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Full Length Article
Efficacy of five anthelmintics against a natural Fasciola species
infection in cattle
K.M. Shokiera, S.M. Aboelhadid b'*, M.A. Waleedb
a Animal Health Institute, Beni-Suef Branch, Beni-Suef, Egypt
b Parasitology Department, Faculty of Veterinary Medicine, Beni-Suef University, Egypt
ARTICLE INFO
ABSTRACT
Article history: Received 15 August 2012 Accepted 16 December 2012 Available online 13 September 2013
Keywords: Fasciola species Cattle
Albendazole
Rafoxanid
Oxyclozanide
Oxyclozanide and levamizole combination Triclabendazole
This study aims to evaluate the efficacy of five different anthelmintics against Fasciola species in naturally infected cattle in Beni-Suef, Egypt. In a randomized trial, 55 cattle were divided into 5 treated groups and untreated one. On zero day each group was administered one of the five anthelmintics; albendazole, rafoxanid, oxyclozanide, oxyclozanide and levamizole in combination and triclabendazole. Faecal samples from each animal in the groups were taken on zero day of treatment then on 1, 3, 7, 14, 28, 56 and 84 days post-treatment. The efficacy of the used anthelmintics was decided by fecal egg count reduction percent in which the fecal egg was investigated by sedimentation technique. The results in groups of oxyclozanide, oxyclozanide and levamizole combination and triclabendazole indicated that, these anthelmintics were found of high efficacy against fascioliasis through, 100% reduction in fecal egg count in the day 14 post-treatment. However, the results for albendazole and rafoxanid groups yielded lower efficacy levels, with faecal egg count reductions between 75% and 80.58%, over the period 7—84 days post-treatment. These results are highly indicative for albendazole and rafoxanid resistant Fasciola in cattle in this area of Egypt.
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1. Introduction
Fasciola species, the etiological agent of fasciolosis, is commonly found in sheep and cattle. Although many fas-cioliscides are available on markets, it is believed that some of them are not completely effective in treating liver fluke.
Bovine fasciolosis usually lacks visible clinical signs. However, the cattle infection was chronic which it causes weight gain loss, reduction in the milk yield and fertility problems (Genicot et al., 1991; Schweizer et al., 2005; Charlier et al., 2007 and Loyacano et al., 2002). Liver cirrhosis was determined (Marcos et al., 2007). Vercruysse and Claerebout (2001) reported that
* Corresponding author.
E-mail address: shawky.abohadid@vet.bsu.edu.eg (S.M. Aboelhadid). Peer review under the responsibility of Beni-Suef University
2314-8535/$ — see front matter Copyright 2013, Beni-Suef University. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bjbas.2013.09.006
significant production losses occur in Fasciola gigantica infections.
Basically, fascioliasis control in ruminants depends on the administration of anthelmintics. Using flukicides effective against immature flukes, previous studies have shown that strategic dosing and administration of these flukicides at certain times of the year cause destruction of the parasite life cycle achieving successfully control of bovine fasciolosis (Richards et al., 1990; Parr and Gray, 2000; Mezo et al., 2008).
However, since the variety of flukicides is very limited and animals are usually treated year after year without any monitoring of the efficiency of treatment, flukicide-resistant parasite populations may arise, making the control of fasciolosis difficult. This problem is particularly relevant considering that various authors have reported the development of parasite populations resistant to triclabendazole (Moll et al., 2000; Coles, 2005; Alvarez-Sanchez et al., 2006), the only anthelmintic drug known to be effective against different stages of Fasciola species (immature and mature) (McKellar and Kinabo, 1991).
The present study aims to investigate the efficacy of five different anthelmintics against a naturally acquired Fasciola species infection in cattle in the middle Egypt.
