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SUMMARYThe use of DNA markers to track the development of anthelmintic resistance in parasites of livestock would allow informed choices for the management of this important problem. We describe a genetic mapping approach for the discovery of DNA markers for anthelmintic resistance inHaemonchus contortus. We crossed a multi-drug resistant field isolate ofH. contortuswith a well-characterized laboratory strain susceptible to 4 drug classes. The F2were separately selected with 5 anthelmintics from 4 drug classes, producing drug-resistant populations carrying gene variants derived from both the field isolate and the laboratory strain. Individual F2worms were analysed using amplicon length polymorphisms (ALPs). We looked for field isolate alleles over- or under-represented in F2populations compared to the unselected F2and/or the laboratory strain. The data we obtained suggest that marker association can be used to link neutral markers with resistance, but also that more markers and perhaps more inbred laboratory strains would make the procedure more likely to succeed.

The number of eggs from gastrointestinal nematodes per gram of faeces (worm egg count WEC) is commonly used to determine the need for anti-parasite treatments and the breeding value of animals when selecting for worm resistance. Diarrhoea increases faecal moisture and may dilute the number of worm eggs observed. To quantify this effect, egg counts in sheep at pasture were simulated by dosing 15 animals with chromic oxide particles. The simulated WEC diminished as faecal moisture increased. When faeces were dried, simulated WEC per unit dry matter was not influenced by the amount of faecal moisture present prior to drying. The results suggest that adjustment for faecal moisture may provide an improved estimate of FEC. Drying faeces to calculate the WEC per unit dry matter would provide such an adjustment but may not be practical for industry application. In the past, the CSIRO McMaster Laboratory has used an adjustment factor developed by Gordon based on the classification of faecal consistency derived from the morphology of faeces. To examine the utility of an adjustment factor based on faecal consistency score (FCS), the relationships between FCS and simulated WEC and dry matter were examined. Dry matter and simulated WEC exhibited an exponential decline as FCS increased. The relationship between FCS and dry matter was further examined in 368 samples collected over 12 months from sheep at pasture, where it was observed that dry matter showed a linear decline as FCS increased. Adjustment factors based on dry matter were similar to those proposed by Gordon however adjustment factors predicted from simulated WEC diverged from the remainder for FCS>4. As no samples scored FCS 5 in the study of simulated FEC, the adjustment factors based on the larger study that included samples with FCS 5 was therefore considered more robust. Adjustment factors were given by the equation: WEC(estimated)=(WEC(observed)/(34.21-5.15 FCS))x29.06. This equation estimates for samples with FCS>1 the WEC that would be expected if the samples were FCS 1, the faecal consistency score for normal faeces. The impact of adjustment of observed WEC for faecal moisture predicted by FCS on decision points for treatment and on estimated breeding values requires further examination.

Eradication can be defined as the permanent reduction to zero of the incidence of infection caused by a specific agent as a result of deliberate efforts. Eradication strategies must be designed to increase access to and utilization of veterinary and animal health support services beyond that normally achieved by routine services. Consequently, before undertaking an eradication program, proponents must be certain that they can be sure of a commitment of resources sufficient to complete the project. A helminth eradication campaign would require the application of anthelmintics to all hosts at a level aimed at killing heterozygote resistant individuals. This strategy runs counter to the prevailing opinion that resistance to chemicals will always occur and control schemes should accept some production loss by letting a proportion of parasites escape selection (in refugia) and thereby increase the useful life of the chemical. Under certain circumstances, environmental constraints may make some species of parasitic nematodes vulnerable to an eradication program. Anthelmintics have been used to exploit these constraints in past control programs and have lead to local eradication of several species of gastro-intestinal parasitic nematodes. Also, there are at present eradication programs for nematode parasites of human that are based on anthelmintic treatment. However, the suppressive pesticide treatment required to bring about the virtual elimination of species is a high-risk strategy and should only be undertaken if eradication is an achievable goal. It is also important to plan an exit strategy if eradication fails.

We have developed a modified migration assay system in 96-well plate format which is able to detect resistance to the macrocyclic lactone group of drugs in Haemonchus contortus. The assay involves exposure of infective stage larvae to drug for a 24 h period, then counting the numbers of larvae that are able to migrate through an agar and filter mesh system over a further 48 h. The agar barrier greatly increased the sensitivity of the assay for resistance detection compared to use of filter mesh alone. The assay was able to detect the presence of 10% resistant worms in an otherwise susceptible background. However, the assay was ineffective with Trichostrongylus colubriformis and Ostertagia circumcincta indicating that its usefulness for field monitoring will be restricted to situations where H. contortus is of most significance. A small-scale drug screening exercise showed that the assay identifies some anthelmintic activities distinct from those identified by larval development assays. The assay therefore also has a potential role in drug discovery programmes in screening for new anthelmintics.

The development of moxidectin resistance (MOX-R) in sheep parasitic gastrointestinal nematodes already carrying multiple resistances to other anthelmintic groups has made control of these strains very difficult. The anthelmintic resistance patterns of MOX-R strains of Trichostrongylus colubriformis and Haemonchus contortus were characterized to provide an insight into the remaining role of anthelmintics in the control of such strains. Homozygous MOX-R individuals of both genera were unaffected by moxidectin. For MOX-R heterozygotes a dose rate of 200 microg/kg abamectin (ABA) given orally removed 25% of H. contortus while 200 microg/kg MOX given orally achieved a 72% reduction. Doubling the dose rate of ABA improved the mean efficacy to 37%. Consequently, in H. contortus, the degree of dominance differs markedly between the two anthelmintics. A dose rate of 8 mg/kg levamisole and 185 mg/kg napthalophos achieved95% reduction in worm count of the MOX-R homozygous H. contortus but only 85 and 7%, respectively against the MOX-R homozygous T. colubriformis.

