Therapy and anthelmintic resistance

Treatment against nematode parasites in sheep and goats is reviewed. The main risk factors for parasitic infection in these hosts are briefly outlined. The mechanism of action of the most important chemical groups (imidazothiazoles and pyrimidines, benzimidazoles/ pro-benzimidazoles and macrocyclic lactones) to which the modern anthelmintic drugs belong are illustrated and discussed with particular emphasis on possible selection of anthelmintic resistance. The need for strategic integrated control based also on the epidemiological patterns of parasitism, the composition and the production of the herd (milk, meat, wool) and the potency of the drugs are discussed. The importance of diet and potential alternative control measures (nematophagus fungi and natural compounds such as tannins) are illustrated.     

Oxidase activities in macrocyclic-resistant and -susceptible Haemonchus contortus

The role of oxidative metabolism in resistance to macrocyclic lactones in Haemonchus contortus was examined by measuring activities toward 2 model cytochrome P450 substrates, aldrin and ethoxycoumarin, in a susceptible and a resistant isolate of the parasite. Microsomal preparations from larvae and adults of the 2 isolates showed no differences in levels of NADPH- or cumene hydroperoxide-supported aldrin epoxidase or ethoxycoumarin O-deethylase activities. Intact adult nematodes showed an ability to catalyze the epoxidation of significant amounts of aldrin, although the nature of the enzyme group responsible was unknown. This epoxidase activity was greater in adults of the susceptible isolate. It is apparent that oxidase activities toward the 2 substrates are not enhanced in the resistant isolate, suggesting that the observed resistance to macrocyclic lactones may not involve enhanced oxidative metabolism.     

Field evaluation for anthelmintic-resistant ovine gastrointestinal nematodes by in vitro and in vivo assays

A coprological survey to analyze the presence of flock resistance to benzimidazoles (BZ) and macrocyclic lactones (ML) was performed in sheep under field conditions. Fecal samples were collected from 2,625 sheep in 72 commercial farms from Galicia (NW Spain). The in vitro (FECRT, fecal egg count reduction test) and in vivo (EHA, egg hatch assay, and LFIA, larval feeding inhibition assay) tests were used to assess the efficacy of these anthelmintics. Coprocultures were also developed to obtain knowledge on the main genera of trichostrongylid nematoda prior to, and after, the administration of the anthelmintics. By using the FECRT, BZ resistance was observed in 13 (18%) flocks, whereas ML resistance was only detected in 2 (3%) farms. The number of resistant flocks to BZ was 21 (29%) by using the EHA and 7 (10%) by means of the LFIA. None of the flocks used in this study showed simultaneous resistance to both employed anthelmintics. The results from the in vitro and in vivo tests revealed that 92% of the flocks FECRT resistant to BZ were also resistant with the EHA. The LFIA confirmed all the farms resistant to ML by using the in vivo test. After the administration of BZ, nematode larvae belonging to Teladorsagia circumcincta (32.2%), Trichostrongylus spp. (29%), Nematodirus spp. (6.5%), and Chabertia ovis (3.2%) were identified. In the flocks receiving ML, only T. circumcincta was identified (57%). We recommend the use of in vitro tests because they are more efficient. As the use of macrocyclic lactones is increasing in this region, further investigation is needed for detecting resistance to the anthelmintic family compounds by the LFIA.     

Dermacentor variabilis: resistance to ticks acquired by mast cell-deficient and other strains of mice

The acquisition of resistance to ticks was monitored in mice of six different strains. Mice were subjected to repeated infestations with Dermacentor variabilis larvae, different skin sites being used for each successive infestation. In the third and fourth infestations, resistance was expressed in three strains of mice (WBB6F1-W/Wv, WBB6F1-+/+, and CFW), as demonstrated by significant reductions in percentages of larvae engorging and in mean weights of fed larvae. Both WBB6F1-W/Wv mice, which are mast cell-sufficient strain attained significantly higher levels of resistance. It is suggested that mast cells may play a relatively minor role in the mechanisms of resistance in this strain of mice. C57B1 mice also expressed tick resistance in their third and fourth infestations as measured by reduced percentages of engorged larvae, but not by reduced mean larval weights. Possibly, the mechanisms of tick resistance in this strain differ from those in other strains. Two other mouse strains (C3H-HeJ and C3H-HeSn) remained relatively susceptible to tick feeding throughout five infestations. In secondary infestations of all strains tested, no resistance was evident. Instead, enhanced feeding of larvae appeared to occur. A new objective measurement of tick resistance is the mean weights of detached, unengorged larvae taken from resistant animals at the end of the infestation period. These were found to be consistently less than those from susceptible animals.     

