New genetic and practical implications of selection for anthelmintic resistance in parasitic nematodes

Investigations into genetic mechanisms underlying anthelmintic resistance in parasitic nematodes have indicated that even at moderate levels of resistance, reversion to the initial level of susceptibility may be impossible. Alleles that confer the higher levels of susceptibility may be lost from the population in the early stages of selection. Therefore close monitoring of early steps in the selection for resistance is needed, as discussed here by Marleen Roos. Marcel Kwa and Warwick Grant.     

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.     

Contrasting genetic structures of two parasitic nematodes,determined on the basis of neutral microsatellite markers and selected anthelmintic resistance markers

For the first time, the neutral genetic relatedness of natural populations of Trichostrongylid nematodes was investigated in relation to polymorphism of the β‐tubulin gene, which is selected for anthelminthic treatments. The aim of the study was to assess the contribution of several evolutionary processes: migration and genetic drift by neutral genetic markers and selection by anthelminthic treatments on the presence of resistance alleles at β‐tubulin. We studied two nematode species (Teladorsagia circumcincta and Haemonchus contortus) common in temperate climatic zones; these species are characterized by contrasting life history traits. We studied 10 isolated populations of goat nematode parasites: no infected adult goat had been exchanged after the herds were established. Beta‐tubulin polymorphism was similar in these two species. One and two β‐tubulin alleles from T. circumcincta and H. contortus respectively were shared by several populations. Most of the β‐tubulin alleles were ‘private’ alleles. No recombination between alleles was detected in BZ‐resistant alleles from T. circumcincta and H. contortus. The T. circumcincta populations have not diverged much since their isolation (F_ST <0.08), whereas H. contortus displayed marked local genetic differentiation (F_ST ranging from 0.08 to 0.18). These findings suggest that there are severe bottlenecks in the H. contortus populations, possibly because of their reduced abundance during unfavourable periods and their high reproductive rate, which allows the species to persist even after severe population reduction. Overall, the data reported contradict the hypothesis of the origin of β‐tubulin resistance alleles in these populations from a single mutational event, but two other hypotheses (recurrent mutation generating new alleles in isolated populations and the introduction of existing alleles) emerge as equally likely.     

Development of anthelmintic resistance in nematodes from sheep in Australia subjected to different treatment frequencies

Development of anthclmintic resistance in nematodes from sheep in Australia subjected to different treatment frequencies. International Journal for Parasitology13: 125–132. In a three year (1978, 1979, 1980) field experiment in south-eastern Australia Merino weaner sheep were exposed to one of four worm control programmes. Sheep were treated either 50–51 times, 9–12 times, 3–6 times or 1–3 times annually with anthelmintic. Thiabendazole (TBZ) was used throughout 1978 and for the initial dose in 1980, levamisole (LEV) throughout 1979 and 1980. Sheep were replaced annually each December.During 1978 in vitro egg hatch tests indicated that populations of Haemonchus contenus with resistance factors to TBZ of 10·7, 10·0, 2.5 and 1·8 respectively were selected by these treatment frequencies. No resistance to LEV was detected in 1979 but during 1980 in vivo egg count reduction tests showed that highly resistant populations of Ostertagia spp. were selected in sheep dosed either 49 or 11 times. This was confirmed by an in vitro larval paralysis test. Partial resistance occurred where sheep were dosed 5 times but not where one dose was given. The only population of H. conlortus still present at the end of 1980 in sufficient numbers to allow eggs to be tested in vitro was from the sheep dosed once. This had retained its resistance to TBZ.     

Evaluation of the nuclear DNA Diffusion Assay to detect apoptosis and necrosis

We applied the nuclear DNA Diffusion Assay, described as an accurate tool to estimate apoptotic and necrotic cells [N.P. Singh, A simple method for accurate estimation of apoptotic cells, Exp. Cell Res. 256 (2000) 328-337] to tobacco root and leaf cells. In this assay, isolated nuclei are embedded in an agarose microgel on a microscope slide and low molecular-weight DNA fragments diffuse into the microgel. Exposure of the roots to hydrogen peroxide significantly increased the average nuclear area of isolated nuclei. After 4 and 24 h of recovery, all DNA damage was repaired. The data clearly demonstrate that the manifestation of diffused nuclei upon exposure to hydrogen peroxide is not the result of non-repairable apoptotic or necrotic DNA fragmentation, but represents repairable genotoxin-induced DNA damage. In contrast, treatment with the alkylating agent ethyl methanesulphonate (EMS) followed by 24 h of recovery produced a significant increase in the average nuclear area. The contribution of apoptosis to this increase cannot be excluded. Heat treatment of leaves at 50 degrees C for 1-15 min leading to necrosis, and treatment of isolated nuclei with DNase-I, which digests DNA to nucleosome-sized fragments as during apoptosis, also led to a dose-dependent increase in the nuclear area. The use of different fluorochromes (ethidium bromide, DAPI or YOYO-1) for DNA staining yielded similar results in the DNA Diffusion Assay. As all types and sizes of diffused nuclei were observed after EMS and hydrogen peroxide treatments, we were unable to differentiate, on the basis of the structure of the nuclei, between apoptotic or necrotic DNA fragmentation and other types of genotoxin-induced DNA damage in plants.     

