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

Resistncia mltipla anti-helmnticos num rebanho ovino no sul do Brasil

Anthelmintic multiple resistance in sheep flock in southern Brazil
Fernanda C.C. Santos*; Andria Buzatti1;
Marciele M. Scheuermann1; Victor F.B. Roll2 ; Fernanda S.F. Vogel1
1

Laboratrio de Doenas Parasitrias (LADOPAR), Universidade Federal de Santa Maria (UFSM),

Avenida Roraima, 1000, Bairro Camobi, Santa Maria, Rio Grande do Sul (RS), Brasil.
Departamento de Zootecnia, Faculdade de Agronomia Eliseu Maciel, Universidade Federal de Pelotas (UFPel),
Campus Universitrio, Pelotas, RS, Brasil.

Summary: The control of gastrointestinal endoparasites in livestock relies on antihelmintic drugs administration, being this
performed inadequately, resulting in stimulation of parasite resistance. The aim of this experiment was to evaluate the efficiency of 5 active principles against gastrointestinal endoparasites in a sheep flock in the southern Brazil. For this, 54 sheep were
divided into 6 groups with 9 animals in each. The active principles tested were moxidectin (T1), ivermectin 1% (T2), levamisol
disofenolat (T3), oxfendazol (T4) and levamisole hydrochloride
(T5), administered orally according to the manufacturers instructions. Examinations of egg counts per gram of feces (EPG)
and fecal culture were performed at days 0, 7 and 14. For statistical evaluation, analysis of variance for repeated measures and
comparisons of means by LS Means test (p <0.05) were performed. 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 levamisole 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 resistance, pharmacological
Resumo: O controle de endoparasitas gastrintestinais em animais de produo baseado na administrao de anti-helmnticos, sendo estes realizados de forma inadequada, resultando em
estmulo resistncia parasitria. O objetivo deste experimento
foi avaliar a eficincia de 5 princpios ativos comerciais contra
a endoparasitas gastrintestinais em um rebanho ovino no Sul do
Brasil. Para isto, 54 ovinos foram distribudos em 6 grupos com
9 animais em cada um. Os princpios ativos testados foram moxidectina (T1), ivermectina 1% (T2), disofenolato de levamisol
(T3), oxfendazole (T4) e cloridrato de levamisole (T5), administrados via oral conforme as indicaes do fabricante. Os exames
de contagem de ovos por grama de fezes (OPG) e coprocultura
foram realizados nos dias 0, 7 e 14. Para avaliao estatstica foi
realizado anlise de varincia para medidas repetidas e as comparaes das mdias pelo teste LS Means (p<0.05). A eficincia
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. Moxidectina, ivermectina, oxfendazole e cloridrato de levamisole apre*Correspondncia: carlini@portoweb.com.br
Tel: +55 (55) 3220-8071

sentaram baixa eficcia. O disofenolato de levamisol foi o nico

medicamento eficiente. A baixa eficcia da maioria dos produtos
testados demonstra a importncia da orientao tcnica no uso
racional dos anti-helmnticos e difuso de medidas de controle
apropriado de parasitas.
Palavras-chave: endoparasitas, farmacologia, parasitas gastrintestinais, resistncia parasitria

The worlds 1.8 billion small ruminant population

comprising 0.7 billion goats and 1.1 billion sheep provide 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 diseases 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 recognized 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 Report, 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 parasite 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 Haemonchus contortus, Trichostrongylus spp., Fasciola
hepatica and Nematodirus spp. (Jackson, 1993).
Parasitism is an important limiting factor in livestock production and, although the history of the broadspectrum anthelmintics started in early 1960s with
a strong empirical knowledge and superior outcomes,
the present situation is alarming and is clearly worsening, with consequences arising from the development
of multidrug resistant parasite populations (Hughes et
al., 2007). Multiple anthelmintic resistance involving
ivermectin closantel, albendazol, levamisol and mo33

Santos F. et al.

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

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 operative factors that lead to the occurrence of this phenomenon (Fao, 2003).
The aim of this experiment was to evaluate 5 active
principles administered orally in a sheep flock naturally infected in the southern Brazil.
The experiment was conducted in the Agricultural College of the Federal University of Santa Maria
(UFSM) installations, localized in the Central Depression 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 principle to be tested. The active principles tested were
moxidectin (T1), ivermectin 1% (T2), levamisol disofenolat (T3), oxfendazol (T4) and levamisol hydrochloride (T5), respectively. The T6 was the untreated
control group. The doses followed the manufacturers
recommendation and were all orally administered
(Table 1).
Table 1 - Dose of active principles tested according to treatment.

