Study on utilization and efficacy of commonly used anthelmintics against gastrointestinal nematodes in naturally infected sheep in North Gondar , North-Western Ethiopia

Anthelmintic utilization and efficacy of commonly used anthelmintics against gastrointestinal nematodes in naturally infected sheep were assessed in North Gondar, North-Western Ethiopia. Anthelmintic utilization data were collected with a semi-structured questionnaire. An efficacy trial was conducted on 28 naturally infected sheep. The animals were randomly allocated into seven groups (four in each). Groups were treated with albendazole, ivermectin, tetramisole, levamisole, albendazole, ivermectin plus albendazole, albendazole plus levamisole, and a no-treatment control group. The faecal egg count reduction test (FECRT) was used to evaluate the efficacy of anthelmintics and identification of parasites was done by the faecal culture examination method. Data from the survey showed that different anthelmintics were used and some improper utilization was also recorded. Livestock owners had a tendency to deworm their animals throughout the year, but most commonly at the beginning of the rainy season. The highest FECRT (100%) was observed in animals receiving combined therapy, followed by albendazole (99.08%), ivermectin (96.69%), levamisole (90.06%) and the lowest reduction percentage was observed in the tetramisole group (89.51%). Parasite species surviving treatment were: albendazole, Trichuris; ivermectin, Trichuris and Haemonchus; levamisole and tetramisole, Trichuris, Haemonchus and Oesophagostomum. In summary, the different anthelmintics used in the study area did not have equal efficacy. Therefore, proper utilization and selection of anthelmintics are necessary for effective control of these parasites in Ethiopia.


INTRODUCTION
In Ethiopia, small ruminants are important sources of income for rural communities whose livelihood is largely based on livestock production (Abebe and Esayas, 2001;Biffa et al., 2006).However, sheep production in the country is hindered by many factors including animal health constraints, inadequate nutrition and poor husbandry systems (Sissay et al., 2006).Studies in different parts of the country have shown that gastrointestinal nematodes are major problems in sheep production, causing mortality and production losses (Tembely et al., 1997;Abebe and Esayas, 2001;Biffa et al., 2006).Livestock owners mainly rely on the utilization *Corresponding author.E-mail: achenefmela@yahoo.com.Tel: +251-918065724.Fax: +251-581141240.
of anthelmintics to control these parasites.Several anthelmintic preparations are imported and distributed in the country via legal or illegal agents without proper efficacy testing, control or registration.Furthermore, majority of anthelmintic preparations used today have been utilized for two or more decades.These conditions favour the development of resistance by the parasites (George et al., 2011).Thus, there is concern among professionals and livestock owners about the efficacy of anthelmintic products available in this region.
Anthelmintic resistance in sheep is a global problem (Kaplan and Vidyashankar, 2012;Chandrawathani et al., 1999;Sangster, 1999;Papadopoulos, 2008;George et al., 2011).In Ethiopia, it has been reported by Sissay et al. (2006) in Eastern, and Eguale et al. (2009) and Kumsa and Abebe (2009) in Southern part of the country.There is no published report on the utilization and efficacy of commonly used anthelmintics to control gastrointestinal nematodes of sheep in North Gondar.This study was designed to assess the pattern of anthelmintic utilization and evaluate the efficacy of commonly used anthelmintics against gastrointestinal nematodes of naturally infected sheep in North Gondar.

Study area
This study was conducted from January to September, 2011 in North Gondar, Amhara regional state, North-Western Ethiopia.The area includes highland, midland and lowland agro-ecologies.These diverse agro-ecological zones are attributable to the high range of altitudinal differences, which varies from 4620 m at the Simen mountains in the North to 550 m in the West.The mean minimum and maximum temperatures vary with altitude.In the highlands, temperature varies from 11 to 32°C, whereas in the lowlands, temperature may reach 44.5°C especially in the month of April or May.Rainfall varies from 880 to 1772 mm.The area has two main seasons: the wet season from June to September when the area receives its major rainfall and the dry season from October to May with sparse and erratic rainfall.The humidity also varies with altitude.Many of the dwellers, both rural and urban, are involved in animal production and the area has an estimated sheep population of 524,087 (CSA, 2009).

Questionnaire survey
A questionnaire-based survey was conducted in three districts which represent highland, midland and lowland areas of the zone.From these districts, data were collected from 15 randomly selected governmental veterinary clinics and 12 private veterinary pharmacies.Ninety livestock owners (farmers) having 5 to 25 animals randomly selected and included in the study.Veterinary professionals in the selected districts and owners or workers of private veterinary pharmacies were informed about the purpose of the study and data collection was started after getting their permission and co-operation.Farmers and veterinary professionals were asked to fill the questionnaire about the pattern of anthelmintic use including details of the estimated number of doses used per year, the estimated cost of anthelmintics used annually and the names of products that they usually used.

