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.

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.

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.

Practical aspects of equine parasite control: a review based upon a workshop discussion consensus.

Development of resistance of several important equine parasites to most of the available anthelmintic drug classes has led to a reconsideration of parasite control strategies in many equine establishments. Routine prophylactic treatments based on simple calendar-based schemes are no longer reliable and veterinary equine clinicians are increasingly seeking advice and guidance on more sustainable approaches to equine parasite control. Most techniques for the detection of equine helminth parasites are based on faecal analysis and very few tests have been developed as diagnostic tests for resistance. Recently, some molecular and in vitro based diagnostic assays have been developed and have shown promise, but none of these are currently available for veterinary practice. Presently, the only reliable method for the detection of anthelmintic resistance is a simple faecal egg count reduction test, and clinicians are urged to perform such tests on a regular basis. The key to managing anthelmintic resistance is maintaining parasite refugia and this concept is discussed in relation to treatment strategies, drug rotations and pasture management. It is concluded that treatment strategies need to change and more reliance should now be placed on surveillance of parasite burdens and regular drug efficacy tests are also recommended to ensure continuing drug efficacy. The present review is based upon discussions held at an equine parasite workshop arranged by the French Equine Veterinary Association (Association Vétérinaire Equine Française, AVEF) in Reims, France, in October 2008.

Natural postnatal Neospora caninum infection in cattle can persist and lead to endogenous transplacental infection.

A serological follow-up study of 3.5 years duration was done of a dairy herd that had experienced a mass seroconversion to Neospora caninum following a point source exposure shortly before the 17th of January 2000. A total of 913 blood samples of 244 animals at seven sampling dates were used to investigate the seroprevalence dynamics in the herd. Most postnatally infected cattle remained seropositive during the period of investigation but 11 animals became seronegative after 6-27 months indicating transient infection. Six animals seroconverted later than the main group of 45 animals and 5 animals became seronegative after at least two seropositive records possibly due to a low infection dose or difference in the haplotypes of the infected animals. In total 58% (14/24) of the offspring of postnatally infected dams was seropositive. Nine of 16 (56%) daughters originating from inseminations after the postnatal infection of their dams were seropositive indicating endogenous transplacental infection.

The use of age-clustered pooled faecal samples for monitoring worm control in horses.

A study was performed on two horse farms to evaluate the use of age-clustered pooled faecal samples for monitoring worm control in horses. In total 109 horses, 57 on farm A and 52 on farm B, were monitored at weekly intervals between 6 and 14 weeks after ivermectin treatment. This was performed through pooled faecal samples of pools of up to 10 horses of the groups 'yearlings' (both farms), '2-year-old' (two pools in farm A), '3-year-old' (farm A) and adult horses (four pools on farm A and five pools on farm B), which were compared with the mean individual faecal egg counts of the same pools. A very high correlation between the faecal egg counts in pooled samples and the mean faecal egg counts was seen and also between the faecal egg counts in pooled samples and larval counts from pooled faecal larval cultures. Faecal egg counts increased more rapidly in yearlings and 2-year-old horses than in older horses. This implied that in these groups of young animals faecal egg counts of more than 200 EPG were reached at or just after the egg reappearance period (ERP) of 8 weeks that is usually indicated for ivermectin. This probably means that, certainly under intensive conditions, repeated treatment at this ERP is warranted in these young animals, with or without monitoring through faecal examination. A different situation is seen in adult animals. Based on the mean faecal egg counts on both farms and on the results of pooled samples in farm A, using 100 EPG as threshold, no justification for treatment was seen throughout the experimental period. However, on farm B values of 100 EPG were seen at 9 and 11, 13 and 14 and 14 weeks after ivermectin treatment in pools 10, 12 and 13, respectively. This coincided with the presence of one or two horses with egg counts above 200 EPG. The conclusion is that random pooled faecal samples of 10 adult horses from a larger herd, starting at the ERP and repeating it at, for instance, 4-week intervals, could be used for decisions on worm control. However, there would be a certain risk for underestimating pasture contamination through missing high-egg excreters. An alternative use of pooled samples would be as a cheap first screening to detect which adult horses really contribute to pasture contamination with worm eggs on a farm. All horses should be sampled and subsequently animals from 'positive' pools can be reexamined individually.

