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ORIGINAL ARTICLE
Adv Biomed Res 2022,  11:72

Environmental contamination of different areas of Isfahan province of Iran with Toxocara spp. eggs using molecular methods


1 Department of Medical Parasitology and Mycology, Isfahan University of Medical Sciences, Isfahan, Iran
2 Department of Genetics and Molecular Biology, Isfahan University of Medical Sciences, Isfahan, Iran
3 Department of Community Medicine, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
4 Departmant of Medical Parasitology, School of Medicine, Shahrekord University of Medical Sciences, Shahrekord, Iran

Date of Submission31-Jan-2022
Date of Decision03-Apr-2022
Date of Acceptance05-Apr-2022
Date of Web Publication30-Aug-2022

Correspondence Address:
Prof. Hossein Yousofi Darani
Infectious Diseases and Tropical Medicine Research Center, Isfahan University of Medical Sciences, Isfahan
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/abr.abr_36_22

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  Abstract 

Background: Toxocariasis is a parasitic disease caused by the larval stage of Toxocara canis and Toxocara cati. Infective stage of this parasite for human develops on soil. So, in this work contamination of the soil of public environments in five geographical areas of Isfahan province of Iran has been investigated. Materials and Methods: In this descriptive study, 355 soil samples were collected from parks, children's playgrounds, student dormitories, and university environments, and examined by Flotation method. The samples were then inspected using microscopic and molecular methods. Results: From the 355 examined soil samples in 77 (21.69%), and 87 (24.50%) cases Toxocara eggs were detected by microscopic and molecular methods, respectively. In the molecular method, 31 (8.70%) cases of T. cati and 44 (12.39%) cases of T. canis were identified. Conclusion: Toxocara eggs were identified in all areas of Isfahan province, although contamination rate was higher in Fereydun Shahr and Semirum counties.

Keywords: Environmental pollution, Iran, Isfahan, polymerase chain reaction, Toxocara


How to cite this article:
Pourshahbazi G, Khanahmad H, Khadivi R, Pestehchian N, Yousefi HA, Abdizadeh R, Mobarakeh SM, Boldaji FH, Darani HY. Environmental contamination of different areas of Isfahan province of Iran with Toxocara spp. eggs using molecular methods. Adv Biomed Res 2022;11:72

How to cite this URL:
Pourshahbazi G, Khanahmad H, Khadivi R, Pestehchian N, Yousefi HA, Abdizadeh R, Mobarakeh SM, Boldaji FH, Darani HY. Environmental contamination of different areas of Isfahan province of Iran with Toxocara spp. eggs using molecular methods. Adv Biomed Res [serial online] 2022 [cited 2022 Sep 30];11:72. Available from: https://www.advbiores.net/text.asp?2022/11/1/72/355233




  Introduction Top


Toxocara canis and Toxocara cati are nematode helminths of dogs and cats, respectively.[1]

Ova are excreted on the soil along with the animal's feces. After development on soil, if the eggs are eaten by humans, their larvae release in the intestine, migrate to different tissues, especially liver and cause human toxocariasis.[2] Several epidemiological studies have shown that soils of different areas such as parks, tourist regions, children's play stations, crowded and busy areas, slaughterhouses, and student dormitories are contaminated with (dogs and cats' Toxocara eggs). These contaminated soils can remain as sources of human infection.[3],[4],[5],[6],[7],[8],[9],[10] Toxocara eggs are very resistant and usually survive in the cold winters for 6–12 months.[11] However, development and survival of the eggs are dependent on different environmental factors such as soil type, pH, light, temperature, humidity, and vegetation. The eggs on the soil are physically dispersed by rainfall, birds, beetles, earthworms, snails, and flies.[12],[13],[14],[15]

