Soil Contamination With Oocysts of Cryptosporidium spp . in Isfahan , Central Iran

1Department of Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, IR Iran 2Skin Diseases and Leishmaniasis Research Center, Isfahan University of Medical Sciences, Isfahan, IR Iran *Corresponding author: Rasool Jafari, Department of Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, IR Iran. Tel: +98-9143084002, E-mail: rasooljafariii@gmail.com


Background
Cryptosporidium is amongst important enteric pathogens and is the causative agent of cryptosporidiosis, which is characterized by diarrhea in humans, especially children.The disease is almost self-limiting, yet it can develop as a severe infection in case of immunodeficiency such as acquired immune deficiency syndrome (AIDS) and intentional immune suppression in organ transplantation (1)(2)(3).Cryptosporidium spp. is present all over the globe.It is estimated that 1% of immunocompetent humans in developed countries and 5 -10% in poor countries are infected by the protozoon.The disease causes prolonged (7 -14 days) and sometimes persistent diarrhea (≥ 14 days) (4).
Cryptosporidiosis is considered as an important waterborne zoonotic disease (5,6).It is believed that the transmission of the organism is not limited to water (7).High temperature and humid conditions are associated with an increased risk of infection with Cryptosporidium (8).Some outbreaks of cryptosporidiosis have been reported worldwide, which were mostly transmitted by water (9,10).Cryptosporidium is transmitted to humans and animals by consumption of food and water contaminated by the parasites' oocysts.Also direct contact with infected hosts and contaminated soil can be another source of the infection (11,12).Humans are in contact with soil, directly through the soil, or indirectly via food, water, and the air.Thus soil can be considered as a source of some important human diseases (13), such as toxoplasmosis (14,15).Soil can be contaminated by manure and become a potential source for human and animal infection.Cryptosporidium spp.are responsible for infection in a variety of hosts, has low infectious dose and infects immunocompromised people more than others.These features of the parasite explain the high level of environmental contamination and consequently facilitate waterborne and foodborne transmission disease (16).
Routine diagnostic methods for the infection are not Int J Enteric Pathog.2015;3(3):e29105 efficient for the detection of all cases of cryptosporidiosis.On the other hand, its treatment is suboptimum (4), thus, preventive measures should be applied especially for immunosuppressed/deficient patients.For achieving this goal, we need updated information about the contamination of soil in every region.There are a few data available about soil contamination in urban areas of Iran with Cryptosporidium oocysts, especially parks where children play.

Objectives
This study aimed to determine the rate of soil contamination with Cryptosporidium spp. of the parks of Isfahan city, central Iran.

Study Area and Sample Collection
Isfahan is an industrial and one of the largest cities of Iran, which is located in the central part of the country.The city has a dry and temperate climate with average temperature of 16.3°C with average annual rainfall of 122.7 mm (17).
The city is divided to 14 regions (Figure 1); two parks were selected from each region and included in the study.

Figure 1. Fourteen Regions of Isfahan
In total, 140 soil samples from 28 public parks were collected during summer 2014.Soil samples were collected from the loose part of the soil, mainly from flowerbeds.Flowerbeds are wet and away from direct sun light, which is suitable for the survival of the oocysts of the parasite.The samples were collected randomly from five different areas of the parks, including one central as well as four different directions from the center.

Parasitological Procedures
The soil samples were transferred to the intestinal parasites research laboratory of Isfahan University of Medical Sciences (Isfahan, Iran), for parasitological investigation.The samples were concentrated using the sucrose floatation technique as described by Kuczynska et al. (18).Microscopic slides were prepared from floating materials and stained using the modified Ziehl-Neelsen method (19).
The prepared slides were studied under the light microscope using 100X oil immersion objective.Cryptosporidium spp.oocysts were observed as reddish spherules against a pale blue background (4 -6 μm in diameter) with crescent shaped internal structures (Figure 2) (20).

Results
In total, 140 soil samples from 28 public parks of Isfahan city were collected.Oocysts of Cryptosporidium spp.were observed in 31 out of 140 soil samples (22.14%).All 14 regions (100%) and 17 out of 28 (60.71%)public parks of the city were contaminated with Cryptosporidium spp.oocysts (Table 1).

