Effectiveness of ivermectin and moxidectin against cyathostomins in four horse breeding farms in Mexico

This study aimed to evaluate the effectiveness of oral ivermectin and moxidectin against natural cyathostomin infection in four horse farms located in the central regions of Mexico. 445 horses of the Warmblood (145), Thoroughbreds (100), and Quarter Horses (200) breeds, aged between 6 months and 27 years, were used. Data on horses and parasite control methods were collected through interviews with farm owners and veterinarians. Using the McMaster technique, fecal samples were processed from all 445 horses, 180 of which were positive for cyathostomins. On each farm, a selection was made of 45 animals meeting the criteria of a Fecal Egg Count Reduction Test yielding results exceeding 150 eggs per gram of strongylid-type nematodes. Subsequently, three separate experimental groups were formed for each farm, each consisting of 15 horses The first group was treated with oral ivermectin 1.87 %; the second group with oral moxidectin 2 %; and the third was the non-treatment control group. Coprocultures were also performed to identify the presence of nematode species. The data obtained were analyzed with RESO.exe©️. Three of the four farms achieved a 100 % reduction in eggs per gram with both macrocyclic lactones. One farm achieved 93 % reduction with ivermectin and 87 % with moxidectin. This study demonstrates that macrocyclic lactones effectively reduce cyathostomins in three of the four farms studied. The results suggest potential cyathostomin resistance to macrocyclic


Introduction
Cyathostomins are the most frequently reported nematodes in horses, and anthelmintics (AH) have been the main method used to control them. (1,2) n Mexico, the total estimated equine population is over 6.3 million (3) It has been assessed that only 300 000 horses receive nutritional and medical care (B.Monroy-Hérnandez, personal communication, October 21th, 2021).Approximately, 150 000 horses receive basic treatments including AH, and 45 000 comprise the high-performance group, which is subjected to more continuous deworming (either monthly or every other month) with macrocyclic lactones (ML), as they offer a pharmacologically approved endectocide action. (4)Regular and non-technical use of AH, derived from customary clinical practice, has favored the selection of cyathostomin populations capable of surviving, thus promoting the anthelmintic resistance (AHR) phenomenon. (5,6) or more than 50 years, horses have been conventionally dewormed using high-intensity short-term schemes.This practice originally served the purpose of eliminating the somatic larvae of Strongylus vulgaris, which causes arteritis and aneurysms in horses. (7,8) lthough it is not common to find serious cases of S. vulgaris, these deworming practices have prevailed, subjecting the cyathostomin populations to high selection pressure, enhancing resistant or multi-resistant parasitic populations because of decreased AH effectiveness. (9,10) 3)(14)

Ethical statement
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.Feces were obtained while farm veterinarians were performing breeding evaluations or from the ground in the case of younger animals.As well as the usual application of deworming pastes.No handling involved stress or injury to the animals.

Type of study and farm location
A longitudinal cohort study with convenience sampling was conducted in four horse breeding farms located in the central and central-western regions of Mexico, specifically in the states of San Luis Potosi (farm 1), Guanajuato (farm 2), both with a dry climate BSw Köppen (15) scale and the State of Mexico (farms 3 and 4) with a temperate-subhumid climate: Cw Köppen scale. (15)

Animals
The study was conducted using a cohort of 445 horses distributed as follows: farms 1 and 3 held a total population of 70 and 75 racing Thoroughbreds, respectively; farm 2 held 100 racing Quarter Horses and farm 4 held 200 show jumping Warmbloods with ages ranging from birth to 27 years (Table 1).

Questionnaire
Interviews were conducted with horse-owners and veterinarians of four farms to retrieve information about the breed, age, intended use, frequency of AH administration, type of AH used, and criteria used for parasite control in the past seven years.Geographic location (municipality and state) and climate were also recorded (Table 1).

Fecal Egg Count Reduction Test
No AHs had been administered to the horses for at least 60 days before this study.
The effectiveness of IVM and MOX against cyathostomins was determined with a Fecal Egg Count Reduction Test (FECRT) (methodology approved by international organizations). (16)It consists of 3 stages and its rationale is to measure the reduction of the eggs count per gram (EPG) after AH treatment (Figure 1).

Stage 1: Pre-treatment
Fecal samples of the total population of horses (n = 445) were processed using a modified McMaster quantitative technique with a sensitivity index of 50 EPG. (18,19) ples of 2g of feces were homogenized in 28 ml of saturated NaCl solution of 1.250 density.The solution was then filtered and transferred to a McMaster chamber to perform an EPG count using an optical microscope (10x) to determine the parasite load.
Forty-five horses with a fecal egg count reduction test ≥ 150 eggs per gram of strongylidtype nematodes and who were 6 months or older were randomly selected per farm.

