Implications of between-isolate variation for climate change impact modelling of Haemonchus contortus populations.

The impact of climate change on parasites and parasitic diseases is a growing concern and numerous empirical and mechanistic models have been developed to predict climate-driven spatial and temporal changes in the distribution of parasites and disease risk. Variation in parasite phenotype and life-history traits between isolates could undermine the application of such models at broad spatial scales. Seasonal variation in the transmission of the haematophagous gastrointestinal nematode Haemonchus contortus, one of the most pathogenic helminth species infecting sheep and goats worldwide, is primarily determined by the impact of environmental conditions on the free-living stages. To evaluate variability in the development success and mortality of the free-living stages of H. contortus and the impact of this variability on future climate impact modelling, three isolates of diverse origin were cultured at a range of temperatures between 15°C and 37°C to determine their development success compared with simulations using the GLOWORM-FL H. contortus model. No significant difference was observed in the developmental success of the three isolates of H. contortus tested, nor between isolates and model simulations. However, development success of all isolates at 37°C was lower than predicted by the model, suggesting the potential for overestimation of transmission risk at higher temperatures, such as those predicted under some scenarios of climate change. Recommendations are made for future climate impact modelling of gastrointestinal nematodes.

The occurrence and genetic characterization of Cryptosporidium and Giardia species in foals in Belgium, The Netherlands, Germany and Greece.

Faecal samples were collected from foals between the age of 1 week and 6 months in Belgium, The Netherlands, Germany and Greece. A quantitative direct immunofluorescence assay based on the commercial MERIFLUOR Cryptosporidium/Giardia kit was performed to evaluate the presence of (oo) cysts. Parasite positive samples were genotyped, based on the 18S ribosomal DNA gene and the heat shock protein (HSP70) gene for Cryptosporidium and on the ß-giardin gene and the triose phosphate isomerase (TPI) gene for Giardia. In total, 134 foals from Belgium, 44 foals from The Netherlands, 30 foals from Germany and 190 foals from Greece were examined. No Cryptosporidium oocysts were identified in faecal samples from foals in Germany and The Netherlands. In Belgium and Greece, 4.5% and 1.1% of the foals examined were Cryptosporidium positive, respectively, all with a low oocyst excretion ranging from 100 to 2450 oocysts per gram of faeces. For Giardia, 14.2%, 11.4%, 10.0% and 11.6% of the foals in Belgium, The Netherlands, Germany and Greece, respectively, were found to excrete cysts, with a range of 50 up to 4,000,000 cysts per gram of faeces. Younger animals secreted significantly more Giardia cysts than older horses (p<0.05), but no significant correlation between Giardia infection and diarrhoea was observed. Most Giardia positive samples belonged to assemblage AI and/or BIV, but also assemblage E was detected in two samples. Together with the identification of Cryptosporidium horse genotype, this suggests only a low risk for zoonotic transmission.

Practices to optimise gastrointestinal nematode control on sheep, goat and cattle farms in Europe using targeted (selective) treatments.

Due to the development of anthelmintic resistance, there have been calls for more sustainable nematode control practices. Two important concepts were introduced to study and promote the sustainable use of anthelmintics: targeted treatments (TT), where the whole flock/herd is treated based on knowledge of the risk, or parameters that quantify the severity of infection; and targeted selective treatments (TST), where only individual animals within the grazing group are treated. The aim of the TT and TST approaches is to effectively control nematode-induced production impacts while preserving anthelmintic efficacy by maintaining a pool of untreated parasites in refugia. Here, we provide an overview of recent studies that assess the use of TT/TST against gastrointestinal nematodes in ruminants and investigate the economic consequences, feasibility and knowledge gaps associated with TST. We conclude that TT/TST approaches are ready to be used and provide practical benefits today. However, a major shift in mentality will be required to make these approaches common practice in parasite control.

Real-time PCR detection for quantification of infection levels with Ostertagia ostertagi and Cooperia oncophora in cattle faeces.

