Molecular Detection of Leptospira interrogans and Borrelia burgdorferi in Wild Rodents from Mexico

Abstract

Leptospirosis and Lyme borreliosis are zoonotic emerging diseases of global importance and wide distribution. The aim of this study was to detect by molecular testing to Leptospira interrogans and Borrelia burgdorferi sensu lato in wild rodents from Nuevo Leon, Quintana Roo, and Campeche, Mexico. This study is the first in report to Chaetodipus nelsoni, Dipodomys merriami, and Peromyscus eremicus infected with L. interrogans in Mexico. Besides, Chaetodipus hispidus, Heteromys gaumeri, Heteromys irroratus, Neotoma micropus, Peromyscus leucopus, Peromyscus maniculatus, and Sigmodon hispidus infected with B. burgdorferi s.l. in Mexico. Also, is the first report in identify coinfection of L. interrogans and B. burgdorferi s.l. in wild rodents such as H. irroratus and S. hispidus in Nuevo Leon, and H. gaumeri in Quintana Roo, Mexico. These wild rodent species infected represent a risk factor for the exposed population in these sylvatic and rural areas of Mexico.

Introduction

Leptospirosis and Lyme borreliosis (also known as Lyme disease) are zoonotic emerging diseases of global importance and wide distribution. The natural enzootic cycles of Leptospira interrogans and Borrelia burgdorferi s.l. are complex and differ by geographical location and time. Many wild vertebrate animals may act as reservoirs of L. interrogans and B. burgdorferi s.l. especially rodents (Steere et al. 2017, Vieira et al. 2018).

In Mexico, L. interrogans has been documented in wild terrestrial mammals (Cantú et al. 2008, Sotomayor-Bonilla 2009, Méndez et al. 2013, López-Pérez et al. 2017). Within wild animals, it has been found in rodent species as Baiomys musculus, Heteromys irroratus, Oryzomys alfaroi, Peromyscus leucopus, and Sigmodon hispidus in the Tamaulipas State (Méndez et al. 2013); Peromyscus maniculatus, Peromyscus levipes, and Reithrodontomys megalotis in the Mexico State (González-Dardayrol 2004); Oryzomys couesi cozumelae in Quintana Roo State (Sotomayor-Bonilla 2009); Heteromys gaumeri and Ototylomys phyllotis in Campeche State (Espinosa-Martínez et al. 2015); and H. gaumeri in Yucatan State (Torres-Castro et al. 2018). In contrast, in Mexico, B. burgdorferi s.l. has been reported in wild animals such as white-tailed deer (Martinez et al. 1999), and rodents Neotomodon alstoni and Peromyscus melanotis (Gordillo-Pérez et al. 2010).

Zoonotic infections such as leptospirosis and Lyme borreliosis require a strong understanding of their ecology and epidemiology. Thus, the study of wild reservoir hosts is necessary for understanding these emerging infectious diseases in the north and south of Mexico. In this way, the aim of this study was to detect L. interrogans and B. burgdorferi s.l. in wild rodents from Nuevo Leon, Quintana Roo, and Campeche, Mexico.

Materials and Methods

Study sites

In the Nuevo Leon State, the study was carried out in five different municipalities: (1) Linares (24°40′08″N, 099°36′16″W; 446 meters asl), (2) Santiago (25°22′39″N, 100°14′31″W; 1304 meters asl), (3) Escobedo (25°48′22″N, 100°17′17″W; 447 meters asl), (4) Cadereyta Jimenez (25°25′35″N, 099°58′23″W; 325 meters asl), and (5) Mina (26°00′04″N, 100°30′40″W; 574 meters asl), from April to August 2010. Whereas in the Quintana Roo State, the sampling was in “Limones” (18°59′13.1″N, 088°09′32.5″W; 24 meters asl) from October 2013 to July 2014. And in the Campeche State, the field work was in “Union 20 de Junio” (18°49′11″N, 89°17′59″W; 212 meters asl) during the months of December 2014, and February and April 2016.