In merino sheep bred for either increased or decreased resistance to Haemonchus contortus, faecal worm egg counts (FEC) were lower in the resistant line (6,831 vs 17,645 epg, P0.01), and circulating eosinophils (EOS) were higher, but not significantly so (3.40 x 10(4) ml(-1) vs 1.40 x 10(4) ml(-1), P = 0.1 1). Another flock was artificially infected with Trichostrongylus colubriformis and significant genetic variation was found in both FEC (heritability 0.40 +/- 0.11) and EOS (0.19 +/- 0.08). In a third flock comprising tropical sheep breeds, a natural challenge with T. colubriformis resulted in significant sire effects on FEC (heritability 0.20 +/- 0.10) but not EOS (heritability inestimable). We conclude that EOS offers no advantage over FEC as it selection criterion for resistance.

Plasma levels of fenbendazole (FBZ) and its sulphoxide (OFZ) and sulphone (FBZ.SO2) metabolites were measured in goats and sheep during low-level administration of FBZ given by intraruminal infusion or formulated into a urea-molasses feed supplement block (UMB). In experiment 1, 6 goats and 6 sheep were offered UMB containing 0.5 g FBZ/kg (MUMB) and individual block consumption was measured daily for 18 days. In experiment 2, some of the same animals (n = 4 for each species) received FBZ by intraruminal infusion at 1, 1.5 and 3 mg/kg liveweight per day for 7 days at each dosage. FBZ, OFZ and FBZ.SO2 levels were determined in plasma collected every 3 days in experiment 1 and on days 4, 5 and 6 of each infusion period in experiment 2. In both experiments, higher equilibrium levels were observed for the three metabolites in sheep than in goats. Significant linear relationships were observed between the daily FBZ dosages and the plasma levels of the three metabolites in both species. The regression coefficients were significantly higher in sheep than in goats for FBZ and OFZ but not for FBZ.SO2, and they were also significantly higher during MUMB administration than during infusion for all three metabolites in both species. FBZ is a suitable anthelmintic for incorporation into a MUMB formulation for use in livestock production systems where responses to molasses urea supplementation have been demonstrated and gastrointestinal parasitism impairs productivity. The results indicate that target dose rates for goats should be 0.75 mg/kg per day compared with 0.5 mg/kg per day for sheep.

A preliminary experiment involving contamination of pasture plots with eggs of Haemonchus contortus, Trichostrongylus colubriformis and Oesophagostomum columbianum every month for a year established that in the tropical environment of the Pacific island of Tongatapu, hatching and development of all species was rapid and continuous, with a short survival on pasture (3–7 weeks) of the resulting infective larvae. These results indicated that a rotational grazing system consisting of ten paddocks grazed in sequence for 3.5 days at a time may permit a reduction in the frequency of anthelmintic treatment of goats. In comparison with an adjacent set-stocked flock which required treatment on three occasions during the year when mean flock egg counts exceeded 2000 eggs per gram (EPG), rotationally grazed goats generally maintained mean egg counts of less than 1000 EPG. Anthelmintic treatment was only given to rotationally grazed goats individually as they kidded, and there were indications that even this precaution was unnecessary. Because of the expense of frequent anthelmintic treatment and the resulting selection of strains of anthelmintic-resistant nematodes, rotational grazing of small ruminants through fencing, tethering or herding deserves further investigation as a nematode control option in wet tropical environments.

Gastrointestinal helminth parasites impact on livestock production systems throughout the world, and the use of anthelmintics to control this problem has lead to the inevitable development of populations of helminths resistant to these treatments. This, coupled with consumer desires for minimal chemical inputs into food and fibre production, has prompted research into non-chemical approaches to helminth control. Scientists of the “Novel Approaches to the Control of Helminth Parasites of Livestock” group met for the 6th time in August 2010 and this paper summarises that meeting. Six scientific sessions addressed current approaches and topics of interest through formal presentations and discussion of issues raised by the contributing authors. Close interaction between researchers and extension specialists during the meeting has contributed to enhanced prospects for field application of research outcomes in the future.

A trial to determine the seasonal pattern of egg hatching and larval survival on pasture was carried out in representative wet and dry zones of Fiji. Fourteen plots were established on parasite-free pasture at each of two sites. One plot at each site was contaminated every month with faeces from naturally infected goats containing a known proportion of Haemonchus contortus and Trichostrongylus colubriformis eggs. Pasture was sampled at regular intervals after contamination and infective larvae identified and counted. Larvae of both species developed throughout the year in the wet zone but development was more sporadic in the dry zone. Larval counts generally declined to below detectable levels within 9 weeks of contamination between September and March but longevity increased during the cooler weather from April to August. The comparatively short larval survival times noted in this experiment may present opportunities for manipulation of parasite population dynamics in the wet tropics.

