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PCR-Based Molecular Characterization of Toxocara spp. Using Feces of Stray Cats: A Study from Southwest Iran

Feces of stray cat are potential sources of gastrointestinal parasites and play a crucial role in spreading and transmitting parasite eggs, larvae, and oocysts through contamination of soil, food, or water. In this study, we investigated the prevalence of Toxocara spp. infection in stray cats in Ahvaz city, southwest Iran. Eggs of Toxocara spp. in feces of stray cats were detected by the sucrose flotation method, and identification was conducted by polymerase chain reaction (PCR) and DNA sequencing. Of the 140 fecal samples that were randomly collected from public environments during the months of January to May 2012, 45% were found to harbour Toxocara spp. eggs. The highest prevalence of Toxocara spp. eggs was found in the central area of Ahvaz city (28.6%). T. canis eggs were found in 4 (6.34%) of the 63 positive samples. Stray cats are found in parks, playgrounds, and other public places and may be a potential contamination risk. Identification of Toxocara spp. using molecular methods is sufficiently sensitive to detect low levels of parasites and identify the different Toxocara spp. in feces. The relatively high prevalence of Toxocara spp. infection may continue to increase due to lack of effective environmental hygiene control in Iran. Consequently, there is a need to plan adequate programs to detect, identify, and control this infection as well as stray cats in the region.

In many areas, accurate information about the prevalence of Toxocara spp. either does not exist or is assessed based on epidemiology of T. canis infection in dogs. Nevertheless, differentiation between the eggs of T. canis and T. cati has not often been attempted; therefore, it is probable that T. cati plays a more important role in human toxocariasis than previously suggested [1,2]. In research based upon coprological examination of fecal samples, parasite eggs are often identified as Toxocara spp., rather than classified to the exact species [4]. Although differentiation among Toxocara spp. is clinically and epidemiologically important, identification of species within the genus is complicated [13]. According to Uga et al. (2000) measurement of egg dimensions was not helpful in the differentiation of Toxocara spp. because approximately 90% of the eggs measured were of similar size. Later, it became possible to differentiate eggs of T. canis from T. cati based on their characteristic surface structure identified using scanning electron microscopy [14]. Various studies have demonstrated that the polymerase chain reaction (PCR)-based molecular approach could provide reliable markers for more accurate identification of Toxocara spp. [2,15,16]. PCR with specific primers can identify the eggs of one Toxocara spp. in the feces of a definitive host.
The populations at greatest risk for Toxocara infection include toddlers and small children, since they are most likely to be exposed to contaminated soil or sandpits while playing outdoors [17] [18]. In addition, several occupational groups such as gardeners, farmers, construction workers, and veterinarians are at elevated risk due to their contact with heavily contaminated soil [19].
In Iran, cats are often allowed to pass freely in and out of houses as predators of rats. Stray cats feast on rubbish and leftover food countless times and discharge helminth eggs and protozoan cysts into public environments. Because of the close association of cats with humans in urban areas, stray cats are important sources of a variety of zoonotic parasites, including Toxocara [20]. Since comprehensive studies about Toxocara spp. have received little attention in Iran, the aim of this study was to identify and determine the prevalence of T. cati infection in stray cats found in urban areas of Ahvaz city, southwest Iran.

Study Area, Design, and Population
Ahvaz city, the capital of the Khuzestan Province, is located in southwest Iran (latitude 31u509N and longitude 49u119E). It covers an area of more than 200 km 2 , and has a population of 1,080,955 inhabitants. During the summer, Ahvaz city has an extremely hot and humid climate and the temperature ranges between 48 and 50uC. During the winter, Ahvaz city has a warm climate, with light to moderate rain. A total of 140 fecal samples were collected from open public spaces in five regions of Ahvaz city (north, south, east, west, and central) during the months of January to May 2012. The sampling procedure required no specific permissions for all five regions of Ahvaz city, and the field studies did not involve endangered or protected species. Approximately 50 g of cat feces was collected; the unused portions of samples were hygienically discarded.

Sample Collection and Fecal Analysis
Fecal samples were put in plastic bags and stored at 4uC until processing, which was performed within 24 h. Isolation of the eggs of Toxocara spp. from each sample was performed by the sucrose flotation method as previously described [21]. In brief, fecal samples were suspended in distilled water, centrifuged at 1000 rpm for 5 min, and approximately 30 ml distilled water was added to the sediment. The suspensions were layered with 15 ml of sucrose (Merck, Germany), with specific gravity of 1.40, and centrifuged at 8006g for 5 min. The upper layer of the liquid was separated and centrifuged at 10006g for 5 min. The sediment was then resuspended in 50 ml distilled water and centrifuged at 5000 rpm for 5 min. Finally, the samples were washed twice with distilled water and sediments were examined under light microscopy (106 and 406).

