Resistência múltipla á anti-helmínticos num rebanho ovino ... · disofenolat (T3), oxfendazol (T4) and levamisole hydrochloride (T5), administered orally according to the manufacturer’s

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RCPV (2014) 109 (589-590) 33-37

Resistência múltipla á anti-helmínticos num rebanho ovino no sul do Brasil

Anthelmintic multiple resistance in sheep flock in southern Brazil

Fernanda C.C. Santos*; Andréia Buzatti1; Marciele M. Scheuermann1; Victor F.B. Roll2 ; Fernanda S.F. Vogel1

1Laboratório de Doenças Parasitárias (LADOPAR), Universidade Federal de Santa Maria (UFSM), Avenida Roraima, 1000, Bairro Camobi, Santa Maria, Rio Grande do Sul (RS), Brasil.

2Departamento de Zootecnia, Faculdade de Agronomia Eliseu Maciel, Universidade Federal de Pelotas (UFPel), Campus Universitário, Pelotas, RS, Brasil.

Summary: The control of gastrointestinal endoparasites in li-vestock relies on antihelmintic drugs administration, being this performed inadequately, resulting in stimulation of parasite re-sistance. The aim of this experiment was to evaluate the effi-ciency of 5 active principles against gastrointestinal endoparasi-tes in a sheep flock in the southern Brazil. For this, 54 sheep were divided into 6 groups with 9 animals in each. The active princi-ples tested were moxidectin (T1), ivermectin 1% (T2), levamisol disofenolat (T3), oxfendazol (T4) and levamisole hydrochloride (T5), administered orally according to the manufacturer’s ins-tructions. Examinations of egg counts per gram of feces (EPG) and fecal culture were performed at days 0, 7 and 14. For statis-tical evaluation, analysis of variance for repeated measures and comparisons of means by LS Means test (p <0.05) were perfor-med. The efficiency of treatments T1 to T5 was 54 and 62%; 21 to 33%; 98% and 84; 1 and 0%; 88 and 68% for day 7 and 14, respectively. Moxidectin, ivermectin, oxfendazol and levamiso-le hydrochloride presented low efficacy. Levamisol disofenolat was the only drug highly effective. The low efficiency of most drugs tested demonstrated the importance of technical guidance in rational use of anthelmintics and dissemination of appropriate control parasites measures.

Keywords: endoparasites, gastrintestinal parasites, parasite re-sistance, pharmacological

Resumo: O controle de endoparasitas gastrintestinais em ani-mais de produção é baseado na administração de anti-helmínti-cos, sendo estes realizados de forma inadequada, resultando em estímulo á resistência parasitária. O objetivo deste experimento foi avaliar a eficiência de 5 princípios ativos comerciais contra a endoparasitas gastrintestinais em um rebanho ovino no Sul do Brasil. Para isto, 54 ovinos foram distribuídos em 6 grupos com 9 animais em cada um. Os princípios ativos testados foram mo-xidectina (T1), ivermectina 1% (T2), disofenolato de levamisol (T3), oxfendazole (T4) e cloridrato de levamisole (T5), adminis-trados via oral conforme as indicações do fabricante. Os exames de contagem de ovos por grama de fezes (OPG) e coprocultura foram realizados nos dias 0, 7 e 14. Para avaliação estatística foi realizado análise de variância para medidas repetidas e as com-parações das médias pelo teste LS Means (p<0.05). A eficiência dos tratamentos T1 a T5 foi de 54 e 62%; 21 e 33%; 98 e 84%; 1 e 0%; 88 e 68%, para os dias 7 e 14, respectivamente. Moxidec-tina, ivermectina, oxfendazole e cloridrato de levamisole apre-

sentaram baixa eficácia. O disofenolato de levamisol foi o único medicamento eficiente. A baixa eficácia da maioria dos produtos testados demonstra a importância da orientação técnica no uso racional dos anti-helmínticos e difusão de medidas de controle apropriado de parasitas.

