- Email: [email protected]
Moxidectin-resistant nematodes in cattle in Brazil
Veterinary Parasitology 161 (2009) 213–217
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Moxidectin-resistant nematodes in cattle in Brazil G.K. Condi a, R.G.V. Soutello b, A.F.T. Amarante a,* a b
UNESP - Universidade Estadual Paulista, Departamento de Parasitologia, Instituto de Biocieˆncias, Caixa Postal 510, Botucatu, SP, CEP 18618-000, Brazil Faculdade de Cieˆncias Agra´rias de Andradina, SP, CEP 16901-160, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 November 2008 Received in revised form 22 January 2009 Accepted 26 January 2009
The frequent use of anthelmintics in cattle has contributed to the emergence of gastrointestinal nematode populations resistant to the drugs available in the market. This study aimed to evaluate moxidectin efficacy in a property with suspected presence of Oesophagostomum spp. resistant to such anthelmintic. The accuracy of fecal egg count reduction test (FECRT) was also assessed through comparison with the data obtained in the controlled efficacy test (worm burden determination after necropsy). Twenty worm free steers were placed together with cattle of the farm with suspected moxidectin resistance. They became naturally infected with the parasite species present in the farm, and when fecal egg counts (FEC) were higher than 200 eggs/g, the animals were randomly distributed into two groups. One group (n = 10) was treated with moxidectin (0.2 mg/kg; Cydectin1, Fort Dodge) and the other was the control group (n = 10). Fecal samples from each steer were collected on the treatment day and three, seven, 10 and 14 days later for FEC and fecal cultures. At 14 days after treatment, all animals were sacrificed and the gastrointestinal nematodes were recovered, identified and counted. Mean FEC reduction in the treated group (compared with control group) was of 88, 85, 88 and 92% at days three, seven, 10 and 14 after treatment, respectively. In fecal cultures, third stage larvae of Cooperia spp. and Oesophagostomum spp. were predominant. As regards the controlled efficacy test, moxidectin had 100% efficacy against the genera Haemonchus spp. and Trichostrongylus spp., and efficacy of 65.2% for Cooperia spp. (Cooperia punctata and Cooperia pectinata), 44.8% for Oesophagostomum radiatum, and 81.4% for Trichuris spp. Most of the Cooperia spp. females obtained from the control animals showed eggs inside the uterus (98.5%); conversely, only 48.2% of the females from the treated group had eggs (P < 0.001). Considering the suppression in egg output following moxidectin treatment, FECRT has to be employed with caution to avoid under estimation of the anthelmintic resistance. From necropsy results, it can be concluded that C. punctata, C. pectinata, O. radiatum and Trichuris spp. showed resistance to moxidectin. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Anthelmintic resistance Macrocyclic lactone Oesophagostomum Trichuris Cooperia
1. Introduction The anthelmintic resistance has become a threat to the prophylaxis of parasitic gastroenteritis in cattle worldwide. In New Zealand, there are several reports on bovine
* Corresponding author. Tel.: +55 14 3811 6239; fax: +55 14 3815 3744. E-mail address: [email protected] (A.F.T. Amarante). 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.01.031
anthelmintic resistance (Mason & McKay, 2006; Waghorn et al., 2006). In North Island, for example, 92% of the evaluated properties showed resistance to ivermectin; 76% to albendazole; 74% to the association ivermectin and albendazole; and 6% to levamisole. Cooperia spp. was the most prevalent parasite in resistant populations, followed by Ostertagia spp. (Waghorn et al., 2006). In Argentina, Cooperia spp. has also been the main genus involved in cases of resistance to ivermectin in cattle
214
G.K. Condi et al. / Veterinary Parasitology 161 (2009) 213–217
(Suarez and Cristel, 2007). In addition, there are also reports of benzimidazole-resistant Ostertagia spp. (Suarez and Cristel, 2007) and Haemonchus spp. with simultaneous resistance to ivermectin and benzimidazoles in bovine herds (Anziani et al., 2004). In Brazil, a high frequency of ivermectin-resistant Haemonchus spp. and Cooperia spp. has been detected in farms of Sa˜o Paulo and Santa Catarina States, and in some farms, albendazole- and levamisole-resistant nematodes were also found (Soutello et al., 2007; Souza et al., 2008). Recently, Soutello et al. (2007) have reported the suspected presence of moxidectin-resistant Oesophagostomum spp. in a cattle farm located in the northwest region of Sa˜o Paulo State, Brazil. This fact stimulated the development of the present study, which aimed to further investigate the presence of moxidectin-resistant gastrointestinal nematodes in the cattle of such farm. The accuracy of fecal egg count reduction test (FECRT) for detection of anthelmintic resistances was also evaluated in comparison with the results of the controlled efficacy test. 2. Material and methods 2.1. Property description and history of anthelmintic utilization This study was carried out from April to June 2007 in a farm located in the municipality of Castilho, northwest region of Sa˜o Paulo State, Brazil. The property has 1210 hectares used for Nelore beef cattle production in pastures of Brachiaria decumbens, Brachiaria humidicula and Panicum maximum. During the experiment, approximately 100 sheep and 80 horses were also being kept in the farm. Macrocyclic lactone anthelmintics have been used in this farm since the beginning of the 1980s. At first, ivermectin was employed, followed by abamectin, mainly at castration and in animals acquired from other properties on their arrival at the farm. However, at that time, levamisole was the most frequently used anthelmintic. In 1992, the weight gain of animals from two properties in that region was comparatively evaluated after moxidectin (Cydectin1, Fort Dodge), ivermectin or levamisole treatment. As the former anthelmintic led to higher weight gain (data not published), moxidectin started to be used in all animals of the farm for approximately five years. Then, to reduce costs, levamisole was chosen to be administered to cows, whereas the remaining bovine categories continued receiving moxidectin treatment. Briefly, anthelmintic application has been carried out as follows: - Adult cows: levamisole once every year when gestation is diagnosed (May to July); - Calves before weaning: moxidectin at three or four months of age, in February; - Steers and heifers: moxidectin twice every year (May and September); - -Adult bulls: moxidectin before and after the breeding season (April and September); - Male calves at castration: abamectin (generally in March);