Discussion
Humans are in contact with soil, directly through the soil, or indirectly via food, water, and the air.Soil can be considered as a source of some important agents of human disease (13).Soil can be contaminated by manure and become a potential source for human and animal infection (16).Entamoeba histolytica, Giardia intestinalis and Cryptosporidium spp.are the most common pathogenic protozoa found in sewage.These pathogenic parasites are the causative agents of diarrhea in humans.Also Ascaris lumbricoides, Trichuris trichiura, Necator americanus and, Strongyloides stercoralis are the most frequently found helminthes of wastewater (13,21).Among the named infectious agents, Cryptosporidium is the one with a very low infection dose; as low as 10 oocysts can cause disease in humans.Interestingly it is mathematically estimated that even one oocyst can cause infection in some humans (22).
The present study aimed to determine the contamination rate of the soil of the public parks of Isfahan, Iran.Isfahan city was divided to 14 regions, with all having the Cryptosporidium spp.oocysts in their parks' soil samples.Cryptosporidium spp.are widely distributed all around the world except Antarctica (4), and Isfahan is not an exception.Parks are among the main play grounds for children, who are at a higher risk of developing cryptosporidiosis (23).The disease is considered as a waterborne and soil-related infection (11,13), thus soil of the parks may be a source of human infection.
Consuming soil or geophagia is still seen in many parts of the world.Geophagia is one of the ways by which some parasites, such as soil transmitted ones, are transmitted to humans (24).Our findings suggest that a large number of soil samples from Isfahan carried Cryptosporidium oocysts.On the other hand, because its infectious dose is very low and is resistant to adverse environmental conditions such as freezing temperature (22,25), the transmission of Cryptosporidium spp.from the soil to human is potentially possible.
Cryptosporidiosis has been reported in a variety of regions of Iran in diarrheic children, patients with HIV/ AIDS, renal transplant recipients, immunocompetent people and livestock (19,(26)(27)(28)(29).There are few data available about the soil contamination of urban areas with Cryptosporidium.Tavalla et al. studied the prevalence of parasites in soil samples of public places of Tehran, Iran.They observed Cryptosporidium spp.oocysts in 10% of soil samples.They suggested that soil might play a significant role in transmission of parasitic diseases with zoonotic importance to humans.They also proposed that, controlling the population of animals in urban areas, such as stray cats and dogs is fundamental in order to reduce the distribution of zoonotic parasites (30).
In Iran Cryptosporidium oocysts have been reported in tap water samples of Hamadan city (31).It is believed that the transmission of the organism is not limited to Int J Enteric Pathog.2015;3(3):e29105 water (7), and the presence of a high prevalence of oocysts in soil samples from Isfahan indicates another possible transmission route for cryptosporidiosis.In the floatation technique, only the oocysts that are intact in terms of appearance can float, while the damaged ones are not able to do so.These intact oocysts may be infective (32).Thus, the observed Cryptosporidium spp.oocysts in the soil samples from the public parks of Isfahan may contain potentially infective oocysts and might be the source of infection for the residents of the city.
Based on the findings of the present study, 22.14% of the soil samples and 60.71% of public parks were contaminated by Cryptosporidium spp.oocysts.Oocysts of the parasite were observed in the soil samples of all 14 regions of Isfahan.It seems that the contamination rate of soil of the parks of Isfahan is high and considering that the parasite is zoonotic, stray animals that are roaming around the parks are potential contaminants of the soil.Since the prevalence of cryptosporidiosis in lambs, goats and especially calves is very high in Iran (26), the manure used for fertilizing planted areas of cities, especially parks, is contaminated by the parasites' oocysts.Thus, this might be one of the important reasons for the high prevalence of Cryptosporidium spp.oocysts in the soil of the parks of Isfahan.
The prevalence of soil contamination with oocysts of Cryptosporidium spp. was high in the public parks of Isfahan, thus preventive measures, especially for high risk people, seems to be necessary.

Figure 2 .
Figure 2. Oocysts of Cryptosporidium spp. in Soil Samples, Stained by the Modified Ziehl-Neelsen Method

Table 1 .
Frequency of Cryptosporidium spp.Oocysts Among Soil Samples From Public Parks of Isfahan