Gastrointestinal Helminths in Horses in Serbia and Various Factors Affecting the Prevalence

Data on gastrointestinal infections in horses in Southeastern Europe are limited; thus, this study aimed to add to the existing knowledge on this topic by reporting on the prevalence of intestinal parasites of horses in the territory of the Republic of Serbia. In the period from April 2017 to December 2018, parasitological examination of 548 samples from horses of different breed, age and sex from four regions and 18 districts of the country was performed. Coprological diagnostic was done by using qualitative methods without concentration and qualitative methods with concentration of parasitic elements. Quantification of the obtained results was performed using semi-qualitative faecal egg count. Four helminthoses were detected in the examined samples: P. equorum (8.57%), O. equi (3.65%), strongylid eggs (71.17%) and Anoplocephala spp. (0.91%). The total prevalence of helminthoses was 77.19%. Monoinfections were significantly more present 70.07% compared to coinfections (7.12%). The highest prevalence of helminthoses was detected in free-ranging horses (93.10%—27/29), in autumn 86.67% (117/135) and winter 79.71% (165/207), in Šumadija and West Serbia region (100%), and in the youngest category (100%). Significant difference (p < 0.001) was detected in the prevalence of monoinfections by strongylids and O. equi and also coinfections by strongylid/P. equorum between horses of different age categories. Obtained results are of great contribution to clinical parasitology and pathology, especially from the aspect of animal health, welfare and preservation of horse population.


Introduction
Unlike many other countries, where the number of horses on pastures, in horsemanship and the production of horse meat and milk is satisfactory, the number of horses in Central Serbia in the past decades has been declining or stagnant. Industrialization and mechanization in agriculture have contributed to this state of horse husbandry in Serbia, but also a very small number of marked and registered animals in individual farms [1]. Nonetheless, the breeding quality of animals in mountainous areas and on individual farms is unsatisfactory [2,3], while certain equid breeds are endangered in terms of genetic preservation [4].
Parasitic infections negatively affect the health and welfare of horses. Depending on the manner of husbandry, the most prevalent helminthoses in horses are large (Strongylus spp.) and small (Cyathostominae) strongylids, ascarid Parascaris equorum [5], tapeworm Anoplocephala perfoliata [6], and pinworm Oxyuris equi [7]. These infections of horses are generally treated with anthelmintics from the same chemical groups, and uncontrolled use of these drugs has resulted in the development of resistance to strongylides and ascarids [8,9].
In the past 20 years, there have been only a few reports regarding the prevalence of endoparasitoses in horsesfrom the West, East and South Serbia in the period 1999-2004. [10], and in autochthonous horses and donkeys in East Serbia in the period 2012-2018 [11][12][13][14][15]. Data regarding the influence of various factors, i.e., age, sex, manner of keeping, season and region, on the occurrence, maintenance, spread and prevalence of certain helminthoses in horses in Serbia, are still scarce.
The aim of this study was an epizootiological screening and determination of parasitological state of horses in the territory of the Republic of Serbia. Obtained results can be used as guidelines for the proposal of effective therapy. Also, information on risk factors of individual categories of horses will facilitate targeted selective treatments of individual animals.

Study Area
In the period from April 2018 to December 2018, parasitological examination of 548 samples from horses of different manner of holding (free ranging, semi-free ranging and stabled), age and sex from four regions (Fig. 1A) and 18 districts of the Republic of Serbia (Fig. 1B) was performed. According to the manner of holding and in relation to the time spent in the pasture, the examined horses were divided into three groups: free ranging (horses that spend 24 h outside), semi-free ranging (horses that have the possibility of going out of the canopy and stay outside from early morning to late evening, i.e. longer than 12 h; during the warmer and drier part of the day, these horses stay under the canopy) and stabled (horses that are permanently in the stable and do not have the possibility of going outside). Horses that have the opportunity to go out to pasture stay in the pasture during the entire grazing season, that is, during the active vegetation period from April to November. Grazing was carried out immediately after the fastest vegetative growth of the dominant species of plants in the pasture, and before heading/ brushing and flowering/creating grass seeds, because the mentioned period represents the best time for grazing [16].
According to the report of the Statistical Office of the Republic of Serbia, the total number of horses by years of research was 17.000 in 2017 and 15.000 in 2018 (Table 1).
Serbia is a continental country in Southeastern Europe in the central area of the Balkan Peninsula (41°53′ and 46°11′ N and 18°49′ and 23°00′ S). Due to the part of the Pannonian plain in the north it also belongs to the region of middle Europe, and geographically and climatically its southern part belongs to the Mediterranean area.

