Horse and donkey parasitology: differences and analogies for a correct diagnostic and management of major helminth infections

Abstract

In June 2022, at the XXXII Conference of the Italian Society of Parasitology, the parallels of the main endoparasitic infections of horses and donkeys were discussed. Although these 2 species are genetically different, they can be challenged by a similar range of parasites (i.e. small and large strongyles, and Parascaris spp.). Although equids can demonstrate some level of resilience to parasites, they have quite distinct helminth biodiversity, distribution and intensity among different geographical locations and breeds. Heavily infected donkeys may show fewer clinical signs than horses. Although parasite control is primarily provided to horses, we consider that there may be a risk of drug-resistance parasitic infection through passive infection in donkeys when sharing the same pasture areas. Knowing the possible lack of drug efficacy (<90 or 80%), it is advocated the use of selective treatment for both species based on fecal egg counts. Adult horses should receive treatment when the threshold exceeds 200–500 eggs per gram (EPG) of small strongyles. Moreover, considering that there are no precise indications in donkeys, a value >300 EPG may be a safe recommendation. We have highlighted the main points of the discussion including the dynamics of helminth infections between the 2 species.

Introduction: early data and similarities

Equids (horses, donkeys, mules and hinnies) originated about 55 million years ago in North America from a browsing species named Hyracotherium (Librado and Orlando, 2021), which had the size of a large dog. All living species of equids belong to the genus Equus, composed of 2 lineages, caballine and non-caballine animals, that are split into 3 phylogenetic clades. The domestic horse (Equus ferus caballus) and the Przewalski's horse (Equus ferus przewalskii) belong to the first clade (caballine horses), while zebras and wild asses belong to the other 2 clades (non-caballine horses) (Cucchi et al., 2017). The modern horse has been intensely raised and selected (kinship) for their athletic potential focusing on morphological parameters of body weight (BW), withers height and also sports performance (Brown-Douglas et al., 2009; Schrurs et al., 2022; Dall'Anese et al., 2023).

It is commonly believed that donkeys are short horses with long ears; however, these 2 animal species are different not only in their physical features and behaviour but also in their genetic and physiological characteristics (Lizzaraga et al., 2004). The chromosome number of horses and donkeys is 64 and 62, respectively, making their hybrids (mules and hinnies/asses) infertile animals. There are approximately 175 different breeds of donkeys worldwide (DAD-IS, 2017; https://www.fao.org/dad-is), characterized by their ability to survive in mountainous and semi-arid environments with scarce water availability (Burden and Thiemann, 2015). Donkeys can survive even having a water loss of 20–30% of their weight (Matthews and van Loon, 2019), whereas horses are less tolerant of water deprivation (Matthews et al., 1997). Furthermore, even with the availability of water, the urine output of donkeys is lower than horses (Grosenbaugh et al., 2011). Contrary to what happens in horses, donkeys have a greater ability to digest fibres of low-nutritional value. These animals are characterized by their lower energy requirement (about 50–75%) than that needed by horses of the same size (Smith and Burden, 2013).

Different responses to pain and fear by donkeys, compared to horses, led to the belief that donkeys could have superior tolerance to distress. For this reason, they are considered stoic animals that do not need regular veterinary care, vaccinations or anthelmintic treatments (Molento and Vilela, 2021). However, donkeys may respond to discomfort and pain more subtly than horses. As an example, sick donkeys (i.e. parasites and bacterial infections) often show dullness and depression but the clinical diagnosis of the disease is performed only at an advanced stage (Burden and Thiemann, 2015).

Parasitic infections are the most important limiting factors for equids’ health and performance, as all common parasites of horses infect donkeys (Matthews and Burden, 2013). In comparison, scientific publications on parasites in horses are 20 times higher than those in donkeys (Molento and Vilela, 2021). Figure 1 shows a map of the main parasite studies only in donkeys with their geographical location. In this review, the differences between the main helminthic diseases affecting donkeys and horses, and the different treatment and management strategies will be examined. Some recommendations will be also highlighted.

Figure 1. Choropleth map of the main epidemiological studies on major helminth infection in donkeys. Numbers in square brackets [*] represent the references reported in Table 1. Africa: Egypt [1–2], Ethiopia [3–38], Kenya [39–40], Morocco [41], Nigeria [42–43], Sudan [44–45], South Africa [46–47], Uganda [48–49]; America: Mexico [50–52]; Asia: India [53–57], Iran [58–62], Iraq [63], Mongolia [64]; Europe: Denmark [65], Germany [66–68], Italy [69–73], Macedonia and Thessalia-Greece [74], Portugal [75–76], Serbia [77], Turkey [78–82], Ukraine [83–84].

Table 1. Epidemiological studies of major helminth infections in donkeys

+, positive animals; [*], see Fig. 1.

Helminth infections in horses and donkeys: head to head!

Horses and donkeys not only share similar parasites but also hold high helminth biodiversity (Gianfaldoni et al., 2020; Sousa et al., 2021). Both species can act as reservoirs for each other; however, in donkeys parasitized with a high helminth burden, clinical signs such as diarrhoea and poor body condition are less common than in horses (Matthews and Burden, 2013). Therefore, donkeys appear to be healthy even in the presence of a high-parasitic load (Maestrini et al., 2020). The prevalence of major helminth infections in donkeys is reported in Table 1.

Intestinal strongyles: epidemiology, diagnostic and innovative research

As in horses and also in donkeys, the most common nematode parasites are intestinal strongyles (large and small strongyles) (Buono et al., 2021; Dias de Castro et al., 2022). In both species, parasitic infections are caused mainly (>90%) by small strongyles (Buono et al., 2021; Jota Baptista et al., 2021; Nielsen et al., 2021). A recent meta-analysis reported that Cylicocyclus nassatus, Cylicostephanus longibursatus and Cyathostomum catinatum represent about 55% of the specimens in horses (Bellaw and Nielsen, 2020). The most common genera for donkeys were Cylicostephanus spp. and Cylicocyclus spp. (Maestrini et al., 2020; Perrucci et al., 2021).

