Leishmania (Leishmania) mexicana Infection in Wild Rodents from an Emergent Focus of Cutaneous Leishmaniasis in Yucatan, Mexico

In 2015, emergent cases of localized cutaneous leishmaniasis (LCL) were reported in Tinum, Yucatan, Mexico. As part of an eco-epidemiological study to characterize the elements that trigger Leishmania infection in that area, we conducted a field study to investigate the occurrence of Leishmania infection in wild rodents. From November 2019 to February 2020, rodents were caught from three sites located in the municipality of Tinum, Yucatan. For each specimen, clinical signs suggestive of Leishmania infection were recorded. Samples from the tail, liver, and spleen were taken for the identification of Leishmania DNA by PCR. Twenty rodents belonging to two species were caught including Heteromys gaumeri (55%, 11/20) and Ototylomys phyllotis (45%, 9/20). Fifty-five percent of the animals presented white spots on the tail, 15% had splenomegaly, and 5% had hepatomegaly. Fifty-five percent (11/20) of the animals were found infected by Leishmania. Heteromys gaumeri was caught in all trapping sites and was the most infected species (63.6%, 7/11). The percentage of infection for O. phyllotis was 44.4% (4/9). Leishmania (Leishmania) mexicana was identified as the infecting species in two H. gaumeri. This study provides, for the first time, evidence of Leishmania infection in wild rodents from the Yucatan state. Heteromys gaumeri and O. phyllotis may be involved in the transmission cycle of L. mexicana in this emergent focus; however, further longitudinal studies are needed to confirm their role as primary reservoirs.


Introduction
Parasites from the genus Leishmania (Kinetoplastea: Trypanosomatida) are the causal agents of the leishmaniases, a group of tropical diseases transmitted by the bite of Phlebotomine sand flies (Diptera: Psychodidae). Approximately, 53 Leishmania species have been described from different regions of the world; of these, 31 species are known to be parasites of mammals and 20 species are pathogenic for human beings [1]. e disease is manifested by three major clinical forms: localized cutaneous leishmaniasis (LCL), mucocutaneous leishmaniasis (MCL), and visceral leishmaniasis (VL). Today, more than 1 billion people in 98 countries live in endemic areas and are at risk of infection. In addition, an estimated 0.2-0.4 million VL cases and 0.7-1.2 million CL cases occur each year [2,3].
Leishmaniases are zoonotic diseases where mammals play a crucial role in the transmission cycle [4]. Many natural hosts of Leishmania parasites have been identified including foxes, coatis, raccoons, opossums, bats, monkeys, dogs, and rodents [4,5]. Some of them have been incriminated as potential reservoirs due to their abundance, life expectancy, temporal, and spatial co-occurrence with vectors and their long co-evolution with Leishmania species [6]. e identification of Leishmania carriers and the study of their role as potential reservoirs are very important to understanding the epidemiology of the disease, especially in areas where leishmaniasis is emerging [7].
Localized cutaneous leishmaniasis (LCL) is endemic in Mexico, mainly in the states of Veracruz, Tabasco, Oaxaca, Chiapas, Campeche, and Quintana Roo [8]. e disease could be asymptomatic or manifest as an ulcer with raised borders located mainly in the ear (39.8%) [8][9][10]. Most studies to characterize the eco-epidemiological features of LCL have been conducted in the state of Campeche [10]. According to those studies, the main causal agent is Leishmania (Leishmania) mexicana and the only proven vector is Lutzomyia olmeca olmeca. However, Lu. cruciata, Lu. panamensi, and Lu. shannoni have been incriminated as potential vectors of L. (L.) mexicana [11][12][13]. In Campeche, Heteromys gaumeri, Peromyscus yucatanicus, and Ototylomys phyllotis were incriminated as L. (L.) mexicana reservoirs based on their geographical and temporal distributions and their overlap with vector and human habitats [14][15][16].
In the Yucatan State, cases of LCL had been restricted to sylvatic areas of the southern cone, close to the well-known endemic areas of Campeche and Quintana Roo [17,18]. Nevertheless, since 2015, the Health Services of Yucatan have reported emergent cases in rural communities of the municipality of Tinum, Yucatan, located in the eastern part of the state [19]. is emergency has prompted us to carry out an eco-epidemiological study to identify all the transmission components (Leishmania, vectors, and reservoir species) in this area. As a first step to identifying potential reservoirs in Yucatan, we investigate the occurrence of Leishmania infection in wild rodents from two villages with autochthonous cases of LCL in the municipality of Tinum, Yucatan, Mexico.

