EFFICACY OF A MOXIDECTIN/TRICLABENDAZOLE ORAL FORMULATION
AGAINST MIXED INFECTIONS OF Fasciola hepatica AND GASTROINTESTINAL
NEMATODES IN SHEEP
M. Martínez-Valladares1, C. Cordero-Pérez2, L. Castañón-Ordóñez2, M.R. Famularo2, N.
Fernández-Pato2, F.A. Rojo-Vázquez1*
1
Instituto de Ganadería de Montaña, CSIC-ULE. Finca Marzanas. 24346 Grulleros, León,
Spain
2
Departamento de Sanidad Animal, Parasitología, Facultad de Veterinaria, Universidad de
León, 24071 León, Spain
* Corresponding author. Tel.: 0034 987 291338
E-mail address: francisco.rojo@unileon.es
1
Abstract
We have evaluated the efficacy in sheep of a combination drench formulation at the
recommended dose rate of 0.2 mg moxidectin/kg bodyweight and 10 mg triclabendazole/kg
bodyweight against an experimental infection with Fasciola hepatica and a natural infection
with gastrointestinal nematodes. We confirmed that the efficacy of reducing fecal egg output
was 98.3% for trichostrongyle eggs and 100% for F. hepatica eggs. Based on adult worm and
fluke recovery, the efficacy varied according to the target species. A reduction was found in the
number of Teladorsagia circumcincta, Trichostrongylus spp., Nematodirus spp., and Trichuris
spp. greater than 95%, but the efficacy for Oesophagostomum spp. varied, with values below
90%. The reduction in F. hepatica was higher than 95% for all stages. The effectiveness of the
formulation was also confirmed by an increase in total proteins and albumin following
treatment.
Keywords: Efficacy; Moxidectin; Triclabendazole; Fasciola hepatica; Sheep
2
1. Introduction
Helminth infections of the digestive system are the most common parasitic infections in grazing
ruminants worldwide and the most economically important, resulting in production losses due to
reduced milk yield, decreased weight gain, and liver condemnations. The prevalence of
gastrointestinal nematodes (GIN), mainly trichostrongyles, in sheep in northwest Spain has been
estimated to be nearly 100%, while the prevalence of fasciolosis in the country is lower, at
between 14.7 and 23.7% (Manga et al., 1990, Ferre et al., 1995).
The most common method of controlling these parasitic infections is through anthelminthic
drugs. Most of the available anthelminthics for controlling GIN in sheep fall into three main
families: benzimidazoles, imidazothiazoles, and macrocyclic lactones. Moxidectin, a
macrocyclic lactone, at a dosage of 0.2 mg/kg bodyweight, has been shown to be effective
against pulmonary infections and GIN in sheep (Dorchies et al., 1996; Cringoli et al., 2009).
Moxidectin has a longer persistence in sheep and maintains higher milk production than
ivermectin (Cringoli et al., 2009). The most widely used drug to combat F. hepatica in sheep is
a benzimidazole derivative, triclabendazole (TCBZ), due to its high efficacy against adult and
immature flukes (Boray et al., 1983; Fairweather 2005).
There is a constant increase in anthelmintic resistance in ruminant livestock; therefore new
formulations should be tested with the currently available drugs. In this study we evaluated the
efficacy in sheep of a combination drench formulation, comprising the recommended dose of
0.2 mg moxidectin/kg bodyweight and 10 mg TCBZ/kg bodyweight, against an experimental
infection of F. hepatica and natural infection with GIN. The combination increases the spectrum
of activity of the formulation, combining a nematocide with a flukicide.
2. Material and methods
The study was carried out on a group of 48 female sheep age 6-8 months, naturally infected
with GIN and free from F. hepatica infection. The test was a blind clinical trial using a
randomized and controlled parallel group design, conducted in accordance with the VICH
guidelines for “Technical requirements for registration of veterinary medicinal products” and
3
for “Determining the efficacy of anthelmintics in ruminants”. The study was conducted at the
experimental farm of the University of León in Spain. According to the eggs per gram (epg)
values, sheep were divided into 6 homogeneous groups of 8: T1 and C1, treated and control
groups 1; T2 and C2, treated and control groups 2; T3 and C3, treated and control groups 3. On
day 0 of the trial, 200-300 metacercariae of F. hepatica were administered orally to each sheep.
