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. 2021 Jul 28;46(1):80–86. doi: 10.1007/s12639-021-01422-w

Therapeutic efficacy of triclabendazole in comparison to combination of triclabendazole and levamisole in sheep naturally infected with Fasciola sp.

Mohaddeseh Abouhosseini Tabari 1, Seyed Aria Fallah Vahdati 2, Shohre Alian Samakkhah 1, Atefeh Araghi 1, Mohammad Reza Youssefi 3,4,✉
PMCID: PMC8901840  PMID: 35299911

Abstract

Fascioliasis is an important parasitic disease which affects productivity of ruminants, and imposes significant economic losses. Benzimidazoles are effective in the treatment of fascioliasis; however, there are several reports on benzimidazoles resistant flukes. Combinational therapy is an approach to delay the emergence of resistant flukes. The present study aimed to compare the efficacy of triclabendazole (TBZ) and combination of triclabendazole and levamisole (TBZ + LVM) in the treatment of sheep naturally infected with Fasciola sp. For this purpose, 40 ewes infected with Fasciola sp. in three groups received TBZ, and TBZ + LVM, or remained untreated as CON. Fecal egg count (FEC), fecal egg count reduction (FECR), liver enzymes activity, albumin, globulin, and total protein levels were measured on day 0, 7, 14, and 28 post treatments. Obtained results showed that treatment with TBZ and TBZ + LVM resulted in significant reduction in FEC (P < 0.05), and FECR reached to values of higher than 90% on 28 day post treatment. The FEC for TBZ + LMV on day 7 and 14 were 12.25 ± 3.82 and 3.08 ± 1.03, respectively which was significantly lower in comparison to TBZ and CON (P < 0.05). Efficacy of TBZ + LMV was higher than TBZ on day 7 and 14 post treatment; however, no significant difference was observed on 28 day. The liver enzyme activities on days 7 and 14 were lower in the TBZ + LVM sheep in comparison to the TBZ and CON. Treatment with TBZ or TBZ + LVM resulted in an increase in albumin and a decrease in globulin. Over all, the present study clarified the importance of combinational therapy, and demonstrated that combination of TBZ + LVM resulted in higher efficacy and earlier improvement of liver conditions in sheep naturally infected with Fasciola sp.

Keywords: Fasciola sp., Triclabendazole, Levamisole, Synergism

Introduction

Fascioliasis is one of the most important parasitic zoonotic diseases in tropical and subtropical countries which is a public health concern, and affects productivity of ruminants, especially sheep and cattle. It has been estimated that more than 550 million animals (cattle and sheep) are infected throughout the world. Economic losses of fascioliasis, in the mid-1990s just in the US livestock industry, were estimated at $3 billion per annum which is expected to be much higher by now (Fairweather et al. 2020) causative agent of this disease, Fasciola hapatica, probably possesses the widest geographic distribution of any helminthic parasite (Ibrahim 2017). Meanwhile, in parts of the world, the prevalence of fascioliasis is increasing due to climate changes or man-made environmental alterations (María Martínez-Valladares et al. 2013). Fascioliasis can be manifested in acute, subacute, or chronic forms. Pathologic changes may include multiple irregular edematous areas distributed randomly in the hepatic parenchyma, fibrosis in the parenchyma, and hemorrhage in the bile ducts (Pérez-Caballero et al. 2018). In Iran, the average infestation of sheep to fascioliasis was reported as high as 32% (Eslami et al. 2009) in some regions; distribution of this disease is so high that one third of livers at abattoirs are condemned due to the presence of this infection (Khosravi and Babaahmady 2012). Fasciola hepatica is the predominant species of trematodes infecting sheep livers in north of Iran (Moghaddam et al. 2004).

Benzimidazoles are the most important anti-parasitic drugs effective in the treatment of fasciolosis. Currently, the most effective drug for the control of fascioliasis is triclabendazole whose efficacy is not limited to adult flukes, and is also effective on immature ones (Martínez-Valladares et al. 2014). Repeated administration of benzimidazoles especially at sub-therapeutic doses is one of the main factors contributing in the development of resistant liver flukes. Several reports on benzimidazoles resistant F. hepatica have been demonstrated in many parts of the world (Kelley et al. 2016; Martínez-Valladares et al. 2014; Moll et al. 2000; Novobilský et al. 2016).