Evaluation of OptiMAL Assay test to detect imported malaria in Italy

This study evaluated a newly developed rapid malaria diagnostic test, OptiMAL Assay, to detect "Plasmodium falciparum malaria" and "non Plasmodium falciparum malaria" in blood samples from 139 individuals with a presumptive clinical diagnosis of imported malaria in Italy. OptiMAL Assay utilizes a dipstick coated with monoclonal antibodies against the intracellular metabolic enzyme, plasmodium Lactate Dehydrogenase (pLDH) present in and released from parasite-infected erythrocytes. Blood samples from 56 cases out of 139 were found "Plasmodium falciparum malaria" positive by microscopy; with these samples OptiMAL Assay and the ParaSight-F test, which is a kit detecting the P. falciparum histidin-rich protein 2 (HRP-2), showed an overall sensitivity of 83% and 94%, respectively, in comparison with microscopy. Parasitemia levels tested in the 56 P. falciparum positive blood samples by microscopy ranged from <0.004% to 20%. A correlation between sensitivity and parasitemia was evident and OptiMAL Assay and ParaSight-F test were more sensitive (96-100%; 100%) with samples with 0.1%-20% levels of parasitemia, while proved less sensitive (0-44%; 50-88%) with <0.004-0.01% levels of parasitemia.     

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.     

Macrocyclic lactone resistance in nematodes of cattle in Brazil: Blame it to the ticks!

Strategic helminth control in adult cattle would hardly impose sufficient selection pressure to parasite populations but reports of resistance against macrocyclic lactone (ML) based-products have been confirmed worldwide. The objective of this study was to evaluate the scientific literature of ML resistance (< 90.0% efficiency) in helminths of cattle from 2001 (the first report) to 2020 in Brazil. Additional to the data, we studied the correlation of parasite control practices based on a questionnaire given to 32 farmers. The search returned 246 reports and 21 full articles were selected. From these, a Wordcloud and a Keyword Co-occurrence Network graph were created. The published data revealed that most of the studies (19/21) reported multi-species (Cooperia spp., Haemonchus sp., Oesophagostomum radiatum, Trichostrongylus sp.) resistance to ML. None of the reports described the treatment frequency in the tested farms. As for the questionnaire, the majority of farmers (> 70.0%) responded that they rotate products after treatment, animals are treated monthly or biweekly (58.0%), treatments are based on visual evaluation (coat condition, ectoparasite infestation), and that in 94.0% of the times farmers treat all animals. Moreover, farmers use ML in association with potent acaricides (cypermethrin, chlorpyriphos, fluazuron) in more than 90.0% of the times (15/16). It was observed that this regimen was used to prevent and control the cattle-tick Rhipicephalus microplus (90.0%) and the horn-fly, Haematobia irritans (30.0%) infestations. We conclude that the most important factor for nematode resistance was the high level of ML exposure of up to 16 times/year, in combination with acaricides to control ticks and to a lesser extent to control horn-flies. Therefore, selection of helminth populations in cattle in Brazil can be considered secondary to ectoparasite control. The present analysis is critical, as one the most widespread recommendations to avoid drug resistance is to reduce the use of long-acting compounds, due to their extended persistent periods, increasing parasite selection. Moreover, a more serious attitude must be taken regarding parasite control strategies for livestock, reinforcing that health protocols should be based on single acaricidal products whenever possible. Complementary, selective evaluations based on transient threshold population abundance must be enforced to reduce treatment frequency, reducing parasite selection and animal distress.     

Effect of chemoprophylaxis on immunity to gastrointestinal nematodes in cattle

Control of gastrointestinal nematodes in first-season grazing cattle is largely based on either strategic anthelmintic treatment or use of anthelmintic-release devices. Control is evolving towards more efficacious anthelmintics and delivery systems, almost annihilating parasite contact. Here, Jozef Vercruysse, Hans Hilderson and Edwin Claerebout focus on the inverse relationship between the intensive use of modern anthelmintics and the build-up of immunity to gastrointestinal nematodes.     

Inhibition patterns and seasonal availability of nematodes for beef cattle grazing on argentina''s western pampas

The seasonal population trends of cattle nematodes in tracer calves are described from 1981 to 1987. Successive worm-free calves were grazed with growing beef cattle for 20–30 days at 6 week intervals and then slaughtered for nematode counts 2 weeks after their removal from pasture. Ostertagia, Cooperia, Trichostrongylus and Haemonchus were the main genera recovered. O.ostertagi was the most often found and acquisition of inhibition-prone larvae began in late winter and reached a peak in spring, while maximum larval availability was in autumn. The seasonal inhibition and larval availability pattern for T. axei was similar to that of Ostertagia. Cooperia showed greatest inhibition during winter with maximum larval availability in autumn and spring. Haemonchus was more prevalent during summer to early autumn and no inhibition was observed. It was concluded that infective larval availability for tracer calves was highest during autumn, and most reduced in summer. All predominant species were able to survive over summer on pasture,     