Biogeographically, Nuevo Leon is part of the Nearctic region with four different provinces: Altiplano Mexicano, Tamaulipas, Sierra Madre Oriental, and the Gulf of Mexico. Whereas Quintana Roo and Campeche are part of the Yucatan Peninsula as the biotic province, Caribbean subregion, and Neotropical region.

Rodents sampling and processing

Rodents were collected using collapsible live Sherman traps^® (HB Sherman Traps, Inc., Tallahassee, FL), which was baited with a mix of oat flakes impregnated with vanilla essence. All traps were set at ground level at intervals of 25 meters on a single transect per site. The capture effort of Nuevo Leon, Quintana Roo, and Campeche was 450, 342, and 1200 trap-nights, respectively. All rodents were sexed, weighed, measured, and taxonomically identified (Ceballos and Oliva 2005, Ramírez-Pulido et al. 2014). Each rodent was manipulated with the proper biosafety precautions, and all rodents were euthanized with inhaled ether, according to the guidelines of the American Society for the Use of Mammalogists of Wild Mammals in Research (Sikes et al. 2011). Samples of kidney and spleen were obtained of everyone with disinfected surgical material. All tissues were coded and preserved in 2 mL sterile Eppendorf vials containing 70% ethanol. Each voucher specimen was transported and placed in a plastic bottle with 70% ethanol and sealed with tape, and after those were deposited at the Autonomous University of Nuevo Leon. The collection permit was issued by the General Directorate of Wildlife of Mexico (SGPA/DGVS/004717/11 and SGPA/DGVS/04631/14).

Molecular testing of L. interrogans and B. burgdorferi from rodents

All tissues were kept in a −20°C freezer until they were processed for molecular analyses. Tissue breakage was performed from the intestine samples using the technique of FastPrep^®-24 (MP Biomedicals). The extraction of DNA was carried out by the method proposed by Ferrer et al. (2001). The PCR was carried out using the 50 μL Master Mix commercial kit in a thermal cycler MJ Research PTC 1000 for each strain. In the search for L. interrogans primers were used that amplify the LipL32 gene described by Ferreira et al. (2014). 45F (5′-AGG CAT TAC CGC TTG TGG-3′) y 286R (5′-GAA CTC CCA TTT CAG CGA TT-3′). The amplification program used was a predenaturation step of 95°C for 2 min, followed by denaturation of 95°C for 15 s, alignment temperature of 60°C for 1 min and an extension of 72°C for 20 s for a total of 35 cycles and a final extension of 72°C for 7 min (Ferreira et al. 2014).

For the PCR of B. burgdorferi, the sequence of the Flagellin B (FlaB) region was amplified, using the primers for species Bbs 1-1 (5′AACACACCAGCATCACTTTCAGG-3′), and Bbsl-3c (5′-GAGAATTAACTCCGCCTTGAGAAGG-3′) (Jaulhac et al. 2000). For the detection of the 230 bp fragment of B. burgdorferi, the conditions used were 40 cycles of denaturation at 93°C for 30 s, alignment at 57°C for 30 s, and extension at 72°C for 30 s, with an initial incubation at 94°C for 1 min and a final extension at 72°C for 1 min. In each amplification reaction, control tubes were placed in which DNA was not added and the final volume was adjusted with sterile deionized water. The analyses of the results of the PCR products were prepared by 1.5% agarose gels. Five microliters of PCR product was placed with 5 μL of GelRedTM Nucleic Acid Gel Stain and 5 μL of bromophenol blue. The gel was placed underwater electrophoresis cuvette at a voltage of 90 V (Sambrook et al. 1989) for an hour.

