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Annals of Parasitology 2015, 61(4), 283–289
Copyright 2015 Polish Parasitological Society
Evidence of Fasciola spp. resistance to albendazole,
triclabendazole and bromofenofos in water buffaloes
(Bubalus bubalis
)
Virginia M.Venturina1, Ma. Antonette F. Alejandro1, Cyril P. Baltazar2,
Nancy S. Abes2, Claro N. Mingala2,3

1College of Veterinary Science and Medicine, Central Luzon State University, Science City of Muñoz 3120, NuevaEcija, Philippines2Animal Health Unit, Philippine Carabao Center National Headquarters and Gene Pool, Science City of Muñoz 3120,Nueva Ecija, Philippines3Affiliate Faculty, Department of Animal Science, College of Agriculture, Central Luzon State University, ScienceCity of Muñoz 3120, Nueva Ecija, Philippines Corresponding author: Claro N. Mingala; e-mail: cnmingala@hotmail.com ABSTRACT. Fasciolosis caused by Fasciola spp. is considered the most important helminth infection of ruminants in
tropical countries. Anthelmintic resistance has become a global concern. This study compared the efficacy of the
commonly used anthelmintics, determined the toxicity level and any indication of resistance. Thirty two water buffaloes
naturally-infected with Fasciola spp. were used to determine the efficacy of triclabendazole (TBZ), albendazole (ABZ),
and bromofenofos (BRO) using Fecal Egg Count Reduction Test (FECRT). To test the toxicity of the drugs given, serum
glutamic-pyruvic transaminase (SGPT) was evaluated before and within one week after treatment. One dose
administration of ABZ registered an efficacy of 79.17%, 73.33% for TBZ and 70.83% for BRO. Efficacy in two dose-
treatment group was 83.33% for both BRO and ABZ, and 90.00% for TBZ. Two dose-treatment was effective for TBZ
(90%), ineffective for BRO and ABZ. SGPT levels were not significantly different between pre-treatment and post-
treatment across all treatments. Giving one or two doses of anthelmintics, at one month interval, does not increase the
efficacy of the three drugs tested. The study also implies that anthelmintic resistance may have developed in the animals.
Key words: Fasciola spp., albendazole, bromofenofos, triclabendazole, anthelmintic resistance
tropical countries [2]. It is the leading cause ofmorbidity and mortality in ruminants in the The total water buffalo population in the Philippines and classified as a disease of farm Philippines as of 2014 was 2.86M with more than concern by the Bureau of Animal Industry. 99.5% raised by smallhold farms. The number of Fasciolosis has been considered as the most commercial farms went up by 5.92% from last destructive parasitic disease of farm animals in the year's inventory while the backyard sector dropped Philippines. In a review reported by Copeman and by 1.03% [1]. This scenario suggests that there is a Copland [3], the prevalence of fasciolosis in need to improve the production management of buffaloes in various parts of the country ranged smallhold-raised farms that represent the large segment of the industry.
Surveys in some Asian countries have shown One of the problems that beset the carabao farms that amongst domestic animals, buffaloes suffer is the occurrence of fasciolosis. The disease is more frequently from fasciolosis [4]. This may be caused by Fasciola spp. which is considered the attributed to the high risk of infection to these most important helminth infection of ruminants in animals in view of their wallowing habit that V.M. Venturina et al.
increases their chance of access to the infective according to their FEC such that heavy, moderate, stage of the worm [2]. Immature Fasciola spp.
and low burdens of fasciolosis were equally parasitizes the liver while adults damage the bile distributed in the groups for each set. The animals ducts [5]. The worms compete with host nutrients were treated with triclabendazole (TBZ), leading to slow and poor development, liver albendazole (ABZ), bromofenofos (BRO) in their condemnation and poor carcass [6].
respective designated groups according to the The most common method of worm control in manufacturer's recommended dose.
ruminants is the use of chemical anthelmintics.
