Canine intestinal parasitic infections and soil contamination by Toxocara spp. in selected areas of Sri Lanka.

INTRODUCTION: Contaminated environment with canine parasitic eggs is the main source for toxocariasis and other geohelminth infections of humans. This study aimed to determine the prevalence and associated risk factors for intestinal parasitic infections in dogs and the degree of soil contamination with Toxocara eggs in Kandy and Gampaha districts in Sri Lanka.
METHODS: A total of 188 fecal samples from both stray and domestic owned dogs from Gampaha district and Kandy district were collected. In addition, soil samples were collected from home gardens and public places. Hypochlorite recovery technique and zinc sulfate floatation method were used to concentrate eggs in soil samples.
RESULTS: The prevalence of intestinal parasitic infections of domestic and stray dogs in Gampaha district was 38.2% and 42.9%, respectively, while in Kandy district, it was 41.1% and 50%, respectively. Hookworms were the most common parasitic infection among domestic dogs (18.5%), while Toxocara spp. were the most common among stray dogs (17.2%). Intestinal parasitic infections were significantly more prevalent among female dogs than males. Age, deworming, immunization, immunization time, and living in rural areas were not significantly associated with intestinal parasitic infections in domestic dogs. A total of 4% and 4.5% soil samples were contaminated with Toxocara spp. eggs in Gampaha and Kandy districts, respectively.
CONCLUSIONS: The higher prevalence of intestinal parasitic infections in dogs could be a major public health issue. Implementations of programs to improve the awareness of parasitic infections among pet owners and control strategies need to decrease the risk of infections to both animal and human health. Copyright:

INTRODUCTION

The existence of viable eggs and larvae in superficial layers of soil represents a potential public health hazard. The eggs of geohelminth are extremely resistant to adverse weather conditions.[1] Thus, soil contamination seems to be the most direct indicator of the risk of soil-transmitted geohelminth infection among human.[2]

Toxocariasis is a zoonotic disease caused by the larva of Toxocara canis and Toxocara cati, whose definitive hosts are dogs and cats, respectively.[3] Soil contamination can occur through feces of infected dogs. Adult parasites live in the small intestine of the dogs, producing many eggs, thus contaminating the environment.[4] In urban areas where a relatively large number of dogs share a limited area for their activities and in areas with poor hygiene and pollution, the contamination with eggs of soil in public areas are expected to be high.[35] Soil contamination with Toxocara eggs has been reported in sandpits in public parks, backyards, and gardens from various parts of the world.[67]

Understanding the epidemiology of zoonotic parasitic infections is important for the minimization of risk to humans and dogs. Roaming non immunized dogs are the main vector for toxocariasis in Sri Lanka and the knowledge about prevalence and soil contamination by Toxocara spp. is sparse. Therefore, this study was designed to determine the prevalence, related risk factors of canine intestinal parasitic infections, and the degree of soil contamination with Toxocara ova in selected areas of Sri Lanka.

METHODS

Study area

This study was a cross-sectional study and was conducted in randomly selected urban and rural areas in Gampaha and Kandy districts from May to August 2017. The study sites were selected to represent urban and semi-urban settings from each district.

Geographically, Kandy district is situated in the central part of the island and it is a mountainous region altitude ranging between 300 and 1500 m above sea level. However, Gampaha district is situated in the Western part of the island and it is mainly flat lowlands <30 m above sea level. Gampaha district covers an area of 1387 km2. The climate is hot, humid subtropical climate showed in Gampaha district, and the average annual temperature and rainfall is 27.3°C and 2398 mm, respectively. Kandy district has an area of 1096.3 km2. The average annual temperature and rainfall in Kandy is 24.5°C and 2083 mm, respectively.

Study population

Sample size was calculated using the statistical formula = z2 P (1 − P)/d2 where n = required sample size, P = estimated prevalence, d = 95% confidence level at 95%, and d = margin of error at 5%. The estimated prevalence of canine toxocariasis was taken as 28.8% based on a study conducted by Perera et al. (2013).[8] The calculated minimum sample size was 188. Samples were collected using simple random sampling method.