We believe this study is the first to consider the genetic and phenotypic divergence between isolates of Haemonchus contortus in Australia. Microsatellite markers have been used to investigate genetic divergence, whilst phenotypic divergence has been considered through individual worm morphology, isolate life history traits and the effect of isolates upon the host. The results are discussed in the context of the likely introduction of H. contortus to Australia, its recent isolation, and the characteristics of sheep and goat farming which might act to either isolate or distribute parasites. We conclude that there is significant observable genetic divergence between isolates of H. contortus in Australia. The divergence may have been under-estimated in this study due to a variety of factors. Phenotypic divergence is also observed, and potentially has significant implications for both economic losses due to haemonchosis on individual properties and for decisions regarding the regulation of stock movements in Australia.

Identification of the presence and abundance of species is important when choosing therapies and control strategies for internal parasitism of livestock. Here we examine lectin binding characteristics of eggs isolated from sheep faeces as a means for identifying the parasite genera contributing to infection. The intensity of lectin staining varied with incubation time, incubation volume, concentration of lectin and concentration of eggs. Formalin fixed eggs had greater autofluorescence but exhibited the same lectin staining pattern as fresh eggs. The stage of egg development did not influence staining. Eggs from Haemonchus contortus, H. placei, Trichostrongylus colubriformis, T. vitrinus, Ostertagia circumcincta, Nematodirus spathiger and the cestode Monezia expansa were incubated with a panel of fluoroscein isothiocyanate (FITC)-labelled lectins. Lectin binding exhibited a genus specific pattern. Haemonchus spp. stained strongly positive with peanut agglutinin (PNA), and were positive for concanavalin A (ConA), Ricinus communis agglutinin (RCA) and Maclura pomifera lectin (MPA). Trichostrongylus spp. were PNA-, ConA-, RCA- and strongly MPA+. O. circumcincta were weakly positive for PNA, MPA, ConA and negative for RCA. N. spathiger were weakly positive for the four lectins, and M. expansa were weakly positive for PNA, RCA and MPA and were strongly ConA+. The genus specificity of lectin staining was used to identify the presence of Trichostrongylus and Haemonchus eggs in faeces from sheep with mixed field infections, however correspondence between lectin staining and larval differentiation for identifying a low prevalence of Ostertagia in the field infection was poor. Refinements in methods for rapid egg isolation may improve egg differentiation on the basis of lectin staining, which could be undertaken by flow cytometry or microscopy.

Objective To provide principles for the appropriate use of avermectin/milbemycin or macrocyclic lactone (ML) anthelmintics in sheep, to ensure effective worm control and to minimise selection for ML resistance. Strategy The principles were based on an assessment of the information currently available. The MLs were categorised into three groups (ivermectin [IVM], abamectin [ABA] and moxidectin [MOX]) based on structural differences, persistence and efficacy against ML resistant strains. The reported order of activity or efficacy against ML resistant worm strains was IVM

To understand the factors that influence selection for anthelmintic resistance, it is necessary to examine the impact of drug treatment, particularly persistent drugs, on all phases of the worm life cycle. The efficacy of various avermectin/milbemycin anthelmintics was determined against resident worms, incoming larvae (L3) and development of eggs in faecal culture. Homozygote-resistant and maternal and paternal F1-heterozygote genotypes of Haemonchus contortus were used to infect sheep before or after treatment with ivermectin (IVM) oral, IVM capsule, moxidectin (MOX) oral or MOX injectable. Total worm count and quantitative larval culture were used to determine efficacy against parasitic and free-living stages, respectively. Selection for resistance by IVM capsules occurred at the adult and L3 stages because of poor efficacy against these stages for all resistant genotypes. However, the selective advantage of these surviving worms was reduced due to the low development of their eggs to L3 in faecal culture. For MOX, selection for resistance predominantly occurred after treatment because of high efficacy against resident adult worms of all resistant genotypes but poor efficacy against resistant L3 ingested after drug administration. The results indicated no evidence of sex-linked inheritance for IVM resistance. Mean IVM efficacies against homozygous and heterozygous resistant adult worms were not different, and IVM capsule efficacy against incoming L3 was approximately 70% for all resistant genotypes, consistent with a dominant trait. MOX was highly effective against adults of all resistant genotypes and approximately 76% effective against incoming L3 regardless of resistance genotype, also consistent with a dominant trait. These results will enable the impact of persistent drugs on worm control and anthelmintic resistance to be estimated. The results indicate that IVM capsules should not be used in populations where avermectin/milbemycin resistance is present.

Chromium 51 labelled erythrocytes were used to measure blood loss in sheep infected with either H. contortus, H. placei or the F 1 of their reciprocal cross hybrids ( H. contortus males × H. placei females = CXP, H. placei males × H. contortus females = PXC). CXP consisted of sterile males and semi-sterile females and sheep infected with this hybrid had nil egg counts; while PXC consisted of semi-sterile males and females and their hosts had some eggs in their faeces. Immediately after their blood loss was estimated the sheep were killed and their worms counted. One hundred male and 100 female worms from each sheep were dried at 60°C overnight and then weighed to give an estimate of the biomass in each infection. Total daily egg production was calculated from egg counts made on 3 random 2 g samples from the 24 h faecal collection. The relationship of the log blood loss to log worm number, to log biomass of infection in each sheep and to log total egg production in the parental species was linear. Biomass was no better an estimator of blood loss than was worm number. There was no improvement in the estimation of blood loss by regressing the male and female worms separately. In all of the analyses, there was no improvement by regressing H. contortus and H. placei or their hybrids separately. It appeared that egg production was not a major factor in the requirement for blood by Haemonchus . The size of individual female worms was negatively correlated with the number of worms in the infection. The linear nature of the relationship of blood loss to worms number, worm biomass and worm egg production indicated that sheep have very little control of blood loss and that the limitation of the pathology of Haemonchus infections is likely to be exerted at the establishment stage of the infection.

