Epidemiology and Multidrug Resistance of Strongyle Nematodes in Ordos Merino Sheep

Background: Regular whole-ock treatments and long-term use of similar anthelmintics have led to the development of anthelmintic resistance and widespread epidemics of nematodiasis in sheep. This study was performed to understand the prevalence of gastrointestinal nematodes in Ordos Merino sheep and evaluate the ecacy of currently used anthelmintics. Results: Between March 2017 and April 2019, a total of 4014 fresh fecal samples were collected from sheep, and fecal nematode eggs were qualitatively and quantitatively analyzed to understand the prevalence of sheep nematodiasis. The anthelmintic ecacy of currently used drugs was evaluated in naturally infected sheep. Severe infection by gastrointestinal nematodes was observed in the Ordos Merino sheep. The infection rates of the nematodes in 3 consecutive years were 84.3%, 36.9%, and 42.3%. Haemonchus contortus and Nematodirus sp. were the predominant nematode species, with infection rates of 84.3% and 65.6%, respectively, in 2017. Moreover, these species had acquired high resistance to ivermectin, doramectin, albendazole, and levamisole; the fecal egg count reduction percentages were 6.9%, 1.7%, 3.5%, and 79.0%, respectively, after a single administration. Nitroxynil and closantel showed strong anthelmintic ecacy against the predominant species Haemonchus contortus and other nematodes, but they had almost no effect on Nematodirus sp. Conclusions: The prevalence of gastrointestinal nematodes in Ordos Merino sheep and their resistance to commonly used anthelmintics were comprehensively evaluated, and the drugs with high ecacy against the predominant species were identied. The ndings of this study will provide a good foundation for the appropriate use of anthelmintics.


Background
Ordos ne-wool sheep is a type of Merino sheep mainly distributed in the southwestern region of Ordos, Inner Mongolia, and it plays an important role in animal husbandry in this region because it provides both wool and meat. The total number of Ordos ne-wool sheep in the Inner Mongolia Autonomous Region is 1.57 million [1]. However, the sheep in this area are often infected by helminths, particularly gastrointestinal nematodes (GINs); these epidemics have not been effectively controlled to date and seriously affect the production performance of the sheep and hinder the healthy development of the newool sheep industry. In ruminants, GINs parasitize the abomasum, small intestine, and large intestine.
GINs can cause not only chronic diseases [2] but also severe economic consequences directly related to lower milk production, slower growth, and even mortality or indirectly related to treatment cost and associated workload [3].
Because most farmers have insu cient understanding of sheep parasitic diseases, they cannot control these diseases in a scienti c and targeted manner. To date, regular whole-ock treatments with anthelmintics are the most commonly used measure to control GINs in sheep, without a correct diagnosis or scienti c evaluation of the deworming effects [4]. Therefore, deworming programs have repeatedly failed in the main distribution areas for Ordos ne-wool sheep, resulting in serious epidemics of GINs in the sheep. Few epidemiological studies of sheep parasitism have been conducted in Inner Mongolia.
In this study, rst, an epidemiological investigation of GINs in Ordos Merino sheep was performed to understand the current status of nematode diseases. Second, the anti-nematode e cacies of several anthelmintics in naturally infected sheep were compared to screen for the most effective drugs against predominant nematode species. Third, on the basis of the epidemiological investigation and anthelmintic experiments, scienti c and effective prevention and control measures were proposed for early and effective prevention and treatment of GINs in sheep in the study area.  Table 1. Therefore, the prevalence of sheep nematodiasis in this area is relatively severe.

Results
In 2017, the epidemic of GINs in the ne-wool sheep in the investigated area was very severe; however, in 2018 and 2019, the survey results showed that the nematode infection rate decreased signi cantly in the sheep ( Table 2). The main reason is that, during the rst epidemiological investigation, we performed a comparative study of the deworming effects of several different anthelmintics; 2 types of antihelminthics with high e cacy against the predominant nematode species were screened, and the farmers were encouraged to use these drugs.  Because of the large differences in pasture characteristics and landform types in the southwestern region of Ordos, the species and distribution of nematodes in this region are affected to some extent. Therefore, the experimental sheep were divided into 4 different groups: sheep that grazed freely in sandy pasture, low-lying pasture, and hilly pasture as well as sheep raised in a pen. Then, the epidemiology of sheep nematodiasis was investigated ( Table 4).
The survey results for 2017 showed that the nematode infection rates in the sheep from 4 different investigated areas were quite high, and the highest nematode infection rate (88%) was found in sheep that grazed in low-lying pastures (Table 4). However, the nematode infection rate in the sheep raised in a pen was relatively low. The survey results for 2018 and 2019 showed that the nematode infection rates in all 4 sheep groups decreased signi cantly, mainly because of the administration of suitable anthelmintics under our guidance.