- Animals purchased from other farms were treated with moxidectin on their arrival at the property. Sheep did not have access to most of the pastures grazed by cattle and moxidectin has also been used in these animals for more than 10 years, twice every year. 2.2. Experiment description Initially, 30 crossbred steers (Bos taurus) of European breeds, aged from eight to 12 months, were obtained from other farms. Before entering the farm, they were identified with numbered earrings, weighed and treated with levamisole phosphate (4.7 mg/kg; Ripercol1 150F, Fort Dodge) in order to be introduced free of nematode infections. Fecal samples from the animals were examined to confirm the efficacy of such treatment. Thus, the animals were naturally reinfected only with the nematode species present in the farm. The steers were placed together with other 200 animals of the farm, which aged between eight and 15 months, in two paddocks of approximately 48.4 hectares each. The animals had free access to troughs with mineral salt and natural watering. Before the beginning of the experiment, they were vaccinated against foot-and-mouth disease (Aftobov1, Intervet) and clostridioses (Polisinto-Vac1, Fort Dodge). After the animals had been kept for 40 days in the contaminated paddocks of the farm, fecal samples were weekly collected directly from their rectum to determine fecal egg counts (FEC) by a modified McMaster technique, where each egg counted represented 50 eggs/g. Fecal cultures were prepared for obtaining and identifying third stage larvae (Ueno and Gonc¸alves, 1998). Of the 30 animals, those 20 with the highest counts of eggs per gram of feces (EPG) (superior to 200 EPG) were selected. On the treatment day (day 0), the animals were classified in pairs and in increasing order of FEC. Then, one animal from each pair was randomly allocated to the treated group and the other to the control group. These groups had weight means of 120.1 kg 25.64 and 116.6 kg 21.34, respectively, on day 0. Animals from the treated group were subcutaneously injected with moxidectin 1%, according to the manufacturer’s instructions (0.2 mg/kg; Cydectin1, Fort Dodge). Control animals received a placebo (saline solution). One day before slaughtering, the animals were gathered in a corral, where they were kept for 24 h with access only to drinking water and no solid food. Twelve hours before slaughtering, water supply was also interrupted. Fecal samples from each animal were collected on the treatment day and again at 3, 7, 10 and 14 days later. Samples were individually processed for FEC and fecal culture preparation. At 14 days after treatment, all animals from both groups were sacrificed for removal of the gastrointestinal tract. Material collection for parasitological analyses was carried out according to the descriptions of Ueno and Gonc¸alves (1998). Samples with 5% of each organ content were preserved in 4% formaldehyde and stored in identified containers. Such material was later processed for quanti-
G.K. Condi et al. / Veterinary Parasitology 161 (2009) 213–217
215
however, the group mean kept low, with a maximum of 0.7% at 10 days after treatment. As regards the results of the controlled efficacy test, moxidectin presented 100% efficacy against the genera Haemonchus and Trichostrongylus; 65.2% efficacy against Cooperia spp.; 44.8% against Oesophagostomum radiatum; and 81.4% against Trichuris spp. (Table 2). Considering all control animals, 150 Haemonchus adult males could be recovered and their spicules measured. From these, 107 (71.33%) were identified as Haemonchus placei, five (3.33%) as Haemonchus similis, and 33 (22%) as Haemonchus contortus. The mean length (standard deviation) of spicules, right hooks and left hooks was, respectively: 460.8 25.9, 53.4 3.7 and 25.9 4.9 mm in H. placei; 449.3 14.2, 47.7 4.3 and 21.7 1.7 mm in H. contortus; and 345.8 11.6, 70.3 2.7 and 54.8 2.3 mm in H. similis. Other five specimens (3.33%) were not identified to species level as they presented intermediate measures between H. placei and H. contortus. Nineteen adult male specimens of the genus Trichostrongylus were recovered, 18 Trichostrongylus axei and one Trichostrongylus colubriformis, all from the control group. Regarding Cooperia genus, 3927 male specimens were identified in the control group. Of these, 3242 were Cooperia punctata and 685 Cooperia pectinata. In the treated group, 1074 C. punctata and 124 C. pectinata were identified. Thus, anthelmintic resistance was confirmed involving these two Cooperia species. The identified O. radiatum accounted for 69 male specimens, 40 in the control and 29 in the treated group. Therefore, this parasite also presented resistance to moxidectin. Fecal examination did not indicate the presence of Trichuris spp. eggs; however, after necropsy, nematodes of this genus were found in the animals. In the treated group, no male adult specimen was recovered. In the control group, however, 10 male adult specimens were found:
fication and identification of nematode species. Speciation of Cooperia spp., Trichostrongylus spp. and Oesophagostomum spp. adult males was according to descriptions by Ueno and Gonc¸alves (1998). Trichuris spp. specimens were identified based on Vicente et al. (1997) and Haemonchus spp., on Achi et al. (2003). Adult females of Cooperia genus were evaluated for the presence of eggs inside their uterus. The statistical analysis was carried out using the RESO software as recommended by Coles et al. (1992). Fecal egg count reduction (FECR) was calculated through the following formula: FECR (%) = 100 (1 arithmetic mean of the treated group FEC/arithmetic mean of the control group FEC). Presence of anthelmintic resistance was confirmed by a FECR lower than 95% and an inferior limit of the confidence interval lower than 90%. Spearman’s correlation coefficient between worm burden and FEC was assessed by using the statistical software Minitab (version 11.21). The same software was employed in the Chi-square analysis related to the presence of eggs in the uterus of female Cooperia spp. 3. Results The FECRT results indicated the presence of moxidectin resistance after treatment, since FECR was lower than 95% on the evaluated days, with inferior limit of the confidence interval lower than 90% (Table 1). From the seventh to the 14th day after treatment, the genera Cooperia and Oesophagostomum showed resistance to moxidectin, based on the larval identification (Table 1). The nematode genera found in fecal cultures of the treated group were: Cooperia, ranging from 34% (10 days after treatment) to 80.7% (3 days after treatment), and Oesophagostomum, from 18.7% (3 days after treatment) to 65.3% (10 days after treatment). In some animals of the treated group, Haemonchus spp. larvae were detected;
Table 1 Fecal egg count reduction (FECR) after treatment with moxidectin and mean percentage of third stage larvae (L3) in fecal cultures of the treated and control groups of cattle (10 animals per group). Days after treatment 0
FEC
Mean FEC SEM FECR (%) L3 Cooperia spp. Haemonchus spp. Oesophagostomum spp.
