Leishmania major Infection in Synanthropic Rodents: Evidence for the Urbanization of Zoonotic Cutaneous Leishmaniasis (ZCL) in Southern Iran

Cutaneous leishmaniasis is of particular importance in southern Iran. This study aimed to investigate the infection of rodents with Leishmania major in an urban area of Fars Province, located in southern Iran. Rodents were trapped and samples from the liver, spleen, and skin were collected. Impression smears were prepared from these tissues and any skin lesions and were examined microscopically. In addition, a portion of the samples were preserved for subsequent DNA extraction. A total of 41 rodents belonging to three species were caught from 10 trapping stations in gardens or houses within the area. The caught rodent species were Rattus rattus (n = 25, 60.97%), Mus musculus (n = 15, 36.58%), and Meriones persicus (n = 1, 2.5%). Leishmania amastigotes were seen in the spleen tissue smear of 6 (2.43%) of the rodents, including 4 of R. rattus and 2 of M. musculus. Skin lesions were observed on the muzzles of two R. rattus and one M. musculus. Samples taken from these lesions tested positive for Leishmania infection. Leishmania DNA was detected in 18 (43.9%) rodents, including 11 R. rattus, 6 M. musculus, and one M. persicus, based on DNA sequencing of the ITS2 gene and PCR of the kDNA. Phylogenetic reconstruction revealed that the parasite infecting the rodents was L. major. The detection of Leishmania infection in these rodents in urban areas raises concerns about the urbanization of cutaneous leishmaniasis caused by L. major. This urbanization poses unique challenges for control and prevention efforts.


Introduction
Leishmaniasis, as one of the most neglected tropical diseases [1,2], is a group of diseases including cutaneous, visceral, and mucocutaneous leishmaniasis caused by infection with protozoan parasites from over 20 Leishmania species.Te disease is transmitted through the bite of infected sandfies.Cutaneous leishmaniasis (CL) is the most common form of the disease which causes skin lesions, especially in hands, feet, and faces [3][4][5].CL is one of the most important parasitic diseases in Asian and Middle Eastern countries [6][7][8][9].
CL is a signifcant public health concern in Iran, with a long history of reported cases and a high prevalence rate [10][11][12][13][14]. Te disease poses a considerable burden on the healthcare system, economy, and overall quality of life for afected people [15,16].
CL holds particular signifcance in Fars Province, located in southern Iran.Tis region has consistently reported a high number of cases, making it one of the most afected areas in the country [8,17,18].Te prevalence of the disease in Fars Province is attributed to several factors, including the suitable climate and environmental conditions that favor the breeding and survival of sandfies and the vector responsible for transmitting the parasite [17,19].In addition, the presence of rural and agricultural communities in Fars Province increases the risk of exposure to infected sandfies.
Rodents serve as important reservoirs for CL in Iran [20][21][22][23].Several species of rodents, including gerbils and jirds, have been identifed as reservoirs of the Leishmania parasite.Tese rodents act as hosts for the parasite, allowing it to multiply and spread within their populations.Te proximity of rodents to human settlements increases the risk of transmission, as infected sandfies can easily bite both humans and rodents [24,25].
Te existence of suitable environmental conditions for the life of rodents, especially gerbils, is the main reason for the endemicity of CL in southern Iran [8,[26][27][28][29].Shiraz, located in the southern region of Iran, is a heavily populated city and serves as the capital of Fars Province.Until recent years, this city was considered one of the important foci of anthroponotic cutaneous leishmaniasis (ACL), but the epidemiological picture of the disease has changed in this city in recent years, in such a way that zoonotic cutaneous leishmaniasis (ZCL) has become the dominant form of the disease in this urban area [17,18,[30][31][32].
Understanding the prevalence of CL in any endemic area is crucial.Equally important is having information related to the disease's reservoirs, agents, and vectors, as well as understanding the genetic diversity of the parasite.Tese elements are vital for implementing efective prevention and control measures.
Te present study was designed and carried out to investigate the L. major infection of rodents, based on the molecular datasets of Leishmania kDNA and ITS2 genes, in an urban area (Shiraz city), in southern Iran.