A quantitative real-time polymerase chain reaction (qPCR) based on hydrolysis (TaqMan) probes is described for robust and sensitive detection of the infection levels with eggs and third stage larvae (L3) of Cooperia oncophora and Ostertagia ostertagi isolated from cattle faeces. The current microscopic method for identification of strongyle nematodes in cattle faeces is labour-intensive where reliable species determination also requires trained expertise, which is increasingly lacking. The goal of this study was to develop a sustainable non-labour intensive diagnostic qPCR assay to detect and determine the levels of infection with the two most common gastro-intestinal nematodes (GIN) in cattle faeces targeting the second internal transcribed spacer of nuclear ribosomal DNA (ITS2) region (rDNA). According to our results, this procedure allows to reliably detect the relative proportions of eggs and L3 for each of the two species. This assay produced consistent results when mixtures with known numbers of L3 of both species were tested, although it was also demonstrated that the calculated copy numbers of ITS2 between single L3 sometimes varied very much. In addition, a positive correlation (r(2)=0.23) between the proportion of eggs and L3 in different paired samples collect in the field was observed for both species. Thus, for the first time a qPCR assay is reported, which can discriminate between the two most important cattle nematode parasites in temperate regions. This is of major importance to the livestock sector as it can be used with great precision to demonstrate strategic treatment efficacy that is important for the detection of anthelmintic resistance (AR).

Gene expression analysis of ABC transporters in a resistant Cooperia oncophora isolate following in vivo and in vitro exposure to macrocyclic lactones.

Members of the ATP-binding cassette (ABC) transporter family (P-glycoproteins, Half-transporters and Multidrug Resistant Proteins) potentially play a role in the development of anthelmintic resistance. The aim of this study was to investigate the possible involvement of ABC transporters in anthelmintic resistance in the bovine parasite, Cooperia oncophora. Partial sequences of 15 members of the ABC transporter protein family were identified, by mining a transcriptome dataset combined with a degenerate PCR approach. Reverse transcriptase PCR showed that most of the ABC transporters identified were constitutively transcribed throughout the life cycle of C. oncophora. Constitutive differences in gene transcript levels between a susceptible and resistant isolate were only observed for Con-haf-9 and Con-mrp-1 in eggs of the resistant isolate, while no differences were observed in L3 or the adult life stage. Analysis of resistant adult worms, collected from calves 14 days after treatment with either ivermectin or moxidectin, showed a significant 3- to 5-fold increase in the transcript levels of Con-pgp-11 compared to non-exposed worms. Interestingly, a 4-fold transcriptional up-regulation of Con-pgp-11 was also observed in L3 of the resistant isolate, after in vitro exposure to different concentrations of ivermectin, whereas this effect was not observed in exposed L3 of the susceptible isolate. The results suggest that the worms of this particular resistant isolate have acquired the ability to up-regulate Con-pgp-11 upon exposure to macrocyclic lactones. Further work is needed to understand the genetic basis underpinning this process and the functional role of PGP-11.

Future consequences and challenges for dairy cow production systems arising from climate change in Central Europe - a review.

