Distribution of trichostrongylid nematodes in the abomasum of sheep

The distributions of Haemonchus contortus, Ostertagia circumcincta and Trichostrongylus axei during their development in lambs were studied by freezing and sectioning the abomasum and then recovering the worms from the contents and mucosa of each section. In previously worm-free lambs, the distribution of fourth stage H. contortus was very regular, the frequency of larvae being highest in the anterior fundic area and then decreasing in each section towards the pylorus. The distributions of the early larval stages of O. circumcincta and T. axei were variable but tended to be biomodal, with one peak of larvae in the fundic area and a second in the pyloric area. It is suggested that the difference in distribution between H. contortus and the other two species may be due to a difference in the rate of response of exsheathed third stage larvae to stimuli in the abomasum. In the later stages of development, H. contortus and O. circumcincta tended to migrate towards the pyloric area. In previously infected lambs, the distribution of inhibited early-fourth stage H. contortus was similar to that of normally developing fourth stage larvae in previously worm-free lambs and appeared to be unaffected by the host response which caused an altered distribution and rejection of late-fourth stage and adult worms. The rate of rejection of adult O. circumcincta differed between the fundic and pyloric areas of the abomasum, which suggests that the host response in these two areas may be different.     

The statistical distribution of trichostrongylid nematodes in grazing lambs

Barger I. A. 1985. The statistical distribution of trichostrongylid nematodes in grazing lambs. International Journal for Parasitology15: 645–649. Worm counts of the genera Haemonchus, Ostertagia, Trichostrongylus and Nematodirus from 104 naturally-infected lambs were found to follow negative binomial distributions. Although mean counts covered a 10-fold range, k values did not differ significantly among the four genera. It was concluded that all genera were equally overdispersed. Some implications of this overdispersion for flock productivity and for parasite control were examined     

The relevance of in vitro anthelmintic screening tests employing the free-living stages of trichostrongylid nematodes

The response of the free-living stages of Nippostrongylus brasiliensis, Nematospiroides dubius, Haemonchus contortus, Trichostrongylus colubriformis and Ostertagia ostertagi to a wide variety of antiparasitic agents in vitro was investigated. All the major broad spectrum veterinary anthelmintics showed good activity against each of these worms with EC50 values varying from about 0.0002 mg/l for certain benzimidazoles and ivermectin to about 6.5 mg/l for febantel. Of 22 known narrow spectrum anthelmintics useful only against H. contortus and/or helminths other than trichostrongyles, only 10% showed good activity at concentrations equal to or less than 10.0 mg/l. Further, only one of 15 antiprotozoal agents showed good activity in these tests at the 10.0 mg/l level. The screening test employing free-living Nippostrongylus brasiliensis was selected for an extended trial where the evaluation of 1400 miscellaneous organic chemicals was undertaken. Approximately 10% of these showed activity at concentrations equal to or less than 10.0 mg/l. It is concluded that in vitro screening tests employing the free-living stages of these five genera of nematodes afford simple yet effective means for selecting relevant compounds for further evaluation as possible leads to new broad spectrum anthelmintics for use in ruminants. However, tests using the free-living stages of these worms, including H. contortus, are unsuitable for detecting narrow spectrum 'specifics', e.g., for the treatment of haemonchiasis.     

