First histopathological study in kidneys of rodents naturally infected with Leptospira pathogenic species from Yucatan, Mexico

Marco Torres-Castro, Leonardo Guillermo-Cordero, Silvia Hernández-Betancourt, Edwin Gutiérrez-Ruíz, Piedad Agudelo-Flórez, Ronald Peláez-Sánchez, Jorge Zavala-Castro, Fernando I. Puerto

1Centro de Investigaciones Regionales ‘Dr. Hideyo Noguchi’, Universidad Autónoma de Yucatán, Mérida, Yucatán, México

2Campus de Ciencias Biológicas y Agropecuarias, Universidad Autónoma de Yucatán, Mérida, Yucatán, México

3Facultad de Medicina, Universidad CES, Medellín, Antioquia, Colombia

First histopathological study in kidneys of rodents naturally infected with Leptospira pathogenic species from Yucatan, Mexico

Marco Torres-Castro1*, Leonardo Guillermo-Cordero2, Silvia Hernández-Betancourt2, Edwin Gutiérrez-Ruíz2, Piedad Agudelo-Flórez3, Ronald Peláez-Sánchez3, Jorge Zavala-Castro1, Fernando I. Puerto1

1Centro de Investigaciones Regionales ‘Dr. Hideyo Noguchi’, Universidad Autónoma de Yucatán, Mérida, Yucatán, México

2Campus de Ciencias Biológicas y Agropecuarias, Universidad Autónoma de Yucatán, Mérida, Yucatán, México

3Facultad de Medicina, Universidad CES, Medellín, Antioquia, Colombia

ARTICLE INFO

Article history:

Received 15 November 2015

Received in revised form 20 December 2015

Accepted 15 January 2016

Available online 20 February 2016

Leptospira spp.

Renal histopathological lesions

Mus musculus

Rattus rattus

Objective: To report the renal histological lesions in synanthropic rodents, Mus musculus and Rattus rattus, naturally infected with Leptospira spp., captured in a rural community in Yucatan, Mexico. Methods: Kidney samples of synanthropic rodents were collected from a rural community in Yucatan, Mexico. Polymerase chain reaction was used to detect Leptospira spp. infection. Tissue kidney was fixed in 10% buffered formalin, processed according to the usual techniques for paraffin inclusion, cut and stained with hematoxylin and eosin, and examined using a conventional electronic microscope. Results: A total of 187 rodents were captured. Nine individuals (4.8%) were positive for Leptospira spp. in the molecular analysis. All renal lesions observed in the histopathological study had been reported previously for Leptospira spp. infection. Conclusions: The histopathological lesions are present in the kidneys, plus the results of the polymerase chain reaction confirm that these rodents are true carriers of Leptospira spp.

1. Introduction

Leptospirosis is a zoonosis of worldwide importance caused by multi-host pathogens within the genus Leptospira. Renal carriage is the key to the persistence and epidemiology of leptospirosis; Leptospira spp. colonizes the renal tubules of animal kidneys and are released into the environment with urine[1,2]. The infection may be transmitted to human beings and other animals by direct contact or indirect exposure to urine from mammalian hosts suchas synanthropic rodents and farms, wild and domestic animals[3]. It is well known that rodents are the main reservoir for Leptospira because they are persistent renal carriers, but rarely develop symptoms and are not impaired by the infection of their kidneys[4,5]. The kidney is a primary target of Leptospira during both acute and chronic infection, where conditions in the renal tubules favor Leptospira survival[6,7]. After infection, damage produced by spirochetes depends on several variables, such as pathogenicity and virulence of the serovar to the host and stage of the disease[8]. Histopathological lesions caused by Leptospira in naturally infected rodent reservoirs have been poorly described around the world[3,9].

2. Materials and methods

The mice for this study were collected from the rural communityof Molas, Yucatan, Mexico (20°40’N, 89°38’W). The predominant vegetation type was tropical low deciduous forest and the climate was warm sub-humid with summer rains (Aw0). The site was divided into four grids, imagining two perpendicular axes that crossed at its center. In each grid, ten households were selected for convenience and sampled for three consecutive nights each month. The sampling period was from October 2011 to March 2012. Twelve Sherman traps (7.5 cm×23 cm×9 cm, HB Sherman Traps Inc?, Tallahasse, Florida, USA) were set in each household. Traps were baited with oatmeal and vanilla flavoring, set in the morning and examined the following morning.