Area of the Study and Sampling of Rodents.
Te study was carried out in Shiraz City, situated in Fars Province (Figure 1).Fars Province, located in the southern part of Iran, is one of the country's provinces.Shiraz, the provincial capital, is divided into 10 districts and is positioned between 29 °and 30 °North and 51.5 °-52.5 °East (Figure 1).Te city is considered as the most populous city in this province and the ffth most populous city in the country [33].
All rodents were captured in 10 stations, in a newly emerged focus of cutaneous leishmaniasis in the northeast of Shiraz, District 6 (Figure 1).To capture rodents for the study, Sherman live traps were used and placed in gardens and houses within the city during the evening (Figure 1).To minimize the pain and distress of the rodents, they were euthanized with CO 2 [34].External measurements, sex, and other characteristics were recorded before dissection, and samples were taken from the spleen, liver, and skin.If there was a lesion in the skin, an impression smear sample was prepared from the skin lesion.All samples were kept at −20 °C for subsequent DNA extraction.

Smear Preparation and DNA Extraction.
For direct visualization of Leishmania amastigotes, multiple slides were prepared from the liver, spleen, sole, and any visible skin lesions of the captured rodents using the stamp-smear method.Te smears were air-dried, fxed by methanol, and then stained with 10% Giemsa stain for 20 minutes and examined under a light microscope.
Te Favorgen Biotech Corp. Kit (Taiwan) was used to extract total genomic DNA from tissue samples of rodents, including liver, spleen, and skin and patent lesions, according to the manufacturer's guidelines.
DNA electrophoresis was carried out on a 2% agarose gel for 45 min at 80 V by adding 3.5 μL of the PCR products, a 100 bp molecular marker (SMOBIO, Hsinchu, Taiwan), and positive controls (reference strains of L. infantum, L. tropica, and L. major).Te PCR products were sequenced for the ITS2 fragment, using the same pair of primers, used in the PCR assay.

Phylogenetic Analyses.
Te raw nucleotide sequences (forward and reverse directions) and chromatograms were checked and analyzed, and the consensus sequences were aligned, using Clustal W. Te fnal sequences were registered in GenBank with accession numbers of ON398771, ON3987781-87, ON3987789, and ON3987892.Te phylogenetic analysis involved 26 partial ITS2 gene sequences of Leishmania comprising 19 sequences of L. major.A total of 16 sequences were selected from the GenBank database (Table 1).Te ITS2 sequence of Crithidia mellifcae was considered as outgroup.Phylogenetic relationships between Leishmania species were reconstructed using a Bayesian inference (BI) tree in BEAST, version 2.6.7 (https://www.beast2.org/).Te reliability of nodes was assessed using Bayesian posterior probability for the Bayesian.Te neighbor-joining tree with 100000 bootstrap generations was conducted using the Kimura 2-parameter (K2P) model [46] in MEGA X software [47].

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Canadian Journal of Infectious Diseases and Medical Microbiology

Rodent Fauna and Leishmania Infection.
A total of 41 rodents, belonging to three species were caught from 10 trapping stations.Te trapping stations were in District 6 of Shiraz (Figure 1) which is not far from a densely populated area in the city.Te caught rodent species were Rattus rattus (n � 25, 60.97%), Mus musculus (n � 15, 36.58%),and Meriones persicus (n � 1, 2.5%).Amastigotes of Leishmania were seen in the spleen smear tissue of 6 (2.43%) of the (Figure 2) rodents, including 4 of R. rattus and 2 of M. musculus (Figure 3, Table 2).Skin lesions on the muzzle of two of R. rattus and one Mus musculus were seen.Samples that were taken from these lesions were PCR-positive for Leishmania (Figure 4).Centers for disease control and prevention (CDC).

