Serosurvey and molecular detection of the main zoonotic parasites carried by commensal Rattus norvegicus populations in Tehran, Iran


 Background: Rattus norvegicus are reservoirs of various zoonotic parasites that have become a global public health concern. Considering the distribution of Rattus norvegicus throughout Tehran, this study aims to assess the frequency of zoonotic parasites carried by commensal rodents in Tehran, Iran.Methods: The study considered five regions (North, South, West, East, and center) of Tehran as case studies. The serological method was used for detecting antibodies against Trichomonas vaginalis , Babesia spp, and Cryptosporidium spp using a commercial qualitative rat ELISA kit. The frequency of Toxoplasma gondii was surveyed by the conventional PCR method. Furthermore, nested PCR was used to detect Giardia spp and Leishmania spp in commensal Rattus norvegicus in Tehran.Results: Approximately, 76% of 100 Rattus norvegicus tested were infected with at least one zoonotic parasite, which demonstrates the significant frequency of parasites within the study areas. Seroreactivity against Trichomonas vaginalis , Babesia spp, and Cryptosporidium spp was detected in 5%, 0%, and 1% of Rattus norvegicus tested, respectively. Toxoplasma gondii DNA was detected in 32 out of 100 (32%) Rattus norvegicus , and Leishmania spp and Giardia spp DNA were found in 18 out of 100 (18%) and 76 out of 100 (76%) Rattus norvegicus investigated, respectively.Conclusion: The findings indicate a wide geographical dissemination of Giardia spp, Toxoplasma gondii , and Leishmania spp DNA in Rattus norvegicus within five districts of Tehran. In contrast, other parasites such as Cryptosporidium spp infection rarely occurred in Rattus populations. No evidence for the circulation of Babesia spp was found in this study.


Introduction
Zoonotic parasites cause a significantly high rate of human infectious diseases [1]. It is predicted that sixty-one percent of pathogens, which are recognized to infect individuals, can cause zoonotic diseases [2]. Zoonotic parasites are transmitted between animals and persons with or without vectors; however, eating foods contaminated by rodent feces or urine and inhaling the germ in feces of rodents are considered the most important pathways for parasite transmission [3,4,5]. Rattus norvegicus globally live and feed in close proximity to human populations and are known to carry various pathogens including bacteria, viruses, and parasites [6]. In urban areas, Rattus norvegicus act as a reservoir of zoonotic pathogens, especially zoonotic parasites, and hold a connection to various important hygienic problems; they are also responsible for human morbidity and mortality, worldwide [7]. Many of these zoonotic parasites including Leishmania spp, Giardia spp, Toxoplasma gondii, Trichomonas vaginalis, and Cryptosporidium spp are assumed to be endemic in Rattus norvegicus populations around the world [8,9,10,11]. Currently, approximately seventy-nine species of rodents have been recognized in Iran; among these previously identified rodents, Rattus norvegicus have shown greater frequency in the urban area and occupied widespread habitats in cities [12]. Although it has been proven that these rats have a potential role in the transmission of a large number of zoonotic parasites, the prevalence and diversity of parasites in urban Rattus norvegicus populations remain unknown and, also, the data concerning zoonotic parasites of Rattus norvegicus are quite insufficient. So far, a comprehensive parasitological assessment of Rattus norvegicus populations in the case of Tehran, Iran has not been conducted. Therefore, the present study performs a comprehensive survey of Rattus norvegicus collected in five districts of Tehran for zoonotic parasites.
These are the first informative data on zoonotic parasites related to Rattus norvegicus in urban areas of Tehran, Iran.

Site selection and sample collection
The study was conducted in five regions (North, South, West, East, and center) of Tehran. All the trapping locations were selected in urban areas in alleys behind the residential dwellings. The sampling strategy was designed to trap a similar number of rats between October 2018 and June 2019. The rodent samplings were carried out using Sherman live traps and suitable baits through the convenient sampling method. The aggregated places of rats were found around dumping garbage sites along the water open canal and gardens. Due to the physical and chemical intervention of Tehran Municipality aimed at controlling rats, the catching of rodents is problematic; therefore, a prebaiting procedure is preferable for improving the efficiency of traps. The trapping was carried out after sundown in each selected region and processed during midnight or the next morning. The traps were distributed in order to cover the present situation. The collected rodents were transferred to a guaranteened special laboratory in animal houses and were euthanized by the intramuscular injection of Ketamine and Xylazine (0.1 mg /kg) followed by bilateral thoracotomy. Finally, faecal samples were collected and blood was obtained by cardiac puncture using a 5mL syringe; then, serum was recovered after centrifugation and stored at −80°C until serological analysis. The subsequent parasitological examination was performed at the Department of Microbiology of Shahid Beheshti University of Medical Sciences.

Enzyme-linked immunosorbent assay (ELISA)
Serum samples were screened for antibodies against Trichomonas vaginalis, Babesia spp, and Cryptosporidium spp using commercial qualitative rat ELISA kit (Shanghai Crystal day Biotech Co., Ltd) according to the manufacturer's instructions. The optical density (OD value) of each well was measured immediately using a microplate reader set at 450 nm (OD450) within 15 minutes after adding the stop solution (sulfuric acid).

DNA extraction and Polymerase Chain Reaction (PCR)
Genomic DNA was extracted from fecal samples using the DNA extraction kit (AllPrep DNA minikit (Qiagen, Inc.) according to the manufacturer's guidelines, and each DNA sample was eluted in 200 ϻl of elution buffer preserved at -80oC until the future use. PCR was conducted for the detection of Toxoplasma gondii using specific primer pairs. The sequence of primers used for PCR reaction is shown in Table 1 PCR products were screened on a 1%-1.5% agarose gel, visualized by DNA safe stain (SinaClon Co., Iran), and photographed under UV light. Moreover, PCR amplified products were confirmed by sequencing analysis (Macrogen Korea), and the obtained sequence results were examined by the NCBI BLAST program (Primer blast).

