First report of multiple anthelmintic resistance in nematodes of sheep in Colombia

This study aimed to report the presence of parasites resistant to the most used anthelmintic drugs in sheep in Colombia. Four farms (denominated farm 1, 2, 3 and 4) were selected where the animals were not treated with anthelmintics for two months before the trial. Animals with faecal egg count (FEC) above 150 and of different ages were allocated into six groups, each consisting of at least 5 animals. The drugs and dosages used were: ivermectin 1% (0.2 mg/kg), albendazole 25% (5 mg/kg), fenbendazole 10% (5 mg/kg), levamisole 10% (5 mg/kg), and moxidectin 1% (0.2 mg/kg). Anthelmintic efficacy was determined by the FEC reduction test (FECRT) with a second sampling 14 days post-treatment. The efficacy of albendazole and fenbendazole at farm 1 was above 95%, which was different from the others farms. The FECRT indicated the presence of multidrug resistance in the other farms where no tested drugs showed activity higher than 79% (albendazole: 0 to 55%, fenbendazole: 51.4 to 76.6%, ivermectin: 67.3 to 93.1%, levamisole: 0 to 78.1%, and moxidectin: 49.2 to 64.1%). Haemonchus contortus was the predominant (96%) species, followed by a small presence of Trichostrongylus sp. (3%) and Cooperia sp. (1%). Therefore, we report for the first time the existence of multiple anthelmintic resistance in gastrointestinal nematodes of sheep in Colombia.


INTRODUCTION
Colombia is considered a livestock country and currently a great interest in sheep farming is occurring (Ospina et al. 2011).The production is mainly focused on meat with a marginal milk industry (González et al. 2011).The population of sheep was estimated at 3.4 million, located mainly in the states of Boyacá, Magdalena, Córdoba, Cundinamarca and Cesar (FAOSTAT 2008).There are many factors favorable to this activity, including the great adaptability of the animals to different climates and environments, and the ease of management (Benavides-Ortiz et al. 2010).
However, there are also many barriers for sheep production.Gastrointestinal parasitism is one of the most important negative economic factors of the small ruminant industry of Colombia CARLOS M.B.GÁRCIA, LEW K. SPRENGER, EFRAÍN B. ORTIZ and MARCELO B. MOLENTO (Pulido-Medellín et al. 2014).In Colombia there are only a few studies relating small ruminants and parasite infections.Herrera et al. (2013) have reported that Haemonchus contortus is the most prevalent nematode, followed by Trichostrongylus spp., Cooperia spp., Strongyloides spp.and Oesophagostomun spp..These infections have been described as being responsible for decreasing weight gain, delaying puberty, causing anemia and even death (Ensuncho-Hoyos et al. 2014).
Parasite control is usually carried out with the use of anthelmintics, often indiscriminately and without any epidemiological knowledge (Lara 2013).The use of such suppressive regime added to inaccurate management strategies, contributes to the selection and establishment of parasites that are resistant to the anthelmintics (Almeida et al. 2010).As parasite resistance is an inherited trait, after every generation there may be an increase in individual parasites that would be able to survive drug treatment (Molento 2004).Parasite resistance is an alarming scenario in small ruminants worldwide, but there are no data on the magnitude of parasite resistance in sheep in Colombia.Therefore, the aim of this study was to report the lack of effi cacy of the most used anthelmintics against gastrointestinal nematodes of sheep in Colombia.

MATERIALS AND METHODS
The experiment was performed from January to July of 2011 on four selected sheep Farms, two located in the state of Cundinamarca and two in the state of Valle del Cauca.The drug test protocol was based on Coles et al. (1992).The animals used in this experiment were of mix ages, breeds and were not treated to a minimum of 60 days prior to the study.Initial faecal samples were taken from all animals, to perform parasite egg counts per gram (EPG) according to Gordon and Whitlock (1939).
Groups with a minimum of five animals were treated with Ivermectin (IVM) 1% 0.2 mg/ kg (Next Mk ® , Technochemistry, Cali, Colombia), Albendazole (ABZ) 25% 5 mg/kg (Insubenzol ® , Insuvin, Bogota, Colombia), Fenbendazole (FBZ) 10% 5 mg/kg (Panacur ® , MSD Salud Animal, Bogota, Colombia), Levamisole (LEV) 10% 5 mg/ kg (Levamisol ® , Vicar Pharmaceutical, Bogota, Colombia), Moxidectin (MOX) 1% 0,2 mg/ kg (Cydectin ® , Wyeth Inc., Fort Dodge, Bogota, Colombia) and a Control group that received no treatment.The animals were distributed so that the mean EPG was similar between the groups.The animals were weighed before treatment to adjust the correct dose according to the manufacturer recommendations.Although the ideal number of animals would be 6 per group, typical farms in Colombia do not have large numbers of animals and we had to fi nd naturally infected animals with acceptable EPG.
Faecal samples were collected from each animal directly from the rectum, on days: -3, 0 and 14 after treatment.Samples were placed in individually sealed containers and sent to the Laboratory of Parasitology at the La Salle University, located in Bogota for EPG counts.A stool culture was performed using the method described by van Wyk et al. (2004), identifying 100 larvae per farm.Farmers from each property completed a questionnaire related to treatment with antiparasitic products in order to better describe the situation of parasite control.All procedures were carried out according to the international practices for animal use and care under the control of an internal committee of the University of La Salle, Bogota, Colombia.
Drug effi cacy was determined by the faecal egg count reduction test (FECRT) and the percentage reduction was calculated according to Coles et al. (1992).The efficacy of the anthelmintics was analyzed using the RESO 2.01 Analysis Software (Wursthorn and Martin 1990).Data was analyzed considering resistance when the effectiveness of the anthelmintic was lower than 95% and when the confi dence interval was below 90% (Coles et al. 2006).