Genetic parameters for resistance to trichostrongylid infection in dairy sheep

In sheep, the traditional chemical control of gastrointestinal nematode (GIN) parasites with anthelmintics has led to the widespread development of anthelmintic resistance. The selection of sheep with enhanced resistance to GIN parasites has been suggested as an alternative strategy to develop sustainable control of parasite infections. Most of the estimations of the genetic parameters for sheep resistance to GIN parasites have been obtained from young animals belonging to meat- and/or wool-specialised breeds. We present here the estimated genetic parameters for four parasite resistance traits studied in a commercial population of adult Spanish Churra dairy ewes. These involved two faecal egg counts (FECs) (LFEC0 and LFEC1) and two serum indicator traits, the anti-Teladorsagia circumcincta fourth stage larvae IgA (IgA) and the pepsinogen (Peps) levels. In addition, this study has allowed us to identify the environmental factors influencing parasite resistance in naturally infected Spanish Churra sheep and to quantify the genetic component of this complex phenotype. The heritabilities estimated for the two FECs analysed (0.12 for LFEC0 and 0.09 for LFEC1) were lower than those obtained for the examined serum indicators (0.19 for IgA and 0.21 for Peps). The genetic correlations between the traits ranged from 0.43 (Peps-IgA) to 0.82 (LFEC0-LFEC1) and were higher than their phenotypic counterparts, which ranged between 0.07 and 0.10. The heritabilities estimated for the studied traits were lower than previously reported in lambs. This may be due to the differences in the immune mechanisms controlling the infection in young (antibody reactions) and adult (hypersensitivity reactions) animals/sheep. In summary, this study demonstrates the presence of heritable variation in parasite resistance indicator traits in the Churra population studied, which suggests that genetic improvement is feasible for this complex trait in this population. However, further studies in which the experimental variables are controlled as much as possible are needed to identify the best trait that could be measured routinely in adult sheep as an indicator of parasite resistance.     

Quantitative trait loci for resistance to trichostrongylid infection in Spanish Churra sheep

Background

For ruminants reared on grazing systems, gastrointestinal nematode (GIN) parasite infections represent the class of diseases with the greatest impact on animal health and productivity. Among the many possible strategies for controlling GIN infection, the enhancement of host resistance through the selection of resistant animals has been suggested by many authors. Because of the difficulty of routinely collecting phenotypic indicators of parasite resistance, information derived from molecular markers may be used to improve the efficiency of classical genetic breeding.

Methods

A total of 181 microsatellite markers evenly distributed along the 26 sheep autosomes were used in a genome scan analysis performed in a commercial population of Spanish Churra sheep to detect chromosomal regions associated with parasite resistance. Following a daughter design, we analysed 322 ewes distributed in eight half-sib families. The phenotypes studied included two faecal egg counts (LFEC0 and LFEC1), anti-Teladorsagia circumcincta LIV IgA levels (IgA) and serum pepsinogen levels (Peps).

Results

The regression analysis revealed one QTL at the 5% genome-wise significance level on chromosome 6 for LFEC1 within the marker interval BM4621-CSN3. This QTL was found to be segregating in three out of the eight families analysed. Four other QTL were identified at the 5% chromosome-wise level on chromosomes 1, 10 and 14. Three of these QTL influenced faecal egg count, and the other one had an effect on IgA levels.

Conclusion

This study has successfully identified segregating QTL for parasite resistance traits in a commercial population. For some of the QTL detected, we have identified interesting coincidences with QTL previously reported in sheep, although most of those studies have been focused on young animals. Some of these coincidences might indicate that some common underlying loci affect parasite resistance traits in different sheep breeds. The identification of new QTL may suggest the existence of complex host-parasite relationships that have unique features depending on the host-parasite combination, perhaps due to the different mechanisms underlying resistance in adult sheep (hypersensitivity reactions) and lambs (immunity). The most significant QTL identified on chromosome 6 for LFEC₁ may be the target for future fine-mapping research efforts.     

The molecular nature of benzimidazole resistance in helminths

The economic importance of benzimidazole (BZ) resistance has resulted in the isolation of resistant populations of helminths and their study (see pp 127-129 this issue). Recent research indicates that BZs act by binding to free beta-tubulin in the cell and inhibiting the formation of microtubules. The effects of BZs on other processes in the cell, such as transport and anaerobic metabolism, probably result from the inhibition of one or more of the functions of tubulin (see pp 112-115, this issue). In this article, Marleen Roos examines the evidence for changes in the beta-tubulin structure and the rate of its synthesis in BZ-resistant parasitic nematodes.     

