tail and Head + Tail being stronger selectors than Head. Computer simulations were used to estimate the rate at which resistance developed in the field using the information generated in the present study. The anthelmintic treatments used in the simulation followed a strategic parasite control program for H. contortus in which all sheep receive three Closantel (CLS) treatments in summer. all sheep receive a broad-spectrum (BS) drench or capsule at weaning and lambs receive an additional two BS drenches insummer or no further treatment in the case of the capsule. Moxidectin, IVM-capsule and IVM were the broad spectrum anthelmintics simulated. All simulations were run four times assuming high or low efficacy against resident resistant worms and in the presence or absence of CLS resistance. The simulations indicated that the presence of CLS resistance hastened selection for macrocyclic lactone (ML) resistance. While the IVM-capsule will select most rapidly for ML resistance, IVM oral is expected to be least selective. Moxidectin treatment is intermediate, except in simulations with no CLS resistance and when MOX is assumed to be highly effective against resident ML-resistant worms, in which case MOX can be expected to select more slowly than IVM oral treatments.     

Variation in resistance to haemonchosis: selection of female sheep resistant to Haemonchus contortus.

Seventy female lambs (6-7 months old) which were exposed to natural infections of Haemonchus contortus were designated as responders or non-responders on the basis of 10 weekly cumulative faecal egg counts. Selected responder and non-responder lambs were treated with ivermectin, housed separately and 6 weeks post-housing, seven lambs from each group were given a trickle infection of Haemonchus contortus at 1000 L3 daily for 5 days per week up to 2 weeks and examined weekly for 10 weeks after first infection. Analysis of data revealed significantly lower mean faecal egg counts and non-significantly less weight loss in responder than non-responder lambs. Mean values of haemoglobin, packed cell volume, total serum protein and peripheral eosinophil counts were significantly higher in responders than non-responders. In contrast, serum pepsinogen concentration was significantly less in responders than in non-responders. At 10 weeks post-infection, there were fewer pathological lesions and significantly lower worm burdens in responders than in non-responders. These results demonstrate a distinct resistance in responders to Haemonchus contortus infection.     

Genetic mapping of quantitative trait loci for resistance to Haemonchus contortus in sheep

This paper presents results from a mapping experiment to detect quantitative trait loci (QTL) for resistance to Haemonchus contortus infestation in merino sheep. The primary trait analysed was faecal worm egg count in response to artificial challenge at 6 months of age. In the first stage of the experiment, whole genome linkage analysis was used for broad-scale mapping. The animal resource used was a designed flock comprising 571 individuals from four half-sib families. The average marker spacing was about 20 cM. For the primary trait, 11 QTL (as chromosomal/family combinations) were significant at the 5% chromosome-wide level, with allelic substitution effects of between 0.19 and 0.38 phenotypic standard deviation units. In general, these QTL did not have a significant effect on faecal worm egg count recorded at 13 months of age. In the second stage of the experiment, three promising regions (located on chromosomes 1, 3 and 4) were fine-mapped. This involved typing more closely spaced markers on individuals from the designed flock as well as an additional 495 individuals selected from a related population with a deeper pedigree. Analysis was performed using a linkage disequilibrium–linkage approach, under additive, dominant and multiple QTL models. Of these, the multiple QTL model resulted in the most refined QTL positions, with resolutions of <10 cM achieved for two regions. Because of the moderate size of effect of the QTL, and the apparent age and/or immune status specificity of the QTL, it is suggested that a panel of QTL will be required for significant genetic gains to be achieved within industry via marker-assisted selection.     

No adaptation of Haemonchus contortus to genetically resistant sheep.

A composite population of Haemonchus contortus was established with larvae from seven diverse sources, then maintained in Merino sheep bred to have either increased or decreased resistance to Haemonchus. After five, seven and 14 parasite generations, the two resulting lines of parasites were used to infect sheep from the increased resistance line, an unselected control line and the decreased resistance line. Line of sheep had a highly significant effect on average faecal egg counts 4 and 5 weeks after infection (geometric means 329, 735 and 1490 epg, respectively after the 14th generation), but the two lines of parasites yielded similar egg counts. There was no significant interaction between line of sheep and line of parasite, indicating that the parasite populations had not diverged significantly in their reproductive fitness, as measured by faecal egg count.     

Detection of resistance to ivermectin in Haemonchus contortus.

