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Introduction
The trematode Fasciola hepatica has been reported in multiple countries worldwide to exhibit resistance to triclabendazole, which has long been the preferred drug for combating fascioliasis in both animals and humans (Cabada et al., 2016; McMahon et al., 2016; Ramadan et al., 2019). Additionally, instances of resistance in specific regions have also been documented against albendazole (Sanabria et al., 2013; Novobilský et al., 2016; Ceballos et al., 2019), as well as closantel (Novobilský and Höglund, 2015), and rafoxanide (Rapic et al., 1988; Elitok et al., 2006).
Association of metabolites with comparable activity and distinct mechanisms of action becomes imperative when pharmacological principles alone do not exhibit efficacy. This approach broadens the spectrum of activity of individual drugs, facilitating the treatment of mixed parasitosis or parasites belonging to the same phylum. Furthermore, such combinations can potentially delay the development of resistance to anthelmintics (Bartram et al., 2012). Incorporation of active principles with differing mechanisms of action from diverse chemical groups enhances the likelihood of achieving synergistic effects (Geary et al., 2012). Consequently, triclabendazole -the primary fasciolicide of choice- has been combined with various anthelmintics, including clorsulon, ivermectin, levamisole, luxabendazole, moxidectin, nitroxynil, oxfendazole, oxyclozanide, among others. Such combinations have demonstrated improved efficacy (Fairweather and Boray, 1999; Geurden et al., 2012; Martínez-Valladares et al., 2014; Khan et al., 2017).
The World Association for the Advancement of Veterinary Parasitology (WAAVP) recommends methods and techniques for assessing antiparasitic efficacy. In controlled trials, efficacy is determined by comparing the number of live parasites in treated animals with that in untreated controls. However, in clinical trials involving live animals, efficacy is ascertained by comparing fecal egg count of treated animals to that of the same untreated animals shortly before or at the time of treatment and within a period not less than 3 weeks later (Wood et al., 1995).
Similar to many regions worldwide, Cajamarca serves as a prominent cattle-raising area where dairy cattle breeds like Holstein Friesian, Brown Swiss, and Jersey are extensively reared. Nonetheless, numerous provinces within Cajamarca are marked by endemic fascioliasis, affecting both animals and humans (Cornejo et al., 2010; Rodríguez-Ulloa et al., 2018; Torrel et al., 2023). This scenario has prompted prolonged anthelmintic usage, ultimately leading to the emergence of anthelmintic resistance due to persistent use of the same active ingredient. This situation is exemplified by triclabendazole, specifically within the Cajamarca district's valley, impacting dairy cattle (Ortiz et al., 2013). This situation is unknown in other provinces, where evaluation of alternative fasciolicides has been unexplored. Thus, the present study assessed the efficacy of four chemical products, namely triclabendazole, nitroxynil, clorsulon/ivermectin, and closantel in four cattle farms within the Cajamarca provinces -Cajamarca, San Marcos, and San Miguel.
Materials and methods
Ethical considerations
Farm owners were informed and gave written authorization for the use of their animals. In addition, all procedures were in accordance with the European ethical regulations for the use of animals in scientific research (European Directive 2010/63/EU).
Location
The study was conducted in four cattle farms located in three provinces of the Cajamarca region: Cajamarca (P-I and P-II), San Marcos (P-III), and San Miguel (P-IV) (Figure 1). Processing and diagnostic tests were performed at Laboratorio de Parasitología Veterinaria y Enfermedades Parasitarias, Facultad de Ciencias Veterinarias of Universidad Nacional de Cajamarca, Perú.
Experimental design
A cross-sectional study was conducted were initial sampling included all the animals within the farms, aiming to confirm positive cases and prevalence rates. From this, a cohort of 60 female cows, each exceeding eight months of age, was meticulously chosen. These cows were positive for presence of Fasciola hepatica eggs in fecal matter, with a parasite load equal to or exceeding 1 egg per gram of feces (EPG). The selection process involved animals naturally infected within each farm; specifically, Jersey cows in the first farm and Holstein cows in the remaining three. Furthermore, the selected cows had not been subjected to anthelmintic administration during four months. They were maintained under similar conditions in terms of management and feeding, in an extensive breeding system.