Feeding behaviour and efficiency of banana weevil (Cosmopolites sordidus) larvae on banana cultivars of varying resistance levels

Nutrient composition, secondary plant substances and other host quality characteristics govern resistance and influence herbivore feeding. The current study was carried out to compare aspects of feeding behaviour and food utilization of banana weevil (Germar) (Coleptera: Curculionidae) larvae feeding on banana cultivars with different levels of resistance. In the first study, penetration times of larvae into tissue of seven banana cultivars (two susceptible, three intermediate and four resistant) were compared. Day‐old larvae were placed on corm or pseudostem tissue of these cultivars and time taken to penetrate the tissue was recorded. Larvae generally took longer to tunnel into resistant cultivars, although Yangambi Km5 (resistant) did not fit this pattern. In the second set of studies, food consumption and growth of larvae were compared on cultivars: (i) Kisubi (resistant) vs. Atwalira (susceptible); and (ii) Mbwazirume (intermediate) vs. Kayinja (resistant). Day‐old larvae were offered weighed pieces of corm that were changed every 4–7 days. At each corm change, weights of the larvae, corms and frass were measured. Changes in corm and larval weight were calculated to determine larval growth and amount of tissue consumed. Larvae feeding on resistant cultivars had lower consumption and growth rates and longer developmental duration. It was concluded that low food consumption rates contribute to delayed development of larvae feeding on resistant cultivars. Differences between resistant and susceptible cultivars in food utilization may be of limited consequence for the cultivars tested. This is one step in identifying the factors influencing weevil development rates in cultivars of different resistance levels. If these factors are identified, they can be targeted in breeding for resistance.     

Parasitological and immunological responses of genetically resistant Merino sheep on pastures contaminated with parasitic nematodes.

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.     

What canCaenorhabditis elegans tell us about nematocides and parasites?

Nematode infections compromise human health and reduce agricultural productivity. Experiments that exploit the powerful molecular genetics of the free-living nematodeCaenorhabditis elegans have contributed to our understanding of how the major classes of anthelmintic nematocides kill worms and how worms might evolve resistance to these drugs. InC. elegans, as in parasites, benzimidizoles interfere with microtubule polymerization, the imidazothiazoles/tetrahydropyrimidines activate nicotinic acetylcholine receptors, and the macrocyclic lactones activate glutamate-gated chloride channels. Mutant alleles of genes that encode drug targets often confer resistance inC. elegans. Preliminary evidence suggests that alleles of homologous genes in parasites will, in many cases, also play a role in resistance. Thus, information acquired fromC. elegans can be usefully applied to understand the mechanisms of drug sensitivity and the genetics of resistance in parasites.     

Impact of plant resistance on southwestern corn borer (Lepidoptera: Crambidae) biology and plant damage

Southwestern corn borer, Diatraea grandiosella Dyar (Lepidoptera: Crambidae), is a major insect pest of corn, Zea mays L., in the southern United States. Germplasm lines with resistance to southwestern corn borer have been developed and released by the USDA-ARS. Two single-cross hybrids produced by crossing germplasm lines with resistance to southwestern corn borer and a susceptible single-cross hybrid were infested with southwestern corn borer larvae in a 2-yr field test conducted in Mississippi. The susceptible hybrid sustained significantly more leaf damage and stalk tunneling than either resistant hybrid. The number of tunnels and the length of tunneling were significantly lower on the resistant hybrids. In 2003, up to 15 times more tunneling was observed on the susceptible hybrid. Larvae feeding on the resistant hybrids were delayed in their movement from the whorl to the stalk and larval survival was 50% lower on the resistant hybrids than on the susceptible hybrid. Larvae recovered from the susceptible hybrid 7-14 d after infestation weighed twice as much as those recovered from the resistant hybrids. Similar differences in larval weight were observed in the laboratory when larvae were reared on diets prepared from lyophilized tissue from the three hybrids. These results provide a foundation for other investigations designed to identify and determine the roles of specific genes and gene families associated with southwestern corn borer resistance in corn.     

