Location

The study was conducted in one farm located in Tenosique, Tabasco, Mexico, at 17° 28’ 21" N 91° 25’ 36" W. The climate in the region is warm and humid, with an average annual temperature of 27.6 °C and an average rainfall of 2131 mm per year (^{CONAGUA 2019}).

Animal Management

One herd of 30 horses from the same farm (12 females and 18 males) were followed for 9 months from December to August 2017, which correspond to the cold (December, January, February), dry (March, April and May) and rainy (June, July and August) season of the region. The whole herd was studied so that animals of different ages were used, classifying them as young (0-1 year), mature (1 to 7 years) and old (> 7 years); the females were kept for breeding and some males were gelds or stallions depending on their size and the decision of the owner according table 1.

The horses are used in activities related to the cattle herd management. They grazed as a single herd together with beef cattle on 50 hectares of low-sized grasses like: Paspalum notatum and Pas- palum conjugatum pastures, some paddocks have African star grass (Cynodon nlemfuensis) in Gmelina (Gmelina arborea) plantations where animals had the function of keeping the grass controlled in a silvopas- toral system. The farmer reported that the horses were not supplemented and three months before starting the study they had not been dewormed. On the farm it is customary to deworm with ivermectin paste every year without making a previous diagnosis, so this study is important to determine the genera of nematodes that prevail in the area. The animals were used for work activities (cattle herding) but were mostly allowed to spend their time grazing.

Determination of anthelmintic efficacy

At the beginning of the study, 20 horses were dewormed with Pyrantel Pamoate (Exodus-Paste, Bimeda) at a dose of 3 mg kg^-1 body weight (BW) to determine the efficacy by the fecal egg count reduction test (FECRT). One group of 10 horses without de- worming represented the control group. The FECRT percent efficacy used by ^{Dobson et al. (2012)} were calculated as: FECRT=1-T2T1xC1C2 . Where T1 represents the mean pre-treatment and T2 the fecal nematode egg counts performed at 15 and 45 days post treatment of a treated group, and C1 and C2 represent the mean pre and post-treatment counts of an untreated control group, respectively.

Determination of re-infection level

At the end of the anthelmintic efficacy study (45 days), the horses were not dewormed again. The horses were enclosed in a handling sleeve and the feces sample was obtained directly from the rectum of the animals monthly for nine months (December to August). The number of EPG in feces was determined using a saturated solution of sodium chloride with the McMaster technique (^{Thienpont et al. 2003}) with a sensitivity of 50 EPG. Stool cultures were performed on positive samples for nematode eggs, keeping feces at room temperature in containers, covered with gauze and adding water every third day to maintain the moisture. After eight days, the feces were placed in Baermann apparatus to obtain the infective larvae (Thienpont et al. 2003) and subsequently the taxonomic identifying was based on the number of intestinal cells as the main characteristic, since the size and shape are very similar (^{Bowman 2011}). To obtain pinworms (Oxyuris equi), the Graham Test Technique was used, briefly, the anal mucus sample is collected on rubberized tape and deposited on slides.

The blood samples were obtained by puncture of the jugular vein and collected in tubes with ethylenediamine tetraacetic acid (EDTA). With whole blood the packed cell volume (PCV) was processed by the micro-hematocrit method (%), also plasma protein (TPP) was determined with a refractometer. Also, thin blood smears were stained with a staining kit (rapid blood stain, Hycel) to determine the differential leukocyte count in which 100 leukocytes were classified as neutrophils, eosinophils, basophils, monocytes and lymphocytes and expressed as percentage.

Antigen preparation

Crude worm antigen (CWA) from adult nematode of Strongylus spp. and cyathostomins was prepared. Adult nematodes were collected from the intestine of one horse in a post mortem nematode diagnosis. The worms were washed in PBS with 0.01% sodium azide and grinding with 10% 1 mM phenylmethylsulphonyl fluoride (PMSF), and 2% (w/v) reduced Triton X-1060. Protein samples were concentered by centrifugation at 20 000 x g per 20 min at 4 °C. The supernatant, containing CWA was collected and stored at -20 °C. The protein concentration was estimated by the ^{Bradford method (Bradford, 1976}) constructing a calibration curve and determining the protein concentration of the sample with Coomassie Protein Assay Kit (Thermo Fisher Scientific, USA).