Early in the morning (6 a.m.) cows were weighted using bovine-specific metric tape tailored for the Jersey/Holstein breed. Simultaneously, fecal samples (approximately 100 g each) were directly retrieved from the rectum using veterinary obstetrical gloves. These samples underwent processing on the same day, employing the Rapid Sedimentation Technique as outlined by Lumbreras et al. (1962). Briefly: a homogenization procedure was employed by mixing four grams fecal matter with 40 mL running water within a conical-bottomed tube. This amalgamation was sieved into a 250 mL glass beaker, completing the volume with running water. The solution was then left to rest for 30 min. Subsequently, two-thirds of the supernatant was decanted and replenished with water for an identical resting period. This sequence was repeated until the supernatant exhibited apparent clarity. The ultimate sediment was augmented with two drops of methylene blue and placed within a Petri dish for examination using a stereomicroscope (3X, 4X).
Three days after obtaining the coproparasitological outcomes four groups were constituted per farm, each with the same number of individuals (n = 15). Each of these groups underwent administration of an antiparasitic agent. The composition of these groups was structured as follows: triclabendazole at a dose of 12 mg/kg of BW, VO (Bilevon® 12%, Bayer S.A. Lab. CIFARMA S.A., Peru); clorsulon at 2 mg/kg of BW, SC, and ivermectin at 0.2 mg/kg of BW, SC (Ivomec® F, Boehringer Ingelheim Animal Health do Brasil Ltda, Brazil); closantel at 10 mg/kg of BW, VO (Fasintel®10, Quimtia, Peru); and nitroxinil at 10 mg/kg of BW, SC (Nitromic 34%, Lab. Microsules Uruguay S.A., Uruguay). The volume administered was calculated by multiplying animal live weight by therapeutic dose of each active ingredient, and then dividing by the concentration of the product. Administration of clorsulon in conjunction with ivermectin did not intend to link these two substances; rather, it was due to absence of a commercially available product in the local market with solely the clorsulon active ingredient.
Sampling and analysis
At a third visit on day 30 post-dosing, fecal samples were extracted with the same procedure conducted during the first visit, and coproparasitological analyses were performed again by rapid sedimentation, and the Fecal Egg Count Reduction Test (FERCT) was applied.
Fecal samples were obtained using the identical procedure as employed during the initial sampling. Subsequently, coproparasitological analyses were reiterated utilizing the rapid sedimentation technique, resulting in the quantification of fecal egg count (FEC) which was expressed in eggs per gram (EPG).
Consequently, the anthelmintic effectiveness of each medication was assessed in accordance with the protocols stipulated by the WAAVP, employing the formulation for a fecal egg count reduction test (FECRT): %reduction = [(X̅ FEC day 0 - X̅ FEC day 30) / X̅ FEC day 0] x 100, where X̅ represents the arithmetic mean. Categorization of product efficacy was defined as highly effective (>98%), effective (90-98%), moderately effective (80 - 89%), and insufficiently active (<80%) (Wood et al., 1995). Simultaneously, the percentage of cattle positive by coprology reduction (CPCR) was also determined.
To establish more precisely the efficacy of flukicides in clinical trials involving F. hepatica, the WAAVP recommends calculating the FEC of treated animals over a period not less than 3 w and comparing it to the FEC of the same animals prior to treatment or at the exact dosing time. This time frame is justified by the biological cycle of the trematode. In the early immature stage (1-4 w), the trematode is migrating to the parenchyma, in the late immature stage (6-8 w), it is in the prepatent period within the biliary duct, and in the mature stage (12-14 w) it resides in the biliary ducts (Wood et al., 1995). In other words, immature parasites do not mature and produce eggs within 30 d, which could introduce bias during the analysis.