3
7
10
14
Control
Treated
Control
Treated
Control
Treated
Control
Treated
Control
Treated
1150 800 650 500 450 450 350 350 250 250 520 89
1200 800 750 500 450 400 350 300 250 250 525 96
400 1600 1100 600 1000 250 200 50 350 100 565 161
250 0 50 0 150 100 0 50 50 0 65 26 88 (96; 68)
950 750 3250 3100 1500 450 600 100 250 350 1130 363
700 50 100 300 50 250 0 0 200 50 170 68 85 (95; 56)
1800 350 3500 2750 1750 950 1050 50 100 1450 1375 357
1250 350 50 0 0 0 0 0 0 0 165 125 88 (98; 35)
3450 2600 1950 2100 2050 900 1200 600 700 4300 1985 384
700 0 0 100 200 50 0 0 100 350 150 71 92 (97; 78)
55.1% 32.8% 12.1%
35.5% 57.1% 7.4%
32.2% 47.0% 20.5%
80.7% 0.6% 18.7%
37.3% 19.7% 43.0%
56.0% 0.2% 43.8%
46.8% 21.6% 31.6%
34.0% 0.7% 65.3%
67.8% 22.7% 9.5%
50.0% 0.0% 50.0%
FECR (%) = 100 (1–treated group mean/control group mean). Upper confidence interval limit (95%) and lower confidence interval limit (95%) are in parenthesis. SEM = Standard error of the mean.
G.K. Condi et al. / Veterinary Parasitology 161 (2009) 213–217
216
Table 2 Worm burden 14 days after treatment of cattle with moxidectin. Control and treated groups with 10 animals each. Group
Nematode genera Cooperia
Haemonchus
Trichostrongylus
Oesophagostomum
Trichuris
Control
27180 18740 30880 40620 18240 14060 11300 5120 700 10060 17690 3873 13060 2300 11120 2560 20120 680 860 640 4560 5660 6156 2072 65% (85; 19)
2240 1640 2040 1820 1180 380 1300 140 360 720 1182 238 0 0 0 0 0 0 0 0 0 0 0 100%
60 40 80 60 60 100 60 160 60 80 76 11 0 0 0 0 0 0 0 0 0 0 0 100%
800 360 360 120 120 240 180 140 20 200 254 69 240 140 40 140 380 200 80 20 140 20 140 36 45% (75;
20 100 60 20 160 120 120 40 0 220 86 22 20 0 0 0 0 20 0 0 80 40 16 8 81% (95; 37)
Mean SEM Treated
Mean SEM Efficacy
21)
Efficacy (%) = 100 (1–treated group mean/control group mean). Upper confidence interval limit (95%) and lower confidence interval limit (95%) are in parenthesis. SEM = Standard error of the mean.
three were identified as Trichuris globulosa and seven as Trichuris discolor. As regards the presence of eggs inside Cooperia spp. uterus, 4305 (98.5%) specimens had eggs and 67 (1.7%) did not, concerning the control group. In the treated group, eggs were found in the uterus of 800 (48.2%) female adult specimens but not in that of 859 (51.8%) adult females. The difference between groups in relation to female fecundity was significant (x2 = 2336; P < 0.0001). The correlation coefficient between worm burden and FEC was 0.652 (P < 0.05) in the control group and 0.503 (P > 0.05) in the treated group. 4. Discussion This trial confirmed the suspicion reported by Soutello et al. (2007) that the studied herd was bearing moxidectinresistant Oesophagostomum spp. Trichuris spp. and two resistant Cooperia species were also detected (C. punctata and C. pectinata). Cooperia species are involved in most of the reports about anthelmintic resistance in cattle. Reduced moxidectin efficacy against larval stages (L4) of C. punctata (Williams and DeRosa, 2003) and against adult stages of C. punctata (88.4%), Cooperia spatulata (93.2%), Cooperia oncophora (88.7%), Cooperia surnabada (89.1%) and Strongyloides papillosus (79%) (Ranjan and DeLay, 2004) was reported in cattle in the United States. Conversely, moxidectin showed high efficacy (99.2%) against adult stage of C. pectinata (Ranjan and DeLay, 2004). Reduced efficacy against C. oncophora infections was also observed in Belgium (Geurden et al., 2004). The presence of moxidectin-, ivermectin- and doramectin-resistant
Cooperia spp. in cattle was also reported in New Zealand (Vermunt et al., 1996). These studies indicate that Cooperia capacity of surviving or developing resistance to the macrocyclic lactone anthelmintics is apparently higher than that of the remaining nematode species. However, besides Cooperia spp., moxidectin-resistant O. radiatum and Trichuris spp. were found in cattle of the present study, which is the first report involving controlled efficacy test. Moxidectin injectable, at 0.2 and 0.3 mg/kg, showed efficacy, respectively, of 99.4 and 97.8% against Trichuris spp. (adult parasites) in cattle in USA (Ranjan and DeLay, 2004). It is possible that nematodes of this genus maturing over the 14 day interval between treatment and necropsy could survive and contribute to reduced efficacy in the present trial. This would also be consistent with the lack of evidence of resistance in Trichuris based on FECR and only on worm counts at necropsy. Moxidectin showed to be effective against Haemonchus spp. Conversely, in sheep raised in Brazil, Haemonchus contortus is the main parasite involved in cases of moxidectin resistance (Thomaz-Soccol et al., 2004; Silva et al., 2008). Although several different tests have been used for the detection of anthelmintic resistance in nematodes of veterinary importance, the FECRT is considered the major method for the detection of resistance. However, a controlled efficacy test is the most reliable method of confirming anthelmintic resistance but expenses usually excludes its use. Nevertheless, it is the gold standard for detecting anthelmintic resistance (Coles et al., 2006). In the present study, the FECRT allowed the resistance detection regarding Cooperia and Oesophagostumum genera, but not Trichuris. In addition, a problem concerning FECRT was the
G.K. Condi et al. / Veterinary Parasitology 161 (2009) 213–217
