New wildlife hosts of Leptospira interrogans in Campeche, Mexico.

Leptospira interrogans has been identified to cause leptospirosis, a widespread zoonotic disease that has been identified in domestic and wild animals. This work analyzed kidneys from two species of wild rodents from the state of Campeche, Mexico. Analyses were made by PCR using specific primers for detection of Leptospira interrogans DNA. The rodent species that tested positive were Heteromys gaumeri and Ototylomys phyllotis, both of which are new hosts for the bacteria in Southeastern Mexico. These records provide new insights into the disease's transmission that should be studied carefully in order to identify other potential host species, including humans, which are at risk of becoming infected if they are in contact with infected wildlife.

Several species of the genus Leptospira cause leptospirosis, a zoonosis of urban distribution3 , 6. Wild and domestic mammals (160 species) have been identified as hosts for these bacteria worldwide2 , 6. Leptospira interrogans has mainly been identified in domestic mammals because they have direct contact with humans4 , 5 , 6. However, in Neotropical areas, such as Panama21, the Peruvian Amazon6 , 8 and the city of São Paulo16, some wild mammals (bats, carnivores, marsupials and rodents) have been identified as hosts of L. interrogans.

In Mexico, records of wildlife hosts for L. interrogans are scarce and widely scattered across different states (e.g. Didelphis virginianus in Yucatán25 [Southeastern Mexico], Odocoileus virginianus in Coahuila9 [Northern Mexico] and Zalophus californianus in the Gulf of California1 , 18). A study carried out in Cozumel, Quintana Roo, identified a 21.5% seroprevalence of Oryzomys couesi cozumelae 24. In Tamaulipas, Northeastern Mexico, five species of wild rodents (Baiomys musculus, Liomys irroratus, Oryzomys alfaroi, Peromyscus leucopus and Sigmodon hispidus) tested positive for different serovars of L. interrogans by Microscopic Agglutination Technique (MAT)22. However, there are no records of wildlife hosts reported in Campeche, and in the Yucatan Peninsula only one species of rodent has been previously reported24. For this reason, the aim of this paper is to report two new species of wild rodents that are hosts of L. interrogans in Calakmul, Campeche, Mexico.

Ten rodents were collected (collection permit FAUT-0170) on August 17th, 2013 from the Yaax'che camp, Calakmul, Campeche, Mexico (located 43 km SSE from the archeological zone of Calakmul, 18° 29' 14" N, 89° 53' 57" W). These specimens were killed in compliance with the guidelines of the American Society of Mammalogy for the Use of Wildlife Mammals in Research17. All specimens were identified and deposited at the Museo de Zoología "Alfonso L. Herrera" in the Facultad de Ciencias (MZFC) of the Universidad Nacional Autónoma de México.

For the identification of Leptospira DNA in these rodents, one kidney was aseptically collected and deposited in 70% ethanol. A portion of 25 mg of kidney tissue was processed for DNA extraction using the QIAamp® DNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer's specifications (using the Purification of Total DNA from Animal Tissues Protocol). After extractions were done, a multiplex PCR was performed using primer sets G1/G2 (specific for the detection of pathogenic leptospires) and B64I/B64II (specific for Leptospira kirschneri) with expected products of 285 bp and 563 bp, respectively19. Additionally, the positive samples were analyzed using specific primers for the identification of pathogenic leptospira species23. The reaction mixture consisted of 12.5 μL of GoTaq® Green Master Mix, 2X of Promega Corporation (Madison, WI, USA), using a pair of primers, Intergroup A fwd and Intergroup A rev (100 ng each), 6.5 μL nuclease-free water and 200 ng DNA in a final volume of 25 μL.

In order to minimize cross-contamination and to avoid false positive results, a negative control (i.e. reaction mix without DNA) and a positive control (i.e. reaction mix and L. interrogans serovar Pomona DNA) were both included. Each PCR reaction was performed in triplicate.

The PCR products were analyzed by electrophoresis on 1.5% agarose gels, using a 100 bp molecular weight marker (Nucleic Acid Markers, LMW DNA Ladder of BioLabs) in 1X TAE buffer. Gels were stained with SYTO® 60 nucleic acid stain (Invitrogen by Life Technologies CA, USA) and visualized using an ODYSSEY CLx Imaging System (LICOR Biosciences).

Two rodents collected at Yaax'che camp, Calakmul, Campeche, Mexico that tested positive using the G1/G2 primers were identified as Heteromys gaumeri (temporary catalog RAVGA014) and Ototylomys phyllotis (temporary catalog RAVGA013).

These tests were confirmed positive with primers of Intergroup A designed by REITSTETTER23, which specifically amplify a segment of 396 bp of L. interrogans DNA. Leptospira kischneri was not detected in any of the samples analyzed, and the DNA of L. interrogans was not found in any of the negative controls (Fig. 1).

Climate affects the timing and intensity of outbreaks of infectious diseases14 , 15. It has been stated by several authors3 , 6 , 20 , 26 that adverse climatic events, such as hurricanes and floods, are related to the timing and intensity of Leptospira outbreaks. In the case of the present study, the presence of two tropical storms that occurred before and after the specimen's collection12 , 13, allowed for speculation regarding the study's findings of L. interrogans.

This is the first work that identifies Heteromys gaumeri and Ototylomys phyllotis as new hosts for L. interrogans, by using the set of primers designed by REITSTETTER23 to identify pathological samples. Moreover, the study area in which the specimens were collected corresponds to a new locality in Mexico, where the presence of the bacteria had not been previously reported. The presence of L. interrogans in wild rodents from the same locality should be studied carefully in order to identify the possibility of other species and particularly humans of this area being infected. The author's suggestion is based on previous studies made on domestic animals and humans. In the case of domestic animals (bovines, pigs and dogs) a study revealed a general positivity of 30.5%10, while a more recent study showed a general positivity of 21.3% registered in dogs of Campeche city7. Particularly in the case of human leptospirosis, incidence varied from 0.7-2.2/100,000 inhabitants, with a general seroprevalence of 14.2%11 , 25.

Since extreme weather events have been reported to promote the presence of Leptospira outbreaks6, it is essential to further analyze potential reservoirs of several pathogenic species of Leptospira in order to identify the dynamics of the transmission between wild mammals and peri-urban human populations, in order to reduce the risks of a potential leptospirosis outbreak in vulnerable groups such as biologists, national and foreign campers and tourists that visit the study area.