Levamisole has been widely used against gastrointestinal nematodes of sheep (Ciuffa et al. 2017). Besides its anthelmintic effects, immunostimulatory activity of levamisole in the parasitic infections has also been reported (Sajid et al. 2006). Although levamisole is mainly effective against nematodes, possible positive impacts of this drug in the prevention and treatment of fasciolosis by its immunomodulatory effects has been demonstrated (Goraish et al. 1988).

Combinational therapy, particularly with drugs of different chemical classes, is one of the fundamental strategies for preventing development of resistant parasites, and preserving antiparasitics efficaciy. The combination of two or more drugs is commonly used for controlling gastrointestinal nematode and liver fluke infections in sheep (Martínez-Valladares et al. 2014).

Bearing above in mind, the present study aimed to evaluate and compare triclabendazole and triclabendazole + levamizole therapeutic efficacies for the treatment of sheep naturally infected with fascioliasis. Moreover, serum biochemical analysis and liver enzymes activity measurements were done in order to assess effect of treatments on improving sheep liver condition.

Material and methods

Infected sheep

Two sheep flocks, being kept in Babol lowland suburb pastures (Babol, Mazandaran), based on the clinical signs and fecal sample analyses were diagnosed as infected with Fasciola sp. Number of 40 ewes from infected herds aged 2–4 years was included in the present study. The ewes had no prior anti-helminthic control programs during the recent 6 months. Infected ewes were randomly divided into three groups. TBZ group (n = 16) were administered triclabendazole (Triclaz®, 250 mg, Zagrospharmed, Tehran, Iran) at the dose of 10 mg/kg orally; TBZ + LVM group (n = 16) received combination of triclabendazole and levamisole in the form of oral suspension containing 5% triclabendazole and 3.75% levamisole (Flunil 8.75%®, Zagrospharmed, Tehran, Iran) at the dose of 1 ml for each 5 kg body weight. The remaining 8 infected sheep received no medication, and were kept separately as the control group (CON). All the sheep were weighted and identified individually by a number as ear tag before initiation of the experiment. All the study procedures was approved by AUSMT animal welfare and ethical committee with approval number of 99/1/23.

Fecal egg count (FEC)

The numbers of Fasciola sp. eggs in the feces samples were counted by the standard sedimentation method using McMaster chambers according to the method previously described (Conceição et al 2002). In brief, 10 g of feces were mixed with tap water and the supernatant discarded. The sedimentation process was done three times. The sediment (0.15 ml) was poured on chambers of a McMaster slide and the eggs on the bottom of the chambers were counted. EPG was determined according to the following equation:

EPG=totalnumberofobservedeggs/numberofchambers×50ml/10g/0.15ml

Feces samples were directly collected from rectum on day 0 before initiation of the treatments, day 7, 14, and 28 post treatments. The calculation of the reduction of eggs in feces for each drug was done on days 7, 14 and 28 post treatments. For calculation of the fecal egg count reduction (FECR), the fallowing method was used:

FECR=100×1-Tx/T0

Tx = arithmetric mean EPG of each group on days 7, 14, 28.

T0 = arithmetric mean EPG of each group on day 0 (Cabaret and Berrag 2004).

Biochemical analysis

Blood was collected from jugular vein of infected sheep pre and post treatment at day 0, 7, 14, and 28. Sera were collected at 1500 g centrifugation for 10 min, and the samples were stored at − 20 °C until further analysis. Serum activities of aspartate aminotransferase (AST) and gamma glutamyl transferase (GGT) and concentrations of total protein (TP), and albumin (ALB) were measured by using automatic analyzer with commercial kits. The globulin (GLB) concentration was calculated by subtraction of ALB from TP.

Statistical analysis

The obtained data were subjected to the Shapiro–Wilk test for normality. Fecal egg counts were analyzed by non-parametric Friedman test and for multiple comparisons between time points (0, 7, 14, 28 day), post hoc analysis with the Wilcoxon signed-rank tests were applied with a Bonferroni adjustment. Comparisons between treatment groups at each time point were assessed using the non-parametric Kruskal–Wallis test, and the Mann–Whitney U-test with a Bonferroni adjustment applied as post hoc test.

Percentage of Fasciola sp. eggs in infected sheep on days 7, 14 and, 28 post treatment was calculated where sheep with 0 egg/g were assumed negative, and those with ≥ 1 egg/g were assumed positive (Coles et al. 1992).