Fecal collection and fecalysis. About 5g of
However, evidence of resistance to various feces were collected directly from the rectum of anthelmintics has led to treatment failures [7].
each animal and properly labeled into zipped plastic Anthelmintic resistance has become a global bags. Collected samples were transported to the concern with the recent report of problems in the laboratory for analysis. Individual FEC was done efficacy of a number of anthelmintic preparations.
using the standard Sedimentation Technique [8].
Local outbreaks of fasciolosis have recently been Fecal Egg Count Reduction Test (FECRT).
reported in some towns in Nueva Ecija (Abes, Data for the average pre-treatment FEC was personal communication, 2013). This was alarming determined at 7 and 0 day post-treatment. Post- because there is a deworming program for treatment analysis of fecal egg counts were done on fasciolosis in the affected areas. Assessment of the the 14th day after treatment. Efficacy rate was based efficacy of available anthelmintics will provide a on the pre- and post-treatment FEC using this basis on planning a sustainable control program against fasciolosis. Options may be developed in order to preserve the efficacy of the drug by = number of egg × amount of fecal suspension ×100 judicious use and integration with other biological amount examined × amount of feces used worm control methods. where: EPG = egg per gram The study determined the efficacy of The efficacy of the anthelmintic compound was albendazole, triclabendazole, and bromofenofos determined based on reduction of egg excretion at against Fasciola spp. of naturally-infected water 14 days post-treatment using the formula below buffaloes in one-dose and two-dose treatments.
Indication of resistance to the drugs was based on the efficacy. The level of toxicity to these drugs = pre-treatment FEC - post-treatment FEC ×100 following treatment was also measured.
Pre-treatment FEC Liver Enzyme Assay. The effect of
Materials and Methods
anthelmintics on the liver function was determinedby the Serum glutamic-pyruvic transaminase Identification of experimental animals.
(SGPT) level determination. Initial SGPT level
Animals owned by farmer cooperatives in the were determined on day 0, prior to drug province of Nueva Ecija, Philippines were used in administration, and succeeding test were done on the study. These animals were utilized to determine 1st week post-treatment. A 10ml blood sample was the efficacy of three types of fasciolicides as reports collected from the jugular vein of the animal using of high morbidities and mortalities have been vacutainer tubes. Samples were kept on ice until accounted in this area (Abes, personal commu - SGPT analysis.
nication, 2013).
Statistical analysis. Analysis of Variance
A total of 32 buffaloes naturally-infected with (ANOVA) was used for the overall comparison of Fasciola spp. regardless of sex, non-pregnant with the different treatments at 95% confidence interval ages ≥8 months were used in the study. Each of the and P value of<0.05.
two treatment sets: one dose-treatment (Set 1) andtwo dose-treatment (Set 2), and the control group Results and Discussion
consisted of four animals for each experimental set.
The average individual pre-treatment fecal egg count Efficacy of TBZ, ABZ, and BRO against Fasciola
(FEC) using standard sedimentation technique was spp. infection using FECRT
taken two weeks before administration of The efficacy of the three drugs used namely anthelmintics. At day 0, all 32 animals were ranked TBZ, ABZ, and BRO that were given one dose- Evidence of Fasciola spp. resistance
Table 1. Efficacy of different anthelmintics against Fasciola spp. infection in buffaloes with one-dose treatment Faecal egg count (eggs per gram)
Efficacy (%)
Means of the same letter are not significantly different at P<0.05 treatment is shown in Table 1. No significant efficacy considering that the preponderant species differences were found in the efficacy of ABZ in the Philippines is Fasciola gigantica [7].
(79.17%), TBZ (73.33%), and BRO (70.83%).
