Introduction

Sheep are susceptible to numerous parasites, and among the gastrointestinal (GI) parasites, the most important strongyle responsible for parasitic gastroenteritis (PGE) in tropical countries is Haemonchus contortus (Bal et al. 2007).

Benzimidazoles, imidazothiazoles, and macrocyclic lactones are the three important broad-spectrum anthelmintic groups available for the treatment and control of nematodes in grazing animals (Coles et al. 2006; Yadav and Singh 2011). However, the prolonged and indiscriminate use of these anthelmintics has led to the emergence of anthelmintic resistance (AR), which is a major constraint for nematode control throughout the world, including India (Butter et al. 2012). Both spatial and temporal evaluation for AR is important. In India, most of the studies done on AR were under farm conditions involving limited number of sheep (Gill 1996; Swarnkar et al. 1999; Amulya et al. 2015). A study under field conditions in larger populations of sheep is essential to know the actual status of AR. The only such survey on AR in India has been done in Rajasthan (Swarnkar and Singh 2010). The present study was planned in field flocks of native Madras Red sheep in the North-eastern agro-climatic zone of Tamil Nadu, India.

Some of the factors which lead to emergence of AR include frequent deworming, treating every animal in the flock, allowing treated animals immediately onto a clean pasture and under-dosing. Among these factors, continuous use of anthelmintic has been incriminated as the most important cause of onset of AR among GI nematodes in sheep from organized farms (Gill 1996; Easwaran et al. 2009; Jaiswal et al. 2013). To our knowledge, no such studies have been conducted in field flocks. The present study was carried out to evaluate AR in field flocks under persistent anthelmintic treatment using common anthelmintic drugs.

Materials and methods

Study design

The present study was undertaken in the field flocks of Network Project on Sheep Improvement (NWPSI), functioning for the improvement of Madras Red sheep in Kanchipuram district of Tamil Nadu, India. The region falls under the North-Eastern agro climatic zone of Tamil Nadu, where the climate is hot and humid. The region gets an average annual rainfall of 1159 mm in about 47 to 60 rainy days. Relative humidity ranges between 58 and 84 %. The minimum and maximum temperatures are 21.5 and 41 °C, respectively.

The NWPSI, funded partly by the Indian Council of Agricultural Research (ICAR), has been functioning at the Postgraduate Research Institute in Animal Sciences (PGRIAS), Kattupakkam, Kanchipuram, since 1990, with the objective of conservation and genetic improvement of Madras Red breed of sheep. About 114 farmers rearing native Madras Red sheep in 16 villages of Kanchipuram district of Tamil Nadu, India, are the beneficiaries of the scheme (Fig. 1). The scheme covers around 9000 sheep, which are maintained by the farmers under extensive system of management. Grazing is the only source of nutrition. PGRIAS acts as the ram-rearing center for the scheme. Purebred Madras Red rams are provided to beneficiary farmers and the flocks are provided complete health cover, including four-times-a-year deworming, as scheme benefits. The drug to be used, frequency and dose (based on manufacturer’s recommendation) of deworming are decided from time to time, based on suggestions from experts of the Tamil Nadu Veterinary and Animal Sciences University, Chennai, to which this station is attached. The details of deworming carried out since 2001 were available in the form of registers maintained in the scheme. These records were perused to identify beneficiaries based on drug-wise deworming schedule. Based on this evaluation, sheep from five beneficiary villages (Kondamangalam, Kayarambedu, Ponmar, Madhurapakkam, and Otteri), which had been continuously dewormed for more than 10 years, were identified to represent the beneficiary animals from field.

Fig. 1
figure 1

Area of coverage under network project on sheep improvement (NWP), Madras Red Field Unit: villages identified for the study are indicated by stars

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In each village, three beneficiary flocks and one non-beneficiary flock (not coming under NWPSI scheme) were selected. Deworming in the non-beneficiary flocks were apparently done on need basis through local veterinary aid available. In all, 20 flocks comprising of 15 beneficiary flocks, and five non-beneficiary flocks were studied. The anthelmintics studied were fenbendazole (5 mg/kg per OS), ivermectin (0.2 mg/kg per OS), and tetramisole (15 mg/kg of 30 % w/w powder per OS), administered once in winter and once in summer. In each flock, four groups, including three treatment groups as above and one untreated control group were selected. Eight adult ewes were allotted randomly to different treatment groups.

