Nematode resistance to five anthelmintic classes in naturally infected sheep herds in Northeastern Brazil

This study aimed to evaluate the occurrence of nematode resistance to anthelmintics on sheep herds from the semi-arid region of Paraíba, Northeastern Brazil. Fecal Egg Count Reduction Test (FECRT) was carried out on 20 properties. In each herd, 30 animals were divided into five groups containing six animals each: group I, treated with albendazole 10%, 4 mg/kg; group II, ivermectin 0.08%, 0.2 mg/kg; group III, closantel 10%, 10 mg/kg; group IV, levamisole hydrochloride 5%, 5 mg/kg; and group V, monepantel 2.5%, 2.5 mg/kg. All treatments were administered orally as a single dose. Fecal samples were collected on days zero and 10 after treatment, to perform FECRT and coprocultures. Multiresistance was observed in all the properties; 95% had high resistance to albendazole, 85% to ivermectin, 80% to closantel, 40% to levamisole, and 45% to monepantel. On property 15, where monepantel was ineffective, a second FECRT for this anthelmintic was carried out 4 months after the first, resulting in 75% efficacy. Immediately after the FECRT result, two ewes were euthanized and necropsied, and Haemonchus contortus, Trichostrongylus axei, Trichostrongylus colubriformis, Oesophagostomum columbianum, and Trichuris ovis were recovered. It was concluded that the resistance of sheep gastrointestinal nematodes to antthelmintic, including monepantel, is high.


Introduction
Sheep production occupies a prominent place in the Brazilian Northeast, where producers use their products and by-products in food and commerce, generating stability and development for the region.However, several factors restrict the production and productivity of these animals, including nutritional problems and inadequate management practices in sanitary hygiene, especially the poor hygiene conditions that favor the increase of the population of gastrointestinal parasites, mainly H. contortus.This parasite is considered of great epidemiological importance due to its high resistance to anthelmintics (IDRIS et al., 2012).
The irrational use of anthelmintics has been contributing to the resistance of gastrointestinal endoparasites of small ruminants, whose drugs are mostly available in the market.Monepantel, in turn, belongs to a new class of anthelmintics, the Amino-Acetonitrile Derivatives (DAA), which acts on gastrointestinal parasites resistant to all classical anthelmintic agents (KAMINSKY et al., 2008).According to Baker et al. (2012), monepantel showed a 100% efficacy against multiresistant H. contortus in sheep.Mederos et al. (2014) and Van den Brom et al. (2015) observed early resistance involving H. contortus in flocks of sheep from Uruguay and Holland, respectively.Likewise, Martins et al. (2017) observed that sheep helminths, especially H. contortus, are already resistant to monepantel in a sheep herd located in the state of São Paulo.
However, there were no studies in Brazil that evaluated the efficacy of monepantel in different herds of naturally infected sheep.Therefore, the present study aimed to evaluate the action of monepantel and other anthelmintics on gastrointestinal nematodes of sheep of the semi-arid region of Brazil.