2. Materials and methods
2.1. Animals
This work was carried out on reared cattle in an endemic zone by fasciolosis. This area located in the Nile river basin and surrounded by water from each side. Also, the animals were fed regularly from water grasses that grow in river basin at the time of examination. Fifty females' crossbred cattle, over the age of two years, and weighing from 300 to 400 kg (mean/350 kg) were selected for inclusion in the study. During the experiment duration (84 days), all animals were kept in the same conditions and ration (dry ration).
2.2. Experimental design and procedure
Before starting of drug application, fecal examination was carried out to report the egg count using the sedimentation test. The 55 animals represent the sum of the experimental groups (n = 50) and the control group (n = 5). The groups were divided randomly to 5 experimental groups (10 animals each); group 1 was treated by albendazole, group 2 was treated by rafoxanide, group 3 was treated by zanil (oxy-clozanide); group 4 was zanide treated (oxyclozanide and levamisole combination) and group 5 was treated by Fluke-scan (triclabendazole). The control group (n = 5) received no treatment. Individual fecal samples were collected on days 0, 1, 3, 7, 14, 28, 56 and 84 days post-treatment. Each faecal sample was taken per rectum for all animals. The fecal egg count of Fasciola eggs per gram (epg) was decided using a sedimentation technique. Two blood samples were also obtained on days 0 and 28 days post-treatment, from which liver enzyme concentration and total protein were meas ured.
2.3. Sedimentation technique
For each individual faecal sample, 3 g of faeces was added to 42 ml of water in a graduated cylinder. The contents were then mixed thoroughly, using a glass rod, and were poured through a tea strainer to remove large debris. The solution was then further passed through a sieve (mesh aperture 210 mm) into a conical flask and water was run through the sieve to ensure no eggs remained attached to the sieve. The filtrate was then allowed to sediment for 3 min after which the supernatant was siphoned off taking care not to disturb the precipitated matters. The latter was stained with two drops of methylene blue and the entire sediment placed on slide covered with a cover slip and viewed under a dissecting microscope (Labomed). The number of Fasciola eggs observed was counted and from this the number of epg was calculated (Mooney et al., 2009).
2.4. Evaluation of drug efficacy
The efficacy of the drugs was evaluated by measuring egg shedding. For each animal, fecal samples were collected on day 0, 1, 3, 7, 14, 28, 56 and 84 days (end of the study) after treatment. For detection of egg count per gram of feces (epg), the sedimentation technique was conducted, as previously mentioned above, according Mooney et al. (2009). The efficacy of the drugs was assessed by the reduction of mean egg excretion at each measurement point mentioned above, following the formula described by Foreyt (1988):
The anthelmintic efficacy percent was calculated by the equation:
Eggs number in untreated control group - eggs number in the treated one/Eggs number in untreated control group
2.5. Blood sampling and biochemical analyses
Puncturingthe jugular vein of cattle was carried out to obtain the blood samples in days 0, and 28 days post-treatment. Serum samples were analyzed for total protein (TP) concentrations; aspartate aminotransferase (AST), alanine transaminase (ALT). These were measured by standard methods using commercial kits supplied from Roche Diagnostics (Germany).
2.6. Statistical analyses
The eggs per gram feces are statistically analysis by using SPSS 17. Differences were considered significant for p < 0.05. The eggs per gram of faeces were uniformly distributed according to the Kolmogorov-Smirnov's Normality Test and were expressed as the mean ± SEM (standard error of the mean).
3. Results
3.1. Faecal egg count (epg) reduction in the treated groups
In day zero, the results showed that, the mean epg between different groups (treated and control non-treated) was non-
Table 1 - Anthelmintic treatments administered to cattle.