Stage 2: Treatment
A total of 180 horses (45 horses per farm) were included in the treatment phase.On each farm, three groups of 15 horses were randomly selected (Figures 1 and 2).
The first group was treated with a single dose of IVM 1.87 % oral paste (200 µg / kg BW); the second group was treated with a single dose of MOX 2 % oral gel (400 µg / kg BW); and the third group was the non-treatment control group (Figure 1).The weight was estimated using a morphometric tape according to Wright's recommendations. (17)

Stage 3: Post-treatment
McMaster's modified quantitative technique (20) was performed in all animals (n = 180) fourteen days after treatment (Figure 1).Two assumptions were considered as part of the methodology: 1) if the result was SUSCEPTIBLE, the test would not be repeated and 2) if the result was RESISTANT, the test would be repeated (Figure 2).

Larval culture
Larval culture was performed in fecal samples preserved at 4 °C from stage 2 (treatment) and stage 3 (post-treatment) of all 180 horses (Figure 1).Following the technique of Corticelli and Lai, (21) larvae were collected using a Baermann device to identify the nematode genera. (2,22) arval viability was analyzed following two criteria for species counting and identification: 1) larvae were shed in their infective stage (L3) and 2) larvae had vigorous motility.A total of 600 larvae were analyzed per farm, following the recommendations and taxonomic keys of Santos et al. (2) and Bevilaqua et al. (22) to classify the species found.

Data analysis
The data obtained were analyzed using the program RESO.exe©; CSIRO, 1993, Animal Health Division of Microsoft Excel©.Data were considered RESISTANT when the percentage of reduction was < 95 %. (23)

Questionnaire
Basic information about the farms (location and climate), total horse population, individual horse information (breed and intended use), and deworming program (frequency, type of AH used, and main criteria) was gathered from horse-owners and farm veterinarians (Table 1).

Fecal egg count reduction test
The results of stage 1 performed in the total population (n = 445) are shown in Table 2.
The percentage of horses that had ≥ 150 EPG were farm 1, 67.The effectiveness of IVM and MOX against strongylid-type nematode populations is shown in Table 3 and Figure 3. Farms 1, 2, and 4 showed 100 % efficacy for both ML, indicating that the nematodes were susceptible to IVM and MOX molecules.The arithmetic mean of the EPG released pre-and post-treatment, showed the effectiveness of both ML only in farms 1, 2, and 4 (Figure 4).
A lack of efficacy due to parasite resistance (93 % for IVM and 87 % for MOX) was observed on farm 3. Therefore, 6 months after the first test, a second FECRT was performed on farm 3 (3b) (Table and Figure 3) according to the American Association of Equine Practitioners (AAEP) guidelines. (24)No horse on farm 3 received any anthelmintic treatment during that 6-month period while starting the second trial.With the same initial population (100 horses) at that moment 72 animals met the same inclusion criteria for testing (≥ 150 EPG).Randomly, 15 horses were treated with oral IVM 1.87 %; 15 horses with oral MOX 2 % at the same initial dosages, and the third group of 15 horses was the non-treatment control group (Figure 3).The effectiveness of 100 % for IVM and 94 % for MOX was then observed (Table 3, Figures 3 and 4).

Larval culture
In both regions (dry and temperate-humid climate), 2 400 L3 (600 per farm) were identified, and 100 % of these larvae were of the cyathostomin group (non-migratory strongyles) in the pre-treatment and post-treatment samples.The morphological characteristics found were sheathed larvae with long and acute larval tails and 6-8 intestinal cells.No larvae with 18 or more than 20 intestinal cells or morphology suggestive of migratory strongylids, such as Strongylus species, were found.

