Prevalence of Potentially Zoonotic Endoparasites in Domestic Dog Puppies

1. Introduction

Some gastrointestinal parasites are of interest from a One Health perspective because of their veterinary and public health relevance. Visceral Larva Migrans and Cutaneous Larva Migrans caused by Toxocara spp. and Ancylostoma spp., respectively, are examples of such parasites [1,2,3,4]. Helminth eggs and protozoan cysts excreted in the environment by domestic animals should be a matter of concern to their owners due to the importance that animals can have for emotional support and their role in social and physical development of people, particularly children and the elderly [5,6,7,8]. Close co-habitation of humans and domestic animals, which are sometimes view as members of the family, favors zoonotic transmission [9,10,11].

Various parasite species are diagnosed with moderate to high frequency in companion animals, despite the existence of therapeutic and prophylactic measures [8,12]. Diarrhea resulting from parasitic infections and from other causes is one of the most frequent disorders in canine puppies and negatively interferes with their growth [8,13]. Thus, it is essential to prevent or minimize parasite transmission. Screening for the presence of gastrointestinal parasites [2,14] informs treatment options and the adoption of environmental management practices for infection control [10,13,15]. In contrast with surveys which include a wide age range, the aim of our study was to investigate the occurrence of gastrointestinal parasites in domestic dogs up to six months of age.

2. Materials and Methods

2.1. Study Population

The minimum sampling required for the execution of this project, at the 95% confidence level and with absolute precision of 10%, was determined to be 96 samples, based on a prevalence of 50% [16]. Thus, we collected fecal samples from 100 randomly selected dogs, 60 males and 40 females, all domestic and of mixed breed, aged less than six months. Within this age range, 20 animals were one to two months old, 44 three to four months old and 36 five to six months old. Puppies aged between 2 and 3, and 4 and 5 months were not represented in the sample. The study was approved by the animal use ethics committee of the Faculty of Dentistry of Araçatuba - São Paulo, under protocol number FOA-00312-2016.

2.2. Degree of Dehydration and Fecal Consistency

The degree of dehydration was assessed as not apparent, mild, moderate, severe and shock [17]. Fecal consistency was assessed visually and defined as liquefied, pasty, semi-solid and solid, according to Coelho et al., 2012 [18].

2.3. Collection of Fecal Material and Parasite Detection

With the aid of urethral catheter (nº 6 - 8 - 10) and a 10-mL syringe, feces were collected directly from the rectal ampulla. Samples were stored in sterile vials and refrigerated between 4º and 8ºC until processed. Subsequently, Faust’s flotation technique in saturated sodium chloride solution [19] and Willis’ centrifuge-flotation technique in zinc sulfate [20] were applied. The presence of parasites eggs and cysts was also examined by negative malachite green staining [21]. All samples were examined with the three diagnostic techniques.

3. Results

Based on at least one positive test, 34% of dogs were positive for Toxocara spp., 28% for Isospora spp., 22% for Ancylostoma spp. and eight for Giardia spp.. In 21% of the animals mixed infections were detected. One or more parasite species were detected in 62% of dogs with at least one technique. Among the 100 dogs evaluated in the study, with the Willis technique we detected 32% positive animals for Toxocara spp., 19% for Ancylostoma spp. and 16% for Isospora spp., 16% of which presented mixed infections with Ancylostoma spp., Isospora spp. and Toxocara spp. Faust's technique identified 30% of dogs with Toxocara spp., 26% with Isospora spp., 10% with Ancylostoma spp. and eight with Giardia. Based on this technique, 21% of the animals were infected with more than one parasite. For 82% of the samples, Faust and Willis gave concordant results in the sense that both tests were negative or both were positive. Of the 18 (18%) discordant results, 12 were positive according to Willis and negative according to Faust. For 6 samples the results were reversed (Chi-square=39.0, p<0.001). Six of 6 Willis-negative/Faust-positive sample were Faust-positive for Isospora spp., indicating that the Willis technique may underestimate the prevalence of this parasite. Conversely, of the 12 samples positive by Willis and negative by Faust, Ancylostoma spp. was detected by itself or in combination with a second parasite in 10 samples, suggesting that the Faust method may undercount this nematode. Of the 44 double-positive samples, 12 were diagnosed with different parasites or a different combination of parasites where more than one pathogen was detected. The diagnosis for the remaining 32 samples was fully concordant.

