Parascaris spp. eggs in horses of Italy: a large-scale epidemiological analysis of the egg excretion and conditioning factors

Background Equine ascariosis, caused by Parascaris spp., is a worldwide endoparasitic disease affecting young horses in particular. Despite the great number of horses reared in Italy, large-scale epidemiological surveys dealing with ascariosis prevalence in the country are not reported in the current literature. For this reason, the present survey aims to describe, for the first time, the spread and infestation of Parascaris spp. in a large population of Italian horses (6896 animals) using faecal egg counts, and further to identify risk factors associated with ascarid egg shedding. Methods Individual rectal faecal samples collected during routine veterinary examinations were used and Parascaris spp. prevalence was tested against the animal’s age, sex, housing conditions, geographic provenance as well as the respective sampling season. Results Among the examined stables, 35.8% showed at least one horse to be positive for Parascaris spp. eggs and an overall prevalence of 6.3% was found. Ascariosis rates tended to decrease significantly with age and, proportionally, 80.0% of the recorded Parascaris spp. eggs were found in 0.7% of the examined animals. Statistically significant differences among prevalence rates were found between the different geographic areas of provenance and prevalence was found to be higher in horses reared outdoors compared to those raised indoors. Analysis of data based on sex and season did not show any significant differences. Despite the lower prevalence found compared to other European countries, ascariosis was concluded to represent a significant health challenge for horses reared in Italy, especially foals. Age (foals and yearlings) and outdoor rearing were identified to be significant risk factors for Parascaris spp. egg shedding. Furthermore, the relevance of the infected horses over 6 years of age should not be underestimated as these represent a significant source of contamination for younger animals. Conclusions The development of improved treatment protocols based on regular faecal examination combined with follow-up assessment of the efficacy of integrated action plans would prove beneficial in regard to animal health and anthelmintic resistance reduction in the field. Graphic Abstract

nasal discharge, coughing, a rough or bristly hair coat, slow growth and occasional small intestine rupture following the heavy accumulation of worms [5][6][7]. Nevertheless, the main clinical finding related to Parascaris spp. infection is small intestinal impaction [6,7].
Typically, ascariosis is defined as an infection caused by Parascaris equorum; however, in the USA, Switzerland and Sweden, Parascaris univalens has recently been recognized as the dominant species affecting horses [8][9][10]. In any case, the two species are considered morphologically identical and can only be distinguished through karyotyping; P. univalens possesses one pair of germ line chromosomes with respect to the two of P. equorum [9,11]. Additionally, recent mitochondrial genome analysis revealed these two to be very closely related and may represent the same species after all [12].
The life cycle of Parascaris spp. initiates with the ingestion of parasite eggs containing infective L2 larvae (larvae develop within 9-14 days after egg excretion within the faeces of infected horses) by a suitable host [7]. After hatching in the host's intestine, larvae embark on a migration following the hepato-tracheal migratory route before finally returning to the small intestine [13,14]. Adult development occurs in the jejunum where females will start laying eggs within about 10-15 weeks of infection [5,15,16].
Mature Parascaris spp. females are particularly fertile, which can lead to high faecal egg counts (FEC) and significant soil and pasture contamination (requiring adequate management), even in the case of limited parasite burden [17,18]. Overall, a single infected horse has been reported to be able to output upwards of 50 million eggs per day [19]. Furthermore, eggs of Parascaris spp. can survive in the external environment (pastures, paddocks, boxes, etc.) in excess of 1 year [13,18,20].
Foal ascariosis control programs are based on yearround monthly rotational anthelmintic treatment with benzimidazole (e.g. fenbendazole-FBZ), tetrahydropyrimidine (e.g. pyrantel-PYR) and macrocyclic lactones (e.g. ivermectin-IVM and moxidectin-MOX) [21]. In Italy, the treatment of foals is predominantly carried out using IVM and PYR starting from 2 months of age and is repeated every 2 months up to 1 year of age [22].
