Toxocara infection in dogs and cats in Isfahan province of Iran in 2021
Gholamreza Pourshahbazi1, Hossein Khanahmad2, Reza Khadivi3, Hossein A Yousefi2, Somayeh Mobarakeh1, Fatemeh Hossini Boldaji2, Hossein Yousefi Darani1
1 Department of Medical Parasitology and Mycology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
2 Department of Genetics and Molecular Biology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
3 Department of Community Medicine, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
|Date of Submission||12-Mar-2022|
|Date of Acceptance||08-May-2022|
|Date of Web Publication||27-Jul-2023|
Dr. Hossein Yousefi Darani
Infectious Diseases and Tropical Medicine Research Center, Isfahan University of Medical Sciences, Isfahan
Source of Support: None, Conflict of Interest: None
Background: Toxocariasis is an acute or chronic disease caused by parasites of the Ascaridae family, especially Toxocara of dogs and cats. Eggs are excreted out by feces of these animals on soil. Infective eggs develop on soil which can be infective to human. In this study, infection rate of Toxocara spp. in dogs and cats of urban and rural areas of Isfahan province of Iran has been investigated.
Materials and Methods: Three hundred and seventy-five stray dog feces and 230 stray cat feces were collected from the public environment (slaughterhouses, parks, children's playgrounds, student dormitories, university environment, streets and squares) in Isfahan province of Iran. At first, dogs' and cats' feces were examined for the presence of Toxocara spp. eggs using formalin ether method. In the second stage, by using molecular methods, Toxocara eggs spp. (Toxocara canis or Toxocara cati) were identified.
Results: From 375 dog fecal samples, 39 (10.40%) and from 230 cat fecal samples, 38 (16.52%) were positive for presence of the Toxocara eggs.
Conclusion: Dogs and cats in Isfahan province of Iran were infected with Toxocara parasite. These infections can be potential risk for human toxocariasis.
Keywords: Cat, dog, Toxocara
|How to cite this article:|
Pourshahbazi G, Khanahmad H, Khadivi R, Yousefi HA, Mobarakeh S, Boldaji FH, Darani HY. Toxocara infection in dogs and cats in Isfahan province of Iran in 2021. Adv Biomed Res 2023;12:201
|How to cite this URL:|
Pourshahbazi G, Khanahmad H, Khadivi R, Yousefi HA, Mobarakeh S, Boldaji FH, Darani HY. Toxocara infection in dogs and cats in Isfahan province of Iran in 2021. Adv Biomed Res [serial online] 2023 [cited 2023 Sep 26];12:201. Available from: https://www.advbiores.net/text.asp?2023/12/1/201/382402
| Introduction|| |
Toxocariasis is a worldwide parasitic infection, transmitted to human by ingestion of Toxocara canis (T. canis) and Toxocara cati (T. cati) eggs., The definitive hosts of T. canis and T. cati are mainly dogs and cats respectively, both species are of zoonotic importance. Mature worms lay eggs in the intestine of the definitive hosts. The eggs are excreted via defecation into the environment and develop into infective eggs in optimum soil and climate conditions. When the infective eggs are ingested by human, the larvae release from the eggs and invade to the intestinal mucosa and migrate to different viscera, especially liver and lungs.,, Infections with Toxocara spp. in humans can result in a variety of clinical symptoms known as visceral larva migrans (VLM), ocular larva migrans (OLM), covert toxocariasis (CT) and neural larva migrans (NLM). Toxocariasis is an acute or sub-acute parasitic disease that causes dangerous complications such as granuloma, pneumonia, epilepsy and even blindness. Toxocara larvae may also transmit dangerous bacteria or viruses to the brain and cause involvement of the meninges. Fever, cough, wheezing, pulmonary edema, enlarged liver, stiff and painful liver on touch, unexplained nausea and vomiting, abdominal pain, weight loss, muscle pain, ataxia, seizures, coma and myocarditis are some of the complications of this disease. Ocular toxocariasis, including serious eye disorders such as strabismus and loss of central vision and causes retinal detachment and keratitis may also see in children over 4 years. Diagnosis of visceral migratory larvae should be considered in patients with eosinophilia, leukocytosis and hepatomegaly.
In a meta-analysis study, pooled prevalence of T. cati infections in Iran were 42.6%. In a review work, the global pooled prevalence of Toxocara infection in cats has been reported to be 17%, but it was higher among stray and young cats in low-income tropical countries. The overall prevalence of 11.1% has been reported for Toxocara infection in dogs. In Iran in Isfahan 1.4% of children sera samples were serologically positive using the ELISA method. In Chaharmahal and Bakhtiari using ELISA method, 2% of the children were serologically positive. In Khuzestan on the rural population using Western blot method, 2% of the patients were serologically positive. In Iran, including Isfahan province, there is a large population of stray dogs that roam in the parks, streets, and farms. In addition, many stray cats often live freely in parks, streets and human houses. Dogs and cats are considered as a public health problem for human because they may have zoonotic helminths including Toxocara species.
