Georg von Samson-Himmelstjerna

P-glycoprotein in helminths: function and perspectives for anthelmintic treatment and reversal of resistance

Infestation with parasitic helminths is a common problem in human populations of third world countries and is ubiquitous in livestock and other domestic animals. The cell-membrane efflux pump, P-glycoprotein (Pgp), appears to contribute to anthelmintic resistance. Pgp have been identified from both phyla of parasitic helminths, Platyhelmintha and Nematoda, and alterations in expression levels and allele frequencies of Pgp in anthelmintic-resistant populations have been observed in nematodes. Localisation of Pgp has been studied in the free-living nematode Caenorhabditis elegans and in the sheep parasite Haemonchus contortus using specific monoclonal antibodies or lectins. Reversing agents used in human studies, such as the calcium-channel blocker verapamil (VPL), appear to have similar effects in helminths as they do in human cancer cells: the efficacy of drug treatment is increased in drug-resistant parasites when reversing agents are co-administered with the anthelmintic. The functional role of the Pgp glycosylation was also studied using a lectin specific for the alpha-mannosyl residues and showed that resistance can be associated with a decreased affinity of the lectin for Pgp sites and that up to 50% reversion in the resistance to benzimidazoles (BZ) can be obtained using this lectin. Furthermore, the current knowledge on the role of Pgp in molecular mechanisms of drug resistance in the parasitic protozoan genus Trypanosoma is discussed. In some Trypanosoma species it was shown that drug resistance was associated with reduced uptake and in other ones with increased efflux. Several trypanosome Pgp-coding sequences have been described. In contrast to earlier data, most recent observations, based on experimentally overexpressed Pgp in Trypanosoma brucei, indicate a possible involvement in the mechanism of drug resistance in this parasite.

Gastrointestinal nematode infections in adult dairy cattle: impact on production, diagnosis and control.

Due to the intensification of dairy herds and the recognition of subclinical infections with a negative impact on production as disease, control of gastrointestinal (GI) nematodes in adult cows is becoming established in an increasing number of dairy herds. The objectives of this paper are to review the aspects related to the impact on production, diagnosis and control of GI nematodes in adult dairy cattle. During the last decade substantial evidence has been generated that GI nematodes can have a negative impact on the performance of adult animals. The milk-yield response to anthelmintic treatment in recent studies in pastured dairy herds was observed to be around 1kg/cow per day, whereas effects on reproductive performance remain equivocal. GI-nematode infections can be monitored based on Ostertagia ostertagi-specific antibody measurement, which provides information on the level of larval exposure and an indication of the associated production losses. Other diagnostic parameters are considered of limited use in adult cattle. Control relies on anthelmintic treatment and grazing management, which can be used complementary to each other. There are three critical points that need to be considered when developing anthelmintic control recommendations in adult cows: the unpredictability of the treatment response, the timing of treatment and the risk for developing anthelmintic resistance. As a consequence, monitoring of GI-nematode infections is desirable in order to focus anthelmintic treatments on those herds with a high larval challenge and associated production losses. For the future, more studies are needed to evaluate the effects of different control approaches in terms of financial benefits for the farmer and sustainability on the long term.

Anthelmintic resistance in cyathostomin populations from horse yards in Italy, United Kingdom and Germany.