Based on the European regulatory guidelines, we have attempted to define efficacy of anthelmintic drugs calculated by this formula:

%Efficacy=C-TC×100

where: C = mean number of parasite in the control group; T = mean number of parasite in the treated group.

Data from each of the blood biochemical measurements (AST, GGT, TP, ALB, and GLB) were subjected to the parametric repeated measures test. Comparisons between time points (0, 7, 14, 28 day) were done using the Bonferroni post hoc test, and between treatment groups at each time point were assessed using the One-way analysis of variance (ANOVA) and the Bonferroni post hoc test. The statistical analysis was performed using the SPSS Version 25 software (SPSS Inc., Chicago, IL, USA). All results were expressed as Mean ± SD. For all analyses, P < 0.05 was considered as statistically significant.

Results

The mean number of Fasciola sp. eggs per gram of feces, and the percentage of reduction in egg count were determined for each anthelmintic at each time point (Table 1). Fecal egg counts were not significantly different in all the groups of ewes on day 0 (P = 0.54). In the TBZ group, the FEC reduced from 28.25 ± 7.37 on day 0 to 18.75 ± 7.62 on day 7, but this decrease was not significant (P > 0.05). The FEC for TBZ treated ewes reached to 6.93 ± 3.37 and 1.12 ± 1.31 on day 14 and 28, respectively (P < 0.05). The final FECR in the TBZ group was 96.01% on day 28. In the TBZ + LMV group, the FEC decreased significantly through the study at all of the time points (P < 0.05). The FEC for TBZ + LMV on day 7 and 14 were 12.25 ± 3.82 and 3.08 ± 1.03, respectively which was significantly lower in comparison to TBZ (P < 0.05). TBZ + LMV showed FECR value of 52.54 and 84.77% on day 7 and 14, respectively. On day 28, FECR of TBZ + LMV reached to 97.33%.

Table 1.

Fecal egg counts and FECR before and after treatment with triclabendazole and triclabendazole + levamisole in the sheep naturally infected with Fasciola sp

Treatment Item Day
0 7 14 28
CON1 Mean ± SD (EPG) 27.12 ± 5.98a,A 25.87 ± 5.27a,A 26.75 ± 4.65a,A 25.62 ± 4.95a,A
FECR4 (%) – 4.60 1.38 5.52
TBZ2 Mean ± SD (EPG) 28.25 ± 7.37a,A 18.75 ± 7.62a,B 7.99 ± 2.37b,B 1.12 ± 1.31c,B
FECR (%) - 33.63 75.44 96.01
TBZ + LVM3 Mean ± SD (EPG) 25.81 ± 5.89a,A 12.25 ± 3.82b,C 3.08 ± 1.03c,C 0.68 ± 0.94d,B
FECR (%) – 52.54 84.77 97.33
Open in a new tab

1Control

2Triclabendazole

3Triclabendazole + Levamisole

4fecal egg count reduction

a,bThe different superscripts letters in the same row indicate significant differences (P < 0.05)

A,BThe different superscripts letters in the same column indicate significant differences (P < 0.05)

Table 2 showed that TBZ efficacy was 27.53% on 7 day post-treatment which increased to 95.60% at the time period of 28 days. For TBZ + LVM, therapeutic efficacy was 52.65% on day 7, and 97.31% on day 28. Efficacy of treatment with TBZ and TBZ + LVM were significant all over the study period (P < 0.05). TBZ + LVM showed higher efficacy in comparison to TBZ on days 7 and 14 (P < 0.05), however, no significant difference was observed on day 28 (P > 0.05).

Table 2.

Efficacy of triclabendazole and triclabendazole + levamisole in the sheep naturally infected with Fasciola sp. on 7, 14, and 28 days post treatment

Treatment Day
7 14 28
TBZ1 27.53a 74.06b 95.60c
TBZ + LVM2 52.65a 84.34b 97.31c
P value3 0.01 0.03 0.40
Open in a new tab

1Triclabendazole

2Triclabendazole + Levamisole

3P < 0.05 was considered as statistically significant in each column

a,bThe different superscripts letters in the same row indicate significant differences (P < 0.05)

The mean of liver enzymes activities (AST and GGT) decreased over time in TBZ and TBZ + LVM groups (P < 0.05) (Fig. 1). Treatment of sheep with TBZ and TBZ + LVM resulted in a significant decrease in liver enzyme activities in comparison to the untreated sheep (P < 0.05). The activity of AST, at time points of 7 and 14 day, showed a significant decrease in TBZ + LVM treated sheep in comparison to other groups (P < 0.05). The GGT activity on day 14 and 28 was significantly lower in the TBZ and TBZ + LVM (P < 0.05). On day 14, the GGT activity was significantly lower in the TBZ + LVM sheep in comparison to the TBZ and CON (P < 0.05), but difference between two treatments was not significant on day 28 (P > 0.05).