Discussion
Results from this study show that the AHR phenomenon in the central and centralwestern regions of Mexico occurs mainly due to non-migratory strongylid populations.
Only larvae of the cyathostomin group or non-migratory strongylids were found in all four farms.Migratory strongylids need a prepatent period of at least 6 months, and when deworming is frequent (3 to 4 times a year), the development of the larvae to an adult stage is negatively affected, probably due to the high susceptibility of these nematodes to ML, as mentioned by Grice et al. (24) In addition, we found low or no prevalence of migratory strongylids such as Strongylus spp, which coincides with previous studies. (7,25) ine cyathostomosis could be caused by at least 53 different species, and approximately 11-15 species have a higher prevalence in the cecum and large colon. (26)Due to the abundance and richness of cyathostomins, AH efficacy tests may not consider this a significant factor, as shown in this study.FECRT is a field test, not a molecular or serological test.It has not been possible to distinguish between species of cyathostomins in equines through coproculture, and therefore, the resistance that each of their species may present is not detected.This can be evidenced in nematode species of ruminants.All these studies were initiated in these animal species and not in equines.The results obtained are limited because we cannot distinguish between several cyathostomin species.However, it is the test recommended by international organizations. (4,6,10,16) Seology, molecular biology, and proteomics technologies need to be developed and implemented for the identification of these species in horses. (6,19,27) I addition, factors such as the age of the horses, the time of the year related to early or late L3, cyathostomins hypobiosis processes, hosting types, feeding practices, and group randomization procedures, must be considered, as mentioned by Nielsen et al. (28) The inadequate management of farm 3 while administering ML without any established criteria or pretreatment diagnosis resulted in the lack of effectiveness of MOX.The continuous use of IVM and MOX molecules resulted in a greater number of resistant strongyles-type nematodes (Figure 4).The effectiveness of IVM (93% in the first test and 100% in the second) (Table 3 and Figure 3) highlights the existence of horses with parasite resistance to this endectocide (Figure 4). the confidence intervals of both samples [CI 52-99 (3a) and CI 12-97 (3b)] indicate that only a couple of animals were excreting resistant nematodes and must be treated as potential high shedders.ML can continue to be used in this farm if strategies to preserve both molecules are implemented.In addition, to avoid high shedders spreading resistant parasites in the pasture, a resistance management strategy (refuge) could be implemented.This strategy involves deliberately allowing the survival of cyathostomin populations that have not been recently exposed to any treatment.The progeny of unselected parasites provides a source of susceptible nematodes that can dilute resistant nematodes that survive AH, thereby reducing the rate of AHR development.In farm 3, two fecal samples were obtained six months apart; therefore, animals were grazing in different areas when each sample was obtained.This would explain why in the first FECRT, the results showed lower and upper limits pointing toward IVM resistance (Table 3 and  3 and Figure 4).In this sense, the appearance of the impending AHR is a factor that should lead to immediate action to change deworming practices, with strategic and selective deworming being a possible solution to the problem. (29)Since the use of AH remains the irreplaceable method in terms of efficacy and practicality, every horse farm should first monitor the need for treatment and subsequently its effectiveness by monthly coproparasitological analysis.
Macrocyclic lactone resistance to cyathostomins was first reported in 2005. (30)nce then, multiple cases of this resistance have been reported in Italy, (31,32) France, (14) Germany, (32) England (32) and Lithuania. (14)In Latin America, ML effectiveness studies have been conducted mainly in Brazil (12,30) and Mexico. (33)Canever et al. (12) evaluated the effectiveness of IVM and MOX against cyathostomins and found levels of 5-65 % and 16 %, respectively.Rosado-Aguilar et al. (33) reported 60 % resistance to IVM in five horse farms located in the southern region of Mexico in 2014.To the best of our knowledge, no other AHR studies have been published in Mexico.We believe that the AHR phenomenon could be an ongoing health problem in several farms in Mexico mainly due to misuse and overuse of ML because of the ease of acquiring deworming products by horse owners without a veterinary prescription and its frequent administration to treat any type of parasites without a proper diagnosis.This information is fundamental to limiting the negative impact on animal health and welfare and constitutes the first step for the rational use of AH.In central and central-western Mexico horse breeding farms, it is necessary to implement integrated parasite management (IPM), which is a non-renewable and effective resource to amplify the effectiveness of ML.To achieve this objective, it is necessary to include actions such as: 1) strategic deworming based on coproparasitoscopic diagnosis, 2) improvement of feeding management practices, 3) manure management in the pasture to reduce the source of contamination, and 4) biological control methods, such as pathogenic fungi, bacteria, and mites or entomopathogenic nematodes. (6,34,35) IM is an approach to a strategy that may change consciousness toward horse breeding farms under sustainable tools.

Conclusions
The present study demonstrates high levels of effectiveness of IVM and MOX in treating horses against cyathostomins; however, we report an increasing and impending resistance to MOX in one farm.IPM of nematodes is mandatory for prolonging the effectiveness of ML in the region.

Figure 1 .
Figure 1.Diagram of the Fecal Egg Count Reduction Test (FECRT) methodology

Figure 2 .
Figure 2. Progressive sampling scheme, the selection of animals, and distribution of

Figure 3 .
Figure 3. Diagram of Fecal Egg Count Reduction Test (FECRT) results showing

Figure 4 .
Figure 4. Pre-and post-treatment arithmetic mean of eggs per gram (EPG) from four horse-breeding farms.

Figure 3 )
Figure3), whereas in the second FECRT, the animals were in a different pasture that

Table 1 .
Farm and horse information and deworming programs at four breeding farms in central and west-central Mexico.

Table 2 .
Number of animals that met the selection criteria of ≥150 EPG on each farm and from which they were selected for stage 2.

Table 3 .
Ivermectin and moxidectin effectiveness in four horse-breeding farms in different ecological regions.