In this study, dogs were categorized into domestic and stray. Domestic dogs were the ones that were kept under a caretaker and vaccinated against diseases and dewormed. Dogs in streets and parks without an owner, not vaccinated against rabies and not dewormed were considered as stray dogs. Both domestic and stray dogs were randomly selected without considering age and gender. Age approximately <1 year was classified as puppies, and more than 1 year was classified as adults.[9]

Data collection

Fecal sample collection of domestic dogs was carried out by visiting each house individually. The purpose of the study was explained to the dog owners and obtained their consent to participate in the study. A pretested interviewer-administered questionnaire was used to collect data.

Individual records for each dog was made on an information sheet and included the following; age, gender, breed, history of worm treatment (age of starting and frequency of worm treatment), defecation habits (within or outside the premises or in the cage), and method of feces disposal. Of the sheltered dogs, age was calculated by examining teeth. Details on gender and place of inhabitance were recorded during the sample collection from stray dogs. Data collection was not done for the fecal samples collected from the open fields.

Collection of fecal samples

Fresh fecal samples were collected into a clean leak proof wide-mouthed screw-capped container from each dog. Sampling of stray dogs was done in places away from residential areas. Fresh fecal samples were collected from places such as roadsides, playgrounds, and public parks soon after defecation. All the samples brought to the laboratory were stored at 4°C in a refrigerator until they were examined.

Detection of Toxocara eggs in fecal samples

Fecal samples were analyzed using three methods. Wet fecal smears were prepared using Iodine and examined for Toxocara eggs under high power lens (×40) of the microscope. All the wet smear-negative samples were subjected to fecal concentration using the Formal-Ether sedimentation technique. All the procedures were done according to methods described in the manual for a health laboratory published by the World Health Organization, Geneva in 2003.[10] The identification of parasitic ova was based on the morphological features described by Soulsby (1982).[11] The result was considered positive when at least one parasite egg is present on the smear.

Collection of soil samples

Soil samples were collected from home gardens of dog owners, roadsides, play areas of children, and public parks in Gampaha and Kandy districts. Sampling was done from May to August 2017. All the soil samples were collected using cluster random sampling. Soil samples were collected using a 15 cm × 15 cm template and with 2 cm depth inside the template were scraped using a metal hand shovel into a clean plastic bag or bottles. The metal hand shovel was cleaned after each sampling to avoid cross-contamination, using tissue paper. Random samples were collected from places of domestic children play areas and from the backyards. Soil samples were collected from four randomly selected places in a particular area. All those samples were transported in a cool box (at 4°C) to the Department of Parasitology, Faculty of Medicine, University of Peradeniya. Samples were kept at 4°C until examined.

Detection of Toxocara eggs in soil samples

The centrifugal flotation technique described by Kegie (APCO Research Group; 1983), with a slight modification of replacement of 30% sodium hypochlorite with two drops of detergent (“Teepol”) was applied for the analysis of soil samples (Edirisinghe and Weligama, 1997).[12]

One gram of soil from each sample was measured, and two volumes of “Teepol” was added into the centrifuge tube and stirred vigorously and frequently and allowed to stand for 30 min. Solution was diluted in 10–20 parts of water and mixed well. Floating particles were removed and centrifuge the tubes at 2000 rpm for 2 min. The supernatant was discarded and the precipitate was resuspended in floatation fluid (Saturated MgSO4(aq)) (Sugiyama et al., 1958).[13] The solution was recentrifuged at 2000 rpm for 2 min. The flotation fluid was topped up to positive meniscus in the centrifuged tube and the coverslips were superimposed for 5 min. These coverslips were placed on glass slides and were examined for the presence of helminth ova. Each sample was analyzed in triplicate to increase the precision of the study and two consecutive coverslips were prepared to recover the floating eggs.

Slides of each sample were analyzed under an optical microscope at × 10, with confirmation at × 40. The larvae, oocysts, and eggs were identified based on the morphology of each species.