Study Area.
Rodents were caught in two villages of the municipality of Tinum, Yucatan; located between 20°40′ and 20°53′ latitudes North and 88°21′ and 88°33′ longitudes West. Trapping sites ( Figure 1) were selected according to previous reports of human Leishmania infection in the municipality of Tinum [19] including a forest fragment located at 20.66°N, 88.49°W; an abandoned cornfield located at 20.67°N, 88.47°W; and a perirural site located at 20.64°N, 88.51°W. e area has an annual rainfall that reaches 85.9 mm mostly during the summer, an average temperature of 26.3°C, and vegetation corresponding to the dry mediumheight semideciduous forest with a predominance of cedar (Cedrus odorata), mahogany (Swietenia macrophylla), ceiba (Ceiba pentandra), chacá (Bursera simaruba), and pochote (Bombacopsis quinata) [20].

Collection of Rodents and Tissue Sampling.
Trapping was carried out in November and December 2019 and February 2020 (a period where a strong correlation was demonstrated between the abundance and rates of infection of both reservoirs and vectors in endemic areas from Southeastern Mexico) [21]. Small rodents were caught alive in Sherman traps baited with corn and sunflower seeds. Ninety traps were laid at 10 m intervals during seven nights in the perirural site (where most clinical and asymptomatic human Leishmania infections were recorded), during four nights in the abandoned cornfield, and during three nights in the forest fragment. Traps were set in the evening in common rodent microhabitats such as burrows hidden in rocks and buttresses of trees and checked the following morning. e captures were conducted with the permission of the Mexican Secretariat of Environment and Natural Resources (SEM-ARNAT, license no. 12783) and approved by the Ethics Research Committee of the Autonomous University of Yucatan with the ID: CEI-22-2018.
Each rodent was identified into species according to its external characteristics using the keys of Hall [22] and Jones [23]. en, they were examined searching for clinical signs suggestive of Leishmania infection such as ulcers, scars, white spots, and/or swelling of the tail [14]. Animals were euthanized by intraperitoneal injection of sodium pentobarbital (100 mg/Kg). Needle aspirates were taken from the base of the tail of each rodent, since it has been reported to be the site where ulcers commonly appear [14,16]. Aspirates were cultivated in RPMI 1640 medium and incubated at 23.5°C. A culture was considered positive when at least one promastigote was observed microscopically and negative if no parasites were found within one month. Additionally, skin, spleen, and liver fragments were collected and frozen to −70°C until used.