Group Anthelmintic Trade name Route Concentration Dosage/body
of use weight (kg)
1 Albendazole Albendazole Oral 2.5% 30 ml/100 kg
2 Rafoxanide Rafoxanide Oral 3% 2.5 ml/10 kg B.wt.
3 Oxyclozanide Zanil Oral 3.4 g/l 30 ml/100 kg
4 Oxyclozanide 15 mg/kg Zanide Oral Oxyclozanide 15 mg/kg 50 ml/100 kg
Levamisole 7.5 mg/kg Levamisole 7.5 mg/kg
5 Triclabendazole Fluke-scan Oral 10% 6 ml/50 kg
significant (before treatment). Also there was no difference between all groups in the epg feces count on first and third days after treatment in the treated groups. In the 7th day after treatment, the fecal egg count in the treated groups began to decrease. At the day 14 post-treatment, the egg count per gram feces in the albendazole and rafoxanide groups decreased significantly (p < 0.05) than the control one but the eggs did not disappear till the end of the experiment (84 days) except in few individuals the egg count was zero. Mean fecal egg output was moderately higher on day 56 in albendazole and rafoxanide treated groups when compared to the previous period (those of days 14 and 28). So the efficacy of these drugs (albendazole and rafoxanide) was 75% & 80.58% respectively. Results are shown in Table 1.
In the other hand, the zanil- and zanide-treated groups showed a significant (p < 0.05) reduction in the egg count from the day 7 post-treatment in the treated animals. Although the efficiency of the used therapeutics was observed on days 14 and 28 in all the treated groups but zanide and zanil were the most effective drugs in reducing the mean epg during the same period of the study to zero. Finally, in zanil- and zanide-treated groups, the eggs disappeared from feces after 14 days post-treatment and did not return again till the end. So, the efficiency of zanil and zanide was 100% reduction of eggs depending on the last reading of egg count at the end of the experiment (84 days post-treatment) (Table 2). Nearly the same results were reported in triclabendazole treated group.
3.2. Results of liver enzymes before and after treatment
In regard to the hematologic and biochemical evaluations, cattle orally treated with albendazole, rafoxanide, zanil and
zanide and triclabendazole developed moderate liver damage when compared to that of the control group (GPT & GOT) (Table 3). Meanwhile the total protein in the treated groups and untreated was similar.
4. Discussion
Fasciolosis remains one of the most important helminthic diseases of livestock in many countries in the world. However, the cattle infection was chronic causing a major economic impact due to reductions in weight gain milk yield and fertility and to liver damage.
As regards to anthelmintic use, we observed in the present study that albendazole and rafoxanide were the treatments that had a limited effect on fasciolosis, as their administration decreased the egg count number but the eggs did not disappear. This may be referred to that albendazole and rafoxanide affect only the adult stages of Fasciola species while the immature stages developed to reach adult stages so that it may be explained why eggs still discharge in this treated group. Also this may explain the efficacy of these therapeutics were 75 & 80.58% respectively. Even though, the treatment by albendazole and rafoxanide was done, most treated cases gave eggs in feces after treatment; this may be attributed to the presence of resistance to these drugs. Albendazole and rafoxanide were used common in Egypt from many years and it applied in random manner by farmers. Their use in treatment and prophylaxis against gastrointestinal helminths was repeated in manner may be lead to give strain resistant to it by time. Similar results were obtained in cattle naturally infected with Fasciola hepatica by Rapic et al. (1988). This result also
Table 2 - Means of fecal egg count/3 g of feces before and after administration of the five anthelmintics used.
Drugs' times Albendazole Rafoxanide Zanil Zanide Triclabendazole
Zero day 30 ± 2.2 30.9 ± 2.3 28.0 ± 1.98 29.4 ± 1.4 30.9 ± 2.3
One day 31.5 ± 2.3 30.9 ± 2.3 27.2 ± 1.87 29.4 ± 1.4 30.9 ± 2.3
3 Days 15.5 ± 1.2a 16 ± 1.2a 12.8 ± 0.7a 13.1 ± 0.9a 16 ± 1.2a
7 Days 1.2 ± 0.6a 3.5 ± 1.0a 1.3 ± 0.5 0.6 ± 0.3a 1.2 ± 0.6a
14 Days 1 ± 0.5a 3.5 ± 1.0a 0a 0a 0a
28 Days 1.1 ± 0.5a 3.1 ± 0.8a 0a 0a 0a
56 Days 5.3 ± 1.3a 4.8 ± 1.5a 0a 0a 0a
84 Days 7.5 ± 1.8a 6.0 ± 1.5a 0a 0a 0a
Efficacy (75%) (80.58%) (100%) (100%) (100%)
a Indicates a value that is statistically significant different from the corresponding value in "zero day" means significant with zero day.