The schedule of infection and treatment was as follows:
Trial week
0
Fluke
infection
4
8
Sacrifice
T1/C1
Treatment T2
Treatment T1
12
Sacrifice
T2/C2
Treatment T3
16
Sacrifice
T3/C3
The anthelminthic was given as a single oral dose in a formulation comprising 0.2 mg
moxidectin/kg bodyweight and 10 mg TCBZ/kg bodyweight, prepared by Fort Dodge Animal
Health (Spain).
A modified McMaster technique was used for nematode egg counts, and epg was measured to
test the efficacy of the formulation 14 days post treatment (PT). Fecal samples from each group
were collected and cultured to obtain third stage larvae (L3) on days 0 and 14 PT. F. hepatica
epg values were determined in groups C3 and T3 at 14 and 28 days PT, by means of a standard
sedimentation method using McMaster chambers.
Blood samples were taken weekly directly from the jugular vein into Venoject vacuum tubes.
Serum samples were sent to the Laboratorio de Técnicas Instrumentales (LTI) of the University
of León to evaluate total protein (TP), albumin, and the hepatic enzymes gammaglutamyltransferase (GGT), aspartate-aminotransferase (AST), and alanine-aminotransferase
(ALT).
Animals were sacrificed with an intravenous overdose of embutramide and mebezonio ioduro
(T-61, 10 ml/sheep) 4 weeks PT. The abomasum, small and large intestines, and liver of each
animal were removed and parasites were recovered following Wood et al. (1995). The number of
immature and mature flukes in the liver as well as the number of adult nematodes in the
abomasum and small and large intestines was determined. When partial flukes were recovered,
4
the count was the higher number of the region collected. The contents of the abomasum, small
intestine, and large intestine were collected up to a total volume of 5, 2, and 10 L respectively.
Aliquots of 10%, 2.5%, and 5% of the total volume from the abomasum, small intestine, and
large intestine, respectively, were examined to identify and count adult nematodes. Nematode
counts were estimated from the average number of worms present in each group multiplied by
the dilution factor.
The identification of adult nematodes and 100 L3, from coprocultures, was carried out
following MAFF keys (MAFF, 1986).
For a new product, the percentage efficacy must be higher than 90%, and the differences
between treated and control groups statistically significant. The number of worms and
immature/mature flukes was counted at necropsy, and the arithmetic mean calculated for each
group. The efficacy was determined using the reduction in the number of parasites at 8, 12, and
16 weeks post infection (PI), using the formula:
% efficacy = 100 × (mean control group-mean treated group)/mean control group.
The same formula was used to measure efficacy according to epg reduction 14 days after
treatment. Differences between treated and control groups were determined using Student’s test
(SPSS for Windows). Values of P < 0.05 were considered significant.
3. Results
Efficacy of the formulation against nematodes in terms of egg output reduction is shown in
Table 1. The first fluke eggs were observed at week 9 post infection (PI) in those experimental
groups remaining. The efficacy of the formulation against F. hepatica could be calculated only
for group T3, based on epg, since, on the treatment day, egg excretion was sufficient to apply
the formula (Table 2).
After necropsy, nematodes and flukes were identified and counted at the same time. The
efficacy of the formulation against Teladorsagia circumcincta, Trichostrongylus axei,
Trichostrongylus spp, Nematodirus spp, and Trichuris spp was between 96 and 100% for all
5
groups However, the maximum value against Oesophagostomum spp was 85% in group 2
(Table 3). Data regarding efficacy against F. hepatica are shown in Table 4.
L3 were found in all groups and identified in Table 5. In T1, there were fewer than 100 L3 and
were expressed as a percentage of the total.