Study animals and experimental design
Twenty-eight sheep of the same sex (male), breed (local) and age (about 6 months) were purchased from the market at Gondar town.The sheep were brought to the Faculty of Veterinary Medicine, University of Gondar.The animals were then acclimatized for one month and allowed to graze in parasite-contaminated pasture.Each animal was identified by ear tag.The animals were randomly allocated into seven groups (four in each).The first group was treated with albendazole, the second with ivermectin, the third with tetramisole, the fourth with levamisole, the fifth with albendazole plus ivermectin, the sixth with albendazole plus levamisole and the last group was left untreated (control).All anthelmintics were purchased from private veterinary pharmacies in and around Gondar town.Each animal was weighed by automatic balance for dose calculation.Dose rate and route of administration were based on manufactures' recommendation and anthelmintics were chosen based on the frequency of utilization in the study area (Table 1).

Faecal sample collection and examination
Faecal samples were collected from each experimental sheep for coproscopic examination by using pre-labelled universal bottles on day 0 (before treatment) and again on day 10 post-treatment (Coles et al., 1992).Samples were examined for parasite eggs using saturated salt solution as a flotation fluid in the Veterinary Parasitology Laboratory.Faecal eggs per gram (EPG) of strongyle-type nematodes were determined for each sample using the modified McMaster technique according to Coles et al. (1992).The efficacy of the drugs was assessed by the percentage reduction of mean egg excretion on the 10th day of post-treatment, following the methods described by Kochapakdee et al. (1995) and Arece et al. (2004).

Larval identification
About 10 g faecal samples from each sheep were collected before and after treatment (11 days).Samples were finely disrupted using a mortar and pestle, a small amount of water was added to moisten, and the samples left for 14 days at room temperature in a Petri dish (Waghorn et al., 2006), adding small amounts of water as necessary.Third stage, larvae (L 3 ) were collected using the Baerman technique as described by Urquhart et al. (2003).L 3 were identified based on the morphological keys given by MAFF (1977) and van Wyk et al. (2004).

Data analysis
Data were recorded in Excel spreadsheet and descriptive statistics (means, standard error and percentages) were calculated.Posttreatment faecal egg counts (FEG) were transformed to the natural logarithm (count plus one) and means were compared among groups through analysis of variance (ANOVA) and difference between treatments was compared using least square method of multiple comparisons.All data were analyzed using Statistical Package for Social Sciences (SPSS) version 17 statistical software.Probability (P) value less than 0.05 was used to determine the level of significance.

Questionnaire survey
The survey indicated that the most commonly utilized anthelmintics were albendazole, followed by ivermectin,     were utilized during the dry season (Figure 1).Livestock owners also responded that deworming was needed if the animal showed decreased appetite (66.67%), loss of body condition and emaciation (56.67%), diarrhoea (20.00%), coughing (23.33%) and finally if there was sneezing and/or nasal discharge (13.33%).None of the farmers interviewed weighed their animals to calculate the dose.Simple guessing and information obtained from the professional were the basis for determining the dose.

Pre-treatment faecal examinations and larval identification
Faecal examination for the presence of parasite eggs in the pre-treatment faecal samples revealed that all sheep (100%) were positive for strongyle eggs.Other gastrointestinal parasites, including Trichuris species (28.57%) and Nematodirus species (17.85%), were also observed.
Haemonchus was the dominant parasite identified after larval culture, occurring in all experimental sheep.

Faecal egg count reduction test (FECRT)
The results of the FECRT for each anthelmintic are shown in Table 4.No parasite egg was recorded on the 10th day of post-treatment in groups receiving anthelmintic combinations.Considerable numbers of eggs were found in groups treated with levamisole and tetramisole.
There was a significant difference (P<0.05) in mean FEC among the groups of sheep treated with different anthelmintics.Pair wise comparisons revealed that there were no significant (P>0.05)differences among the albendazole, ivermectin and anthelmintic combination groups, whereas post treatment FEC were significantly (P<0.05)lower in the albendazole, ivermectin and anthelmintic combination groups than in the tetramisole and levamisole groups.

Post treatment parasite identification
The types of gastrointestinal nematode parasites that survived in each group after treatment are listed in Table 5.No parasite was recovered in groups receiving combined anthelmintics.Only Trichuris was found in faecal samples of animals treated with albendazole.The most pathogenic gastrointestinal nematode (Haemonchus) was observed in animals that were treated with ivermectin, levamisole and tetramisole.