Susceptibility of different strains of mice to various levels of infection with the eggs of Taenia taeniaeformis

Susceptibility of different mouse strains to varying levels of Taenia taeniaeformis eggs has been studied. C3H are shown to be susceptible to any quantity of eggs. However C57 and NMRI are only susceptible to 1–2 eggs, while larvae from an infection of 30–50 eggs are precociously destroyed. Sometimes fertile larvae can also develop in resistant strains of mice infected with some hundred eggs. In C3H the challenge larvae are unable to survive even from an infection given 24–48 h post-first inoculum. The hypothesis is proposed that in resistant strains, infection with 30–50 eggs induces a more rapid immune response which becomes effective while the larva is still vulnerable; in massive infections, however, immune paralysis may occur. Although susceptible strains allow primary infections to develop, they show resistance to challenge infections because larvae are destroyed before they become insusceptible to host attack.     

Relative fitness of Cry1A-resistant and -susceptible Helicoverpa armigera (Lepidoptera: Noctuidae) on conventional and transgenic cotton

Glasshouse and laboratory experiments were conducted to evaluate the relative fitness of Cry1A-susceptible and laboratory-selected resistant strains of Helicoverpa armigera (Hübner). Life history parameters of H. armigera larvae feeding on young cotton plants showed a significant developmental delay of up to 7 d for the resistant strain compared with the susceptible strain on non-Bacillus thuringiensis (Bt) cotton. This fitness cost was not evident on artificial diet. There was no developmental delay in the F1 hybrid progeny from the reciprocal backcross of the resistant and susceptible strains, indicating that the fitness cost is recessive. In two cohorts tested, survival to pupation of resistant larvae on Bt cotton expressing Cry1Ac was 54 and 51% lower than on non-Bt cotton, whereas all susceptible and F1 larvae tested on Cry1Ac cotton were killed. Mortality of susceptible larvae occurred in the first or second instar, whereas the F1 larvae were able to develop to later instars before dying, demonstrating that resistance is incompletely recessive. The intrinsic rate of increase was reduced by >50% in the resistant strain on Cry1Ac cotton compared with the susceptible strain on non-Bt cotton. There was a significant reduction in the survival of postdiapausal adults from the resistant strain and the F1 strains, indicating that there is a nonrecessive overwintering cost associated with Cry1A resistance in H. armigera.     

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.     

In vitro bioassay of sheep gastrointestinal mucus for nematode paralysing activity mediated by substances with some properties characteristic of SRS-A

A bioassay is described for determining the inhibition of nematode larval migration from agar by substances exerting a paralysing action.In the assay, larval migration was completely inhibited by the anthelmintic levamisole (25 μg/ml) whereas biogenic amines, and prostaglandins E₁ and E₂ at 50 μg/ml, were without effect. Mucus from the gastrointestinal tract of sheep resistant to nematode infection inhibited larval migration by up to 93% whereas mucus from heavily infected sheep or sheep reared helminth free did not significantly inhibit larval migration. Mucus from sheep resistant to Trichostrongylus colubriformis inhibited the migration of larvae of other nematode species.The larval migration inhibitory (LMI) activity of mucus from resistant sheep was associated with components having some properties of slow reacting substance of anaphylaxis (SRS-A).Faecal samples from resistant sheep possessed significantly more LMI activity than faecal samples from heavily infected sheep or sheep reared helminth free. The level of LMI activity in the faeces of sheep undergoing challenge infection may be a useful indicator of the sheep's resistance status.The presence of the larval migration inhibitory activity in sheep mucus is discussed in relation to resistance to infection.     