Fecal egg count reduction test (FECRT) and coproculture

FECRT was done as per the procedure described by Coles et al. (1992). Approximately 10 g of dung samples were collected from the rectum of all the study animals on day 0 (pre-treatment sample) and again on the 14th day after treatment (post-treatment sample) for assessing the gastrointestinal strongyle nematode infections in terms of eggs per gram (EPG). A modified McMaster method was used to count the EPG. Fecal egg count reduction (FECR) was calculated using resistance software RESO, which calculates different parameters of FECRT as described in Coles et al. (1992).

The pre-treatment and post-treatment dung samples from beneficiary group and non-beneficiary group were cultured for the identification of the third stage infective larvae (L3) of strongyles as per the procedures adopted by Van Wyk and Mathew (2013). The total length, breadth, length of esophagus, tail, and sheath of the L3 were measured by micrometry and the species identified with the help of the keys provided by Soulsby (1982), MAFF (1971), Moredun Research Institute (2000) and Van Wyk and Mathew (2013).

Larval paralysis assay (LPA)

The LPA was done according to the procedure described by Martin and Le Jambre (1979). L3 of H. contortus obtained from dung samples of beneficiary and non-beneficiary flocks were evaluated for their resistance status using LPA, by comparing the LD50 and LD95 values between these groups. A serial aqueous dilution of commercially available levamisole hydrochloride was prepared in the concentration range from 32 to 0.0039 μg/ml. About 0.1 ml of a suspension containing 10–20 larvae was mixed with 0.1 ml of levamisole solution, in a clean cavity microscopic slide. The mixture was incubated for 15 min at room temperature. After 15 min, the slide was focused under low power objective (10×) of the microscope and the larvae were classified as normal (moving) or paralyzed (no observable motion for 5 s). The assay was repeated three times. Per cent paralyzed were analyzed by probit regression and log-dose were plotted against probit values to obtain LD50 and LD95.

Allele specific-PCR (AS-PCR)

Larvae of H. contortus from pooled pre-treatment samples of beneficiary (75 larvae) and non-beneficiary flocks (25 larvae) were used for AS-PCR. The method adopted by Silvestre and Humbert (2000) for DNA extraction from single larva was followed. A drop of larval suspension was kept in a clean microscopic slide, focused under low power objective (4×). Single larva of H. contortus was identified and pipetted out using micropipette 5 μl volume into a sterile autoclaved PCR (200 μl) tube. Ten microliters of lysis reagent (Viagen Biotech Ltd.) was added along with 0.25 μl of proteinase K (>600 mAU/ml) into the PCR tube and incubated in a water bath at 55 °C overnight. The next day, proteinase K was inactivated by heating the tube to 90 °C for 2 min. The tube was then kept at −20 °C for 20 min. For amplification of β-tubulin gene, this larval digest containing DNA was directly used as template. The method adopted by Tiwari et al. (2007) was followed for AS-PCR using the following primers.

  • Hc Sus Forward- 5′-TAG AGA ACA CCG ATG AAA CAT T -3′

  • Hc Res Forward – 5′-G TAG AGA ACA CCG ATG AAA CAT A -3′

  • Hc common Reverse - 5′-GGA ACC ATG TTC ACG GCT AAC -3′

PCR reactions for susceptible and resistant alleles were carried out separately in a reaction volume of 10 μl. PCR products were electrophoresed in a submarine gel electrophoresis. The products were visualized using UV transilluminator and photographed using video gel documentation system (Bio-Rad Gel Documentation System with Image Lab Software).