It is well documented that global warming is unequivocal. Dairy production systems are considered as important sources of greenhouse gas emissions; however, little is known about the sensitivity and vulnerability of these production systems themselves to climate warming. This review brings different aspects of dairy cow production in Central Europe into focus, with a holistic approach to emphasize potential future consequences and challenges arising from climate change. With the current understanding of the effects of climate change, it is expected that yield of forage per hectare will be influenced positively, whereas quality will mainly depend on water availability and soil characteristics. Thus, the botanical composition of future grassland should include species that are able to withstand the changing conditions (e.g. lucerne and bird's foot trefoil). Changes in nutrient concentration of forage plants, elevated heat loads and altered feeding patterns of animals may influence rumen physiology. Several promising nutritional strategies are available to lower potential negative impacts of climate change on dairy cow nutrition and performance. Adjustment of feeding and drinking regimes, diet composition and additive supplementation can contribute to the maintenance of adequate dairy cow nutrition and performance. Provision of adequate shade and cooling will reduce the direct effects of heat stress. As estimated genetic parameters are promising, heat stress tolerance as a functional trait may be included into breeding programmes. Indirect effects of global warming on the health and welfare of animals seem to be more complicated and thus are less predictable. As the epidemiology of certain gastrointestinal nematodes and liver fluke is favourably influenced by increased temperature and humidity, relations between climate change and disease dynamics should be followed closely. Under current conditions, climate change associated economic impacts are estimated to be neutral if some form of adaptation is integrated. Therefore, it is essential to establish and adopt mitigation strategies covering available tools from management, nutrition, health and plant and animal breeding to cope with the future consequences of climate change on dairy farming.

Advances in laboratory diagnosis of parasitic infections of sheep.

Parasitic infections constitute an important group of diseases in sheep concerning the health status, welfare and productivity. On a global scale, there are considerable differences concerning the epidemiological situation with respect of the various parasite species. However, there are also numerous species, which occur on all continents and, potentially, in every country. Accordingly, the present review aims to providing an overview about the recent developments in methods and technologies for the laboratory diagnosis of parasite infections in sheep. Following in principle a systematic order the review encompasses publications addressing the diagnosis of helminthes (i.e., trematodes, cestodes and nematodes) and arthropod species. New approaches using conventional (e.g., microscopic), immunological and molecular techniques are being considered. The diagnosis of anthelmintic resistance is highlighted separately, due to its significant importance. The review ends with an outlook into the future by discussing most recent technological advances, which might become of use for the diagnosis of parasite infections in sheep in the future.

Standardization of the larval migration inhibition test for the detection of resistance to ivermectin in gastro intestinal nematodes of ruminants.

Resistance to anthelmintics is an increasing problem in sheep, goat and cattle industries worldwide. For parasite management on farms reliable methods for the detection of resistance are required and it is important that tests give comparable, reproducible and reliable results in different laboratories. The ability of the larval migration inhibition test (LMIT), to detect ivermectin resistance in cattle and sheep nematodes has been evaluated through ring testing in up to six different laboratories in Europe, supported by an EU sixth Framework Project (PARASOL). Third stage larvae of Ostertagia ostertagi, Cooperia oncophora, and Haemonchus contortus with a known resistance status were obtained from faecal cultures of experimentally infected calves and sheep. Following a series of ring tests using identical protocols, reproducible results were obtained within and between participating laboratories. In all tests dose-response curves with R(2) values >0.90 were obtained by all laboratories. Resistance ratios of 8.3 and 8.4 were found when susceptible and IVM-resistant isolates of C. oncophora and H. contortus were compared and differences in the EC(50) values were highly significant (p<0.0001). Protocols for the LMIT and the preparation of ivermectin solutions are described in a supplementary file.

Adaptation and evaluation of three different in vitro tests for the detection of resistance to anthelmintics in gastro intestinal nematodes of cattle.

