The Przewalski’s Horse (
Since 2005, ivermectin (IVM) has been used as the single equine anthelmintic once a year in winter to combat parasitic diseases on all of the horses kept in captivity without preexamination. Long-term use of single deworming drug may cause anthelmintic resistance (AR) in highly adaptable parasitic nematodes (Leath-wick, 2013). Decreased IVM efficacy against important intestinal parasites of horses, especially
Given that the efficacy of anti-parasitic drugs against gastrointestinal nematode is key to control parasite that prevalent in captive population and prevent the spread of resistant strains to released population, the present study evaluated the efficacy of IVM against gastrointestinal nematode parasitizing captive PHs based on FECRT method and could serve for the deworming work in species reintroduction program.
All PHs were maintained at the Xinjiang Uygur Autonomous Region wild horse Breeding Research Center located in Jimsar County, Changji City, Xinjiang Uygur Autonomous Region, China (44°12’12”N, 88°44’26”E). Environmental conditions of temperature were -14 °C ±6 °C during sampling period in November and December. The horses were bred outdoor in different size of irony stalls all day long. Captive population were divided into breed, female or bachelor groups. There is no vegetation in the stalls. Alfalfa and carrots were offered daily. No water were provided as the water freeze, and horses could replenish water by eating snow.
Sixteen horses were randomly selected for an infection survey, of which two horses with typical histories of parasitic nematode disease were selected as representatives to explore the change pattern of egg counts number in 20 successive days during November and December 2015. One representative horse (#105) is an adult female, born in 1998; the other (#345) is a young male foal, born in 2013. Furthermore, the EPG were compared in the same animals both before and after treatment in 3 foals (<1 year of old) and 8 sub-adults (<2 years of old) in 2017 January. The larvae culture was applied to verify the composition of strongyles. Only small group of animals were enrolled (11 individuals including 3 foals and 8 sub-adult) because EPG before treatment in most horses was too low to take into account. Another practical justification for this small group experimental design is that drug treatment was only allowed on certain days in winter due to restrictions of management measures. The limited time and cold weather made it hard to obtain enough samples by individual track in deworm process or use unpaired design which need extra control group. Although control groups were not adopted, its influence can be eliminated since pre-treatment counts can be used as the compared baseline (McKenna, 1990).
An oral paste of IVM (Beijing Wanfeng Pharmaceutical Laser Target Designator, Beijing 100193, China; 2 mg/g i.m) was mixed with corn meal and fed to all captive PHs at a dosage of 100 μg/kg following the IVM deworming instructions reported before ( Zhang, 2006). Records of body weight of the horses was offered by the Xinjiang Uygur Autonomous Region wild horse Breeding Research Center. Deworming was carried out by a qualified veterinary surgeon. No dose errors were reported.
The surface and interior of each fecal pellet feces were randomly mixed. For fecal egg counts (FEC), a total of 1.0 g feces was ultimately sampled and fixed in 10 mL centrifuge tube with 2.0 g 10 % formalin solution. Each sample was replicated three times at least. For larval culture, about 5.0 g feces were collected from each horse before treatment. All feces samples were collected from ground immediately or within two hours after they were naturally excreted, without disturbing PHs. All PHs lived in their assigned paddocks during and after collection and all acted normally. Samples were stored at 4 °C in order to preserve the morphological characteristics of the nematode eggs and maintain accurate egg counts (Nielsen et al., 2010).
The FEC were performed based on a Modified Wisconsin centrifugal-flotation technique (Cox & Todd, 1962) with analytical sensitivity of 1.0. 6 mL saturated NaNO3 were added to each sample tube. Pellet was vortexed vigorously using Vortex Genie 2 mixer (Scientific Industry, Vortex Genie 2) for 3 minutes, and then filtered with a 60-mesh sieve. The filtrate was poured back into the centrifuge tube and centrifuged for 5 minutes at 950 r/min. Saturated NaNO3 solution was added until a meniscus was formed on the top of the tube. The samples were stand for 10 minutes and then placed a 24 mm × 24 mm coverslip on the top. After an additional 5 minutes, the coverslip was removed and placed on a microscope slide. The numbers of eggs of each parasite type were counted under a light microscope (OLYMPUS CX22). Then the process was repeated with new coverslips until no more eggs were found on the cover-slip. Larval cultures were performed to determine the composition of strongyles. The fecal samples collected from each horse were pooled and humidified with sawdust and water. The cultures were incubated in 28 °C for 14 days. The L3 larval were recovered using Baermann apparatus and identified using morphological keys by Bevilaqua et al. (1993).
The FEC reduction (FECR) of individual PH was calculated by using arithmetic means according the following formula: FECR(%)=[(FEC–FEC)/FEC] × 100 (Francisco et al., 2011), 121where FEC1 is the mean of FEC before treatment and FEC2 is the FEC at the 15th post-treatment day. Since only small groups of horses were available and FEC1 in the current study were low, a reliable FECR-result can hardly been calculated by regular formula based on the arithmetic mean of pre- and post-treatment FEC (Levecke et al., 2018). An alternative Bayesian based methodologies was applied in a user friendly web interface, egg Counts (Torgerson et al., 2014), to assess the FECR and coverage probability of 95 % highest posterior density (HPD) intervals of group. We adopted the paired design (fecal samples obtained before and after the treatment from each horse). Resistance is identified if both the FECR is less than 90 % and the 95 % lower confidence level is less than 90 %, following the typical criteria recommended by the World Association for the Advancement of Veterinary Parasitology (Coles et al., 1992).
The authors declared that all sample procedures in this study were approved by the Xinjiang Uygur Autonomous Region Wild Horse Breeding Research Center (Under the jurisdiction of Forestry Department of Xinjiang Uyghur Autonomous Region) and Beijing Forestry University. Sample collection were performed in accordance with the IUCN Policy Statement on Research Involving Species at Risk of Extinction (Approved by the 27th Meeting of IUCN Council, Gland Switzerland, 14 June 1989). Non-lethal and responsible collections were applied in the case of species listed as endangered under criterion C.
Before the administration of IVM, 75 % of the 16 selected PHs tested positive for
As is shown in Figure 1, before fed with IVM, the
A common pattern is observed in
Strongyles FEC was generally low among horses. The infection level of strongyles in both the adult and the foal was lower than 200 EPG, which caused the experiment of inadequate sample with low pre-FEC and led to a reduction of credibility (Levecke et al., 2018). A low analytic sensitivity method and “egg Counts” package, which allowed for a much smaller pre-FEC threshold, were employed to compensate the negative impact and hence provide a more reliable result. This high efficacy still needs to be validated in the further study if a higher number of pre-FEC was observed.
Feces of all tested PHs were negative for both larval and adult nematode parasites by the 15th post-treatment day, which means the efficacy of IVM is 100 %. It is indicated that IVM, a broad-spectrum antiparasitic agent used against both nematode and arthropod domestic animal parasites since 1979 (Chabala et al., 1980), remains highly effecacy against both
Given failures of IVM treatment to decrease
The potential role of captive population of reintroduced species serve as vectors of resistant parasites to wild animals, make it important to keep a high anthelmintic efficacy. The managers should consider the importance of slowing down the occurrence of drug resistance instead of wiping out all nematodes. Leaving foals untreated should be avoided as they might be infected with