Differences in reaction to a drug may occur at the Nevertheless, the three drugs are all significantly species level, which may be the case in the present different from the control in one dose treatment.
study. However, the eggs counted in the present The drugs used in the study have claims of efficacy study were not identified to the species level. It is to different developmental stages of Fasciola spp.
possible that the worm load of the animals were TBZ is effective against early immature, immature, predominated by Fasciola gigantica which may not and mature stages [11,12]. ABZ, a broad spectrum have high susceptibility to TBZ.
dewormer, is effective against adult stages of the The present findings suggest the development of parasite [13]. BRO on the other hand is known to be resistance of Fasciola sp. against the three drugs effective against mature and immature fluke stages used. According to the WAAVP standards, a highly [11]. The present study did not establish the specific effective anthelmintic should have an efficacy of stage at which the drugs used were effective as the >98% [14]. An efficacy of less than 90% is only basis for the calculation of efficacy is the considered an indication of resistance development.
FECRT. Nevertheless, this method is considered Several studies have established development of reliable in field trials of drug efficacy. Resistance or resistance of Fasciola spp. against different types of failure of treatment with the tested drug was defined anthelmintic. A recent study on Fasciola hepatica as efficacy of less than 90% based on the standards sheep isolate showed resistance against ABZ while set by the World Association for the Advancement being susceptible to TBZ [20]. On the contrary, of Veterinary Parasitologists (WAAVP) [14].
resistance of liver flukes to TBZ was observed in a Others have also used 90% reduction in FECRT as clinical trial in a cattle population [21]. the threshold for resistance based on a set country An earlier study demonstrated resistance of liver standard. For example, Brockwell et al. [15] flukes to TBZ in experimentally-infected sheep followed 90% reduction as cut off based on the with very low efficacy of 10%. In buffaloes, Australian flukicide efficacy trials.
resistance of liver flukes to flukecides have also Guidelines and standard tests to determine drug been reported [22].
resistance in F. hepatica is currently not available The mean pre-treatment and post-treatment FEC [16]. However, previous studies in experimentally- after one-dose administration of ABZ, TBZ, and infected cattle [17] and in sheep [18] show that the BRO are shown in Table 1. Except for the control, FEC have suitable characteristics for resistance there was a decreasing trend from pre-treatment to post-treatment FEC in all of the three treatments.
One dose treatment with TBZ was found to be Data shows a mean pre-treatment FEC of 675 EPG ineffective with efficacy of 72.22%. This is in ABZ, 450 EPG in TBZ, 325 EPG in BRO, and contrary to the reports of efficacy of TBZ against 450 EPG in the control animals. Both ABZ and liver flukes in the country [19]. There are factors TBZ groups registered a FEC of 150 EPG 14 days that may be associated with the inefficacy of the after treatment. Post-treatment FEC of 100 EPG and drug. It is worth mentioning that TBZ's claim of 600 EPGwere recorded for BRO and Control efficacy specifically refers to Fasciola hepatica.
groups, respectively. This may be considered a limiting factor in the drug Differences in the pre-treatment and post- V.M. Venturina et al.
Table 2. Efficacy and faecal egg counts of different anthelmintics against Fasciola spp. infection in buffaloes withtwo-dose treatment Faecal egg count (eggs per gram)
Efficacy (%)
Means of the same letter are not significantly different at P<0.05 treatment FEC of the different treatment groups can difference (P<0.05) between TBZ and the two other be possibly attributed to the fact that these animals were owned by different farmers and were located Administration of ABZ and BRO against liver in different areas. It is likely that they have flukes in buffaloes two times at one month interval differences in the level of infection and different was effective with efficacy rates of 83% for both amount of eggs excreted in the feces. Statistical drugs. The efficacy of TBZ (90%) indicates that the analysis reveals no significant difference P=0.05 drug was effective if given at two-dose treatment between the FEC after using ABZ, TBZ, and BRO regimen. TBZ is claimed to be effective against in treatment animals. early immature until the adult stage. On the other Efficacy of TBZ, ABZ, and BRO against
hand, BRO and ABZ are known to be effective Fasciola spp. infection using FECRT in two dose-
against adult and immature stages. It is possible that the immature stages were killed at first dose of TBZ The efficacies of ABZ, TBZ, and BRO in and hence reduced the number of egg-laying adults.
buffaloes after two dose-treatments with interval of It seems likely as well that immature flukes were one month in-between treatments are shown in not affected by ABZ and BRO allowing them to Table 2. ABZ had an efficacy of 83.33% which was mature and lay eggs.