Results and Discussion

With this study, the number of wild rodent species increases to 14 species infected with Leptospira spp. in Mexico (González-Dardayrol 2004, Sotomayor-Bonilla 2009, Méndez et al. 2013, Espinosa-Martínez et al. 2015). Of the six rodent species infected with L. interrogans in Nuevo Leon, only two species (P. maniculatus and S. hispidus) were the most abundant and occurrence throughout the select locations of Nuevo Leon (Table 1). To our knowledge, there is only one study in Tamaulipas state that reported the presence of Leptospira in five wild rodents' species (Méndez et al. 2013) and coincides in part with this study. Three species (H. irroratus, P. leucopus, and S. hispidus) in both studies were captured, but only 1 species (P. leucopus) was not detected with infection to L. interrogans in 12 specimens from Nuevo Leon of this study, but in Tamaulipas state it was detected with Leptospira in 3 of 6 specimens (Méndez et al. 2013). One possible explanation is that Méndez et al. (2013) sampled in an extensive beef cattle production center, where also found to bovine, equine and canine infected with serovars of Leptospira. This close contact of domestic species with wild rodents in modified environments makes the maintenance relevant to this pathogenic Leptospira. Whereas in this study, P. leucopus was captured in pine-oak forest from Santiago, and crops (sorghum and oats) from Cadereyta Jimenez, Nuevo Leon. Another possible explanation is the sampling time, Méndez et al. (2013) do not mention the sampling period, whereas in this study was in June (Santiago) and August (Cadereyta Jimenez).

Table 1.

Species of Wild Rodents from Nuevo Leon, Quintana Roo, and Campeche, Mexico

Rodent species	Sample size	Leptospira interrogans			Borrelia burgdorferi s.l.
Rodent species	Sample size	Nuevo Leon, n (%)	Quintana Roo, n (%)	Campeche, n (%)	Nuevo Leon, n (%)	Quintana Roo, n (%)	Campeche, n (%)
Chaetodipus hispidus	3	0/3 (0)	0	0	1/3 (33)	0	0
Chaetodipus nelsoni	3	1/3 (33)	0	0	0/3 (0)	0	0
Dipodomys merriami	1	1/1 (100)	0	0	0/1 (0)	0	0
Heteromys gaumeri	17	0	3/11 (27)	0/6 (0)	0	3/11 (27)	0/6 (0)
Liomys irroratus	1	1/1 (100)	0	0	1/1 (100)	0	0
Neotoma micropus	2	0/2 (0)	0	0	1/2 (50)	0	0
Ototylomys phyllotis	15	0	0/1 (0)	0/14 (0)	0	0/1 (0)	0/14 (0)
Peromyscus eremicus	1	1/1 (100)	0	0	0/1 (0)	0	0
Peromyscus leucopus	12	0/12 (0)	0	0	3/12 (25)	0	0
Peromyscus maniculatus	30	5/30 (17)	0	0	8/30 (27)	0	0
Peromyscus yucatanicus	8	0	0	0/8 (0)	0	0	0/8 (0)
Sigmodon hispidus	21	5/21 (24)	0	0	4/21 (19)	0	0
Total (%)	114	14/74 (19)	3/12 (25)	0/28 (0)	18/74 (24)	3/12 (25)	0/28 (0)
N° species	12	9	2	3	9	2	3
N° species infected		6	1	0	6	1	0

With molecular prevalence data (%) of L. interrogans from kidney samples and B. burgdorferi s.l. from spleen samples.

Of the two species of wild rodents collected in Quintana Roo, only one (H. gaumeri) presented infection with L. interrogans, thus the first report in the state. But not like that in Campeche, none of the three species of rodents (H. gaumeri, O. phyllotis, and Peromyscus yucatanicus) were detected with L. interrogans. The first study in detect L. interrogans in wild rodents (H. gaumeri and O. phyllotis) from Calakmul, Campeche was by Espinosa-Martínez et al. (2015). This does not coincide with the data of this study. A possible explanation is the time of sampling and the sampling place. The sampling by Espinosa-Martínez et al. (2015) was in August; whereas in this study was in the cold season (December, February, and April). And the area of this study is 105 km to north with reference to Espinosa-Martínez et al. (2015).