Swamp buffalo (Bubalus bubalis) and Droughtmaster cattle (Bos indicus x B. taurus), fitted with gastrointestinal cannulae, were dosed intraruminally with fenbendazole at 7.5 mg/kg liveweight, together with a chromium oxide capsule and a pulse dose of NaCoEDTA, to estimate the flow dynamics of the digesta in the rumen and duodenum. The concentrations of fenbendazole (FBZ) metabolites were measured in plasma and duodenal fluid collected over 120 h. In plasma, significantly lower peak concentrations and earlier disappearance of FBZ and its sulphoxide (OFZ) metabolite were observed in buffalo, which considerably reduced systemic availability in comparison with cattle. The availability of OFZ in the duodenal fluid of buffalo was significantly lower, whereas FBZ disposition was similar to that in cattle. The turnover rate of fluid in the rumen was higher in buffalo than in cattle, while the flow parameters for other digesta were similar in the two species. It is concluded that the decreased absorption of drug in buffalo was attributable to the shorter residence time of the dose in the rumen, and probably in the entire gastrointestinal tract. This may reduce the efficacy of treatment and indicate the need for higher dose rates for benzimidazole anthelmintics in buffalo than in cattle.

Broad-spectrum anthelmintics were tested for their efficacy in either single active or combination formulations against multiple anthelmintic-resistant strains of Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus colubriformis. Three efficacy trials based on post mortem worm counts showed that the triple and quadruple combinations of benzimidazoles, levamisole, macrocyclic lactones and closantel were effective against most strains that were resistant to each of the components when drenched individually. Consequently, use of any one of these classes of anthelmintics by itself against multiple-resistant strains would lead to a rapid increase in the level of resistance. When used in combinations, the anthelmintics reduced the survival rate of most strains to

One hundred and twenty lambs were grazed continuously from weaning until 9 months of age on 12 plots contaminated with larvae of three nematode species (Haemonchus contortus, Trichostrongylus colubriformis and Ostertagia circumcincta). The lambs were sired by either a genetically resistant ram or susceptible rams (determined by the response of previous progeny to artificial H. contortus infection). Half the resistant and half the susceptible lambs were given strategic anthelmintic treatment and the remainder remained untreated. Faecal egg counts and blood packed cell volume were measured frequently in all animals. One and 5 months after weaning, two lambs from each plot were slaughtered, and worm burdens and larval establishment rates of the three species of nematode were estimated. At the second slaughter, leukotriene levels and larval migration inhibitory (LMI) activity were measured in mucus collected from the small intestine. The dominant species in all faecal samples and the gastrointestinal tract was T. colubriformis. Lambs of the resistant genotype had lower faecal worm egg counts, lower worm burdens and higher levels of resistance to larval establishment. There were no differences in larval migration inhibition (LMI) activity, but resistant lambs had higher levels of the leukotriene LTC4/D4/E4. Further, the resistant genotype, identified on responsiveness to artificial infections with H. contortus, was more resistant to infections of three important species acquired naturally from contaminated pastures. All these genetic differences were maintained while the lambs were subject to strategic anthelmintic treatment.

A number of haematological parameters were monitored in eight-month-old Merino wethers, six infected with 10,000 Haemonchus contortus and 13 controls. Blood loss caused by infection was reflected in a decrease in packed cell volume (r = —0 78; P

The paper describes a method for detecting levamisole and morantel tartrate resistance by determining the percentage of paralysed third stage larvae in serial dilutions of anthelmintic. Levamisole resistantOstertagia spp. had a smaller proportion of larvae undergoing tonic paralysis in either levamisole or morantel tartrate than did a non-resistant strain. The dose response thus obtained for a strain of worms can be tested statistically against strains which are known to be resistant or non-resistant.

Adult worms from a levamisole resistant population ofOstertagia consisting ofO. circumcincta andO. trifurcata showed no dose response to morantel tartrate. The lowest dose rate of 2.2 mg/kg removed 30.8% of worms while the highest dose rate of 17.6 mg/kg removed 39.2%. A dose response was obtained against naphthalophos and the LD50 and LD95 were determined as 12.54 mg/kg and 23.25 mg/kg respectively. It was concluded that the strain was cross resistant to morantel tartrate but not to naphthalophos.

