Presence of Antibodies to Leptospira spp. in Black-tailed Prairie Dogs (Cynomys ludovicianus) and Beavers (Castor canadensis) in Northwestern Mexico

Leptospirosis is a global infectious disease of animals and humans caused by spirochetal bacteria belonging to the genus Leptospira (Adler and de la Peña-Moctezuma 2010). The infection is usually transmitted through direct contact with infected animals or by exposure to contaminated water or soil (Haake and Levett 2015). Twenty species of Leptospira are currently described, of which at least 14 are considered pathogenic. Among these, 24 serogroups and 300 pathogenic serovars of Leptospira with broad mammalian host range have been reported (Zhang et al. 2015). Domestic and wildlife species can act as reservoirs and/or incidental hosts of Leptospira spp., and different species of rodents have been recognized as important maintenance hosts and common sources of infection for livestock, pets, and humans (Vado-Solís et al. 2002; Meerburg et al. 2009). Although rodent reservoirs often maintain leptospirosis as a chronic infection in the renal tubules without any clinical signs (Monahan et al. 2009), the disease can appear in rodents species, such as beavers, especially when their immune system is compromised (Nolet et al. 1997), or as incidental hosts (Schreiber et al. 2015). In the context of epidemiology, infection can vary with some individual attributes such as sex and age of rodent hosts (Cosson et al. 2014). Prevalence of Leptospira antibodies in rodents is reported worldwide, but little is known about their presence in prairie dogs (Cynomys spp.) and beavers (Castor spp.) in Mexico.

Prairie dogs and beavers are considered keystone species and ecosystem engineers. Both rodent species contribute to creating, maintaining, and diversifying habitats (Rosell et al. 2005; Bangert and Slobodchikoff 2006), and every single factor affecting their populations, such as infectious diseases, can have severe consequences on ecosystem structure and function. The study of potential pathogens in wildlife, particularly in those species of conservation concern, is important for the development and implementation of policies and management programs for the prevention and control of these infectious diseases.

We investigated the presence of Leptospira spp. antibodies in two wild rodents and how population structure (age and sex) might be related to the prevalence of this bacteria in the San Pedro River Basin (SPRB) in northern Sonora, Mexico. The SPRB extends 9,800 km² from the headwaters in Cananea, Sonora to the confluence with the Gila River in Arizona. In Mexico (Fig. 1), the upper basin includes 1,895 km² (30°37′ to 31°19′N, 109°48′ to 110°37′W; Boykin et al. 2012). Between the Chihuahuan and the Sonoran Deserts, the SPRB is in a transitional zone and comprises a mosaic of grassland, mesquite woodland, oak forest, and riparian vegetation. The SPRB provides habitat for several species of conservation concern such as the black-tailed prairie dog (Cynomys ludovicianus) and the North American beaver (Castor canadensis). Both are considered as threatened species by the Mexican federal government (Secretaria de Medio Ambiente y Recursos Naturales 2010). Two colonies of prairie dogs have been recognized in SPRB; however, the largest colony was extirpated in recent years, and the remaining colony (Palmitas) contains only about 300 prairie dogs (Moreno 2013). Palmitas is a genetically unique colony, and has no connectivity with the colonies from Chihuahua and the US (Castellanos-Morales et al. 2015). On the other hand, little is known of the ecology and viability of beaver populations in Mexico.

We captured rodents during fall 2015 (October–November). Black-tailed prairie dogs were captured using Tomahawk traps (47.5×15×15 cm; Tomahawk Live Trap Co., Hazelhurst, Wisconsin, USA) and baited with corn, oats, wheat, and molasses, and handled in canvas bags. Beavers were live-trapped with landing nets and were chemically restrained with an intramuscular injection of ketamine hydrochloride (10 mg/kg, Anesket®, Pisa, Atitalaquia, Hidalgo, Mexico) and xylazine hydrochloride (2 mg/kg, Procin®, Pisa). From each individual, we recorded sex and age (adult or juvenile). We collected blood samples from the caudal (beaver) and femoral veins (prairie dog), which we centrifuged at 1,400 × G for 5 min to obtained serum that was frozen at −20 C until laboratory analysis. After handling, animals were ear-tagged (National Band and Tag Co., Newport, Kentucky, USA). Our procedures for handling rodents were done in accordance with the guidelines of the American Society of Mammalogists (Sikes and Gannon 2011) and were approved by the Ministry of Environment and Natural Resources of Mexico (permits DGVS/12532/14 and DGVS/12431/15).

