Development of methods to understand the epidemiology of tick infestation, tick-borne diseases, and acaricide resistance in sub-tropical Ecuadorian livestock | Développement de méthodes pour comprendre l'épidémiologie de l’infestation par les tiques, les maladies transmises par les tiques et la résistance aux acaricides dans le bétail équatorien subtropical

anglais. BACKGROUND: The livestock sector is an important source of income for the development of the Ecuadorian economy. In the rugged land of Ecuador, livestock production is concentrated in three geographical areas. Andean highlands have a temperate climate and use an intensive model for milk production. The other two regions, i.e. the Coastal and Amazon regions, have a tropical environment and use an extensive and semi-extensive model for milk and meat production on dual-purpose farms (Sánchez et al., 2019; Torres et al., 2014). Around 75% of livestock herds are found in the tropical and subtropical areas (Pourrut et al., 1983; Torres et al., 2014). However, the introduction of short-cycle pastures and environmental conditions make cattle susceptible to tick infestations, mainly Rhipicephalus microplus. To combat tick infestations, farmers use acaricides, but the misuse and overuse of these chemicals has led to resistance, which worsens the problem (Betancur & Giraldo-Ríos, 2019). In the quest of Ecuador for a sustainable agricultural development, research, interdisciplinary collaboration, investment in research, and surveillance initiatives are essential to improving the management of ticks and tick-borne diseases (TBDs). This approach will protect farmers' livelihoods while promoting the interconnected health of humans, animals, and the environment within the One Health paradigm. METHODOLOGY: This work was conducted as part of a project called "Socio-eco-epidemiology of ticks, tick-borne parasites, acaricide resistance and residual effects of acaricides in tropical Ecuadorian livestock: environmental, animal, and public health impacts" funded by the Academy of Research and Higher Education (ARES). This study consists of 4 parts, for which 6 field activities were carried out (Appendix 1 and Table 1). In field activity I, a cross-sectional questionnaire survey was conducted on 139 farms in two tropical and subtropical areas of Ecuador. In field activity II, the level of tick infestation was evaluated, blood samples were taken, and ticks were collected for resistance studies and morphological identification and to investigate the presence of tick-borne pathogens. In field activity III, agricultural warehouses were interviewed to determine the cost of agricultural inputs. As part of field activity IV, participatory meetings were conducted with farmers in the study areas. Field Activity V was carried out in local farms where milk, urine, and faeces samples were collected to measure ivermectin residues. In addition, meat and liver samples were collected from local slaughterhouses to determine ivermectin residues. Finally, in field activity VI, a cross-sectional survey was conducted to determine the amount of consumption of bovine products and by-products. With the information collected, five studies were conducted. Study 1 identified the tick species present in the study area, determined the level of tick infestation, and identified risk factors associated with the level of tick infestation. Study 2 reported the level of resistance to three acaricides (amitraz, alpha-cypermethrin, and ivermectin), the production typology of study farms was carried out, and the influence of acaricide treatment on the production costs of the dairy farm was studied. Study 3 described the perceptions, knowledge, and common and uncommon tick control practices employed and related these variables to the level of tick infestation and the presence of acaricide resistance. Study 4 determined the prevalence of bovine anaplasmosis and the proportion of animals naturally immunized against this disease. This study also allowed us to determine the diagnostic characteristics (sensibility and specificity) of three tests used to diagnose anaplasmosis: multiplex polymerase chain reaction (mPCR), competitive inhibition enzyme-linked immunosorbent assay (cELISA), and blood smear (BS) by using a probabilistic model and a Bayesian approach. In study 5, a high-performance liquid chromatography (HPLC) was used to determine the presence of acaricide residues in bovine meat, liver, milk, urine, and faeces. In addition, the risk assessment was estimated on the basis of the amount of ivermectin present in milk, liver, and meat, the consumption of bovine products in the study areas, and the dietary intake data established by the Food and Agriculture Organization of the United Nations (FAO). RESULTS: Study 1 identified R. microplus as the main tick species present on cattle in the study areas. The study also showed a high level of tick infestation at both farm (41%) and individual (animal) (38%) levels. Studies 2 and 3 described that, to combat tick infestations, the main control method was the spraying of chemicals (by using a backpack sprayer). The acaricides used in spraying (amides, organophosphates, and alpha-cypermethrin) were often overdosed due to their affordability. In contrast, pour-on acaricides were often underdosed due to their high cost. When the chemical treatment was failing, some farmers resorted to overdosing, mixing different acaricides, and applying other risky practices such as mixing different commercial acaricides or applying acaricides by swabbing. Alternative methods such as grazing management, manual tick removal, biological control, or herbal extracts were demonstrated to be helpful in reducing high tick infestation levels on animals and the level of acaricide resistance in farms. Study 2 revealed in R. microplus resistance rates of 49%, 37%, and 53% to amitraz, ivermectin, and alpha-cypermethrin, respectively. The study also found that at the animal level, age (old), body condition (thin), and lactation were associated with high tick infestations, while Bos indicus cattle and their crosses reduced the likelihood of high tick infestations. At the farm level, cattle farming as the main activity, and the use of external paddocks, the use of amitraz, and the lack of farm technification were associated with high tick infestations. When knowledge and farmers’ perceptions were considered (Study 3), a high level of tick infestation was related to the use of organophosphate acaricides, fair levels of knowledge about ticks and TBDs, extensive grazing practices, report of TBDs cases in the farm, and a frequent application (every 1 or 2 weeks) or an irregular application (every 5 or more weeks) of acaricide treatments. Acaricide resistance was related to delegating the preparation of acaricide treatments to employees and not supervising them, and by applying acaricides by swabbing. To quantify the indirect losses associated with the presence of ticks, Study 2 found that the acaricide treatment varied according to the level of technification, herd size, and practices carried out in the farms. Technified farms had a lower expenditure on acaricide treatments (1.30% of production budget) compared to semi-technified (3.43%) and non-technified farms (6.24%). Highly infested farms spend more (4.28%) than farms with a reduced tick infestation rate (2.74%). Lastly, Study 4 determined that the actual prevalence of A. marginale infection was estimated at 32% and that 70% of cattle harboured protective antibodies against A. marginale. The high seroprevalence and infrequent clinical outbreaks in these areas suggest that the pathogen has reached an endemic stability. Study 5 evidenced the presence of ivermectin residues in 68% of the faeces samples and 3% in milk, urine, and liver samples. No residues were found in meat samples. The results obtained from the estimated daily intake of ivermectin show that the consumption of ivermectin is low, and the risk is assessed as negligible. CONCLUSIONS: Initially, our research carried out a survey to gather information about the livestock practices on the farm, economic management, and measures for sanitary and tick control. This helped us identifying the common (chemical control: acaricides by spraying) and uncommon practices (alternative control: medicinal plants, entomopathogenic fungi, manual removal of ticks, among others) employed by farmers to control tick infestations and determining the factors that contribute to the presence of tick infestation and acaricide resistance. The findings of this study have provided valuable insights into the situation of tropical livestock and can serve as a foundation for future research on the impact of mismanagement of acaricides on the environment. We have shared our work widely with academic institutions and farmers in Ecuador through workshops, conferences, and the distribution of manuals (Appendix 2) on ticks and TBDs. Additionally, this research has resulted in the publication of three articles, and we have one more submitted, which will contribute to research in this field.

Socio-eco-epidemiology of ticks, tick-borne parasites, acaricide resistance and residual effects of acaricides in tropical Ecuadorian livestock: environmental, animal and public health impacts