Antigen preparation of Strongylus spp. and Cyathostomins and immune-enzymatic assay were according to a widely description in other study (^{Gonzalez-Garduno et al. 2017}). Results were expressed as the percentage of the Optical density (OD) of the positive control serum and employing the following formula (^{Cardoso et al. 2013}):

Statistical Analysis

The data analysis considered the gender of the animal, the season of sampling, and the age of the animal as the main factors and some interactions were tested using de SAS program (^{SAS 2017}) with the following model:

Where: Y _ijkl is the response variable (EPG leukocyte count, IgA), µ is the general mean, p; is the fixed effect of the season (i = cold, rainy, dry), ζ_i is the fixed effect of the j^th horse age (young, mature, old), and Ψ^k is the fixed effect of the k ^th gender (male or female). The interactions are p * ζ_ij, p * Ψ_ik, ζ * Ψ_jk, and p * ζ *Ψ_ijk. ɛ_ijkl is experimental error. The means separation was carried out using Tukey test. Spearman correlations were made to known the relation between variables in overall data. Only for correlations, the age was recorded as a discrete variable in days.

Eggs per gram of feces and immune response

In rainy and cold seasons, the number of EPG shows the highest values when high humidity was recorded in the region (Table 2). In addition, differences in number of EPG and TPP were found by the gender of the animals (p < 0.01). Females had higher number of EPG and TPP values (1224 ± 1269 EPG and 7.6 ± 0.6 g dl^-1, respectively) than the males (623 ± 671 EPG and 7.4 ± 0.5 g dl^-1, respectively), and no differences were observed with the age of the animals (p > 0.05).

In the humoral immune response determined by iga levels (percentage with respect to the positive standard; % RPS), the lowest values were observed with cyathostomins antigens used during the dry season (45.3% RPS). Conversely, the highest levels of iga for both antigens were observed in mature and old animals (> 50% RPS, Table 2) while young animals (younger than 1.0 years old) showed the lowest iga value (16.9%). No differences were observed by animal gender (p < 0.05).

The main GIN species identified belonged to the Cyathostominae subfamily, they exhibited eight intestinal cells (97.4%). A smaller proportion was classified into the L3 of Strongylus genus (2.6%), and three species of this genus were identified as follows: S. Equinus (16 intestinal cells), S. Edentatus (20 intestinal cells), and S. Vulgaris (30 intestinal cells). The presence of Oxyuris equi eggs was also recorded in three horses.

The season did not affect the numbers of eosinophils (1.1-1.4 cells x 10³ jl) and similar values were observed between seasons, ages, and genders (p > 0.05). The highest levels of leukocytes and neutrophils were observed during rainy season (18.8 and 10.5 cells x 10³ jl), while the lowest values were observed in the cold season (12.5 and 6.0 cells x 10³ jl). On the other hand younger animals had higher leukocyte and lymphocyte counts (Table 3).

Positive correlations were observed between age and iga levels, while negative correlations occurred with white cells (leukocytes, lymphocytes and eosinophils). On the other hand, leukocytes were positively correlated with the number of lymphocytes, neutrophils and eosinophils and there were only negative correlations against iga in both antigens in dry season. The number of leukocytes also correlated positively with the number of EPG in the cold season and in the total data (Table 4).

Fecal egg count reduction test

In the present study the anthelmintic drug was efficient to control GIN infection in horses, this means that the parasites maintain their susceptibility to the drug (^{Matthews 2014}). In a previous study carried out in this tropical region of Mexico, an anthelmintic efficiency higher than 90% in horses treated with fenbendazol was reported (^{Gonzalez-Garduno et al. 2015}).

The high efficiency of Pyrantel Pamoato obtained in the present study could be due to the low frequency of deworming in herds in this region. In addition, a low number of EPG was recorded after 45 days post-deworming (519 EPG), which could be attributed to the long prepatent period (2-3 months) of cyathostomins (^{Andersen et al. 2013}); so, establishing the anthelmintic treatments in rainy and cold season could be a good strategy to reduce the number of EPG during the seasons of the highest risk of re-infection. This fact would allow achieving a low number of EPG after the last deworming and thus prolonging the frequency between anthelmintic treatments. This action could improve the health of the herd, which would avoid the clinical symptoms such as diarrhea, chronic weight loss, colic and an acute larval cyathostominosis often characterized by fatal diarrhea (^{Von Samson-Himmelstjerna 2012}). However, the strongyle egg counts won’t contribute to increase the EPG total counts; since their prepatent period could be as long as 6 to 12 months (^{Andersen et al. 2013}).