The Wilcoxon test (for paired samples) was employed using the IBM SPSS Statistics 27.0.1 software to assess whether a statistical difference exists between EPG on day 0 and day 30. Differences or similarities in egg count between day 0 and day 30, as well as efficacies among the antiparasitic groups within each farm, were analyzed using the Kruskal-Wallis statistic. Following this test, the Mann-Whitney U test was employed to identify distinct groups within each farm in cases where the Kruskal-Walli’s test detected statistical differences.
Results
Through FERCT and CPCR assessments, triclabendazole demonstrated insufficient activity across all farms. The clorsulon/ivermectin and closantel groups exhibited high efficacy in all farms, while nitroxynil showed varying efficacy results in both types of analysis (Table 1).
Table 1 Anthelmintic efficacy against F. hepatica within naturally infected cattle across three provinces within Cajamarca region.
| Province | Prevalence (%) | Drug | n | FERCT | CPCR | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| x̄ EPG ± CI95% (day 0) | x̄ EPG ± CI95% (day 30) | Efficacy (%) | Condition | Efficacy ± CI95% | Condition | |||||
| Cajamarca: P-I | 80/117 (68.38 ± 8.43) | Triclabendazole | 15 | 6.53 ± 3.62ax | 2.47 ± 1.32b | 62.24 ± 9.60x | IA | 33.33 ± 23.86x | IA | |
| Clorsulon/Ivermectin | 15 | 6.27 ± 3.03ax | 0.00 ± 0.00b | 100 ± 0.00y | HE | 100 ± 0.00y | HE | |||
| Closantel | 15 | 5.93 ± 3.74ax | 0.00 ± 0.00b | 100 ± 0.00y | HE | 100 ± 0.00y | HE | |||
| Nitroxynil | 15 | 7.13 ± 2.82ax | 0.07 ± 0.13b | 99.07 ± 1.82z | HE | 93.33 ± 12.63z | E | |||
| Cajamarca: P-II | 62/76 (81.58 ± 8.72) | Triclabendazole | 15 | 7.93 ± 5.78ax | 6.47 ± 5.65b | 18.49 ± 6.98x | IA | 20 ± 20.24x | IA | |
| Clorsulon/Ivermectin | 15 | 7.4 ± 3.17ax | 0.00 ± 0.00b | 100 ± 0.00y | HE | 100 ± 0.00y | HE | |||
| Closantel | 15 | 7.8 ± 3.99ax | 0.00 ± 0.00b | 100 ± 0.00y | HE | 100 ± 0.00y | HE | |||
| Nitroxynil | 15 | 9.13 ± 3.73ax | 0.06 ± 0.13b | 99.27 ± 1.43z | HE | 93.33 ± 12.63z | E | |||
| San Marcos: P-III | 65/95 (68.42 ± 9.35) | Triclabendazole | 15 | 6.13 ± 3.44ax | 3.07 ± 2.50b | 50.00 ± 10.22x | IA | 26.67 ± 22.38x | IA | |
| Clorsulon/Ivermectin | 15 | 8.13 ± 3.49ax | 0.00 ± 0.00b | 100 ± 0.00y | HE | 100 ± 0.00y | HE | |||
| Closantel | 15 | 7.07 ± 3.63ax | 0.00 ± 0.00b | 100 ± 0.00y | HE | 100 ± 0.00y | HE | |||
| Nitroxynil | 15 | 5.73 ± 2.92ax | 0.4 ± 0.32b | 93.02 ± 5.39z | E | 66.67 ± 23.86x | IA | |||
| San Miguel: P-IV | 69/124 (55.65 ± 8.74) | Triclabendazole | 15 | 18.47 ± 5.01ax | 16 ± 5.43a | 13.36 ± 4.30x | IA | 0.00 ± 0.00x | IA | |
| Clorsulon/Ivermectin | 15 | 20.07 ± 5.71ax | 0.00 ± 0.00b | 100 ± 0.00y | HE | 100 ± 0.00y | HE | |||
| Closantel | 15 | 19.13 ± 7.81ax | 0.00 ± 0.00b | 100 ± 0.00y | HE | 100 ± 0.00y | HE | |||
| Nitroxynil | 15 | 18.07 ± 8.08ax | 0.00 ± 0.00b | 100 ± 0.00y | HE | 100 ±0.00y | HE | |||
*EPG: Eggs per gram o feces
a,bDistinct letters within the same row indicate statistical differences in EPG counts between day 0 and day 30 (Wilcoxon test, p<0.05).
x,y,zDistinct letters within the same column within each farm indicate statistical differences in efficacies (Kruskal-Wallis + Mann-Whitney U post hoc, p<0.05).
