A controlled study on efficacy and egg reappearance period of Ivermectin in donkeys naturally infected with small strongyles

Donkeys (Equus asinus) are very important working animals as they provide vital support to human beings in many developing countries. Moreover, they are increasingly used for recreational activities (agritourism, trekking), onotherapy (Borioni et al., 2012), meat production (Polidori et al., 2008), and mostly milk production, since jenny’s milk can be used in children with allergy to cow’s milk (Polidori et al., 2013). In addition, the potent anti-proliferative activity of whey protein of donkey milk against human lung cancer cells has been reported (Mao et al., 2009). Therefore, improving the donkey health status is important to owners, farmers, and consumers.

Small strongyles, also commonly called cyathostomins, are among the most important intestinal parasites of donkeys due to the high prevalence values commonly reported in several countries around the world (Matthee et al., 2000; Bu et al., 2009; Getachew et al., 2010; Matthews et al., 2013; Ismail et al., 2016; Jajere et al., 2016; Dibaba et al., 2017). They include more than 40 species and several genera, they belong to the subfamily Cyathostominae and to the family Strongylidae, and they are found in the cecum and colon of domestic equids. It is known that small strongyles can be a threat to donkeys’ health and welfare (Matthee et al., 2002; Yoseph et al., 2005; Orian et al., 2015; Waqas et al., 2015).

The control of endoparasites, including small strongyles, in donkeys depends mainly on the use of anthelmintics at doses determined for horses (Grosenbaugh et al., 2011). These include macrocyclic lactones (i.e. Ivermectin and Moxidectin), benzimidazoles (i.e. Fenbendazole), and tetrahydropyrimidines (i.e. Pyrantel embonate) (Grosenbaugh et al., 2011; Matthews et al., 2013). However, the first report of resistance to moxidectin in cyathostomins was observed in donkeys (Trawford et al., 2005). High levels of widespread resistance to fenbendazole have been reported in cyathostomins worldwide (Matthews et al., 2013), including in a donkey farm in Italy (Buono et al., 2018). Cases of pyrantel resistance of cyathostomins have been reported in two herds of donkeys in the UK (Lawson et al., 2015) and in one donkey farm in Italy (Buono et al., 2018). Conversely, it has repeatedly been shown that the efficacy of ivermectin (IVM) against intestinal strongyles in donkeys is as high as 100 % (Buono et al., 2018; Seri et al., 2005; Imam et al., 2010; Arias et al., 2013; Fangama et al., 2013).

The time interval between the last effective anthelmintic treatment and the resumption of significant strongyle egg shedding is called the egg reappearance period (ERP). The ERP is calculated as weeks post-treatment and this parameter differs for each drug (Nielsen et al., 2019). Thus, it is important to know the ERP of each anthelmintic drug after treatment in the different equine species. However, the ERP after treatment with IVM in donkeys still remains poorly investigated. Indeed, the majority of experimental studies evaluating the efficacy of IVM in donkeys did not investigate the corresponding ERP (Seri et al., 2005; Imam et al., 2010; Fangama et al., 2013). To the best of the authors’ knowledge, only two studies have reported data about the ERP in donkeys treated with IVM so far (Buono et al., 2018; Arias et al., 2013). Thus, in order to give further insights, the aim of the present study was to investigate the efficacy and ERP of IVM in donkeys found naturally infected by intestinal small strongyles.

For this study, formal consent is not required. The research related to animals complied with all the relevant national regulations and institutional policies for the care and use of animals.

On day 0 of the study, all the 14 selected donkeys resulted to be coprologically positive for stongyle eggs and were classified as low (n=2), moderate (n=7), or heavy (n=5) shedders. Their FEPGCs ranged from 180 to 770 eggs in faeces of treated donkeys and from 210 to 640 eggs in untreated ones with AMs of 446 and 385 eggs, respectively (Table I).

Neither adverse drug reactions nor other clinical signs were observed in any of the donkeys treated with IVM during the study period. Table 1 shows individual and mean FEPGCs with percent of efficacy of IVM as determined by results of FECRTs at each sampling time. Overall, IVM showed FECRT of 100 % and, thus, 100 % efficacy in each of the treated donkeys from day 14 to day 42 (6 weeks) post-treatment. Later, the efficacy of IVM started to slightly decrease but, nonetheless, the drug still continued to be highly effective (i.e. efficacy >95 %) during four additional weeks. Mean FECRTs varied as follows: 97.7 % (individual FECRT range = from 97.9 to 100 %), 97.4 % (from 89.6 to 100 %), 97.5 % (from 94.8 to 100 %), and 96.7 % (from 93.2 to 99.2 %) on days 49, 56, 63, and 70 after treatment, respectively. Thereafter, IVM progressively lost its efficacy since the mean percent of FECRTs drastically dropped to 89.8 % (from 75 to 95.1 %), 80.9 % (from 64.6 to 93.9 %), and finally 70.8 % (from 38.8 to 85.4 %) on days 77, 84, and 91, respectively. On day 49, 2/10 of the treated donkeys were found coprologically positive and then the number of donkeys becoming coprologically positive slowly increased to 6/10 on day 56, 8/10 on day 63, and finally 10/10 from day 70 onwards. The ERP after treatment was determined to be 11 weeks, as the mean percent of FECRT decreased below the ERP-threshold chosen in the present study, i.e. a cutoff value of 90 % efficacy, on day 77 of faecal sampling.