Three different in vitro methods, the Larval Development Test (LDT), the Larval Migration Inhibition Test (LMIT) and the Micromotility Meter Test (MMT) have been adapted to detect anthelmintic resistance in cattle nematodes. Nematode eggs and third stage larvae of different Ostertagia ostertagi and Cooperia oncophora isolates were obtained from faecal cultures of experimentally infected calves. Additionally, adult C. oncophora were evaluated in the MMT for the detection of resistance to ivermectin (IVM). For all three in vitro tests standard operating procedures (SOPs) were established and successfully used for the detection of responses of non-parasitic and parasitic stages to different anthelmintic substances and the description of dose-response curves. In the LDT ivermectin (IVM) and thiabendazole (TBZ) were tested, in the LMIT IVM and levamisole (LEV) and in the MMT only IVM was evaluated. Susceptible isolates of C. oncophora and O. ostertagi, an IVM-resistant isolate of C. oncophora and a TBZ-selected isolate of O. ostertagi were used in all (C. oncophora) or only some of these tests (O. ostertagi). For all isolates sigmoidal dose-response curves and EC(50) values for the tested substances were obtained using a four-parameter logistic model. For the LDT, the previously reported problem in development of larvae was successfully overcome with mean development rates between 80% and 87% in negative controls. Following optimization of incubation times, temperatures, mesh sizes (LMIT only), nutritive medium (LDT only) and group size (MMT only) all three test systems reliably detected significant differences in the response to IVM between the susceptible and IVM-resistant isolate of C. oncophora (p<0.0001), resulting in an resistance ratio (RR) value of approximately 5 for IVM and 2.8 for LEV in C. oncophora. The LDT also detected differences in the response to TBZ between the susceptible and BZ-selected O. ostertagi isolates (p<0.001) with an RR of 2 for TBZ. With the standardization of the described tests we report reproducible and reliable in vitro methods for the detection of resistance to IVM (LDT, LMIT and MMT) and TBZ (LDT) for cattle parasitic nematodes.

The use of a simplified faecal egg count reduction test for assessing anthelmintic efficacy on Belgian and German cattle farms.

Anthelmintic resistant cattle nematodes have been reported in different regions around the world. However, in Western Europe the assessment of the problem relies largely on case reports and no prevalence data based on wide-scale surveys are available. Therefore, we performed a survey to (1) screen for reduced anthelmintic efficacy in Belgian and German cattle farms; (2) evaluate the usefulness of a simplified faecal egg count reduction test (FECRT), where efficacies are based on the mean FECs of 10 at random collected faecal samples pre- and post-treatment per farm and (3) identify possible risk factors for reduced anthelmintic efficacy. Of 88 farms included in this study, 84 farms used macrocyclic lactones (MLs). A FECR <95% was observed on 39% of these 84 farms. However, using a Markov chain Monte Carlo simulation analysis, to correct for the used McMaster FEC technique with a detection limit of 50 epg, reduced efficacy could only be confirmed in 25% of the farms (21/84). Only Cooperia spp. were found in significant numbers in the coprocultures post-treatment. Reduced efficacy was significantly associated with farm type and with a lower efficacy in beef herds compared to dairy herds. Four farms were revisited and a standardized FECRT was performed to confirm anthelmintic resistance (AR). Surprisingly, macrocyclic lactone resistance against Cooperia oncophora was only confirmed in one of four farms. In conclusion, our results show that a reduced efficacy observed in a FECRT are not only caused by AR but that the detection limit of the FEC technique used and the (in)correct administration of the anthelmintic drugs are confounding factors of major importance.

Monitoring the efficacy of ivermectin and albendazole against gastro intestinal nematodes of cattle in Northern Europe.

Faecal egg count reduction tests (FECRT) using ivermectin (IVM) and benzimidazole (BZ) were conducted to investigate the prevalence of anthelmintic resistance in gastro-intestinal nematodes on cattle farms in Germany, Belgium and Sweden in 2006 and 2007. Based on sufficient numbers of eggs prior to the study, between 3 and 10 farms per country were selected. 10-15 animals were randomly selected per farm and subcutaneously treated with 0.2 mg IVM/kg bodyweight (Ivomec, Merial). Faecal samples were collected individually from every animal on day 0 (treatment), day 7 (Belgium & Sweden) or 14 (Germany), and day 21 (Germany, Belgium and Sweden). Faecal egg counts (FEC) were performed at each sampling occasion to estimate the eggs per gram of faeces (EPG) and the reduction of eggs after treatment. The FECRT using IVM in 2006 revealed mean reduction of egg counts between 69-100% on day 7/14 (95% confidence interval (CI) 19-102) and 35-96% (95% CI 0-102) on day 21. Farms with a suggested problem of anthelmintic resistance have been re-visited in 2007 and except for one case all results obtained in 2006 were confirmed in 2007. Larvae obtained from faecal cultures were identified using microscopic identification keys or genus-specific real time PCR. Cooperia oncophora was the predominant species detected after treatment, but Ostertagia ostertagi was found in samples on 3 farms in Germany and 3 farms in Sweden post-treatment. In 2007 additionally a FECRT using benzimidazoles was conducted in Germany and Sweden. In Germany oral Valbazen (albendazole, 10%, Pfizer) was used at a concentration of 7.5 mg albendazole/kg bodyweight; in Sweden Valbazen Vet (albendazole, 10%, Orion Pharma) at a dose of 8 mg/kg was used. For benzimidazoles an efficacy of 100% was obtained on all tested farms in both countries. This is the first report of a multinational anthelmintic efficacy investigation in cattle in Europe. The results suggest that testing of anthelmintic efficacy should be performed more intensively due to possible insufficient efficacy of current drugs.

