Initial assessment of the economic burden of major parasitic helminth infections to the ruminant livestock industry in Europe.

We report a European wide assessment of the economic burden of gastrointestinal nematodes, Fasciola hepatica (common liver fluke) and Dictyocaulus viviparus (bovine lungworm) infections to the ruminant livestock industry. The economic impact of these parasitic helminth infections was estimated by a deterministic spreadsheet model as a function of the proportion of the ruminant population exposed to grazing, the infection frequency and intensity, the effect of the infection on animal productivity and mortality and anthelmintic treatment costs. In addition, we estimated the costs of anthelmintic resistant nematode infections and collected information on public research budgets addressing helminth infections in ruminant livestock. The epidemiologic and economic input data were collected from international databases and via expert opinion of the Working Group members of the European Co-operation in Science and Technology (COST) action COMbatting Anthelmintic Resistance in ruminants (COMBAR). In order to reflect the effects of uncertainty in the input data, low and high cost estimates were obtained by varying uncertain input data arbitrarily in both directions by 20 %. The combined annual cost [low estimate-high estimate] of the three helminth infections in 18 participating countries was estimated at € 1.8 billion [€ 1.0-2.7 billion]. Eighty-one percent of this cost was due to lost production and 19 % was attributed to treatment costs. The cost of gastrointestinal nematode infections with resistance against macrocyclic lactones was estimated to be € 38 million [€ 11-87 million] annually. The annual estimated costs of helminth infections per sector were € 941 million [€ 488 - 1442 million] in dairy cattle, € 423 million [€ 205-663 million] in beef cattle, € 151million [€ 90-213 million] in dairy sheep, € 206 million [€ 132-248 million] in meat sheep and € 86 million [€ 67-107 million] in dairy goats. Important data gaps were present in all phases of the calculations which lead to large uncertainties around the estimates. Accessibility of more granular animal population datasets at EU level, deeper knowledge of the effects of infection on production, levels of infection and livestock grazing exposure across Europe would make the largest contribution to improved burden assessments. The known current public investment in research on helminth control was 0.15 % of the estimated annual costs for the considered parasitic diseases. Our data suggest that the costs of enzootic helminth infections which usually occur at high prevalence annually in ruminants, are similar or higher than reported costs of epizootic diseases. Our data can support decision making in research and policy to mitigate the negative impacts of helminth infections and anthelmintic resistance in Europe, and provide a baseline against which to measure future changes.

Alarming levels of anthelmintic resistance against gastrointestinal nematodes in sheep in the Netherlands.

In a survey involving 34 sheep flocks spread over the Netherlands anthelmintic resistance (AR), based on a fecal egg count reduction (FECR) test, was determined for six different products. The study was conducted in ewes shortly after lambing during spring 2015. A FECR of less than 90%, indicating presence of AR against one or more nematode genera producing strongylid eggs, was found in 22 of 30 (73.3%) flocks against oxfendazole, 18 of 23 (78.3%) flocks against ivermectin, 15 of 32 (46.9%) flocks against moxidectin, and 2 of 26 (7.7%) flocks against monepantel. No AR was observed against levamisole. If oxfendazole resistance was observed, Haemonchus contortus was involved in 90.5% of the cases. If resistance against ivermectin, moxidectin or monepantel was observed, it invariably involved H. contortus. In the majority of cases resistance was also observed for Teladorsagia circumcincta and/or Trichostrongylus spp, between which no distinction was made in this study. Based on FECR 9 of 15 (60.0%) flocks showed resistance against closantel, which was mainly due to closantel not being effective against most other nematode species than H. contortus. However, in 44.4% of flocks showing reduced FECR it did involve H. contortus as well. Multi-drug resistance (excluding closantel) was found in 16 flocks, of which 8 showed resistance against 2 products, 7 against 3 products and 1 flock showed resistance against 4 products. If resistance against 3 or 4 products was present, there invariably was resistance against both ivermectin and moxidectin. Overall, of the 22 flocks in which both macrocyclic lactones (ML) were tested, 4 (18.2%) showed no resistance against both products, 9 (40.9%) showed resistance against ivermectin only, and 9 (40.9%) showed resistance against both MLs. It is concluded that AR is widespread in sheep in the Netherlands and involves products from all major anthelmintic classes, with possibly the exception of levamisole. It appears that the macrocyclic lactones have lost much of their efficacy against sheep nematodes over the last decade.