In horses (Nielsen et al., 2021) and donkeys (Perrucci et al., 2021), small strongyle infections (cyathostominosis) are often asymptomatic. However, cyathostomins can cause colic, reduced nutrient absorption, diarrhoea and weight loss, and in some cases intestinal infarction and death in horses (Molento, 2005; Steuer et al., 2018; Walshe et al., 2021). In donkeys, clinical signs due to parasitic infections are somewhat rare (Getachew, 2006; Burden et al., 2010). It is known that a high-parasitic fecal egg count (FEC) (>1000 eggs per gram – EPG) may cause quantitative and qualitative milk reduction in asymptomatic lactating jennies (Perrucci et al., 2021). Small strongyles can be responsible for a clinical manifestation known as larval cyathostominosis in horses. This demonstration is caused by the synchronous emergence of the encysted larvae from the mucosa of the colon and caecum causing severe typhlocolitis. Although it is quite uncommon in both animal species, in the horse, the fatality rate of this syndrome can reach 50% (Love et al., 1999). Even though larval cyathostominosis has been reported in working donkeys (Oryan et al., 2015), the fatality rate of this syndrome is not known for the species. Matthews and Burden (2013) consider that the fatality would be lower than that for horses. Although horse foals (<1 year), yearlings (1–2 years) and young horses (3–5 years) are responsible for the majority of intestinal strongyle egg excretion (Lester et al., 2018; Scala et al., 2020), in donkeys this phenomenon still needs to be determined. Until recently, no differences in parasite FEC have been observed among age categories (da Costa et al., 2018; Maestrini et al., 2020; Buono et al., 2021). Moreover, it has been reported that intestinal strongyle FEC is higher in donkeys than in horses (Mezgebu et al., 2013). This trend is not quite implicit but perhaps it could be due to different reasons, such as the lower frequency of anthelmintic treatment in donkeys than in horses (Buono et al., 2021). Another interesting observation is the smaller amount of feces produced by donkeys when compared to an average-sized horse. Assuming a similar infection density, horses that produce a higher amount of feces (15–20 kg day⁻¹) than donkeys (6–10 kg day⁻¹) would have a dilution of eggs on EPG, showing a lower FEC than donkeys (M. Molento, personal observation). More data need to be gathered (i.e. performance, EPG, necropsy) to demonstrate this biological condition.

Large strongyles encompass Strongylus spp., Triodontophorus spp., Craterostomum acuticaudatum, Oesophagodontus robustus and Bidentostomum ivaschkini (Lichtenfels et al., 2008). Of these, the most pathogenic are those belonging to genus Strongylus (Cav et al., 2013). Three species of Strongylus spp. infect horses and donkeys (Strongylus vulgaris, Strongylus equinus and Strongylus edentatus). In donkeys, Strongylus asini has also been reported, whereas horses are less susceptible to this nematode (Malan et al., 1982). Strongylus asini is morphologically more similar to S. vulgaris than S. equinus and S. edentatus; however, the internal transcribed spacer-2 sequence of S. asini proved to be more similar to S. equinus and S. edentatus (Hung et al., 1996). In donkeys and zebras, S. asini develops in the lumen of the portal vein, having a comparable life cycle to S. vulgaris (Malan et al., 1982). Generally, large strongyles are less abundant than small strongyles in horses and donkeys and these parasites are less common in farms that make constant use of macrocyclic lactones (MLs) [i.e. ivermectin (IVM), abamectin, moxidectin (MOX)]. It has been reported that the prevalence of S. vulgaris may increase if selective therapy is adopted in horses (Tydén et al., 2019) and in donkeys (Sousa et al., 2021).

In horses, the overwhelming majority of intestinal strongyle egg excretion is concentrated in certain animals. In general, 15–30% of adult horses shed approximately 80% of eggs (80:20 distribution rule) (Tzelos and Matthews, 2016; Lester et al., 2018), and this pattern is known as overdispersion. In donkeys, this behaviour has been less investigated and the levels of egg excretion are quite different, as about 40% of adult donkeys can shed approximately 80% of eggs, suggesting an 80:40 distribution rule (Buono et al., 2021). These differences in the overdispersion of intestinal strongyle eggs between horses and donkeys have important practical implications. In horses, the anthelmintic treatment of 20% of the animals is adequate to limit pasture contamination, whereas in donkeys the number of treated animals to obtain the same result would be considerably higher (Buono et al., 2021).

As selective therapy and other on-farm management (i.e. reproduction) focuses on individual animal performance, it has been shown that invasion of the intestinal mucosa by parasites activates a defensive mechanism involving the solute-like carrier family 11a1 (SLC11a1) gene, also known as NRAMP1 (Pires et al., 2021). The authors were looking to determine the DNA methylation profile of this gene in cyathostomin-infected horses correlating with FEC. First, they have shown that in the core of this gene, there were 2 cytosines adjacent to guanine (CpG) islands. The data presented a positive epigenetic correlation between the hypermethylation of island 2 of CpG of the gene and FEC. This information can help explain the differences in FEC detected among animals raised under similar conditions. Further research is being undertaken, looking to elucidate the host-specific aspects and the involvement of certain genes across different horse categories (foals, yearlings and adult horses) that may be related to host resilience to parasitic infections. In the future, epigenetic processes shall be used as biomarkers to identify and target animals for anthelmintic treatments, as well as other breeding purposes (Pires et al., 2021). Epigenetic studies are still rare for horses and no such data have been produced for donkeys. As seen above, cyathostomin infections may be measured by individual FEC to be used in selective anthelmintic therapy. The difference in FEC is a phenotypic condition that might be explained by the genetic effects of horses. A new research approach was used by Dias de Castro et al. (2022) that focused on genome-wide association studies (GWASs) using the Illumina Equine 70K BeadChip to correlate genomic variants and FEC of 90 thoroughbred horses. The GWAS used a panel containing 65 157 single-nucleotide polymorphism (SNP) markers. The analysis revealed 33, 21, 30, 21 and 19 genes related to FEC, packed cell volume, eosinophils, neutrophils and lymphocyte count, respectively. The data demonstrate a good correlation between important phenotypic health traits and the potential-specific SNP markers.