Nested PCR
Nested PCR was used for the detection of Giardia spp and Leishmania spp using specific primer pairs. Briefly, Leishmania DNA was amplified and detected using the first-round primer pairs including 5′-CTGGATCATTTTCCGATG-3′ and 5′-TGATACCACTTATCGCACTT-3′ and the second-round primers including 5′-CATTTTCCGATGATTACACC-3′ and 5′-CGTTCTTCAACGAAATAGG-3′. The PCR conditions for the first step were based on a previously published study by Poonam et al. [13]. On the other hand, Giardia spp DNA was amplified using the first round and second round primers, as shown in Table 2.
The PCR conditions for the first step were based on a previously published study by Adnan et al [14].

Statistical analysis
The data was formatted in an SPSS file, and the frequency of each surveyed parasite was analyzed by the statistical package SPSS v.23.0 (SPSS Inc., Chicago, IL, USA) using descriptive statistic tests.

Detection of Trichomonas vaginalis, Babesia spp, and Cryptosporidium spp
A total of 100 live Rattus norvegicus (20 rats from each district of Tehran) were captured and surveyed in order to determine their zoonotic parasites. To evaluate the seroprevalence of Trichomonas vaginalis, Babesia spp, and Cryptosporidium spp in the trapped rats, the presence of rat IgG antibodies was examined by ELISA kit. In total, results of serological assay revealed that of the 100 rats captured in Tehran, 5% (n = 5/100) and 1% (n = 1/100) were positive for

Detection of Toxoplasma gondii, Giardia spp, and Leishmania spp
In this study, PCR method was used to screen the presence of Toxoplasma gondii in fecal samples, collected from Rattus norvegicus. Moreover, Nested PCR was used for detecting Giardia spp and Leishmania spp using specific primer pairs. The number of Rattus norvegicus and sample types positive for zoonotic parasites in five districts of Tehran are shown in Table 3. Results showed that the percentage of positive animals in the five regions of Tehran for Toxoplasma gondii was 32%. Among

Discussion
In general, in the urban area, rodents such as Rattus norvegicus exist in large populations and represent a significant reservoir of a range of human pathogens including bacteria, viruses, and parasites [6,16]. In comparison to other mammalian species, Rattus norvegicus live and feed in closer proximity to humans. These rodents harbor and disseminate zoonotic parasites through their ectoparasites or via biological materials; therefore, they play an active and main role in the transmission of various zoonotic diseases [17,18]. Tehran as the capital of Iran is a large city in the north of the country that features a continental-influenced Hot-summer Mediterranean climate. Home to a population of about 10-12 million in the city and 15 million over the larger metropolitan area of Greater Tehran, Tehran is the most populous city in Iran and Western Asia and has the second largest metropolitan area in the Middle East [19,20,21]. However, the prevalence and diversity of parasites in Rattus norvegicus populations in Tehran remain unknown, and a comprehensive parasitological assessment of Rattus norvegicus populations has not been conducted so far. The result of our study revealed that Giardia spp was the main parasite that was frequently (76%; n = 76/100) isolated from the Rattus population of Tehran. In addition, the frequency of Giardia spp was very high in the eastern (95%, n = 19/20) part of Tehran. This result was in contrast to those of published studies of Carolina and Tiwari in Brazil and India, respectively. These studies found that the frequency of Giardia spp in the Rattus norvegicus population was 17.1% and 42.9%, respectively [22,23]. On the other hand, the result of our study revealed that Toxoplasma gondii had the highest frequency (70%; n = 14/20) among Ratti captured from the northern part of Tehran. The total frequency of Toxoplasma gondii was 32%. These results were similar to those of published studies conducted by Dellarupe and Chao Yan.
They revealed that the frequency of Toxoplasma gondii in the Rattus norvegicus population in Argentina and China was 32.8% and 23.9%, respectively [24,25]. However, Pellizzaro et al. in Brazil [8], Saki et al. from Ahvaz province of Iran [10], Gennari from Brazil [9], and Cheng Yin from China [26] showed that the frequency of Toxoplasma gondii in Rattus population was 4.6%, 6%, 8.6%, and 3.2%, respectively. Generally, the high frequency of Giardia spp and Toxoplasma gondii in the Rattus population in Tehran is an important concern. Accordingly, humans and animals have been infected mainly by the ingestion of oocysts in the environment or consumption of foods containing cysts of Toxoplasma gondii [11]; therefore, sanitary control is extremely important to observe in Tehran. Moreover, these data will help veterinarians and physicians to better plan diagnostic and preventative Leishmania (11.11%) in the Rattus population in Morocco [31]. Leishmaniasis is a vector-borne infectious disease and is considered to be a major public health problem in the urban environment.
The diagnosis of natural hosts of Leishmania spp in urban areas is critical and will facilitate a better understanding of the epidemiology of the Leishmaniasis [32].
In conclusion, this finding indicates that the Rattus norvegicus population is a significant reservoir of

Ethics approval and consent to participate
The present study was approved by the Ethics Committee of National Institutes for Medical Research Development (NIMAD) with reference number IR.NIMAD.REC. 1396.323. All authors of this research paper have directly participated in the planning, execution, or analysis of this study.

Consent for publication
All authors made substantial contributions to the conception and design, acquisition of data, or analysis and interpretation of data. They played an active role in drafting the article or revising it critically to achieve important intellectual content, gave the final approval of the version to be published, and agreed to be accountable for all aspects of the work.  Table 2. Primers used for the detection of the Giardia lamblia using nested PCR method.