All the donkeys of the untreated control group were constantly found coprologically positive for strongyle eggs throughout the study period, with individual FEPGCs ranging from 195 to 1575 eggs and mean FEPGCs ranging from 285 to 1043.7 eggs (Table I). No one of them showed clinical signs referable to intestinal strongylosis or to any other pathologic condition. Statistical analysis by Student’s t test showed that differences between the AMs of FEPGCs in the treated group and those in the control group after treatment were extremely significant (P=0.0000) at each sampling time.

Results of pre-treatment (day 0) and post-treatment (day 70) faecal cultures and morphological identification showed that only small strongyle larvae were present in samples from all the enrolled donkeys, both from those assigned to the treated group and from those assigned to the control group.

Results of our study provide information on the efficacy of IVM against small strongyle infection in donkeys. The Mini-FLOTAC technique in combination with Fill-FLOTAC was used for FEPGCs in the present study. This technique can be considered as the most accurate egg counting method nowadays available in Veterinary Medicine (Bosco et al. 2014) and, thus, it allowed an accurate interpretation of the results of FEPGCs which were very reliable. Our findings show that IVM efficacy was very high (100 %) against Cyathostominae larvae in all orally treated donkeys. These findings are supported by the high FEPGCs constantly detected in untreated control donkeys which rule out any influence on the worms’ burden in the examined donkeys caused by season or weather conditions occurred during the study period. The present findings are in agreement with those of other reports where an efficacy of 100 % was stated in donkeys at 14 days after administration of a dose of 200 mcg/kg of IVM both in oral (Buono et al., 2018; Imam et al., 2010; Arias et al., 2013) and in injectable formulation (Seri et al., 2005; Fangama et al., 2013). Similar findings were also detected in horses (McFarlane et al., 2010; Larsen et al., 2011). Nevertheless, lower efficacy and complete AR of cyathostomins to IVM are important reasons for concern in horses (Canever et al., 2013; Molena et al., 2018). Moreover, donkey breeders and equine veterinary practitioners working with donkeys have a reduced number of anthelmintics to use, which can lead to continuous and frequent use of a single drug, subjecting cyathostomins to higher selection pressure. This inappropriate approach is considered responsible for the rapid appearance of AR.