Data analysis

Data were entered into Microsoft Excel 2007 sheet and checked again for the accuracy (compared with questionnaires). Statistical analyses were performed using SPSS version 17 (SPSS, Chicago, IL, USA) statistical package. The descriptive data of continuous variables and proportions of categorical variables were expressed to get a brief understanding of sociodemographic characteristics of the study community.

Chi-square analysis was performed to find the associated risk factors. The presence of Toxocara was the dependent variable and the demographic details such as age, sex, habitat, place of living, presence of other parasitic diseases, and breed were the independent variables to obtain the P value. For the determination of soil transmission, the following equation was used; degree of soil transmission = number of positive samples/total number of samples analyzed × 100%. The differences were considered to be statistically significant when the value of P < 0.05.

Ethical consideration

This study was approved by the Ethical Review Committee, Faculty of Allied Health Sciences, University of Peradeniya, Sri Lanka. Each of the dog owners was informed regarding voluntary participation, and they conversed that they could withdraw from the study at any time. Informed written consents were obtained from dog owners for the participation of their children in the study. All data were kept confidential.

RESULTS

A total of 188 fecal samples (92 from Kandy and 96 from Gampaha) were examined. Of them, 124 fecal samples were collected from domestic pets and 64 were collected from stray dogs. The majority (50) of stray dogs was adults and only 14 were puppies. The age range of the domestic dogs was 3 months to 12 years (mean age = 4.2 years). A large proportion of domestic and sheltered dogs were males (99, 52.6%). Of the total dog population, most were mongrels (153) followed by purebred (35). The breeds of purebred dogs included Rottweiler (12), German Shepard (6), Retriever (4), RidgeBack (3), Boxer (2), Mastiff (2), Lion Shepard (2), Beagle (2), and Doberman pinscher (2). The highest number of dogs was from suburban areas (135, 71.8%) with more or less equal numbers participated from urban and rural areas.

The positivity of intestinal parasitic infections of dogs in Kandy and Gampaha districts were 27.1% and 38%, respectively. Hookworms were the predominant helminth infections in domestic dogs while Toxocara spp. was the most common in stray dogs. Toxocara egg positivity was high in Kandy district (14.6%) compared to Gampaha district (12.5%) [Table 1]. Toxocara egg positivity reported from Kandy district was 15.2% (n = 14) and 12.5% (n = 12) fecal samples positive from Gampaha district. The majority of the stray dogs had Toxocara infection (17.2%). However, there was no statistically significant difference in domestic and stray dogs.

Table 1

Parasitic infections reported from fecal samples of dogs (n=188)

Parasite	Gampaha (n=96), n (%)		Kandy (n=92), n (%)
	Domestic (n=68)	Stray (n=28)	Domestic (n=56)	Stray (n=36)
Hook worms	9 (13.2)	2 (7.1)	14 (25.0)	5 (13.9)
Toxocara spp.	7 (10.3)	5 (17.9)	8 (14.3)	6 (16.7)
Spirocerca spp.	3 (4.4)	4 (14.3)	1 (1.8)	4 (11.1)
Strongyloides spp.	3 (4.4)	0	0	2 (5.5)
Giardia lamblia	2 (2.9)	0	0	0
Entamoeba coli	2 (2.9)	0	0	0
Diphylobothrium spp.	0	1 (3.6)	0	0
Spirometra spp.	0	0	0	1 (2.7)
Total	26 (38.2)	12 (46.2)	23 (41.1)	18 (50.0)

Intestinal parasitic infection was predominantly observed in female domestic dogs. The positivity of intestinal parasitic eggs was higher in puppies than adult dogs. High parasitic infections were reported in dogs living in rural areas than in urban areas. There was a reduction of intestinal parasitic infection among domestic dogs which were dewormed and immunized regularly. Domestic adult dogs showed significantly high hookworm egg positivity, while stray puppies had a higher prevalence of hookworm infections compared to stray adult dogs [Table 2].

