Prevalence of intestinal parasite infections in stray and farm dogs from Spain

Prevalence of intestinal parasite infections in


Introduction
Dogs are hosts to a large number of gastrointestinal parasites and can shed helminth eggs and protozoan oocysts in their faeces (Balassiano et al., 2009;Traversa et al., 2014). The potential role of dogs as reservoirs for zoonotic parasites (such as Giardia spp., Cryptosporidium spp., Toxocara spp., hookworms -Ancylostoma and Uncinaria-and Echinococcus granulosus) has been recognized as a significant public health problem worldwide (Benito et al., 2006;Traversa et al., 2014;Felsmann et al., 2017;Mateus et al., 2014;Saldanha-Elias et al., 2019). Uncontrolled dog populations, such as stray and farm dogs, have access to populated areas, representing a potential risk to public and animal health (Martínez-Moreno et al., 2007;Liberato et al., 2018), mainly in rural areas where there is a close contact between dogs and humans or livestock (Pierangeli et al., 2007;Soriano et al., 2010;Cardoso et al., 2014). However, there are very few investigations on gastrointestinal parasites present in such outdoor dogs that might also present risk as zoonotic reservoirs for human infections, since they are not routinely tested for parasitic infections. The objective of the present study was to determine the prevalence of intestinal parasites in stray and farm dogs in northern Spain.

Material and Methods
A total of 233 dogs sampled in 2013 from areas located in northern Spain were included in this study: 102 stray dogs from a peri-urban area (La Rioja) and 131 dogs from dairy farms located in one of the main cattle-producing regions in Spain (Lugo, Galicia) ( Figure 1). Stray dogs were euthanised according to stray dog control programs of the Municipality, and necropsies were performed. Faecal samples were collected directly from the rectum of the necropsied stray dogs or from the ground after defecation by farm dogs, and submitted refrigerated (4 °C) to our laboratory facilities (SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Spain). Samples were processed by the Telemann method (Thienpont et al., 1979) followed by sucrose flotation. One wet mount and one flotation slide were observed for each faecal sample. Identification of parasite cysts, oocysts and eggs was performed by morphological observation at 100-400× magnification. Furthermore, to detect Cryptosporidium oocysts, DNA was extracted from faeces (72 stray dogs and 119 farm dog samples) using a method described previously (McLauchlin et al., 1999). Briefly, this method uses a disruption with zirconia beads in the presence of guanidinium thiocyanate, followed by purification with activated silica. Subsequently, faecal DNA samples were analysed using a Cryptosporidium specific PCR as described previously (Xiao et al., 2000). To identify false-positive results, negative control samples (without template) were added to each set of DNA extractions. In farm dogs, data regarding age and housing (free or controlled access to the farm) were collected. Prevalence data were compared by Chi-square test (χ 2 ) at a significance level of 95% (P<0.05). Statistical analysis was carried out using GraphPad Prism 7 v.7.04 (San Diego, CA, USA) software.

