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 altitude of 1600 to 2100 m above sea level with the mean annual temperature and relative humidity of 18°C and 65%, respectively and the surrounding farming areas are semi-arid. Haramaya, it is located 041º 59' 58" N latitude and 09º 24' 10"S longitudes.
Study Animals
Apparently health goats maintained in Haramaya University (HU) goat farm were used to identify the GI nematodes of goats and anthelmintic resistance (AR) to commonly used anthelmintic drugs. HU goat flock of approximately 150 heads of different breeds; Abergel, Somale, Cross breed and Hararghe highlander, either sexes, different age, and weight groups was used. Each goat was individually marked with ear tag before the start of experiment. The goats allowed grazing pasture at Haramaya University pasture during the day time. In addition supplement hay and concentrate provided during the dry season. Approximately 20 to 30 goats are bedding in group for night shade and house cleaning is done every day during the morning after goats released for grazing. Sick goats and goats with newborn separated for especial treatments. Since experimental parameters don’t affect the health and production parameters of goats the study goats after experimental work rejoined the remaining groups of goats and continues to use for production at Haramaya University. All considered factors/variable evaluated based on criteria of ARRIVE guideline.
Determination of EPG
Around 15g of faecal samples were collected directly from the rectum of each goat, early morning before sent out for grazing; before and after treatment of anthelmintic drug. 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 modified McMaster technique with saturated sodium chloride solution as the floating medium (14, 15). 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 then grouped randomly in to five treatment groups then four group received drug treatments while one group left untreated control in the same day of laboratory analysis. At ten day intervals faecal samples was collected from the goats to determine the concentrations of the parasite eggs (EPG) as described above and anthelmintic resistance was evaluated based on the standard guideline.
Faecal Culture and Larval Recovery
The unexploited faecal materials after EPG determination was incubated at room temperature (approximately 22–27°C) for 10 days, 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 (15). The larvae was 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 according. Where possible, 100 larvae were identified and counted for each experimental group of animals (16).
Experimental Design
The study goats were divided into five treatment groups; four treated and one untreated control groups; ideally 20 goats were included in each group and naturally exposed to GI nematodes. Each goat’s weight measured, in order to administer the correct dose of the anthelmintic drug and the drug given to animals based on the manufacturer recommendation. On day 0, fecal samples collected from each goat enrolled in the study, and then the goats are either treated with an anthelmintic or left untreated. Subsequently, faecal samples collected 10days after treatment. Use of larval cultures in pre and post treatment samples helps to determine specific nematode genera involved, in order to identify AR within specific parasitic genera. AR was assessed based on FECRT% explained by Coles et al. (17) in which AR is occur when FECRT% is less than 95% and the lower 95% confidence level is lower than 90%. If only one of these 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).
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. All considered variable meet criteria of ARRIVE guideline.
Fecal Egg Count Reduction Test (FECRT)
FECRT was done based on reduction of the concentration of eggs per gram of faeces (EPG) by more than 95 percent; measured ten days after treatment, in comparison with the EPG measured at the time of treatment and failure to do so was indicative of resistance. The EPGs count of goats in the pre-treatment group exceed 150 to 200 included in the research (17). The distribution of parasites within the experimental groups before administration of drugs 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 calculation as described by Coles et al. (18) method.
FECRT (%) = 100 (1-[T2 ̸ C2]).
Where
T2 arithmetic mean FEC after treatment,
C2 arithmetic mean FEC control group at day 10.
Approximate 95 % confidence interval for R 100 = [(1-(Xt/Xc)) exp (± 2.1√v)]
Variance of reduction (on log scale v=[(s2 t /(ntX2t))]+[( s2 c /(ncX2c))])
Upper confidence limit 100[1-(Xt/Xc)exp(-2.1√v)]
Lower confidence limit 100[1-(Xt/Xc)exp(+2.1√v)]
Where Xt is the mean egg count of the treated group at 10days and Xc is that of the control group at 10days.
Based on Coles et al., (18) 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 pretreatment and post treatments groups was separately estimated to identify resistant nematode (19).
Data Analysis
The efficacy of anthelmintics 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. (18). 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.