Climatic Conditions
There are three climate types in Serbia. Continental climate is present in the Pannonian plain and in the peripheral part, up to an altitude of about 800 m characterized by hot summers and harsh winters, the average annual temperature is lower than 10 ℃, the annual amount of precipitation is between 600 and 700 mm. Temperate continental climate is present in areas up to an altitude of about 1400 m that is characterized by moderately warm and dry summers and cold winters, and autumn is warmer than spring. Average annual temperatures decrease with increasing altitude (for every 100 m, average temperatures decrease by 0.6 ºC), and the amount of precipitation amounts to 1,000-1,350 mm. Altered Mediterranean climate is present in Metohija basin, foothills of Šar Mountain and Accursed Mountains to the Beli Drim Valley. In the southern parts of the Metohija basin, it is the warmest (11.8 ℃), and during the winter months, the average temperature does not drop below zero degrees. The amount of precipitation is higher in the western part (1170 mm) than in the eastern part (600 mm [17,18].

Sampling
Faeces were sampled because it was known that these horses had never been dewormed before or had been dewormed irregularly. The intention was to check their parasitological status. Faecal samples were collected immediately after defaecation as individual samples from single animal. The samples were packed in plastic bags and marked according to the animal, date and location. Perianal tape samples were collected from all examined animals. All samples were kept at temperature + 4 ℃ and transported to the parasitological laboratory and examined in the next 24 h.

Macroscopic Examination
Faecal samples collected from horses were placed in a clean glass Petri dish and examined for the presence of adult nematodes or of tapeworm proglottids. It was also examined to determine the consistency and any type of abnormality of the faecal sample.

Microscopic Examination
Qualitative methods without concentration and quantitative methods with concentration of parasitic elements were used for microscopic examination.

Faecal Examination by Qualitative Method
Coprological diagnostic was done by using qualitative methods without concentration of parasitic elements (native slide by Pataki, Vayda method and perianal tape) and qualitative methods with concentration of parasitic elements-gravitational flotation using saturated NaCl aqueous solution

Faecal Examination by the Semi-quantitative Method
Quantification of obtained results was performed using semi-qualitative faecal egg count-FEC [19]. This method is based on the conventional faecal gravitational flotation and determined egg count is evaluated as: "minus ( −)" for negative sample or "plus ( +)" for positive sample (with three levels of gradation: " + , + + , + + + "). It is necessary to examine the entire amount of faecal suspension under the two microscopic cover glasses to estimate the number of present parasite eggs. According to Pittman et al. [20], in 5-10 g of faecal sample parasite eggs can be semi-quantitatively counted using a scale for rough determination of infection intensity (low: 1-10 ( +), medium: 11-49 (+ +) and high ≥ 50 (+ + +) eggs/oocysts counted under two cover glasses).

Treatment
Examined horses were not dewormed, or were randomly and sporadically dewormed and there is no veterinary data about used drugs.