Dehydration was not apparent in 50 dogs and the same number was found to be mildly dehydrated. Regarding the consistency of fecal samples, 43 samples were scored as liquefied, 7 as pasty, 8 as semi-solid and 42 as solid. Dogs excreting liquefied and solid feces presented more positive than dogs with pasty and semi-solid feces. In particular, it is important to note that 40 of 53 puppies excreting solid feces were positive for gastrointestinal parasites (Table 1). There was no significant association between fecal consistency and presence of parasite (Fisher’s Exact Test, p=0.8589).

No association between age and parasite prevalence was observed using linear regression (r=0.05, p=0.56). Similarly, sex and infection were not significantly associated. A total of 65% of males and 57.5% of females were positive by at least one diagnostic technique (Chi-square=0.30, p=0.58). Toxocara was more prevalent in the 2 younger age groups (45% and 34%) and equally prevalent as Ancylostoma spp. and Isospora spp. in the oldest group (28%) (Table 2). An opposite trend was observed for Ancylostoma spp. which increases in prevalence with age from 5% to 25% and 28%.

4. Discussion

Our results extend the epidemiological study of common gastrointestinal helminths and protozoa of dogs to puppies up to six months of age. Typically, coproparasitological surveys include animals of any age [2,14,22]. Other studies have reported the occurrence of gastrointestinal parasites, but they differ from this study since they frequently examine stray dogs regardless of age [15,23,24,25].

The main finding of our survey is the frequent detection of Isospora spp., Giardia spp., Toxocara spp. and Ancylostoma spp. in puppies with varied fecal consistencies, including animals showing no symptoms typically associated with intestinal parasites. This observation is relevant to public health as the latter three species are potentially zoonotic [3,4,26,27,28,29]. As puppies are more likely to excrete gastrointestinal parasites [23,28] deworming and other measures to reduce transmission are particularly important to reduce the risk of infection, which can have severe consequences in immunocompromised children and adults. Such measures are also expected to benefit the health of puppies [23,38]. Mixed infections with two, or even three, parasites were relatively common in our survey. This observation emphasizes the need for adequate medications to treat helminth and protozoan co-infections.

The use of two flotation techniques supports the conclusion that both methods have similar sensitivity. This observation is consistent with the fact that both methods concentrate parasite eggs and cysts by flotation on a high-density salt solution. The difference in spp. and Isospora spp. prevalence based on Faust and Willis raises interesting questions about the buoyant properties of these eggs and cysts and may justify the use of both methods where the presence of these parasites is suspected [2,14,30]. This recommendation should be easy to implement as fecal flotation is cheap and easy to perform [14]. Veterinarians should make owners aware of the importance of diagnosing these parasites, particularly given the high prevalence of Toxocara spp. and its potential for zoonotic transmission [11,31].

Malachite green staining did not reveal the presence of Cryptosporidium spp. oocysts. This stain has the advantage of being cheaper and easier to perform than immunological and molecular assays [32], but its disadvantage is low sensitivity, with the possibility of false negative results [33]. The oocysts have small dimensions, being hardly observed in fecal smears after staining, which requires time and experience from the examiner [34].

5. Conclusions

We investigated for the first time the occurrence of gastrointestinal parasites in domestic puppies less than six months of age. The high prevalence of intestinal helminths, and the diagnosis of Toxocara spp. in asymptomatic dogs highlights the risk of zoonotic transmission.