Molento and Vilela (2021) have proposed a susceptible–infected–recovered (SIR) predicted model for cyathostomins under limited (low host infection rate), moderate and severe (high host infection rate) infectivity scenarios for donkeys. The process data confirmed that once the infection level is low, disease recovery would be much faster than at the high-risk level. The larval numbers on pasture, parasite challenge and development in the host, parasite lifespan and the return of host susceptibility shall be considered when interpreting the complexity of the host–parasite model. The SIR model simulations should be taken into consideration when proposing herd management and health control programmes for donkeys and horses in distinct climatic zones (Molento and Vilela, 2021).

Considering the widespread anthelmintic resistance (AR), the use of selective therapy has been advocated to control parasitic infections, avoiding parasite selection. In horses, a cut-off of 200–500 EPG has been adopted (Nielsen et al., 2014), whereas in donkeys there are no specific indications, and a value of 300 EPG has been suggested (Matthews and Burden, 2013). However, considering that donkeys with >1000 EPG are often asymptomatic this safe FEC limit could be increased. As drug treatment is a human-made perturbation, the problem is that the lack of efficacy maintains the parasitic infection, increasing the risk of heavy parasitic infections of equids.

The diagnosis of intestinal strongyles is routinely based on copromicroscopic examination (Dias de Castro et al., 2017) and fecal culture for larval identification (Bevilaqua et al., 1993; Lichtenfels et al., 2008; Madeira de Carvalho et al., 2008; Santos et al., 2016, 2018) to differentiate between large and small strongyles. However, larval cultures can result in false negative for S. vulgaris but real-time polymerase chain reaction (PCR) (Nielsen et al., 2008), conventional PCR and other PCR-based methods (Gasser et al., 1996; Hung et al., 1999) are available for S. vulgaris diagnosis in horses and donkeys (AbouLaila et al., 2020).

Parascaris spp.

Generally, equine ascarids refer to only 1 species: Parascaris equorum. However, in the literature other 2 species have been described: Parascaris univalens and Parascaris trivalens. These parasite species differ in having 1 (P. univalens), 2 (Parascaris bivalens also named P. equorum) (Goday and Pimpinelli, 1986) or 3 (P. trivalens) pairs of chromosomes (Li, 1937). Hybrids between P. univalens and P. equorum have been described; however, they are sterile (Goday and Pimpinelli, 1986). Parascaris univalens and P. equorum are quite similar morphologically and they differ only in their spicula, which appear distally truncated in P. univalens and rounded in P. equorum respectively (Biocca et al., 1978). Different ascarid populations were karyotyped and identified as P. univalens suggesting that this ascarid species and not P. equorum is the predominant one in horses (Martin et al., 2018, 2021a). There are few phylogenetic analyses for donkeys; however, a recent study on mitochondrial genes COX1 and NADH1 showed that Parascaris spp. isolated from donkeys formed a distinct clade compared to those collected from mountain zebras and domestic horses (Peng et al., 2019). Furthermore, a recent whole-genome analysis performed in horses, donkeys and zebras showed that P. univalens found in horses belong to distinct clades than those reported in donkeys and zebras (Han et al., 2022).

Parascaris spp. in horses represents the most important parasite infecting young animals, causing coughing, nasal discharge, lethargy, poor appetite, diarrhoea and colic (Clayton and Duncan, 1978). In working equids, poor body condition has been associated with ascarid infection (Getachew et al., 2010; Seyoum et al., 2015). However, horse foals and donkeys bred under optimal farm management programmes may not show this pattern (Bellaw et al., 2016; Buono et al., 2021; Nielsen et al., 2021). In horses, the most important ongoing complication of this parasitosis is small intestine impaction and intussusception, which often requires surgery with a poor prognosis (Tatz et al., 2012), but there is no evidence of clinical complications in donkeys.

In horses, ascarid eggs are excreted mainly by foals and yearlings (<1.4 years old) (Hautala et al., 2019; Scala et al., 2021), whereas in donkeys a high prevalence of Parascaris spp. can be seen regardless of the age of the animals (Getachew et al., 2008b, 2010). These differences could be justified by the poor farming conditions of the donkeys that may not have developed an acceptable degree of immunity, associated with the lack of suitable nutrition (Getachew et al., 2010). A recent paper showed that even if ascarid eggs were reported in all age groups, a significantly higher prevalence was found in younger donkeys confirming that, as in horses, the main contaminators of roundworm eggs are juvenile animals (Buono et al., 2021).

The most common diagnostic method in horses and donkeys is by FEC. However, it is not possible to find eggs during the prepatent period and FEC may not correlate well with the worm burden (Nielsen et al., 2010). No statistical association has been seen between FEC and adult worms in horses (Fabiani et al., 2016). Transabdominal ultrasound may be used in routine diagnostics, but veterinarians must have sufficient practice to identify adult Parascaris spp. worms in foals (Nielsen et al., 2016).

The control of Parascaris spp. represents a key point in breeding farms and all foals under the age of 6 months should be considered potentially infected and at risk of developing acute clinical signs. Therefore, selective therapy is not recommended for juvenile animals if the stud has a history of Parascaris spp. infection. For this reason, anthelmintic treatments must be administered to horses and donkeys younger than 1 year of age. Moreover, it is important to evaluate the presence of other intestinal parasites (i.e. strongyles, Strongyloides westeri, Anoplocephalidae) and the efficacy of the anthelmintic against Parascaris spp. (Nielsen, 2016a). The widespread resistance of Parascaris spp. and small strongyles towards MLs and tetrahydropyrimidines (THPs)/benzimidazoles (BZDs), respectively, has complicated the pharmacological control of these helminths considering that in case of mixed parasitic infections, the efficacy of any active agents could be reduced (Molento et al., 2008; Morris et al., 2019).

