Anthelmintic efficiency of doramectin, fenbendazole, and nitroxynil, in combination or individually, in sheep worm control

The anthelmintic efficiency of doramectin, fenbendazole, and nitroxynil, used individually or in combination, was determined by the Fecal Egg Count Reduction (FECR) test and cultivation of larvae of anthelminthic-treated sheep grouped as follows: G1 (doramectin), G2 (fenbendazole), G3 (nitroxynil), G4 (doramectin + fenbendazole), G5 (doramectin + nitroxynil), G6 (fenbendazole + nitroxynil), G7 (doramectin + nitroxynil + fenbendazole), G8 (untreated). In addition to individually used doramectin and fenbendazole, the helminths were also resistant to the combination of doramectin + fenbendazole; nitroxynil + fenbendazole; and doramectin + nitroxynil + fenbendazole, with their FECR rates ranging from 62-83%. The helminths showed possible nitroxynil-resistance, but had low resistance when the drug was administered in combination with doramectin. The evaluation of individual helminth species revealed that fenbendazole was fully effective against Cooperia; doramectin (G1), moderately effective against Haemonchus and insufficiently active against Cooperia; nitroxynil, effective against Haemonchus and insufficiently active against Cooperia. It was concluded from the results that herd nematodes are resistant to doramectin, fenbendazole, and nitroxynil, and that the combined use of the drugs not only fails to significantly improve the anthelmintic efficiency against Haemonchus and Cooperia, but is also cost-ineffective.


Introduction
According to the data from the Brazilian Institute of Geography and Statistics (IBGE), sheep farming in Paraná, Brazil, has been observed to be slowly increasing in recent years. In the penultimate livestock census, in 2009, the herd size was estimated to be 599,925 head of sheep, which corresponded to 12.5% of the total sheep population in southern Brazil (IBGE, 2013). The last census estimated a 6.7% increase in the number of sheep a small increment, considering the effective increase in the sheep population over the last four years in other states of southern Brazil, which was estimated to be 12.97% and 7.72% in Santa Catarina and Rio Grande do Sul, respectively.
The main obstacle to sheep production is the negative economic impact due to health problems that affect the sheep, among which, infections with gastrointestinal parasites stand out. Chemical method of control remains the most used among the endoparasite control methods. However, failure in using this method effectively has favored the emergence of resistant strains of parasites (SANGSTER, 2001).
Drugs with benzimidazoles and avermectins are commonly used for deworming sheep. However, due to reports of development of resistance in parasites to these anthelmintics, less common alternatives have been investigated, such as compounds with phenolic group substitutions and salinomycins, which are active against Fasciola, and are also used to treat haemoncose in sheep and cattle. Oxyclozanide is active mainly against adult worms, while nitroxynil, rafoxanide, and closantel are active against parasites in both adult and immature stages (BISHOP, 2005).
The resistance of helminths to anthelmintic drugs can be defined as an increase in the ability of certain strains of parasites to resist or survive doses of a drug that would normally destroy most individuals of the same species. The various mechanisms of resistance include modifying the action of the target molecule, increasing the number of targets receptors, increasing the rate of drug excretion, increasing the metabolism of the active ingredient, or simply, drug sequestration (KELLY & HALL 1979;TORRES-ACOSTA & HOSTE, 2008). A large variation in the degree of resistance of various nematode populations against anthelmintics have been widely reported the world over for several decades (JACKSON, 1993;MELO et al., 2003;COLES, 2005;BORGES et al., 2008;CONDI et al., 2009;SCZESNY-MORAES et al., 2010).
The aim of this study was to evaluate the anthelmintic efficacy of doramectin, fenbendazole, and nitroxynil, individually or in combination, in sheep that were naturally infected by gastrointestinal nematodes in a rural property in northern Paraná.

Materials and Methods
The study was conducted in a rural property in the municipality of Ribeirão Claro, north of Paraná State, between April 2012 and June 2012. Of the total 420 sheep of farm, 180 weaned Suffolk ewes were selected for this study. The number of eggs per gram of feces (EPG) was enumerated for all the 180 animals, and from these, only those animals with EPG counts below 1000 were selected for the final study. Treatment groups were formed after randomization based on the EPG values, such that the mean EPG of the animals in each group was more than 150, which corresponds to the threshold for anthelmintic resistance and efficiency tests in sheep (COLES et al., 2006).
The fecal samples from all of the animals were collected on day zero, prior to the treatment, and on days 7, 14, and 21 post-treatment, and evaluated by the technique developed by Gordon & Whitlock (1939). To calculate the fecal egg count reduction (FECR), the samples collected on day 7 were evaluated, and the corresponding anthelmintic efficiency was established according to the guidelines given by Coles et al. (1992). The larvae from the samples collected on day 7 were cultivated according to the technique developed by Roberts & O'Sullivan (1950), in order to verify the efficacy of the anthelmintics against each of the nematode genera identified in the samples. The FECR values were calculated using the following formula: FECR= 1 -(mean EPG treated group on day 7/mean EPG control group on day 7) x 100. The larvae (L3) were identified according to the criteria put forth by Keith (1953).