impossibility of evaluating egg production suppression after treatment, which was observed in Cooperia spp. females of the moxidectin-treated group. This phenomenon was also reported in goats infected with Ostertagia circumcincta, in which egg output was inhibited for 10–14 days after ivermectin treatment (Jackson, 1993). When egg elimination is inhibited, the anthelmintic efficacy can be overestimated; for this reason, samples are recommended to be collected at 14–17 days after moxidectin treatment (Coles et al., 2006). However, the present results indicate that when FECRT is employed, a period even longer than 17 days may probably be necessary to accurately determine moxidectin resistance. It is also necessary the development of more sensitive diagnosis methods, capable of detecting resistance when the frequency of genes that determine such characteristic is low in the parasite population. The present study confirms that anthelmintic resistance is not anymore a problem restricted to small ruminants but a concern also in cattle industry in Brazil. It must also be emphasized the need of alternative strategies for the prophylaxis of parasitic gastroenteritis less dependent on anthelmintic utilization, such as selection of resistant cattle (Bricarello et al., 2007). Acknowledgement We thank Sa˜o Paulo State Research Foundation (FAPESP) for providing scholarship to the first author. References Achi, Y.L., Zinsstag, J., Yao, K., Yeo, N., Dorchies, P., Jacquiet, P., 2003. Host specificity of Haemonchus spp. for domestic ruminants in the savanna in northern Ivory Coast. Vet. Parasitol. 116, 151–158. Anziani, O.S., Suarez, V., Guglielmone, A.A., Warnke, O., Grande, H., Coles, G.C., 2004. Resistance to benzimidazole and macrocyclic lactone anthelmintics in cattle nematodes in Argentina. Vet. Parasitol. 122, 303–306. Bricarello, P.A., Zaros, L.G., Coutinho, L.L., Rocha, R.A., Kooyman, F.N.J., De Vries, E., Gonc¸alves, J.R.S., Lima, L.G., Pires, A.V., Amarante, A.F.T., 2007. Field study on nematode resistance in Nelore-breed cattle. Vet. Parasitol. 148, 272–278. Coles, G.C., Bauer, C., Borgsteede, F.H.M., Geerts, S., Klei, T.R., Taylor, M.A., Waller, P.J., 1992. World Association for the Advancement of
217
Veterinary Parasitology (W.A.A.V.P.) methods for the detection of anthelmintic resistence in nematodes of veterinary importance. Vet. Parasitol. 44, 35–44. Coles, G.C., Jackson, F., Pomroy, W.E., Prichard, R.K., Von Samson-Himmelstjerna, G., Silvestre, A., Taylor, M.A., Vercruysse, J., 2006. The detection of anthelmintic resistance in nematodes of veterinary importance. Vet. Parasitol. 136, 167–185. Geurden, T., Claerebout, E., Deroover, E., Vercruysse, J., 2004. Evaluation of the chemoprophylactic efficacy of 10% long action injectable moxidectin against gastrointestinal nematode infections in calves in Belgium. Vet. Parasitol. 120, 331–338. Jackson, F., 1993. Anthelmintic resistance—the state of play. Br. Vet. J. 149, 123–138. Mason, P.C., McKay, C.H., 2006. Field studies investigating anthelmintic resistance in young cattle on five farms in New Zealand. N.Z. Vet. J. 54, 318–322. Ranjan, S., DeLay, R., 2004. Therapeutic and persistent efficacy of moxidectin 1% nonaqueous injetable formulation against natural and experimentally induced lung and gastrointestinal nematodes in cattle. Vet. Parasitol. 120, 305–317. Silva, B.F., Amarante, M.R.V., Kadri, S.M., Carrijo-Mauad, J.R., Amarante, A.F.T., 2008. Vertical migration of Haemonchus contortus third stage larvae on Brachiaria decumbens grass. Vet. Parasitol. 158, 85–92. Soutello, R.G.V., Seno, M.C.Z., Amarante, A.F.T., 2007. Anthelmintic resistance in cattle nematodes in northwestern Sa˜o Paulo State, Brazil. Vet. Parasitol. 148, 360–364. Souza, A.P., Ramos, C.I., Bellato, V., Sartor, A.A., Schelbauer, C.A., 2008. Resisteˆncia de helmintos gastrintestinais de bovinos a anti-helmı´nticos no Planalto Catarinense. Cienc. Rural 38, 1363–1367. Suarez, V.H., Cristel, S.L., 2007. Anthelmintic resistance in cattle nematode in the western Pampeana Region of Argentina. Vet. Parasitol. 144, 111–117. Thomaz-Soccol, V., Souza, F.P., Sotomaior, C., Castro, E.A., Milczewski, V., Mocelin, G., Silva, M.C.P., 2004. Resistance of gastrointestinal nematodes to anthelmintics in sheep (Ovis aries). Braz. Arch. Biol. Technol. 47, 41–47. Ueno, H., Gonc¸alves, P.C., 1998. Manual para diagno´stico das helmintoses de ruminantes, 4th ed. Japan International Cooperation Agency, Tokyo, pp. 143. Vermunt, J.J., West, D.M., Pomroy, W.E., 1996. Inefficacy of moxidectin and doramectin against ivermectin-resistant Cooperia spp. of cattle in New Zeland. N.Z. Vet. J. 44, 188–193. Vicente, J.J., Rodrigues, H.O., Gomes, D.C., Pinto, R.M., 1997. Nemato´ides do Brasil. Parte V: Nemato´ides de mamı´feros. Rev. Bras. Zool. 14, 1– 452. Waghorn, T.S., Leathwick, D.M., Rhodes, A.P., Jackson, R., Pomroy, W.E., West, D.M., Moffat, J.R., 2006. Prevalence of anthelmintic resistance on 62 beef cattle farms in the North Island of New Zealand. N.Z. Vet. J. 54, 278–282. Williams, J.C., DeRosa, A., 2003. Dose confirmation of moxidectin 0.5% pour-on against adults and fourth-stage larvae of various Cooperia spp. and Trichostrongylus colubriformis in Louisiana. Vet. Parasitol. 114, 295–303.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Moxidectin-resistant nematodes in cattle in Brazil G.K. Condi a, R.G.V. Soutello b, A.F.T. Amarante a,* a b
UNESP - Universidade Estadual Paulista, Departamento de Parasitologia, Instituto de Biocieˆncias, Caixa Postal 510, Botucatu, SP, CEP 18618-000, Brazil Faculdade de Cieˆncias Agra´rias de Andradina, SP, CEP 16901-160, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 November 2008 Received in revised form 22 January 2009 Accepted 26 January 2009
The frequent use of anthelmintics in cattle has contributed to the emergence of gastrointestinal nematode populations resistant to the drugs available in the market. This study aimed to evaluate moxidectin efficacy in a property with suspected presence of Oesophagostomum spp. resistant to such anthelmintic. The accuracy of fecal egg count reduction test (FECRT) was also assessed through comparison with the data obtained in the controlled efficacy test (worm burden determination after necropsy). Twenty worm free steers were placed together with cattle of the farm with suspected moxidectin resistance. They became naturally infected with the parasite species present in the farm, and when fecal egg counts (FEC) were higher than 200 eggs/g, the animals were randomly distributed into two groups. One group (n = 10) was treated with moxidectin (0.2 mg/kg; Cydectin1, Fort Dodge) and the other was the control group (n = 10). Fecal samples from each steer were collected on the treatment day and three, seven, 10 and 14 days later for FEC and fecal