Dictyocaulus arnfieldi

Dictyocaulus arnfieldi is the lungworm parasite of equids that infects the respiratory tract. Animals became infected by ingesting the infective larvae during grazing. The donkeys represent the competent host, and they are permissive of the entire life cycle of this nematode, whereas in horses, D. arnfieldi rarely develops into an adult, although the transmission between horses has been reported (Matthews, 2002).

Donkeys can be infected by a high number of adult helminths without showing clinical signs (Matthews and Burden, 2013) or only slight hyperpnoea and harsh lung sounds (Matthews, 2002). In donkeys, persistent infections are very common and patent infections can persist for at least 5 years. In this regard, donkeys can act as reservoirs and main pasture contaminators, representing an important risk factor for co-grazing horses (Beelitz et al., 1996). Horses may show signs of persistent cough, tachypnoea, chronic pneumonia and pulmonary oedema. Infections can easily develop secondary bacterial infections. In horses, patency is shorter than in donkeys which can range from 6 weeks to 8 months, in foals and older horses, respectively (Matthews, 2002). Furthermore, young horses are at a major risk of infection than adult horses and are also characterized by a higher larval output (Jenkins et al., 2020).

Considering the clinical signs that can occur in horses (light coughing), it is advisable to pay attention to those animals that share the same pasture with donkeys. It has been reported that donkeys co-grazing with horses are less infected than those that do not co-graze. Thus, the diagnosis of donkeys that share pasture with horses is essential for controlling this parasite and for reducing the risk of infection in horses (Buono et al., 2021). In infected donkeys, the diagnosis is performed through the finding of first-stage larvae (L1), whether free or inside the eggs, in the feces by modified Baermann technique (Rode and Jorgensen, 1989). Considering that temperature fluctuations during the first 48 h following fecal collection could adversely affect the recovery of L1, it is mandatory to perform the parasitological examination of fresh samples (Rode and Jorgensen, 1989).

Horse lungworm infections do not often reach patency and fecal diagnosis is more difficult than in donkeys. Trans-tracheal aspirates can be useful for demonstrating the increase in eosinophil numbers and endoscopic examination may reveal the presence of larvae in the airways, lymphoid follicular hyperplasia and exudate in the trachea and bronchioles (Dixon et al., 1995). Furthermore, an enzyme-linked immune sorbent assay (ELISA) test can demonstrate the presence of antibodies from 5 weeks after infection, and it could be useful during high-risk seasonal (late winter) infections (Tagesu, 2018). Resistance to MLs was determined in Dictyocaulus viviparus of cattle (Molento et al., 2006) but no reports have confirmed the expected high efficacy in donkeys and horses.

Anoplocephala spp.

Tapeworm infection represents a serious worldwide parasitic disease of equids caused by the species Anoplocephala perfoliata, Anoplocephala magna that infect horses and donkeys and Paranoplocephala mamillana that occurs only in horses. These tapeworm species differ from each other in size, location and pathogenicity (Nielsen, 2016b). Anoplocephala magna and P. mammillana are less frequent and also have an uncertain or marginal pathogenic role (Gasser et al., 2005). Some biological behaviour of A. perfoliata includes preferential adhesion sites, consisting of the caecum wall and the ileo–caeco–colic ostium. The parasite has the tendency to cluster with numerous specimens in this region causing serious clinical intestinal disorders (Edwards, 1986; Fogarty, 1994; Trotz-Williams et al., 2008). The significant association between heavy parasitic burdens (greater than 100 specimens) of A. perfoliata and both medical (i.e. spasmodic) and surgical colics of the ileo–caecal–colic tract (i.e. ileocaecal, caecal–caecal and caecal–colic intussusceptions, ileal impaction) has been demonstrated (Proudman and Edwards, 1993, 1998; Reinemeyer and Nielsen, 2009; Veronesi et al., 2009; Pavone et al., 2011) in horses. The pathogenesis and the clinical impact of this infection in donkeys are still limited. However, colic might be considered an occasional and exceptional onset of infection in both hosts. Animals in good health can tolerate a high-parasitic load, and tapeworm infections are usually suspected due to vague clinical signs – such as weight loss, the opacity of the coat and generic digestive disorders, such as constipation alternating with moderate diarrhoea.

Anoplocephalidae infections are considered a typical parasitosis of equids on pasture, although infections can occur in stabled animals, through the feeding of forage contaminated with oribatid mites, the intermediate hosts of the life cycle. In most of the developed countries, the prevalence of infection has been increasing over time, due to the absence of efficacy of the most used modern anthelmintics (i.e. IVM and MOX). The parasite also reduces the competitive pressure of further parasites of the digestive tract such as strongyles (Bello and Abell, 1999; Gasser et al., 2005). The worldwide prevalence of infection in horses ranges from 18 and 82% with the highest positivity rates observed in humid and rainy areas that favours the development of the intermediate hosts (i.e. Sweden, UK, Germany, etc.) (Fogarty, 1994). Infection occurs in animals of all ages and patent infections can be described starting between the 16th and 20th weeks of life (Gasser et al., 2005). However, there were some reports showing the highest prevalence and intensity of infection in animals younger than 3 years of age (Campigli et al., 2009). In donkeys, epidemiological studies have reported high prevalences in Africa (>80%) (Matthews and Burden, 2013), and low in Europe (<10%) (Buono et al., 2021).