Table 2 - Percentage of reduction, minimum, average and maximum values of EPG from sheep treated with moxidectin (T1),
ivermectin (T2), levamisol disofenolat (T3), oxfendazol (T4),
levamisol hydrochloride (T5) and control group.
TREATMENT
1

DAY
0

14

2800 A, a

1262 B, a

1044 B, a

54%

62%

% reduction
Mininum
values

200

Maximum
values

4500

4700

4400

3183 A, a

2483 B, b

2125 B, b

21%

33%

% reduction
Mininum
values

200

100

Maximum
values

5500

5700

7000

2800 A, a

33 B, b

288 B, b

98%

89%

% reduction
Mininum
values

300

Maximum
values

4900

100

800

2166 A, a

2150 B, b

3187 B, b

1%

0%

Treatment

ACTIVE
PRINCIPLE

DOSE

Moxidectin

1mL/10 kg BW

Ivermectin 1%

2,5mL/10 kg BW

Levamisol
disofenolat

1mL/10 kg BW

Mininum
values

200

Oxfendazol

1mL/9 kg BW

Maximum
values

4500

4600

3300

Levamisol
hydrochloride

1mL/10 kg BW

3888 A, a

457 B, a

1212 B, a

88%

68%

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 culture 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 transformed 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
34

were tested: (1) auto-regressive first order, (2) compound symmetry and (3) unstructured. In choosing the
matrix of variance and covariance, it was used Akaike
Information Criterion to select the one possessing lowest value.
EPG means, efficiency of treatment and fecal cultures are demonstrated in Table 2 and 3.

% reduction

% reduction
Mininum
values

200

Maximum
values

5200

1800

2200

Control

2044 A, a

2787 A, a

3133 A, a

0%

0%

% reduction
Mininum
values

300

Maximum
values

5000

4900

6800

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 Chisquare test. The interaction between treatment and time
was significant (p<0.001).

Santos F. et al.

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

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

Control

GENRES

DAY

EFFIENCY

EFFIENCY

14

0-7

0-14

Haemonchus

84

88

100

Cooperia

Trichostrongylus

100

100

Ostertagia

10

12

100

Haemonchus

87

79

74

14

Cooperia

100

Trichostrongylus

13

100

Ostertagia

100

100

Bunostomum

26

Haemonchus

99

22

37

77

62

Cooperia

10

Trichostrongylus

76

53

Ostertagia

Bunostomum

Haemonchus

90

48

86

46

Cooperia

100

100

Trichostrongylus

14

Ostertagia

47

Bunostomum

100

100

Haemonchus

82

70

99

14

Cooperia

Trichostrongylus

16

25

100

Ostertagia

Bunostomum

Chabertia

100

100

Haemonchus

92

96

99

Cooperia

Trichostrongylus

Ostertagia

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 indicates 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 anthelmintic, a FECR of at least 95% (and a lower 95% confidence 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 Information 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.
35

Santos F. et al.

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

Table 4 - Variance analysis of repeated measures of moxidectin

(T1), ivermectin (T2), disofenolato of levamisole (T3), oxfendazole (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.
Treatment

DAY
0

14

49,28 A, a

22,60 B, a

23,30 B, a

30,57 A, a

58,30 B, b

55,98 B, b

38,10 A, a

4,01 B, a

10,38 B, a

39,45 A, a

37,64 A, b

49,44 A, b

56,60 A, a

13,22 B, a

22,10 B, a

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. 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).
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 Gonalves, 1967; Echevarria,
1996; Waller et al., 1996; Farias et al., 1997). Multiple
resistance to the three main classes of drugs with anthelmintic action (avermectins/milbemicins, benzimidazoles
and imidazothiazoles) is not rare in this region (Waller et
al., 1996).
Drug combinations could possibly restore the effectiveness 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 chemotherapy, 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 infecting 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 within 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 selection through exposure of the worm population to anthelmintic drugs, especially when it is performed inadequately. Intensive exposure to antiparasitic drugs and using
this as the only control method accelerates the process
36

considerably. Side resistance between drugs with similar

mechanisms of action is also common (McKellar e Jackson, 2004).
A difficult situation regarding gastrointestinal nematodes 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. Levamisol 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 importance of establishing programs of parasite control.
In this context, treatments should be applied at the minimum frequency that is feasible and management measures 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 effective anthelmintic action. Levamisol disofenolat was the
only antihelmintic with high efficiency, being this a
new molecule. The present study illustrates the alarming 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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