cultures. At 14 days after treatment, all animals were sacrificed and the gastrointestinal nematodes were recovered, identified and counted. Mean FEC reduction in the treated group (compared with control group) was of 88, 85, 88 and 92% at days three, seven, 10 and 14 after treatment, respectively. In fecal cultures, third stage larvae of Cooperia spp. and Oesophagostomum spp. were predominant. As regards the controlled efficacy test, moxidectin had 100% efficacy against the genera Haemonchus spp. and Trichostrongylus spp., and efficacy of 65.2% for Cooperia spp. (Cooperia punctata and Cooperia pectinata), 44.8% for Oesophagostomum radiatum, and 81.4% for Trichuris spp. Most of the Cooperia spp. females obtained from the control animals showed eggs inside the uterus (98.5%); conversely, only 48.2% of the females from the treated group had eggs (P < 0.001). Considering the suppression in egg output following moxidectin treatment, FECRT has to be employed with caution to avoid under estimation of the anthelmintic resistance. From necropsy results, it can be concluded that C. punctata, C. pectinata, O. radiatum and Trichuris spp. showed resistance to moxidectin. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Anthelmintic resistance Macrocyclic lactone Oesophagostomum Trichuris Cooperia
1. Introduction The anthelmintic resistance has become a threat to the prophylaxis of parasitic gastroenteritis in cattle worldwide. In New Zealand, there are several reports on bovine
* Corresponding author. Tel.: +55 14 3811 6239; fax: +55 14 3815 3744. E-mail address: [email protected] (A.F.T. Amarante). 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.01.031
anthelmintic resistance (Mason & McKay, 2006; Waghorn et al., 2006). In North Island, for example, 92% of the evaluated properties showed resistance to ivermectin; 76% to albendazole; 74% to the association ivermectin and albendazole; and 6% to levamisole. Cooperia spp. was the most prevalent parasite in resistant populations, followed by Ostertagia spp. (Waghorn et al., 2006). In Argentina, Cooperia spp. has also been the main genus involved in cases of resistance to ivermectin in cattle
214
G.K. Condi et al. / Veterinary Parasitology 161 (2009) 213–217
(Suarez and Cristel, 2007). In addition, there are also reports of benzimidazole-resistant Ostertagia spp. (Suarez and Cristel, 2007) and Haemonchus spp. with simultaneous resistance to ivermectin and benzimidazoles in bovine herds (Anziani et al., 2004). In Brazil, a high frequency of ivermectin-resistant Haemonchus spp. and Cooperia spp. has been detected in farms of Sa˜o Paulo and Santa Catarina States, and in some farms, albendazole- and levamisole-resistant nematodes were also found (Soutello et al., 2007; Souza et al., 2008). Recently, Soutello et al. (2007) have reported the suspected presence of moxidectin-resistant Oesophagostomum spp. in a cattle farm located in the northwest region of Sa˜o Paulo State, Brazil. This fact stimulated the development of the present study, which aimed to further investigate the presence of moxidectin-resistant gastrointestinal nematodes in the cattle of such farm. The accuracy of fecal egg count reduction test (FECRT) for detection of anthelmintic resistances was also evaluated in comparison with the results of the controlled efficacy test. 2. Material and methods 2.1. Property description and history of anthelmintic utilization This study was carried out from April to June 2007 in a farm located in the municipality of Castilho, northwest region of Sa˜o Paulo State, Brazil. The property has 1210 hectares used for Nelore beef cattle production in pastures of Brachiaria decumbens, Brachiaria humidicula and Panicum maximum. During the experiment, approximately 100 sheep and 80 horses were also being kept in the farm. Macrocyclic lactone anthelmintics have been used in this farm since the beginning of the 1980s. At first, ivermectin was employed, followed by abamectin, mainly at castration and in animals acquired from other properties on their arrival at the farm. However, at that time, levamisole was the most frequently used anthelmintic. In 1992, the weight gain of animals from two properties in that region was comparatively evaluated after moxidectin (Cydectin1, Fort Dodge), ivermectin or levamisole treatment. As the former anthelmintic led to higher weight gain (data not published), moxidectin started to be used in all animals of the farm for approximately five years. Then, to reduce costs, levamisole was chosen to be administered to cows, whereas the remaining bovine categories continued receiving moxidectin treatment. Briefly, anthelmintic application has been carried out as follows: - Adult cows: levamisole once every year when gestation is diagnosed (May to July); - Calves before weaning: moxidectin at three or four months of age, in February; - Steers and heifers: moxidectin twice every year (May and September); - -Adult bulls: moxidectin before and after the breeding season (April and September); - Male calves at castration: abamectin (generally in March);
- Animals purchased from other farms were treated with moxidectin on their arrival at the property. Sheep did not have access to most of the pastures grazed by cattle and moxidectin has also been used in these animals for more than 10 years, twice every year. 2.2. Experiment description Initially, 30 crossbred steers (Bos taurus) of European breeds, aged from eight to 12 months, were obtained from other farms. Before entering the farm, they were identified with numbered earrings, weighed and treated with levamisole phosphate (4.7 mg/kg; Ripercol1 150F, Fort Dodge) in order to be introduced free of nematode infections. Fecal samples from the animals were examined to confirm the efficacy of such treatment. Thus, the animals were naturally reinfected only with the nematode species present in the farm. The steers were placed together with other 200 animals of the farm, which aged between eight and 15 months, in two paddocks of approximately 48.4 hectares each. The animals had free access to troughs with