Palavras-chave: endoparasitas, farmacologia, parasitas gastrin-testinais, resistência parasitária

The worlds 1.8 billion small ruminant population comprising 0.7 billion goats and 1.1 billion sheep pro-vide milk, meat and fiber (Faostat, 2010). Brazil is the 8th largest sheep and goat producer in the world, with 25.8 million animals. One of the most important dise-ases that limit the production of small ruminants in the tropics and sub-tropics is parasitism by gastrointestinal nematodes (Fao, 1998) and in Brazil this disease is re-cognized as a major problem in increasing production (Ramos et al., 2002).

Parasiticides account for over 28% of the global spend on animal health products (IFAH Annual Re-port, 2009) and for the last half century, it have been used almost exclusively to control these diseases and limit their effects. Unfortunately, the extensive use of chemicals has inevitably led to the emergence of para-site populations resistant strains. To date, anthelmintic resistance has been recorded in many of the common parasites of sheep and it is particularly prevalent in the most economically important parasites, such as Hae-monchus contortus, Trichostrongylus spp., Fasciola hepatica and Nematodirus spp. (Jackson, 1993).

Parasitism is an important limiting factor in lives-tock production and, although the history of the bro-adspectrum anthelmintics started in early 1960s with a strong empirical knowledge and superior outcomes, the present situation is alarming and is clearly worse-ning, with consequences arising from the development of multidrug resistant parasite populations (Hughes et al., 2007). Multiple anthelmintic resistance involving ivermectin closantel, albendazol, levamisol and mo-

*Correspondência: [email protected] Tel: +55 (55) 3220-8071

Santos F. et al. RCPV (2014) 109 (589-590) 33-37

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xidectin have been reported previously (Waruiru et al., 1997; Molento e Prichard, 1999; Armson et al., 1995). The inadequate management of anthelmintics and their indiscriminate use are among the main ope-rative factors that lead to the occurrence of this phe-nomenon (Fao, 2003).

The aim of this experiment was to evaluate 5 active principles administered orally in a sheep flock natu-rally infected in the southern Brazil.

The experiment was conducted in the Agricultu-ral College of the Federal University of Santa Maria (UFSM) installations, localized in the Central De-pression of Rio Grande do Sul, Brazil. It was used 54 sheep, both sexes, aged from 2 to 6 years, Texel x Ile de France. The flock remained in the same paddock of native grassland, continuous grazing, with water ad libitum. The last administration of anthelmintic was performed 45 days before the beginning of the experiment.

The sheep were randomly divided into 6 groups of 9 animals, each group representing an active prin-ciple to be tested. The active principles tested were moxidectin (T1), ivermectin 1% (T2), levamisol di-sofenolat (T3), oxfendazol (T4) and levamisol hydro-chloride (T5), respectively. The T6 was the untreated control group. The doses followed the manufacturer’s recommendation and were all orally administered (Table 1).

Table 1 - Dose of active principles tested according to treatment.

TreaTmenTaCTIVe

PrInCIPLeDOSe

1 Moxidectin 1mL/10 kg BW

2 Ivermectin 1% 2,5mL/10 kg BW

3Levamisol disofenolat

1mL/10 kg BW

4 Oxfendazol 1mL/9 kg BW

5Levamisol

hydrochloride1mL/10 kg BW

6 Control -

The animals received all treatments on the same day, which was considered day 0. Fecal samples from all sheep were collected at day 0, 7 and 14 for the exams egg counts per gram of feces (EPG) and fecal cultu-re in pool of the groups, according the methodology described by Gordon e Whitlock (1939) and Roberts’ Sullivan (1950), respectively. Treatment efficiency was calculated by fecal egg count reduction test (FECR).

Statistical analysis was performed with data transfor-med in square root (x+1) by repeated measures analysis. It was obtained the main effects means, interactions of pairs (treatment x time) and the mean comparison test was performed by LS Means (p <0.05). For modeling the variance and covariance matrix, three structures

were tested: (1) auto-regressive first order, (2) com-pound symmetry and (3) unstructured. In choosing the matrix of variance and covariance, it was used Akaike Information Criterion to select the one possessing lo-west value.