Results and Discussion
Microscopically, gastrointestinal parasite forms found in the faeces of the dogs were identified as nematodes (Ancylostomatidae, Toxocara canis, Trichuris spp. and Toxascaris leonina), cestodes (Taeniidae) and protozoa (Cystoisospora spp. and Giardia). Cryptosporidium DNA was not detected in any of the faecal samples analysed by PCR. Table 1 shows the prevalence of the different gastrointestinal parasites found in stray and farm dogs. From the 233 dogs analysed, 63.5% (148) were positive for at least one intestinal parasite, indicating a high degree of intestinal parasitism in these animals. In accordance to our study, similar prevalence rates of intestinal parasites were reported in Spanish rural (52.4%; Ares-Mazas et al., 1987) and shelter dogs (ranging from 61.8 to 71.6%; Martínez-Moreno et al., 2007;Ortuño et al., 2014) and in Portugal (58.8%; Cardoso et al., 2014;Mateus et al., 2014).
In tropical countries such as Brazil, urban stray dogs also showed high prevalence rates of intestinal parasites (e.g. 87.1%; Saldanha-Elias et al., 2019). However, lower prevalence (≤ 25%) were described in other European countries, such as Poland (Felsmann et al., 2017) or Italy Scaramozzino et al., 2018). All these differences may be explained by the climate, dog populations studied, geographical location, sampling protocols, anthelmintic usage or diagnostic techniques employed (Adell-Aledón et al., 2018). The parasites most frequently identified in our study were Ancylostomidae (35.6%; 83/233) and Trichuris (35.2%; 82/233) and co-infections of both parasites were present in 14.1% (33/233). From biological and epidemiological standpoints, a possible association between the presence of Trichuris and hookworms has also been described in infected dogs (Traversa et al., 2014). This fact could be accounted for by the accumulation of infective larvae or eggs in the environment that has a crucial role in the transmission of these dog intestinal nematodes. Likewise, Ancylostomatidae and Trichuris were the parasites more frequently detected in stray and shelter dogs in Italy Scaramozzino et al., 2018), Poland (Felsmann et al., 2017) or Brazil (Saldanha-Elias et al., 2019). In the case of hookworms, the predominant agricultural environment where most of these dogs roam provides protection to larvae from direct sunlight and dehydration, especially in unpaved areas where sanitation processes are ineffective. Moreover, the trans-mammary route is favoured by the lack of controls and treatments in these dog populations . Interestingly, the prevalence in farm dogs with free access to the farm was significantly lower (42.8%; 24/56) than in dogs with controlled access (66.1%; 37/56) (P<0.05), especially for Ancylostomidae (16% vs 50%; P<0.05). This may be explained by the accumulation of faecal material (and therefore potential sources of infection) in a confined area inside the farm, since faeces are not usually collected and disposed of. Controlled-access dogs would have a more frequent contact with this faeces accumulation than those that freely enter and exit the farm.
Moreover, this potential environmental contamination of hookworms eggs (e.g. Ancylostoma caninum and Uncinaria stenocephala in Mediterranean area) poses a high risk of zoonotic human infection, as these species are known to cause cutaneous larva migrans syndrome. Although identification of Ancylostomidae species was not carried out, the high prevalence observed here points out that the presence of stray or farm dogs would lead to an increased exposure of humans to this zoonosis. In line with this, other potential zoonotic parasites were detected in our study, although the prevalence was low for Giardia (3%), Taenidae (3%; which could potentially be Echinococcus granulosus, the causative agent of hydatidosis) and T. canis (11.6%, one of the causative agents of visceral larva migrans). In previous studies, canine giardiasis has been reported in a variety of dog populations (owned, sheltered, stray) with prevalence ranging from 1 to 38% in Spain (Dado et al., 2012;Ortuño et al., 2014;Gil et al., 2017) or 25% in Brazil (Saldanha-Elias et al., 2019. Taenidae eggs were scarcely detected. Although we were not able to gather information regarding anti-parasitic treatment records, the low prevalence of cestodes observed in our study could be explained by the prevention campaign against E. granulosus conducted by the Spanish Government and local councils in the last decades, consisting in a bi-annual praziquantel administration (Jiménez et al., 2002). As a matter of fact, in a previous study in the province of Álava (northern Spain), a low prevalence (0.5%, 5/1040) of E. granulosus infection in dogs was also found (Benito et al., 2006). Prevalence rates ranging from 0.2 to 1.3% have also been reported in dogs from La Rioja (Jiménez et al., 2002). T. canis infections are found worldwide (including in the European canine populations) and it is one of the most widespread public health and economically important zoonoses in humans (Schnieder et al., 2011). Toxocara was the second more prevalent parasite detected in our study. However, the actual prevalence could be higher because adult dogs show lower prevalence and worm burdens (Schnieder et al., 2011). In our study, age data were collected for 112 farm dogs. When dogs ≤1 year old were compared to dogs >1 year old, no significant differences were found. Nevertheless, dogs younger than 1 year comprised only 14.3% (16/112) of the studied population. Another important potential zoonotic parasite is Cryptosporidium, which was not detected in this study. We used PCR to detect its presence because this technique allows to detect a low number of parasites in faeces that would otherwise be difficult to detect using coprological examination. Nevertheless, the presence of PCR inhibitors in faecal samples hinders PCR detection. In this sense, we employed a previously described method for nucleic acid extraction (modified Boom's method), which has been demonstrated to avoid co-purification of PCR-inhibitors present in faeces (McLauchlin et al., 1999).
On the other hand, when the different dog populations were compared, the overall prevalence of intestinal parasites was significantly higher in stray dogs (72.5%, 74/102) than in dairy farm dogs (58.8%, 77/131) (P<0.05). Specifically, the presence of Ancylostomatidae, Toxocara, Toxascaris and Taenidae were more often detected in stray dogs (P<0.05). These results show that stray dogs are heavily parasitized, which could be related to the uncontrolled access to potentially infection sources and to the absence of anti-parasitic treatments. This fact is relevant because peri-urban stray dogs can easily spread different gastrointestinal parasites with zoonotic potential posing a high risk for humans in urban areas.
In summary, the high overall prevalence of intestinal parasites found in the present study may account for the little access to veterinary care and high exposure to infection of these dog populations. Environmental contamination by dog faeces is considered a risk factor to public health, as dogs can act as carriers of pathogenic agents transmissible to humans (Martínez-Moreno et al., 2007). Thus, a strict control of at-risk dog populations is crucial in order to limit public health problems derived from parasitic infections. A combination of strategic anthelmintic treatment programs, control of stray and farm dogs, and enhanced owner education and awareness is highly recommended for the control of intestinal parasites in dogs.