similar to those treated with BRO. The efficacy of There is an indication based on the present TBZ (90%) was higher compared to the two other findings that resistance to ABZ and BRO may be treatments. However, there was no significant present. ABZ, a broad spectrum dewormer, is Fig. 1. Efficacy (%) of albendazole (ABZ), triclabendazole (TBZ), and bromofenofos (BRO) against Fasciola spp. inbuffaloes Evidence of Fasciola spp. resistance
Table 3. Comparison of SGPT values in triclabendazole On the other hand, BRO group had an initial (TBZ), albendazole (ABZ), and bromofenofos (BRO) FEC average of 250 EPG which was reduced to 50 treated buffaloes and Control (CON) in one-dose EPG 14 days after treatment. Meanwhile, the control group had an initial FEC of 375 EPG which Mean SGPT Level (U/L)
slightly reduced to 300 EPG after 14 days. Thedifference in the FEC of the different treatment groups from the pre-treatment to the 14th day post-treatment can be possibly attributed to the different farm locations of the farms. Reduction of FEC in the untreated control group can be attributed to thepossible natural death and expulsion of worms that reach their life span. Statistical analysis reveals no significant difference between the FEC after using ABZ, TBZ, and BRO in treatment animals P=0.05. Means with the same letter within rows are not Comparison of efficacy of TBZ, ABZ, and BRO
significantly different at P<0.05 against Fasciola spp infection using FECRT in
commonly used in ruminants. Indication of ABZ one-dose treatment and two dose-treatment
resistance in nematodes of buffaloes has been Comparison on the efficacy of the three drugs reported in the Philippines [19]. Frequent use of the given at one dose and two dose- treatment at one same anthelmintic has been proven to be one of the month interval is shown in Fig. 1. Analysis of the factors that contribute to resistance development.
data shows that the efficacy of ABZ is comparable Abes (personal communication, 2013) had between one dose and two-dose treatments with all disclosed that ABZ had been in use for more than three anthelmintics used. There was no significant five years in most cooperator farms. Hence, the difference between the two treatment sets for ABZ probability that selection pressure for resistance at P =0.80. Similarly, one-dose and two-dose development has increased throughout the years.
treatments with TBZ (P=0.41) and BRO (P=0.62) TBZ may be considered effective at 90% but since were not significantly different. These findings the drug is expected to be highly effective (98%) indicate that administration of one or two-dose with single dose as a commercial drug, the current treatments of any of the three drugs used have efficacy shows a tendency for the drug to develop comparable levels of efficacy. Results also suggest that giving a subsequent dose of any of the three The mean pre-treatment and post-treatment FEC anthelmintics after a month will not increase their after two-dose administration of ABZ, TBZ, and efficacy. Some farmers have disclosed that they tend BRO with interval of one month are shown in Table to give a follow-up dose of anthelmintics if they 2. The data shows a mean pre-treatment FEC of 725 observe that the animal's weight based on visual EPG in ABZ, 475 EPG in TBZ, 250 EPG in BRO, estimation do not improve after one treatment dose.
and 375 EPG in the control animals. Both TBZ and This study proves that a follow-up treatment at one BRO groups registered a FEC of 50 EPG 14 days month interval will not improve the efficacy of the after treatment, while a post-treatment FEC of 100 drug. Thus, it only shows that it is impractical to EPG and 300 EPG respectively were recorded for give a second dose of the respective anthelmintics as ABZ and the control groups.
it will be an added cost without additional benefit.
Table 4. Comparison of SGPT values in triclabendazole (TBZ), albendazole (ABZ), bromofenofos (BRO) and CON(Control) SGPT (U/L) 1st Treatment
SGPT (U/L) 2nd Treatment
Means with the same letter within rows are not significantly different at P<0.05 V.M. Venturina et al.