Statistical Analysis
The results were classified and statistically analysed according to the following parameters: manner of keeping (stable, semi-free, free-ranging horses), age (young-< 3 years; adult-3-10 years; old-> 10 years), sex, region and season (spring = April and June; summer = September; sutumn = October, November and December 2017 and winter = January, February and March; spring = April and June; summer = September; autumn = October, November and December 2018). Statistical analysis was performed using the software GraphPad Prism. The descriptive statistics reported the data analysis results and were presented as the mean and standard deviation. The statistical significance between the horses from different districts, which were positive for the presence of certain endoparasites, was determined using the Chi-squared test (χ 2 ). The confidence level was maintained at 95% and statistical analyses were considered significant at p < 0.05 and p < 0.001.

Results
Four helminthoses were detected in the examined samples-P. equorum ( Fig. 2A)

3
Anoplocephalidosis was detected in one horse (0.50%) in the Vojvodina region and in each of examined regions in one horse in coinfection with strongylids ( Table 6).

Semi-quantitative Diagnostic Results
Strongylidosis was the most prevalent as low-intensity infection in male ( In free-ranging horses that spend 24 h outside, the highest prevalence of helminthosis was determined (93.10%), with the dominant presence of strongylids (72.41%). The highest prevalence of strongylidosis was detected in free-ranging horses younger than 3 years during summer and autumn. These nematodes also prevailed in semi-free-ranging horses (52.87%), which have the possibility to go into the outlet from the canopy and stay outside from early morning to late evening. This finding is expected, considering that in the mentioned conditions of keeping, horses have a greater possibility of being infected with L3 larvae during the night and at dawn. Monoinfections were the most prevalent in the Šumadija and West Serbia region (74.71%), while coinfection by P. equorum was dominant in the South and East Serbia region (8.41%).    Table 7 Prevalence of helminthoses in horses in relation to season  The prevalence of strongylidosis is in concordance with studies in other countries such as Strongylus spp. 77.10% in Turkey [27], S. vulgaris 64% in Denmark [28], and large strongylids 62% in Island [29] and 61% in Sweden [30]. The findings of the authors who used qualitative methods of coprological research (flotation, sedimentation, Baermann method) [27,29], larval culture [28,30] and PCR [30] are in accordance with the results of our research. However, authors from India [24] and Pakistan [26], who also used the flotation method, report finding lower prevalences of strongylidosis (25% and 17.95%), as well as authors from Brazil [31] who found a prevalence of 40% at the parasitological necropsy.
During the whole period and all the seasons in this study (2017 and 2018), low-intensity infections caused by strongylids were diagnosed constantly in a large number of horses (38.69%). These infections were the most prevalent in male stable horses in the Belgrade region during the spring season. This finding suggests that subclinical strongylidosis is continuously present in the epizootiological area of Serbia. The disease is usually asymptomatic and neglected by horse owners, which cause an indirect effect in infected animals (behavioural changes, lower fertility, low physical condition and reduction of working ability, reduction of resistance and higher susceptibility to infections of other aetiology) [7][8][9]. Therefore, from the economic aspect and the aspect of animal welfare, timely diagnosis and proper therapy of the disease are very important in horse husbandry.
Strongylus vulgaris can cause blood clots and clogging of arteries and arterioles of the intestine, causing ischaemia of the intestine and septic peritonitis [32]. Small strongylids can cause larval cyathosomosis in young animals, followed by serious inflammatory reaction in colon and caecum, which may cause fatal diarrhoea [33]. Very complex aetiopathogenesis and problems with diagnostic, therapy and control make this parasitosis a constant challenge for parasitologists and cause serious problems for field veterinarians and horse owners.
Parascaris equorum was diagnosed as a monoinfection (2.37%) and coinfection with strongylids (6.20%) and the highest prevalence was detected in horses younger than 3 years (16.98%) during autumn. Monoinfections were the most prevalent in stable horses (3.48%) in the Vojvodina region (3.02%), while coinfections were dominant in free-ranging horses (20.69%) in the South and East Serbia regions (8.41%). Low-intensity infections (in horses younger than 3 years in winter) and medium-intensity infections (in free-ranging horses) prevailed.