Results

The results of FECRT for fenbendazole, tetramisole, and ivermectin are presented in Table 1. Mean FECR percent of beneficiary group during winter and summer season were 77.77 and 76.04 % respectively for fenbendazole. Mean FECR per cent of non-beneficiary group during winter and summer season were 74.82 and 81.09 %, respectively. Mean FECR percentages of beneficiary group during winter and summer seasons were 93.65 and 92.12 %, respectively, for tetramisole, while those for non-beneficiary group during winter and summer seasons were 96.05 and 97.40 %, respectively. Ivermectin was effective, with values of FECRT greater than 95 % and there was no significant difference between beneficiary and non-beneficiary flocks for AR in both the seasons for this drug.

Table 1 Efficacy of anthelmintic drugs in beneficiary and non-beneficiary groups during winter and summer
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Coproculture

The frequencies of different species of strongyles obtained through coproculture of dung samples collected before and after treatment are presented in Table 3. Haemonchus contortus, Trichostrongylus spp., and Strongyloides papillosus were present in pre-treatment coproculture of both beneficiary and non-beneficiary groups. In post-treatment coproculture of beneficiary group, H. contortus was the predominant larva with others, including Trichostrongylus spp., S. papillosus and Oesophagostomum spp. being present. In contrast, in post-treatment coproculture of non-beneficiary groups, H. contortus was the only larvae found, except in Madurapakkam village, where Trichostrongylus spp. was also present.

Larval paralysis assay (LPA)

Response in terms of paralysis to levamisole concentration was studied as probit regression. The LD50 for beneficiary and non-beneficiary group were 0.0152 and 0.0150, while the LD95 were 0.0886 and 0.0863, respectively.

Allele-specific PCR (AS-PCR)

Of the 75 pre-treatment samples from beneficiary flock analyzed by AS-PCR, amplification was noticed in 32 samples. The distribution of SS and rS alleles were 6 (18.75 %) and 26 (81.25 %), respectively. Homozygous resistant (rr) alleles were not encountered. In the non-beneficiary group, amplification was noticed in 11 out of 25 samples, with four (36.36 %) SS, 3 (27.27 %) rS and four (36.36 %) rr, respectively. Comparison of beneficiary and non-beneficiary group pre-treatment results showed that there was significant difference between them (Table 2).

Table 2 Frequencies of β-tubulin genotypes among pre-treatment Haemonchus contortuslarvae between beneficiary and non-beneficiary groups
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Discussion

Madras Red sheep, native to the Kanchipuram district of Tamil Nadu is one of the distinct tropical hair sheep known for its adaptability to the region and utility in the form of meat. The region is classified under the north-eastern agro climatic zone, where the climate is hot and humid. This study on anthelmintic resistance is the first of its kind in actual farmers’ condition covering a wide area in the region (Fig. 1).

In this study, resistance to fenbendazole was observed in both beneficiary and non-beneficiary groups and their mean FECRT was 76.9 and 77.95 %, respectively (Table 1). Animals in beneficiary group have been dewormed with benzimidazole group of drugs continuously for more than 10 years from 2001 to 2011 in all the villages. This might be the reason for beneficiary group to have developed resistance to fenbendazole.

Similar incidences of resistance to fenbendazole were also reported by Chaudhri et al. (2007) where FECR was found to be 76.9 % and Yadav et al. (1995) who found the FECR to be 81 % Jaiswal et al. (2013) also reported resistance to fenbendazole by FECRT in organized farms under continuous treatment.

Resistance to fenbendazole was also observed in animals maintained by the non-beneficiary group. Benzimidazoles have been in use for decades in India, the first thiabendazole having been discovered as early as 1961 (Yadav and Singh 2011; Anon 2004). In India, the first report of AR in sheep dates back to 1976 against phenothiazine and thiabendazole (Varshney and Singh 1976). Benzimidazoles being the most popular group of broad spectrum anthelminthic drugs supplied by both state animal husbandry department and local veterinary practitioners, the animals of the non-beneficiary group could have been indiscriminately dosed by the flock owners themselves, leading to the emergence and fixing of anthelmintic resistance. Fenbendazole is safe for pregnant animals (Bowman 2014) and farmers readily use this drug for deworming. Such reports of frequent and indiscriminate deworming are more common in flocks where regular deworming is not practiced and records are not maintained. This could have led to the establishment of anthelmintic resistance in the flocks.