Pitfalls and opportunities of teaching veterinary parasitology within an integrated curriculum.

The Faculty of Veterinary Medicine at Utrecht University has seen three major curriculum changes, in 1995, 2001 and 2007. The last change was made because of the European change to a Bachelor-Master system. Almost each time teaching hours tagged for veterinary parasitology have been reduced to currently a minimum of between 46 and 51.5 h, which is much less than the WAAVP-recommended minimum of 70-90 h. This results in a challenge to maintain a qualitatively adequate veterinary parasitology program in a curriculum. Following a brief historic account of previous curricula and implemented curriculum changes, experiences, limitations and opportunities are discussed, including the potential of introducing new teaching materials based, for example, on digital technologies and gaming.

Presence and species identity of rumen flukes in cattle and sheep in the Netherlands.

The purpose of the study was to gain knowledge about the prevalence and identity of rumen flukes (RF) in cattle and sheep in the Netherlands. Routine faecal examinations of diagnostic submissions between May 2009 and September 2014 showed a mean annual herd or flock RF prevalence of 15.8% for cattle and 8.0% for sheep. Prevalence in cattle was higher after 2012 than before, which may reflect a change in detection method as well as an increase in true prevalence. During November and December 2014, an abattoir survey was conducted to allow for scoring of rumen fluke burden and to obtain specimens for molecular species characterization. Over 8 visits to 5 abattoirs in areas deemed to pose a high risk for trematode infection, 116 cows and 41 sheep from 27 herds and 10 flocks were examined. Prevalence of RF was higher in beef cattle than in dairy cattle and higher in cattle than in sheep. Median fluke burden was >100 specimens per animal for most positive animals. Using a semi-quantitative RF density score as a gold standard, sensitivity and specificity of a modified quantitative Dorsman egg counting method were estimated at 82.6% and 83.3%, respectively. Of 14 collected adult rumen flukes, twelve (8 bovine and 4 ovine specimens) were identified as Calicophoron daubneyi. The other two, of bovine origin, were identified as Paramphistomum leydeni, which was unexpected as in other European countries all recently collected rumen flukes in both cattle and sheep were identified as C. daubneyi. The findings implicate that multiple rumen fluke species, intermediate host species and transmission cycles may play a role in rumen fluke infections in the Netherlands.

Perceptions and actions of Dutch sheep farmers concerning worm infections.

