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

Background Rattus norvegicus are reservoirs for transmission of various zoonotic parasites, and they have become a threat to public health worldwide. Given the large number and the significant presence of R. norvegicus throughout the city of Tehran, this study aims to assess the frequency of zoonotic parasites carried by commensal rodents wandering in Tehran, Iran. The study considered the north, south, west, east, and center regions of Tehran for the purposes of this study. The serological tests were applied in order to detect effective antibodies against Trichomonas vaginalis (T. vaginalis), Babesia spp., and Cryptosporidium spp. using a commercial qualitative rat ELISA kit. The frequency of Toxoplasma gondii (T. gondii) was surveyed by using the conventional PCR method. Furthermore, nested PCR was employed to detect the presence of Giardia spp. and Leishmania spp. in commensal R. norvegicus dispersed in Tehran. Results Approximately, 76% of the 100 R. norvegicus tested were infected with at least one zoonotic parasite, indicating the significant frequency of parasites within the study areas. Seroreactivity against T. vaginalis, Babesia spp., and Cryptosporidium spp. was detected in 5%, 0%, and 1% of the R. norvegicus tested, respectively. T. gondii DNA was detected in 32 out of 100 (32%) R. norvegicus. In addition, Leishmania spp. and Giardia spp. DNA were found in 18 out of 100 (18%) and 76 out of 100 (76%) R. norvegicus investigated, respectively. T. vaginalis with 15% and T. gondii with 70% had the highest frequency of parasites among the R. norvegicus collected from the western and northeastern regions of Tehran, respectively. Moreover, Giardia spp. with 95% and Leishmania spp. with 30% had the highest frequency in the east and center districts, respectively. Conclusion The findings showed a wide geographical dissemination of Giardia spp., Toxoplasma gondii, and Leishmania spp. in R. norvegicus within five districts of Tehran. In contrast, other parasites such as Cryptosporidium spp. infection were rarely detected in Rattus populations. No evidence for the circulation of Babesia spp. was found in this study.


Background
Zoonotic parasites cause a significantly high rate of human infections [1]. It is predicted that 61% of pathogens, which are recognized to have infected individuals, can cause zoonosis [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 found 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, R. norvegicus represent a reservoir for transmission of zoonotic pathogens, especially zoonotic parasites, and they are linked 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., T. gondii, T. vaginalis, and Cryptosporidium spp. are assumed to be endemic in R. norvegicus populations around the world [8][9][10][11]. Currently, seventy-nine species of rodents have been approximately recognized in Iran; among these previously identified rodents, R. norvegicus are frequently populated in the urban areas as their habitats [12]. Although these rats potentially transmit a large number of zoonotic parasites, the prevalence and diversity of parasites in urban R. norvegicus population remain unknown and the data concerning zoonotic parasites of R. norvegicus are quite insufficient.
Tehran, the capital of Iran, is the largest city in the northern part of Iran that features a continentalinfluenced 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 ranks as the second largest metropolitan area in the Middle East [13][14][15]. However, the prevalence and diversity of parasites in R. norvegicus populations in Tehran remain unknown, and a comprehensive parasitological assessment of R. norvegicus populations has not been conducted so far. Therefore, the present study conducts a survey of the R. norvegicus collected from five districts of Tehran for the main zoonotic parasites. The survey provides the first informative data on the traces of zoonotic parasites existing in R. norvegicus in the urban areas of Tehran, Iran.
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 (Fig. 1).
Males (n = 80) were trapped more often than females (n = 20). The distribution of surveyed parasites among male and female R. norvegicus is shown in Table 1.
To detect T. 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 T. vaginalis and Cryptosporidium spp., respectively. Among the five different districts, T. vaginalis had the highest frequency (15%, n = 3/20) among the R. norvegicus collected from the western part of Tehran. However, this parasite was not detected in the northern and central parts of Tehran. On the other hand, Cryptosporidium spp. was detected only in one rat, collected from the central part of Tehran. Babesia spp. was not detected in any of the 100 serum samples of all 100 animals examined.
In this study, the PCR method was employed to screen the presence of T. gondii in the fecal samples collected from R. norvegicus. Moreover, nested PCR was used to detect Giardia spp. and Leishmania spp. using specific primer pairs. Table 2