In Parascaris spp., anthelmintic resistance (AR) to macrocyclic lactones, probably due to intensive use of these drugs, has been widely reported since 2002 [23][24][25]. Additionally, recent reports have suggested AR to pyrantel salts and benzimidazole drugs to be present as well [1,26,27]. In Italy, AR to PYR and IVM in ascarids in horses has been reported since 2009 [22].
Recently, a survey on the therapeutic plan applied by Italian horse breeders for the control of ascariosis highlighted that anthelmintic treatment is administered without prior faecal examination in 85.3% of cases, and in 57.8% with a dosage based solely on a visual weight estimation of the animal. Furthermore, only 21.3% of the farmers generally removed horse faeces from pastures as part of ascariosis prophylaxis [28].
Despite the large number of horses (154,505 individuals) reared in Italy, to the best of our knowledge, no large-scale epidemiological surveys dealing with ascariosis prevalence in horses are reported in the present literature. Moreover, studies which do graze this subject are mostly related to anthelmintic efficacy trials [22,29].
Against this background, the present survey aims to describe, for the first time, the spread and infestation of Parascaris spp. in a large population of horses throughout Italy using faecal egg counts (FEC), and further to identify risk factors associated with ascarid egg shedding.

Sampling and coprological examination
The study population for this research had been defined in a parallel study evaluating the excretion of gastrointestinal strongyle eggs [30].
A total of 6896 randomly selected horses, ranging from 6 months to 36 years old, were examined through coproscopic analysis for the presence of ascarid eggs between 2015 (n. 4164) and 2016 (n. 2732). Animals from 548 different stables located throughout all regions and islands of Italy were included.
Individual rectal faecal samples collected during routine veterinary examinations were sent, vacuum-packed, in refrigerated containers, to the parasitology laboratory of the Department of Veterinary Medicine of the University of Sassari through an express courier within 3 days after collection.
Each faecal sample was accompanied by a form filled in by the sampling veterinarian with the horse's data including age, sex, housing conditions and stable. Coprological examination was carried out through the modified McMaster method as described by Raynaud [31], using a sodium chloride (NaCl) supersaturated solution (specific gravity = 1.2) for flotation and an egg detection limit of 15 eggs per gram (EPG) of faeces. Parascaris spp. eggs were identified by morphology [7].

Statistical analysis
Data were collected on a spreadsheet (Microsoft Excel ® ) and subsequently processed using Minitab ® ( Results were considered statistically significant at a P-value lower than 0.05 (P < 0.05).
The mean intensity (MI) was calculated as the arithmetic mean of EPG values on the total number of infected horses in their respective category.
Kolmogorov-Smirnov normality test showed the EPG values attained from the examined horses not to be normally distributed (KS = 0.472; P = 0.010).
Data processed according to age groups for 5065 horses are shown in Table 1. Prevalence rates tended to decrease significantly with age (χ 2 = 386.202, df= 6, P < 0.001) as well as EPG means (Kruskal-Wallis test: H = 491.09, P < 0.001), MI of EPG (H = 25.79; P < 0.001) and OR values; the correlation between age and EPG levels was in effect negative (Pearson Correlation r = − 0.079; P < 0.001).
Proportionally, 80.0% of the recorded Parascaris spp. eggs were found in 49 horses, representing 0.7% of the total examined animals and having EPG values between 750 and 19,065. Moreover, 48.5% (95% CI 0.36-0.63) of these horses were younger than 1 year of age.
Analysis of data based on sex (on a subset of 5137 animals: 2366 females and 2771 males) did not show any significant differences (χ 2 = 0.1663; P = 0.683) ( Table 2). Neither did Mann-Whitney comparison between EPG means (U = 6,088,128; P = 0.851) and MI EPG (U = 20,702; P = 0.074) show significantly different values in females compared to males.