[Frequent deworming in horses; it usually does not do any good, but it often harms]. / Vaak ontwormen bij paarden; het baat meestal niet maar het schaadt vaak wel.

Due to excessive and inappropriate use of dewormers anthelmintic resistance has developed as a significant problem in horse parasites in the Netherlands. Since it is unlikely that new classes of anthelmintics against horse nematodes will be introduced in the near future, it is important to use the present drugs wisely. Veterinarians should advice horse owners about worm control programs with a more targeted approach. The number of anthelmintic treatments should be reduced and, through selective anthelmintic treatments, further development of anthelmintic resistance should be delayed. Preferably, horses with a low faecal egg count should not be treated at all to ascertain a reduction of the selection pressure for anthelmintic resistance. The propensity for low faecal egg counts is hereditary. This implies that mature horses with consistent low egg counts can be detected by faecal examination and that it is not necessary to repeat faecal examination each time in these animals. New horses on the farm should always be dewormed on arrival and should be introduced only after the efficacy of treatment has been determined. Anthelmintic resistance can also be introduced with the arrival of a new animal that is infected with drug-resistant parasites.

The impact of daily Duddingtonia flagrans application to lactating ewes on gastrointestinal nematodes infections in their lambs in the Netherlands.

Two experiments were performed in 2002 and 2003 to evaluate the effect of biological control of gastrointestinal nematodes in sheep through the daily feeding of 500,000 chlamydospores of Duddingtonia flagrans/kg bodyweight to lactating ewes during the first 9 weeks with their young lambs on pasture. In both experiments four groups of eight ewes and their April-borne lambs were used. They were turned out on four separate plots (plots A) at the beginning of May, moved to similar separate plots after 3 (plots B) and 6 weeks (plots C), respectively, and weaning occurred after 9 weeks. In both experiments, two groups were fed spores daily while the two other groups served as controls. The effect of D. flagrans application was evaluated through faecal egg counts of ewes and lambs, the yield of faecal cultures in ewes, pasture larval counts and worm counts of lambs and tracer lambs. The results demonstrated no effect of D. flagrans application during the first 5 (2002) or 4 (2003) weeks. Subsequently, fungus application strongly reduced the yield in faecal cultures of the ewes. This was, however, not reflected in the pasture larval counts, but lower worm burdens were observed in tracer lambs of 'treated' plots C in 2002 than on those of 'control' plots. In 2003 worm burdens in 'treated' lambs returned to plots B were lower than those of 'control' lambs and a tendency for the same was observed for plots C. However, in all groups, lambs and tracer lambs developed severe haemonchosis.

[Resistance of Teladorsagia circumcincta in goats to ivermectin in the Netherlands]. / Ivermectineresistentie bij teladorsagia circumcincta bij geiten in Nederland.