Poverty, ignorance, eating soil, poor sanitation, presence of stray dogs and cats, and suitable climatic conditions are the factors in favor of transmission of Toxocara infection to humans.[16],[17] Children are at a higher risk for toxocariasis due to more contact with the soil and the habit of eating and transfer of objects to their mouths.[18] Mice and birds feed on soil, so, they are easily infected as intermediate hosts. Final hosts become infected by eating these intermediate hosts.[19] Therefore, in the control program of Toxocara infections study of soil contamination is extremely important.[20] Numerous studies have been performed so far about soil contamination with Toxocara eggs in different parts of the world[21] and in Iran.[22] In the world, prevalence of T. canis in dogs and T. cati in cats were 1.2% and 3.2% in Australia, 4.4% and 4.6% in the Netherlands, and 6.1% and 4.7% in Germany respectively.[23] In Iran according to Maleki et al.[24] study, the average rate of soil contamination with Toxocara spp eggs was 16%, with the highest and lowest level of contamination in Tehran (38.7%), and Qazvin (3.15%) provinces, respectively. However, the situation of soil contamination in Isfahan province of Iran is not clear. Isfahan province with population of over five million located in the center of Iran and serves as a pass way from North to South and East to West of the country. Annually many domestic, and foreign tourists visit this province. Previous studies about soil contamination with Toxocara spp. in Isfahan were limited to certain areas. So, in this work soil of different counties and different locations such as parks, playgrounds, slaughterhouses, squares, and streets together have been examined.

Molecular methods were applied for better differentiation of T. canis and T. cati eggs.


  Materials and Methods Top


In this descriptive research, study population consisted of soil samples that were collected from different areas of Isfahan province. The sample size for the whole province was 355, in which 115 were allocated to Isfahan (115 samples), and the remaining to 4 other selected counties including Ardestan, Semirum, Naein, and Fereydun Shahr (each county 60 samples).

Samples collection

Samples were collected from residential areas, tourist regions, busy places, parks, universities, student dormitories, slaughterhouses, and children's playgrounds. Isfahan is a large province, so considering climate, and geographical locations, five counties including; (Ardestan in the north), (Semirum in the south), (Naein in the east), (Fereydun Shahr in the west) and (Isfahan in the center) were selected.

Microscopic examination

In this study, for each sample, at least 250 gms of soil were taken from a depth of 5 cm and transferred to a plastic bag, and then transferred to the parasitology laboratory at Isfahan University of Medical Sciences. The soil samples were then filtered to remove the coarse particles. In the next step, the eggs of Toxocara parasite were isolated by flotation method (Sheather) and wet mount of each sample was observed under the microscope.

Molecular examination

DNA extraction

The parasite's eggs, which were collected, using saturated sugar solution, were broken using freeze-thaw and homogenizing methods (Bertin Instrument, Precelleys). Thereafter, DNA extraction was performed using the phenol–chloroform method.[24],[25],[26]

Polymerase chain reaction

For T. canis, forward primer (NC5: 5′-ATTAACGCGCAAG GTTGTGG-3′) and reverse primer (NC2: 5′- TGGCCATGCATTCCTCATTC-3′) and for T. cati forward primer (NC5: 5′-CTTCTGGTGCATTCTTT CGC-3′) and reverse primer (NC2: 5′- CCAAGCAACAA CAAACTACGC-3′) were designed by NCBI database and Genius Prime (Version 2019.2.1) software. The polymerase chain reaction (PCR) reactions were carried out in a 25 μL final volume, comprised 12.5 μL of PCR master mix (Amplicon, Denmark), 1 μL of each primer, 5 μL of template DNA, and 6.5 μL distilled water. Denaturation at 95 for 15 s, annealing at 61 for 30 s, and activation at 72 for 30 s were all used in the PCR procedures (Bio-RAD T100 thermal cycler, USA). After that, the PCR products were run on 1.5% agarose gel and visualized with UV detect equipment.


  Results Top


In this study, 355 soil samples were examined by microscopic and molecular methods for detection of Toxocara eggs. In microscopic method, 77 samples were positive, details of these results have been reported in [Table 1]. Following molecular analysis (PCR), 87 samples were positive for Toxocara spp, 12 soil samples had mixed contamination [Table 2]. Semirum and Fereydun Shahr counties had the highest rate of contamination. Shape of Toxocara eggs in soil samples collected from Isfahan province following microscopic examination have been shown in [Figure 1]. Using the Chi-Square test, it was shown that Toxocara spp. The contamination rate was different in different counties and the difference was statistically significant (P value = 0.002).
Figure 1: Toxocara parasite egg observed in soil samples collected from Isfahan province of Iran

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Table 1: Prevalence of Toxocara spp eggs in soil samples in Isfahan province of Iran based on microscopic methods

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Table 2: Prevalence of Toxocara spp eggs in soil samples in Isfahan province of Iran based on molecular method