Based on the WAAVP recommendations (World Association for the Advancement of Veterinary Parasitology) helminths are considered to be resistant to a particular drug when their FECR values seven days post-treatment are less than 95%, and the lower limit of the confidence interval at 95% is less than 90%. In the absence of these results, the helminths are categorized as having suspected resistance (COLES et al., 1992). In accordance with these recommendations, we categorized the resistance as proposed by Vizard & Wallace (1987): the presence of resistance when the FECR was less than 95%, and the lower limit of the confidence interval at 95% was less 90%; low resistance when the FECR exceeded 95%, and the lower limit of confidence interval at 95% was less than 90%; and sensitive when the FECR exceeded 95%, and the lower limit of confidence interval at 95% was greater than 90%.
The efficacy of the treatment on different days of evaluation and that for the nematode genera that were identified were calculated using the formula below: where: LPG: larvae per gram of feces, calculated by multiplying the value of EPG with the percentage of each sex of the larvae found in the culture X: genus of larvae identified GC: control group GT: treated group The efficacies were categorized as: highly effective when Efficacy of X > 98%; effective when 90-98%; moderately effective 80-89%; and insufficiently active < 80% (Ordination 79/1996MERCOSUL, GMC, 1996. The cost of each of the treatments was evaluated by the analysis of variance and the comparison of the means by the Tukey's test. The values of EPG were log-transformed (log x + 1), and the means compared by paired and unpaired t-tests. Statistical analyses were performed using the statistical program GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA).

Results and Discussion
The EPG of feces of the animals of all of the groups was observed to have decreased seven days after treatment. The mean EPG values of only those animals treated with a combination of doramectin and fenbendazole (G4) remained significantly lower (P = 0.002) than those of animals given treatment combinations throughout the experimental period. As expected, there was no significant decrease in the EPG values in the control animals ( Table 1).
The comparison of the EPG values between the groups revealed that the animals treated with nitroxynil (G3), a combination of doramectin and fenbendazole (G4), and a combination of doramectin and nitroxynil (G5), had EPG values significantly lower than those of the control group (P = 0.22, 0.0456, and 0.0203, respectively), respectively, seven days after treatment. On day 14, the only groups that showed no significant decrease in the EPG values in comparison with those of the control group were those that were individually treated with doramectin (G1) and nitroxynil (G3). Until day 14 of the experiment, none of the treatment groups showed any significant differences in the EPG values between themselves. The EPG values of all of the treated groups were lower than those of the control group, on day 21. However, when comparing the treated groups with each other, it was found that the group of animals treated with doramectin in combination with fenbendazole and nitroxynil (G7) showed an FEC value significantly lower (P < 0.0001) than that of the group of animals treated with the same combination, but without doramectin (G6), thus, demonstrating an additive effect of doramectin. This effect might be attributed to two causes: a synergistic action by summation, since the three drugs have different modes of action (NIES & SPIELBERG, 1996), and a slow rate of absorption (2.71 days) and more persistent plasma concentrations of the drug (MARRINER & BOGAN, 1981;ATTA & ABO-SHIHADA, 2000). This hypothesis is strengthened by comparing the FECR rates of the animals of G3 (nitroxynil) and G5 (doramectin + nitroxynil) (93% and 95%, respectively), which implies that the increase in the FECR rate of group G5 by 2.15% might have been due to doramectin treatment (Table 2).
Despite the significant decrease observed in the mean EPG values evaluated on several days in virtually all the treatment groups, the helminths were categorized as being resistant to treatment with doramectin (G1) and fenbendazole (G2) individually, and to treatment with combinations involving doramectin + fenbendazole (G4), fenbendazole + nitroxynil (G6), and doramectin + fenbendazole + nitroxynil (G7), as could be evaluated based on their FECR rates (83%, 80%, 83%, 83%, and 62%, respectively). Nitroxynil resistance was suspected in helminths from the animals of G3, which showed an FECR rate of 93%, with a 91.2% lower limit Different letters right of EPG averages represent significant difference (p <0.05) between days in the same group (lines), calculated by "pared t" test. Different letters left of EPG averages represent significant difference (p <0.05) between groups in the same day (column), calculated by "unpared t" test. ***most significant difference (p<0.0001). of the confidence interval at 95%; the helminths also showed low resistance when nitroxynil was used in combination with doramectin (G5). Groups G5 and G3 had the best FECR rates. The animals of both the groups were administered nitroxynil, with only those in G5 being administered doramectin additionally. However, a group very similar to G3 and G5 in terms of the treatment, the G7 (doramectin + nitroxynil + fenbendazole), had the worst FECR rates (62%) among all the groups in the study. This was because of one particular animal in G7, which presented EPG values much higher than those of the rest of the animals in the other two groups (G3 and G5). If this animal were removed from the group, the reevaluated FECR rate would increase to 94%, which is comparable to those of G3 and G5 (93% and 95%, respectively), suggesting that fenbendazole neither contributed to the improvement of the efficacy of the treatment, nor was it responsible for any antagonism (Table 2). Cezar et al. (2010) proposed that random combinations of drugs are not advisable as they might increase the treatment costs without adding to the efficacy of their anthelminthic activity, and might also speed up the problem of development of cross-parasitic resistance.