Resistance of Teladorsagia circumcincta in goats to ivermectin In 2005, suspected ivermectin resistance of gastrointestinal nematodes was investigated in 17 goats on a farm in the Netherlands, using a faecal egg count reduction test combined with faecal larval cultures. Eight goats were treated orally with 0.2 mg/ kg ivermectin on 1 August 2005 and the other nine goats served as untreated controls. Faeces were collected from all goats on 1 August and on 12 August. Faecal egg counts were reduced by 80.6% in goats receiving ivermectin. On the basis of faecal larval cultures and the egg counts, ivermectin was found to be 100% effective against Haemonchus contortus, 15.0% effective against Teladorsagia circumcincta, and 93.3% effective against Trichostrongylus spp. The effect of levamisole and oxfendazole on gastrointestinal nematodes was also evaluated. Nine goats were treated subcutaneously with 5 mg/kg levamisole and seven goats orally with 4.5 mg/kg oxfendazole on 12 September On the basis of faecal egg counts performed on 12 and 22 September, levamisole reduced the total egg count by 95.4% and oxfendazole reduced the egg count by 85.8%. Differentiation of pooled faecal larval cultures revealed that levamisole was 99.9% effective against H. contortus, 81.9% effective against T. circumcincta, and 99.5% against Trichostrongylus spp. Oxfendazole was 75.5% effective against H. contortus, 33.2% effective against T. circumcincta, and 100% effective against Trichostrongylus spp. On 28 October the nine goats that were still present on the farm were orally treated with 0.4 mg/kg ivermectin. Faecal egg counts and differentiation of pooled larval cultures of faecal samples collected on 28 October and 9 November, respectively, revealed that total egg counts were reduced by 93.1%. Ivermectin was 49.1% effective against T. circumcincta but 100% effective against H. contortus and Trichostrongylus spp. Thus H. contortus appears to be resistant to oxfendazole and T. circumcinta appears to be resistant to oxfendazole and ivermectin. This is the first documented case of ivermectin resistance of a gastrointestinal nematode of small ruminants in the Netherlands.

Effect of biological control through the daily application of spores of Duddingtonia flagrans in lambs kept under an evasive grazing system in the Netherlands.

In 2004, an experiment was carried out to evaluate the effect of biological control through feeding spores of Duddingtonia flagrans on parasitic gastroenteritis in lambs, kept under an evasive grazing system. In total 66 lambs were used. Forty naturally infected 3-month old ram lambs were weaned in mid June, and divided into four groups of 10 lambs. On 21 June, G1-G4 were moved to four separate virtually clean plots, they were moved after 4 and 8 weeks to similar plots, and housed after 12 weeks to be necropsied 16 days later. The other 26 lambs had been raised helminth-free, and were used as pairs of tracer lambs. All but one of these pairs, were grazed during the last 2 weeks on each plot. The remaining pair (TA) was grazed during the last 2 weeks on pasture (30 August to 13 September) on the plot that had been grazed by G3 between 19 July and 16 August, to study inhibited development in Haemonchus contortus. All lambs were fed 200 g of concentrates daily throughout the whole period, and those of G1 and G2 were also fed 500,000 spores of D. flagrans/kg bodyweight daily. The faecal cultures demonstrated a high reduction in yield as a result of fungal application. However, no differences between groups were seen in weight gain, faecal egg counts, pasture larval counts, worm counts and tracer worm counts. H. contortus was the dominant species, and it is obvious that the moves at 4-week intervals prevented the development of severe haemonchosis. This is in particular demonstrated by the much higher worm counts in the two TA tracer lambs grazed. Nevertheless, increases to high faecal egg counts 3 weeks after the first and second moves, indicated acquisition of infection before these moves and at least subclinical haemonchosis. This was supported with the worm counts of lambs and tracer lambs. A higher proportion of inhibited early L4 than in other tracers and than in the permanent lambs were found in the pair of TA tracer lambs. This indicates that moves to new pastures in late summer and autumn delays the onset of inhibition.

Consequences of the unusually warm and dry summer of 2003 in The Netherlands: poor development of free living stages, normal survival of infective larvae and long survival of adult gastrointestinal nematodes of sheep.

During 2003 a grazing study was performed at Utrecht University to evaluate evasive grazing and application of Duddingtonia flagrans for the control of parasitic gastroenteritis in sheep. However, the summer of 2003 (June-August) was unusually warm and dry. As a result the patterns of gastrointestinal nematode infections deviated from those observed in more average years. The proportion of eggs that developed to infective larvae was far lower than normal in July-August. On the other hand, survival of larvae that had developed before the middle of July was not affected compared to other years. In fact, severe haemonchosis was observed in (tracer) lambs grazed at the end of July on pastures that had been contaminated from 26-05 to 16-06 and from 16-06 to 07-07. Moreover, tracer lambs grazing in September on some of these plots still acquired large Haemonchus contortus burdens. Over 60% of H. contortus that had established before the middle of July appeared to be able to survive until October in the virtual absence of re-infection. That may have consequences for the application of evasive grazing as a control option in that suppression of adult burdens might still be necessary, through a limited use of anthelmintics or through alternative deworming strategies.