The results of the survey conducted by Easwaran et al. (2009) indicated multiple resistances in H. contortus and Teladorsagia spp. to benzimidazoles in three farms. In all the three farms, albendazole and fenbendazole had been used over the past 20 years.

The FECRT per cent among animals in beneficiary group dewormed with tetramisole was 92.88 %, indicating suspect resistance. However, the animals in the non-beneficiary group were highly susceptible to tetramisole, with an average mean FECRT of 96.72 % (Table 1).

It could be inferred that the emergence of suspect resistance among animals of the beneficiary group may be due to the fact that these animals were dewormed with tetramisole for the past 4 years. Similar results of H. contortus resistance towards the levamisole were observed by Yadav et al. (1993) and Easwaran et al. (2009). Manikkavasagan et al. (2013) reported suspect resistance to levamisole in Cauvery delta zone of Tamil Nadu.

In striking contrast, animals in non-beneficiary group were susceptible to tetramisole. Deworming in most of the non-beneficiary animals is likely to have been decided by the farmers on their own. Swarnkar and Singh (2010) conducted a survey in Rajasthan and found that 70 % farmers selected anthelmintics based on their own perception or sought advice of chemists, while only 27.6 % of the farmers consulted local veterinarian for selection of anthelmintics. Due to the awareness among farmers about the low safety margin of “tetramisole hydrochloride,” they usually do not use this drug routinely. Hence, this might be the reason for less exposure and susceptibility of worms in non-beneficiary group animals to tetramisole.

In this study, susceptibility to ivermectin was detected in both beneficiary and non-beneficiary groups and the mean FECR was 96.7 and 97.74 %, respectively (Table 1).

Animals of the beneficiary group were initially dewormed with ivermectin in 2006, and the same drug was used again only in 2012 and 2013. Less frequent use and rotation with different anthelmintics might be the reason behind the susceptibility to ivermectin.

The non-beneficiary flocks were also found to be susceptible to ivermectin. Increased productivity in ruminants through the control of helminth parasites will depend upon the availability of low cost, effective anthelmintics (Maharshi et al. 2011). It is possible that most of the local farmers preferred benzimidazoles over both tetramisole and ivermectin by virtue of its lower cost and high margin of safety. Moreover, ivermectin became commercially available relatively late in India (Yadav et al. 1995) and thus the duration of usage of this drug was comparatively short. Records of deworming indicate meager use of ivermectin in beneficiary flocks. The usage of this drug in general has been less frequent due to various reasons such as lack of awareness among farmers, higher cost, lesser availability in nearby medical retail outlets and lower margin of safety as compared to benzimidazoles. It is probable that helminths were not exposed frequently to ivermectin and were thus susceptible. Geurden et al. (2014) reported lower than expected efficacy for ivermectin based on the reduction in egg excretion after treatment on European cattle farms, with confirmed anthelmintic resistance on 12.5 % of the farms. Holsback et al. (2016) found both fenbendazole and doramectin to be ineffective against helminths in Suffolk ewes, when administered individually and as a combination.

Varshney and Singh (1976) documented that frequent and indiscriminate use of anthelmintic is often responsible for development of resistance in gastrointestinal nematodes. Similarly, in the present study, resistance to fenbendazole in beneficiary group due to continuous use of the same drug for the over 10 years was documented.

The post-treatment larval profiles from beneficiary and non-beneficiary animals revealed that in non-beneficiary group, only H. contortus larvae were present (except in Madhurapakkam village, where Trichostrongylus spp. were present), whereas in beneficiary groups H. contortus, S. papillosus, Trichostrongylus spp., and Oesophagostomum spp. were present (Table 3). This indicates that in the beneficiary group, resistance is established not only to H. contortus, but also in the other species of GI nematodes. In the non-beneficiary groups, while resistance was fixed in H. contortus alone, the other GI nematodes being still susceptible to anthelmintics. Similar results were observed in studies elsewhere in India conducted in organized farms of sheep and goat, where it was reported that H. contortus was the most predominant larvae identified in farm flocks followed by Trichostrongylus spp. (Das and Singh 2005; Jaiswal et al. 2013) in post-treatment profiles.