The kinetics of TP and albumin were similar throughout the study. Values were within normal
limits (6-7.5 and 2.7-3.7 g/dl) (Benjamín, 1984) for all groups until 8 weeks PI when values
decreased below the normal range. However, following the treatment of T3, TP increased to
values higher than in C3 and showed significant differences (P 0.01) at the conclusion of the
study. The treatment also caused a significant increase in albumin (P 0.01) in T2, as well as in
T3, compared to the controls.
The mean value for hepatic enzymes at day 0 ranged from 64.5 to 77.5 U/L (GGT), 13.6 to
18.6 U/L (ALT), and 76.1 to 93.8 U/L (AST). After infection, these values increased,
becoming significantly different (P0.01) for GGT and ALT. Throughout the study the
maximum enzymatic activity was found at 3 weeks PI for AST, at 4 weeks PI for ALT, and
between weeks 8 and 10 PI for GGT with a second peak for AST.
4. Discussion
The present study demonstrates the efficacy of a combination of 2 mg moxidectin/kg
bodyweight and 10 mg TCBZ/kg bodyweight against natural GIN infection and concurrent
experimental F. hepatica infection in sheep. Moxidectin has been satisfactorily tested against
GIN in sheep previously; 100%, 99.98%, 100% and 100% efficacy values have been reported
against adults H. contortus, T. circumcincta, Trichostrongylus columbriformis, and
Trichostrongylus vitrinus, respectively (Uriarte et al., 1994). The efficacy of the tested
formulation against GIN was based on egg output reduction and post-mortem adult worm
counts, comparing control and treated groups. Concerning the GIN epg values, the efficacy at 2
weeks PT was 98.3%, after calculating the mean of the data obtained for the three groups. This
value is in accordance with the efficacy described based on recovery of adult worms.
6
Moxidectin was 100% effective against adult T.circumcincta and T.axei found in the
abomasum, 97.6% against Trichostrongylus spp established in the small intestine, 98.6% against
Nematodirus spp, and 100% against Trichuris spp. These results are in agreement with assays
carried out by Uriarte et al. (1994). However, efficacy against Oesophagostomum spp. was low,
between 23 and 85%, although the reduction based on epg values was higher than 95% in all
groups. Previous trials have reported moxidectin to show 100% efficacy against
Oesophagostomum venulosum and Trichuris ovis (Dorchies et al., 1996), based on worm
recovery at 14 and 35 days PT. In this study, moxidectin could have produced a temporary
suppression of the egg output of surviving worms, therefore anthelminthic resistance may have
been developed. In this case, the efficacy of moxidectin against Oesophagostomum spp should
be recalculated.
In an attempt to include treatment for a second target species for the formulation, TCBZ was
added to control F. hepatica infection. Our data have confirmed TCBZ efficacy of 100%, based
on the reduction of epg values and numbers of mature and immature flukes. The actual
effectiveness should be calculated following adult fluke recovery to determine whether parasites
have been removed. The mean efficacy against flukes of 1-4 mm, 5-10 mm, and >10 mm
length, and head/tail was 100%, 94.4%, 99.4%, and 98.9% respectively. We have not taken into
account the efficacy in group 2 (75%) against flukes of 1-4 cm, because of the limited number
(3) recovered in C2. It is worth noting that the efficacy against mature flukes is almost 100%
and in immature more than 95%, in accordance with Boray et al. (1983).
One of the clinical signs of fasciolosis is hypoproteinemia as a result of hypoalbuminemia in
chronic infections (Rojo-Vázquez and Ferre-Pérez, 1999). Both parameters were altered from
week 8 PI, when flukes had reached maturity. Thus, the effectiveness of the formulation was
also confirmed since these values increased following treatment to normal values.
The hepatic enzymes have been described as in vivo indicators of the hepatic damage produced
by F. hepatica. GGT is associated with penetration into the bile ducts by migrating flukes and
causes hyperplastic cholangitis. In our study this enzyme remained elevated from week 8 PI, as
has been confirmed in previous trials (McConville et al., 2009). Regarding the enzymes
7
associated with the parenchymal migrations of immature stages, AST and ALT were increased
by week 3 and 4 PI, respectively.