Dosage: Triclabendazole 12 mg/kg, clorsulon 2 mg/kg, closantel 10 mg/kg, nitroxynil 10 mg/kg, ivermectin 0.2 mg/kg.
Categorization: Insufficiently active (IA), effective (E), and highly effective (HE).
Discussion
According to the WAVVP, in clinical trials conducted on animals naturally infected with Fasciola hepatica the control period must extend for at least 21 d (Wood et al., 1995) because adult flukes could be dying and yet continue to release eggs, or the eggs stored in the bile duct could be eliminated. Upon reaching the 30-d mark -as in the present study- it is assured that adult trematodes have either died or become infertile as a result of the antiparasitic treatments. Furthermore, the possibility of a juvenile parasite developing into an adult and initiating egg laying within a 30-d timeframe is not likely. If any juvenile worm were to migrate to the bile ducts, it would perish due to the early blood consumption induced by the administered drugs. If it were to survive, it would take between two to three months to reach sexual maturity and commence egg production.
Although the WAAVP envisions studies on antiparasitic efficacy with control groups, it was not feasible in the present study since livestock farmers in the Cajamarca Valley follow a health calendar that entails three to four mandatory deworming treatments per year. They strictly adhere to this schedule and do not allow their animals to exceed these intervals, even though they do not perform diagnostic studies to confirm parasitic infections. If, for any reason, they detect that their animals are confirmed to have parasites, they become distressed and do not cooperate in establishing a control group, making it difficult to conduct comprehensive studies including control groups. Therefore, working with control groups is nearly impossible in this location unless the study involves the use of the researchers’ own animals. Nonetheless, clinical studies comparing fecal egg counts between post-treatment and pre-treatment stages yield satisfactory results.
The prevalence of Fasciola hepatica in the farms ranged from 55.65 ± 8.74% in San Miguel to 81.58 ± 8.72% in Cajamarca. These results are not novel, as Cajamarca, particularly the Cajamarca Valley, is recognized as an endemic area for fasciolosis due to favorable environmental conditions for the intermediate host and extensive breeding. Records of trematodes in animals exist, even predating 1998 (Claxton et al., 1998). Various districts in Cajamarca have reported diverse prevalences of F. hepatica in cattle. In more distant districts from the Cajamarca Valley, such as Chota, a prevalence of 20.3 ± 4% has been found, 45.5 ± 5% in Celendín, 50 ± 5% in San Juan, and in close areas, 80.7 ± 4.1% in La Encañada, 61.2 ± 5.6% in Los Baños del Inca, and 49.5 ± 5% in hamlets of the Cajamarca district (Torrel et al., 2023).
Due to high prevalence of this trematode in the Cajamarca Valley, livestock farmers have the habit of deworming their animals against F. hepatica every three or four months. Due to the fact that triclabendazole acts on various stages of the trematode (Cwiklinski and Dalton, 2018), it has been indiscriminately used for a long time without technical considerations throughout the local livestock sector. Reports of its use even precede 1998 (Claxton et al., 1998). The average livestock owner does not conduct clinical efficacy tests of the antiparasitics used. This responsibility falls on non-professional livestock personnel who often fail to accurately calculate therapeutic doses and neglect drug rotation. This situation is consistent with observations by other researchers who have mentioned that resistance tends to develop when the parasite control relies exclusively on the same active ingredient over an extended period, with high frequency of deworming, suboptimal dosing, indiscriminate use of antiparasitics, lack of drug rotation, and the absence of comprehensive technical criteria (Márquez, 2003; Anziani and Fiel, 2015).