We performed microscopic agglutination tests for the detection of both immunoglobulins M and G on all serum samples. The serum samples were assayed using 12 live antigens of Leptospira spp. serogroups: Autumnalis, Bataviae, Australis (var. Bratislava), Canicola, Celledoni, Grippotyphosa, Sejroe (var. Hardjo prajitno), Icterohaemorrhagiae, Pomona, Pyrogenes, Tarassovi, and Serjoe (var. Wolffi). We diluted each serum sample to 1:25 dilutions (60 μL of serum and 1,440 μL of saline solution). We placed 50 μL of 1:25 serum dilution in each well in flat, 96-well plastic microtiter plates (Sarstedt, Nümbrecht, Germany), organized in columns by serotype; 50 μL aliquots of the antigen were added for the screening test. The negative control consisted of saline solution with the antigen. Plates were incubated at 30 C for 2 h, and the serum-antigen reactions were observed in a dark-field microscope. Reactive samples were retested in several twofold dilutions beginning at 1:10 to determine the final titer of serum for each serotype. The reciprocal of the highest dilution with 50% agglutination of Leptospira was considered the sample titer. Based on practical approaches, we considered titer ≥1:100 as seropositive (Faine et al. 1999). Fisher's exact test was used to evaluate Leptospira associations with rodent sex and age. We considered a significance level of α=0.05. The analyses were performed using R software (R Development Core Team 2014).

We detected antibodies to Leptospira spp. in 12 of 26 (46%) black-tailed prairie dogs and in two of the three (67%) beavers sampled. We did not find differences between sex and age concerning Leptospira positive rodents. We identified seven serogroups: Autumnalis, Australis, Bataviae, Canicola, Celledoni, Grippotyphosa, and Sejroe (Table 1). Serum positivity for antibodies against multiple serogroups occurred in 17% of tested samples. The antibody titers for antibody-positive rodents varied from 1:100 to 1:200 (Table 1); however, none of the animals showed clinical signs of leptospirosis disease.

This is the first evidence of Bataviae, Grippotyphosa, and Sejroe serogroups in prairie dogs. Our finding of Australis as the most prevalent Leptospira serogroup in black-tailed prairie dogs was consistent with others serological studies in free-ranging (Montiel-Arteaga et al. 2015) and captive black-tailed prairie dogs (Olds et al. 2015). The only one Canicola-positive rodent was a beaver caught in the surroundings of an agricultural area located at 4.7 km from the nearest human settlement. This might be explained by the presence of stray dogs, which are the main reservoir of Canicola serogroup. The two beavers that were not positive to Canicola serogroup were caught upstream from the positive one, far from any human settlement and agricultural areas. In addition, we found antibodies against Autumnalis and Australis serogroups in beavers. Other studies have described the occurrence of several Leptospira serogroups, including those we report, in North American beavers from Louisiana (Stuart et al. 1978), and in Eurasian beavers (Castor fiber) from Norway (Goodman et al. 2012). Additionally, it is important to note that Icterohaemorrhagiae serogroup can cause mortality in captive North American beavers (Mettler 1975) and in free-ranging Eurasian beavers (Nolet et al. 1997; Goodman et al. 2012). The deaths of the free-ranging beavers were attributed to an immune system suppression associated to stress of trapping and translocation in different reintroduction programs. Although we did not find the Icterohaemorrhagiae serogroup, we found Leptospira serogroups circulating in beavers from SPRB that might represent a potential risk of infectious disease to animals during the translocation process. In order to avoid immunosuppression and therefore an increase in the prevalence of diseases, it is imperative to lessen the stress associated with the management of beavers in this reintroduction and conservation program. The finding of Leptospira spp. in black-tailed prairie dogs from Sonora suggest endemic infections but it is still important to identify how Leptospira bacteria affect these two species and their role as competent reservoirs or incidental hosts. Our study shows the importance of a long-term monitoring project to understand the dynamics of Leptospira in the prairie dog and beaver systems.

This study is part of the health survey of black-tailed prairie dogs and the beaver reintroduction program funding by the Recovery Programs for species at risk of The National Commission of Natural Protected Areas of Mexico (PROCER-CONANP). The Alianza World Wildlife Fund–Fundación Carlos Slim sponsored the work.