Material and Methods
The experiment was carried out in 20 sheep-producing properties of the Paraíba semi-arid region between July and December 2016 (Figure 1).Thirty animals from each farm were used amounting to a total of 600 sheep of both sexes, with no defined racial pattern, with no anthelmintic treatment for at least 3 months and presenting a count of eggs per gram of feces (EPG) > 500.Subsequently, the animals were identified individually and randomly distributed into five groups composed of six animals each: group I, treated with albendazole 10% (4 mg/kg; Ibazole 10%  ; Ibasa); group II, treated with ivermectin 0.08% (0.2 mg/kg; Ivomec Solução Oral  ; Merial); group III, treated with closantel 10% (10 mg/kg; Diantel  ; Hipra); group IV, treated with levamisole hydrochloride 5% (5 mg/kg; Ripercol L Solução  ; Fort Dodge); and group V, treated with monepantel 2.5% (2.5 mg/kg; Zolvix  ; Novartis).All treatments were carried out with a single dose, administered orally, according to the manufacturers' recommendations.
Fecal samples were collected individually and directly from the rectal ampulla, and were subsequently placed in plastic bags, identified, kept refrigerated in an isothermal box, and promptly sent to the Veterinary Parasitology Laboratory (VPL) of the Instituto Federal da Paraíba (IFPB), Sousa-PB, for analyses.
Similarly, counting of EPG was performed according to Gordon & Whitlock (1939) and coprocultures according to Roberts & O'Sullivan (1950).The evaluation of the Fecal Egg Count Reduction Test (FECRT) was performed according to Coles et al. (1992).Such procedures were performed on day 0 and on day 10 after administration of anthelmintics.
The evaluation of the efficacy of the drugs was based on the Technical Regulation of Ordinance No. 48/1997 of the Ministry of Agriculture, Livestock and Food Supply (MAPA) for chemical substances with antiparasitic activity, using the following criteria: (1) highly effective, when it reduces the parasite load by more than 98%; (2) effective, with a 90-98% reduction; moderately effective, with a 80-89% reduction; and insufficiently active, with a reduction of < 80% (BRASIL, 1997).
The reduction percentage (PR) of the fecal egg count was estimated by the following equation: Where X d10 is the arithmetic mean of the egg count for the group on day 10, and X d0 is the arithmetic mean for the group on day 0 (before treatment).
Information related to the size of the properties and the number of animals in the herds were obtained for the calculation of the stocking rate (Animal Unit (AU)/ha; 1 AU = 450 kg of animal body weight).To associate the stocking rate to anthelmintic resistance, univariable analysis was performed using the Fisher's exact test, and variables that presented P ≤ 0.20 were used for multivariable logistic regression, performed using the BioEstat 5.0 software (AYRES et al., 2007).
In one of the properties that presented high resistance to monepantel (Prop.15), new FECRT for this drug was performed 4 months later in 20 sheep for resistance confirmation purposes.
On the day following the confirmation of resistance, the two animals with the highest parasitic load were purchased, slaughtered, and necropsied according to international standards established by the World Association for the Advancement of Veterinary Parasitology (WAAVP) and described by Vercruysse et al. (2001).Therefore, the gastrointestinal tract of sheep was separated by its anatomical segments (abomasum, small intestine, and large intestine), isolated, and subsequently separated by double ligatures with cotton thread.The abomasum was opened at its greatest curvature and the contents stored in a container, where a 10% aliquot was preserved in 5% formol.The mucosa of the whole abomasum was submerged in saline solution at 39 °C for 6 h.Subsequently, an aliquot of 10% of the digested material was preserved in 5% formalin.Similar procedures were also performed with respect to the small and large intestines.The counting and identification of the recovered helminths were carried out according to Ueno & Gonçalves (1998).
When categorizing properties based on drug efficacy, we observed that levamisole was highly effective in 30% (6/20) of the properties, while albendazole showed the least effectiveness, being classified as insufficiently active in 90% (18/20) of the properties.It was noted that monepantel was highly effective in only 15% (3/20) of the properties (Table 2).
It was observed the presence of three gastrointestinal nematodes genera after larval recovery from the coprocultures, Haemonchus sp., Oesophagostomum sp. and Trichostrongylus spp., with no great variation of their percentages between the groups, nor between the samples before and after treatments (Day 0 and Day 10, respectively), as demonstrated in Figure 2.
At property 15, new TRCOF for monepantel was performed 4 months after the first, resulting in a 75% efficacy.It was observed that the two necropsied animals had a high number of nematodes in their gastrointestinal tract after monepantel treatment (Table 4).H. contortus presented the highest infection intensity, with an average of 505 adult nematodes, followed by T. colubriformis, with an average of 175 nematodes.