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The most contaminated areas, were parks and children's playgrounds for T. cati eggs and slaughterhouses for T. canis eggs [Table 3]. Using the Chi-Square test, there was no significant relationship between Toxocara eggs in soil and the sample location (P value = 0.083). Soil contamination with Toxocara eggs based on climatic conditions in different counties has been shown in [Table 4]. Using the Chi-Square test, it was shown that there is a difference in soil contamination rate among different climate conditions and this difference was statistically significant (P value = 0.001). Frequency map of soil contamination with Toxocara spp eggs has been shown in [Figure 2]. Results of PCR experiment following gel electrophoresis indicating 260bp band for T. canis and 204bp for T. cati have been shown in [Figure 3].
Figure 2: Frequency map of soil contamination with Toxocara eggs in different counties of Isfahan province

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Figure 3: Results of PCR experiment following gel electrophoresis indicating 260bp band for T. canis and 204bp for T. cati, (M = Marker, P = positive control, N = negative control for T. canis and cati), and Lines 1, 2, 3, 4, 5 with a length of 260bp indicate T. canis samples and lines 6, 7, 8 with a length of 204bp indicate T. cati samples

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Table 3: Prevalence of Toxocara spp eggs in soil samples in Isfahan province of Iran based on location of sample collection

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Table 4: Prevalence of Toxocara spp eggs in soil samples in Isfahan province of Iran based on climate condition

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  Discussion Top


Human toxocariasis is caused by the ingestion of infective Toxocara eggs in soil or soil materials. Different studies have also shown that the prevalence of toxocariasis in humans directly depended on the degree of soil contamination.>[15],[26],[27] So, in this study the contamination rate of soil samples collected from different parts of Isfahan province has been investigated using both microscopic and molecular methods.

In different investigations in Iran, contamination rates of 6.3%, 22.2%, and 38.7% were reported for soil samples collected from Urmia, Khorramabad, and Tehran, respectively.[27],[28],[29] While in our work contamination rates of 21.69% and 24.5% were achieved using parasitological and molecular methods, respectively.

Soil contamination rates may vary in different regions due to climate and presence of stray animals.[30] In this regard, contamination rates of 9.7%, 6.7%, 28.1%, 11.57%, 55%, 14.03%, 11.87% has been reported for North America, Latin America, Europe, Asia, the Middle East, Australia and Turkey, respectively.[31] These different results are due to several different factors such as climate, poverty, social status, type of soil, and number of stray dogs and cats in these areas.[32] In a work in Turkey, soil contamination with Toxocara spp in Fenced parks and Fenceless parks were different.[33] Differential diagnosis of Toxocara spp. is very important in epidemiological studies. Microscopic examination is not suitable for distinguishing between two species of Toxocara, especially for soil samples. Molecular methods are usually considered as an accurate method for differentiation of Toxocara species. Most studies have shown that dog's Toxocara is the predominant species, however, other studies have also mentioned the cat's species in some areas is the dominant strain. Our work is the first report of soil contamination with Toxocara spp eggs in dogs and cats in Isfahan province in central Iran. According to our results in Fereydun Shahr and Semirum counties, higher contamination rates were seen. So, in these counties Toxocara infection may easily happen in human. Control measures, especially control of stray dogs and cats should be applied by health authorities in these two regions.


  Conclusion Top


According to results of this work, Toxocara parasite eggs exist in the soil of public areas of Isfahan province. The contamination rate was higher in Fereydun Shahr and Semirum counties.

Ethics approval

This work was approved by Isfahan University of medical sciences research ethical committee with code number of IR.MUI.MED.REC.1400.529

Consent to participate

Is not relevant.

Consent for publication

All authors approved the manuscript.

Data availability

All the data of this manuscript will be provided in case of request.

Acknowledgments

Thanks to Dr. Vahid Moghaddas (Isfahan General Veterinary Administration) for his support in collecting soil samples from slaughterhouses.