Multiple anthelmintic resistance in gastrointestinal nematodes of small ruminants in Bangladesh

Anthelmintic resistance (AR) against gastrointestinal nematodes (GINs) of sheep and goats is a global concern. To address the problem, this study assessed the status of AR in different government and private sheep and goat farms in Bangladesh. We conducted fecal egg count reduction test (FECRT) and Egg hatch assay (EHA) experiments. For the detection of resistant larvae, pooled fecal samples from treated and non-treated groups were subjected to coproculture. Furthermore, 195 adult Haemonchus parasites were genotyped to ascertain benzimidazole (BZ) resistance allele from seven topographic zones of Bangladesh using allele specific PCR (AS-PCR). In FECRT, the percentage reduction along with 95% confidence intervals indicated that GINs were resistant to albendazole (ABZ), levamisole (LEV) and ivermectin (IVM). Coproculture revealed that Haemonchus spp., Oesophagostomum spp. and Trichostrongylus spp. were resistant to anthelmintics. ABZ resistance was also confirmed by in vitro EHA in all the farms except the private goat farm in Mymensingh. The genotype frequencies were 6% for homozygous resistant (rr), 59% for heterozygous (rS) and 35% for homozygous susceptible (SS) among different topographic zones. The allelic frequency of the mutation conferring resistance (r) ranged from 25% to 47% signifying resistance to BZ in nematodes of sheep/goats. The genotype frequencies (rr, rS and SS) and allelic frequencies (r and S) varied significantly (p˂0.05) in different zones in Bangladesh. Overall, the data suggest an alarming condition created by multiple AR in Bangladesh.     

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.     

Efficiency of a genetic test to detect benzimidazole resistant Haemonchus contortus nematodes in sheep farms in Quebec,Canada

Haemonchus contortus is a hemophilic nematode which infects sheep and causes anemia and death to lambs. Benzimidazole drugs are used to remove these parasites, but the phenomenon of resistance has arisen worldwide. A sensitive test to detect resistance before treatment would be a useful tool to enable farmers to anticipate the efficiency of the drug before drenching the flock. In this study, we compared a test for benzimidazole resistance based on detection of genetic markers in H. contortus before treatment with the common method of fecal egg count reduction test (FECRT). We recruited 11 farms from different regions of Quebec for this study. Fecal samples from animals were collected per rectum before and after treatment in control and treated groups (10 animals per group). The 10 sheep were treated with fenbendazole at the recommended dose rate. Among the 11 farms participating in the study, we found H. contortus in 8 of them and it was the most predominant nematode species detected by egg count. Using the genetic test, we found benzimidazole resistance in each of these 8 farms. In 5 of these 8 farms there were sufficient sheep with an egg count for H. contortus above 150 eggs per gram to allow the FECRT test to be conducted. Benzimidazole resistance was observed in each of these 5 farms by the FECRT. When we compared the results from the genetic test for samples off pasture and from individual sheep, with the results from the FECRT, we concluded that the genetic test can be applied to samples collected off pasture to estimate benzimidazole resistance levels before treatment for H. contortus infections.     

Screening of cassava varieties for resistance to root-knot nematodes

Twenty cassava cultivars were screened in micro-plot experiments in the green house of Institute of Agricultural Research and Training, Obafemi Awolowo University, Moor Plantation, Ibadan for root-knot nematode resistance. Four weeks after planting, each plant was inoculated with 5000 eggs of Meloidogyne incognita. There were three replications per cultivar. The plants were uprooted and assessed for resistance after 60 days of inoculation. Assessment of plant damaged was determined by gall index and reproductive factor. Based on host status rating, 3 cultivars were found to be resistant, 4 cultivars were tolerant, 10 cultivars ere susceptible while .3 cultivars were hypersusceptible. All the screened cultivars are resistant to cassava mosaic disease and other major pests of cassava like bacterial disease, anthracnose disease, cassava green mite and cassava mealy bug. They are all high yielding, suitable for food, industry and livestock feed.     