Acquired resistance in sheep to infection with larvae of the blowfly, Lucilia cuprina

Acquired resistance in sheep to infection with larvae of the blowfly, Lucilia cuprina. International Journal for Parasitology16: 69–75. Resistance to blowfly larvae infections developed in sheep exposed to at least four consecutive infections. Half of the sheep treated showed significant levels of resistance, the others remained susceptible. This resistance took the form of a decreased yield of third instar larvae in comparison to controls and sheep which remained susceptible. In addition an increased sensitivity to larvae developed, as shown by the area of wound obtained per maggot recovered, by the early appearance in resistant sheep of exudate from the infection site and by skin reactions to larval products. Radioimmunoassays demonstrated high levels of serum antibody against larval excretory/secretory antigens, though the response did not peak until after four infections. Resistant animals showed somewhat lower antibody titres than susceptible sheep. Consecutive infections of only 50 larvae failed to induce resistance to larger challenge infections. It is suggested that consecutive infections of larger numbers of maggots induce a hypersensitivity response which may effect larval survival especially of first and second instar maggots.     

Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) larval feeding behavior on transgenic maize (MON 863) and its isoline

Diabrotica species (Coleoptera: Chrysomelidae) larval behavior studies have posed a challenge to researchers because of the subterranean life cycle of this pest. To fully understand how the western corn rootworm, Diabrotica virgifera virgifera LeConte, injures the maize, Zea mays L., root system, its behavior must be studied. For example, larvae that can detect an area of the root that has a lower amount of toxin, whether from an insecticide or a transgenic maize plant, have an increased chance of survival. This study assessed D. v. virgifera larval feeding behavior on rootworm-susceptible maize and maize containing a biotechnology-derived trait (MON 863) with resistance to D. v. virgifera first instar feeding. Maize plants were grown in a medium that allowed for direct observation and measurements during feeding of larval stadia. Neonates were placed on maize seedlings, and data were taken at 3, 6, 9, and 12 d postinfestation on resistant and susceptible maize. On rootworm-susceptible maize, neonate larvae aggregated at the root tips and began actively feeding, and then they moved to older root tissue. Conversely, some larvae that ingested Cry 3Bb1 from the resistant maize exhibited no movement. Other larvae on the resistant maize moved continuously, sampling root hairs or root tissue but not actively feeding. The continuously moving larvae had visibly empty guts, suggesting possible nonpreference for the resistant root. This study contributes to our understanding of D. v. virgifera larval behavior and provides insight into questions surrounding the potential evolution of behavioral and biochemical resistance to Cry3Bb1.     

Haemonchus contortus: characterization of a glutamate binding site in unselected and ivermectin-selected larvae and adults.

A specific ivermectin-sensitive, glutamate binding site has been identified in the parasitic nematode Haemonchus contortus. Glutamate binding in H. contortus was saturable and occurred in a single class of high-affinity binding sites which appeared to have pharmacological properties different from those of mammalian glutamate receptors. Adult and larval forms of H. contortus had dramatically different glutamate binding kinetics, the larvae showing nearly up to 200-fold higher Bmax values and up to 9-fold increases in Kd values compared to adults. Treatment of adult H. contortus with the anthelmintic, ivermectin, decreased the Bmax value for glutamate binding in the susceptible strain but not in the resistant parasites. Furthermore, selection for ivermectin resistance was associated with a significant increase in Bmax for glutamate binding in adults and a similarly significant increase in glutamate binding affinity in larvae. These results suggest that the H. contortus glutamate binding site identified in this study may be involved in the phenomenon of ivermectin resistance.     

Oviposition choice and larval survival of Dasineura marginemtorquens (Diptera: Cecidomyiidae) on resistant and susceptible Salix viminalis

Abstract.