Gastrointestinal (GI) nematode infections are considered among one of the toughest challenges sheep farmers face worldwide. Control still is largely based on the use of anthelmintics, but anthelmintic resistance is becoming rampant. To facilitate implementation of alternative nematode control strategies and to reduce anthelmintic usage, the purpose of this study was twofold: (i) to gain insight in common practices, knowledge gaps and perceptions of farmers regarding nematode control, and (ii) to provide foci of attention for improving parasite control practices and transfer of knowledge within the sheep husbandry. An internet-based questionnaire was made available to all sheep farmers pertaining to the year 2013, resulting in 450 entered questionnaires for analysis. The two most important nematodes mentioned, were Haemonchus contortus and, to a lesser extent, Nematodirus battus. Of all respondents, 25.6% said they did not have any worm problems. Of these, almost a third did notice clinical signs that can be related to worm infections and about three quarters did use anthelmintics. Overall, clinical symptoms mentioned by farmers matched the worm species they identified as the cause of problems. Ewes and lambs were treated up to 6 times in 2013. On average, ewes were treated 1.53 and lambs 2.05 times. Farmers who treated their ewes more often, also treated their lambs more often (P<0.001). Both ewes and lambs were frequently treated based on fixed moments such as around lambing, at weaning and before mating, rather than based on faecal egg counts. Treatments based on faecal egg counts were practiced, but on a minority of the farms (32.7%). The majority of the farms (75.6%) did not leave 2-5% of the sheep within a flock untreated. About 74% of farmers keep newly purchased animals quarantined for at least 10days, but some (13.4%) leave quarantined animals untreated nor check faecal egg counts. Of farmers who do treat their quarantined animals, just 12.6% check the efficacy of the treatment. Slightly over 40% of the respondents said they did not experience bottlenecks in parasite control. Yet, over half of these said having problems with worm infections, over half did see clinical signs related to worm infections and over three quarters used anthelmintics. Within the group of farmers experiencing difficulties in parasite control, the most often mentioned bottleneck concerned pasture management (75.8%). When asking farmers for solutions, 90% of all respondents indicated they are willing to adjust their pasture management. Farmers are also interested in other methods to reduce the risk of worm infections, such as possibilities to enhance the immune system of sheep in general (71%), to increase specific genetic resistance to worms and to apply anti-parasite forages, both about 40%. Results of this study gave the following potential foci of attention: (1) making complex scientific knowledge more accessible to farmers through simple tools and applicable in the daily farming process; (2) changing the mindset of farmers about their current worm control practices, i.e. breaking long-standing habits such as treating ewes and lambs at fixed moments rather than based on actual worm infection monitoring data; (3) demonstrating effective pasture rotation schemes on specific farms and using these in extension work; (4) making farmers more aware that checking anthelmintic efficacy is important; (5) improving quarantine procedures; (6) creating a wider array of applicable alternative control measures from which individual farmers can choose what fits them most; and finally, (7) improving mutual understanding among farmers, veterinary practitioners and parasitologists alike.

Prevalence and risk factors for patent Toxocara infections in cats and cat owners' attitude towards deworming.

The prevalence of and risk factors for shedding Toxocara eggs in cats older than 6 months were determined by examining 670 faecal samples collected in 4 cross-sectional studies in the Netherlands. Additionally, cat owners provided information on their attitude towards routine deworming. Samples were examined using the centrifugal sedimentation flotation method. Overall Toxocara prevalence was 7.2 %. Multivariable logistic regression analysis revealed that young age and living in rural areas were significant risk factors for shedding Toxocara eggs. Moreover, the more time a cat was allowed to roam outdoors, the higher was its risk to shed Toxocara as compared to cats with no outdoor access at all. For 199 cats (81.6 % of cats subjected to a deworming regimen) owners provided the reason for treatment. The main reason for routine deworming (80.4 %) concerned the cat's health and only 10.6 % of the cats were treated for public health reasons. Moreover, the generally advocated four-times-a-year deworming advice was applied on only 24.5 % of cats. We concluded that free roaming is a key factor in the acquisition of patent Toxocara infections leading to the environmental contamination with Toxocara eggs. Additionally, the knowledge of cat owners is still insufficient to expect them to make sound decisions on routine deworming.

Species composition of larvae cultured after anthelmintic treatment indicates reduced moxidectin susceptibility of immature Cylicocyclus species in horses.