Capture, management and euthanasia of the rodents was conducted in compliance with the specifications of the American Society of Mammalogists. The captured rodents were transferred to the Zoology Laboratory of the Campus de Ciencias Biológicas y Agropecuarias, Universidad Autónoma de Yucatán. After being anaesthetized with ether, the rodents were euthanized by cervical dislocation. Autopsies were conducted in order to collect both kidneys. One kidney was preserved to -70 ℃ until it was used in the polymerase chain reaction (PCR) test, and the other was fixed in 10% buffered formalin and processed according to the usual techniques for paraffin inclusion. Next, 3 μm paraffin sections were cut and stained with hematoxylin and eosin (H-E) and examined using a conventional electric microscope with the lens 10×, 40× and 100×. The lesions were classified according to severity levels as mild (＜30% affected), moderate (30%-50%) and severe (＞50%). For the identification of Leptospira DNA in rodents, the kidneys preserved -70 ℃ were processed according the method established by Torres-Castro et al[10].

3. Results

A total of 187 rodents were captured: 57 (30.48%) Rattus rattus (Rr) and 130 (69.51%) Mus musculus (Mm). All rodents appeared healthy under external physical examination. Nine (9/187, 4.81%) individuals were positive for Leptospira spp. in the PCR: seven Rr and two Mm. The H-E histopathological findings of the positive rodents to Leptospira spp. by PCR were described in the Table 1.

4. Discussion

A broad range of morphological alterations were detected in the kidneys of rodents naturally infected with Leptospira spp., captured in a rural community of Yucatan, Mexico. In the persistent colonization cases as in reservoir animals, Leptospira spp. cause systemic infection but are subsequently cleared from all organs except the renal tubules. The renal tubule is an immune-privileged site, a feature that may contribute to high-grade persistence of the pathogen[11].

The lesions observed in the kidneys of these rodents have been reported previously for Leptospira spp. infection[4]. The most common lesion we detected was the lymphoplasmacytic inflammatory infiltrate, which has been reported by Agudelo-Florez et al[9], Athanazio et al[12] and Monahan et al[6]. The inflammatory infiltrate is a primary lesion during acute renal injury in leptospirosis and can be caused by direct damage to host tissue by Leptospira or the presence of leptospiral antigen, initiating a renal immune response[13]. However, this lesion cannot be attributed solely to Leptospira spp. since these rodents could also be exposed to other unknown environmental infectious agents that could cause renal lesions[9].

Other present lesions like edema and cell hyperplasia are also reported by others authors like Tucunduva de Faria et al[3]. Also, tubular necrosis, have been reported in kidneys from human beings affected with Leptospira spp. infection[14,15], slaughtered pigs[16], and experimentally infected hamsters[17]; therefore our findings are most likely to be caused by the spirochete present in the kidneysof reservoir rodents. The histopathological lesions present in the kidneys of the rodents, plus the positive results of the PCR, confirm that these rodents are true carriers of Leptospira spp., and this make it a likely source of infection for the people in the study site.

Table 1Description of H-E histopathological lesions in kidneys of Rr and Mm positives by PCR for Leptospira spp.

This is the first study to describe and report the renal histological lesions in synanthropic rodents, Mm and Rr naturally infected with Leptospira spp, captured in a rural community in Yucatan, Mexico.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgements

Our sincere thanks go to PROMEP-México-Proyecto 103.5/09/1258 (Red Epidemiológica de Enfermedades Zoonóticas y Transmitidas por Vector de Importancia en Salud Pública) and to the Consejo Nacional de Ciencia y Tecnología de México, for the financial support granted to this Project (No. grant, 320021).

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ent heading

10.1016/j.apjtm.2016.01.018

*Corresponding author: Marco Torres-Castro, Laboratorio de Enfermedades Emergentes y Reemergentes, Centro de Investigaciones Regionales ‘Dr. Hideyo Noguchi’, Universidad Autónoma de Yucatán, Mérida, Yucatán, México.

Tel: (999) 9245809

Fax: (999) 9236120

E-mail: antonio.torres@correo.uady.mx

Foundation project: Itis supported by PROMEP-México-Proyecto 103.5/09/1258 (Red Epidemiológica de Enfermedades Zoonóticas y Transmitidas por Vector de Importancia en Salud Pública) and Consejo Nacional de Ciencia y Tecnología de México (No. grant, 320021).