DNA Isolation and PCR
Genomic DNA from the eggs of Toxocara spp. was extracted using the QIAamp DNA Mini Kit (Qiagen AG, Hombrechtikon, Switzerland) according to the manufacturer's instructions. The samples were first subjected to three freeze-thaw cycles, and proteinase K digestion was performed overnight (,16 h) as suggested by Borecka and Gawor [15] Species-specific oligonucleotide primers were selected from internal transcribed spacer 2 (ITS2) gene sequences that were previously described as Tcan1 (59-AGTATGATGGGCGCGCCAAT- 39) and NC2 (59-TAGTTTCTTTTCCTCCGCT-39) for T. canis and Tcat1 (59-GGAGAAGTAAACTC-39) and NC2 for T. cati [22].
PCR amplification was performed using 50 mL of reaction mixture containing 250 mM of each deoxynucleotide, 100 pmol of each primer, 50 mM KCl, 10 mM Tris-HCl (pH 9), 3 mM MgCl 2 , 10% dimethyl sulfoxide (DMSO, Sigma), 2 U of Taq DNA polymerase (Fermentas), and 10-15 ng of template DNA. The PCR reaction was conducted using a thermal cycler (MyCycler; Bio-Rad, Hercules, CA) and the amplification cycle consisted of an initial cycle at 94uC for 30 s; followed by 35 cycles of denaturation at 94uC for 60 s, annealing at 58uC for 30 s, and extension at 72uC for 30 s; and a final extension cycle at 72uC for 10 min. Finally, 20 mL of PCR products were run on a 1.8% agarose gel containing ethidium bromide in 16Tris-borate EDTA buffer along with 1444-80 bp DNA ladder (Fermentas).

Sequencing Method
In the present study, PCR amplification of the ITS2 region of nuclear ribosomal DNA (rDNA) was used to identify the eggs of Toxocara spp. isolated from stray cats. The results were confirmed by DNA sequencing. DNA derived from 20 infected feces was sequenced by MWG (Germany) using the primers employed during PCR. The resulting data were analyzed using Chromas software (http://technelysium.com.au/).

Evaluation of the ITS2 Motif Specificity using BLAST
The Toxocara ITS sequences were used to query the nonredundant GenBank database of the National Center for Biotechnology Information (nr at NCBI) using BLAST. The BLAST output was then analyzed to find only the exact or perfect pairwise matches showing high percent identity scores and low expect (E) values for each species.

Phylogenetic and Data Analysis
DNA sequences were initially aligned with ClustalW for multiple sequence alignment, using the default gap and extension penalties from this program [23]. Next, the ITS sequences were entered in PAUP* [24] to generate phylograms for each dataset using the methods of Dettman et al. [25,26], i.e., maximum parsimony, NNI branch swapping, and 500 random sequence additions. Three or four locus-combined datasets were made and subjected to weighted parsimony with the weight for each locus calculated as the number of parsimony informative characters for the least variable locus divided by the parsimony informative characters of that locus. Bootstrapping was performed using

Statistical Analysis
Statistical analysis was performed by SPSS 16.0 software. Data are expressed as number and percentage. Difference between  Table 3. Sequence analysis of 20 randomly selected eggs of Toxocara spp. in the five regions of Ahvaz city.

Results
In this study, all collected feces were processed by the sucrose flotation method, which showed that 63 (45%) of 140 samples were contaminated with the eggs of Toxocara spp. Determining the prevalence of Toxocara spp. in cats from each of the five regions of the city (north, south, east, west and central) revealed that the highest prevalence was in the central region (28.6%, 95% CI: 20-38.6) and the lowest (14.3%, 95% CI: 8.3-23) was in both the west and south (P = 0.026, Table 1 and 2).
All Toxocara-positive samples were examined by PCR using Tcan/NC2 and Tcat/NC2 primers. From 63 positive samples, 96.8% showed 370-bp fragments from T. cati and only four showed 380-bp fragments from T. canis. However, no mixed infections were observed, and no amplification was observed in the negative controls (Figure 1). Sequence analysis was performed using 16 randomly selected Tcan/NC2 PCR products and four randomly selected Tcat/NC2 PCR products. All sequences obtained were submitted to NCBI GenBank; their reference numbers are given in Table 3. GenBank was searched for similar published sequences using BLAST, and significant homology was detected with other Toxocara spp. sequences. The sequences of all Toxocara spp. isolates were homologous with a small number of differences corresponding to punctual base substitutions (Table 3).
Multiple sequence alignment revealed conserved nucleotides among the obtained ITS2 sequences (Figure 2). Phylogenetic trees were obtained by comparing our ITS sequences with those of other Toxocara spp. Phylogenetic analyses using various distance methods and character methods like maximum parsimony showed that the topology is similar among the trees obtained with significant bootstrap support for the clades. The values of 75% and above in the bootstrap test of phylogenetic accuracy indicate reliable grouping among different members of Toxocara spp. (Figure 3).