Two decades ago, there were no detailed studies or available data regarding the prevalence and intensity of infection by this ascarid in horses in Europe. The prevalence of 41.3% was detected in Lithuania [34], and in Belgium only 5.33% [35]. Studies in Poland have shown that 26% of horses were infected with a maximum number of 142 parasites per animal [36], and in Germany in 56% positive horses a maximum of seven nematodes per animal [37]. Borgsteede and Beek [38] detected P. equorum in 28.6% in the Netherlands with a maximum parasite burden of 308 per animal.
In certain areas, no association was found between the age of horses and the prevalence of P. equorum, such as 21.6% in older horses in Lesotho (South Africa) [45], 16.3% in horses older than 8 years in Ethiopia [46], and in Pakistan 22% in horses older than 2 years [47] and 33.33% stable horses [26].
The usual pattern of acquisition of immunity against P. equorum may differ in some developing countries, which may be influenced by management strategies [48,49]. Although younger horses are true hosts for adult ascarids, patent infections may also be present in older horses [45]. In the current study in Serbia, low prevalences of P. equorum were found in horses 3-10 years of age (8.16%) and horses older than 10 years (3.38%).
Parascariosis is usually asymptomatic and found in younger horses, while it is rarely diagnosed in older animals due to acquired immunity [49]. The most common negative effect is perforation of the intestine by the large number of parasites [50].
The high prevalence of helminthoses in winter (79.71%) and summer (72.11%) may be because 2017/18 was one of the warmer winters in Serbia, with an average temperature of 14.5 ℃, while the summer of 2018 was humid and rainy, with the temperatures above 30 ℃ [16][17][18]. The established differences in the prevalence of GI parasites during winter (73.43%) and spring (52.54%) can be because horse faeces were sampled during 3 months of the winter period (January, February and March) compared to 2 months of the spring period (April and June). As a result, horses sampled in winter had an additional 30 days to be parasitized.
Anoplocephalid eggs were detected in 0.18% horses as monoinfection in winter, while 0.73% horses were coinfected with strongylids during all the seasons. The highest prevalence of Anoplocephala spp. was diagnosed in horses 3-10 years old (1.02%), exclusively in stable horses and semi-free-ranging horses, mainly as high-intensity infections during spring and summer. The spring of 2018 was extremely warm for Serbia climate, with an average temperature of 18.2 ℃. Together with the rainfall, bioecological preconditions were made for the development of intermediate host (nonparasitic mites from Oribatidae family) and infection of horses during summer. Individual cases of Anoplocephala spp. infections were detected in all examined regions.
Epidemiological studies of equine anoplocephalidosis conducted in Central Spain shown significant humiditydependent seasonality [51]. The highest prevalence was detected in autumn (37.5%) and winter (32.3%), while the prevalences were significantly lower in spring (9.2%) and summer (10.8%). During the obduction of horses in Belgium, Agneessens et al. [52] detected the prevalence of tapeworms of 28.9% and no significant differences during seasons.
High-intensity infection of horses with A. perfoliata can cause spastic colic of various degrees [53,55]. Localization of parasites in the ileocaecal junction can cause ulcers at the place of fixation of parasites, intestinal invagination, acute catarrhal enteritis, physical obstruction and/or change of intestinal motility [54][55][56][57].
The low prevalence of Anoplocephala (0.91%) in the examined horses can be because they were mainly kept in stables, under controlled conditions. In that manner, the contact with the transient hosts is disabled. It is assumed that the occasional use of individual horses on pastures for work or maintaining physical conditions, as well as consumption of grass or hay, was a potential risk factor for their contact with oribatid intermediate host.
Proglottids of these tapeworms are not excreted daily and continuously, which is why it is necessary to sample faeces from suspected animals for several consecutive days. In this research, faecal samples were collected only once, due to technical reasons, which may be one of the causes of such low prevalence of anoplocephalidosis.
Out of the total prevalence of oxyuriosis, 3.47% were monoinfections diagnosed in December using the perianal tape method. The eggs of O. equi were detected in faeces of only one horse (0.18%) as coinfection with strongylids in November.