Tapeworm infection in equids can be detected by direct parasitological methods based on qualitative tools. Differently from the other Cyclophyllidea, the search for proglottids in feces is of scant diagnostic value. The proglottids are rarely detectable since before they are released with feces they are broken down directly in the intestine (Nielsen, 2016b). The traditional techniques of concentration by flotation show an overall poor sensitivity (between 11 and 40%) for A. perfoliata infection. This is due to the inconstant egg output even in massive infestations (>100 specimens), and cannot be considered predictive of colic risk (Williamson et al., 1998; Abbott and Barret, 2008). To improve the sensibility of detection, specific techniques have been developed in horses that can be used also in donkeys, i.e. Proudman's test (flotation concentration test) (Proudman and Edwards, 1992) and the Cornell–Wisconsin test (sediment concentration test) (Egwang and Slocombe, 1982). These techniques can attain sensitivities between 61 and 92% for parasitic loads greater than 20 specimens (Williamson et al., 1998). Immuno-diagnostic assays have also been developed and validated in horses for the detection of antibodies (immunoglobulin G) stimulated by somatic or excretory/secretory antigens of A. perfoliata with a mean sensitivity of 68% and a specificity of 95% (Proudman and Trees, 1996a). A semi-quantitative capture antigen-ELISA test, using a purified 12/13 kDa antigen of A. perfoliata, is available in the UK and USA and can be used both for large-scale epidemiological screening and in the clinic as an early diagnostic test to prevent colic episodes (Proudman and Trees, 1996b). In addition, a commercially available ELISA test has also been validated for the detection of antibodies in saliva with a sensitivity of 83% and a specificity of 85% (Lightbody et al., 2016, 2018). Such assay seems to be more useful than those conducted on sera to evaluate the efficacy of drug treatment, since post-treatment antibody levels in saliva decrease faster than in the blood. A coproantigen test in ELISA was also validated to detect the excreted/secreted proteins of A. perfoliata, showing a sensitivity of 74% and specificity of 92% (Kania and Reinemeyer, 2005) but is not currently available. Neither of these immunological tests has been validated in donkeys but several experiments have been conducted showing good sensibility (Getachew et al., 2012).

Anthelmintics in horses and donkeys

Donkeys are characterized by the greater activity of some cytochrome p450 isoenzymes than horses, giving them a greater ability to metabolize certain drugs (Peck et al., 1997). For this reason, there may be differences in the drugs’ disposition and availability, which may require higher concentrations or shorter dosing intervals than those used in horses to obtain effective drug concentrations for optimum parasite control (Horspool et al., 1994; Mealey et al., 1997; Grosenbaugh et al., 2011).

The most common anthelmintic drugs used in equids are BZDs, THPs, MLs and praziquantel (PZQ) (Gokbulut and McKellar, 2018). Only a few drugs are registered for use in donkeys (none exclusive). Thus, extra-label administration of products registered for horses or ruminants is the norm. Although there is less than a hand-full data on the pharmacokinetics and pharmacodynamics of anthelmintic drugs in donkeys and mules (Gokbulut et al., 2014, 2016a), anthelmintic treatment is performed using the same doses and regimens suggested for horses without considering species, breed, nutritional status or individual (height and weight) differences (Lizzaraga et al., 2004; Veneziano et al., 2011, 2013) leading to a reduction in effectiveness (Molento et al., 2008), and an increased risk of toxic signs due to overdosing (Grosenbaugh et al., 2011). Therefore, there is a great need for more data on the donkey as well as new horse anthelmintic drugs and treatment strategies to support welfare (Senior, 2013).

Tetrahydropyrimidines

Pyrantel (PYR) is the only molecule of its class, licensed for horses and is available as salt PYR pamoate (syn. embonate), insoluble in water and as PYR tartrate (soluble in water). PYR pamoate is available as paste or granule formulations, and it is poorly absorbed in the gastrointestinal (GI) tract, which increases its persistence in the intestine (Bjorn et al., 1996). The mechanism of action of THP is by binding to nicotinic acetylcholine receptors of the nematode muscle cells causing spastic paralysis and subsequent elimination from the host (Martin and Robertson, 2007).

A pharmacokinetic study showed that PYR pamoate administered to horses at 13.2 mg kg⁻¹ BW was poorly absorbed and the plasma concentration of the parent drug was very low (C _max = 0.09 ± 0.02). Moreover, its persistence in feces was 48 h and the highest dry fecal concentration (1.034 mg g⁻¹) was detected at 24 h (Gokbulut et al., 2001a). In donkeys, PYR pamoate paste formulation at 6.94 mg kg⁻¹ BW showed a lower C _max and a smaller area under the curve (AUC) than the granule formulation administered at the same dose (Gokbulut et al., 2014) (Table 2). This difference in plasma levels can be attributed to lower intestinal absorption of the paste rather than granule formulation. The pharmacokinetic parameters of PYR in donkeys are quite different from those reported in horses, showing in the latter a lower concentration and a short mean residence time (MRT) than in donkeys and, consequently a lower bioavailability (Gokbulut et al., 2014). These differences were attributed to a different diet as donkeys were fed with hay while horses were kept in grass pastures. The process promoted a decrease in the gut transit time resulting in a lower bioavailability of PYR in horses (Gokbulut et al., 2001a). Moreover, residues of PYR were found in the feces of donkeys and horses for up to 120 h (Gokbulut et al., 2014) and 48 h (Gokbulut et al., 2001a), respectively.

Table 2. Comparative studies on pharmacokinetic parameters of THPs and BZDs in horses and donkeys

Adapted from Gokbulut and McKellar (2018).

C _max, peak plasma concentration; T _max, time to reach peak plasma concentration; AUC_last, area under the (zero moment) curve; AUMC_last, area under the first moment curve; MRT, mean residence time; T _1/2, terminal half-life; PYR, pyrantel; FBZ, fenbendazole; FBZSO, fenbendazole sulphoxide; FBZSO₂, fenbendazole sulphone; ABZSO, albendazole sulphoxide; ABZSO₂, albendazole sulphone; MBZ, mebendazole.

In equids, PYR is licensed at a dosage of 6.6 mg kg⁻¹ BW (Gokbulut and McKellar, 2018) and it is highly effective against adults of small strongyles, S. vulgaris and Parascaris spp. but shows moderate activity against S. edentatus and Oxyuris equi (Mirck, 1985). Moreover, a high dose, twice the nematocidal dose of PYR (13.2 mg kg⁻¹ BW) was effective in controlling A. perfoliata infection (Slocombe, 1979; Höglund et al., 1998). In donkeys, PYR showed high efficacy against cyathostomins and S. vulgaris (Napoli et al., 2013; Gokbulut et al., 2014; Buono et al., 2018). Although it has been reported that PYR-administered high dose used for intestinal strongyles was effective against tapeworms in horses (Höglund et al., 1998), there are still no studies on the effectiveness of PYR pamoate against cestodes of donkeys.