Le Jambre I. F. , Southcott W. H. and Dash K. M. 1977. Resistance of selected lines of Ostertagia circumcincta to thiabendazole, morantel tartrate and levamisole. International Journal for Parasitology7: 473–479. A strain of Ostertagia circumcincta was isolated from a field in which all sheep had been treated in sequence every 7–10 days from September 1970 to January 1974 with either thiabendazole, morantel tartrate or levamisole. Thiabendazole had not been used after the first 15 months. The LD 95 for this strain was 88 mg/kg thiabendazole, 6.9 mg/kg morantel tartrate and 5-4 mg/kg levamisole. Another strain of O. circumcincta isolated from an area where anthelmintics had been used much less frequently was divided into four lines for exposure to selection in the laboratory. The first line was selected with 50 mg/kg thiabendazole, the second with 5 mg/kg morantel tartrate, and the third with 3.2 mg/kg levamisole; the fourth line was not selected for drug resistance. After eight generations the three lines selected with thiabendazole, morantel tartrate and levamisole had (Spld)(in95) of > 200, 5.7 and 6.2 mg/kg for the selecting drugs respectively, compared with corresponding values of 20, 2.9, and 1.8 in the unselected line. That is, the field strain had about the same levels of resistance to morantel tartrate and levamisole as the respective laboratory strains selected with these individual drugs. However, the field strain, which had been exposed to thiabendazole for only 15 months, was less resistant to thiabendazole than the laboratory strain selected with this drug. These results show that giving of several drugs in sequence cannot be relied upon to prevent the development of resistance to the individual drugs. The dose responses of adult worms showed low, but significant resistances to morantel tartrate and levamisole and a relatively high resistance to thiabendazole. Levamisole was found to select for inhibition of development with approx. 8.0% of the inhibited larvae showing no dose response above 1.6 mg/kg. Levamisole was also associated with an increase from 0.1 % to 9.0% in the O. trifurcata component of an Ostertagia population.

Angora goats grazing on a paddock contaminated with Ostertagia ostertagi and O. circumcincta were found to have infections consisting of equal numbers of both species. Subsequently, goats were infected with O. ostertagi larvae of bovine origin. The infection became patent by day 28 and egg counts reached a plateau about 7 weeks after infection. Worm numbers did not decline during the 12 weeks of the experiment. It was concluded that Angora goats are suitable hosts for O. ostertagi .

Laboratory strains of Haemonchus contortus and Ostertagia circumcincta selected for thiabendazole resistance were found to have a strong side resistance to a single dose of oxfendazole. The LD50 and LD95 in mg of drug per host liveweight were respectively 4.28 and 18.46 mg/kg for H contortus and 3.61 and 11.20 mg/kg for O circumcincta. A field strain of Trichostrongylus colubriformis that had not been selected with thiabendazole for seven years also had a strong side resistance to oxfendazole with approximately 50 per cent of its population resistant to the recommended dose rate of 5 mg/kg. Prolonged administration of oxfendazole by intraruminal controlled release capsules was found to be effective against both susceptible and resistant strains of the above parasites. The first observed effect of oxfendazole, from controlled release capsules, on resistant worms was a decrease in the percentage of eggs developing to third stage larvae. This was followed by a decrease in egg count and in worm numbers.

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.

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 ld 95 to be 18 mg/kg morantel tartrate; the reported ld 50 and ld 95 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.

Sera from hosts infected with a variety of nematodes were examined for the presence of antibodies against nematode acetylcholinesterase (AChE). Antibodies were detected in the serum from hosts infected with Oesophagostomum spp., Ostertagia spp. and Trichostrongylus spp., but not in serum from hosts infected with Cooperia pectinata or Haemonchus spp., Toxocara spp. or Trichuris vulpis . In those infections in which anti-AChE antibodies were found, some individual animals failed to produce detectable antibodies. The enzyme appeared to possess antigenic specificity at the genus but not the species level.

Hybridization experiments between Haemonchus contortus from sheep and H. placei from cattle indicate that the H. placei vulvar morph type and the inability of the eggs to hatch and develop at 11°C were inherited as dominant traits in the hybrids. The size of the third stage larvae was similar to H. placei in hybrids from the mating of male H. placei × female H. contortus while larvae from the reciprocal mating were intermediate in size. Hybrids produced by the mating of thiabendazole resistant female H. contortus × non-resistant male H. placei were also resistant. The F 1 males of the mating between male H. contortus and female H. placei were sterile. Male sterility did not occur in the reciprocal cross until the F 2 generation. Female hybrids from these generations had a low level of fertility when backcrossed to males of either parent species. Cytological studies of the hybrid males indicated that sterility was due to several kinds of meiotic disturbance and that spermatogenesis stopped during metaphase I. The chromosomes in eggs of unfertilized females did not undergo meiosis and polar body formation; instead they increased in number by a process of endomitosis. Unfertilized eggs in the faeces were characterized by uneven cytoplasmic division and abnormal shape. It is proposed that hybrid sterility can lead to the local eradication of one species of Haemonchus by the other. Furthermore, the outcome of which species is eradicated can be influenced by such variables as initial population sizes, climate, and the local ratio of sheep to cattle.

The effect of changes in genetic constitution (associated with developing benzimidazole resistance) on the physiological characteristics of Haemonchus contortus was investigated. Benzimidazole resistant H contortus were significantly more infective for sheep (20 per cent) than benzimidazole susceptible strains. Pathological changes were more severe in sheep infected with resistant H contortus as measured by packed cell volume, plasma protein concentration and haemoglobin levels. Faecal egg output was significantly higher for resistant strains as was the development and survival of eggs and free-living stages on pasture. Infective third stage larvae of benzimidazole resistant H contortus had a significantly faster exsheathment rate in rumenal fluid than did susceptible strains. These adaptive changes in response to continuing anthelmintic selection of resistant worm populations are discussed and the implications of these findings for worm control programmes indicated.