Table 3 Pre and post-treatment coproculture percentage of strongyle larvae in beneficiary and non-beneficiary groups
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Larval paralysis assay confirmed that H. contortus was susceptible to levamisole in beneficiary group as well as non-beneficiary groups, contrary to the results from FECRT, which indicated suspect resistance to tetramisole.

A single nucleotide polymorphism in codon TTC to TAC resulting in phenylalanine to tyrosine substitution is widespread and often at high frequency in many countries (Redman et al. 2015). AS-PCR results in this study revealed the complete absence of homozygous resistant (rr) alleles in the pre-treatment samples of beneficiary groups, while the same was present to the level of 36 0.36 % in the non-beneficiary group. The beneficiary group had a higher percentage (81.25) of heterozygous alleles (rS) when compared to that of the non-beneficiary group, in which only 27.27 % were heterozygous. The predominance of heterozygous alleles in beneficiary group might be due to selection pressure exerted by continuous use of anthelmintics at regular intervals. If the selection pressure due to benzimidazole continues, after some generations, the population of homozygous resistant nematodes, resulting from mating among heterozygous susceptible worms, can lead to fixation of the resistant allele. It has been documented that increasing dose of fenbendazole decreases homozygous susceptible (TTC/TTC) individuals, and increases heterozygous susceptible (TTC/TAC) and homozygous resistant (TAC/TAC) nematodes (Pape et al. 2003). An increase in the homozygous resistant population of H. contortus has been observed in studies on anthelmintic resistance from other parts of India (Tiwari et al. 2007; Chandra et al. 2014). However, more samples from field flocks will have to be genotyped to confirm the result on frequencies obtained in this study.

Humbert et al. (2001) tested the role of BZ under-dosing in the development of BZ resistance. With the recommended dose, only homozygous resistant worms (rr) survived the BZ treatment. But, they found that in under-dosing conditions, heterozygous worms (rS) were less susceptible to BZ than homozygous (SS) worms. After development of resistant strains, they do not revert to susceptibility to BZs, even when the selection pressure has been removed for many years (Palcy et al. 2010).

The study reveals that regular and continuous deworming at the manufacturer’s recommended dose can lead to the emergence of anthelmintic resistance against the most commonly used drug. The results confirm that drugs that are not frequently used retain their ability to control the helminths, as evident by the sustained efficacy of tetramisole in the non-beneficiary flocks in comparison to the beneficiary flocks.

Fenbendazole has been used more frequently in beneficiary group which can be attributed the development of resistance in strongyle helminths. Therefore, this particular drug should not be used in the near future for the beneficiary groups.

Emergence of tetramisole resistance was detected in beneficiary group probably due to its continuous use for 4 years. So, usage of this drug should be withheld for the future years. Ivermectin was still found to be effective among all groups. It is concluded that ivermectin along with newer generic groups of drugs (salicylanilide groups like closantel and tetrahydropyrimidine group like morantel) should be used judiciously in future to prolong their efficacy among the beneficiary groups.

The frequency of deworming followed in NWPSI from its inception included four times a year deworming with a random anthelmintic, without carrying out FECRT. The results of the present study unequivocally prove that this practice leads to onset of AR against the drugs used. Thus, it becomes mandatory to reduce the frequency of dosing. However, a drastic reduction in the frequency of anthelmintic treatment is also not advisable as it might end up in loss for the farmers, due to lack of baseline data on the prevalence of helminths among these flocks. So, to balance these two elements viz. prevention of onset of AR and loss to farmers due to worm load, twice a year deworming with FECRT being carried out to evaluate the deworming efficacy each time and rotational use of anthelmintics, is being implemented among NWPSI flocks. Targeted deworming (Busin et al. 2014) could be considered in future, to prevent selection of resistance against commonly used drugs within this setting.