Anthelmintic e cacy
According to the epidemiological survey for GINs in the sheep, nematode infections were very severe in the study area. Therefore, to examine the deworming effects of extensively used anthelmintic drugs and screen for drugs that effectively kill predominant GIN species in the investigated area, a total of 140 severely infected sheep (PGE > 2000) were selected and randomly allocated to 7 groups and administrated different drugs (Table 1). On the 14 th day after administration, fecal samples were collected and quantitatively analyzed using the modi ed McMaster technique (Table 5). The fecal egg count reduction (FECR) on the 14 th day after administration showed that albendazole, ivermectin, and doramectin had no anthelmintic effects on GINs predominant in the investigated area ( Table 5). The average FECR in these 3 sheep groups was less than 10%. Levamisole also had a poor deworming effect on the nematodes, with an FECR of 79% after administration. However, nitroxynil and closantel injections had strong anti-nematodal effects, and the FECR was more than 99% after administration.

GIN drug resistance
The anthelmintic e cacy indicated that avermectins, which are widely used anthelmintics, have no The results showed that doubling the ivermectin dose had almost no deworming effects on GINs predominant in the sheep of the examined area (Table 6). On the 14 th day after ivermectin administration, the FECR in both the therapeutic dose group and increased dose group was less than 15%. Therefore, GINs in this area have become highly resistant to ivermectin. The therapeutic dose and increased dose of doramectin had no deworming effects on the predominant GIN species in the examined area ( Table 7). The anthelmintic effect of the therapeutic dose of doramectin was less than 10%, and that of the increased dose of doramectin was less than 20%. This indicates that, although doramectin has no history of clinical use in the area, it also has no anthelmintic effects on digestive tract nematodes because both doramectin and ivermectin belong to the same class of avermectins with the same anti-parasitic mechanism. Thus, the predominant GIN species already possess high cross-resistance to ivermectin and doramectin. Control programs for GINs are mainly based on a combination of animal management practices, pasture management, and, especially, anthelmintic drugs [10]. However, intensive use of synthetic anthelmintics for the treatment and control of GINs in sheep farms has led to the widespread development of resistance to one or more anthelmintic drug classes [11][12][13].
The deworming experiment results show that the dominant nematode species have developed high resistance to the most extensively used anthelmintics, ivermectin and albendazole, and the average FECR was less than 10%. The dominant species also acquired strong resistance to levamisole (FECR = 79%). However, nitroxynil and closantel, which are not widely used locally, had strong antinematodal effects, and the FECR was more than 99% after a single administration. According to Yong Rong [5], a comparative deworming test of sheep nematodes in the same area showed that the GINs in sheep had developed resistance to albendazole (FECR = 33%), but ivermectin had a strong antinematodal effect (FECR = almost 100%). However, the results of this study showed that an increased dose of ivermectin did not have any deworming effect, and even doramectin, which has not been widely used in this area, had a very poor deworming effect. This indicates GINs endemic to this area have developed high crossresistance to ivermectin and doramectin.
A wide range of GINs in small ruminants have been proven to be resistant to anthelmintics in a number of countries, such as England, France, Germany, Norway, Sweden, Denmark, Netherlands, Austria, Switzerland, Lithuania, and Slovakia [4]. Mickiewicz [14] published the rst report of the resistance of some GIN species (namely, Haemonchus contortus) to benzimidazole anthelmintics in a goat population in Poland. Bartley [15] emphasized that the anthelmintic resistance of GINs in small ruminants has been observed worldwide. Francisca Flávia da Silva [16] reported multi-resistance in all evaluated farms, wherein 95% of the farms showed high resistance to albendazole; 85%, ivermectin; 80%, closantel; 40%, levamisole; and 45%, monepantel. In another study involving 35 sheep farms in the state of São Paulo, resistance to albendazole and ivermectin was observed in 100% of the farms, whereas 92% showed resistance to closantel and 53% to levamisole [17]. High anthelmintic resistance has also been reported by Melo [18], who evaluated the e cacy of ivermectin and levamisole in 13 sheep farms in Agreste of the State of Paraíba, the FECR was only 30.9% and 93%, respectively.
The main cause of anthelmintic resistance in nematodes is the prophylactic and repeated use of a limited number of anthelmintics to control GIN infestation without a diagnosis. In most sheep farms, whole-ock treatments are commonly administered once or twice annually, with a very limited rotation of applied anthelmintics and no fecal sampling for e cacy control [19]. To reduce the resistance of the nematodes and achieve effective control of sheep nematodiasis, farmers must use comprehensive measures and a combination of animal management practices, pasture management, and anthelmintic drugs. It is essential to minimize dependency on anthelmintics alone and stop their preventive use as much as possible without prior checking for worm eggs in the feces. In addition, sheep farmers are strongly recommended to check the e cacy of the anthelmintic products every time before use [20].

