Prevalence of Toxocara Species Eggs in the Soil of Public Parks in Hamedan City, Western Iran

Background: Toxocariasis is one of the most commonly reported zoonotic helminth infections in the world which is caused by the parasitic roundworms of dogs or cats. This survey aimed to provide data on the contamination of the soil of public parks by Toxocara spp. eggs in Hamedan city, capital of Hamedan Province in the west of Iran. Methods: During April and May 2019, a total of 120 soil samples were collected from 20 public parks (4 to 10 soil samples from each park) from all geographical areas of the city. For detection of eggs, soil samples were dried overnight at room temperature and passed through a 150 μm mesh sieve. After performing Sheather’s flotation technique using 2 g of powdery soil, light microscopic examinations were carried out. Results: Microscopic evaluation of specimens revealed that 14 samples out of 120 (11.67%) collected from 10 public parks out of 20 in Hamedan (50.0%) were contaminated with Toxocara spp. eggs. Various developmental stages of Toxocara spp. eggs including (1) non-developed, (2) 2-cell to tadpole stage, and (3) larvated eggs were observed. Furthermore, in two public parks, Trichuris spp. eggs were found in the soils. Conclusions: This study provides the first information about the contamination of public areas in Hamedan 0rovince and suggests that public awareness about this fact is needed. Education of citizens especially parents with children, who have commonly mouthing behavior, is recommended. Further molecular-based studies for the identification of parasite species in the province are suggested. All of the published literature about contamination of the soils of public areas in Iran with Toxocara eggs until July 2020 were also reviewed.

Toxocara eggs in the soil of public parks in Hamedan, Iran different WHO regions ranged from 13% to 35% (9). According to the published reports, 18% of soil samples from the Middle East and North Africa were contaminated with Toxocara spp. (range: 11%-24%) (9). In Iran, a recent meta-analysis article reported that 16% of soil samples from public areas are contaminated with the eggs of Toxocara spp. (10). Considering (i) notable prevalence of toxocariasis is in both stray and owned dogs (11,12) and cats (first author's personal experience), (ii) presence of antibodies against T. canis in blood sera of 8.8% of children in the urban and rural areas of Hamedan (13), and (iii) the public health importance of contamination of parks with Toxocara spp. eggs, this study aimed to examine soil samples from different public parks of the city for the presence of Toxocara eggs.

Study Area
Hamedan city (34° 48′ 0″ N, 48° 31′ 12″ E) is the capital of Hamedan province in the west of Iran with around 600 000 inhabitants. It has a cold semi-arid climate with an annual rainfall of 318 mm and an annual average temperature of 11.3°C. In the city, there are 130 public parks under different categories such as pocket, neighborhood, regional, district, and city parks with various sizes distributed in four districts (14).

Sampling and Detection of Helminths Eggs
From each park, four to ten soil samples were taken based on the area of the parks i.e., separate samples were collected from every 50 steps. Soil samples of approximately 200 g were collected at a depth of 3 cm, stored in plastic bags, and taken to the laboratory. For detection of ova, soil samples were dried overnight at room temperature and passed through a 150 μm mesh sieve. Exactly 2 g of powdery soil was subjected to a normal sucrose flotation method (15). Light microscopic examinations were carried out by trained personnel and photomicrographs were taken from positive samples. Eggs were counted and classified according to their developmental stage i.e., nondeveloped, 2-cell to tadpole stage, and larvated eggs (16).

Results
Fourteen out of 120 samples (11.67%) collected from 10 public parks out of 20 in Hamedan (50.0%) were contaminated with Toxocara spp. eggs.
The ratio of the number of positive specimens to the total number of collected soil samples from each park ranged from 12.5% to 50.0%.

Discussion
In this study, 11.67% of soil samples were contaminated with eggs of Toxocara spp. Several studies in different cities of Iran have reported contamination rates ranging between 4 and 63.3% in public areas. All of the studies  except two are based on light microscopy examination of soil specimens (Table 1). Furthermore, in the present study, 50.0% of the public parks were found to be contaminated with Toxocara spp. eggs. In similar studies different rates were reported i.e., 80% in Piranshahr (32) (15), 15% in Shiraz (18), and 3.9% in Urmia (26). It has been shown that Toxocara eggs in public places are prevalent in regions with a high relative humidity, which are located at high longitudes and low latitudes (2). However, as sampling seasons and soil examination methods are different, it is not possible to reach a clear conclusion on the contamination of public areas in different localities of the country. A countrywide survey at one time period and a single method is suggested.
In the present study, the molecular examination was not performed to define the distribution pattern of Toxocara species in soil samples; however, there are two studies in Iran in which PCR followed by sequencing were used for this purpose. In Ahavz, 28.0% and 5.7% of the soil samples contained eggs of T. cati and T. canis, respectively (22). And, in Shiraz, 15.3% and 0.7% of soil samples were contaminated with T. cati and T. canis (19). There are controversies in the literature concerning Toxocara spp. in human patients. Some authors state that most cases of human toxocariasis have been associated with parasitism caused by T. canis (44), while some others believe that T. cati has been underestimated and as cats have more access to places where children go, T. cati should be considered responsible for the majority of larva migrans cases (7). In Iran, since the first report of larva migrans in 1976 (45), more than 25 confirmed cases of human toxocariasis including VLM, OLM, and NT have been documented as reviewed in a previous study (3). However, the species of Toxocara infecting patients in the country has not been investigated so far. In human populations, serologic tests are commonly used for diagnosis of toxocariasis; however, the problems of cross-reactivity in polyparasitism and indistinguishability between T. canis and T. cati (or possibly T. malayensis) still exist (2). Species-specific diagnostic tools are required to support future epidemiological investigations. Moreover, surgeons should be encouraged to send the freshly removed worm body for molecular identification.
It is generally believed that only dogs and cats play role in the contamination of the environment. However, it has been shown that snails have roles in the epidemiology of toxocariasis (46). In a study in an open space in Buenos Aires, Argentina, 20% of Rumina decollata were infected with the third larval stage (L3) of Toxocara cati (46). A clinical trial on experimental infection of dogs/cats with infected snails will clarify the role of snails in toxocarosis of carnivores. Furthermore, the snail-to-snail transmission of nematodes larvae has been described as a novel route (47). It would be valuable to study whether or not "intermediesis" occurs for Toxocara species.
In most of the discussions, stray and pet cats and dogs are mentioned to be responsible for contamination of the environment but several species of wild canids (e.g. red fox, golden jackal, and wolf ) and wild felids (e.g. lynx) are definitive hosts of T. canis and T. cati (48). It is suggested that foxes can be considered as the primary source of Toxocara spp. eggs in the environment because of the high prevalence of patent infections and a total absence of any anthelmintic treatment (49). Phylogenetic analyses of Toxocara spp. recovered from wild canids and felids are required to clarify the role of wild carnivores in contamination of the urban environments via their movements in borders of the cities and their potential to infect stray dogs and cats.

Conclusions
This study provides the first information about the contamination of public areas with Toxocara spp. in Hamedan province and calls for an increased general awareness of public health risk. Parents and pet-owners should get informed about the hazards of toxocariasis by medical and veterinary practitioners. Moreover, the media and science communicators should educate the society on prevention strategies to minimize the risks of zoonotic transmission. Further studies in the province Toxocara eggs in the soil of public parks in Hamedan, Iran especially with the aid of molecular-based studies for the identification of parasite species are suggested.