Evaluation of resistance to permethrin,cypermethrin and deltamethrin in different populations of Musca domestica (L.), collected from the Iranian dairy cattle farms

The house fly, Musca domestica (Linnaeus) (Diptera: Muscidae), is a major pest for human and livestock health and is also resistant to different insecticides. Herein, six M. domestica populations were collected, five of them from industrial cattle farms and the Koohrang population from a remote area as a susceptible population. The resistance/susceptibility of populations to three pyrethroids was evaluated. High levels of permethrin resistance were observed in all field populations and the resistance ratios (RRs) were estimated to vary from 52- to 129-fold. Resistant populations also exhibited resistance to other pyrethroids (cypermethrin and deltamethrin), with RRs ranging between 45- and 180-fold. According to synergistic (piperonyl butoxide, diethyl maleate and triphenyl phosphate) and enzymatic assays, resistant populations exhibited multiple resistance phenotypes. Cytochrome P450 monooxygenases (P450s), glutathione S-transferases (GSTs), and carboxylesterases (CarEs) were found to be involved in pyrethroid resistance in Isfahan population, P450s and GSTs in Mobarake population and CarEs detoxified pyrethroids in Natanz and Alavijeh populations. As substitution of Leucine (CTT) with Phenylalanine (TTT) at position 1014 of the voltage sensitive sodium channel (VSSC) gene is the most common mutation conferring resistance to pyrethroids in M. domestica, we sequenced a partial fragment of IIS6 and L1014F mutation was detected in all resistant populations. The present study provides valuable information for early detection of pyrethroid resistance and developing resistance management strategies in the house fly populations.     

Prevalence and antimicrobial resistance of thermophilic Campylobacter spp. from cattle farms in Washington State

The prevalence of thermophilic Campylobacter spp. was investigated in cattle on Washington State farms. A total of 350 thermophilic Campylobacter isolates were isolated from 686 cattle sampled on 15 farms (eight dairies, two calf rearer farms, two feedlots, and three beef cow-calf ranches). Isolate species were identified with a combination of phenotypic tests, hipO colony blot hybridization, and multiplex lpxA PCR. Breakpoint resistance to four antimicrobials (ciprofloxacin, nalidixic acid, erythromycin, and doxycycline) was determined by agar dilution. Campylobacter jejuni was the most frequent species isolated (34.1%), followed by Campylobacter coli (7.7%) and other thermophilic campylobacters (1.5%). The most frequently detected resistance was to doxycycline (42.3% of 350 isolates). Isolates from calf rearer facilities were more frequently doxycycline resistant than isolates from other farm types. C. jejuni was most frequently susceptible to all four of the antimicrobial drugs studied (58.8% of 272 isolates). C. coli isolates were more frequently resistant than C. jejuni, including resistance to quinolone antimicrobials (89.3% of isolates obtained from calves on calf rearer farms) and to erythromycin (72.2% of isolates obtained from feedlot cattle). Multiple drug resistance was more frequent in C. coli (51.5%) than in C. jejuni (5.1%). The results of this study demonstrate that C. jejuni is widely distributed among Washington cattle farms, while C. coli is more narrowly distributed but significantly more resistant.     

Transglutaminase-catalyzed reactions in the growth, maturation and development of parasitic nematodes

Parasitic nematodes cause several debilitating diseases in humans and animals. New drugs that are parasite specific and minimally toxic to the host are needed to counter these infections effectively. The identification and inhibition of enzymes that are vital for the growth and survival of parasites offer new approaches for developing effective chemotherapeutic agents. Several enzymes in nematodes fall into this category. Here, Ramaswamy Chandrashekar and Kapil Mehta examine in detail the role of transglutaminase, a protein-crosslinking enzyme, in the normal growth and development of nematodes, with an emphasis on filarial parasites.     

Genotyping and antibiotic resistance patterns of Campylobacter fetus subsp.venerealis from cattle farms in India

Bovine genital campylobacteriosis caused by Campylobacter fetus subsp. venerealis (Cfv) is of considerable economic importance to the cattle industry worldwide. Cfv causes syndrome of temporary infertility in female cattle, early embryonic mortality, aberrant oestrus cycles, delayed conception, abortions and poor calving rates. In the present study, a total of 200 samples obtained from vaginal swabs, cervicovaginal mucous (CVM), preputial washes and semen straws were investigated that were obtained from organized cattle farm of MLRI, Manasbal and unorganized sectors. Out of a total of 200 samples, 49 (47·57%) vaginal swabs, 1 (3·33%) preputial wash and 8 (25%) carried out CVM samples were positive for Cfv, whereas none of the semen straws were positive for Cfv. A total of eleven isolates of Cfv were recovered. PFGE (Pulse field gel electrophoresis) analysis revealed four different pulsotypes (I–IV) circulating in the screened farms. A common pulsotype circulating among farms could not be established. Insertion element (ISCfe1), a 233 bp amplicon of Cfv, was sequenced and the sequence was deposited in GenBank (accession no: MK475662).