Cases of reduced cyathostomin egg-reappearance period and failure of Parascaris equorum egg count reduction following ivermectin treatment as well as survey on pyrantel efficacy on German horse farms.

In 2003 and 2004, on a total of 63 different German horse farms, a survey using the faecal egg count reduction (FECR) test was performed to investigate the efficacy of ivermectin (IVM, Ivomec) and pyrantel (PYR, Banminth) treatment against gastro-intestinal nematodes in a total of 767 horses. IVM treatment resulted in 100% reduction of the cyathostomin egg production 14 and 21 days post-treatment (d.p.t.) on 37 farms. On the remaining five farms, the mean faecal egg count reduction ranged between 97.7 and 99.9%. The mean cyathostomin FECR following PYR treatment ranged between 92.2 and 100% on the 25 farms tested. Therefore, based on the 90% FECR threshold suggested for detection of anthelmintic resistance in horses, neither IVM nor PYR anthelmintic resistance was detected. However, if the thresholds recommended for the detection of resistance in small ruminants were applied, on one and four farms signs of reduced IVM and PYR efficacy, respectively, were observed. In 2005, to further investigate these findings, the cyathostomin egg-reappearance period (ERP) following IVM treatment was examined on six selected farms, two of which were found to show less than 99.8% FECR in the previous survey. On these two latter farms, the ERP was less than 5 weeks, while on the other four it was at least 8 weeks. Earlier investigations described IVM cyathostomin ERP of at least 9 weeks. The efficacy of IVM to reduce Parascaris equorum egg excretion was also studied. On one farm in 2 consecutive years, IVM treatment did not lead to a significant reduction in P. equorum faecal egg counts in one and five young horses, respectively.

Resistance as a tool for discovering and understanding targets in parasite neuromusculature.

The problem of anthelmintic resistance prevents efficient control of parasites of livestock and may soon compromise human parasite control. Research into the mechanisms of resistance and the quest for diagnostic tools to aid control has required research that focuses on field resistance. On the other hand, resistant worms, including those kept in the laboratory, provide useful tools for studying drug action, especially at neuromuscular targets in worms. While the needs and directions of these research aims overlap, this review concentrates on research on drug targets. In this context, resistance is a useful tool for site of action confirmation. For example, correlations between molecular expression studies and resistance assays conducted on whole worms can strengthen claims for sites of anthelmintic action. Model systems such as Caenorhabditis elegans have been very useful in understanding targets but give a limited picture as it is now clear that resistance mechanisms in this worm are different from those in parasites. Accordingly, research on parasites themselves must also be performed. Resistant isolates of the sheep nematode parasite Haemonchus contortus are the most widely used for this purpose as in vivo, in vitro, physiological and molecular studies can be performed with this species. Neuromuscular target sites for the anthelmintics levamisole and ivermectin are the best studied and have benefited most from the use of resistant worm isolates. Resistance to praziquantel and the newer chemical groups should provide new tools to explore targets in the future.