DNA Extraction and Detection of Leishmania Infection.
DNA was extracted from the different tissues using the standard phenol/chloroform method. e PCR reactions were performed with the GoTaq ® PCR Core Systems I kit (Promega M7660) in a final volume of 25 μL containing buffer 1x, 1.75 mM MgCl 2 , 0.2 mM dNTP's, 2.5 U/μL Taq polymerase, 1 μM of each primer JW11 (5′-CCTATTT-TACACCAACCCCCAGT-3′), and JW12 (5′-GGGTAGGGGCGTTCTGCGAAA-3′), which amplify a 120 bp fragment of kDNA of the genus Leishmania [24] and 200 ng of DNA. e DNA isolated either from the footpad of an infected hamster or from parasites in culture was used as a positive control. As a negative control, DNA from the skin tail of a non-infected Peromyscus yucatanicus was used. Negative controls that contained water instead of DNA were also included in each PCR experiment. PCR amplification was performed in a SimpliAmp thermal cycler (Applied Biosystems) with an initial denaturation step at 94°C for 1 min to activate the Hot Start Taq polymerase followed by 30 cycles of 94°C for 1 min, 67°C for 30 s, and 72°C for 30 s with a final extension at 72°C for 5 min.
e reaction products were run in 2.5% agarose gel stained with ethidium bromide (0.5 μg/μL) and visualized under UV light. RNAse free water was used as a negative control. e PCR assay used the following amplification cycle: 94°C for 4 min followed by 36 cycles at 94°C for 40 s, 54°C for 30 s, and 72°C for 1 min and a final extension at 72°C for 6 min. en, 3 μL of the PCR product was reamplified following the amplification cycle: 94°C for 4 min followed by 18 cycles at 94°C for 40 s, 54°C for 30 s, and 72°C for 1 min and a final extension at 72°C for 6 min. e PCR products were run on a 1.5% agarose gel stained with SYBR Gold Nucleic Acid Gel Stain (S11494, Invitrogen, USA). e PCR products from the reamplification of ITS were subjected to digestion with HaeIII restriction enzyme (R0108, New England, BioLabs) for Leishmania species identification. A mixture with 2 μL of RNAse free water, 2 μL of buffer, and 1 μL of HaeIII was added to 15 μl of the amplified product. en, the microtube was incubated at 37°C for 3 hours and at 80°C for 20 min to inactivate the enzyme. e species profiles of each sample and reference controls were observed in a 4% agarose gel subjected to electrophoresis for 3.5 hours at 90 V. e gel was stained with SYBR Gold Nucleic Acid Gel Stain (S11494, Invitrogen, USA).