SGPT Levels of buffaloes before and after
mean of 55.00 U/L SGPT before treatment and was treatment with TBZ, ABZ, and BRO in one dose-
reduced to 49.90 U/L SGPT after treatment. TBZ treatment and two-dose treatment
had a mean of 48.77 U/L SGPT before treatment The mean values of SGPT on each treatment are and has increased to 55.67 U/L SGPT after shown on Table 3. ABZ had an average SGPT of treatment. Animals treated with BRO had a mean of 54.83 U/L prior to treatment which was reduced 48.43 U/L SGPT before treatment which was slightly to 50.43 U/L after treatment. Conversely, lowered to 44.67 U/L SGPT after treatment. Control SGPT level before treatment with TBZ was 47.80 group however has a mean of 56.77 U/L SGPT U/L which had a subtle increase after treatment before treatment and 57.00 U/L after treatment.
(49.77 U/L). The group treated with BRO had mean These differences in SGPT levels before and after of 53.13 U/L SGPT before treatment which was treatment in all treatments including the control are reduced to 41.90 U/L after treatment. The SGPT of not significantly different at P<0.05. Non- the control group was 46.87 U/L before treatment significant reduction in liver function tests after which rose to 61.33 U/L after treatment. These anthelmintic treatment against Fasciola spp. is differences in SGPT levels before and after inconsistent with the findings of Pal and Dasgupta treatment in all treatments including the control are [24]. They demonstrated that aspartate amino - not significantly different at P<0.05.
transferase (AST), alanine aminotranferase (ALT) The normal SGPT value in cattle and buffalo and alkaline phosphatase (ALP) decreased may vary from 6.9–35 U/L depending on associated significantly after treatment with TBZ. Others have condition. Except for the post-treatment SGPT shown increase in SGPT following treatment [25].
levels in the control group, the SGPT levels High SGPT in control animals before treatment may obtained in the current study is within the normal be attributed to other unknown physiological range, assuming that the standards set for cattle are difference in these animals. In the same manner, the same with buffaloes. However, there is no SGPT is normally present in large concentrations in significant increase or decrease in the level of the the liver due to worm infestation.
SPGT values in all the animals treated with differentanthelmintics, hence it can be concluded that there was no significant effect on the liver. References There is an indication of resistance of Fasciola show that increase in the SGPT values are related to spp. in Philippine water buffaloes to albendazole, toxicity of the drug to the liver, primarily because an triclabendazole, and bromofenofos based on the low increase in the enzymes means the liver overworked efficacy. One or two-dose treatment with the three for the release of these enzymes in the blood, hence anthelmintics has no effect on efficacy of the drugs.
the detected high values [23]. However, other None of the drugs tested are potentially toxic based physiological processes such as increase exercise, on the SGPT levels after treatment.
pregnancy, and even muscle damage can increaseSGPT values. An extreme fluctuation however isindicative of liver disease.
The mean SGPT on one dose- and two dose- We thank Dr. Arnel N. Del Barrio, Executive treatment with TBZ, ABZ, and BRO, as well as the Director of the Philippine Carabao Center, for his untreated control are shown in Table 4. With one valuable support. We also thank Dr. Peregrino G.
dose- treatment, ABZ showed an average SGPT of Duran for providing support and assistance to 53.13 U/L before treatment which was decreased to conduct the study in the PCC National Impact Zone, 41.90 U/L after treatment. This observation was Nueva Ecija. We extend our appreciation to Prof.
inconsistent with TBZ wherein SGPT level before Misao Onuma and Dr. Fumitoshi Saito of the treatment was 50.10 U/L which increased slightly to Consortium for Japanese Veterinary Medicinal 51.90 U/L after treatment. The group treated with BRO has a mean of 41.00 U/L SGPT beforetreatment which increased very slightly to 42.67U/L SGPT after treatment. However, the control group has a mean of 85.10 U/L SGPT before [1] BAS 2014. Bureau of Agricultural Statistics.
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for evaluating the efficacy of anthelmintics in Received 12 October 2015 ruminants (bovine, ovine, caprine). Veterinary Accepted 17 November 2015

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