Fig. 1.

Fig. 1

Open in a new tab

Kinetics of the liver enzymes activity, aspartate aminotransferase (AST) and gamma glutamyl transferase (GGT), during 28 days post treatment with triclabendazole (TBZ), and combination of triclabendazole and levamisole (TBZ + LVM) in sheep infected with Fasciola sp

In the TBZ group, an increase was observed in the ALB values on day 28, but in TBZ + LVM, a significant increase was noted on days 14 and 28 (P < 0.05) (Table 3). In comparison of two treatments, TP and GLB levels were not different between TBZ and TBZ + LVM at any time points of the study (P > 0.05), but significant difference was noted in comparison to the CON (P < 0.05). ALB/GLB ratio in treated sheep was significantly different in comparison with the untreated animals (P < 0.05), but the ratio between two different treatments was not significant (P > 0.05).

Table 3.

Total protein, albumin, and globulin concentrations and albumin/globulin ratio in the sheep naturally infected with Fasciola sp. on days 0, 7, 14, and 28 post treatments with triclabendazole or triclabendazole + levamisole

Variable Treatment Day
0 7 14 28
Total protein (U/L) CON1 70.88 ± 4.73a,A 68.88 ± 6.17a,A 69.25 ± 5.94a,A 72.50 ± 5.60a,A
TBZ2 69.00 ± 6.50a,A 69.13 ± 7.53a,A 69.00 ± 3.29a,A 68.88 ± 4.35a,A
TBZ + LVM3 70.88 ± 4.73a,A 68.50 ± 10.51a,A 69.75 ± 5.25a,A 70.25 ± 3.28a,A
Albumin (U/L) CON 26.87 ± 4.91a,A 26.25 ± 3.88a,A 28.38 ± 6.52a,A 27.00 ± 6.50a,A
TBZ 25.50 ± 4.27a,A 25.63 ± 4.43a,A 31.75 ± 2.65ab,A 34.00 ± 4.95b,B
TBZ + LVM 26.87 ± 4.91a,A 30.38 ± 3.88ab,A 34.00 ± 3.25b,A 35.50 ± 2.61b,B
Globulin (U/L) CON 44.00 ± 6.84a,A 42.62 ± 6.88a,A 40.87 ± 9.04a,A 45.50 ± 8.83a,A
TBZ 43.50 ± 9.16a,A 43.50 ± 8.48a,A 37.25 ± 2.60a,A 34.88 ± 6.37a,B
TBZ + LVM 44.00 ± 6.84a,A 38.12 ± 13.24a,A 35.75 ± 5.31a,A 34.75 ± 4.30a,B
Albumin/Globulin CON 0.63 ± 0.20a,A 0.63 ± 0.16a,A 0.75 ± 0.33a,A 0.63 ± 0.28a,A
TBZ 0.62 ± 0.24a,A 0.61 ± 0.19a,A 0.85 ± 0.10a,A 1.02 ± 0.28a,B
TBZ + LVM 0.63 ± 0.20a,A 0.92 ± 0.45ab,A 0.97 ± 0.19ab,A 1.04 ± 0.19b,B
Open in a new tab

1Control

2Triclabendazole

3Triclabendazole + Levamisole

a,bThe different superscripts letters in the same row indicate significant differences (P < 0.05)

A,BThe different superscripts letters in the same column indicate significant differences (P < 0.05)