Discussion
In general, in urban areas, rodents such as Rattus norvegicus exist in large populations and represent a significant reservoir of different human pathogens including bacteria, viruses, and parasites [6,16]. The results 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 quite high in the eastern (95%, n = 19/ 20) part of Tehran. This finding illustrates that Giardia spp. is the main gastrointestinal parasite in the Rattus population and these rodents are the reservoir of this parasite. Moreover, this result shows that rats can transmit Giardia spp. to humans and cause severe infections such as giardiasis [17]. Therefore, a number of more effective measures such as appropriate maintenance of hygienic conditions, regular disinfection of urban environments (dumping garbage sites, the open water canal, and gardens), and prevention of the contamination of food and water sources against Giardia spp. need to be taken by the government and healthcare workers to combat zoonosis. Obtained results are in agreement with those of previous studies from Germany [17], Grenada [18], and Poland (two studies) [18,19], which reported that the prevalence of Giardia spp. among rodents was 73%, 55%, 50%, and 70%, respectively. These studies stated that rodents were the significant reservoirs of Giardia spp. However, this result is not consistent with those of published studies by Chagas et al. in Brazil [20], Perec-Matysiak et al. in Poland [21], and Li et al. in USA [22]. These three studies found that the frequency of Giardia spp. in the rodent population was 42.9%, < 35%, and 24.2%, respectively.
The result of our study revealed that T. gondii had the highest frequency (70%; n = 14/20) in the Rattus captured from the northern part of Tehran. The total frequency of T. gondii was 32%. Globally, T. gondii is a common zoonosis which is considered as an obligate intracellular parasite and causes toxoplasmosis [23]. Cats are the main source of toxoplasma eggs and are the definitive hosts that shed eggs (oocysts) in feces. Rats serve as intermediate hosts of T. gondii, and the ingestion of toxoplasma oocysts is the most common way individuals contract toxoplasmosis [24]. It is revealed that naturally infected rodents can act as significant reservoir hosts and have a critical role in spreading T. gondii to other animals including pigs, dogs, and cats [23] [9], and Yin from China [29] showed that the frequency of T. gondii in Rattus population was   [26]. Generally, the high frequency of Giardia spp. and T. gondii in the Rattus population in Tehran is an important concern. Therefore, sanitary control is extremely important to observe in Tehran. Moreover, these data assist veterinarians and physicians with better diagnostic and preventative measures. The frequency of Leishmania-positive Rattus population was 18% lower than what has been found in other studies.  [35].
The small number of positive cases in the current study in comparison to other studies is justified through reasons such as type of sample and methods used to detect Leishmania spp. and the different hygienic levels of countries. However, our results were consistent with the findings of several studies conducted by Echchakery [38]. They revealed that the frequency of Leishmania spp. in the rodent population was 11.1%, 17.46%, 14.6%, and 20%, respectively. 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 a necessity, which will facilitate a better understanding of the epidemiology of the leishmaniasis [39].
The prevalence of Cryptosporidium spp. among Rattus population was 1% lower than what was found by other studies around the world. The findings of other studies conducted in the four above-mentioned countries revealed that the prevalence rate of Cryptosporidium spp. in the rodent population was 38% [40], > 60% [21], 34.2% [41], and 25.8% [42], respectively. However, the results of several other related studies were relatively consistent with our findings, and they reported that the frequency of Cryptosporidium spp. in the rodent population was < 10% [43][44][45]. According to the reports, it is concluded that rodent population can act as a potential reservoir of Cryptosporidium spp. and probably transmit this important enteric pathogen to humans.
This study applied the commercial qualitative rat ELISA kit to the screening of antibodies against T. vaginalis among Rattus serum samples. Our findings revealed that the prevalence of T. vaginalis among the Rattus population was 5%. As far as we are concerned, the present study is the first research to have investigated the prevalence of T. vaginalis in the Rattus population, worldwide.

Site selection and sample collection
This study concentrated on five regions (north, south, west, east, and center) of Tehran. Alleys behind the residential dwellings in urban areas were the trapping locations. A sampling strategy was designed to trap 20 rats in each region between October 2018 and June 2019. Rodent sampling was carried out by using Sherman live traps and alluring baits through convenient sampling method. Rats were found mostly around dumping garbage sites along open water canals and gardens as their aggregated habitats. Given that the Tehran Municipality takes physical and chemical measures to control rats, catching rodents has become challenging and problematic; therefore, a prebaiting procedure is preferable for improving the efficiency of traps. Trapping was set after sundown in each selected region and processed during midnight or the next morning. Traps were distributed in order to manage the present situation. Collected rodents were transferred to a guaranteed special laboratory in animal houses, and then, they were euthanized by the intramuscular injection of ketamine and xylazine (0.1 mg/kg) followed by bilateral thoracotomy. Finally, fecal samples were collected, and blood samples were obtained via cardiac puncture using a 5-mL syringe; then, the serum was recovered after centrifugation and stored at − 80°C prior to serological analysis. The subsequent parasitological examination was conducted at the Department of Microbiology of Shahid Beheshti University of Medical Sciences.