For the control of cyathostomins in horses, the macrocyclic lactones (MLs), moxidectin (MOX) and ivermectin (IVM) are the most commonly used anthelmintics. However, reduced activity, observed as shortening of the egg reappearance period (ERP) has been described. Shortening of the ERP may be caused by a decreased susceptibility of immature worms for MLs. Alternatively, immature worms may develop faster into egg producing adults as a result of repeated ML treatments. The species composition of the larval cultures obtained shortly after ML and pyrantel (PYR) treatment can confirm the hypothesis of decreased ML susceptibility, as this is often class-specific, whereas faster development would also occur after treatment with anthelmintics with a different mode of action. From 3 farms with a known history of shortened ERP, 8 horses per farm were selected and divided into 2 groups. The MOX-PYR-MOX group was treated twice with MOX (day 0 and 126) and once with PYR (day 84) and the IVM-PYR-IVM group was treated twice with IVM (day 0 and 98) and once with PYR (day 56). Cultured infective larvae (L3s) were counted and differentiated with the reverse line blot on pooled samples. Per cyathostomin species, the number of larvae per gram was calculated. The efficacy of all ML treatments was 100% and a shortened ERP was found on all 3 farms. The species composition of the larval cultures after ML treatment did not differ significantly from that after PYR treatment in the IVM-PYR-IVM group, but it did differ in the MOX-PYR-MOX group. The larval cultures obtained after MOX treatment consisted mostly of Cylicocyclus nassatus, while after PYR treatment Cylicostephanus longibursatus was the most abundant species. In the cultures from 42days after MOX treatment 6 cyathostomin species from 3 genera were found on the farm with the lowest activity (farm 1), while on the farm with the highest activity (farm 3) only 3 species from one genus were found in the same number of examined L3s. The high numbers of L3s of Cylicocyclus species 42days after MOX treatment and the low numbers 42days after PYR treatment can be explained by reduced susceptibility of the immature worms to MOX, but not by a faster development. In conclusion, shortening of the ERP following MOX treatment is most likely a process in which an increasing number of immature worms from an increasing number of species is becoming less susceptible to the active compound.

Toxocara canis in household dogs: prevalence, risk factors and owners' attitude towards deworming.

The prevalence of gastrointestinal parasites and risk factors for shedding of Toxocara eggs were determined for 916 Dutch household dogs older than 6 months. Additionally, the owners answered a questionnaire about their dogs and their attitude towards routine deworming was assessed. Faecal samples were examined using the centrifugal sedimentation flotation method. The overall prevalence of dogs shedding Toxocara eggs was 4.6 %. Multivariable logistic regression analysis revealed that the risk for 1-7-year-old dogs to shed Toxocara eggs was significantly lower (OR 0.38) than that of 6-12-month-old dogs. Compared to dogs walking ≤20 % of the time off-leash, those ranging freely 50-80 % and 80-100 % of the time had a significantly higher risk (OR 10.49 and 13.52, respectively) of shedding Toxocara eggs. Other risk factors were coprophagy (OR 2.44) and recently being kenneled (OR 2.76). Although the applied deworming frequency was not significantly associated with shedding Toxocara eggs, there was a trend towards no shedding in dogs under strict supervision that were dewormed 3-4 times a year. Most dog owners (68 %) recognized 'dog's health' as the main reason for deworming. Only 16 % of dogs were dewormed four times a year. It was concluded that the prevalence of Toxocara egg-shedding household dogs is almost unchanged over recent years and that the knowledge of owners is insufficient to expect sound decisions on routine deworming.

Cylicocyclus species predominate during shortened egg reappearance period in horses after treatment with ivermectin and moxidectin.

The normal time interval between treatment of horses with a macrocyclic lactone (ML) and reappearance of strongyle eggs in the feces, or 'Egg Reappearance Period (ERP)', is at its shortest 8 weeks for ivermectin (IVM) and 12 weeks for moxidectin (MOX). Nowadays, it is not uncommon to find shorter ERPs, potentially indicating the beginning of the development of drug resistance. Whether all cyathostomin species contribute equally to a shortened ERP is not known. In the present study a Reverse Line Blot (RLB) on individual infective larvae was used to compare species composition before and after ML-treatment in horses with either a normal or a shortened ERP. Species composition was determined for a total of 21 horses on 4 farms and was based on differentiation of approximately 40 larvae per horse per sampling day. After IVM treatment the shortest ERP was found to be 28-42 days. After MOX treatment the shortest ERP was found to be 42-56 days. The RLB showed that early egg shedding was dominated by species of the genus Cylicocyclus (Cyc.) (Cyc. ashworthi, Cyc. insigne, Cyc. leptostomum and Cyc. nassatus). Before treatment, species composition was generally more diverse with species from several other genera also present. Interestingly, on the farm with the overall shortest ERP and where all horses showed a shortened ERP, more species/genera were present during early egg excretion than on other farms. Results suggest that cyathostomin species within the genus Cylicocyclus contribute to a shortened ERP more than species from other genera. However, if shortening of ERP overall becomes more pronounced and widespread within a farm, species from other genera will also be present during early egg shedding.