Canadian Journal of Infectious Diseases and Medical Microbiology
Multiple lesions were seen simultaneously on the sole, lip, femur, and other parts of the body of one of the R. rattus rodents, while only the sample prepared from the sole was positive for Leishmania infection.
Leishmania's DNA was detected in 18 (43.9%)rodents, including 11 of R. rattus, 6 of M. musculus, and one of M. persicus, based on the PCR of kDNA (Figure 4) and sequencing of ITS2 gene (Figure 5).Te Leishmania infection rate in R. rattus and M. musculus were 44% and 40%, respectively.Most (81.8%) of the R. rattus which were positive for Leishmania infection were male while most of the positive M. musculus (80%) cases were female (Table 2).

Phylogenetic Analysis.
Phylogenetic analysis revealed the parasite infecting the rodents R. rattus, M. persicus, and M. musculus belongs to L. major (Figure 5).Te L. major group was divided into two main clades with a bootstrap of   Canadian Journal of Infectious Diseases and Medical Microbiology 100 and posterior probability value of one (Figure 5).Te average evolutionary divergence over sequence pairs was 0.0344 while within the L. major infecting rodents in the current study was 0.02.

Discussion
Leishmaniasis is one of the important parasitic diseases in diferent countries of the world, especially in tropical and subtropical regions.Iran is one of the important foci of leishmaniasis in the world, and every year a signifcant number of patients with cutaneous and visceral leishmaniasis are reported from this country [8,17].In the Fars Province in southern Iran, both CL and visceral leishmaniasis (VL) are present [49][50][51][52][53][54][55].Vectors and animal reservoirs of CL are two main afective factors in the spread and emergence of the disease.Previous studies demonstrated ACL type of CL in the city of Shiraz, the capital of  Fars Province, especially in the center of the city, while the ZCL is mostly reported from the rural areas in the province or from the outskirts of the city [13,17,30,32].Changes in the profle of CL have been previously reported in a study conducted by Davami et al. in one of the cities of the province, where ZCL has become the dominant form of the disease in the city [56,57].
In this study, Leishmania infection was confrmed in rodents captured in urban gardens and residential areas, indicating a concerning shift in the epidemiology of leishmaniasis.Tis suggests that the disease, traditionally associated with rural areas, has now permeated into city centers.
Te urbanization of ZCL is an escalating issue in numerous countries, including Iran [17,27,58,59].As urban areas continue to expand and encroach upon natural habitats, the risk of transmission and spread of the disease increases.Tis urban growth induces environmental changes that promote the proliferation of both sand fy vectors and reservoir rodents, thereby increasing the potential for disease spread.
One of the main factors contributing to the urbanization of CL is the construction of new settlements and infrastructure.Tese developments often disrupt natural ecosystems, leading to the displacement of wildlife and the introduction of new habitats for sandfies and their rodent hosts.In addition, the infux of people into urban areas can create overcrowded living conditions, poor sanitation practices, and limited access to healthcare, all of which contribute to the spread of the disease.
Another aspect of urbanization that plays a role in the transmission of CL is the increased movement of people and goods.Urban areas are often hubs for transportation and trade, facilitating the movement of infected individuals and potentially infected animals.Tis can lead to the introduction of new Leishmania strains into urban populations, further complicating control eforts.
Te consequences of urbanization on CL are not limited to its transmission dynamics but also impact the burden of the disease on afected communities.Urban areas tend to have better healthcare infrastructure compared to rural areas, which may result in higher rates of diagnosis and reporting.However, the concentration of susceptible individuals in urban settings can also lead to larger outbreaks and more severe disease outcomes if control measures are not efectively implemented.
In Iran, Rhombomys opimus and Meriones libycus (Rodentia: Gerbillidae) serve as the main reservoir hosts for ZCL [20][21][22]60].Te rodents of Rhombomys opimus, T. indica, M. libycus, and Meriones hurrianae have been reported as the major reservoir hosts of ZCL in a diferent area of Iran, while other species like Nesokia indica and Gerbillus nanus have been described as the accidental or probable reservoir hosts in diferent parts of Iran [20,21,[61][62][63][64].In Fars Province, the species of M. libycus, M. persicus, T. indica, M. musculus, and R. rattus have been found to be positive for Leishmania infection [20-22, 26, 27, 60-62, 65, 66].In a recent study, we also reported the infection of Calomyscid rodents with L. major in the mountainous area of the same area of the current study [28].In recent years Leishmania infection in Mus musculus, commonly known as the house mouse, has been reported from diferent areas of the world including Iran [23,64,67,68].One study by Parhizkari and colleagues investigated the role of rodents caught in southern Iran as the reservoir hosts for L. major.Te researchers collected Mus musculus from diferent habitats and examined them for the presence of Leishmania parasites.Tey found that a high proportion of mice (42.9%) were infected with L. major, indicating their importance as a possible reservoir host [23].Moreover, vertical transmission of L. infantum in Mus musculus has been recently documented [69].
In the present study, R. rattus was the most abundant rodent species infected with L. major.Although rodents in the subfamily Gerbillinae are the most likely reservoir hosts in the rural area of Fars Province, it seems that rodents of the genera rattus and Mus are important hosts or probably reservoirs of L. major in the transmission of CL in the urban area, in Fars Province.
Te newly identifed focus of ZCL, as described in this study, is situated in a relatively mountainous and foothill region on the periphery of a densely populated area in the city of Shiraz.Tis area provides an ideal habitat for the Persian jird (M.persicus), which is recognized as a probable reservoir of ZCL in Iran [70].Terefore, it is not unexpected that L. major infection was also detected in this species during our study.
In our research, we not only detected Leishmania molecularly but also observed Leishmania amastigotes in tissue slides prepared from the spleens of infected R. rattus and M. musculus.Tis could serve as evidence for the potential transmission of L. major from these rodents to the parasite vector Ph.papatasi and subsequently to humans.A comprehensive investigation is required to ascertain the role of these rodents in transmitting Leishmania to sandfies.Tis could help clarify the uncertainties surrounding the role of these synanthropic rodents in the epidemiology of ZCL in this region of Iran.