Infective, third-stage (L3) larvae of Haemonchus contortus isolates resistant to ivermectin (IVM) show a decreased sensitivity to IVM-induced paralysis in vitro. The inhibition of larval motility by IVM can be detected in L3 larvae incubated in the dark on an agar matrix containing IVM, by the failure of affected larvae to move when stimulated by exposure to light. Optimally, avermectin (AVM) potency is quantified after three cycles, each involving storage in the dark for 24 h followed by a brief exposure to light. For IVM-susceptible isolates, a 50% inhibition of motility (LP50) was achieved with IVM concentrations between 0.30 and 0.49 microM, while LP50 values in IVM-resistant isolates ranged from 0.8 to 2.6 microM depending on the in vivo resistance status of the isolate. A limited study of structure-activity relationships within the AVM class indicated that in vitro inhibition of L3 motility was consistent with the known in vivo efficacy of each analogue. Resistance factors for IVM-resistant isolates were dependent on AVM structure with the more polar AVM B2 analogue being a particularly sensitive probe of IVM-resistance status.     

Laboratory selection of Haemonchus contortus for resistance to ivermectin

The eighth generation of adult Haemonchus contortus, selected by subjecting infected pairs of sheep to suboptimal ivermectin treatment once per generation from parent (P; BBH isolate) through F7 (IV-A; selected isolate), required an approximate 4-fold increase in the ivermectin dose to produce 95% efficacy compared with its contemporary parent isolate. In a dose titration experiment the dose-response curve of the drug pressure-derived isolate, IV-A, was significantly (0.02 less than P less than 0.05) less steep than was the response curve of the parent, BBH, isolate. Potency estimates based upon these nonparallel dose-response curves would not remain constant over a range of efficacy levels but would decrease rapidly at efficacies greater than 95%. Passage of a closed population of the F8 generation of IV-A sequentially through pairs of sheep for an additional 11 generations (F8A-F8K) without additional drug pressure being applied produced no reversion to sensitivity to ivermectin relative to the F7 generation, thus suggesting that the selected "resistance" was stable.     

Genetic parameters for growth and faecal worm egg count following Haemonchus contortus experimental infestations using pedigree and molecular information

Background

Haemonchosis is a parasitic disease that causes severe economic losses in sheep industry. In recent years, the increasing resistance of the parasite to anthelmintics has raised the need for alternative control strategies. Genetic selection is a promising alternative but its efficacy depends on the availability of genetic variation and on the occurrence of favourable genetic correlations between the traits included in the breeding goal. The objective of this study was twofold. First, to estimate both the heritability of and the genetic correlations between growth traits and parasite resistance traits, using bivariate linear mixed animal models, from the phenotypes and genotypes of 1004 backcross lambs (considered as a single population), which underwent two subsequent experimental infestations protocols with Haemonchus contortus. Second, to compare the precision of the estimates when using two different relationship matrices: including pedigree information only or including also SNP (single nucleotide polymorphism) information.

Results

Heritabilities were low for average daily gain before infestation (0.10 to 0.15) and average daily gain during the first infestation (0.11 to 0.16), moderate for faecal egg counts during the first infestation (0.21 to 0.38) and faecal egg counts during the second infestation (0.48 to 0.55). Genetic correlations between both growth traits and faecal egg count during the naïve infestation were equal to zero but the genetic correlation between faecal egg count during the second infestation and growth was positive in a Haemonchus contortus free environment and negative in a contaminated environment. The standard errors of the estimates obtained by including SNP information were smaller than those obtained by including pedigree information only.

Conclusions

The genetic parameters estimates suggest that growth performance can be selected for independently of selection on resistance to naïve infestation. Selection for increased growth in a non-contaminated environment could lead to more susceptible animals with long-term exposure to the infestation but it could be possible to select for increased growth in a contaminated environment while also increasing resistance to the long-term exposure to the parasite. The use of molecular information increases the precision of the estimates.     

Genome-wide scan reveals important additive and non-additive genetic effects associated with resistance to Haemonchus contortus in Florida Native sheep

Florida Native sheep is among the sheep breeds best adapted to humid and hot climatic conditions such as those of Florida, USA, and have shown a superior ability to regulate nematode burdens. This is one of the oldest sheep breeds in North America and is an endangered species. To ensure genetic diversity and long-term survival of the breed, protection of the current genetic stock is critical and conservation efforts are required to promote its breeding and production. The objective of the present study was to investigate the importance of additive and non-additive genetic effects on resistance to natural Haemonchus contortus infections in Florida Native sheep using a whole genome scan. A total of 200 sheep were evaluated in the present study. Phenotypic records included faecal egg count (FEC, eggs/gram), FAMACHA® score, packed cell volume (PCV, %), body condition score and average daily gain (ADG, kg). Sheep were genotyped using the GGP Ovine 50K SNP chip and 45.2 k single nucleotide polymorphism (SNP) markers spanning the entire genome were available for quality control procedures. Mixed models were used to analyse the response variables and included the identity by state matrix to control for population structure. Bonferroni correction was used to control for multiple testing and a second arbitrary threshold (0.1 × 10^?3) was used. Fifteen SNPs with additive and non-additive genetic effects and located in Ovis aries chromosome OAR1, 2, 3, 6, 8, 10, 11, 12, 13 and 21 were associated with FEC, FAMACHA® score, PCV and ADG. These SNPs could be potential genetic markers for resistance to natural H. contortus exposure in Florida Native sheep.     