This combination can be used in cases of fasciolosis and/or GIN infection since TCBZ and
moxidectin belong to different chemical groups, showing different modes of action. Moreover,
it is thought that the anthelminthic combination reduces the frequency of treatment and delays
the development of resistance (Barnes et al., 1995).
Conclusion
We confirmed that a combination of 2 mg moxidectin/kg bodyweight and 10 mg TCBZ/kg
bodyweight produces a 98.3% reduction in the fecal egg counts of strongyle-like eggs and 100%
of fluke eggs. Based on the nematode and fluke recovery, effectiveness can vary according to
the target species. The trial showed a reduction in the number of T. circumcincta,
Trichostrongylus spp., Nematodirus spp., and Trichuris spp. greater than 95%. However the
efficacy against Oesophagostomum spp. varied widely, with values below 90%. The efficacy of
the formulation against F. hepatica is higher than 95% for all stages, immature and mature
flukes.
Acknowledgments
This study was supported by a research project of Fort Dodge Animal Health (Spain) (Study
number 0987-O-SP-01-06) and by the National Spanish project of the Ministry of Science and
Innovation (ref. No. RTA2006-00183-C03-02).
References
Barnes, E.H., Dobson, R.J., Barger, I.A., 1995. Worm control and anthelmintic resistance:
adventures with a model. Parasitol. Today 11 (1995), pp. 56–63.
Benjamín, M.M., 1984. Manual de parasitología clínica veterinaria. Ed, Limusa, México, pp
135.
8
Boray, J.C., Crowfoot, P.D., Strong, M.B., Allison, J.R., Schellenbaum, M., Von Orelli, M.,
Sarasin, G., 1983. Treatment of immature and mature Fasciola hepatica infections in
sheep with triclabendazole. Vet. Rec. 113, 315-317.
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Salamina, V., 2009. The effect of moxidectin 0,1% vs ivermectin 0,08% on milk
production in sheep naturally infected by gastrointestinal nematodes. BMC Vet. Res. 5,
41.
Dorchies, P., Cardinaud, B., Fournier, R., 1996. Efficacy of moxidectin as a 1% injectable
solution and a 0.1% oral drench against nasal bots, pulmonary and gastrointestinal
nematodes in sheep. Vet. Parasitol. 15, 163-168.
Fairweather I. 2005. Triclabendazole: new skills to unravel an old(ish) enigma. J. Helminthol.
79, 227-34.
Ferre, I., Ortega-Mora, L.M., Rojo-Vázquez, F.A., 1995. Seroprevalence of Fasciola hepatica
infection in sheep in northwestern Spain. Parasitol. Res. 81, 137-142.
MAFF 1986. Manual of Veterinary Parasitology Laboratory Techniques. HMSO, London, pp
1–152.
Manga, Y., Gonzalez-Lanza, C., Del-pozo, P., Hidalgo, R., 1990. Kinetics of Fasciola hepatica
egg passage in the faeces of sheep in the Porma basin, Spain. Acta Parasitol. Pol. 35, 149157.
McConville, M., Brennan, G.P., Flanagan, A., Edgar, H.W., Hanna, R.E., McCoy, M., Gordon,
A.W., Castillo, R., Hernández-Campos, A., Fairweather, I., 2009. An evaluation of the
efficacy of compound alpha and triclabendazole against two isolates of Fasciola hepatica.
Vet, Parasitol. 162, 75-88.
Rojo-Vázquez, F.A., Ferre-Pérez, I., 1999. Parasitosis hepáticas. Parasitología Veterinaria.
Interamericana. McGraw-Hill·Interamericana. España, pp. 260-272.
9
Uriarte, J., Gracia, M.J., Almeria, S., 1994. Efficacy of moxidectin against gastrointestinal
nematode infections in sheep. Vet. Parasitol. 51, 301-305.