• 1 Substantial intraspecific variation exists in Salix viminalis resistance to the gall midge Dasineura marginemtorquens. Earlier work has found this variation to have a large genetic component. Willow clones are stable in their resistances between midge generations and different nutrient levels in both field and laboratory culture.
• 2 This study reports the results of laboratory experiments on female oviposition choice and larval survival on potted plants from clones that are very different in resistance as determined in field studies.
• 3 In choice experiments using pairs of plants, the average female midge did not prefer susceptible willow clones over resistant ones for oviposition. In about one third of the replicates, midges actually laid more eggs on the resistant clone. Further work is necessary to examine the nature of variation among midges in discrimination of these plant types.
• 4 Resistance is manifested as great differences in larval survival. Six days after oviposition survival was 92% on susceptible plants but only 6% on resistant ones. Galls developed on all of the susceptible plants, while in 73% of the resistant plants galls were not even initiated.
• 5 The plant traits causing resistance are enigmatic. Larval behaviour suggests that resistant plants interfere with feeding behaviour. On resistant plants, most larvae wander for more than 24 h without initiating any galls before dying. On susceptible plants many first instar larvae begin feeding and initiate galls within this period.

     

Antixenosis in maize reduces feeding by western corn rootworm larvae (Coleoptera: Chrysomelidae)

SUM2162 is the first known example of a naturally occurring maize, Zea mays L., genotype with antixenosis (nonpreference) resistance to western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), larval feeding. Behavioral responses of neonate western corn rootworm larvae were evaluated in laboratory bioassays with seven maize genotypes selected for native resistance to rootworm feeding damage. Two susceptible maize genotypes and one transgenic (Bacillus thuringiensis) maize genotype were included as controls. In soil bioassays with cut roots, no larvae entered the roots of the resistant variety SUM2162, but at least 75% of the larvae entered the roots of every other maize type. Larvae made significantly fewer feeding holes in the roots of SUM2162 than in all the other maize genotypes, except the isoline control. In feeding bioassays, larval feeding varied significantly among maize genotypes, but there was no significant difference between the resistant varieties and the susceptible controls. There were no significant differences among any of the genotypes in host recognition (search) behavior of larvae after exposure to the roots. Little variation in feeding stimulant blends was observed among maize genotypes, indicating minimal contribution to the observed antixenosis.     

Evaluation of the Egg Hatch Assay and the Larval Migration Inhibition Assay to detect anthelmintic resistance in cattle parasitic nematodes on farms

Resistance to anthelmintic drugs, particularly to the widely used benzimidazoles (BZs) and macrocyclic lactones (MLs) is an increasing problem in cattle industries worldwide. Reliable methods for the assessment of anthelmintic efficacy in the field are required in order to react before resistance becomes an obvious problem on individual properties. The ability of the Egg Hatch Assay (EHA) and the Larval Migration Inhibition Assay (LMIA) to detect anthelmintic resistance under field conditions was evaluated on cattle farms in Northern Germany. As published previously Faecal Egg Count Reduction Test (FECRT) was performed using oral albendazole (Valbazen®) or injectable ivermectin (Ivomec®). Herein the FECRT results described earlier were compared with data from EHAs or LMIAs, respectively, performed with eggs from fresh faeces or larvae from faecal cultures of the tested animals before and after treatment. The obtained EC₅₀ values allowed the assessment of efficacy of albendazole and ivermectin on farm level. The results of the FECRTs and the results of both in vitro assays were comparable. In comparison to the FECRT the in vitro assays are less time, labour and cost intensive and are able to assess the susceptibility status of a worm population without treatment. Therefore both are beneficial alternatives for the reliable detection of reduced efficacy of these two drug classes on farms.     

Genetic variability following selection of Haemonchus contortus with anthelmintics

Genetic diversity in nematodes leads to variation in response to anthelmintics. Haemonchus contortus shows enormous genetic diversity, allowing anthelmintic resistance alleles to be rapidly selected. Anthelmintic resistance is now a widespread problem, especially in H. contortus. Here, I compare the genes involved in anthelmintic resistance in H. contortus with those that confer susceptibility or resistance on the free living nematode Caenorhabditis elegans. I also discuss the latest knowledge of genes associated with resistance to benzimidazoles, levamisole and the macrocyclic lactones and the need for DNA markers for anthelmintic resistance.