EPG means, efficiency of treatment and fecal cultu-res are demonstrated in Table 2 and 3.

Table 2 - Percentage of reduction, minimum, average and ma-ximum values of EPG from sheep treated with moxidectin (T1), ivermectin (T2), levamisol disofenolat (T3), oxfendazol (T4), levamisol hydrochloride (T5) and control group.

TreaTmenTDaY

0 7 14

1 2800 A, a 1262 B, a 1044 B, a

% reduction 54% 62%

Mininum values 200 0 0

Maximum values 4500 4700 4400

2 3183 A, a 2483 B, b 2125 B, b

% reduction 21% 33%



3 2800 A, a 33 B, b 288 B, b

% reduction 98% 89%



4 2166 A, a 2150 B, b 3187 B, b

% reduction 1% 0%



5 3888 A, a 457 B, a 1212 B, a

% reduction 88% 68%



Control 2044 A, a 2787 A, a 3133 A, a

% reduction 0% 0%



Means followed by different capital letters in the same row differ significantly (p<0.05) by Chi-square test. Means followed by different lowercase letters in the same column differ significantly (p<0.05) by Chi-square test. The interaction between treatment and time was significant (p<0.001).


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The results of this study indicate that oxfendazol was completely ineffective in the flock examined, as no FECR was observed in treated animals. Moxidectin and ivermectin 1% have retained some efficacy in the flock described, clinical improvement of the affected animals may be expected in the short term, but the low mean of FECR (21-62%) is a cause of concern and in-dicates a considerably reduced efficacy of this drug. Levamisol hydrochloride presented 88% efficiency at day 7, but at day 14 it droped to 68%, considered low mean. Levamisol disofenolat was the only drug with high efficiency (>95%). In a fully effective anthelmin-tic, a FECR of at least 95% (and a lower 95% confi-dence interval) has to be expected (Coles et al., 1992).

For the repeated measures analysis it was chosen ARH structure due the lowest value for Akaike In-formation Criterion. Based on the repeated measures analysis there was no significant effect of treatment (p = 0.0150), time period (p = 0.006) and interaction between treatment and time (p = 0.0002).

As shown in Table 4, there was no difference in EPG values between groups in day 0. On subsequent days, it was observed that moxidectin, levamisol disofenolat and hydrochloride presented reduction in EPG values on day 7, whereas in the control group and in oxfendazol treatment there was no EPG reduction in the same day. Ivermectin treatment presented a rise in EPG value according to time, indicating in this case parasite resistance to the product.

Table 3 - Percentage of helmint larvae identified in coproculture according to the treatments moxidectin (T1), ivermectin (T2), levamisol disofenolat (T3), oxfendazol (T4), levamisol hydrochloride (T5) and control group.

TreaTmenT GenreSDaY eFFIenCY eFFIenCY

0 7 14 0-7 0-14

Haemonchus 84 88 100 0 0

1 Cooperia - - - - -

Trichostrongylus 6 - - 100 100

Ostertagia 10 12 - 0 100


2 Cooperia 3 7 - 0 100

Trichostrongylus 7 13 - 0 100

Ostertagia 3 - - 100 100

Bunostomum - 1 26 0 0


3 Cooperia - 2 10 0 0

Trichostrongylus 1 76 53 0 0

Ostertagia - - - - -

Bunostomum - - - - -


4 Cooperia 4 - - 100 100

Trichostrongylus 5 5 14 0 0

Ostertagia - 47 - 0 0

Bunostomum 1 - - 100 100


5 Cooperia - - - - -

Trichostrongylus 16 25 - 0 100


Bunostomum - - 1 0 0

Chabertia 2 - - 100 100

Haemonchus 92 96 99 - -

Control Cooperia 2 4 - - -

Trichostrongylus 2 - 1 - -



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Table 4 - Variance analysis of repeated measures of moxidectin (T1), ivermectin (T2), disofenolato of levamisole (T3), oxfenda-zole (T4), levamisole hydrochloride (T5) and control group (T6) in a sheep being evaluated by EPG (square root x+1) on days 0, 7 and 14 post anthelmintic administration.