Enzyme-linked immunosorbent assay
Serum samples were screened for antibodies against T. vaginalis, Babesia spp., and Cryptosporidium spp. by using commercial qualitative rat enzyme-linked immunosorbent assay (ELISA) kit (Shanghai Crystal day Biotech Co., Ltd) according to the manufacturer's instructions. The optical density (OD value) of each well was measured by immediately using a microplate reader set at 450 nm (OD450) within 15 min after adding the stop solution (sulfuric acid).

DNA extraction and polymerase chain reaction
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 ml buffer preserved at − 80°C until further use. Polymerase chain reaction (PCR) was conducted to detect T. gondii using specific primer pairs including F: 5′-GTAGCGTGCTTGTTGG CGAC-3′ and R: 5′-ACAAGACATAGAGTGCCCC-3′. PCR was conducted at a final volume of 25 μl including 0.5 μl of 10 mM of each deoxynucleoside triphosphate (dNTPs), 3 μl of 10× PCR buffer without MgCl2, 2.5 mmol/l MgCl2, 1 unit of Taq polymerase (Cinnagene, Iran), 0.5 μM of each primer (10 mM), 3 μl of template DNA, and 7.5 μL of sterile distilled water. Amplification reactions were performed under the following condition: one cycle at 95°C for 4 min, followed by 36 cycles at 94°C for 45 s, annealing at 56°C for 45 s, and preservation at 72°C for 1 min with the final extension step at 72°C for 10 min following the last cycle. 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 using the NCBI BLAST program (Primer blast).

Nested PCR
Nested PCR was used for detecting Giardia spp. and Leishmania spp. using specific primer pairs. In brief, Leishmania DNA was amplified and detected using the first-round primer pairs including 5′-CTGGATCATT TTCCGATG-3′ and 5′-TGATACCACTTATCGCAC TT-3′ and the second-round primers including 5′-CATTTTCCGATGATTACACC-3′ and 5′-CGTTCT TCAACGAAATAGG-3′. PCR conditions at the first step were set based on a previously published study by Salotra et al. [46]. Giardia spp. DNA was amplified using the first-round primer pairs including G7-F: 5′-AAGC CCGACGACCTCACCCGCAGTGC-3′ and G759-R: 5′-GAGGCCGCCCTGGATCTTCGAGACGAC-3′ and the second round primers including BG1-F: 5′-GAACGA GATCGAGGTCCG-3′ and BG2-R: 5′-CTCGACGAGT TCGTGTT-3′. PCR conditions at the first step were set based on a previously published study by Ayan et al. [47]. In summary, PCR was conducted with the final volume of 50 μl including 10 mM Tris-HCl (pH 8.3) and 50 mM KCl, a 200-μM concentration of each dNTP, 1.5 mM MgCl 2 , 1.25 U of Taq DNA polymerase (Invitrogen), 50 ng of each primer, 5 μl DNA, and 1× PCR buffer (Invitrogen). For the second round, the method provided by Sreenivas et al. was used [48]. Briefly, the second round was performed with a total volume of 50 μl containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 200 mM of each dNTP, 1.5 mM MgCl2, 2 mM of each primer, and 1.5 U platinum Taq DNA polymerase (Invitrogen). Moreover, we used 1 μl of the diluted (1:10) products from the first-round reaction as a template. Amplification reactions were performed under the following condition: initial denaturation at 94°C for 5 min followed by 35 cycles at 94°C for 1 min, annealing at 50-54°C for 1 min, and preservation at 72°C for 90 s with the final extension at 72°C for 3 min. PCR products were screened on a 1% agarose gel, visualized by DNA safe stain (Sina-Clon Co., Iran), and photographed under UV light; they were confirmed by sequencing analysis (Macrogen Korea). The sequencing results were examined by the NCBI BLAST program (Primer blast).

Statistical analysis
The data were 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.