Benzimidazoles and pro-benzimidazoles

The first anthelmintic drug belonging to the BZDs class licensed in 1961 for horses was thiabendazole (Drudge et al., 1981). BZDs are characterized by poor water solubility and are administered as paste or drench formulations in horses (Gokbulut and McKellar, 2018). BZDs bind to the β-tubulin of microtubules, preventing their polymerization, destroying the cellular structure causing the death of the parasite (Martin, 1997).

Fenbendazole (FBZ) represents the most common BZD used for horses that belong to the methylcarbamate group. The pharmacokinetics of FBZ is different in donkeys and horses and influencing its efficacy. FBZ is poorly absorbed from the GI tract of horses, and this would explain why higher concentrations are needed to be effective against migrating larvae and encysted larval stages of cyathostomins (McKellar et al., 2002). Following oral administration in donkeys, FBZ and its metabolites, FBZ sulphoxide (oxfendazole – FBZSO) and FBZ sulphone (FBZSO₂) were not detected in plasma probably due to the lower absorption and greater fecal excretion when compared to horses (Gokbulut et al., 2006). Furthermore, FBZ showed a longer gut transit time in donkeys than in horses (Gokbulut et al., 2006) (Table 2).

In horses, FBZ is licensed at a dosage of 7.5 mg kg⁻¹ BW and it is effective against adult large and small strongyles, O. equi. FBZ appears to be the best option for controlling P. equorum in foals, characterized by large ascarid burdens (Reinemeyer and Nielsen, 2017). Moreover, in horses, a 5-day regimen of FBZ at 10 mg kg⁻¹ BW was effective for controlling the larval stage of small strongyles in enteric mucosa (Duncan et al., 1998). However, a reduced efficacy against early third-stage larvae (EL3), late L3 (LL3) and L4 were reported probably due to the presence of BZD-resistant cyathostomin populations (Reinemeyer et al., 2015; Bellaw et al., 2018). FBZ is registered for use in donkeys, and it seems to have the same spectra of action as in the horse (Gokbulut and McKellar, 2018).

In donkeys, following oral administration of FBZ, 10 mg kg⁻¹ BW, the drug was not detected in plasma probably due to the lower absorption and greater fecal excretion of the drug faster than observed in horses (Gokbulut et al., 2006). The lack of absorption of FBZ supports its ineffectiveness against D. arnfieldi even when administered at high doses (50 mg kg⁻¹ BW) (Taylor and Craig, 1993).

Mebendazole (MBZ) is an anthelmintic drug belonging to the methylcarbamate group and pharmacokinetic studies have shown that the peak plasma concentration and AUC were lower than those of albendazole (ABZ); however, T _1/2 and MRT were longer than FBZSO and ABZ when administered at 10 mg kg⁻¹ BW (Gokbulut et al., 2006, 2016a) (Table 2).

The suggested dose of MBZ is 5–10 mg kg⁻¹ BW for horses (McKellar and Scott, 1990), and when administered at 8.8 mg kg⁻¹ BW it was effective against large and small strongyles (Colglazier et al., 1977). In donkeys, MBZ oral paste administered at 10 and 20 mg kg⁻¹ BW was effective against cyathostomins. Moreover, at 10 mg kg⁻¹ BW, there was no residue found in the milk of donkeys, apart from what was observed when MBZ was administered at 20 mg kg⁻¹ BW. The data suggest that MBZ at 10 mg kg⁻¹ BW proved to be effective for controlling cyathostomins in donkeys and its milk-withdrawal period is zero (Gokbulut et al., 2016a). Furthermore, MBZ administered at 20 mg kg⁻¹ BW for 5 consecutive days was effective in treating D. arnfieldi infection in donkeys (McKellar and Scott, 1990).

ABZ belongs to the methylcarbamate group, and it is recommended orally in horses at a dosage of 5 mg kg⁻¹ BW. The pharmacokinetics parameters of ABZ and its metabolites (albendazole sulphoxide – ABZSO and albendazole sulphone – ABZSO₂) are shown in Table 2. In experimentally infected ponies, ABZ administered at 50 mg kg⁻¹ BW twice a day for 2 days was effective in controlling S. vulgaris larvae in the cranial mesenteric artery showing few toxic signs in 3 of 11 ponies (Georgi et al., 1980). Furthermore, when administered at 50 mg kg⁻¹ BW twice a day for 4 days and at 25 mg kg⁻¹ BW 3 times a day for 5 days resulted in a faster larval kill but more toxic symptoms and death occurred in 3 of 6 ponies (Georgi et al., 1980). An extra-label pellet formulation of ABZ licensed for ruminants and administered to horses at a dose of 7.5 mg kg⁻¹ BW showed a reduced efficacy against small strongyles. However, considering that there are no precise indications on the evaluation of the formulation used, it is not possible to state the certain presence of AR (Salas-Romero et al., 2017). ABZ suspension (25 mg mL⁻¹) was administered in a single oral dose at 10 mg kg⁻¹ BW and 2 oral doses 14 days apart at 10 mg kg⁻¹ BW in donkeys and both doses showed high efficacy against adult stages of large and small strongyles until the end of the study period (Imam et al., 2010).

Macrocyclic lactones

MLs (avermectins and milbemycins) are a class of natural and semisynthetic drugs of which IVM, MOX, doramectin (DRM) and eprinomectin (EPM) are the most commonly administered in equids (Gokbulut and McKellar, 2018). MLs are characterized by a high activity against endo- (i.e. nematodes) and ectoparasites (i.e. mites, flies, lice) in humans and animals and thus are also defined as endectocides (Gokbulut and McKellar, 2018). Apart from that, although IVM and MOX have a similar mechanism of action by binding to glutamate and gammabutyric acid-gated chloride channels, these drugs have profound pharmacological differences (Dent et al., 1997; Feng et al., 2002) and must be further studied in donkeys.

