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Deborah van Doorn

Tuesday 27 May 2014

Macrocyclic lactone resistance in cyathostomin species of horses

Promotor: Prof. dr J.A. Wagenaar
Defence: 27 May 2014

Horses on pasture always acquire helminth infections as parasitic larvae or eggs will be ingested. Cyathostomins are the primary helminths pathogens of young (1-3 years) horses. The most pathogenic form of the disease (larval cyathostominosis) mainly develops in this age class (chapter 1). Worldwide anthelmintic resistance development of cyathostomins against several anthelmintic drug classes has been shown. Since macrocyclic lactones (MLs) is the last group of anthelmintics that was introduced it was considered necessary to determine if resistance against these drugs had developed in the Netherlands and if risk factors and/or the resistance mechanism could be determined.
The aim of the first study described in this thesis (chapter 2) was to look for ivermectin (IVM) resistance on Dutch horse premises and to determine possible risk factors for resistance development. This study was performed according to the guidelines of the World Association of the Advancement of Veterinary Parasitology (WAAVP) using the fecal egg count reduction test (FECRT). Seventy premises of which half were raising farms were investigated through pooled fecal samples and larval cultures. The FECRT was only performed (by the investigators) on horses that showed positive egg counts or larval cultures after IVM treatment by their owners. Questionnaire data showed that the frequency of treatment was high (on average 5 times per year) on most farms. Thirty-four percent of these 70 owners dewormed frequently within the egg reappearance period (ERP). None of the farmers tried to use pasture rotation as worm control measure. Only 9 out of the 15 farms claiming to remove feces from pasture did this at least once every 2 weeks. Fecal examination before deworming (monitoring) was only done on 2 (3%) farms. Only in one horse an efficacy against cyathostomins of IVM less than 95% was found. This implies that resistance of cyathostomins against IVM was not detected in this study and risk factors for ML resistance development could not be established.
To further study ML resistance development with FECRT and to study shortening of the ERP, premises in Italy, Belgium and the Netherlands were selected based on a history of (frequent) deworming with MLs (chapter 3). At least 10 study sites in each country were selected and animals were allocated to one of two treatment groups based on the pre-treatment fecal egg counts (FEC). Animals were treated with a macrocyclic lactone and this was either IVM or moxidectin (MOX). In total, 320 horses on 32 premises were examined. The FECRT 14 days after treatment indicated a 100% efficacy in 59 of the 64 treatment groups and >92% efficacy in the remaining 5 groups. In the Netherlands an efficacy for IVM of 92% was found on one farm and shortening of ERP on 6 premises. In total the ERP for IVM was decreased on 17 out of 32 study sites and for MOX this was 15 out of 32 study sites. On 9 sites the ERP was decreased for both IVM and MOX.

To investigate the species that contributed to a shortened ERP, 4 Dutch horse premises were selected of which 2 came from the ERP studies described in chapter 3. Fecal samples from 21 horses, before and after ML treatment, were cultured and 40 larvae obtained from each culture were individually differentiated with reverse line blot (RLB) (chapter 4). The RLB showed that early egg shedding was dominated by species of the genus Cylicocyclus (Cyc.). Before treatment species composition was generally more diverse with species from several other genera also present and no differences in composition of the larval cultures of horses with a shortened ERP and those who had regular ERPs were found. It is concluded that particularly Cylicocyclus spp. are responsible for a shortened ERP.
In the Netherlands in 2008, as a result of an EU directive, regulatory changes were made which made anthelmintics prescription only medicines (POM). Questionnaire surveys at the beginning of POM and again 4 years later were performed (chapter 5). The aim was to study whether the regulatory changes, post-academic education, the website www.parasietenwijzer.nl and a paper on Good Veterinary Practice regarding helminths and deworming (Sloet van Oldruitenborgh-Oosterbaan et al., 2009) resulted in changes in horse practitionersí practices. Results showed that in 2012 significantly more fecal samples were examined than in 2008, more fecal samples were taken for monitoring purposes, more fecal samples were examined quantitatively and more use was made of pooled fecal samples. The number of veterinarians that routinely examined a fecal sample before deciding whether to treat or not increased by more than three-fold from 2008 to 2012. Results also showed that continuing efforts need to be focused on improving the number of quantitatively processed fecal samples and the interpretation of fecal diagnostics.
In the 1st in vitro study of this thesis (chapter 6) cyathostomin eggs were obtained from 2 populations of horses, one from never treated and the other from regularly IVM treated ponies. The larvae, cultured from eggs, were in vitro selected using a reiterative Larval Migration Inhibition Assay (rLMIA) and were differentiated by RLB. In the rLMIA the proportion of larvae that migrated through 4 sieves increased with each passage, demonstrating that the applied procedure selects for the least IVM susceptible larvae. Adding glutamate to the procedure reversed the selection effect, which suggested that glutamate gated chloride channels (GluCls) play a role in IVM susceptibility of the selected larvae. In both larval cultures the predominant species were Cyathostomum (Cya) catinatum, Cyc. nassatus and Cylicostephanus (Cys) longibursatus. After in vitro selection in the rLMIA in the presence of IVM the predominant species became Cya. catinatum in both cultures, while Cyc. nassatus disappeared in the never treated but not in the regularly IVM exposed larval population. It was concluded that the rLMIA RLB is useful in studying in vitro ML susceptibility of larvae.

In the 2nd in vitro study a two-sieve rLMIA was performed (chapter 7) to study the intra-specific variation in IVM susceptibility and to examine the variation in outcome in the assays using 8 worm populations with different histories of ML exposure. In all 8 populations the pre-dominant species were Cya. catinatum, Cyc. nassatus, Cys. longibursatus and Cya. pateratum. After in vitro separation of larvae in the presence of IVM by rLMIA the most prevalent species were Cya. catinatum and Cys. longibursatus. The combined rLMIA RLB demonstrated a higher migration percentage for the 2nd sieve compared with the 1st sieve, indicating selection of larvae with low IVM susceptibility. The highest migration percentage for the 2nd sieve was found for Cya. catinatum. Overall, a significantly lower migration percentage for the 2nd sieve was observed for Cya. catinatum in the never treated horses compared with the treated horses. It was found that within cyathostomin species there is heterogeneity with respect to in vitro IVM susceptibility and this was also found in larvae obtained from never treated horses. It was concluded that rLMIA using IVM and 2 sieves separated susceptible from less susceptible Cya. catinatum L3s and that their heterogeneity was related to treatment frequency.
In the discussion section (chapter 8) the relationship between the use of anthelmintics and resistance development against several drug classes and how to detect (ML) resistance in cyathostomins was discussed. The three main conclusions of this thesis mentioned in chapter 8 are: although a high efficacy of IVM and MOX was still achieved 14 days after treatment, a shortened ERP after these treatments on a considerable proportion of the investigated farms was detected. Most often Cylicocyclus species were found in horses shortly after ML treatment. On the farm with the most pronounced ERP shortening, species from other genera were also found early after treatment indicating that other genera can be selected as well.

It was learned that reduced susceptibility for IVM of specifically Cya. catinatum appears to be related to anthelmintic treatment history on the horse premises suggesting in vitro resistance at least.
After the introduction of the legislation that made anthelmintics prescription only medicines some significant improvements in the prudent use of anthelmintics by veterinarians were accomplished.