This diagnosis can be explained by the fact that that horse had high-intensity infection, and egg-rich deposits, probably due to gravity, fell on excreted faeces. Since faeces are collected from the ground, immediately after defaecation, it is possible that the contamination with O. equi eggs occurred from these deposits. Infection with O. equi was detected mainly in horses younger than 3 years (10.38%) as high-intensity infection, and at lower prevalence in horses 3-10 years old (3.06%) as medium-intensity infection. The infections were dominant in the region of Šumadija and West Serbia (21.84%) in winter, while sporadic cases were detected in the regions of Vojvodina and Belgrade. Besides stable horses (4.35%), the infections were detected in semi-free-ranging horses (2.87%). Higher prevalences were detected in female horses compared to males, and no statistical significance was detected between sexes.
The analysis of the parasitological status by reproductive categories within the sexes was done in light of the wellknown fact that parasitic diseases have a negative impact on a horse's ability to reproduce [60]. In relation to different reproductive categories of horses, statistically significant differences (p < 0.001; p < 0.01) were discovered in the prevalence of oxyuriosis (11.29%) and coinfection with strongylidosis-parascariosis (14.52%), with the highest prevalence in mares under 3 years of age. The established differences in the prevalence of GI helminths by reproductive category within the sexes indicate how important the prevention and control of parasitic infections are in horses, considering that they affect their reproductive performance [61].
It is known that the development of O. equi is favoured by heavy rainfall, which is confirmed by the obtained results of a higher prevalence of adult parasites in horses from regions with uniformly high rainfall (region of Šumadija and West Serbia) and the highest precipitation in the period of October-December.
By monitoring the current pathology of autochthonous breeds of equids (domestic mountain horse and Balkan donkey) in the region of Braničevo district in the period 2012-2018, high-intensity infections with nematodes were detected [11][12][13][14]. Strongylids and Dictyocaulus arnfieldi were the most prevalent parasites. Dictyocaulus arnfieldi was dominant in all examined donkeys, but also in horses which were not in close contact with the donkeys [15]. A high prevalence of Strongyloides westeri was detected in donkeys and in one herd it was associated with the occurrence of diarrhoea in several foals [15].
Ilić and Dimitrijević [10] detected high prevalences of nematodoses in horses in the districts of Braničevo (88.24%), Zlatibor (66.66%) and Nišava (85.72%). By comparing the results obtained in the current study (from the same localities), a constant maintenance of high prevalences of nematodoses, or increase in prevalences, is observed (Šumadija and West Serbia region). These findings obtained 12 years later indicate that these results are due to non-deworming or unplanned deworming of horses. Since there are no veterinary data regarding the treatment of the examined horses, there is a presumption that random and occasional deworming of horses and inadequate drug selection and dosing could cause resistance to the used anthelmintics. Consequently, according to the guidelines of ESCCAP [9], deworming treatments of horses should be determined according to the age and usage of horses, while respecting the principles of good hygiene in the stable and on pasture. It is recommended that the first treatment during the grazing season be performed before grazing, or 1-2 months after grazing. This is considered strategically important from the aspect of strongylid life cycle and pasture contamination by parasite eggs.

Conclusion
A high prevalence of helminthoses (77.19%) could indicate the existence of resistance of the diagnosed helminthes to the drugs used so far in an uncontrolled manner. Horse owners are advised to perform planned coprological testing first, to define the presence of certain parasites and thereafter to make targeted selective treatments of individual animals.
A study to this extent has not been performed in Serbia so far. The results greatly contribute to clinical parasitology and pathology, especially from the aspect of animal health, welfare and preservation of horse population. Since the number of horses in Serbia has been significantly reduced in the last 10 years, this epizootiological database is extremely important, because it provides data for designing guidelines and more efficient protocols for the treatment and prophylaxis of equine parasitoses.