In horses, the kinetic of IVM was evaluated in several studies showing significant differences probably due to the distinct methods of application. A larger AUC and a longer MRT in donkeys than in horses were reported (Gokbulut et al., 2005). These data suggest that in the GI tract of donkeys, IVM had a longer persistence and greater absorption. However, Marriner et al. (1987) reported a larger AUC and a higher C _max in horses than in donkeys and these differences could be due also to diet, breed and anatomical differences between horses and donkeys (Table 3). No pharmacokinetic data of MOX are available in donkeys.

Table 3. Comparative studies on pharmacokinetic parameters of MLs in horses and donkeys

Adapted from Gokbulut and McKellar (2018).

C _max, peak plasma concentration; T _max, time to reach peak plasma concentration; AUC_last, area under the (zero moment) curve from time 0 to the last detectable concentration; MRT, mean residence time; T _1/2, terminal half-life; P.O., oral route/per os; T., topical.

In horses, IVM and MOX are registered at 200 and 400 μg kg⁻¹ BW, respectively. Both IVM and MOX, only when used orally in combination with PZQ, are active against tapeworms (Gokbulut and McKellar, 2018). Although MLs are highly effective in controlling intestinal strongyles they are not effective to control roundworms (Veronesi et al., 2010). In donkeys, off-label IVM administered at 200 μg kg⁻¹ BW was highly effective for controlling small strongyles (Papini et al., 2020). Even though IVM (200 μg kg⁻¹ BW) and MOX (400 μg kg⁻¹ BW) were effective for controlling intestinal strongyle infection 14 days post-treatment, a shorter egg reappearance period (ERP) was reported for both active drugs in Italy (Buono et al., 2018).

IVM and MOX administered at 200 and 400 μg kg⁻¹ BW, respectively, are the drugs of choice for the treatment of D. arnfieldi in horses and donkeys (Lyons et al., 1985; Coles et al., 1998; Matthews and Burden, 2013).

DRM following oral administration at 200 μg kg⁻¹ BW showed a high persistence and bioavailability than IVM (Gokbulut et al., 2005) in donkeys, and it also showed a larger AUC and a longer MRT than in horses (Table 3). Furthermore, in horses, DRM administered orally at 200 μg kg⁻¹ BW showed a faster absorption than the injectable 1% formulation administered at the same dose by intramuscular route (Pérez et al., 2010).

In horses, DRM administered both orally and intramuscularly at 200 μg kg⁻¹ BW was effective for controlling intestinal strongyles (Pérez et al., 2010). In naturally infected horses, DRM following oral administration at 200 μg kg⁻¹ BW was effective against small strongyles with an ERP of 10 weeks (Cirak et al., 2007). In donkeys, DRM administered at 1 mL per 50 kg by subcutaneous injection was effective for controlling small strongyles until day 28 post-treatment (Elmeligy et al., 2021).

EPM is the last licensed drug belonging to avermectins (Gokbulut and McKellar, 2018) that is 2 or 3 times more effective than IVM and yields zero milk-withdrawal time. Therefore, EPM can be used safely in lactating animals (Shoop et al., 1996). The pharmacokinetic parameters of EPM in horses and donkeys are shown in Table 3 and the low disposition rate of EPM in the milk of animals allows the use of the drug in milk-producing horses (Gokbulut et al., 2016b) and donkeys (Gokbulut et al., 2011). In donkeys, EPM following pour-on administration (bovine dose – 500 μg kg⁻¹ BW) was effective in eliminating D. arnfieldi larvae for 28 days (Veneziano et al., 2011). Moreover, following topical administration, EPM showed high efficacy against large and small strongyles (Gokbulut et al., 2011).

Praziquantel

PZQ is an isoquinoline and it is licensed both in human and animal medicine for controlling cestodes and trematodes (Gokbulut and McKellar, 2018). PZQ acts by binding to the parasites’ glutathione S-transferase (McTigue et al., 1995), altering the concentration of intracellular calcium, causing muscle contractions and tegument rupture (Harnett, 1988). There are no data on the pharmacokinetics of PZQ in horses and donkeys but, it is quickly and widely absorbed following administration in humans (Leopold et al., 1978).

In horses, PZQ is registered at 1.0 mg kg⁻¹ BW and a PZQ paste of 9% was effective for controlling A. perfoliata, A. magna and A. mamillana (Slocombe et al., 2007). Moreover, following PZQ administration, the prevalence of cestodes was reduced by 96% until 10 weeks post-treatment (Lyons et al., 2017). Similarly, PZQ paste administered orally at 1 mg kg⁻¹ BW was effective for controlling A. perfoliata in donkeys (Getachew et al., 2013). In equids PZQ is licensed in association with the MLs (IVM and MOX); however, anecdotal data suggest that PZQ should be poorly tolerated in donkeys (Matthews and Burden, 2013). PYR pamoate paste administered at a dose of 13.2 mg kg⁻¹ BW was safe and highly efficacious for controlling Anoplocephala spp. infection in horses (Marchiondo et al., 2006) and, although there are no safe dosage studies in donkeys, it would be safer to administer PYR (at a double dose of 13.2 mg kg⁻¹ BW) than PZQ for controlling Anoplocephala spp.

AR in equids: horses vs donkeys

Resistance is the ability of a worm population to survive treatments generally effective against the same species and stage of infection (Sangster, 1999). Drug resistance was first reported in horses in the 1960s against phenothiazine in small strongyles (Gibson, 1960). Resistance to anthelmintics persists for many years and is transmitted to parasite populations being a genetic-based trait. In horses, the overuse of anthelmintics has led to the development of resistance, especially against small strongyles and Parascaris spp. (Molento, 2005; Peregrine et al., 2014). The FEC reduction test is the in vivo test for determining the effectiveness of anthelmintic treatment against intestinal strongyles and Parascaris spp. in horses and donkeys and it is based on the percentage of reduction of eggs in the faces after 14 days post-treatment (Nielsen et al., 2019). The first report of cyathostomin resistance to PYR in horses was published in 1996 (Chapman et al., 1996) and nowadays, resistance to this drug class is very common (Zanet et al., 2021; Nielsen, 2022). In horses, resistance to THP was evaluated since 2000 in 37 studies and reported in 34 (92%) (Nielsen, 2022).