mineral salt and natural watering. Before the beginning of the experiment, they were vaccinated against foot-and-mouth disease (Aftobov1, Intervet) and clostridioses (Polisinto-Vac1, Fort Dodge). After the animals had been kept for 40 days in the contaminated paddocks of the farm, fecal samples were weekly collected directly from their rectum to determine fecal egg counts (FEC) by a modified McMaster technique, where each egg counted represented 50 eggs/g. Fecal cultures were prepared for obtaining and identifying third stage larvae (Ueno and Gonc¸alves, 1998). Of the 30 animals, those 20 with the highest counts of eggs per gram of feces (EPG) (superior to 200 EPG) were selected. On the treatment day (day 0), the animals were classified in pairs and in increasing order of FEC. Then, one animal from each pair was randomly allocated to the treated group and the other to the control group. These groups had weight means of 120.1 kg 25.64 and 116.6 kg 21.34, respectively, on day 0. Animals from the treated group were subcutaneously injected with moxidectin 1%, according to the manufacturer’s instructions (0.2 mg/kg; Cydectin1, Fort Dodge). Control animals received a placebo (saline solution). One day before slaughtering, the animals were gathered in a corral, where they were kept for 24 h with access only to drinking water and no solid food. Twelve hours before slaughtering, water supply was also interrupted. Fecal samples from each animal were collected on the treatment day and again at 3, 7, 10 and 14 days later. Samples were individually processed for FEC and fecal culture preparation. At 14 days after treatment, all animals from both groups were sacrificed for removal of the gastrointestinal tract. Material collection for parasitological analyses was carried out according to the descriptions of Ueno and Gonc¸alves (1998). Samples with 5% of each organ content were preserved in 4% formaldehyde and stored in identified containers. Such material was later processed for quanti-
G.K. Condi et al. / Veterinary Parasitology 161 (2009) 213–217
215
however, the group mean kept low, with a maximum of 0.7% at 10 days after treatment. As regards the results of the controlled efficacy test, moxidectin presented 100% efficacy against the genera Haemonchus and Trichostrongylus; 65.2% efficacy against Cooperia spp.; 44.8% against Oesophagostomum radiatum; and 81.4% against Trichuris spp. (Table 2). Considering all control animals, 150 Haemonchus adult males could be recovered and their spicules measured. From these, 107 (71.33%) were identified as Haemonchus placei, five (3.33%) as Haemonchus similis, and 33 (22%) as Haemonchus contortus. The mean length (standard deviation) of spicules, right hooks and left hooks was, respectively: 460.8 25.9, 53.4 3.7 and 25.9 4.9 mm in H. placei; 449.3 14.2, 47.7 4.3 and 21.7 1.7 mm in H. contortus; and 345.8 11.6, 70.3 2.7 and 54.8 2.3 mm in H. similis. Other five specimens (3.33%) were not identified to species level as they presented intermediate measures between H. placei and H. contortus. Nineteen adult male specimens of the genus Trichostrongylus were recovered, 18 Trichostrongylus axei and one Trichostrongylus colubriformis, all from the control group. Regarding Cooperia genus, 3927 male specimens were identified in the control group. Of these, 3242 were Cooperia punctata and 685 Cooperia pectinata. In the treated group, 1074 C. punctata and 124 C. pectinata were identified. Thus, anthelmintic resistance was confirmed involving these two Cooperia species. The identified O. radiatum accounted for 69 male specimens, 40 in the control and 29 in the treated group. Therefore, this parasite also presented resistance to moxidectin. Fecal examination did not indicate the presence of Trichuris spp. eggs; however, after necropsy, nematodes of this genus were found in the animals. In the treated group, no male adult specimen was recovered. In the control group, however, 10 male adult specimens were found:
fication and identification of nematode species. Speciation of Cooperia spp., Trichostrongylus spp. and Oesophagostomum spp. adult males was according to descriptions by Ueno and Gonc¸alves (1998). Trichuris spp. specimens were identified based on Vicente et al. (1997) and Haemonchus spp., on Achi et al. (2003). Adult females of Cooperia genus were evaluated for the presence of eggs inside their uterus. The statistical analysis was carried out using the RESO software as recommended by Coles et al. (1992). Fecal egg count reduction (FECR) was calculated through the following formula: FECR (%) = 100 (1 arithmetic mean of the treated group FEC/arithmetic mean of the control group FEC). Presence of anthelmintic resistance was confirmed by a FECR lower than 95% and an inferior limit of the confidence interval lower than 90%. Spearman’s correlation coefficient between worm burden and FEC was assessed by using the statistical software Minitab (version 11.21). The same software was employed in the Chi-square analysis related to the presence of eggs in the uterus of female Cooperia spp. 3. Results The FECRT results indicated the presence of moxidectin resistance after treatment, since FECR was lower than 95% on the evaluated days, with inferior limit of the confidence interval lower than 90% (Table 1). From the seventh to the 14th day after treatment, the genera Cooperia and Oesophagostomum showed resistance to moxidectin, based on the larval identification (Table 1). The nematode genera found in fecal cultures of the treated group were: Cooperia, ranging from 34% (10 days after treatment) to 80.7% (3 days after treatment), and Oesophagostomum, from 18.7% (3 days after treatment) to 65.3% (10 days after treatment). In some animals of the treated group, Haemonchus spp. larvae were detected;
Table 1 Fecal egg count reduction (FECR) after treatment with moxidectin and mean percentage of third stage larvae (L3) in fecal cultures of the treated and control groups of cattle (10 animals per group). Days after treatment 0
FEC
Mean FEC SEM FECR (%) L3 Cooperia spp. Haemonchus spp. Oesophagostomum spp.