[Drug resistance makes new control measures of stomach parasites in small ruminants necessary]. / Resistentie maakt nieuwe aanpak van de bestrijding van maagdarmwormen bij kleine herkauwers noodzakelijk.

Control of parasitic gastroenteritis in small ruminants is threatened by the worldwide growing problem of anthelmintic resistance. Therefore, alternative approaches for worm control are imperative. Of utmost importance is to slow down selection pressure for anthelmintic resistance by using alternative control measures combined with minimal anthelmintic usage. New control options for the Netherlands are discussed.

The possibilities and limitations of evasive grazing as a control measure for parasitic gastroenteritis in small ruminants in temperate climates.

Between 1999 and 2003 studies were done at Utrecht University on the population dynamics of gastrointestinal nematodes and the potential to use evasive grazing for the control of nematode infections in small ruminants. On most pastures grazed in May and June it took at least 3 weeks before high pasture infectivity levels for Haemonchus contortus developed while it took at least 2 weeks on pastures grazed in July, August and September. Occasionally, larvae emerged earlier on pasture when high temperatures coincided with rain. Pasture infectivity levels for H. contortus subsequently increased and the highest levels were found between 5 and 9 weeks on pastures grazed in May-June and between 3 and 9 weeks on pastures grazed in July, August and September. Pasture infectivity usually had decreased again to low levels after approximately 3 months. Larvae of the other trichostrongylids emerged later on pasture. However, patterns of Teladorsagia circumcincta, Trichostrongylus spp. and Cooperia curticei were basically similar to H. contortus. Strongyloides papillosus larvae emerge within 2 weeks on pasture and survival is short. On the other hand Nematodirus larvae took a lot longer to emerge on pasture than the other trichostrongylids. The results imply that only on a small proportion of farms (mixed dairy cattle/sheep farms; some organic dairy goat farms) evasive grazing can be recommended as only control measure for parasitic gastroenteritis. However, the present data offer possibilities to combine evasive grazing with other methods in an integrated control scheme.

Shedding consistency of strongyle-type eggs in Dutch boarding horses.

Faeces of 484 horses were sampled twice with an interval of 6 weeks while anthelmintic therapy was halted. Faecal eggs counts revealed that 267 (55.2%) horses had consistently low numbers of eggs per gram faeces (EPG) (EPG < 100 or = 100), 155 (32.0%) horses had consistently high EPGs (EPG > 100). Horses with consistently high EPGs were more often mares with access to pasture, aged less than 6 or more than 23 years, that were dewormed at intervals longer than 6 months, and were treated for the last time more than 3 months before the start of the study. Horses with consistently low EPGs were more often male horses with no or limited access to pasture, that were dewormed at maximally 6-month intervals, and were aged between 6 and 23 years. The results are an indication that some horses have consistently low EPGs and perhaps could be used as non-treated animals in a selective anthelmintic treatment scheme aimed at the prevention of the development of anthelmintic resistance.

Individual variation and effect of priming dose level on establishment, growth and fecundity of Cooperia oncophora in re-infected calves.

We investigated the effect of bovine anamnestic immunity on a range of parasitological variables. To this end, calves were primed with a single oral dose of 30000 or 100000 infective larvae (L3) of Cooperia oncophora, drenched with anthelmintic, maintained worm free in the following 2.5 months and subsequently re-infected with 100000 L3. Parasitological profiles of low, intermediate, and high responders were compared. The reduction in establishment of the worms was shown by a lower worm burden and increased percentage of fourth-stage (L4) larvae. Worm length and fecundity were similarly reduced by both priming doses but, the speed by which the effect occurred differed between animals primed with 30000 or 100000 L3. The difference in establishment between the responder types demonstrates that the ability of intermediate responders to mount a more effective and faster immune response compared to low responders is sustained after secondary infection.