Conclusion
Te fndings of the current study reveal that rodents captured in urban areas of Fars Province, southern Iran, are infected with L. major.Notably, the infection of Mus musculus with this parasite has been confrmed in this study.Te presence of Leishmania infection in these rodents in urban areas signals the potential urbanization of ZCL, caused by L. major.Tis urbanization presents unique challenges for control and prevention eforts.Te increased proximity of humans, rodents, and sandfies in urban areas amplifes the risk of transmission.To efectively combat this disease, a multidisciplinary approach is required, focusing on surveillance, vector control, public awareness, and Canadian Journal of Infectious Diseases and Medical Microbiology collaboration between diferent sectors.Only through these concerted eforts can the urbanization of CL be efectively addressed and its impact minimized.

Figure 1 :
Figure 1: Map of Iran and Fars Province showing the sampling localities of rodents in Shiraz City (numbers are related to districts).

Figure 3 :
Figure 3: Leishmania amastigotes (arrows) are seen in the spleen smear of M. musculus (a, b) and R. rattus (c, d).

Figure 5 :
Figure 5: Te evolutionary relationships of Leishmania species (ITS2 gene sequences) inferring based on the Bayesian method [48].Te posterior probability value for the Bayesian tree and the percentage value of bootstrap tests (10000 replicates) for the neighbor-joining tree are shown at the base of the clades before and after the slash symbol, respectively.

Table 1 :
Information about the ITS2 sequences of Leishmania used for the phylogenetic analyses.

Table 2 :
Molecular and microscopic characterization of L. major infection in captured rodents.Te parameters considered include species, sex, PCR (kDNA) test results, presence of lesions, and observation of L. major amastigotes in the rodent tissues.Positive infection (+), negative infection (−), not seen (N), L. major (L.m.).