3
7
10
14
Control
Treated
Control
Treated
Control
Treated
Control
Treated
Control
Treated
1150 800 650 500 450 450 350 350 250 250 520 89
1200 800 750 500 450 400 350 300 250 250 525 96
400 1600 1100 600 1000 250 200 50 350 100 565 161
250 0 50 0 150 100 0 50 50 0 65 26 88 (96; 68)
950 750 3250 3100 1500 450 600 100 250 350 1130 363
700 50 100 300 50 250 0 0 200 50 170 68 85 (95; 56)
1800 350 3500 2750 1750 950 1050 50 100 1450 1375 357
1250 350 50 0 0 0 0 0 0 0 165 125 88 (98; 35)
3450 2600 1950 2100 2050 900 1200 600 700 4300 1985 384
700 0 0 100 200 50 0 0 100 350 150 71 92 (97; 78)
55.1% 32.8% 12.1%
35.5% 57.1% 7.4%
32.2% 47.0% 20.5%
80.7% 0.6% 18.7%
37.3% 19.7% 43.0%
56.0% 0.2% 43.8%
46.8% 21.6% 31.6%
34.0% 0.7% 65.3%
67.8% 22.7% 9.5%
50.0% 0.0% 50.0%
FECR (%) = 100 (1–treated group mean/control group mean). Upper confidence interval limit (95%) and lower confidence interval limit (95%) are in parenthesis. SEM = Standard error of the mean.
G.K. Condi et al. / Veterinary Parasitology 161 (2009) 213–217
216
Table 2 Worm burden 14 days after treatment of cattle with moxidectin. Control and treated groups with 10 animals each. Group
Nematode genera Cooperia
Haemonchus
Trichostrongylus
Oesophagostomum
Trichuris
Control
27180 18740 30880 40620 18240 14060 11300 5120 700 10060 17690 3873 13060 2300 11120 2560 20120 680 860 640 4560 5660 6156 2072 65% (85; 19)
2240 1640 2040 1820 1180 380 1300 140 360 720 1182 238 0 0 0 0 0 0 0 0 0 0 0 100%
60 40 80 60 60 100 60 160 60 80 76 11 0 0 0 0 0 0 0 0 0 0 0 100%
800 360 360 120 120 240 180 140 20 200 254 69 240 140 40 140 380 200 80 20 140 20 140 36 45% (75;
20 100 60 20 160 120 120 40 0 220 86 22 20 0 0 0 0 20 0 0 80 40 16 8 81% (95; 37)
Mean SEM Treated
Mean SEM Efficacy
21)
Efficacy (%) = 100 (1–treated group mean/control group mean). Upper confidence interval limit (95%) and lower confidence interval limit (95%) are in parenthesis. SEM = Standard error of the mean.
three were identified as Trichuris globulosa and seven as Trichuris discolor. As regards the presence of eggs inside Cooperia spp. uterus, 4305 (98.5%) specimens had eggs and 67 (1.7%) did not, concerning the control group. In the treated group, eggs were found in the uterus of 800 (48.2%) female adult specimens but not in that of 859 (51.8%) adult females. The difference between groups in relation to female fecundity was significant (x2 = 2336; P < 0.0001). The correlation coefficient between worm burden and FEC was 0.652 (P < 0.05) in the control group and 0.503 (P > 0.05) in the treated group. 4. Discussion This trial confirmed the suspicion reported by Soutello et al. (2007) that the studied herd was bearing moxidectinresistant Oesophagostomum spp. Trichuris spp. and two resistant Cooperia species were also detected (C. punctata and C. pectinata). Cooperia species are involved in most of the reports about anthelmintic resistance in cattle. Reduced moxidectin efficacy against larval stages (L4) of C. punctata (Williams and DeRosa, 2003) and against adult stages of C. punctata (88.4%), Cooperia spatulata (93.2%), Cooperia oncophora (88.7%), Cooperia surnabada (89.1%) and Strongyloides papillosus (79%) (Ranjan and DeLay, 2004) was reported in cattle in the United States. Conversely, moxidectin showed high efficacy (99.2%) against adult stage of C. pectinata (Ranjan and DeLay, 2004). Reduced efficacy against C. oncophora infections was also observed in Belgium (Geurden et al., 2004). The presence of moxidectin-, ivermectin- and doramectin-resistant
Cooperia spp. in cattle was also reported in New Zealand (Vermunt et al., 1996). These studies indicate that Cooperia capacity of surviving or developing resistance to the macrocyclic lactone anthelmintics is apparently higher than that of the remaining nematode species. However, besides Cooperia spp., moxidectin-resistant O. radiatum and Trichuris spp. were found in cattle of the present study, which is the first report involving controlled efficacy test. Moxidectin injectable, at 0.2 and 0.3 mg/kg, showed efficacy, respectively, of 99.4 and 97.8% against Trichuris spp. (adult parasites) in cattle in USA (Ranjan and DeLay, 2004). It is possible that nematodes of this genus maturing over the 14 day interval between treatment and necropsy could survive and contribute to reduced efficacy in the present trial. This would also be consistent with the lack of evidence of resistance in Trichuris based on FECR and only on worm counts at necropsy. Moxidectin showed to be effective against Haemonchus spp. Conversely, in sheep raised in Brazil, Haemonchus contortus is the main parasite involved in cases of moxidectin resistance (Thomaz-Soccol et al., 2004; Silva et al., 2008). Although several different tests have been used for the detection of anthelmintic resistance in nematodes of veterinary importance, the FECRT is considered the major method for the detection of resistance. However, a controlled efficacy test is the most reliable method of confirming anthelmintic resistance but expenses usually excludes its use. Nevertheless, it is the gold standard for detecting anthelmintic resistance (Coles et al., 2006). In the present study, the FECRT allowed the resistance detection regarding Cooperia and Oesophagostumum genera, but not Trichuris. In addition, a problem concerning FECRT was the