Financial support and sponsorship

This work was supported by a grant from Isfahan University of medical sciences.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Glickman LT, Schantz PM. Epidemiology and pathogenesis of zoonotic toxocariasis. Epidemiol Rev 1981;3:230-50.  Back to cited text no. 1
    
2.
Hotez PJ, Wilkins PP. Toxocariasis: America's Most Common Neglected Infection of Poverty and A Helminthiasis of Global Importance? San Francisco, USA: Public Library of Science; 2009.  Back to cited text no. 2
    
3.
Deutz A, Fuchs K, Auer H, Kerbl U, Aspöck H, Köfer J. Toxocara-infestations in Austria: A study on the risk of infection of farmers, slaughterhouse staff, hunters and veterinarians. Parasitol Res 2005;97:390-4.  Back to cited text no. 3
    
4.
Giudice PAF, Lescano SAZ, Gonzáles WHR, Giuffrida R, Bandeira FN, Kmetiuk LB, et al. Serosurvey and associated risk factors of anti-Toxocara spp. antibodies in bovines from slaughterhouses of southeastern Brazil. Parasit Vectors 2021;14:1-9. doi: 10.1186/s13071-021-04755-w.  Back to cited text no. 4
    
5.
Mahdi N, Ali H. Toxocara eggs in the soil of public places and schools in Basrah, Iraq. Ann Trop Med Parasitol 1993;87:201-5.  Back to cited text no. 5
    
6.
Jarosz W, Mizgajska-Wiktor H, Kirwan P, Konarski J, Rychlicki W, Wawrzyniak G. Developmental age, physical fitness and Toxocara seroprevalence amongst lower-secondary students living in rural areas contaminated with Toxocara eggs. Parasitology 2010;137:53-63.  Back to cited text no. 6
    
7.
Raissi V, Saber V, Bahadory S, Akhlaghi E, Raiesi O, Aslani R, et al. Comparison of the prevalence of Toxocara spp. eggs in public parks soils in different seasons, from 2017 to 2018, Tehran Province, Iran. Clin Epidemiol Global Health 2020;8:450-4.  Back to cited text no. 7
    
8.
Kleine A, Springer A, Strube C. Seasonal variation in the prevalence of Toxocara eggs on children's playgrounds in the city of Hanover, Germany. Parasite Vectors 2017;101-8. doi: 10.1186/s13071-017-2193-6.  Back to cited text no. 8
    
9.
Shin DH, Seo M, Shim S-Y, Hong JH, Kim J. Urbanization and Parasitism: Archaeoparasitology of South Korea. The Bioarchaeology of Urbanization: Springer; 2020. p. 73-89.  Back to cited text no. 9
    
10.
Fecková M, Antolová D, Zaleśny G, Halánová M, Štrkolcová G, Goldová M, et al. Seroepidemiology of human toxocariasis in selected population groups in Slovakia: A cross-sectional study. J Infect Public Health 2020;13:1107-11.  Back to cited text no. 10
    
11.
Tiyo R, Guedes T, Falavigna D, Falavigna-Guilherme A. Seasonal contamination of public squares and lawns by parasites with zoonotic potential in southern Brazil. J Helminthol 2008;82:1-6. doi: 10.1017/S0022149X07870829.  Back to cited text no. 11
    
12.
Beer S, Novosil'tsev G, Mel'nikova L. The role of the water factor in the dissemination of Toxocara eggs and the spread of toxocariasis in a megalopolis. Parazitologiia 1999;33:129-35.  Back to cited text no. 12
    
13.
Webster GA. A report on Toxocara canis Werner, 1782. Can J Comp Med Vet Sci 1958;22:272.  Back to cited text no. 13
    
14.
O'Donnell CJ, Meyer KB, Jones JV, Benton T, Kaneshiro ES, Nichols JS, et al. Survival of parasite eggs upon storage in sludge. Appl Environ Microbiol 1984;48:618-25.  Back to cited text no. 14
    
15.
Mizgajska H. Eggs of Toxocara spp. in the environment and their public health implications. J Helminthol 2001;75:147-51.  Back to cited text no. 15
    
16.
Pozio E. Foodborne nematodes. Foodborne Parasites in the Food Supply Web. Amsterdam, The Netherlands: Elsevier; 2015. p. 165-99.  Back to cited text no. 16
    
17.
Rai SK, Uga S, Ono K, Rai G, Matsumura T. Contamination of soil with helminth parasite eggs in Nepal. Southeast Asian J Trop Med Public Health 2000;31:388-93.  Back to cited text no. 17
    
18.
Van Wijnen J, Clausing P, Brunekreef B. Estimated soil ingestion by children. Environ Res 1990;51:147-62.  Back to cited text no. 18
    