BackgroundA large survey was carried out in 2008 in Europe to evaluate the efficacy of fenbendazole (FBZ), pyrantel (PYR), ivermectin (IVM) and moxidectin (MOX), i.e. the major anthelmintic molecules used in current practice against cyathostomins affecting horses. A total of 102 yards and 1704 horses was studied in three countries: 60 yards and 988 horses from Italy, 22 and 396 from the UK, 20 and 320 from Germany. The survey consisted of Faecal Egg Count Reduction Tests (FECRTs) with a faecal egg count reduction (FECR) categorization of (I) resistance present if FECR <90% and the lower 95% confidence limit (LCL) <90%, (II) resistance suspected if FECR ≥ 90% and/or LCL <90% and (III) no resistance if FECR ≥ 90% and LCL >90%. The calculation of FECR data was performed employing bootstrap analysis of group arithmetic means.ResultsThe testing of FBZ on a total of 80 yards resulted in resistance present on more than 80% of the UK and German yards and on significantly fewer in Italy, i.e. in 38% (p < 0.01). PYR, IVM and MOX were tested on a total of 102 yards. For PYR resistance present was found in 25% of the yards with no significant differences between countries. For IVM resistance present was encountered in one Italian and two UK yards (3%), resistance present to MOX was not found in any yard in any country.ConclusionThe results indicate that single and/or multiple drug resistance in equine cyathostomins is present in the three countries, is widespread particularly for FBZ and/or PYR and in one UK yard multiple resistance present was detected to FBZ, PYR and IVM. Macrocylic lactones proved to be the most effective drugs, with some evidence of resistance to IVM and highest activity of MOX, despite a single case of reduced efficacy in Germany. These data call for the development and implementation, among practitioners, owners and managers, of further plans to reduce the expansion of the anthelmintic resistant populations and to use those anthelmintics that remain effective in a manner that preserves their efficacy as long as possible.

Recent advances in candidate-gene and whole-genome approaches to the discovery of anthelmintic resistance markers and the description of drug/receptor interactions

Graphical abstract

Molecular epidemiology of Cryptosporidium in livestock animals and humans in the Ismailia province of Egypt.

The zoonotic potential of Cryptosporidium was studied in one of the most densely populated provinces of Egypt regarding livestock and people. In a representative survey, faecal samples from cattle, buffalo and stool samples from diarrhoeic children (<10 years) were investigated. Parameters assumed to be related to cryptosporidiosis were recorded for animals and children. Animal samples (804) were examined by the Copro-antigen RIDA(®)QUICK test, followed by PCRs targeting the 18S rDNA and gp60 genes for antigen-positive and 10% randomly selected negative samples. All 165 human samples were tested by both methods. The overall estimated prevalence of Cryptosporidium in ruminants was 32.2%, without significant difference between animal species. PCR identified 65.7% Cryptosporidium parvum, 11.8% Cryptosporidium ryanae, 4.1% Cryptosporidium bovis, and combinations of C. parvum plus C. ryanae (11.2%), C. parvum plus C. bovis (5.3%) and of C. parvum plus Cryptosporidium andersoni (1.8%), also without significant differences in species occurrence between cattle and buffalos. The human Cryptosporidium spp. prevalence was 49.1%, of which 60.5% were Cryptosporidium hominis, 38.2% C. parvum and 1.2% C. parvum plus C. bovis. Analysis of gp60 variants allocated C. parvum found in animals to the zoonotic subtype family IIa (18.9%, subtype IIaA15G1R1 only) and to IId (81.1%, mostly IIdA20G1). In humans 50% were classified as subtype family IIa (IIaA15G1R1 and IIaA15G2R1) and 50% were IIdA20G1. C. andersoni occurred only in cattle older than 1 year. In contrast, mono-infections with one of the three single Cryptosporidium species and the three combinations with C. parvum were more prevalent in cattle and buffaloes younger than 1 year, particularly in those younger than 3 months, and were predominantly subtype family IId. In human samples no Cryptosporidium were identified in children younger than 7 months. Neither place of residence nor the source of drinking-water had measurable effects on prevalence. Remarkably, however, all children with C. parvum subtype family IIa and 86% with subtype family IId had contact to animals. High prevalence and identical genotypes of C. parvum in animals and humans indicate zoonotic transmission due to contact with animals, involving IIdA20G1 as the most frequent subtype.

Standardization of the egg hatch test for the detection of benzimidazole resistance in parasitic nematodes.

The ability to reliably detect anthelmintic resistance is a crucial part of resistance management. If data between countries are to be compared, the same test should give the same results in each laboratory. As the egg hatch test for benzimidazole resistance is used for both research and surveys, the ability of different laboratories to obtain similar results was studied through testing of known isolates of cyathostomins, Haemonchus contortus, Ostertagia ostertagi, and Cooperia oncophora in programs supported by the EU (Cost B16 and FP6-PARASOL). Initial results showed difficulties in obtaining reproducible and similar data within and between laboratories. A series of ring tests, i.e., simultaneous and coordinated rounds of testing of nematode isolates in different laboratories was subsequently performed. By adopting identical protocols, especially the use of deionized water and making dilutions of thiabendazole in dimethyl sulfoxide in the final ring test, laboratories correctly identified both susceptible and resistant isolates. The protocols for the test and preparation of solutions of thiabendazole are described.