Wood, I.B., Amaral, N.K., Bairden, K., Duncan, J.L., Kassai, T., Malone, J.B., Pankavich, J.A.,
Reinecke, R.K., Slocombe, O., Taylor S.M., Vercruysse, J., 1995. World Association for
the Advancement of Veterinary Parasitology (W.A.A.V.P.) second edition of guidelines
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Parasitol. 58, 181–213.
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Table 1
Mean fecal epg values ( standard deviation) and efficacy of the formulation in terms of fecal
egg reduction against gastrointestinal nematodes. The data include strongyle-like eggs,
Nematodirus spp. and Trichuris spp.
Group
Day 0
Treatment day
+ 14 PT
Efficacy (%)
C1
380 ( 187)
434 ( 394)
T1
336 ( 161)
445 ( 212)
463 ( 194)
0
100
C2
414 ( 222)
408 ( 269)
393 ( 434)
T2
498 ( 310)
579 ( 489)
4 ( 11)
C3
479 ( 319)
218 ( 138)
144 ( 99)
T3
569 ( 365)
189 ( 133)
6 ( 17)
11
99
96
Table 2
Mean F. hepatica fecal epg values ( standard deviation) and efficacy of the formulation in
terms of fecal egg reduction in group T3 compared to group C3.
Group
Treatment day
+ 14 PT
+ 28 PT
Efficacy +14 (%)
Efficacy +28 (%)
C3
184 ( 187)
T3
191 ( 66)
409 ( 411)
0
377 ( 210)
0
100
100
12
Table 3
Mean of worms counts ( standard deviation) in each gastrointestinal tract and efficacy of the
formulation per group
Gastrointestinal tract
C1
T1
C2
0
T2
C3
T3
Abomasum
T. circumcincta 1486 ( 496)
1129 ( 534)
0
1099 ( 582)
0
0
(100)
0
-
5 ( 4)
(100)
0
(100)
25 ( 14)
(100)
0
(100)
Trichostrongylus spp.
73 ( 56)
0
55 ( 40)
0
28 ( 23)
2 ( 4)
(% efficacy)
Nematodirus spp.
(% efficacy)
66 ( 52)
(100)
0
(100)
36 ( 55)
(100)
0
(100)
70 ( 80)
(93)
3 ( 7)
(96)
385 ( 510)
750 ( 800)
110 ( 122)
203 ( 541)
157 ( 322)
70 ( 169)
(85)
0
(100)
0
(23)
0
-
(% efficacy)
T. axei
(% efficacy)
Small intestine
Large intestine
Oesophagostomum spp. 560 ( 698)
(% efficacy)
Trichuris spp.
(% efficacy)
8 ( 16)
(31)
0
(100)
13
Table 4
Mean F. hepatica recovery ( standard deviation) and efficacy of the formulation per group
based
Group
1-4 mm
5-10 mm
› 10 mm
Head/Tails
C1
T1
(% efficacy)
3.9 ( 3.6)
0
(100)
18.8 ( 10.3)
0
(100)
1.4 ( 2.4)
0
(100)
8.6 ( 3.9)
0
(100)
C2
0.4 ( 0.7)
7.1 ( 6.9)
68.5 ( 45.4)
21.3 ( 9.5)
T2
(% efficacy)
0.1 ( 0.4)
(75)
0.8 ( 1.8)
(88.8)
1.3 ( 2.2)
(98.1)
0.7 ( 1.1)
(96.7)
C3
0
0
T3
(% efficacy)
0
-
0
-
106.6 ( 187)
0
(100)
12.9 ( 187)
0
(100)
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Table 5
Identification of L3 recovery in coprocultures, pre- and post-treatment (PT)
Treatment day
Group
Tc (%)
Tch (%)
Day 0
C1
T1
C2
T2
C3
T3
C1
T1
C2
T2
C3
T3
95
94
78
94
91
83
97
98
83
50
97
*
5
6
22
6
9
17
3
2
17
50
3
*
Day +14 PT
Tc (T. circumcincta), Tch (Trichostrongylus spp), *(L3 dead)
15