Groups of sheep were infected three times weekly for 15 weeks with infective larvae of Haemonchus contortus at four rates ranging from 600 to 4800 larvae per week. At 3-weekly intervals, sheep from each group were killed for total worm counts after receiving a dose of radiolabelled larvae which enabled a direct measurement of establishment of incoming larvae during the continuing infection. Peak H. contortus burdens were reached between 6 and 9 weeks of infection, and were related to infection rate. In the groups receiving 2400 and 4800 larvae per week, worm numbers then declined rapidly and by the end of the experiment were lower than those in the groups given 600 or 1200 larvae per week. Establishment of incoming larvae was not influenced by infection rate, and declined from 45% in the first 4 weeks of infection to insignificant levels during the final 6 weeks. The proportion of incoming larvae arrested in their development increased as the infection progressed. It was concluded that H. contortus numbers were regulated by development of resistance to infection and by a loss of established worms which was related to the current rate of larval intake, and to the host's previous experience of infection.

Divergent selection of Quackenbush mice, according to survival time following an acute Nematospiroides dubius infection, produced lines that were significantly different for this parameter after only two generations. After eight generations of selection, mean times to death (averaged over sexes) of the high, control and low survival lines were 11.06,9.57 and 7.88 days post infection, respectively (i.e. deviations from the control for the high and low lines of + 15.5 and −17.7%). Survival of males post infection was significantly ( P N. dubius infection was 9.2 ± 3.5%.

Six populations of H. contortus were selected for a study of thiabendazole resistance from a collection of 40 populations made during a survey of the efficiency of thiabendazole on the Northern Tablelands of New South Wales. Based on survey results, 3 of these populations were considered susceptible and the remaining 3 were considered resistant. However, when these populations were compared with a known susceptible strain on the ability of their eggs to hatch in a solution containing thiabendazole and 0.1% NaCl all 6 had significantly greater LC50's. The resistance ratios of the LC50 for each of the 6 populations to that of the known susceptible strain were 5.1, 4.3, 3.2, 3.1, 2.3, and 1.6 respectively. Following dosing of their host with 44 mg/kg thiabendazole the resistance ratios of the survivors increased to 5.4, 5.1, 4.7, 4.4, 3.1 and 2.4. Eggs produced by the F1 generation of the worms surviving 44 mg/kg thiabendazole did not revert back to the lower LC50's of the unselected parents. Rather the LC50 remained at a level near that of a known highly resistant strain of H. contortus.

SUMMARYHaemonchus contortus worm populations isolated from naturally infected sheep at the Pastoral Research Laboratory, Armidale, N.S.W., were found to contain approximately 20% of worms resistant to a 50 mg/kg dose of thiabendazole. Following 3 generations of selection with 50 mg/kg thiabendazole the number of worms removed by the anthelmintic was too small to detect differences between treated and control groups. After more than 15 generations of selection, matings between males from the selected strain and non-resistant females produced resistant males and females in equal numbers. Thus, thiabendazole resistance does not appear to be sex-linked. A dose–response assay on the F2 adults indicated that worms from female resistant × male non-resistant crosses were more resistant than F2 adults of the reciprocal cross. An in vitro technique that identified thiabendazole-resistant eggs by their ability to hatch in a solution containing thiabendazole and 0·1% NaCl solution was also used to study the inheritance of resistance. F1 eggs had similar LC50's to the resistant parents. F2 and back-cross eggs from an original mating of thiabendazole-resistant females × non-resistant males had a higher LC50 than F2 and back-cross eggs from the reciprocal mating, indicating a degree of matroclinous inheritance of resistance. However, the resistant parents had tolerances to thiabendazole exceeding those of F2. F3 eggs had a resistance distribution that ranged from that of the resistant to the non-resistant parent. No significant deviation from linearity was observed in any of the dose–response lines. These results indicate that thiabendazole resistance in H. contortus worms is inherited as an autosomal and semi-dominant trait.

Two strains of Haemonchus contortus with different levels of thiabendazole resistance were developed from the same parent strains by being subjected to different selection intensities. These two strains were each subdivided into two; one selected by thiabendazole in each generation, the other unselected for four generations. Levels of thiabendazole resistance were monitored in each generation by an in vitro egg hatch technique. During this time the unselected strain with the higher level of resistance maintained an LC50 similar to its selected control. The LC50 of the unselected strain with the lower level of resistance actually increased until at the end of the fourth generation it had an 1(50 similar to the thiabendazole selected strains.

A technique is described in which thiabendazole resistance is assayed by hatching nematode eggs in solutions of thiabendazole. Eggs of Haemonchus contortus and Ostertagia circumcincta were hatched in a series of concentrations of thiabendazole in a 0.1% NaCl solution. Thiabendazole resistant strains hatched in higher concentrations of thiabendazole than non-resistant strains. The dose response thus obtained for a strain of worms can be tested statistically against strains which are known to be either resistant or non-resistant.

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.

SummaryTwo lines of guinea-pigs, whose susceptibilities to infection with the parasitic nematode Trichostrongylus colubriformis differ significantly, have been established. This difference is probably based on genetically determined differences between the ability of members of each line to bring about immune expulsion. of the parasite.