Genetic resistance to helminths. The influence of breed and haemoglobin type on the response of sheep to re-infection with Haemonchus contortus.

The influence of genetic factors on acquired resistance to Haemonchus contortus infections in sheep was investigated. Animals whose primary infections were terminated with an anthelmintic failed to develop any immunity against subsequent challenge as judged by worm numbers. Nevertheless, all were better able to retard the development and reduce the fecundity and haematophagic activities of their parasite populations than animals undergoing primary infections. High levels of resistance, as judged by all these parameters, were observed in most animals when the challenge larvae were superimposed on existing worm populations. The patterns of worm establishment and disease indicated that genetic factors operated in determining resistance, since fewer worms became established and less severe clinical and pathophysiological changes were observed in Scottish Blackface than in Finn Dorset sheep with the same haemoglobin type. Similar advantages were displayed by animals with haemoglobin AA and to a lesser extent those with haemoglobin AB over haemoglobin BB types. The importance of breed was further indicated by the occurrence of 'self-cure' in the majority of the Scottish Blackfaces but in only one Finn Dorset. There was no evidence that this reaction was associated with haemoglobin type.     

A search for associations between major histocompatibility complex restriction fragment length polymorphism bands and resistance to Haemonchus contortus infection in sheep

Polymorphic bands were detected within the DQB and DRB regions of the ovine major histocompatibility complex by probing TaqI digested DNA from three large sheep half-sib families derived from a highly resistant ram. All animals were phenotypically assessed for Haemonchus contortus resistance by faecal egg counts and associations with RFLP bands and haplotypes were estimated using mixed model, best linear unbiased prediction statistical methods. Although the highly resistant sire was homozygous at the MHC, no significant associations were found between any band or haplotype and faecal egg count.     

Changes in anthelmintic resistance status of Haemonchus contortus and Trichostrongylus colubriformis exposed to different anthelmintic selection pressures in grazing sheep

This experiment was designed to study, over a 5-year-period, the effect of different frequencies of treatment with three different anthelmintic groups, namely, benzimidazoles, levamisole and ivermectin, and different frequencies of alternation between them, on existing levels of anthelmintic resistance in the nematode parasites Haemonchus contortus and Trichostrongylus colubriformis of grazing sheep. No evidence of ivermectin resistance emerged, even in suppressively treated groups. Likewise, H. contortus failed to develop resistance to levamisole under a similar selection regimen. Thiabendazole was shown to select positively against levamisole resistance in T. colubriformis resulting in significantly greater susceptibility to this drug than for the natural reversion which occurred in the untreated control. There was no evidence that an anthelmintic treatment combined with a movement of sheep to pastures of low infectivity selected more rapidly for resistance than where the same number of treatments were given to set-stocked sheep. Rotation between anthelmintic groups at yearly intervals appeared to be more beneficial in delaying resistance than rotation of drugs with each treatment.     

Observations on the development of Haemonchus contortus in young sheep given a single infection.

Worm free lambs 2-4 months old were given a single infection of 50,000 Haemonchus contortus larvae. The changes in packed red cell volume were compared with those of uninfected sheep and the infected sheep were divided into two groups according to the rate of change of packed cell volume. In group 1, packed cell volume fell sharply from the 10th day of infection and the sheep eventually died. In group 2, the fall was less marked and the packed cell volume returned to normal after about 40 days. The rates of increase of parasite body length and dry weight were compared for the two groups. Parasites in group 1 sheep grew more slowly than their group 2 contemporaries. Accurate planning of metabolic studies on H. contortus was made possible by using this information to predict the course of an infection and the size of the worms. Parasite development was also measured in sheep for which the packed cell volume was not recorded. Computer analysis showed that adult worm length increase did not follow a simple growth pattern starting from the last ecdysis, but consisted of a rapid elongation followed by a simple negative exponential increase. Dry weight increase also followed a negative exponential during the second phase. Measurements of dry weight as a percentage of wet weight indicated that the rapid elongation was possibly linked with cell enlargement by the uptake of water.     

Studies on the abomasal pathology of immunized and non-immunized sheep infected with Haemonchus contortus

Five adult ewes and five lambs were repeatedly immunized with weekly doses of 10,000 irradiated Haemonchus contortus L3 before challenge with 100,000 and 10,000 normal larvae respectively. Two groups of non-immunized ewes and lambs were similarly challenged and one animal from each of the four groups killed on days 3, 5, 7, 10 and 24 post-challenge. The cellular changes in the abomasal mucosa were less marked in the non-immunized groups than in the immunized animals and appeared later in the lambs than in the ewes. Thus, in the immunized ewes increases in the numbers of mast cells and eosinophils were evident within five days of challenge whereas similar changes appeared later in the immunized lambs. Also marked lymphoid aggregates at the base of the mucosa and in the submucosa were detected only in the immunized ewes. However, both immunized and non-immunized ewes showed rises in the numbers of IgA plasma cells after challenge which were not evident in either group of lambs.     

Field studies on inhibition of Haemonchus contortus.

The epidemiology of Haemonchus contortus was studied in north east England in lambs reared under field conditions with infected ewes. The parasite population was monitored by pasture larval counts, faecal egg counts and lamb postmortems. From 25 June to 11 November, eight lambs were slaughtered each month, together with eight worm-free tracer lambs. Postmortem worm counts showed that on 25 June, small numbers of adult H. contortus were present but no inhibited larvae. However, subsequently the number of worms recovered rose rapidly to 1000-5000 and the percentage inhibition increased to 57% in July, 75% in August and virtually 100% in September, October and November. On each sampling occasion no significant differences in worm count or percentage inhibition were recorded between experimental and tracer lambs. These results suggest that neither autumn climatic effects nor host immunity were responsible for inhibition in this strain of H. contortus.     

Comparative physiological responses of stress in sheep selected for resistance or susceptibility to Haemonchus contortus in semi-arid tropical region during different seasons

The present study was conducted to assess the comparative physiological responses of stress in sheep selected for resistance or susceptibility to Haemonchus contortus in the semi-arid region during different seasons. A total of 20 adult Malpura sheep from resistant and susceptibility line were selected for study. The intensity of strongyle infection, blood biochemical and cortisol levels were estimated monthly basis while meteorological data were collected everyday throughout the year. The temperature-humidity index was the highest during dry-hot season. The faecal egg count increased significantly (p < 0.05) during June–August (rainfall season) in susceptible sheep as compared to resistant sheep. The plasma cortisol level increased significantly (p < 0.05) in both the groups during dry hot (March–May) season and it remained higher in the susceptible line till June–August. The total plasma protein reduced significantly during hot-humid with scantly rainfall (September–November) season as compared to other season in susceptible line. It is pertinent to conclude that though sheep remained in stress during dry hot (March–May) season, but effects of stress on the intensity of strongyle infection and blood biochemical were better tolerated by animals of resistant line during hot-humid season compared to susceptible line under semi-arid region.     

Immunity to Haemonchus contortus and the cellular response to helminth antigens in the mammary gland of non-lactating sheep.

Cellular exudates induced by infusion with helminth antigens were examined in non-lactating mammary glands of ewes immune to infection with the abomasal nematode, Haemonchus contortus. Secondary immunological responsiveness was expressed in two ways. Firstly, antigens from adult H. contortus elicited larger eosinophil-rich cellular exudates in immune compared to non-immune ewes. In this situation, secondary responsiveness in the mammary gland must have been generated through abomasal infection with the parasite. Secondly, repeated infusion with the antigens from adult H. contortus increased the size of cellular exudates in both immune and non-immune ewes. Eosinophils predominated but numbers of macrophages and lymphocytes were also increased. In this second situation, secondary responsiveness must have been either supplemented in immune ewes or derived completely in non-immune ewes by contact with helminth antigens through the mammary gland. The helminth antigens which induce eosinophil exudates in the mammary gland may not be potently protective against H. contortus. Furthermore, eosinophil exudation may not be an in vivo correlate of immunity which is directly useful for discriminating protective antigens and applicable to vaccine development. Infusion with antigens from adult forms of either H. contortus or Trichostrongylus colubriformis elicited cellular exudates equally well in immune ewes primed by infusion with H. contortus adult antigens 7 days beforehand. In addition, antigens from infective larvae of H. contortus elicited cellular exudates more potently than antigens from adult worms. However, vaccination with irradiated larvae has shown that species-specific protective immunity for H. contortus is stronger than cross-protective immunity conferred by T. colubriformis.(ABSTRACT TRUNCATED AT 250 WORDS)     

A study of the mortality and fecundity of Haemonchus contortus in sheep following experimental infections.

The regulation of the fecundity and mortality of H. contortus in sheep was examined using a series of mathematical models. Six-month-old Dorset crossbred lambs were infected once only with various doses of infective H. contortus larvae (500-20,000 larvae). Parasite mortality was found to be an increasing linear function of the magnitude of the initial infection over the range of doses examined. Parasite fecundity was found to remain constant over the intensity and duration of the infection. The average fecundity for H. contortus at the time of slaughter was found to be 7037 eggs per female worm per day. There was no evidence of time-dependent changes in fecundity or density-dependent regulation of fecundity.