Coprophagy in dogs interferes in the diagnosis of parasitic infections by faecal examination.

Many dogs display coprophagic behaviour. Helminth eggs can passively pass the dog's digestive tract and this may result in a false positive diagnosis of infection with gastrointestinal helminth parasites. For a period of one year, faecal samples of dogs were examined monthly using the Centrifugal Sedimentation Flotation (CSF) technique with a sugar flotation solution (s.g. 1.27-1.30 g/cm(3)). If a sample tested positive for canine helminth eggs, the owner was asked to submit another sample after preventing the dog from eating faeces for 3 days. If the second sample again tested positive for the same type of helminth egg, the dog was considered to have a patent infection. If the second sample tested negative, the first sample was considered a false positive due to coprophagy. The focus of this study was on dogs shedding Toxocara eggs. At the first examination, 246 samples (out of 308 samples testing positive for canine-specific helminth eggs) tested positive for Toxocara spp. Of these, 120 (49%) tested negative at the second examination. Coprophagic behaviour was recognized by 261 of the 564 owners that answered the accompanying questionnaire. This concerned 391 dogs. Coproscopical examination also provided proof of coprophagy (e.g. oocysts of Eimeria spp. or non-dog typical helminth eggs) in dogs belonging to owners that did not report coprophagic behaviour in their dogs. Results indicate that coprophagy in dogs may result in an overestimation of the prevalence of patent helminth infections and that dogs may serve as a transport host for helminth eggs.

Comparison of two serum and bulk-tank milk ELISAs for diagnosing natural (sub)clinical Dictyocaulus viviparus infection in dairy cows.

Lungworm antibody ELISAs developed in Germany (DE) and The Netherlands (NL) were compared using four sets of serum (S) and bulk-tank milk (BTM) samples from adult dairy cows. The samples originated from 37 farms with or without a suspected clinical lungworm infection during August-October 2010 (Dataset 1), from cows excreting lungworm larvae or not during August-October 2010 (n=59) or May-June 2011 (n=164) (Dataset 2), from 305 farms in a national survey during October 2010 (Dataset 3), and 14 zero-grazing farms during February-April 2011 (Dataset 4). During August-October 2010, covering the second half of the grazing season, the NL-S and NL-BTM ELISA outperformed the DE-S and DE-BTM ELISAs in terms of sensitivity. For at least the NL-S and DE-S ELISA the opposite was found during May-June 2011, covering the end of the winter housing period and the early grazing season. Of the 305 farms in the survey 62.6% were found positive with the NL-BTM ELISA, whereas 2.6% was found positive with the DE-BTM ELISA. ODR values for the zero-grazing farms indicated that a cut-off value of 30% for the DE-BTM ELISA might be more appropriate than the currently used 41%. Results suggest that the NL ELISAs also respond to lungworm antigens other than Major Sperm Protein as used in the DE ELISAs. It is concluded that the generally higher sensitivity of the NL-BTM ELISA makes it better suited for large-scale prevalence studies and herd health monitoring programmes than the DE-BTM ELISA, positivity of which is more associated with the presence of clinical lungworm infection. Reducing the cut-off value of the DE-BTM ELISA from the original 49.3% to the current 41% or the possibly more appropriate 30% increased its sensitivity for detecting lungworm infections, but did not lead to similar sensitivity estimates as found for the NL-BTM ELISA.