DISCUSSION
The study revealed that anthelmintic drugs are quite commonly but improperly utilized in the area.Three group of anthelmintics namely benzimidazoles End of rainy season (Albendazole), imidazothiazole (Tetramisole and Levamisole) and macrocyclic lactone (Ivermectin) were used.Other alternative anthelmintic were not available in the market.Utilization of limited group of drugs for a long period may favour the development of resistance (Papadopoulos, 2008).Benzimidazoles group of anthelmintics especially albendazole was the most commonly used by livestock owners to deworm their sheep.Similar finding was also reported by Kumsa et al. (2010a, b) in the Southern part of the Ethiopia and Arece et al. (2004) in Cuba.
Availability of anthelmintics in the hand of livestock owners and selling together with other commodities in supermarkets and ordinary shops may also promote resistance as under dosing is eminent.Most farmers deworm their animals twice a year and spent about 3.00 US dollars for anthelmintic purchase.This is a rough estimate as most of them did not keep proper records.Anyway, deworming practice should not be routine task.It has to be conducted strategically based on the epidemiology of the parasites and after confirming the presence of the parasite in a given animal.Syndromes mentioned by livestock owners for deworming may not be induced by gastrointestinal parasites alone in which use of anthelmintic may be useless.
The anthelmintic trial study revealed that the anthelmintic drugs used in the area does not have equal efficacy on reduction of helminth infection the sheep.Albendazole and ivermectin formulations were found to be more efficacious for gastrointestinal nematodes in sheep at a recommended dose than others.Similar finding was also reported by Arece et al. (2004).
Haemonchus was also identified from the coproculture samples after treatment with ivermectin, tetramisole and levamisole.This finding was supported by many previous studies that reported the association between Haemonchus species and reduced efficacy of anthelmintics (Chandrawathani et al., 1999;Arece et al., 2004).Albendazole has a very good efficacy in reducing gastrointestinal nematodes.Only one parasite (Trichuris) was found in one of the treated sheep.Other most pathogenic nematodes like Haemonchus were effectively treated.This finding is in agreement with previous research outputs like Chaka et al. (2009) as they confirmed that most anthelmintics sold on Ethiopian markets were effective to treat Haemonchus in experimentally infected sheep.Good state of efficacy of albendazole was also reported by Sheferaw and Asha (2010) in the Wolaita (Southern Ethiopia).However, resistance of Haemonchus against albendazole were reported by Kumsa and Abebe (2009) in goat in Hawassa (Southern Ethiopia) and a high level of anthelmintic resistance to albendazole in goat was also reported by Sissay et al. (2006) in Eastern Ethiopia.These may be related with the difference in anthelmintic utilization in different parts of the country.
Ivermectin was also effective in reducing FEC but in lesser extent than albendazole (99.08 versus 96.69%, respectively).Especially the recovery of Haemonchus in ivermectin treated group needs a special attention.Similarly, Sissay et al. (2006) reported ivermectin resis-tance in Haemonchus contortus and Trichostrongylus species dominated infection in goats.In contrast to this finding Arece et al. (2004) reported a 100% efficacy of different formulations of ivermectins in Cuba.This may be related to the difference in the frequency and ways of utilization of the drug among localities.
Tetramisole and levamisole were less efficacious than others.The lower efficacy may also be related to the fact that the resistance can be developed against one anthelmintic in a group which may also occur in the other members of the group (Sangster, 1999) since these two anthelmintics are related drugs (Imidazothiazoles).Lowest efficacy of imidazothiazoles was also reported by Arece et al. (2004) and Sissay et al. (2006).The quality of the preparation of these anthelmintics may also the contribution factor for their lowest efficacy which necessitates further investigation and comparison with standard products.
The 100% efficacy result obtained by the use of two anthelmintics together is promising.It can be recommended to be used by farmers despite the cost and safety.Reducing resistance by the administration of combined anthelmintics was also mentioned by Papadopoulos (2008).However, the benefit and cost has to be thoroughly investigated.
In conclusion, the results of this study showed the high efficacy of albendazole and ivermectins; however, the low efficiency of levamisole and tetramisole.For a more sustainable control of helminth parasites, the strategy has to be directed to the integration of different parasite control methods.To maintain the efficacy of anthelmintics, proper utilization should be practiced.

Figure 1 .
Figure 1.Pattern of deworming by livestock owners in relation to season.

Table 1 .
Detail information about anthelmintics used in the trial.

Table 2 .
The most commonly utilized anthelmintics to deworm sheep in North Gondar.
anthelmintics in their possession during the time of interview.Most farmers (44.44%) dewormed their animals twice a year (Table3) and spent about 51.67 Ethiopian birr (equivalent to 3 US dollars) on average per year to purchase anthelmintics.They have a tendency to

Table 3 .
Response of livestock owners on their deworming practices (frequency per year, number and percent of respondents).
deworm throughout the year but most commonly at the beginning of rainy season, followed by end of rainy season and during rainy season.Fewer anthelmintics

Table 4 .
Mean faecal egg count and percent reduction after treatment of sheep using different anthelmintics or their combinations.

Table 5 .
Survivor parasites after treatment with different anthelmintics.