Discussion
The present study was the first to test the anthelmintic efficacy of monepantel against gastrointestinal nematodes in different sheep herds in Northeastern Brazil.This drug was insufficiently active (efficacy < 80%) in 25% (5/20) of the analyzed properties, showing resistance in the region.Cintra et al. (2016) reported for the first time the occurrence of resistance to monepantel in Brazil associated with T. colubriformis, in naturally infected sheep of the Experimental Farm of the Catholic University of Paraná (PUC-PR), with an efficacy of -21.5%.Mederos et al. (2014) reported for the first time the occurrence of resistance to monepantel in Latin America, associated with Haemonchus sp. in two properties in Uruguay.Van  Monepantel was introduced in the Brazilian market with the promise of 100% efficacy in the treatment of gastrointestinal strongyles in sheep, including those that have already developed resistance to other active principles.However, even with the use of the drug for the first time in the studied properties, anthelmintic resistance cases were found.It is well described that the entire herd treatments at short intervals, leads to the selection of strains of resistant parasites, generates contaminant residues in the environment, and increases production costs (SUTHERLAND et al., 2008;ZAROS et al., 2014).Bartley et al. (2004) emphasized that the anthelmintic resistance of gastrointestinal nematodes of small ruminants has been observed worldwide.In this study, multiresistance was observed in all evaluated farms, wherein 95% of farms had high resistance to albendazole, 85% to ivermectin, 80% to closantel, 40% to levamisole, and 45% to 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 studied, while 92% showed resistance to closantel and 53% to levamisole (VERÍSSIMO et al., 2012).High anthelmintic resistance was also reported by Melo et al. (2013), when evaluating the efficacy of ivermectin and levamisole in 13 sheep farms in the Agreste of the State of Paraíba, with a reduction of only 30.9% and 93%, respectively.When categorizing properties regarding drug efficacy, it was observed that levamisole was highly effective in 30% of the analyses.Albendazole was the one that showed the least effectiveness, being classified as insufficiently active in 90% of the properties.It was also noted that monepantel was highly effective in only 40%.Although monepantel is a novel molecule with no history of use in the animals of this herd, this drug has not shown to be effective on gastrointestinal nematodes.According to Oliveira et al. (2017), who evaluated the anthelmintic resistance in sheep flocks in southern Brazil, the resistance to at least three anthelmintic agents was reported on all properties, being Monepantel not effective in 18% of the tested flocks, confirming that parasitic resistance can be established very soon after the anthelmintic treatment with new molecules, mainly when alternative program of parasite control is not performed.
In all coprocultures, the most prevalent helminth genus was Haemonchus sp., corroborating with Vieira et al. (2014), who identified (79.9%) of Haemonchus sp. in sheep in the semi-arid region of Paraíba State.The successive anthelmintic treatments performed on the properties lead to a higher selection pressure for Haemonchus, making it the most prevalent in most researches (VILELA et al., 2012(VILELA et al., , 2016)).It is plausible that this worm acquires faster resistance due to its high biotic potential, great genetic variability, and for having the allele that causes the increase of the resistance to a drug (BLACKHALL et al., 1998).Vieira et al. (2014) observed that the most relevant risk factor for the development of helminthiasis in their study was the farm area, because it defines the area available for grazing animals.Properties with many animals and little pasture area, which are the most abundant type in the Sertão region of Paraiba, tend to have high prevalence of gastrointestinal helminthiasis, because the animals are more prone to reinfection.On the other hand, in the present study, because so many mistakes of anthelmintic management were observed in the evaluated herds, especially underdoses and fast alternation of active principles, the resistance was so high that the stocking rate may not have influence it.
In the second stage of the study, after new FECRT for monepantel at property 15, it was again insufficiently active (75% efficacy).After the recovery of adult helminths, it was possible to diagnose resistance of the species H. contortus, T. axei, T. colubriformis, O. columbianum and T. ovis.In São Paulo, Martins et al. (2017) diagnosed a resistance to monepantel 2.5% using a critical test (parasitological necropsy) in sheep, in which the compound was ineffective against H. contortus (24.65%).Cintra et al. (2016) have detected resistance (-21.5%) of T. colubriformis to monepantel.When evaluating sheep in New Zealand, Scott et al. (2013) described the first report of resistance to monepantel of Teladorsagia spp., Trichostrongylus spp., and Oesophagostomum spp., being also ineffective against Capillaria spp., Trichuris spp., and Strongyloides spp.Still in that study was reported that the continued presence after treatment of Capillaria spp., Trichuris spp.and Strongyloides spp.do not necessarily indicate resistance in these parasites since they have never been included in any registered claim for Zolvix  .There may in fact be no intrinsic activity of monepantel against these nematodes (RUFENER et al., 2010).
Several factors contribute to the development of resistance to the drugs used to control gastrointestinal nematodes, such as the uncontrolled use of anthelmintics due to the lack of knowledge of the producers, the poor management practices, and the ease of acquiring these medicines.According to Vilela et al. (2012), the high anthelmintic resistance observed in studies carried out in the semi-arid region of the Brazilian Northeast is due to the fact that it is a common practice to treat massively the entire herd of small ruminants four to six times a year.

Conclusion
The resistance of the gastrointestinal nematodes of sheep from the semi-arid region of Paraíba, Northeastern Brazil, to the anthelmintics, including monepantel, is high.The most resistant helminth was H. contortus.The incorrect management that is commonly practiced in the herds of the region tends to aggravate the multiresistance observed in all evaluated properties.
Den Brom et al. (2015) demonstrated a 0% efficacy by FECRT in a sheep farm in the Netherlands, related to the genus Haemonchus sp.

Figure 2 .
Figure 2. Percentage of nematodes by genus, farm and treatment recovered from coprocultures (Days 0 and 10) of sheep in the semi-arid region of Paraíba, Brazil.

Table 1 .
Efficacy of anthelmintics on sheep gastrointestinal nematodes in the semi-arid region of Paraíba, Brazil.

Table 2 .
Percentage of properties by category of efficacy of anthelmintics on gastrointestinal nematodes of sheep in the Paraíba semi-arid region, Brazil, according to Brazilian Ministry of Agriculture, ordinance No. 48/1997.

Table 3 .
Number of insufficiently active (efficacy < 80%) anthelmintics according to the area and stocking rate of sheep producing properties in the Paraíba semi-arid region.

Table 4 .
Species of gastrointestinal nematodes recovered from sheep submitted to necropsy after Fecal Egg Count Reduction Test for monepantel in the Paraíba semi-arid region, Brazil.