Drug resistance in nematodes of veterinary importance: a status report

Reports of drug resistance have been made in every livestock host and to every anthelmintic class. In some regions of world, the extremely high prevalence of multi-drug resistance (MDR) in nematodes of sheep and goats threatens the viability of small-ruminant industries. Resistance in nematodes of horses and cattle has not yet reached the levels seen in small ruminants, but evidence suggests that the problems of resistance, including MDR worms, are also increasing in these hosts. There is an urgent need to develop both novel non-chemical approaches for parasite control and molecular assays capable of detecting resistant worms.     

A PCR-based method to characterise and identify benzimidazole resistance in Helminthosporium solani

Control of Helminthosporium solani, the cause of silver scurf in potato tubers, has been impaired by selection of benzimidazole-resistant strains as a result of repeated use of the fungicide thiabendazole. Identification of thiabendazole-resistant strains of H. solani by conventional techniques takes several weeks. Primers designed from conserved regions of the fungal β-tubulin gene were used to PCR amplify and sequence a portion of the gene. A point mutation was detected at codon 198 in thiabendazole-resistant isolates causing a change in the amino acid sequence from glutamic acid to alanine or glutamine. Species-specific PCR primers designed to amplify this region were used in conjunction with a restriction endonuclease to cause cleavage in sensitive isolates only and thus provide a rapid diagnostic test to differentiate field isolates.     

Analysis of putative inhibitors of anthelmintic resistance mechanisms in cattle gastrointestinal nematodes

Effects of the cytochrome P450 inhibitor piperonyl butoxide and the P-glycoprotein inhibitor verapamil on the efficacy of ivermectin and thiabendazole were studied in vitro in susceptible and resistant isolates of the cattle parasitic nematodes Cooperia oncophora and Ostertagia ostertagi. The effects of combined use of drug and piperonyl butoxide/verapamil, respectively, were investigated in the Egg Hatch Assay, the Larval Development Assay and the Larval Migration Inhibition Assay. The effects of piperonyl butoxide and verapamil as inhibitors of thiabendazole and ivermectin responses were particularly marked for larval development, where both inhibitors were able to completely eliminate all differences between susceptible and resistant isolates. Even the lowest concentrations of anthelmintics used in combination with inhibitors caused complete inhibition of development. Differences and/or similarities among responses in different isolates were only obtained in the two other assays: in the Egg Hatch Assay piperonyl butoxide caused a shift in concentration–response curves obtained with thiabendazole to the left for all isolates tested, changing relative differences between isolates. In contrast, an effect of verapamil in the Egg Hatch Assay was only apparent for benzimidazole-resistant isolates. In the Larval Migration Inhibition Assay only ivermectin was tested and piperonyl butoxide shifted the concentration–response curves for all isolates to the left, again eliminating differences in EC₅₀ values between susceptible and resistant isolates. This was not the case using verapamil as an inhibitor, where curves for both susceptible and benzimidazole-resistant isolates shifted to the left in Ostertagia isolates. In Cooperia the picture was more complex with ivermectin-resistant isolates showing a larger shift than the susceptible isolate. Single nucleotide polymorphisms in the β-tubulin isotype 1 gene were investigated. Significantly increased frequencies of resistance-associated alleles were observed for the codons 167 and 200 in one benzimidazole-resistant isolate but not in an isolate selected for benzimidazole resistance at an early stage of selection.     

Breeding cattle and sheep for resistance to gastrointestinal nematodes

Gastrointestinal nematodes are an important cause of reduced production of meat, milk and wool in domestic livestock. It is generally believed that problems caused by these parasites have increased owing to the intensification of animal husbandry(1-3) of resistance to anthelmintics, current research is focussed on alternative control strategies that do not rely on anthelmintics. Here, Bram Kloosterman, Henk Parmentier and Harm Ploeger review work on the genetic resistance of domestic ruminants to these nematodes and discuss the practicality of breeding programmes.