The control of small strongyles in donkeys depends largely on the routine administration of anthelmintic drugs at specific times of the year to remove adult parasites, to prevent or minimize contamination of the environment with eggs and larvae, and thereby to disrupt the seasonal cycle of transmission. Knowing the ERP of each anthelmintic drug is of pivotal importance for parasite control in equines, including donkeys, since anthelmintic treatments at intervals equal to or shorter than ERP can provide a selective advantage to resistant strongyle populations (Saeed et al., 2019). Indeed, the frequent exposure of small strongyles to an individual anthelmintic may result not only in the elimination of susceptible adult worms but also in the lack of parasite stages in refugia (encysted larvae, free-living larval stages in environment) that escape the effects of the treatment, though they are still susceptible to the anthelmintic. The refugia subpopulation is not under selection for AR because it is not exposed to the drug at the time of treatment. This subpopulation of worms does not have genes for AR, as such it may dilute and delay the proliferation of those worm populations having resistance alleles and allow the effectiveness of an anthelmintic drug to be prolonged by providing a reservoir for drug-susceptible genes (Saeed et al., 2019). Therefore, treatment intervals with any anthelmintic should be based on its ERP and a shortened ERP is the first indicator of the development of resistance to an anthelmintic drug (Nielsen et al., 2019). Accurate knowledge of the ERP of an anthelmintic is also essential to assess the potential for shedding and contamination of eggs by each animal within any group of adult equines. For this purpose, indeed, individual fecal samples should be examined by FEPGC a minimum of 4 weeks beyond the ERP for the last drug used (Nielsen et al., 2019). Unfortunately, ERP definitions have been reported exclusively for horses but researchers are not in agreement with a common threshold beyond which animals should be retreated, not even in horses. In fact, the criterion determining the ERP varies between authors. Some authors have calculated the ERP as the first week after treatment when eggs reappear in the stool (Little et al., 2013). Other authors have considered the ERP as the week after treatment when the AM of FEPGCs is equal to or greater than a fixed threshold such as 100 (Van Doorn et al., 2012) or 200 (Mercier et al., 2001) eggs. Unfortunately, using all these methods, results are highly biased by the pre-treatment FEPGC levels, since some imply that an anthelmintic must reduce the FEPGC of 100 % to be considered effective, whilst others do not take into consideration that low shedder donkeys can take longer to reach the fixed threshold or may never reach the threshold. A third group of authors have defined the ERP based on results of FECRTs. The ERP is thereby measured by performing FECRTs at weekly intervals and is calculated as the first week after treatment when the mean percent FECRT is lower than a predetermined cutoff level of 80 % (Tarigo-Martinie et al., 2001) or 90 % (Larsen et al., 2011) efficacy. In the present study, the ERP was considered as the week when a <90 % FECRT was observed. This definition was chosen among the different methods reported in literature to calculate ERP because we agree with other authors (Larsen et al., 2011) that it represents a reasonable and reliable approach. Indeed, as the ERP is a measure of the efficacy of an anthelmintic during the weeks after treatment, this method is adapted to the drug under evaluation and sets the cutoff level approximately 10 % lower than the expected 99 – 100 % efficacy of IVM (Larsen et al., 2011). The present study was performed for a 13-week period to determine the ERP of IVM in the treated donkey group as ERP rates of 9 – 13 weeks for IVM have been reported in horses (Nielsen et al., 2019), and the ERP was determined to be 11 weeks. Thus, we found no evidence of shortened small strongyle ERP in donkeys as the ERP we achieved for IVM is consistent with guidelines of the AAEP for parasite control in horses. Moreover, our study with IVM indicates a much longer ERP for cyathostomins in donkeys than the range of ERP (6 – 8 weeks) usually reported in horses in the last ten years, when the drug results to be effective (Nielsen et al., 2019). The present cyathostomin ERP for IVM in donkeys falls within the range of rates previously reported in other studies. One study relied on the first post-treatment positive coprological flotation and showed that the ERP after treatment with IVM plus Praziquantel was 2 and 3 months in a group of 6 European donkeys and 6 African donkeys kept in a zoological park in Spain, respectively (Arias et al., 2013). Another study estimated the ERP based on FECRT calculation and reported that the ERP for IVM was 8 or 12 weeks in two groups of 6 farmed donkeys each in Italy (Buono et al., 2018). Therefore, combining results of the present study with those of similar studies previously reported (Buono et al., 2018; Arias et al., 2013), it can be argued that the ERP after treatment with IVM in donkeys range from 8 to 12 weeks. This is very close to ERP rates (9 – 13 weeks) documented for IVM when the drug was first introduced in horses (Nielsen et al., 2019). However, it is somewhat difficult to compare our results with those of other studies assessing the ERP after treatment with IVM in donkeys due to the lack of a uniform method to calculate ERP. Moreover, additional reasons for different results may be due to level of parasite burden and age of donkeys as well as to season and weather conditions, since intestinal strongyles are known to reduce the egg shedding when environmental conditions are less favorable for their transmission.

Small strongyles can be a threat to health and welfare of donkeys. The findings of the present study (i.e. 100 % efficacy in all treated donkeys on day 14 after treatment and ERP as long as 11 weeks) show that a single oral dose of IVM paste formulation administered at an estimated dose rate of 200 mcg/kg BW was highly effective and safe to control naturally acquired small strongyle infection in donkeys and ruled out any emerging resistance to IVM in the studied donkey group bred in Italy. This corroborates results of previous studies on efficacy of IVM and its ERP after treatment against small strongyles in donkeys. Therefore, our findings may be useful to veterinary practitioners and breeders as they show that IVM can be still considered as a valuable pharmacological tool to use for deworming programmes in donkeys. Given the increased number of reports of the emergence of AR worldwide (Tarigo-Martinie et al., 2001; Kaplan and Nielsen, 2010; Van Doorn et al., 2012; Canever et al., 2013; Little et al., 2013; Matthews et al., 2013; Molena et al., 2018), including cases in donkeys (Trawford et al., 2005; Lawson et al., 2015 ; Buono et al., 2018), and mostly given the lack of new anthelmintics, our findings are a strong reminder that all strategies needed to prevent and minimize the development of AR to IVM in donkeys must be undertaken. Furthermore, since ERP may be an early indicator of suspected AR, it is advisable that researchers and practicing veterinarians reach an agreement on the definition of ERP in donkeys.