With respect to B. burgdorferi, only one study (Gordillo-Pérez et al. 2010) has documented the existence of B. burgdorferi sensu stricto in wild rodents (N. alstoni and P. melanotis) from Central Mexico. This study confirms the presence of B. burgdorferi s.l. in wild rodents and joins the list to seven species of rodents infected naturally with B. burgdorferi in Nuevo Leon (P. maniculatus, P. leucopus, Chaetodipus hispidus, Neotoma micropus, H. irroratus, S. hispidus) and Quintana Roo (H. gaumeri), Mexico. Thus, it is added in total nine species of wild rodents in Mexico.

Of the seven species of rodents that were found infected naturally with B. burgdorferi s.l. in this study, only P. leucopus has been identified as a reservoir host (or reservoir competence) of B. burgdorferi in the northeastern United States (e.g., Anderson et al. 1987, Donahue et al. 1987). The other six species of rodents (P. maniculatus, C. hispidus, N. micropus, H. irroratus, S. hispidus, and H. gaumeri) are called carrier hosts since it would be necessary to do more studies to incriminate as reservoir hosts. However, species such as P. maniculatus and S. hispidus with putative to reservoir host have been found infected with B. burgdorferi s.l. in the United States (e.g., Burgdorfer and Gage 1987, Clark et al. 2002) and Canada (e.g., Bouchard et al. 2011).

Remarkably, of nine rodent species captured in Nuevo Leon, six were found infected with B. burgdorferi s.l. In three (Escobedo, Santiago y Cadereyta) of five localities of Nuevo Leon, B. burgdorferi s.l. was found in all rodent species captured. This represents a high number of rodent species infected with these bacteria in Nuevo Leon, Mexico. One study of United States found only three species positive to B. burgdorferi s.l. of eight rodent species tested (Clark et al. 2002). In line, it is necessary to know the richness of rodent species in these sites of Nuevo Leon, and as Ostfeld and Keesing (2000a) mentioned diversity could play an important role in the maintenance of disease and in the probability of its transmission to humans. Also, the Dilution Effect model, which predicts that high species diversity in the community of hosts reduces the infection prevalence (Ostfeld and Keesing 2000a, 2000b). Of the rodent species that is present in Mexico, there is no knowledge about the role of reservoir hosts of B. burgdorferi. It is necessary to have more studies that document the presence of pathogen, vector, and mammal hosts.

In this study, three species of rodents (H. irroratus, S. hispidus, and H. gaumeri) were found with coinfection to L. interrogans and B. burgdorferi in Nuevo Leon and Quintana Roo. Also, P. maniculatus was found infected with both bacteria (L. interrogans and B. burgdorferi) but in different specimens captured in Escobedo, Santiago, and Cadereyta Jimenez in Nuevo Leon, Mexico. There is evidence that shows the coinfection of Leptospira and Borrelia in wild mammals from the United States (Magnarelli et al. 1986) and this coincides with our results.

In conclusion, we documented that L. interrogans share the same or a similar enzootic cycle with B. burgdorferi in Cadereyta Jimenez, Santiago, and Escobedo, Nuevo Leon, a border state to the United States located in northeastern Mexico; and in southern Mexico, particularly in Limones, Quintana Roo. In line with this and the light of One Health, it is necessary to do long-term longitudinal studies with the objective of improving the understanding of the ecology of these diseases in population animals, vectors, and human cases. And can determine possible areas of transmission to humans.

Footnotes

Acknowledgments

We extend our recognitions to the people of different communities of Nuevo Leon, Quintana Roo, and Campeche, Mexico. We express our gratitude to Dr. Eduardo Alfonso Rebollar-Téllez for all the support logistic in the field. We also thank the reviewers for helping to improve the article.

Author Disclosure Statement

No conflicting financial interests exist.

Funding Information

This study received funding from the Universidad Autonoma de Nuevo Leon through the Facultad de Medicina Veterinaria y Zootecnia.

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