G.K. Condi et al. / Veterinary Parasitology 161 (2009) 213–217
impossibility of evaluating egg production suppression after treatment, which was observed in Cooperia spp. females of the moxidectin-treated group. This phenomenon was also reported in goats infected with Ostertagia circumcincta, in which egg output was inhibited for 10–14 days after ivermectin treatment (Jackson, 1993). When egg elimination is inhibited, the anthelmintic efficacy can be overestimated; for this reason, samples are recommended to be collected at 14–17 days after moxidectin treatment (Coles et al., 2006). However, the present results indicate that when FECRT is employed, a period even longer than 17 days may probably be necessary to accurately determine moxidectin resistance. It is also necessary the development of more sensitive diagnosis methods, capable of detecting resistance when the frequency of genes that determine such characteristic is low in the parasite population. The present study confirms that anthelmintic resistance is not anymore a problem restricted to small ruminants but a concern also in cattle industry in Brazil. It must also be emphasized the need of alternative strategies for the prophylaxis of parasitic gastroenteritis less dependent on anthelmintic utilization, such as selection of resistant cattle (Bricarello et al., 2007). Acknowledgement We thank Sa˜o Paulo State Research Foundation (FAPESP) for providing scholarship to the first author. References Achi, Y.L., Zinsstag, J., Yao, K., Yeo, N., Dorchies, P., Jacquiet, P., 2003. Host specificity of Haemonchus spp. for domestic ruminants in the savanna in northern Ivory Coast. Vet. Parasitol. 116, 151–158. Anziani, O.S., Suarez, V., Guglielmone, A.A., Warnke, O., Grande, H., Coles, G.C., 2004. Resistance to benzimidazole and macrocyclic lactone anthelmintics in cattle nematodes in Argentina. Vet. Parasitol. 122, 303–306. Bricarello, P.A., Zaros, L.G., Coutinho, L.L., Rocha, R.A., Kooyman, F.N.J., De Vries, E., Gonc¸alves, J.R.S., Lima, L.G., Pires, A.V., Amarante, A.F.T., 2007. Field study on nematode resistance in Nelore-breed cattle. Vet. Parasitol. 148, 272–278. Coles, G.C., Bauer, C., Borgsteede, F.H.M., Geerts, S., Klei, T.R., Taylor, M.A., Waller, P.J., 1992. World Association for the Advancement of
217
Veterinary Parasitology (W.A.A.V.P.) methods for the detection of anthelmintic resistence in nematodes of veterinary importance. Vet. Parasitol. 44, 35–44. Coles, G.C., Jackson, F., Pomroy, W.E., Prichard, R.K., Von Samson-Himmelstjerna, G., Silvestre, A., Taylor, M.A., Vercruysse, J., 2006. The detection of anthelmintic resistance in nematodes of veterinary importance. Vet. Parasitol. 136, 167–185. Geurden, T., Claerebout, E., Deroover, E., Vercruysse, J., 2004. Evaluation of the chemoprophylactic efficacy of 10% long action injectable moxidectin against gastrointestinal nematode infections in calves in Belgium. Vet. Parasitol. 120, 331–338. Jackson, F., 1993. Anthelmintic resistance—the state of play. Br. Vet. J. 149, 123–138. Mason, P.C., McKay, C.H., 2006. Field studies investigating anthelmintic resistance in young cattle on five farms in New Zealand. N.Z. Vet. J. 54, 318–322. Ranjan, S., DeLay, R., 2004. Therapeutic and persistent efficacy of moxidectin 1% nonaqueous injetable formulation against natural and experimentally induced lung and gastrointestinal nematodes in cattle. Vet. Parasitol. 120, 305–317. Silva, B.F., Amarante, M.R.V., Kadri, S.M., Carrijo-Mauad, J.R., Amarante, A.F.T., 2008. Vertical migration of Haemonchus contortus third stage larvae on Brachiaria decumbens grass. Vet. Parasitol. 158, 85–92. Soutello, R.G.V., Seno, M.C.Z., Amarante, A.F.T., 2007. Anthelmintic resistance in cattle nematodes in northwestern Sa˜o Paulo State, Brazil. Vet. Parasitol. 148, 360–364. Souza, A.P., Ramos, C.I., Bellato, V., Sartor, A.A., Schelbauer, C.A., 2008. Resisteˆncia de helmintos gastrintestinais de bovinos a anti-helmı´nticos no Planalto Catarinense. Cienc. Rural 38, 1363–1367. Suarez, V.H., Cristel, S.L., 2007. Anthelmintic resistance in cattle nematode in the western Pampeana Region of Argentina. Vet. Parasitol. 144, 111–117. Thomaz-Soccol, V., Souza, F.P., Sotomaior, C., Castro, E.A., Milczewski, V., Mocelin, G., Silva, M.C.P., 2004. Resistance of gastrointestinal nematodes to anthelmintics in sheep (Ovis aries). Braz. Arch. Biol. Technol. 47, 41–47. Ueno, H., Gonc¸alves, P.C., 1998. Manual para diagno´stico das helmintoses de ruminantes, 4th ed. Japan International Cooperation Agency, Tokyo, pp. 143. Vermunt, J.J., West, D.M., Pomroy, W.E., 1996. Inefficacy of moxidectin and doramectin against ivermectin-resistant Cooperia spp. of cattle in New Zeland. N.Z. Vet. J. 44, 188–193. Vicente, J.J., Rodrigues, H.O., Gomes, D.C., Pinto, R.M., 1997. Nemato´ides do Brasil. Parte V: Nemato´ides de mamı´feros. Rev. Bras. Zool. 14, 1– 452. Waghorn, T.S., Leathwick, D.M., Rhodes, A.P., Jackson, R., Pomroy, W.E., West, D.M., Moffat, J.R., 2006. Prevalence of anthelmintic resistance on 62 beef cattle farms in the North Island of New Zealand. N.Z. Vet. J. 54, 278–282. Williams, J.C., DeRosa, A., 2003. Dose confirmation of moxidectin 0.5% pour-on against adults and fourth-stage larvae of various Cooperia spp. and Trichostrongylus colubriformis in Louisiana. Vet. Parasitol. 114, 295–303.