Table 2

Intestinal parasitic infections in domestic dogs (n=124)

Variables	Categories	Total (%)	Positive (%)	OR	95% CI	P
Domestic dogs
Gender	Male	66 (53.2)	21 (31.8)	1
	Female	58 (46.8)	28 (48.3)	2.597	1.146-5.883	0.022
Age	Puppy	33 (26.6)	12 (36.4)	1
	Adult	91 (73.4)	37 (40.7)	2.530	0.746-8.585	0.137
Place of living	Rural	10 (8.6)	6 (60.0)	1
	Sub urban	97 (77.6)	38 (39.2)	0.731	0.151-3.543	0.697
	Urban	17 (13.7)	5 (29.4)	0.315	0.039-2.555	0.279
Breed	Mongrel/mixed bred	92 (74.2)	39 (42.3)	1
	Purebred	32 (25.8)	10 (31.3)	0.682	0.205-2.272	0.533
Deworming	≤6 months	87 (70.2)	32 (36.8)	1
	Irregular	12 (9.7)	6 (50.0)	0.620	0.098-3.925	0.612
	Never	25 (20.2)	11 (44.0)	1.042	0.349-3.115	0.941
Having other diseases	No	106 (86.3)	43 (40.6)	1
	Yes	18 (13.7)	6 (33.3)	0.689	0.183-2.597	0.582
Immunization time	Yearly	83 (66.9)	30 (36.1)	1
	Irregular	23 (18.5)	11 (47.8)	0.149	0.015-1.521	0.108
	Never	18 (14.5)	8 (44.4)	0.976	0.221-4.300	0.974
Immunization	Rabies	15 (12.1)	6 (40.0)	1
	Rabies, parvo	2 (2.0)	1 (50.0)	0.284	0.046-1.745	0.174
	Rabies, DHL, Parvo	88 (71.0)	34 (38.6)	0.144	0.005-4.102	0.257
	Never	19 (15.3)	8 (42.1)	0.533	0.081-3.519	0.514

OR: Odds ratio, CI: Confidence interval

In the present study, 23 and 46 soil samples were collected from home gardens from Gampaha and Kandy districts, respectively, and 27 and 43 were collected from public places in selected districts. Of them, two samples (4%) were positive for Toxocara eggs and six samples (12%) were positive for Hookworm eggs and all positive samples were found in roadsides. In addition, 4 (4.5%) were positive for Toxocara eggs in Kandy district and they were found in roadsides, home gardens, and a playground. In addition, Ascaris, hookworm, and Trichiuris eggs were found in soil samples collected from Kandy district [Table 3].

Table 3

Geohelminth eggs recovery in soil samples

	Places of soil samples collected
	Home gardens	Road sides	Play grounds	Children’s parks	Total
Gampaha district
Number of collected soil samples	23	14	8	5	50
Number of positives for Toxocara spp. (%)	0	2 (14.3)	0	0	2 (4.0)
Number of positives for Hookworms (%)	2 (8.7)	3 (21.4)	1 (12.5)	0	6 (12.0)
Kandy district
Number of collected soil samples	46	24	14	5	89
Number of positives for Toxocara spp. (%)	1 (2.2)	2 (8.4)	1 (7.1)	0	4 (4.5)
Number of positives for Hookworms (%)	1 (2.2)	1 (4.2)	0	0	2 (2.2)
Number of positives for Ascaris eggs (%)	2 (4.4)	1 (4.2)	1 (7.1)	0	4 (4.5)
Number of positives for Trichiuris eggs (%)	1 (2.2)	0	0	0	1 (1.1)

DISCUSSION

Contact or close association with household or stray dogs was high risk for Toxocara and other geohelminth infections. The present study observed that the rate of intestinal parasitic infections egg positivity among dogs was lower than in a suburban area of Sri Lanka and in India.[814]

A higher prevalence of parasitic infections among stray dogs could be the constant exposure to infection and the lack of proper veterinary care, including anthelmintic treatments. In addition, widespread distribution and unrestricted defecations in common places are major reasons to spread this infection. Protozoan infections were more common than helminth infections in more domestic dogs. This could be due to the regular anthelminthic treatments used to control helminth. Hence, the high prevalence of intestinal parasitic infections in dogs subsequently increasing the chance of contaminating the soils through indiscriminate defecation.