Table 3 - Measurements of total protein and liver enzymes in the treated groups (at zero day and 28 days post-treatment).
Time (days)/groups/ Control group Albendazole Rafoxanide Zanil Zanide Triclabendazole
enzymes (before treatment) group group group group group
ASTa (u/l) 24-31 18-21 18-21 18-21 18-21 18-21
ALTb (u/l) 80-90 55-75 55-75 55-75 55-75 55-75
Total protein (g/dl) 6.5-7 6-6.7 6-6.7 6-6.7 6-6.7 6-6.7
a Aspartate aminotransferase (AST). b Alanine transaminase (ALT).
obtained by Keyyu et al. (2008), they reported that one to two (14-33%) of animals treated with albendazole were positive for Fasciola eggs two weeks after treatment. Same results were stated by Elitok et al. (2006) in turkey, albendazole and rafox-anide efficacy was 66.7 and 68.2% and they referred this lower efficiency to the resistance of Fasciola to these drugs.
The results for zanil- and zanide-treated cattle in the present study indicated that these drugs are highly effective on liver fluke therapy. The fecal egg count reductions for zanil and zanide were 100%. The majority of zanil- and zanide-treated cattle had nearly negative fecal egg counts from day 7 till the end of the experiment (84 days after treatment). This clinical field test for egg reduction affords valuable information, even though the number of flukes removed is unknown (Wood et al., 1995). Also they mentioned that a suitable time to evaluate the efficacy of a compound is at days 14 or 21 post-treatment. Athar et al. (2011) used oxyclozanide as flukicide with efficiency of 96%. Also Mooney et al. (2009) recoded that oxyclozanide is effective with faecal egg count being reduced by 100% by day 14 post-treatment. The results of Keyyu et al. (2008) showed that 14-33% of treated animals with oxy-clozanide had Fasciola species eggs and this antagonize with the present work. However, zanil and zanide are new on the market in Egypt. It has never been used in the study area. This may explain why no resistant to F. gigantica strain were observed.
Regarding the results of triclabendazole treated group, the results indicated that all animals treated with triclabendazole didn't give any eggs of Fasciola in faeces two weeks after treatment and its efficacy was 100% at the end of the study. This may be referred to the use of triclabendazole in this area is not common. However, the current results are not in agreement with the findings of Moll et al. (2000) and Gaasenbeek et al. (2001), who reported the presence of triclabendazole resistance. Elitok et al. (2006) reported resistance to triclabendazole in turkey as this drug is commonly used in cattle. These variations with our results may refer tricla-bendazole using in the study area is uncommon. In contrary the fore mentioned authors that using of triclabendazole is common.
In the present study, we used 5 anthelmintics. These therapeutics were divided into two divisions; benzimidazole group which it causes degenerative alterations in the tegument and intestinal cells of the worm by binding to the colchicine-sensitive site of tubulin, thus inhibiting its polymerization or assembly into microtubules. This group includes albendazole and triclabendazole. From our results we found that it may be a resist to albendazole and there is no resist to triclabendazole. We haven't view for this. The other
division, salicylanilides/substituted phenols act by uncoupling of oxidative phosphorylation in flukes. This includes rafoxanide and oxyclozanide. We found that rafoxanide resist but no resist for oxyclozanide.
Consequently, our results indicate that, zanil, zanide and triclabendazole are highly effective compounds for the treatment of Fasciola species infection in cattle under these field conditions in this area of Egypt.
Acknowledgments
The team of this work thanks Dr. Ayman Mostafa for measuring the biochemical enzymes.
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