Martin P. J. , Le Jambre L. F. and Claxton J. H. 1981. The impact of refugia on the development of thiabendazole resistance in Haemonchus contortus . International Journal for Parasitology 11 : 35–41. Beginning with a mixture of 5% thiabendazole-resistant and 95% non thiabendazole-resistant Haemonchus contortus larvae, worm free sheep were artificially infected with either 10,000, 9000, 7000, 2500 or zero larvae. Four weeks later, the infected sheep were treated with 44 mg/kg of thiabendazole. Seven days after anthelmintic treatment the same sheep were infected with zero, 1000, 3000, 7500 or 10,000 larvae respectively so that each sheep was given a total of 10,000 larvae. The larvae given after thiabendazole treatment are referred to as being in refugia (defined as an area where individual members of a population can escape exposure to a drug). This was repeated for six generations of parasites, maintaining the same proportion of larvae in refugia in each generation. The egg hatch assay for resistance indicated that refugia delayed the development of resistance in Haemonchus contortus . Where none or a small proportion of larvae were in refugia, a rapid increase in resistance occurred. With an increased proportion of larvae in refugia, resistance was slower to develop. Population size, as estimated by faecal egg counts done after anthelmintic treatment and corrected for the number of larvae given in each treatment, was lower at the higher levels of refugia. This was due to a lower level of resistance in the higher refugia lines and consequently a greater effect of the drug. However, the estimate of population size, from egg counts done after the larvae introduced from refugia had matured, were similar in all lines.

SUMMARY: Adult worms from a population of Ostertagia consisting of 88%O. circumcincta and 12%O. trifurcata were found to survive a double drench (14 mg/kg) of levamisole. An anthelmintic assay on this population found that against levamisole the LD50 was 11.95 mg/kg and the LD99 was 67.75 mg/kg. Three benzimidazoles were also tested against this population and the LD50 and LD99 were respectively thiabendazole 11.8 and 144.9 mg/kg, oxfendazole 0.17 and 6.10 mg/kg and albendazole 0.08 and 2.55 mg/kg. The percentage of worms killed by the recommended dose rate was levamisole 24%; thiabendazole 91%; oxfendazole 99% and albendazole 99%.

Le Jambre L. F. and Royal W. M. 1979. Meiotic abnormalities in backcross lines of hybrid Haemonchus. International Journal for Parasitology10: 281–286. Hybrid worms from the reciprocal matings between Haemonchus contortus and H. placei were backcrossed to the parent species. Six of the possible eight backcrosses produced fertile eggs. The six backcross lines were passaged through sheep for four generations. The adults of this generation had various levels of meiotic abnormality as evidenced by abnormal variations in chromosome structure and aneuploidy. The eggs from backcross lines with a degree of meiotic abnormality had the lowest percentage hatch. Four of the six backcross lines required a higher temperature for larval development than did H. contortus. The size of the third-stage larvae varied from one with larvae larger than H. placei to one line with larvae smaller than H. contortus.

There were distinct ecotypie differences in the ability to develop to third stage larvae at a constant 11 or 13°C for two weeks. H. conforms cayugensis could develop at both 11 and 13°C; H. contortus from Louisiana could develop at 13°C but not 11°C and H. placei could not complete development at either temperature. Eggs produced from the first cross between ecotypes had the same cold tolerance as their maternal parent. F 1 eggs were intermediate between parental ecotypes, while F 2 eggs, when fertile, developed at the same temperature as the most cold-tolerant parent in the first cross. The H. placei knobbed vulvar morph type was dominant to H. contortus cayugensis smooth morph type and recessive to the H. contortus (Louisiana) smooth morph type. Knobbed morph type of H. contortus (Louisiana) was dominant to smooth of H. contortus cayugensis . Dominance of morph type was enhanced by a matroclinous effect in the between species matings but not in the within species matings. No fertile eggs were produced by the F 1 of the mating between female H. placei × male H. contortus (Louisiana) or by the F 2 of the reciprocal mating. Reciprocal matings between H. placei × H. contortus cayugensis produced an F 1 and F 2 that had reduced fertility but were not completely sterile. No decrease in fertility was observed in the F 2 and F 1 from the H. contortus cayugensis × H. contortus (Louisiana)matings. Cytological studies revealed several kinds of meiotic disturbances in the between species F 1 and F 2 . The most frequent were aneuploidy, failure of chromosome pairing, and pairing between non-homologous chromosomes. Males were more seriously affected than females by meiotic disturbances. The H. placei X chromosome appeared to be selected against by the hybrid genetic background. Within species hybrids showed no meiotic disturbances and the percentage of male offspring from these crosses was the same as for female offspring.

SUMMARY Worm egg counts of yearling Angora or Merino wethers grazing in mixed flocks at either 4/ha or 8/ha were observed over a 5 month period. Based on faecal egg counts infection levels in the sheep increased for the first 3 months and then decreased as the animals developed resistance. Egg counts in the goats did not decrease during the experiment. Post mortem worm counts indicated that there was a significant host species – stocking rate interaction on worm burdens of Haemonchus contortus adults and Ostertagia circumcincta adults and larvae. Goats had more H. contortus and O. circumcinta adults than sheep at the 8/ha stocking rate. At 4/ha goats had fewer O. circumcinta fourth stage larvae than sheep. There was no stocking rate effect on the numbers of Trichostrongylus axei or T. colubriformis. Sheep had more T. axei and fewer T. colubriformis than goats.

