Study area
The study was conducted in Haramaya district, which is located approximately 510 km east of Addis Ababa capital of Ethiopia. The estimated animal population is about 63,723 cattle, 79,950 sheep, and 120,350 goats. Topographically, it is situated at altitudes of 1600 to 2100 m above sea level, which puts the area into the category of a highland. Haramaya is located 9°24′N 42°01′E at an altitude of 1950 meters above sea level. The surrounding farming areas are semi-arid. The mean annual rainfall received in the district ranges from 600 to 1260 mm. The district is representative of a sub-humid mid-altitude agro-climatic zone. The relative humidity varies between 60 and 80%. The minimum and maximum annual temperatures range from 6oC to 12oC and 17oC to 25oC, respectively [17].
Study animals
Apparently healthy goats maintained in Haramaya University (HU) goat farm were used to identify the GI nematodes of goats and their anthelmintic resistance (AR) status to commonly used anthelmintic drugs. A HU goat flock of approximately 150 heads of different breeds (Abergel, Somale, Cross breed and Hararghe highlander), both sexes, different age and weight groups was used. Each goat was individually marked with an ear tag before the start of the experiment. The goats were allowed to graze at Haramaya University pasture land during the daytime. In addition, supplemental hay and concentrate were provided during the dry season. Approximately 20 to 30 goats were bedding in groups for night shade and house cleaning was done every day during the morning after goats were released for grazing. Sick goats and goats with newborns were also kept in a separate room for special treatments.
Determination of EPG
Before and after treatment of anthelmintic drugs around 15 g of faecal samples were collected directly from the rectum of each goat early morning before allowed for grazing. Samples were placed in labeled polythene bags and immediately transported to the Haramaya University Animal Science Parasitology Laboratory for examination. Faecal samples were examined for helminth eggs using a modified McMaster technique in accordance with Urquhart et al. [18]. In each case, 3 g of faeces were mixed in 42 ml of saturated salt solution, and the number of nematode eggs per gram of faces (EPG) was obtained by multiplying the number of nematode eggs counted in two squares of the McMaster slide by a dilution factor of 50. The parasitic burden of the animals was evaluated based on EPG and goats with EPG count greater than 150 counts were included in the research and then grouped randomly into five treatment groups; four groups received drug treatments and one group left untreated control. At ten-day intervals faecal sample was collected from the goats and immediately laboratory analysis done to determine the number of the parasite eggs (EPG) as described above, and anthelmintic resistance was evaluated based on the standard guideline.
Faecal culture and larval recovery
Ova culture was performed on the unexploited faecal materials after EPG determination to obtain larval stages, as described by Van Wyk and Mayhew [19]. Faecal samples containing parasitic eggs were finely crushed with a pestle and mortar and were placed in a petri dish and incubated at room temperature (approximately 22–27°C) for 10 days and subjected to Baermann technique. Centrifuge tubes were filled to two thirds and spin at 1500rpm for 5 minutes in an electrically powered centrifuge to concentrate the larvae to the bottom [20]. The larvae were recovered and pooled together after decantation of the supernatant fluid. A drop of the lugol’s iodine added to kill recovered larvae, then a drop of sample placed in microscope slide and cover-slip applied, then the nematode larvae were identified to genera level and quantified based on morphology described by Van Wyk et al. (21) and John (22). Where possible, 100 larvae were identified and counted for each experimental group of animals [22].
Experimental design
The study goats were divided into five treatment groups; four treated and one untreated groups; ideally 20 goats were included in each group and naturally exposed to GI nematodes. Each goat was weighed to give the correct dose of the anthelmintic drug. The drug given to animals was also based on the manufacturer's recommendation. On day 0, faecal sample was collected from each goat enrolled in the study. Subsequently, faecal sample was collected 10 days after treatment. Larval cultures were also conducted pre- and post-treatment to determine specific nematode genera involved, to identify AR within specific parasitic genera. AR was assessed based on FECRT% explained by Coles et al. [23] in which AR occurs when FECRT% is less than 95% and the lower 95% confidence level is lower than 90%. If only one of these was fulfilled there was suspected AR. The sources of anthelmintic drugs were Chongoing Fangton Animal Pharmaceutical Co.LTD China (albendazole) from, Ashish Life Science Pvt Limited India (tetramisole, and tetraclozan) and Shenyang sunvictor pharmaceutical co.ltd./China (ivermectin). These drugs were chosen because of their availability and commonly used in the country.
Dosage was given as per manufacturer recommendations and considering the heaviest weight of goat in each group. Albendazole, tetramisole and tetraclozan given at the dosage rate of 7.5 mg/kg body weight orally while ivermectin injected subcutaneously at the dosage rate of 200 µg/kg body weight.
Faecal Egg Count Reduction Test (FECRT)
FECRT was done based on reduction of the number of eggs per gram of faeces (EPG) by more than 95 percent measured ten days after treatment, the presence of resistance occurs if the reduction is less than 95% and the lower confidence limit is less than 90%. The EPGs count of goats exceed 150 to 200 in the pre-treatment group were included in the research [24]. The number of EPG from experimental groups before administration of drugs were done by determination of the mean level of egg excretion across animals (mean pre-drug administration (preDA) FEC) then after the administration of the drug (postDA FEC) as described above for the preDA FEC. Subsequently, the FECRT was calculated using the Coles et al. [23] method.
FECRT (%) = 100 (1-[T2 ̸ C2]).
Where
T2 arithmetic mean FEC after treatment,
C2 arithmetic mean FEC control group at day 10.
Based on Coles et al. [23] method, resistance to an anthelmintic was considered to be present if the percentage reduction in egg count was less than 95%, and the lower 95% confidence limit is less than 90. But as most natural infections include a mixture of species, therefore third stage larvae cultured from pre-treatment and post-treatments groups were separately assessed to identify resistant nematodes [24].
Data analysis
Anthelmintics efficacy was evaluated based on the reduction in faecal egg count and percentage of larvae found in the cultures. Calculation of the arithmetic mean, percentage of reduction and 95% upper and lower confidence limits were conducted according to the procedures described by Coles et al. [23]. Resistance was declared when the percentage of reduction was less than 95% and the 95% lower confidence limit was less than 90%. If only one of the two criteria was met, resistance was suspected.