Searching for ivermectin resistance in Dutch horses.

A study was conducted to evaluate the occurrence of resistance against, in particular, ivermectin in cyathostomins in the Netherlands. Seventy horse farms were visited between October 2007 and November 2009. In initial screening, faecal samples were collected 2 weeks after deworming with either ivermectin, moxidectin or doramectin. Pooled faecal samples from a maximum of 10 horses were examined for worm eggs using a modified McMaster technique and for worm larvae after faecal larval cultures. In total 931 horses were involved. On 15 of 70 farms eggs and/or larvae were found. On 8 of these 15 farms a FECRT with ivermectin was performed on 43 horses. Efficacy of ivermectin against cyathostomins of 93% was found in one animal on one farm. Additionally, the strategies and efforts of the horse owners to control cyathostomins, as well as risk factors for the development of macrocyclic lactone resistance were evaluated with a questionnaire. Strikingly, many responders indicated that the control of cyathostomins in horses is achieved through very frequent deworming. Fourteen percent of these owners deworm seven times per year or more. On 34% of the 70 farms treatment was repeated within the Egg Reappearance Period of a product.

Failure to eradicate the lungworm Dictyocaulus viviparus on dairy farms by a single mass-treatment before turnout.

On two dairy farms it was attempted to eradicate lungworm, Dictyocaulus viviparus, by means of a single mass-treatment of all cattle that had been grazed the previous year(s), before turnout in the spring. Both farms experienced two years of lungworm outbreaks in the adult dairy herd prior to this study. Following confirmation that both herds contained lungworm carriers, all animals older than approximately 6 months were treated with eprinomectin in March 2007. One week after treatment none of the animals were shedding lungworm larvae. Subsequently, animals were pastured according to normal farm routine. From August to November all first-calving heifers were coprologically and serologically monitored for lungworm infection. During 2007 both farms remained lungworm-negative and did not report any clinical sign indicative for a lungworm infection. The following year, on one of the farms replacements grazing on cow pastures, started showing signs of parasitic bronchitis which was serologically confirmed. The other herd remained free of parasitic bronchitis until at least the fourth year after the mass treatment, although some coughing was noticed in 2008 among first-lactation heifers. It was concluded that a single mass-treatment before the grazing season may be useful to break a series of annual lungworm outbreaks. However, it is not a secure method to prevent parasitic bronchitis for more than one year.

The value of a bulk-tank milk ELISA and individual serological and faecal examination for diagnosing (sub)clinical Dictyocaulus viviparus infection in dairy cows.