TreaTmenTDaY

0 7 14

1 49,28 A, a 22,60 B, a 23,30 B, a

2 30,57 A, a 58,30 B, b 55,98 B, b

3 38,10 A, a 4,01 B, a 10,38 B, a

4 39,45 A, a 37,64 A, b 49,44 A, b

5 56,60 A, a 13,22 B, a 22,10 B, a

6 41,70 A, a 45,45 A, b 49,67 A, b

Means followed by different capital letters in the same row differ significantly (p<0.05) by Chi-square test. Me-ans followed by different lowercase letters in the same column differ significantly (p<0.05) by Chi-square test. The interaction between treatment and time was signifi-cant (p<0.001).

The high frequency of varying anthelmintic treatments is very likely to play a significant role in selecting highly resistant worm population (Voigt et al., 2012). Several studies have revealed the existence of gastrointestinal nematodes resistant to antiparasitic drugs in sheep in southern Brazil (Santos e Gonçalves, 1967; Echevarria, 1996; Waller et al., 1996; Farias et al., 1997). Multiple resistance to the three main classes of drugs with anthel-mintic action (avermectins/milbemicins, benzimidazoles and imidazothiazoles) is not rare in this region (Waller et al., 1996).

Drug combinations could possibly restore the effecti-veness of treatments. However, these resources are likely to become ineffective with long-term use, when parasites are resistant to all classes of available drugs. Furthermore, alternative methods of parasites control have been proven to be more efficient when used as a complement to che-motherapy, once it can delay the development of parasite resistance and preserve the usefulness of the antiparasitic drugs (Waller, 2006).

In addition, on the contrary of the appearances (lack of clinical signs), the presence of resistant nematodes infec-ting sheep recently introduced in an farm is very probable, due the anthelmintic regimen which is performed in most areas of Rio Grande do Sul (Molento, 2009). Because of this, these animals may transport and spread resistant strains to a new farm (Wolstenholme et al., 2004) or fair.

The general consensus is that anthelmintic resistance appears to be a pre-adaptive heritable phenomenon with the gene or genes conferring resistance, being present wi-thin the parasite population even prior to the drug being used for the first time (Silvestre e Hubert, 2002). Under these circumstances resistance arises as a result of selec-tion through exposure of the worm population to anthel-mintic drugs, especially when it is performed inadequa-tely. Intensive exposure to antiparasitic drugs and using this as the only control method accelerates the process

considerably. Side resistance between drugs with similar mechanisms of action is also common (McKellar e Jack-son, 2004).

A difficult situation regarding gastrointestinal nema-todes control was observed in the target flock of the study, since the endoparasite population was resistant to most of the classes of anthelmintic drugs current available for ruminants in the Brazilian market. Le-vamisol disofenolat is a new anthelmintic developed recently by a Brazilian company. Up to this date, this drug is still fully effective against nematode strains. In order to maintain a long-term efficacy of anthelmintic, the responsible use of this classical drugs is crucial in fighting further development and spread of resistant worm populations. Present results confirmed the im-portance of establishing programs of parasite control. In this context, treatments should be applied at the mi-nimum frequency that is feasible and management me-asures that decelerate the development of the parasite resistance should be employed. Quarantine measures, veterinary input in parasite control programmes and pre and post-treatment fecal egg counts also needs to be better promoted within the farming community.

In this flock, moxidectin, ivermectin, oxfendazol and levamisol hydrochloride did not present fully effecti-ve anthelmintic action. Levamisol disofenolat was the only antihelmintic with high efficiency, being this a new molecule. The present study illustrates the alar-ming lack of anthelmintic drugs efficiency in sheep. It is important to establish alternative strategies of management in a broad program of parasite control for reducing the selection pressure on parasites by the commercially available anthelmintics.

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Resistência múltipla á anti-helmínticos num rebanho ovino ... · disofenolat (T3), oxfendazol (T4) and levamisole hydrochloride (T5), administered orally according to the manufacturer’s