Results and Discussion
According to the WHO, surveys of mammals in new foci of Leishmania infection are essential to identify potential reservoir hosts and to understand the eco-epidemiology of the disease [6]. In this work, we reported, for the first time, L.
(L.) mexicana infection in wild rodents captured in a rural community of the Yucatan state where autochthonous LCL cases emerged in 2015 [19].
A total of twenty animals belonging to both the Heteromyidae and Cricetidae families were captured. e sample was represented by two rodent species (Figures 2(a) and 2(b)): eleven Heteromys gaumeri (55%) and nine Ototylomys phyllotis (45%). is finding is quite different from previous studies carried out in the sylvatic region of Campeche [14,16], where a great diversity of small mammals has been reported, including Oryzomys melanotis, Or. couesi, Sigmodon hispidus, Reithrodontomys gracilis, H. gaumeri, O. phyllotis, P. yucatanicus, and the marsupial Marmosa mexicana. is result could be related to some ecological aspects of the state of Yucatan that are different from the entities that constitute the Yucatan peninsula, including its drier climate, the predominance of arid vegetation, and the high percentage of deforested areas [25][26][27]. However, limitations due to the catch effort cannot be excluded.
In this study, the same number of males and females were caught, and at the time of inspection, no animal had ulcers suggestive of Leishmania infection. However, 55% (11/20) of the animals presented white spots on the tail, 15% had splenomegaly, and 5% had hepatomegaly (Figures 2(c) and 2(d)).
Parasites were successfully isolated from the base of the tail of one O. phyllotis. Leishmania DNA was detected in 55% (11/20) of the animals including seven H. gaumeri (out of 11 captured, 63.6%) and four O. phyllotis (out of 9 captured, 44.4%). Regarding the presence of parasite DNA in the different organs (Table 1) (Table 1). Leishmania (L.) mexicana was detected as the infecting species in the liver samples from two H. gaumeri (Figure 3).
Ototylomys phyllotis and H. gaumeri are the two most abundant rodent species captured in the sylvatic endemic areas of LCL in Campeche [14,16]. Moreover, several  parasitological and mastozoological studies have incriminated these two species as potential reservoirs of L. (L.) mexicana in that area [11,14,16]. On the other hand, O. phyllotis was one of the first Leishmania carriers identified in sylvatic areas of the Yucatan Peninsula presenting multiple, hairless, white spots on the proximal half of the tail [14]. Later, its ability to retain Leishmania parasites for at least 18 months was demonstrated, confirming their role as a reservoir [15]. Tail depigmentation in H. gaumeri specimens was also frequently reported in surveys conducted between 1997 and 2004 in Calakmul, Campeche [16]. In this study, we found white spots on the tail in 55% (11/20) of the animals, and we detected Leishmania infection by PCR in 54.5% (6/11) of them, confirming that this sign could be considered as a fair indicator of Leishmania infection in wild rodents. Surprisingly, only three animals were positive to Leishmania DNA in the skin, the site where signs commonly appear. e dissemination of several dermotropic Leishmania parasites to visceral organs in rodents has been reported previously. Tonelli et al. [28] found the DNA of L. (V.) braziliensis in the liver and the spleen of rodents captured in an ecotourism area from Brazil. Similarly, the presence of L. (L.) tropica (a species associated mainly with cutaneous manifestations in humans) was detected by PCR in the spleen of wild rodents captured in Ethiopia [29]. e systemic dissemination of L. (L.) mexicana in naturally infected rodents from one of the main LCL foci in Campeche, Mexico, was also documented [15]. Nevertheless, the presence of L. (L.) mexicana amastigotes in the organs (liver, spleen, kidney, and heart) of the rodents was not considered as a visceralization of the disease since its effect was nonpathogenic. In addition, the experimental infection of richomys laurentus, a putative reservoir of L. (V.) braziliensis and L. (L.) infantum in Brazil, did not reveal histological changes in organ's tissues despite the presence of Leishmania DNA, suggesting that the division between cutaneous and visceral tropisms observed in humans is less clear in natural hosts of Leishmania spp. [30]. e association of parasite DNA in the liver and the spleen of rodents captured in the emergent focus of Yucatan with its pathogenic effects in the tissue needs to be investigated. Further studies are needed to understand this phenomenon and its implications in the natural transmission of Leishmania in endemic areas.
Regarding the infection rate by rodent species, here, we report that H. gaumeri is the species most infected by Leishmania (63.6%, 7/11).
is finding is consistent with previous studies done in Campeche, where high infection rates (29-88%) have been documented for this Yucatan Peninsula's endemic species [16]. Due to their high abundances and infection rates, H. gaumeri has been incriminated as one of the three potential reservoirs of L. (L.) mexicana in Campeche [16]. On the other hand, several studies have provided evidence to incriminate O. phyllotis as a potential reservoir of L. (L.) mexicana in Mexico and Belize. In the latter, a prevalence of Leishmania infection ranging from 2% to 40% was reported for this species of animals [31][32][33]. In addition, the high attraction of O. phyllotis to Lu. flaviscutellata (the most important vector in Belize) has been proved, indicating its role as the main reservoir of L. (L.) mexicana in that country [34]. In Campeche, the Leishmania infection rate for O. phyllotis varied from 27% to 100%. Peromyscus yucatanicus, the other wild rodent incriminated as a reservoir of L. (L.) mexicana in Campeche, appear not to be involved in the transmission of the parasite in this new emergent focus of leishmaniasis at the Yucatan State [15,16].
Here, we found H. gaumeri specimens in all trapping sites being more frequent (54.5%, 6/11) in the forest fragment followed by the perirural site (36.3%, 4/11). e peak of the abundance of this species was documented in November. Ototylomys phyllotis specimens were captured with a similar frequency in the abandoned cornfield (55.5%, 5/9) and the perirural site (44.4%, 4/9). e peak of the abundance of this species was documented in February.
Most of the infected animals (54.5%, 6/11) were found in the forest fragment followed by the abandoned cornfield (27.2%, 3/11) and the perirural site (18.2%, 2/11) ( Table 2). Heteromys gaumeri was the most infected species (100%, 6/6) captured in the forest fragment suggesting that cutaneous leishmaniasis remains mainly a sylvatic zoonotic disease in the Yucatan State. e high infection rate for O. phyllotis (60%, 3/5) was observed in the abandoned cornfield (Table 2), and the similar proportions of the animals caught in both the abandoned cornfield and the perirural site suggest that this species could be maintaining the L. (L.) mexicana transmission cycle in human-disturbed areas.

Conclusion
Our

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest.