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.     

Multiple resistance to anthelmintics by Haemonchus contortus and Trichostrongylus colubriformis in sheep in Brazil

The objective of this study was to determine the level of resistance of Haemonchus contortus and Trichostrongylus colubriformis in sheep to levamisole, albendazole, ivermectin, moxidectin, closantel and trichlorfon. The parasites were isolated from sheep naturally infected by gastrointestinal nematodes and were then kept in monospecifically-infected lambs for production of infective larvae (L3) of both species. Forty-two lambs, at three months of age, were simultaneously artificially infected with 4000 L3 of H. contortus and 4000 L3 of T. colubriformis. The animals were allocated into seven groups with six animals each that received one of the following treatments: Group 1--control, no treatment; Group 2--moxidectin (0.2mg/kg body weight (BW)); Group 3--closantel (10mg/kg BW); Group 4--trichlorfon (100mg/kg BW); Group 5--levamisole phosphate (4.7 mg/kg BW); Group 6--albendazole (5.0mg/kg BW); and Group 7--ivermectin (0.2mg/kg BW). Nematode fecal egg counts (FEC) were carried out on the day of treatment and again at 3, 7, 10 and 14 days post-treatment. On the same occasions, composite fecal cultures were prepared for each group for production of L3, which were identified into genus. The animals were sacrificed for worm counts at 14 days after treatment. The efficacy of each treatment was calculated from the arithmetic mean of the FEC or worm burden of the treated group, compared with the values of the control group. Only trichlorfon and moxidectin treatments resulted in a significant reduction of H. contortus recorded at necropsy (73% and 45% respectively). Moxidectin reduced T. colubriformis worm burdens by 82% and albendazole by 19%. All other anthelmintics resulted in no significant reduction in the numbers of worms found at necropsy. In conclusion, the isolates of H. contortus and T. colubriformis showed multiple resistance to all groups of anthelmintics tested. This is the first report, based on the controlled efficacy test, to show resistance of T. colubriformis to macrocyclic lactones in Brazil.     

Resistance of selected lines of Haemonchus contortus to thiabendazole, morantel tartrate and levamisole.

A field strain of Haemonchus contortus isolated from sheep at Armidale N.S.W., was found to be resistant to thiabendazole with approximately 20 per cent of the worms surviving a 50 mg/kg dose. The isolate was selected over six generations for resistance to 50 mg/kg thiabendazole. After this time, selection on one line was continued at 50 mg/kg thiabendazole and selection on a second line was extended to include 8·8 mg/kg morantel tartrate. A drug tolerance assay on the third generation of the thiabendazole-morantel tartrate selected line showed the ld_5o to be 5·3 mg/kg and the ld₉₅ to be 18 mg/kg morantel tartrate; the reported ld₅₀ and ld₉₅ for non-resistant strains of H. contortus to morantel tartrate are 2·5 mg/kg and 8 mg/kg respectively. In the fourth generation the thiabendazole and the thiabendazole-morantel tartrate selected lines together with a recently isolated field strain were assayed for resistance to thiabendazole, morantel tartrate and levamisole. The results indicated that the resistance to thiabendazole was probably due to a single gene. Both selected lines were more resistant to thiabendazole than the field strain. The thiabendazole-morantel tartrate selected line was more resistant to morantel tartrate than either of the other two. Resistance to morantel tartrate appeared to be polygenic in nature and due to increased vigour. The lowest dose of levamisole (1·6 mg/kg) killed more than 95 per cent of all strains of worms. There was no significant increase in effectiveness at higher dose rates indicating that surviving worms were resistant to the drug.     

Further investigation of the primary mechanism of benzimidazole resistance in Haemonchus contortus

The binding of the [³H] benzimidazole carbamates (BZCs)—albendazole (ABZ), oxibendazole (OBZ), parbendazole (PBZ), mebendazole (MBZ), fenbendazole (FBZ) and oxfendazole (OFZ)—to tubulin from three ecologically-related isolates of adult Haemonchus contortus has been examined. The extent of binding of each BZC was inversely proportional to the known resistance status of the isolate. Biochemically, the change in the formation of the BZC-tubulin complex was due to a reduction in the amount of drug bound to resistant tubulin, with no significant change in the association constant of the complex. The resistance factors derived from the binding data support the hypothesis that the complex is ligand-dependent, with the aryl-substituted BZCs—MBZ, OFZ and FBZ—demonstrating lower resistance factors than those of the alkyl-substituted BZCs—ABZ, OBZ and PBZ. Examination of the slope derived from plots of binding against protein concentration demonstrated that the failure of resistant or partially resistant isolates to bind was due to either a decrease in the number of binding sites or, more likely, to reduced stability of the BZC-tubulin complex rendering it unstable to charcoal extraction.     

Utility of a Haemonchus contortus/jird (Meriones unguiculatus) model for studying resistance to levamisole

Trichostrongylid nematodes of sheep commonly are identified as exhibiting resistance to levamisole. In vitro assays have been developed to study levamisole resistance for Haemonchus contortus, but no in vivo model has been identified for this species. To determine the utility of a H. contortus/jird (Meriones unguiculatus) model for examining levamisole resistance, immunosuppressed jirds were inoculated with approximately 1,000 exsheathed infective larvae of H. contortus (resistant or susceptible to levamisole), treated per os on day 10 postinoculation (PI) with levamisole hydrochloride or analogs of the drug, and killed on day 13 PI. Stomachs were removed, opened longitudinally, incubated in distilled water at 37 C for 5 hr, fixed in formaldehyde solution, and stored for subsequent microscopic examination. Doses of levamisole and its analogs, which elicited percentage clearances of greater than or equal to 93.5 for the susceptible strain, cleared less than or equal to 68.9% of the resistant worms. These data are consistent with activities for the drugs against wild-type and levamisole-resistant strains of Caenorhabditis elegans. Thus, the H. contortus/jird model provides a useful in vivo tool to study resistance to levamisole and possibly other anthelmintics.     

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.     

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.     

A simple,nondestructive spraying assay for the detection of an active kanamycin resistance gene in transgenic tomato plants

Summary A simple, nondestructive kanamycin spraying assay for detecting neomycin phosphotransferase II activity in tomato has been developed. This assay does not require the use of tissue culture or biochemical methods, but allows transgenic and non-transgenic tomato plants to be distinguished directly at the plant level in the green-house. Its potential applications in large-scale genetic analyses are discussed.     

Quantitative trait loci for resistance to Haemonchus contortus artificial challenge in Red Maasai and Dorper sheep of East Africa

A genome‐wide scan was performed to detect quantitative trait loci (QTL) for resistance to the gastrointestinal nematode Haemonchus contortus in a double backcross population of Red Maasai and Dorper sheep. The mapping population comprised six sire families, with 1026 lambs in total. The lambs were artificially challenged with H. contortus at about 6.5 months of age, and nine phenotypes were measured: fecal egg count, packed cell volume decline, two weight traits and five worm traits. A subset of the population (342 lambs) was selectively genotyped for 172 microsatellite loci covering 25 of the 26 autosomes. QTL mapping was performed for models which assumed that the QTL alleles were either fixed or segregating within each breed, combined with models with only an additive QTL effect fitted or both additive and dominance QTL effects fitted. Overall, QTL significant at the 1% chromosome‐wide level were identified for 22 combinations of trait and chromosome. Of particular interest are a region of chromosome 26 with putative QTL for all nine traits and a region of chromosome 2 with putative QTL for three traits. Favorable QTL alleles for disease resistance originated in both the Red Maasai and Dorper breeds, were not always fixed within breed and had significant dominance effects in some cases. We anticipate that this study, in combination with follow‐up work and other relevant studies, will help elucidate the biology of disease resistance.     

A hybridisation technique to identify anthelmintic resistance genes in Haemonchus

The identification of genes associated with anthelmintic resistance can be facilitated in Haemonchus contortus by the ability of this species to hybridise with Haemonchus placei. Although the hybrid males are sterile, the lines can be rescued by backcrossing the females to either parental species. Resistance genes can be retained in Haemonchus hybrids, while the unwanted contortus background is removed through backcrossing to H. placei and anthelmintic selection of the progeny. Under this selection, genes involved in resistance would retain the H. contortus nucleotide sequence, while those that are not would either be H. placei or a random mixture of both, depending on the amount of backcrossing that had occurred. The first candidate gene to be tested in this system was a Haemonchus P-glycoprotein, hcpgp-1. hcpgp-1 was amplified, cloned and sequenced from H. contortus and H. placei. Two restriction sites were then identified in the sequenced product; one specific to H. contortus hcpgp-1 and the other found only in the H. placei gene. These genes were identified from macrocyclic lactone selected and non-selected worms by restricting PCR products from individual worms. Fitted occurrence of the H. contortus allele was 49% of unselected worms and 69% of macrocyclic lactone selected worms. The probability of this percentage occurring by chance was P=0.006. Thus macrocyclic lactone selection was acting to increase the percentage of hcpgp-1 from macrocyclic-lactone-resistant CAVRS.     

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.     

A sensitive method for the determination of levamisole in serum by electrochemiluminescence

A novel method was developed for the determination of levamisole by electrochemiluminescence. The method was based on electrochemiluminescence signal enhancement produced by Ru(bpy)₃²⁺, which reacted with the tertiary amine group of levamisole on a platinum electrode in 12 mmol/L borate buffer (pH 9). A linear relationship between the luminous intensity and concentration of levamisole in the range 0–1 × 10^–7 mol/L was obtained and the detection limit was 1.76 × 10^–11 mol/L. The method is sensitive, selective, simple and convenient. The method has been successfully applied to the analysis of levamisole in serum. Copyright © 2013 John Wiley & Sons, Ltd.     

The genetics of resistance and resilience to Haemonchus contortus infection in young merino sheep

Young Merino sheep in 60 half-sib groups, totalling over 1000 lambs, were given a single dose of 11,000 Haemonchus contortus larvae. Resistance was measured, together with productivity during infection and when uninfected. One half-sib group exhibited an extremely high level of resistance; its sire was postulated to be the carrier of a major resistance gene. Genetic parameters were estimated for productivity during infection and in the absence of infection, for resistance as measured by faecal egg count and haematocrit, and for resilience as measured by the depression of productivity due to infection. The heritability of faecal egg count was estimated at 0.3, that for haematocrit after infection at 0.4. The heritability of resilience was too low to allow substantial progress by direct selection for this trait. Genetic correlations between resistance to infection and production traits when uninfected were not significantly different from zero. It was concluded that selection for polygenically controlled resistance would lead to substantial progress for this trait and would also increase productivity of infected animals.     

Localisation of serotonin and dopamine in Haemonchus contortus

Serotonin and dopamine play important roles in the biology of nematodes where they exert their effect on feeding, locomotion and reproductive behavior. Haemonchus contortus, a parasitic nematode which infects small ruminants, is responsible for considerable economic losses in agriculture. In the current study we have mapped the localisation of these two neurotransmitters in this parasite using immuno-staining. Serotonin localised in amphidial and pharyngeal neurons in both adult female and male worms. Serotonin was also found in ray sensory neurons as well as in a few ventral cord motor neurons exclusively in adult males. Surprisingly, dopamine was only detected in the neuronal commissures linking the lateral and sub-lateral nerve cords in both sexes. We also studied the effect of these two molecules on female adult worms in vitro. Serotonin mainly inhibited movement whereas dopamine had a profound paralytic effect on the mid-body of the worms.     

Mating barriers between genetically divergent strains of the parasitic nematode Haemonchus contortus suggest incipient speciation

Haemonchus contortus, in common with many nematode species, has extremely high levels of genetic variation within and between field populations derived from distant geographical locations. MHco10(CAVR), MHco3(ISE) and MHco4(WRS) are genetically divergent H. contortus strains, originally derived from Australia, Kenya and South Africa, respectively, that have been maintained by numerous rounds of in vivo experimental infection of sheep. In order to explore potential pre-zygotic competition or post-zygotic incompatibility between the strains, we have investigated the ability of MHco10(CAVR) to interbreed with either MHco3(ISE) or MHco4(WRS) during dual strain co-infections. Sheep were experimentally co-infected with 4000 infective larvae (L₃) per os of the MHco10(CAVR) strain and an equal number of either the MHco3(ISE) or the MHco4(WRS) strain L₃. The adult worm establishement rates and the proportions of F₁ progeny resulting from intra- and inter-strain mating events were determined by admixture analysis of microsatellite multi-locus genotypes. Although there was no difference in adult worm establishment rates, the proportions of F₁ progeny of both the MHco10(CAVR) × MHco3(ISE) and MHco10(CAVR) × MHco4(WRS) dual strain co-infections departed from Mendelian expectations. The proportions of inter-strain hybrid F₁ progeny were lower than the expected 50%, suggesting either pre-zygotic competition or post-zygotic incompatibility between the co-infecting strains. To investigate this further, both eggs and hatched L₁ of broods from single adult female worms recovered from each dual co-infection were genotyped. Unhatched eggs from the broods revealed no inter-strain hybrid genotype deficit, suggesting there is no pre-zygotic competition between the strains. In contrast, there was a deficit in L₁ inter-strain hybrid genotypes in the broods derived from MHco3(ISE) or MHco4(WRS) maternal parents, but not from MHco10(CAVR) maternal parents. This suggests that hybrid progeny of MHco10(CAVR) paternal parents have reduced post-zygotic development and/or viability consistent with incipient speciation of the MHco10(CAVR) strain. The presence of mating barriers between allopatric H. contortus strains has important implications for parasite ecology, including the ability of newly introduced anthelmintic-resistant parasite populations to compete and interbreed with populations already established in a region.     

Genetics of vulvar morph types in Haemonchus contortus: Haemonchus contortus cayugensis from the finger lakes region of new york

The inheritance of the four most common morph types (smooth, knobbed, linguform A and B) in Haemonchus contortus cayugensis was studied by selection of offspring from females of known morph type and also by mating males from an inbred strain of linguiform A with smooth and linguiform B females. The dominance hierarchy of these characters was found to be linguiform > knobbed > smooth. Linguiform B was recessive to linguiform A but neither character is expressed in the presence of the smooth morph type.     

Evaluation of a recombinant excretory secretory Haemonchus contortus protein for use in a diagnostic enzyme-linked immunosorbent assay

The nematode Haemonchus contortus (H. contortus) is one of the most pathogenic and economically important parasites of sheep. A 24 kDa protein is one of the important components in H. contortus excretory/secretory (ES), which was shown to have important biological function. In our research, the cDNA of its open reading frame (ES24) was obtained and analyzed. Then the ES24 was sub-cloned into pET-30a expression vector. The recombinant vector that codes hexahistidyl peptide fusion protein (His-ES24) was transformed into Escherichia coli BL21 (DE3) strain. After induction, a high expression level of His-ES24 was found at 6h taking about 26% of the total bacterial protein analyzed by gel thin-layer scanning. The expressed His-ES24 was purified and then used in an enzyme-linked immunosorbent assay (ELISA) to detect specific antibodies in serum samples. The ELISA was able to differentiate between H. contortus-infected sheep serum and Fasciola hepatica-infected sheep serum or non-infected sheep serum. No cross-reaction was observed in sheep sera that have been experimentally infected with F. hepatica. A total of 153 field sheep serum samples conserved in our laboratory were examined using the His-ES24 ELISA, and 82 (53.6%) of them were found seropositive to H. contortus. Our results demonstrate that the prokaryotic-expressed His-ES24 might be a useful diagnostic reagent for epidemiological studies of H. contortus in sheep.     

Resistance of exotic and domestic lambs to experimental infection with Haemonchus contortus

Resistance to primary and secondary infections with H. contortus was compared in St. Croix, 3/4 St. Croix, Florida Native, Barbados Blackbelly and domestic crossbred lambs. On the basis of worm counts, fecal egg counts and packed cell volumes, St. Croix and 3/4 St. Croix lambs were the most resistant to secondary infections, Florida Native and Barbados lambs were intermediate in resistance, and the domestic lambs were the least resistant. Breed differences were highly variable during primary infection. Breed differences were most pronounced prior to puberty, with domestic lambs highly susceptible to secondary infections. After puberty, domestic lambs were better able to resist secondary infections. Age apparently had little or no effect on the parasite resistance of exotic breeds. Sex differences were significant only after puberty when ewe lambs were more resistant to secondary infection than ram lambs. In one of three experiments lambs of hemoglobin genotype AB were more resistant to secondary infection than lambs of genotype BB in terms of fecal egg counts, packed cell volumes and the proportion of ‘responder’ and ‘nonresponder’ lambs but not in terms of worm counts, although subtle differences may not have been detected because of small numbers of lambs.