Discussion

Studies have shown that combinational therapies are more effective in the control of liver flukes than individual treatments (Anderson et al. 1988). It was shown that combination of albendazole and cloruson resulted in higher efficacy and significant reduction in EPG with 99.01% FECR after 15 days post treatment. Two anthelminthic with distinct mechanisms of action could have caused synergistic effects against F. hepatica (Martínez-Valladares et al. 2014). It has been shown that LVM has beneficial effects based on its immunostimulatory properties on ruminants infected with helminthic parasites and liver flukes (Mitchell and Armour 1981). This is in line with the findings of the present study which showed that combination of TBZ + LVM resulted in higher efficacy and FECR, especially on days 7 and 14. TBZ + LVM treatment led to FECR value of 83.77% on day 14 which was higher than TBZ alone. In addition, therapeutic efficacy of TBZ + LVM on day 14 (84.34) was higher than TBZ (74.06) (p < 0.05). After 28 days in comparison of TBZ + LVM and TBZ, a slight higher efficacy was noted in TBZ + LVM. In line with our findings, Mavromati et al (2016) reported that combinational therapy with triclabendazole and levamisole, twice a year, in spring and autumn, showed a better effect against F. hepatica than the treatment with triclabendazole or albendazole per se (Mavromati et al. 2016).

Studies have showed that serum GGT and AST activities are elevated in the sheep fascioliasis due to the presence of flukes in the bile ducts and liver parenchyma, and consequent parenchymal degeneration (Matanović et al. 2007). The same results were found in our study that revealed high GGT and AST activities in sheep naturally infected with Fasciola sp. Treatment with TBZ or TBZ + LVM caused a significant decrease in the serum liver enzymes activities of infected sheep. This decrease can be regarded as an indicator of improving sheep’s liver condition. The liver enzymes serum activity values in the sheep treated with TBZ + LVM and TBZ decreased during the study. On the contrary, Kozat and Denizhan (2010) demonstrated that liver enzymes activity of sheep did not show any significant decrease even after receiving fascioliasis treatment (Kozat and Denizhan 2010). It has been reported that measurement of liver enzyme activity is useful for determination of drugs efficacy in fascioliasis, and increase in the liver enzyme activity in spite of anthelminthic treatment can be a sign of limited efficacy or therapeutic failure (Maria Martínez-Valladares et al. 2010). In the present study on days 7 and 14 post treatment, liver enzymes activity was lower in TBZ + LVM than TBZ alone (P < 0.05). This finding is in line with lower EPG and higher FECR values in the TBZ + LVM group. Probably, earlier lowering number of flukes in the liver and bile ducts by TBZ + LVM caused prior decrease in the liver enzymes activities.

Hypoalbuminemia due to damaged liver and hyperglobulinemia as an immunological response are common biochemical manifestations in the sheep fascioliasis (Matanović et al. 2007). The present study showed that treatment with TBZ + LVM caused increase in the albumin levels during 28 days period after treatment. Significant increases were noted on days 14 and also 28 (P < 0.05). Increase in the albumin can be a mark of decrease in the hepatic injuries (Kozat and Denizhan 2010). Some of this improvement in the liver condition of TBZ + LVM treated sheep may be attributed to LVM because its beneficial effects on the healing process. It has been documented that LVM possesses promising ameliorative properties (Waard et al. 1995), and will cause the healing process to occur more rapidly (Findlay et al. 2000). Levels of GLB in the sheep infected with fasciolosis are higher than healthy animals (Matanović et al. 2007). In the present study after treatment with TBZ or TBZ + LVM, GLB levels significantly decreased in the treated sheep in comparison to the control animals. In common with our findings, it has been reported that treatment of fascioliasis in buffaloes with oxyclozanide resulted in TP, ALB, and GLB changes into normal values (Pal and Dasgupta 2006). Scott et al (2005) reported increase in ALB, and decrease in GLB levels in lambs infected with subacute fasciolosis after treatment with TBZ (Scott et al. 2005).

Considering several numbers of reports on benzemidazole resistant F. hepatica from some parts of the word, proposing novel approaches to delay the emergence of resistance, and maintain the efficacy of available anthelmintics seems to be essential by now. To the best of our knowledge, no report has been published in the literature on TBZ resistant F. hepatica from sheep of the Middle East. Combinational therapy can be applied as a strategy to postpone the advent of drug resistant flukes in this region and other regions of the world. Findings of the present study clarified the importance of combinational therapy, and demonstrated that combination of TBZ + LVM resulted in higher efficacy and earlier improvement of liver conditions in sheep naturally infected with Fasciola sp. Further studies are needed to find out the possible positive effects of LVM on TBZ resistant flukes.

Acknowledgements

The authors would like to express their gratitude to Dr. Sooud Rouhani, veterinarian practitioner for his help and supports during the study. This study was partly supported by Amol University of Special Modern Technologies, Amol, Iran.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

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