RESULTS AND DISCUSSION
The results for the effi cacy test in each farm are shown on Table I.The FECRT showed that the response to ABZ and FBZ in farm 1 was different from the others.According to the RESO software, a suspect of resistance was diagnosed for ABZ and FBZ in farm 1 because the treatment revealed an egg count reduction just above 95% (95.5 and 96.5%, respectively) with a confidence interval below 90% (89.6 and 89%, respectively).At the other farms, no tested drug showed activity higher than 95% (ABZ: 55 to -15.1%; FBZ: 76.6 to 51.4%; IVM: 93.1 to 67.3%; LEV: 78.1 to -8.1%; and MOX: 49.2%, confi rming anthelmintic resistance to all tested products. In most parts of Latin American countries there are very few restrictions or even none for buying veterinary medicines and farmers are able to purchase them without prescriptions (Molento 2007, Molento et al. 2011).In Colombia this is not different, where the prophylactic use of triclabendazole to control sheep nematodes led to the emergence of Fasciola hepatica resistance detected by the FECRT (Flanagan et al. 2011).
Parasite resistance was determined in all tested farms in Colombia refl ecting the same situation found in Uruguay (Bonino and Mederos 2003), Argentina (Caracostantogolo et al. 2006) (Caracostantogolo et al. 2006).
The fact that H. contortus was the only nematode found in the pre-treatment stool culture may be due to its high prevalence throughout South America (Suárez andBusetti 2009, Almeida et al. 2010) including Colombia (Pulido-Medellín et al. 2014).Herrera et al. (2013) analyzed 95 stool samples of small ruminants from fi ve municipalities in the state of Antioquia, Colombia, where 66.3% (63/95) were positive to H. contortus.Martinez (2011), in a survey conducted in Oicata, state of Boyaca, examined 100 samples of sheep from fi ve different farms and in 53% (53/100) of them, the presence of H. contortus was detected.In a study conducted in the state of Cordoba, Colombia, 97.7% (170/174) of the stool samples from sheep were contaminated by gastrointestinal Strongylidae.In 40.83% (71/174) of the coprocultures, the authors observed the presence of the infective stage larvae of Haemonchus sp.(Ensuncho-Hoyos et al. 2014).
The resistance to H. contortus to all drug classes have being reported worldwide (Cudekova et al. 2010, Fauvin et al. 2010, Beech et al. 2010).To face this problem, effective new tools need to be developed and implemented for a sustainable control of parasites (Macedo et al. 2012).Colombian research centers are studying new alternative methods, such as nematophagous fungi and herbal medicine (Pino-Benítez andStashenko 2006, Orozco et al. 2009).
To date, no registered cases of multiple anthelmintic resistance to gastrointestinal nematodes have been reported in sheep in Colombia.Thus, it was very important to study the drug effi cacy situation.In the studied regions of the country, there is a predominance of small farms with low levels of investment, which makes the obtained epidemiological information of paramount importance for the local farmers and breeders.We have demonstrated for the fi rst time the existence of multidrug resistant parasites.New studies need to be conducted to further look into other regions of the country and using other compounds.
On the questionnaire, the farmers replied that the anthelmintic use was performed at every 30day interval in two farms and in between two and three month-intervals.On three farms, the animals were weighted without the use of a scale before the treatment, and only one farmer used the FAMACHA method for partial selective treatment.Only one farmer had some knowledge about parasitic diseases and the drug resistance issue.About the choice of anthelmintic drugs, farmers reported that the choice was based just on the availability of products.Sheep producers should not neglect the present management data because a failure in health management could affect the production of meat and wool (Manfredi et al. 2010).We consider that the strategies for parasite control used by the farmers were responsible for drug failure, as the farmers reported the use of the suppressive strategy for parasite control as a standard sanitary protocol.
The use of questionnaires is an important tool to check how the parasite control is made and to better understand the local epidemiological situation (Nielsen et al. 2006).This practice is used by research groups in an attempt to obtain more accurate worm control data (Čerňanská et al. 2008).In our case, it was determined that in general farmers do not know proper methods of parasite control, nor applied different techniques of sanitary management.In an interview conducted in Rio de Janeiro, Brazil, with 34 sheep producers, although a better technical knowledge about parasitic resistance and its implications was observed, the results were not satisfactory (Cruz et al. 2010).Only by understanding all the epidemiological factors involved in a parasite infection will a correct diagnosis be able to be made and the appropriate set of management strategies be used (Aagard-Hansen et al. 2010).