The cultivation of larvae from the feces of the animals of all the groups revealed a higher occurrence of the larvae of the Haemonchus and Cooperia genera, with the former being found at a higher frequency. The efficacy of the treatments against these genera varied from 62%, with the combination of doramectin + fenbendazole + nitroxynil (G7) to 93.2% with nitroxynil (G3) for the Haemonchus genus. Regarding the efficacy of the treatment against particular genera, only fenbendazole proved fully effective (100%) against Cooperia, and predictably, all other combinations containing this drug (G4, G6, and G7) were also effective against the parasite, demonstrating that, despite several years of exposure to this drug in the examined farm, Cooperia had not yet established a resistance to it.
The results indicate that fenbendazole was moderately effective against Haemonchus and highly effective against Cooperia. Doramectin (G1) was found to be moderately effective against Haemonchus and insufficiently active against Cooperia, and nitroxynil (G3) was effective against Haemonchus and insufficiently active against Cooperia. It was not possible to establish the efficacy of the combination of doramectin + nitroxynil (G5) against each of the nematodes genera because the cultivation of the larvae from the feces of the animals of this group yielded no growth (Table 3).
The anthelmintic properties of fenbendazole were first presented in 1975, who reported that the drug eliminated 100% of the Haemonchus contortus larvae in 3-10 days at doses of 3.5 mg/kg of body weight (KIRSCH & DÜWEL, 1975). Santiago et al. (1978) also reported high levels of efficacy of fenbendazole.
The results of this study corroborate those of studies by Chartier et al. (1998) and Palcy et al. (2010) in France, Bartley et al. (2004Bartley et al. ( , 2005 in Scotland, and Farias et al. (1997), Melo et al. (2003), and Cruz et al. (2010) in Brazil, who identified the inefficacy of fenbendazole in most of the surveyed sheep farms.
In Chile, similar studies demonstrated, that in sheep treated with fenbendazole, the FECR rates 7-days post-treatment were 41%, and the genera that had highest resistance to fenbendazole were Trichostrongylus and Teladorsagia (TORO et al., 2014).
The parasitic resistance against doramectin, and its low efficacy against the organisms of the Haemonchus and Cooperia genera found in this study corroborate the findings of the studies by Cruz et al. (2010) in Rio de Janeiro and Borgsteede et al. (2007) in the Netherlands. The latter reported only 15% efficacy of the drug, as indicated by its FECR values. However, the results of a previous study by Echevarria et al. (2000) in Rio Grande do Sul contradict these results.
The results observed in our experiment are consistent with those of several other recently published studies. Cruz et al. (2010) showed nitroxynil efficacies ranging from 76% to 100%, Vila Nova et al. (2014) too identified resistance in nematodes to nitroxynil treatment, with overall FECR rates of 66%, despite those of the larvae of Haemonchus being high (91%). In our study, despite the insufficient reduction in the total nematode count (less than 95%), the reduction in the larvae of the Haemonchus genera was noted to be high (93.2%).
With regard to the efficacy noted with the drugs administered individually or in combination, the results of this study corroborate those presented by Sczesny-Moraes et al. (2010), but contradict those reported by Soccol et al. (2004), Buzzulini et al. (2007), Cezar et al. (2010), and Holsback et al. (2013), all of which found higher efficacies with the administration of drug combinations compared to those obtained with individual drugs.
It is concluded from the results of this study that herd nematodes are resistant to doramectin, fendendazole, and nitroxynil, and that the efficacy levels of these drugs are unsatisfactory despite their administration in combination. The drug combinations of doramectin, fendendazole, and nitroxynil improve neither the anthelmintic efficiency, nor the efficacy against Haemonchus and Cooperia. In addition, the use of drug combinations is more expensive, and the resultant efficacy does not justify the high costs of treatment. Despite the cessation of the use of nitroxynil and fendendazole for several years, it can be concluded from our results that the parasitic resistance acquired by helminths persists for a long period after their use has been stopped. Table 3. The number of larvae per gram (LPG) of feces of the animals of groups G1-Doramectin, G2-Fenbendazole, G3-Nitroxynil, G4-Doramectin + Fenbendazole, G5-Doramectin + Nitroxynil, G6-Fenbendazole + Nitroxynil, G7-Doramectin + Fenbendazole + Nitroxynil, and G8-Control, on days 0 and 7, and the efficiency (%) of the treatments.