Due to these circumstances, triclabendazole was reported to be inadequately effective in controlling F. hepatica in dairy cattle within the Cajamarca Valley as early as 2012. In one farm (Tartar) 2.8% efficacy was observed; 3.1% in the second (El Cortijo); and 68% in a third (San Vicente) (Rojas-Moncada, 2012). A year later in the same valley, in a more rigorous study, triclabendazole achieved 31.05% efficacy on day 14 and 13.63% on day 30 in cattle (Ortiz et al., 2013). Simultaneously, reports of F. hepatica resistance to triclabendazole surfaced in other regions of Peru (Chávez et al., 2012). This phenomenon has also been observed worldwide (Olaechea et al., 2011; Brockwell et al., 2013; Coyne et al., 2020; Kelley et al., 2020). Even at a concentration of 24 mg/kg, double the usual dose, satisfactory results have not been achieved (Romero et al., 2019). Nevertheless, in areas where its use is not widespread or where its introduction is recent, triclabendazole maintains optimal efficacy (Kouadio et al., 2021).
The local livestock owners with better resources engage in improved livestock management and receive guidance from veterinarians to implement strategic antiparasitic administration, including drug rotation, which might explain the high efficacy of clorsulon/ivermectin, closantel, and nitroxinil. Furthermore, these drugs are relatively new in the local market compared to triclabendazole. Other researchers have also found satisfactory efficacy results. Clorsulon has been used as an alternative to eliminate the adult phase of parasites resistant to triclabendazole (Elliott et al., 2015). While closantel has shown excellent results in the present study and in other regions (Borgsteede et al., 2008; Nzalawahe et al., 2018; Bushra et al., 2019), therapeutic failures have also been reported (Novobilský and Höglund, 2015). On the other hand, nitroxinil has yielded optimal results in the therapeutic management of bovine fascioliasis and represents an alternative in cases of triclabendazole resistance in cattle (Wood et al., 1995; Martínez-Valladares et al., 2010).
Several researchers report that antiparasitic products with efficacy below 90-95% still hold value, even if not 100% effective, as they substantially reduce parasite burden or reach an economic threshold, thus not significantly impacting animal health and productivity (Fairweather, 2011; Forbes, 2013). However, in the present study triclabendazole did not reach even 20% efficacy; hence, its use should be ceased to avoid unnecessary losses, given that the cost of bovine fasciolosis infection can be quite high, manifesting as decreased fertility, reduced weight gain, diminished milk production, liver condemnations, and poor carcass performance (Schweizer et al., 2005; Sariözkan and Yalçın, 2011; Charlier et al., 2012; Fanke et al., 2017). Therefore, control schemes must be cost-effective, and drug administration should be judicious, accompanied by regular clinical efficacy studies. Nevertheless, controlling F. hepatica requires an integrated approach considering the epidemiological triad. For instance, cattle are less infected with F. hepatica in sprinkler-irrigated pastures, in contrast to flood-irrigated pastures, which is a common practice in Cajamarca (Torrel-Pajares et al., 2023).
Although emphasis is being placed on current techniques for evaluating F. hepatica resistance and several diagnostic methods are available, recommended guidelines and standardized protocols are lacking (Fairweather et al., 2020). Molecular techniques can be employed to identify molecular markers of resistance, along with simpler methods such as the controlled efficacy test (CET), fecal egg count/reduction test (FEC/FECRT), coproantigen reduction test (CRT), and egg hatching assay (EHA). The CRT has proven to be a solid alternative to FECRT for evaluating triclabendazole resistance of F. hepatica in cattle, and its use involves employing an ELISA kit (Brockwell et al., 2013). However, the CRT and other techniques entail higher costs compared to FECRT, which can be implemented in basic laboratories and field settings, accessible to most professionals with non-sophisticated and cost-effective technology. Nonetheless, further studies comparing these techniques are necessary to define the best method for assessing drug resistance of F. hepatica.
In conclusion, triclabendazole is insufficiently effective against F. hepatica. Antiparasitics based on clorsulon/ivermectin, closantel or nitroxinil show good results for controlling the trematode in the studied zones of Cajamarca region. However, they should be carefully used -including rotation and regular clinical evaluations- to prevent antiparasitic resistance.