[Infections with endoparasites in dogs in Dutch animal shelters]. / Infecties met endoparasieten bij honden in Nederlandse dierenasielen.

Faecal samples from 224 dogs from 23 animal shelters in the Netherlands were examined for endoparasites. In total 20.5% of the faecal sample were positive for helminth and/or protozoa infections. Eggs of Toxocara canis were found in 8.5% of the faecal samples. Other endoparasites found were Toxascaris leonina (0.5%), Trichuris vulpis (4.9%), Uncinaria stenocephala (2.2%), Dipylidium caninum (1.3%), Taenia spp. (0.5%), Cystoïsospora canis (1.3%), and C. ohioensis (1.3%). Dogs younger than 1 year and stray dogs showed the highest prevalence of infection. T. vulpis was found more often in dogs from shelters with a high cleaning frequency.

[Infections with helminths and/or protozoa in cats in animal shelters in the Netherlands]. / Infecties met helminthen en/of protozoën bij katten in asielen in Nederland.

To determine the prevalence of infections with helminths and protozoa in cats in animal shelters, faecal samples from 305 cats from 22 animal shelters in the Netherlands were examined, using a centrifugation-sedimentation-flotation-technique. The association between potential risk factors and the occurrence of an infection was also tested. Infections with helminths and/or protozoa were found in 160 samples (52.5%). Toxocara cati was found in 86 cats (28.2%), Cystoisospora felis in 59 cats (19.3%), Cystoïsospora rivolta in 43 cats (14.1%), Capillaria spp. in 34 cats (11.2%), Ancylostoma tubaeforma in 9 cats (3.0%), Taenia taeniaeformis in 9 cats (3.0%), Aelurostrongylus abstrusus in 8 cats (2.6%), Giardia intestinalis in 3 cats (1.0%), Dipylidium caninum in 2 cats (0.7%) and Toxoplasma gondii in 1 cat (0.3%). The highest prevalence was seen in kittens and stray cats. The main preventive factor against infection was a short stay in a shelter.

Expulsion of small strongyle nematodes (cyathostomin spp) following deworming of horses on a stud farm in Sweden.

This study was conducted on a stud farm in Sweden to investigate the species composition of cyathostomins expelled in the faeces of horses after deworming using three different anthelmintic preparations. Twenty-seven horses excreting > or = 200 strongyle eggs per gram faeces (EPG) were divided into three comparable groups and dewormed on day 0 with either of following compounds: 0.2 mg ivermectin per kg body weight (bw), 19 mg pyrantel pamoate per kg bw or 7.5 mg fenbendazole per kg bw. For each of the 3 days following anthelmintic treatment faeces was collected from individual horses and subsamples were fixed in formalin. Four days after the anthelmintic treatment all horses were re-treated with ivermectin and faeces was collected on day 5. Individual subsamples from each of the four sampling occasions were examined for cyathostomin nematodes. Sixty-three to 270 worms per horse were identified to the species level. The majority of the worms recovered were expelled during the first day from horses treated with ivermectin or pyrantel pamoate, and during the second day from horses treated with fenbendazole. Fifteen cyathostomin species were identified and the six most prevalent were Cylicocyclus nassatus, Cyathostomum catinatum, Cylicostephanus longibursatus, Cylicocyclus leptostomus, Cylicostephanus minutus and Cylicostephanus calicatus. These species composed 91% of the total burden of cyathostomins. The number of species found per horse ranged from 6 to 13, with an average of 9. No significant differences in species composition or distribution were found between the treatment groups. On day 5, i.e. 1 day after the last ivermectin treatment, 93% of the adult worms were recovered from horses in the fenbendazole group. This study showed that it was possible to identify cyathostomins expelled in faeces of dewormed horses, and that the most prevalent species corresponded to those found in autopsy surveys performed in other countries.