Conclusion
We conducted a comprehensive survey and anthelmintic study to understand the epidemiology and resistance of GINs in Ordos ne-wool Merino sheep. The ndings of this study revealed that free-range sheep are severely infected with GINs in the research area, and most of them have become highly resistant to extensively used anthelmintics such as ivermectin, doramectin, albendazole, and levamisole. The results will provide a good foundation for the development of appropriate anthelmintic regimens.

Ethical approval
A total of 10 sheep with severe nematode infections were selected and anesthetized with pentobarbital sodium for autopsy to examine the GINs. All study procedures and animal care activities were conducted in accordance with the Bioethics Committee of the College of Veterinary Medicine, Inner Mongolia Agricultural University (12150000460029509N), Hohhot, China.

Selection of farms
The investigated sheep were raised in a pen or were free-range sheep southwest of Ordos City, Inner Mongolia, China. The free-range sheep were further classi ed into sheep that grazed in sandy, low-lying, or hilly grasslands. Most of the sheep farms had 150 or more sheep that grazed year-round on pastures, with variable provision of supplementary feed during winter and early spring.

Sample collection
The study was conducted for 3 consecutive years: Qualitative and quantitative analyses The qualitative method was used to identify the nematode eggs on the basis of morphological observation of different eggs by otation in a saturated magnesium sulfate solution (speci c gravity, 1.32) [21]. The quantitative method was used to count the nematode eggs. Fecal egg counts were performed using the modi ed McMaster technique with the same solution used for otation, and it is a relatively simple and cheap method [22]. The minimum detectable limit of the McMaster technique is 25 EPG of feces.

Identi cation of nematodes
The identi cation of nematode species was based on morphological observation of the eggs with a microscope [21,22] and typical structures of the adult nematodes during the autopsy, and this was subsequently further veri ed on the basis of the morphology of third-stage larvae (L3) [21].

Anthelmintic e cacy
To assess deworming e cacy, 140 severely infected sheep (PGE > 2000) were selected, labelled, individually weighed to calculate the correct dosage, randomly divided into 7 groups of 20 animals each, and administered different anthelmintics ( Table 2). All the drugs used in this study are commercially available anthelmintics.
Fresh fecal samples were collected directly from the rectum pre-treatment and on the 14 th day posttreatment and analyzed using the modi ed McMaster technique. FECR was calculated according to the following formula: FECR (%) = (EPG pre-treatment -EPG post-treatment )/EPG pre-treatment × 100%

Declarations
Ethics approval and consent to participate The research involved obtaining information from farmers and treating sheep naturally infected with strongyle nematodes. The study was conducted in accordance with the Bioethics Committee of the College of Veterinary Medicine, Inner Mongolia Agricultural University (12150000460029509N), China. For the eld trials, informed verbal consent was obtained from each farmer, and the proceeding was approved by the Ethics Committee.

Consent for publication
Not applicable. Abomasum mucosa is covered completely with Haemonchus contortus in severely infected sheep