The equilibrium of vulvar phenotypes in Haemonchus contortus cayugensis was changed by infecting lambs with either a selected strain of smooth or of linguiform worms. Subsequently the lambs grazed the same pasture and observations were made on changes in worm populations in the lambs, and on pasture it appeared that the seasonal change in the relative frequencies of A and B linguiform worms was related to density-dependent factors in the host. In the spring, when lambs first become infected, the worm population is small and the majority of linguiform worms are A-type. As the season progresses the density of the parasite population increases. Once the population becomes crowded selection begins to favour the B-type females. The effects of crowding on linguiform A females can be seen as a positive correlation between the percentage of linguiform A and their weight. Population density also affected the survival of A-type worms since there was a negative correlation of percentage A-type on total worm numbers. There was a similar negative correlation between percentage A-type on total worm numbers. There was a similar negative correlation between percentage A-type and total worm numbers in both the continuously infected and tracer lambs. Population density did not affect the proportion of smooth worms. However, smooth worms increased in the tracers from approximately 10% at the beginning of the experiment to 55% at its conclusion 18 weeks later. The latter percentage is almost within range of the local subspecies, which has 60% or more smooth worms. It appears therefore that the proportion of smooth to linguiform worms is a stable equilibrium maintained by natural selection.

Established adult infections of Haemonchus contortus in sheep were found to exclude the establishment of H. placei given at dose levels of either 500 or 1500 larvae per week for at least 8 weeks. However, established infections of adult H. placei could not exclude H. contortus. At dose levels of 500 H. contortus larvae per week the existing H. placei infections in sheep were gradually replaced, during which time considerable species hybridization occurred. Dose levels of 1500 H. contortus larvae per week led to the rapid dislodgement of H. placei and much less opportunity for hybridization. A second experiment confirmed that existing H. contortus infections could exclude H. placei and that incoming H. contortus larvae could dislodge H. placei. Furthermore, the exclusion or dislodgement of H. placei was abrogated by injecting the host with dexamethasone. It is proposed that in sheep H. contortus is using a host mediated response to limit their competitor H. placei and that the resulting exclusion and dislodgement of H. placei acts as a major pre-mating barrier to species hybridization.

The F 1 (HX 1 ) from the mating of female Haemonchus placei (Hp) × male Haemonchus contortus (Hc) and the F 2 (HX 2 ) from the reciprocal mating, male Hp × female Hc, consist of sterile males and semi-sterile females. Infections in sheep of HX 2 established from 5000 larvae were found to exclude 90% of a subsequent H. contortus challenge, beginning a fortnight later and consisting of 1600 larvae per week for 6 weeks. When sheep were given an initial infection of 5000 Hc larvae and then challenged a fortnight later with 1600 HX 2 for 6 weeks the mean infection was less than 500 worms, 95% of which were HX 2 . At the end of 6 weeks of larval challenge the resulting infections in both groups of sheep were sterile. The same comparisons between Hc and HX 1 led to mixed infections, with Hc predominating, and the production of fertile eggs. A second experiment indicated that both the exclusion and dislodgement of Hc by HX 2 could be abrogated by injecting the sheep with dexamethasone, a drug with anti-inflammation and immunosuppressing activities. It appears that the HX 2 worms are using a host mediated response to exclude and dislodge Hc similar to the pre-mating barrier used by Hc to prevent its competitor, Hp, from concurrently infecting sheep. The ability of HX 2 to use this response against Hc may be due to hybrid vigour enhancing an existing Haemonchus trait.

Le Jambre L.F., Southcott W.H. and Dash K.M. 1978. Development of simultaneous resistance in Ostertagia circumcincta to thiabendazole, morentel tartrate and levamisole. International Journal for Parasitology8: 443–447. A field strain of Ostertagia circumcincta was divided into five strains based on anthelmintic selection in the laboratory. The first strain was selected with 50 mg/kg thiabendazole, the second with 4 mg/kg morantel tartrate, the third with 3.2 mg/kg levamisole, the fourth was not selected and the fifth strain was selected with all three anthelmintics in each generation. The present paper reports the dose response of the eighth generation of the multi-selected strain to thiabendazole, morantel tartrate and levamisole and compares these results with those from the eighth generation of the single selected and unselected strains. In adult O. circumcincta the LD95 for thiabendazole, morantel tartrate and levamisole was 172.0, 9.2 and 8.4 mg/kg respectively in the multiselected strain, compared with corresponding values of > 200, 6.1 and 6.9 for the single selected strains and 14.5, 2.8 and < 1.6 in the unselected parent strain. Multiple selection with three anthelmintics was associated with an increase in O. trifurcata from less than 0.1 % in the unselected strain to 16% in the multi-selected strain. An increase in inhibition was a feature of both multiple selection and selection by levamisole alone. The parent strain had less than 0.1 % inhibition but the incidence increased to 16% in the levamisole selected strain and to 2% in the multi-selected strain. Approximately 8% of the inhibited larvae in the levamisole selected strain were resistant to dose levels of levamisole from 1.6 to 8.0 mg/kg. Resistance in inhibited larvae was further enhanced in the multi-selected strain and after eight generations 100% of larvae in this strain were resistant to all dose levels up to 100 mg/kg thia-bendazole, 20 mg/kg morantel tartrate and 8 mg/kg levamisole. Apparently when selected with anthelmintics that are less effective against larvae than adults, O. circumcincta responds by increasing the percentage and resistance of inhibited larvae.

Three paddocks were contaminated with Haemonchus contortus eggs from early spring to mid summer by yearling sheep. In Paddock 1, all contamination was derived from worms that had been previously inhibited in their development. The contamination in Paddock 2 was produced only by infections recently acquired from pasture, and in Paddock 3 contamination was attributable to both sources. When the yearling sheep were replaced in mid summer by newly weaned lambs, severe outbreaks of haemonchosis occurred on all paddocks. It was concluded that inhibited H. contortus larvae are capable of producing sufficient eggs, when they resume development, to initiate an outbreak of haemonchosis in susceptible sheep.