Helminth eggs such as those of Toxocara, Ascaris, and Trichiuris possess thick external layers, which provide protection from environmental factors.[1516] Geohelminth eggs are more readily and viable in soil rich with sand compared with other soil types.[17] The open access to the road sides to stray animals and defecate repeatedly and indiscriminately, thus increasing the contamination of soil with intestinal parasitic eggs. On the other hand, very low contamination of soil in public parks with parasitic eggs reflecting that no allowance to stray and domestic dogs for parks as rules and protected them with fencing.

Studies carried out in 1960 reported very high prevalence of (80% and 90%) canine toxocariasis in puppies.[1819] Decreasing prevalence of Toxocara egg positivity in Sri Lanka over the years could be due to improved awareness of zoonotic diseases among pet owners leading to responsible pet ownership. Puppies are congenitally infected while in utero through transplacental transmission.[20] Therefore, the prevalence of T. canis is more common in puppies than in older dogs.

In the present study, the highest infection rate intestinal parasitic infections reported in dogs in suburban areas. This could be the reason of wandering and defecating of hookworm-infected dogs in the public areas increasing the risk of contamination of roads, playgrounds, and public parks. Hookworm needs shady, sandy, and the moist type of soil for its further development.[21] Soils in home gardens and roadsides are shady and sandy in the study areas. This could be another reason for the high prevalence of hookworm infections in dogs and soil contamination.

There was a high prevalence of toxocariasis among female domestic dogs. This could be explained by the stress and altered psychology during pregnancy and lactation, which may reduce the immunity, thus activating dormant larvae in tissues.[22]

High intestinal parasitic infections in stray dogs compared to domestic dogs caused by the constant exposure to the infection and lack of proper veterinary care, including anthelmintic treatment among stray dogs. In addition, widespread distribution and unrestricted defecations in common places are major reasons to spread this infection. In Sri Lanka, pedigree dogs are expensive to purchase. This may be due to better nutrition and sanitation facilities and regular anthelmintic treatment in pedigree dogs. Owners of pedigree dogs are usually from high socioeconomic background and dogs are given properly cooked food and/or commercial pet food. These doges are usually leashed or caged, and free-roaming is not allowed and is given worm treatment and vaccination regularly.

In Sri Lanka, especially in rural areas, dogs do not stay indoors, and they roam freely. Therefore, there is a high possibility of rural dogs to get repeated infection thus contaminating the environment heavily. Most of the dogs, irrespective of whether strays or domesticated, is not restricted but roam at will, fouling open fields, yards, schools, and public playgrounds. Therefore, there is a high possibility of rural dogs to get the repeated infection, thus contaminating the environment heavily.[23]

In Sri Lanka, only one study so far has attempted to determine the degree of soil pollution with geohelminths ova associated with human habitat.[12] The present study was able to detect Toxocara eggs in 4% of soil samples which was less than the reported prevalence in Sri Lanka. In the rainy season, eggs can get washed off, thus reporting low prevalence. In Sri Lanka, domesticated dogs are not kept in cages and they roam free and usually defecate outside dwelling place. This could be the reason for the absence of Toxocara eggs in home gardens. The differences of contamination rate may be due to geographical and climatic changes and soil types of sampling areas.

In the present study, it has been found that adult dogs are more frequent in public places compare to puppies. This is another reason for low prevalence in soil because puppies harbor Toxocara worms and the main source of soil contamination. Future studies with more samples covering the whole country are indicated to assess the degree of soil pollution by Toxocara eggs in Sri Lanka.

CONCLUSIONS

This study showed high rate of intestinal parasitic infection in female dogs. In Sri Lanka, although rabies is a high public health priority, little attention is paid to other zoonotic diseases that humans could acquire from dogs. Toxocariasis has now been documented as a high health hazard in Sri Lanka. Control of stray dogs and improved veterinary services with interval de-worming of bitches and their litter are clearly indicated.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.