Latrophilin-like receptor from the parasitic nematode Haemonchus contortus as target for the anthelmintic depsipeptide PF1022A.

PF1022A belongs to a new class of cyclodepsipeptides with broad anthelmintic activity. Here, we describe a novel target for PF1022A. Using PF1022A ligand immunoscreening of a cDNA library constructed from the parasitic nematode Haemonchus contortus, we identified a 3539 bp cDNA encoding a novel orphan heptahelical transmembrane 110 kDa‐receptor, termed HC110‐ R, similar to the mammalian G‐protein coupled receptor latrophilin. HC110‐R is localized at plasma membranes and in intracellular vesicles of HC110‐R‐transfected HEK‐293 cells. The ligand of latrophilin, a‐latrotoxin (LTX), binds to the extracellular N‐terminal region of HC110‐ R and induces influx of external Ca2+ through Cd2+‐ and nifedipine‐blockable Ca2+ channels. PF1022A also binds to the N‐terminus of HC110‐R and acts as an antagonist to LTX signaling in HC110‐R transfected HEK‐293 cells.

Cyclooctadepsipeptides – a new class of anthelmintically active compounds

Nematode infections are a major cause of human morbidity and loss of disability-adjusted life-years, both in the tropics and in temperate climates (Bundy and Silva 1998). It is suggested that helminth infections in general impair the immune response of the host to HIV and tuberculosis by its permanent stimulation in the direction of the TH2 response and may thus be responsible for the spread of these diseases mainly across Africa and Southeast Asia (Bentwich et al. 1999). In animal health, nematode infections play a crucial role, mainly in cattle and sheep, leading to enormous economic losses (Waller 1997). In addition, companion animals are affected by nematodes. Moreover, some nematode infections of companion animals can afflict man as a zoonosis. For example, Toxocara canis larvae may lead to the so-called toxocariasis in man, which may affect the brain and the eyes (Mehlhorn 2001). Since the early 1960s, three classes of broad-spectrum anthelmintics have been available. These have been described in detail repeatedly by many authors. A recent overview is given by Harder (2001). In short, thiabendazole was the first benzimidazole introduced into the market in 1963. Since then, a wide variety of other benzimidazoles, benzimidazole carbamates and prebenzimidazoles have been marketed for the control of nematode infections in ruminants, swine, other food animals, horses, dogs, cats and humans. Thiabendazole, mebendazole and albendazole are widely used in human medicine. The second class of anthelmintics are the nicotinic acetylcholine receptor agonists. Their action is directed against this target in nematodes. Members of this class are the imidazothiazole levamisole and the tetrahydropyrimidines pyrantel, morantel and oxantel. Only levamisole, pyrantel and oxantel are used in human medicine. Since the middle of the 1970s, the new class of macrocyclic lactones has revolutionized the animal health market (Meinke 2001). They can be divided into two subclasses: the milbemycins and the avermectins. The common feature of the macrocyclic lactones is their simultaneous activity against nematodes and ectoparasites, leading to the term endectocides. To these belong ivermectin, abamectin, eprinomectin, doramectin, milbemycin, moxidectin and selamectin. Macrocyclic lactones are predominantly used in the livestock industry. However, ivermectin and moxidectin are also recommended for the prevention of dirofilariasis in dogs and cats. Selamectin, the most recent macrocyclic lactone introduced into the market, is registered as a companion animal endectocide for the control of intestinal nematodes, ticks and fleas. In human medicine, until now only ivermectin has been used in the control programs for onchocercosis and Wuchereria bancrofti filariasis. There are also first experiences of ivermectin for the treatment of scabies and head lice infections (Wolff and Kock 1998). In the livestock industry, the situation for anthelmintic treatment was dramatically turned worse by the emergence of resistance against all commonly used anthelmintics (Table 1; Sangster 2001). Thus, resistance against benzimidazoles is seen in four trichostrongyle species in cattle, sheep and goats and in cyathostomes in horses. In some parts of the world, the situation is now so severe that benzimidazoles have no efficacy in the treatment of nematode infections in sheep, goats and horses. Resistance against the levamisole, pyrantel and morantel group is reported in three nematode species in sheep and goats, in two nematode species in cattle and again in cyathostomes in horses. Finally, Parasitol Res (2002) 88: 481–488 DOI 10.1007/s00436-002-0619-2