Discussion
The present study confirms that stray cats in all areas of the city were infected and that the prevalence of T. cati was higher in most localities in Iran than elsewhere in the world. Previous studies have shown various prevalences in Toxocara spp. infection in cats in different parts of Iran. The high prevalence of T. cati infection found in the present study is similar to the results of a survey from northern Iran (44%) [29]. Arbabi and Hooshyar in central Iran and Sadjjadi et al. in southern Iran reported 13.3% and 52.8%, respectively [20,30]. One study in Turkey showed that the prevalence of T. cati was 62.5% [31] whereas other studies in Estonia and Ireland reported a prevalence of 48.2% and 42.8% in adult cats, respectively [32,33]. It seems that the higher prevalence of T. cati in our study is probably because of the climatic conditions, which are suitable for the spread and survival of helminth eggs. Interestingly, our results revealed that the highest Previous studies conducted morphological tests to differentiate various Toxocara spp. via egg size, but it seems that egg size is the least specific criteria used to differentiate between T. cati and T. canis [14]. PCR-based approaches using the ITS1 and ITS2 segments of rDNA were employed in several studies to differentiate among the eggs of Toxocara spp. from adult helminths, which are closely related and/or morphologically similar [2,15,16]. Recently, Durant et al. used a duplex real-time PCR (2qPCR) method targeting the ITS2 sequences from 53 adult worms to identify the species of the eggs of Toxocara spp. in fecal and soil samples. They claimed that the newly developed 2qPCR assay could be considered a useful tool for the detection of the eggs of T. canis and/or T. cati in fecal samples as well as in soil samples [34].
In this study, PCR with ITS2 primers was used for the identification of the eggs of Toxocara spp. isolated form stray cats; the results were confirmed by sequencing. The results of the present study indicate that only four (6.34%) of 63 cat feces samples were contaminated with T. canis; DNA sequencing confirms these findings. BLAST analysis at NCBI revealed remarkable homology between our DNA sequence data and the T. canis sequences previously submitted to GenBank with accession numbers Y09489 and AB110034 [35,36]. The phylogenetic studies presented here confirmed that the variation between the sequences obtained herein resulted because they belonged to two different Toxocara spp. The natural and experimental establishment of T. canis adult infections in the intestines of cats was reported previously [37,38]. Fahrion et al. showed that dogs shed considerable amounts of T. cati eggs, but no T. canis eggs have been identified in the feces of cats, because coprophagy is common in dogs, and is usually not observed in cats. They mentioned that true host switching of Toxocara spp. might be an exceptional event, but their attempts to reproduce patent infections by experimental inoculations of cats with T. canis eggs failed [39]. Our results revealed that the highest contamination rate was in the central region of the city, and also three out of four T. canis eggs isolated from feces of cats were found in this region. The growing numbers of stray dogs that tend to discharge their feces into these environments could be an important factor in T. canis infections of cats, and a possible explanation of this finding.
In conclusion, Toxocara spp. can be considered neglected zoonoses, which often occur in areas with poor hygiene and education levels. This study is the first report on the prevalence and identification of the eggs of Toxocara spp. isolated from stray cats using molecular methods in the southwestern region of Iran. Differentiation of Toxocara spp. using molecular methods such as PCR are sufficiently sensitive to detect low levels of parasites and can identify the eggs of Toxocara spp. in feces. There are many stray cats in various parts of the city, which leads to the presence of Toxocara infection that may be consider a potential environmental contamination risk for the parks, playgrounds, and other public places.
The relatively high prevalence of Toxocara spp. infection may continue to increase due to a lack of effective environmental hygiene control in Iran. Consequently, there is a need to plan adequate programs to detect, differentiate and control such infection, as well as stray cats in the region.