Limited local investigations about the situation of Toxcara Infection in Isfahan province have been performed so far, however, there is not a comprehensive work about Toxocara infection in dogs and cats in whole area of province. So, this study investigated the prevalence of Toxocara infection among dogs and cats in Isfahan province of Iran.
| Materials and Methods|| |
In this descriptive study, fecal samples of dogs and cats were collected from Isfahan province. Three hundred and seventy samples of dog feces and 230 samples of cat feces were collected from five different counties (Ardestan, Naein, Fereydun Shahr, Semirom and Isfahan). Fecal sample of every animal was collected in a 50 ml test tube and then formalin 10% was added as preservative.
The formalin ether concentration method was used to detect parasite eggs in stool samples. Briefly, stool suspension was prepared for each sample by adding about 1 g of each animal feces to 7 cc of 10% formalin. After that, 3 ml ether was added, shacked for one minute and centrifuged at 1500 rpm for 5 minutes. The supernatant was then removed and one drop of the sediment was examined under microscope.
Parasite eggs that were collected from formalin-ether stage were broken by homogenizer equipment, (severely homogenized for 3 × 60 s at 6,000 rpm (Bertin Instrument, Precelleys 24). DNA was extracted using phenol-chloroform method. Briefly, 300 μl of homogenized eggs were mixed with 500 μl of lyses buffer in a 2 ml test tube. The tubes were put in 56°C water bath and were mixed every 15 minutes. After 3 hours, the tubes were taken out from the water bath and 250 μl phenol-chloroform and 10 μl isoamide alcohol were added, then mixed until milky color. The tubes were centrifuged at 5000 rpm for 5 minutes and the supernatant moved to 2 ml new tubes. Chloroform, equal volume content of the tube, was added and again centrifuged at 5000 rpm for 5 minutes. The supernatant was moved to a new tube, and 2 times content volume of supernatant, absolute alcohol and 10% content volume of supernatant 3M sodium acetate were added. The tubes were kept overnight at −20°C, and then centrifuged for 20 minutes. The supernatant was then moved to a new tube and 600 μl 70% alcohol was added and centrifuged at 13000 rpm for 10 minutes. This stage was repeated, the supernatant was discarded and the tubes were kept out to achieve dried DNA. To be used in PCR to the dried DNA was added 50 μl distilled water and kept at 56°C for 5 minutes. For PCR, T. canis forward primer (NC5: 5′-ATTAACGCGCAAGGTTGTGG-3′) and reverse primer (NC2: 5′-TGGCCATGCATT CCTCATTC-3′) and T. cati forward primer (NC5: 5′-CTT CTGGTGCATTCTTTCGC-3′) and reverse primer (NC2: 5′-CCAAGCAACAACAAACTACGC-3′) were designed by NCBI database and Genius Prime (Version 2019.2.1) software. The PCR reactions were carried out in a 25 μl final volume, comprised of 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 seconds, annealing at 61 for 30 seconds, and activation at 72 for 30 seconds were all used in the PCR procedures. After that, the PCR products were run on a 1.5% agarose gel and visualized with UV detect equipment.
| Results|| |
Three hundred and seventy-five dog fecal samples and 230 cat fecal samples were examined by microscopic and molecular method. In microscopic examination of stool samples of 10.4% dogs and 16.52% cats, Toxocara eggs were detected. The eggs collected from these positive samples were then subjected to PCR method. In all 38 egg samples isolated from cat feces, a 204 bp band was detected following PCR and electrophoresis. Thirty-nine egg samples which isolated from fecal samples of infected dogs a 260 bp band was detected [Figure 1]. In Fereydun Shahr, 18.4% of dogs and 24% of cats were infected while in Naein the infection rate for dogs and cats were 5.4% and 8%, respectively. So Fereydun Shahr had the highest infection rate and Naein had the lowest infection rate. Details of results have been summarized in [Table 1]. The Chi-square test detected a relationship between Toxocara eggs in dogs' and cats' fecal samples and the county type (p-value = 0.013). Map frequency of Toxocara infection among dogs and cats in 5 different area of Isfahan province has been shown in [Figure 2] and [Figure 3], respectively. Frequency of T. canis eggs in fecal samples based on sampling area has been shown in [Table 2]. The Chi-square test revealed a relationship between Toxocara eggs in dogs' fecal samples and the sampling location (p-value = 0.001). The frequency of T. cati eggs in fecal samples based on sampling area has been presented in [Table 3]. The Chi-square test showed a relationship between Toxocara eggs in cats' fecal samples and the sampling location (p-value = 0.001). Also, different shapes of Toxocara eggs in fecal samples collected from Isfahan province following microscopic examination has been shown in [Figure 4].