Eggs per gram of feces and immune response

Immunological and parasitological parameters showed large inter-season variations. The environmental conditions are important factors that determine the development of the infective stages of nematodes; so that high number of EPG counts in the rainy season promote the contamination of pastures with free-living stages (^{Navarro dos Santos et al. 2012}). Therefore in warm and humid climates like in Brazil, fecal mass plays an important role as a reservoir of larvae that survive with the remaining moisture and that can parasitize animals during the dry season. Data from studies conducted in Germany suggest that the risk of infections with nematodes is relatively small in this country where environmental conditions do not favor the larval development (^{Schneider et al. 2014}). Conversely, in the present study, the lowest number of EPG occurred in the months with the lowest rain (dry season). This coincided with higher values of PCV and TPP and a lower production of IgA against cyathostomins. Probably the low re-infection provokes the reduction of the IgA, because it requires the stimulation of the infection to maintain the high levels of humoral immune response. Interestingly, in another study with African equines a positive correlation between the number of EPG and IgA level, was recorded (^{Tombak et al. 2020}). The lowest numbers of neutrophils were also observed during dry season.

This implies that the lower re-infection during the low rainfall months allows horses improving their health indexes (higher PCV and TPP and lower number of EPG counts), which increases in the dry season compared to the rainy seasons. However, the changes in PCV did not reach extreme levels that represent health problems during the study, since the observed values (39 ± 4.3 %) were within the normal range (^{Lumsden et al. 1980}). In other study, it was shown that parasitic burden did not seriously affect the health of horses and was not correlated with the body condition (^{Burden et al. 2010}), therefore other aspects are likely to be more important; for example, the nutrition of the animals (^{Valdez-Cruz et al. 2013}). It is important to consider not deworming unnecessarily the whole herd; but using other control strategies, such as the selective treatment, that allows the individual treatment, when the animals really need it.

Females had higher fecal GIN egg counts than males, which was also reported in another study in Spain (^{Francisco et al. 2009}). This parasitologi cal response was surely because the females have greater susceptibility during lactation and can be a source of environmental contamination (^{Martins et al. 2018}), and in the study some females were lactating. In this case, only TPP values were different between males and females, which suggests that females produce a greater amount of protein in response to GIN infection; however, no differences in cellular and humoral values were found by gender. Other studies reported greater differences between horse breeds; however, the gender and age of the animals were only partially related to the acquired resistance (^{Gras et al. 2011}).

Horses having a high rate of infection regu larly show serious health problems (^{Tavassoli et al. 2010}). However, it has been indicated that helminths stimulate a strong immune response that lead to reduce their establishment and persistence in their hosts. In addition, helminths are immunomodulatory and are capable of interfering with immune and inflammatory mechanisms of the host (^{Moreau and Chauvin 2010}).

In the present study was observed that young horses had less IgA compared to adults and olds, which coincides with the results by ^{Perkins and Wagner (2015)}, who indicated that the immunoglobulin isotype composition in foal serum showed reduced concentrations during the first year of life. Foal endogenous IgG (4/7) was first detectable by 16-20 weeks and slowly increased during the first year of life. This finding was also confirmed in the present study by obtaining a positive correlation coefficient between age and IgA concentration.

The high number of EPG of the horses in this study was comparable with the values indicated in other countries, where the main species observed were cyathostomins (^{Francisco et al. 2009}; ^{Nielsen et al. 2010}). In Germany, a high prevalence (98.4%) of the Cyathostominae subfamily was observed, and to a lesser extent ascarids (16.7%), tapeworms (14.3%), pinworms (8.7%), and Strongyloides (4.0%) were also observed (^{Hinney et al. 2011}). In donkeys of Ethiopia 94.9% of prevalence in Strongylus genus was re ported (^{Ibrahim et al. 2011}).

The major changes in the white blood cell counts occurred in time by the effect of the nematode infection (^{Hubert et al. 2004}). The reduction in the number of lymphocytes and neutrophils was also explained by the low re-infection originated by the dry season; since, due to the lower NGI infection, the immune response of the animals is low. On this regard is interesting to mention that in another study, the leukocyte, neutrophils and lymphocytes counts were higher in the group of parasitized horses than in their control group (^{Hubert et al. 2004}). In the same way, another study has indicated an increase in neutrophils, monocytes and eosinophils in nematode infected horses (^{Katre et al. 2020}).

Immunoglobulins have been used as a tool for the diagnosis of nematode infections (^{Abo-Aziza et al. 2017}); however, in this study, no relationship was observed between IgA levels and EPG values. The high variability found in the number of EPG between individuals and the local action site of IgA in the mucous membranes and the low periphery levels are some of the explanations for which there was no correlation between IgA and number of EPG. The only positive correlation of EPG was with leukocytes and neutrophils.