Results obtained through stratification based on the geographic provenance (Table 2) showed significant differences between the prevalence rates for the three different areas, increasing from south to north (χ 2 = 14.155; df = 2; P = 0.0008). Non-parametric Kruskal-Wallis test showed no significant differences with respect to the EPG means (H = 5.27; P = 0.072) and EPG MI (H = 1.79; P = 0.617) in the three investigated areas.
Data stratification related to 5696 horses based on housing type highlighted significant differences in Parascaris spp. prevalence (χ 2 = 23.119; df = 2; P < 0.001), with the highest prevalence rates found in horses raised   Table 2.
Data processed according to EPG classes are shown in Table 3. Prevalence rates as well as OR values tended to decrease significantly in the highest EPG levels (χ 2 = 16,231.2; df = 5; P < 0.0001).

Discussion
The present study reports, for the first time, results of an epidemiological survey on Parascaris spp. eggs in Italian horses, including over 6000 animals from all over the country. More than one-third of the examined stables (38.5%) tested positive for Parascaris spp. eggs, which is lower than the prevalence reported in the literature for Finland (47%) [32], the UK (50%) [33] and Kentucky (86%) [34]. The overall Parascaris spp. prevalence (6.3%) found in horses screened in this survey is lower than those reported in similar studies carried out in other countries [1,3,32,33,[35][36][37][38], but higher than that reported in Germany [39], as shown in Table 4. However, the prevalence of Parascaris spp. eggs found in foals (36.7%), which allows for a better comparison of the spread of infestation of these parasites, aligns with the results of a previous study carried out in the UK (38%) [33]. This being said, the prevalence rate of Parascaris spp. in yearlings reported in the present study (17.3%) is higher than the 4% reported by Relf et al. [33]. Furthermore, the prevalence of egg shedding reported in foals in our survey is higher than that reported in Germany (2.9% in horses up to 2 years of age) [39] where the parasitic pressure seems to be particularly low compared to other studies, including this one; this is probably due to the management of outdoor conditions with particular regard to the horse pasture safety and hygiene management in a broad sense [40]. Considerably higher prevalence of egg shedding was found in Normandy, France (30.5% with EPG means of 388) [3] and Finland (50% with EPG means of 360) [2]. Lastly, a study carried out on horses up to 2 years of age in Cuba reported a prevalence of Parascaris spp. of 26.7% [38], while the prevalence found in our study was reported to be 17.7% for the same age category.
Unfortunately, further direct comparison between the present and other studies was not possible due to a lack of data uniformity.
Considering the Parascaris spp. mean FEC value of 209 EPG reported in the UK for foals younger than 1 year [33], a much higher value was found in Italy in the present study (EPG mean 546). In accordance with the results reported by Relf et al. [33], the present EPG mean does decrease with increasing age. Regardless, despite this decreasing trend, the recorded MI in older horses should not be underestimated as this could represent a significant source of environmental contamination and may pose a risk to younger animals. Especially foals within the youngest age group could be at risk as they are likely to ingest ascarid eggs while suckling the mare and exploring their immediate environs [13]. Besides, ascaridiosis is occasionally diagnosed in immunocompetent adult horses as well, even if such infections are typically observed on properties where foals also reside and do show overall low FEC values (< 50 EPG) [41].
In any case, prevalence rates and EPG values reported in this survey agree with previous studies in which ascarids are reported to be more common in horses younger than 2 years [42,43]. Indeed, Parascaris spp. are widely recognized as the most common and pathogenic parasite in foals and yearlings [15,25]. Most likely, this is due to the significant age-dependent and active immunity of foals, which seems to develop at around 6 months of age [19]. In fact, a bimodal pattern of Parascaris spp.  egg shedding has been reported with a primary peak at 3-4 months followed by a secondary more contained peak at 8-10 months of age [44]. Despite the statistically significant differences between the overall prevalence rates of Parascaris spp. eggs recorded in 2015 and 2016 (6.8% in 2015 vs 5.6% in 2016; χ 2 = 4.2412; P = 0.0394), environmental contamination levels seem to be uniform during these two periods as no significant differences in mean EPG and MI values (P < 0.05) could be pointed out. The lower prevalence recorded in 2016 (5.6% vs. 6.8% in 2015) could be explained through differences in climatic conditions in Italy observed between the two years. In 2016, a severe drought was recorded [45,46], possibly leading to lower pasture and paddock ascarid egg loads [43].