|Figure 1: PCR of Toxocara eggs collected from dog and cat feces following gel electrophoresis. Numbers 1 to 6 are dog fecal samples and 7 to 10 are cat fecal samples. Letter P stand for positive control and N for negative control|
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|Figure 2: Map of T. canis infection among dogs in 5 different areas of Isfahan province|
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|Figure 3: Map of T. cati infection among cats in 5 different areas of Isfahan province|
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|Figure 4: Different shapes of Toxocara eggs in fecal samples of dogs and cats collected from Isfahan province following microscopic examination|
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|Table 1: Results of microscopic examination and PCR analysis of dogs and cat's fecal samples for detection of Toxocara eggs in Isfahan province of Iran|
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|Table 2: Frequency of Toxocara canis eggs in fecal samples of dogs according to location of sample collection|
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|Table 3: Frequency of Toxocara cati eggs in fecal samples of cats according to location of sample collection|
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| Discussion|| |
According to the results of this study, 10.4% of dogs and 16.52% of cats in Isfahan province of Iran were infected with Toxocara. Fereydun Shahr had the highest rate of infection and Naein had the lowest rate of infection. Differential diagnosis between T. canis and T. cati infections was performed by PCR method. Dogs and cats either as pets or stray ones can transmit zoonotic helminths such as T. canis and T. cati, by defecating eggs into the human environment. Humans can be infected by ingestion of infective eggs from contaminated soil by unwashed hands, from eating raw vegetables or direct contact with dogs or cats. So, it is important to understand the rate of infection among dogs and cats in each area. In Western Europe infection rates of Toxocara vary from 3.5% to 34% for T. canis in dogs and from 8% to 76% for T. cati in cats,,,,, also it has been reported that Albania has the highest rate of infection (76%) and Germany had the lowest rate of infection (1%). The rate of infection in Spain has been reported to be 34.5%.,,, In America, infection rate of 18% has been reported for T. canis from Cuba. In Argentina Argentina, 61% of cats were infected with T. cati.,,, In Asia, 63% infection with T. canis from Russia and 36.5% infection with T. cati from China has been reported.,,,
Prevalence of 92.9% in Shiraz and 44% in the northern Iran have been reported for T. cati infections in stray cats. In comparison with other parts of the world, the rate of infection of animals in Isfahan province of Iran was moderate and it is in agreement with other reports about the rate of infection in other parts of Iran. In this context, Zibaei et al. in a meta-analysis study, reported a weighted prevalence of 24.2% and 32.6% for T. canis and T. cati, respectively. In our study, Fereydun Shahr and Semirom counties had the highest rate of T. canis and T. cati infections. These two counties had some common features. First, they had the highest rate of rainfall in the province and second in both of them sheep raising is popular. Sheep can act as intermediate hosts by eating larval eggs in the soil, and humans can become infected with Toxocara by eating the meat and liver of these animals. In agreement with these findings, it has been shown that moisture is in favor of Toxocara development in soil. In another work, it has been shown that more Toxocara eggs were found in soil samples from shaded and moist areas. Results of another investigation revealed that moisture is essential for the development of Toxocara eggs in soil.
Considering human infection, the frequency of anti-Toxocara antibodies in human sera in developing countries such as Cuba and Vietnam was 40.1% and 45.2%, respectively, while this rate in developed countries such as Spain and Japan were, 2.1% and 1.6%, respectively. In Iran, infection rates of (29.5%) in Azarbaigan 2.7% in Zanjan, 2% in Khuzestan and (5.6%) in Tehran has been reported for human.
In a study performed by Pestechian et al. in 2012 in Isfahan, 6.25% of stray dogs were infected with Toxocara. Arbabi et al. In 2009, Kashan examined 113 cats and reported that 13.3% of cats were infected. In another work in Isfahan conducted by Torkan, on stray cats, fecal samples were examined by PCR and 17.7% of cats were infected. The results of this study are consistent with what we found about Toxocara infection rates in dogs and cats in Isfahan province. In Iran sheep raising is usually associated with keeping dogs to look after the sheep. These dogs may be responsible for circulation of Toxocara life cycle in these areas.
| Conclusion|| |
Animal fecal samples collected from Isfahan province of Iran were contaminated with Toxocara eggs. The contamination was more prevalent in area with more rainfall.
Thanks of Isfahan General Veterinary Administration for his support in collecting animal fecal samples.
This work was approved by the Isfahan University of Medical Sciences research ethical committee with the code number IR.MUI.MED.REC.1400.529.
Financial support and sponsorship
This work was supported by grant from the Isfahan University of Medical Sciences.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]