Anthelmintic resistance to ivermectin and moxidectin in gastrointestinal nematodes of cattle in Europe.

Anthelmintic resistance has been increasingly reported in cattle worldwide over the last decade, although reports from Europe are more limited. The objective of the present study was to evaluate the efficacy of injectable formulations of ivermectin and moxidectin at 0.2 mg per kg bodyweight against naturally acquired gastro-intestinal nematodes in cattle. A total of 753 animals on 40 farms were enrolled in Germany (12 farms), the UK (10 farms), Italy (10 farms), and France (8 farms). Animals were selected based on pre-treatment faecal egg counts and were allocated to one of the two treatment groups. Each treatment group consisted of between 7 and 10 animals. A post-treatment faecal egg count was performed 14 days (±2 days) after treatment. The observed percentage reduction was calculated for each treatment group based on the arithmetic mean faecal egg count before and after treatment. The resistance status was evaluated based on the reduction in arithmetic mean faecal egg count and both the lower and upper 95% confidence limits. A decreased efficacy was observed in half or more of the farms in Germany, France and the UK. For moxidectin, resistance was confirmed on 3 farms in France, and on 1 farm in Germany and the UK. For ivermectin, resistance was confirmed on 3 farms in the UK, and on 1 farm in Germany and France. The remaining farms with decreased efficacy were classified as having an inconclusive resistance status based on the available data. After treatment Cooperia spp. larvae were most frequently identified, though Ostertagia ostertagi was also found, in particular within the UK and Germany. The present study reports lower than expected efficacy for ivermectin and moxidectin (based on the reduction in egg excretion after treatment) on European cattle farms, with confirmed anthelmintic resistance on 12.5% of the farms.

Anthelmintic resistance in equine parasites – detection, potential clinical relevance and implications for control

During the past two decades anthelmintic resistance in equine parasites has been found in the group of small strongyle species (cyathostomins) and in the ascarid species Parascaris equorum. The ubiquitous nature and possible severe consequences of disease with these nematodes make them the prime targets of current worm control programmes. Traditional control strategies mainly rely on the strategic application of anthelmintics, currently represented by three major drug classes: benzimidazoles (BZ), the tetrahydropyrimidine pyrantel (PYR) and macrocyclic lactones (ML). Following decades of routine and frequent anthelmintic applications, many cyathostomin populations on horse farms in industrialised countries must be considered as resistant to BZ anthelmintics. However, to date no published cases of cyathostomin disease specifically associated with anthelmintic resistance were reported. Possibly this is due to the generally subclinical and unspecific symptoms associated with cyathostomin infections. Nevertheless, exclusive reliance on the ML drug class may increase the threat of clinical disease due to drug-resistant cyathostomins. More recently, P. equorum has been reported as having developed resistance against ivermectin and moxidectin, two representatives of the ML-class. These anthelmintics are currently the most frequently used drug class in horses. This nematode species is mainly found in foals and in younger horses due to the development of immunity following exposure to infection. Infection with P. equorum can result in clinically drastic consequences such as obstruction and/or penetration of the small intestine, the latter usually leading to death. In conclusion, on horse farms the efficacy of anthelmintic treatments should be examined routinely for each drug class. Several factors can influence the rate at which anthelmintic resistance develops; high frequency of treatment being one of the most important. Modern control strategies should therefore attempt to significantly reduce anthelmintic treatments. Several pasture and farm management practices found to be negatively associated with nematode and anthelmintic resistance prevalence will be discussed in the review presented here.