Worm-control practices and prevalence of anthelmintic resistance using in vivo FECRTs on smallholder sheep farms in Lithuania

A questionnaire surveyed 71 sheep farmers. Twenty-nine farms were visited, and the farmers were interviewed personally. Forty-two sheep farmers from the list of the Lithuanian Sheep Breeders Association were interviewed by telephone. All farmers were asked about their practices of farm management: number of animals, sheep breeds, size of pastures, and worm-control practices: treatment times and frequency, products, and dosages of anthelmintic drugs. The surveyed farms were situated in western (12.7 %), central (40.8 %), and eastern (46.5 %) Lithuania. Because of the appropriate farm management and treatment strategy twenty five sheep farms from the questionnaire survey were selected for the further research.

A total of 25 sheep farms, mainly in central and southern Lithuania, were visited between April 2014 and November 2014. Eighteen of these farms were included in the study and seven were excluded due to faecal egg counts (FECs) <140 eggs per gram (EPG). Flocks were selected randomly and identified by GIN FECs. Faecal samples were collected from the rectums of 15 randomly selected animals in each flock and analysed using a modified McMaster technique, with a minimum sensitivity of 20 EPG (Roepstorff & Nansen, 1998). The selected flocks had been regularly treated with anthelmintics (Jackson et al., 2012). All flocks had grazing animals at the time of the study. The last anthelmintic treatment had been given at least eight weeks before the beginning of the study. The selected flocks consisted of approximately 40 – 500 animals

Sheep older than 18 months in each flock were divided into twogroups of 15 animals, marked with different coloured sprays, and treated with two different anthelmintics. The number of animals in each group was based on the recommendation that 10 animals per group were sufficient to detect differences in FECs between groups (Coles et al., 1992). We chose 15 animals per group to guard against any losses of faecal samples (Falzon et al., 2013). We selected the BZ and ML anthelmintic classes for evaluation in this survey, based on the information obtained from the questionnaire. One group in each flock was treated with the BZ fenbendazole (FBZ) at 7.5 mg/kg body weight (Panacur® granules, Intervet International B.V., Boxmeer, Netherlands), and the other group was treated with IVM at 0.2 mg/kg body weight (Biomectin 1 %, Vetoquinol Biowet Sp.zo.o., Gorzow Wlkp., Poland). The doses were based on the heaviest animal in each flock. FBZ was administrated orally over the back of the tongue, and IVM was administrated subcutaneously behind the scapula. Five of the 18 flocks received only one anthelmintic (IVM on three farms and FBZ on two farms), because of the difficulties of management. Individual faecal samples were collected on the day of treatment (T1). Animals with <140 EPG on day zero (T1) were removed from the trial. Eggs of Nematodirus spp. were not included in the counts. Individual post-treatment faecal samples (T2) were collected after 14 days from both groups in each flock treated with both anthelmintics, after 10 days from the flocks treated with only FBZ, and after 14 days from the flocks treated with only IVM. The FEC reduction (FECR) was calculated as:

where T2 is the arithmetic mean FEC post-treatment and T1 is the arithmetic mean FEC pre-treatment.

Post-treatment larval cultures were prepared from pooled faecal samples for the flocks with FECRT efficacies <100 %. The pooled samples were composed of the faeces collected from each animal of the group. Ten grammes of faeces were mixed with 4 g of vermiculite and incubated for 7 d at 27 °C (water was added to maintain an adequate moisture level). Third-stage larvae (L3) were then recovered from the coprocultures by a Baermann technique (Coles et al., 1992). The L3 were morphologically

differentiated and identified according to Maff (1986) and Van Wyk et al. (2004). The first 100 L3, or all L3 when <100 developed, were identified.

The FECR and the lower limit for a 95 % confidence interval were calculated following the WAAVP recommendations (Coles et al.,1992). Flocks with FECRs <95 % and lower limits <90 % were considered as harbouring GINs resistant to an anthelmintic. If only one of these conditions was met, resistance was suspected. Descriptive statistics were calculated using Microsoft® Excel 2007 and IBM SPSS Statistics (Version 21.0).

This study complied with Lithuanian animal welfare regulations (No. B1-866, 2012; No. XI-2271, 2012) and was approved by the Lithuanian Committee of Veterinary Medicine and Zootechnics Sciences (Protocol No.07/2010).

All farms were mainly specialized for meat production and practiced grazing, and 19.7 % of the farms had an ecological status. Rotational grazing was used on 78.3 % of the farms, and 21.7 % of respondents kept sheep on the same pasture with a shelter during the grazing period. Pastures had a mean area of 19.53 ha (1-120 ha). The average number of sheep per farm was 149.9 (1-1700). Lithuanian black-headed sheep was the dominant breed (46.5 %). Other breeds were Romanov (15.0 %), German black-headed (13.3 %), Suffolk (8.3 %), and Berichon du Cher (5.0 %). The remaining 11.9 % consisted of breeds such as Merinofleischschaf, Ile de France, Texel, and Lacaune and crossbreeds. Sheep were usually pastured from March/April to October/November, and all sheep were housed during the winter.

An estimated 71.8 % of sheep farmers used anthelmintics against GINs (Table 1), but 9.5 % of farmers declared that they treated their sheep only with the appearance of clinical symptoms such as diarrhoea, apathy and/or weight loss. The most commonly used classes of anthelmintics were MLs (68.6 %), and BZs (27.5 %). From the BZs group 68.7 % albendazole and 31.3 % FBZ were used. Levamisole was used very sporadically (3.9 %) (Table 1). Spring before turn out and autumn before turn in were the most common times to treat ewes. Yearlings and adults were usually treated together, with a mean annual drenching rate of 1.39. Most of the respondents declared that they treated lambs at the same time as the yearlings and adults to save time, with a mean number of treatments of 1.24 (Table 1). Of the respondents that used anthelmintics, 62.7 % declared that they treated their animals twice every year. All respondents that used anthelmintics once per year treated their ewes in spring. A few farms added a treatment in summer (7.1 %). Anthelmintics were rotated on 4.8 % of the farms. Only one respondent reported four treatments per year. Annual treatments were usually performed without any parasitological analyses, and 11.9 % of the respondents reported only a single coprological analysis during the entire period when sheep were kept. Animal weights were visually appraised on 92.9 % of the farms, and only 7.1 % of farmers weighed their animals. Veterinarians treated the animals on 54.8 % of the farms, and owners or farm workers treated the animals on 45.2 % of the farms. Problems with sheep GINs were declared by 39.2 % of the respondents.

The arithmetic mean FECs, percentages of the FECRs and 95 % confidence intervals are presented in Table 2. The FECRs indicated the presence of FBZ resistance on three of the 15 farms where FBZ was used (FECRs ranged from 44.9 to 85 %), and FBZ resistance was suspected on one farm (94.3 %, CI 93-99). Resistance to IVM was present on two of the 16 farms that used IVM (FECRs of 74.9 and 77.2 %). On one farm (Farm No.7) resistance was detected to both classes of anthelmintics. Mean pre-treatment EPG counts varied from 216 to 3114. The main species of resistant GIN identified after treatment were Teladorsagia spp. On all 6positive farms, with distribution varying from 42 to 100 %. Trichostrongylus spp. was found on five farms, with distribution varying from 4 to 100 %, and 6-56 % of the GINs on seven farms were Cooperia spp. Chabertia ovina was found on two farms (4-18 %), and Haemonchus contortus was found on only one farm (10 %) (Table 3).