Resistance to the BZDs has been commonly reported in the equine industry worldwide (Matthews, 2014) and in wild equids (Kuzmina et al., 2020), showing that resistance against this anthelmintic drug class is not always associated with the intensity of anthelmintic treatments. In horse strongyle infection, resistance to the BZDs has been associated with the polymorphisms of codons 167, 168 and 200 of isotype 1 β-tubulin (Ishii et al., 2017; Özben et al., 2022). In horses, since 2000, AR against BZDs has been evaluated in 58 studies and it was reported in all of them (Nielsen, 2022). Moreover, in horses, multiple resistance to different anthelmintic drug classes has been reported (Flores et al., 2020) and it is quite common to find a population of small strongyles resistant both to BZD and THP (Canever et al., 2013). Another minor mutation was described by Ishii et al. (2017) at codon 172 that deserves further research.

Although MLs are the most commonly administered anthelmintic drugs in horses (Tzelos et al., 2019) as in donkeys (Buono et al., 2021), drug resistance is not so common and it may develop in a more complex way – as more genes are involved. However, in the last few years, several studies have reported a reduced efficacy of MLs against small strongyles in horses, both as a reduced efficacy at 14 days post-treatment and as a shortened ERP (Molento et al., 2008, 2012; Tzelos et al., 2017; Nielsen et al., 2020; Abbas et al., 2021) that it was postulated representing the first sign of emerging resistance (Sangster, 1999). For this reason, in the next few years, an increased numbers of reports of AR to MLs are expected worldwide. A recent study showed that a shortened ERP cannot be explained only by the survival of 4th-stage larvae but probably could be associated also with other factors such as the selection of species or strains that accelerate their life cycle. Thus, a shortened ERP could not indicate the development of drug resistance (Nielsen et al., 2022).

Since 2000, resistance against MLs in horses has been evaluated in 57 studies and reported in 13 (23%) (Nielsen, 2022). In several nematode species, MLs resistance is associated with a group of genes that encode adenosine triphosphate-binding cassette transporters (Tydén et al., 2014; Raza et al., 2015).

In donkeys, drug resistance has not been reported as commonly as in horses and few clinical trials have been performed for evaluating the efficacy of the most common anthelmintic drugs (FBZ, PYR and MLs) (Matthews and Burden, 2013). Resistance to PYR was reported in 2 donkey farms in the UK (Lawson et al., 2015); furthermore, in 1 of these farms, a suspected resistance to MOX was reported 10 years earlier after continuous use of the cattle formulation (Trawford et al., 2005). However, considering that donkeys were treated orally using an injectable formulation licensed for cattle, the data reported for MOX probably did not confirm the presence of AR. Recently, a population of small strongyles resistant to FBZ and PYR was reported in an Italian donkey farm, also associated with a reduction of ERP for IVM and MOX (Buono et al., 2018).

In horses, the first report of MLs resistance in Parascaris spp. was described by Boersema et al. (2002) in the Netherlands. Nowadays, AR to MLs have been reported in 29 out of 29 studies worldwide, while resistance against THP and BZDs was reported in 4 out of 16 and 3 out of 13 studies, respectively (Nielsen, 2022). The sporadic resistance of Parascaris spp. against BZDs and THP should be due to the limited studies on the effectiveness of these anthelmintic drug classes. Thus, drug resistance should be more common than those reported in the literature, as also suggested in some studies (Martin et al., 2018, 2021b; Hautala et al., 2019).

AR of P. univalens against BZDs is not associated with SNP suggesting that the mechanism of AR in this parasite is different from those reported for intestinal strongyles (Martin et al., 2021b). The lack of efficacy of IVM, MOX and PYR has been reported also in donkeys (Matthews and Burden, 2013). Studies on anthelmintic efficacy in donkeys are reported in Table 4.

Table 4. Studies in donkeys reporting anthelmintic efficacy – FECRT (14 days post-treatment)

PYR, pyrantel pamoate; ABZ, albendazole; MBZ, mebendazole; FBZ, fenbendazole; IVM, ivermectin; MOX, moxidectin; EPM, eprinomectin; DRM, doramectin; PZQ, praziquantel; SC, subcutaneous administration; IM, intramuscular administration; inj. for., injectable formulation; PO, per os.

According to American Association of Equine Practitioners Parasite Control Guidelines cut-off (Nielsen et al., 2019), the cut-off values used to interpret the results of FECRT were the following: PYR susceptible (S) >90%, suspected resistance (SR) 85–90%, resistant (R) <85%; FBZ susceptible (S) >95%, suspected resistance (SR) 90–95%, resistant (R) <90%; IVM/MOX susceptible (S) >98%, suspected resistance (SR) 95–98%, resistant (R) <95%.

^a For these anthelmintic drugs, cut-off values are not suggested in AAEP Guidelines.

Conclusions

The host–parasite relationship is a complex process in equids that still needs much attention. Although donkeys and horses are closely related species and share practically the same parasite fauna, they are divergent in their physiological and pharmacological (absorption and distribution of drugs) characteristics. Some features such as breed, geographic location/climate, parasite challenge and intensity, immune response and control methods including chemical use shall be regarded as a priority to help solve important parasitic infections in both species. Target selective treatment can also be adopted, looking mainly for clinical signs, performance (body growth and withers height) and to FEC. Once adopted, the selective strategy may help preserve drug effectiveness and the welfare of both horses and donkeys. Horses and donkeys are ‘different cousins’; for this reason, precise farm management and parasite control programme must take these differences into account.