It is important to note that the proportion of the animals found to be shedding 80.0% of the total eggs in this research (0.7%) is lower than those reported in other countries [33]. Regardless, such Parascaris spp. "high shedders" do not have the same epidemiological importance as those shedding strongyle eggs [47]. In fact, while faecal examination is adequate for the detection of ascarid eggs shedding in general, the magnitude of quantitative egg counts is not correlated to the size of the worm burden [47,48].
Unfortunately, selective treatment strategies widely recommended for strongyle control in adult horses, with treatments based on FEC, cannot be applied in juvenile horses. In these animals (foals), which are all considered susceptible and more prone to ascarid infections, an agedefined approach is recommended [13]. At that stage, egg counts merely reflect the foal's age rather than the actual infection status. Furthermore, leaving a proportion of foals on a given farm untreated is risky and ill-advised as cases of ascarid-associated colic most often occur in previously untreated foals between 4 and 7 months of age [7].
Regarding the seasonal data, in line with previous studies carried out in Europe [32,39] and the USA [15], no statistically significant differences between the prevalence rates as well as the EPG means were found. Absence of seasonality of the ascarid burden in horses from Italy could be related to the high resistance of the infective eggs in the environment [18,20] and the Mediterranean climate characterizing Italy. Indeed, significant variations in seasonal trends of ascariosis have been reported in horses raised in more arid climates such as Saudi Arabia [49].
In line with previous studies, no significant differences were found between the prevalence rates, EPG means and MI between sexes [37,[50][51][52]. The vast majority of positive animals in this study consisted of young horses managed in a uniform manner, independently of their respective sex (fillies or colts). Therefore, any significant correlation between gender and prevalence and intensity of infection reported by Imani-Baran et al. [36] is, as stated by Relf et al. [33], probably due to variations in management strategies rather than differences in susceptibility or receptivity between fillies and colts.
Results regarding the animals' housing suggest keeping animals outdoors (compared to horses being kept in stables) to be an important risk factor for the occurrence of ascariosis, as previously reported [2,33,38]. In this regard, hygienic management of manure, which is applied on a daily basis for stabled horses, may explain the reduced ascarid burden reported in the literature [2]. However, our results point to stabled horses having higher MI and mean EPG values (506.4 EPG) and thus requiring appropriate manure management protocols to reduce environmental contamination of confined spaces such as boxes.
Although very little is known regarding effective procedures for the reduction of Parascaris spp. pressure at present, management of pastures, paddocks and stalls can play a critical role in ascariosis epidemiology. Furthermore, conditions required to reduce ascarid contamination may be substantially different from those known to be effective against strongyles [7]. Next, daily management of the manure in boxes where foals and mares are kept year-round could prove an effective control strategy considering the resistant nature of ascarid eggs. Lastly, little is known regarding which molecules (and their relative dosage) may be appropriate for the safe removal or inactivation of equine ascarid eggs from the environment [7,13].

Conclusions
In conclusion, results of the present survey show, despite the lower Parascaris spp. prevalence found compared to other European countries, ascariosis to represent a health challenge for horses reared in Italy, especially young animals. Furthermore, the relevance of the MI attained from horses over 6 years of age should not be underestimated as these animals represent a possible source of continuous re-infestation responsible for significant environmental contamination and horizontal transmission. Thus, these horses can be considered "egg shedders", posing a severe health challenge for young animals. Finally, housing type (specifically outdoor rearing) and geographical location should be implemented as risk factors within Parascaris spp. monitoring and control programs for Italian horses, with a specific focus on reduction of risk exposure.