To test the value of a recently developed bulk-tank milk (BTM) ELISA for diagnosing (sub)clinical Dictyocaulus viviparus infection in lactating dairy herds under field conditions, bulk milk samples were collected from farms with or without clinical symptoms suspected to be caused by lungworm infection. Results of the BTM ELISA were compared against individual examinations for lungworm larvae in faeces and lungworm antibodies in serum from up to 20 heifers (parity 1) and up to 20 cows (parity ≥ 2) on the same farms. This also allowed, for the first time, to examine the value of individual faecal and serological examinations in the diagnosis of (sub)clinical lungworm infections. In total, 33 farms participated. Of these, 16 reported clinical symptoms possibly related to lungworm infection (defined as a suspected positive clinical status or CS(+)) and 17 reported having no such symptoms (CS(-)). In total, 503 heifers and 649 cows were sampled. Of all faeces samples positive for lungworm larvae, 94 were from heifers (18.9% of all heifers) and 75 from cows (11.7% of all cows) (P<0.001). Of all sera positive for lungworm antibodies, 130 were from heifers (26.1% of all heifers) and 113 from cows (17.5% of all cows) (P<0.001). Of the CS(-) farms 41% had at least one heifer or cow shedding larvae and 71% had at least one seropositive heifer or cow. Of the CS(+) farms this was 81% and 94%, respectively. There were only 4 farms, all CS(-), where none of the animals were found shedding larvae and all animals tested seronegative. This implies that on 76% of the CS(-) farms lungworm infection circulated unnoticed. On all CS(+) farms the suspicion that lungworm caused the respiratory symptoms was confirmed by the individual faecal and serological examinations, whereas the BTM ELISA confirmed presence of lungworm on half of the CS(+) farms. The latter in particular occurred on farms with the more severe outbreaks. Overall, of 32 available BTM samples 10 tested positive (8 of 15 CS(+) and 2 of 17 CS(-) farms). For diagnosing suspected lungworm disease it was concluded that testing a BTM sample might suffice in case of moderate to severe outbreaks. However, in case of a mild outbreak with just a few animals coughing, examining individual animals has to be preferred over testing a BTM sample. The likelihood to detect lungworm infection is higher if heifers are sampled compared to cows. Sensitivity of the BTM ELISA was 35.7% if the presence of at least one seropositive and/or one larvae shedding animal in the herd was used to define lungworm positive farms. On average, at least 30% of the herd had to be seropositive before the BTM ELISA was found positive for lungworm antibodies. Results indicate that the BTM ELISA in its current form does not appear to be suitable for surveys on the prevalence of lungworm presence on farms. However, this BTM ELISA might be used in large-scale surveys to detect, for instance, annual changes in percentage positive farms, as long as it is recognized that positivity is more closely related to incidence of lungworm disease than to prevalence of lungworm infection.

Lungworm outbreaks in adult dairy cows: estimating economic losses and lessons to be learned.

Two lungworm outbreaks in dairy herds were investigated in order to estimate the resulting economic costs. On the two farms, with 110 and 95 cows, total costs were estimated at €159 and €167 per cow, respectively. Overall, milk production reduced by 15 to 20 per cent during the outbreaks. Five cows died on one farm, while on the other farm seven cows died as a result of the lungworm outbreak. On one farm, 51.7 per cent of the total costs was due to reduced milk production and 33.1 per cent was due to disposal of dead animals. On the other farm, it was 36.3 and 50.9 per cent, respectively. The remaining 13 to 15 per cent of the total costs were due to extra inseminations, laboratory diagnosis and treatments. The history and development of the outbreaks are described. One lesson from these outbreaks is that recognising that potentially lungworm-naïve animals are to be introduced into the adult herd allows for timely measures (for example, vaccination) to prevent a lungworm outbreak.

In vitro selection and differentiation of ivermectin resistant cyathostomin larvae.

Cyathostomins are considered to be the primary helminth pathogen of horses and macrocyclic lactones (ML) are the most frequently used anthelmintics. Therefore, ML resistance is a serious threat for the control of these parasites. In the present study ivermectin resistant cyathostomin L3 were in vitro selected, using a reiterative larval migration inhibition assay (rLMIA) and differentiated by reverse line blot (RLB). Larvae were obtained from two populations, one from a never treated, free-roaming horse population in the nature reserve Oostvaardersplassen (OVP) and the other from regularly ivermectin-treated ponies of Utrecht University (UU). In the rLMIA the proportion of larvae that migrated increased with each passage, demonstrating that the applied procedure indeed selects for larvae the least susceptible for ivermectin. This was further supported by the fact that glutamate addition to this procedure reversed the selection effect, which also suggests that glutamate-gated chloride channels (GluCls) play a role in the ivermectin resistance of the selected L3. In both populations the predominant species were Cyathostomum catinatum, Cylicostephanus longibursatus and Cylicocyclus nassatus. After in vitro selection in the rLMIA in the presence of ivermectin the predominant species became C. catinatum in both larval populations, while C. nassatus disappeared in the never treated OVP larval population but not in the regularly ivermectin-exposed UU population. It is concluded that the rLMIA and RLB can be used to study anthelmintic resistance in cyathostomin populations and to study differences and changes in species composition between populations with different anthelmintic exposure histories.