19.
Choi D, Lim JH, Choi D-C, Lee KS, Paik SW, Kim S-H, et al. Transmission of Toxocara canis via ingestion of raw cow liver: A cross-sectional study in healthy adults. Korean J Parasitol 2012;50:23-7.  Back to cited text no. 19
    
20.
Papavasilopoulos V, Pitiriga V, Birbas K, Elefsiniotis J, Bonatsos G, Tsakris A. Soil contamination by Toxocara canis and human seroprevalence in the Attica region, Greece. Germs 2018;8:155-61.  Back to cited text no. 20
    
21.
Fakhri Y, Gasser R, Rostami A, Fan C, Ghasemi S, Javanian M, et al. Toxocara eggs in public places worldwide-A systematic review and meta-analysis. Environ Pollut 2018;242:1467-75.  Back to cited text no. 21
    
22.
Eslahi AV, Badri M, Khorshidi A, Majidiani H, Hooshmand E, Hosseini H, et al. Prevalence of toxocara and toxascaris infection among human and animals in Iran with meta-analysis approach. BMC Infect Dis 2020;20:1-17. doi: 10.1186/s12879-020-4759-8.  Back to cited text no. 22
    
23.
Afshar MJA, Zahabiun F, Heydarian P, Saadati HM, Mohtasebi S, Khodamoradi F, et al. A systematic review and meta-analysis of toxocariasis in Iran: Is it time to take it seriously? Acta Parasitol 2020;65:569-84.  Back to cited text no. 23
    
24.
Maleki B, Khorshidi A, Gorgipour M, Mirzapour A, Majidiani H, Foroutan M. Prevalence of toxocara spp. eggs in soil of public areas in Iran: A systematic review and meta-analysis. Alexandria J Med 2018;54:97-101.  Back to cited text no. 24
    
25.
Aboutalebian S, Ahmadikia K, Fakhim H, Chabavizadeh J, Okhovat A, Nikaeen M, et al. Direct detection and identification of the most common bacteria and fungi causing otitis externa by a stepwise multiplex PCR. Front Cell Infect Microbiol 2021;11:210.  Back to cited text no. 25
    
26.
Trejo CAC, Romero Núñez C, García Contreras AdC, Mendoza Barrera GE. Soil contamination by toxocara spp. eggs in a University in Mexico city. Rev Bras Parasitol Vet 2012;21:298-300.  Back to cited text no. 26
    
27.
Zibaei M, Abdollahpour F, Birjandi M, Firoozeh F. Soil contamination with Toxocara spp. eggs in the public parks from three areas of Khorram Abad, Iran. Nepal Med Coll J 2010;12:63-5.  Back to cited text no. 27
    
28.
Tavassoli M, Hadian M, Charesaz S, Javadi S. Toxocara Spp. Eggs in Public Parks of Urmia City, West Azer baijan Province Iran. Iranian J Parasitol 2008;3:24-9.  Back to cited text no. 28
    
29.
Tavalla M, Oormazdi H, Akhlaghi L, Razmjou E, Lakeh MM, Shojaee S, et al. Prevalence of parasites in soil samples in Tehran public places. Afr J Biotechnol 2012;11:4575-8.  Back to cited text no. 29
    
30.
Marques JP, Guimarães CdR, Boas AV, Carnaúba PU, Moraes JD. Contamination of public parks and squares from Guarulhos (São Paulo State, Brazil) by toxocara spp. and Ancylostoma spp. Rev Inst Med Trop São Paulo 2012;54:267-71.  Back to cited text no. 30
    
31.
Avcioglu H, Burgu A. Seasonal prevalence of Toxocara ova in soil samples from public parks in Ankara, Turkey. Vector-Borne Zoonotic Dis 2008;8:345-50.  Back to cited text no. 31
    
32.
Congdon P, Lloyd P. Toxocara infection in the United States: The relevance of poverty, geography and demography as risk factors, and implications for estimating county prevalence. Int J Public Health 2011;56:15-24.  Back to cited text no. 32
    
33.
Avcioglu H, Balkaya I. The relationship of public park accessibility to dogs to the presence of Toxocara species ova in the soil. Vector-Borne Zoonotic Dis 2011;11:177-80.  Back to cited text no. 33
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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