Detection of anti-Leptospira spp. agglutinins in captive South American river turtles, Podocnemis expansa

Leptospirosis is a zoonosis transmitted by contact with infected urine or water contaminated with the agent. Searches for Leptospira spp. in reptiles are scarce although most species have contact with aquatic environments. We evaluated the presence of anti-Leptospira spp. antibodies in Podocnemis expansa housed at the Amazonian Zoobotanical Garden, in Belém, Pará state, Brazil. We analyzed 74 serum samples through the microscopic agglutination test using 31 live antigens from different Leptospira spp. serogroups. Thirty samples (40.5%) were positive against Leptospira spp., with titrations between 100 and 3,200 for one or more serogroups. The Hebdomadis serogroup was the most prevalent, with 26 (87%) out of the 30 positive samples, followed by Djasiman, with two (7%) and Celledoni and Bataviae with one (3%) sample each. The detection of anti-Leptospira spp. agglutinins in P. expansa suggests that the aquatic environment is a transmission route for this pathogen among chelonians.

Bacteria of the genus Leptospira cause leptospirosis, an anthropozoonosis of great public health concern (Mahajan and Daljeet 2008). This pathogen affects humans and domestic and wild animals via direct or indirect contact with urine of infected animals or contaminated water (Cubas and Baptistotte 2014). Studies on Leptospira spp. infection in chelonians are limited and little is known about the relevance of these animals as reservoirs of the pathogen and their risk to public health (Andrews et al. 1965;Glosser et al. 1974;Lindtner-Knific et al. 2013;Oliveira et al. 2016;Fornazari et al. 2018). In Brazil, there are a few studies conducted in zoos that provide information on the sanitary status of chelonians regarding leptospirosis (Esteves et al. 2005;Brasil et al. 2013;Rocha et al. 2019). In the Brazilian Amazon region, one turtle species, Rhynoclemmys punctularia, was found to test positive for anti-Leptospira spp. agglutinins, also in captive individuals (Rocha et al. 2019).
Serological studies using the microscopic agglutination test are used to evaluate the exposure of animals to pathogens and their susceptibility to infection with different Leptospira spp. isolates, and have already been used to test for exposure to Leptospira in chelonians (Glosser et al. 1974;Lindtner-Knific et al. 2013). In this study, we aimed at detecting the presence of anti-Leptospira spp. antibodies in South American river turtles, Podocnemis expansa (Schweigger, 1812) (Podocnemididae) housed in captivity in a public aquarium in the northern Brazilian city of Belém.
The study was authorized by the Brazilian federal environmental authority Sistema de Autorização e Informação em Biodiversidade -SISBIO license no. 59785-1. Turtles were sampled at Zoobotanical Park Amazonia (Bosque Rodrigues Alves -Jardim Zoobotânico da Amazônia), located in an urban neighborhood of the city of Belém, in the northern Brazilian state of Pará.
The studied specimens inhabit artificial lakes alongside other chelonian species (Rhinoclemmys punctularia and Podocnemis unifilis). Individuals of both sexes and different age groups originate from apprehensions of illegally traded wild animals or from reproduction in captivity in the park itself. Water in the lakes is replaced only when the compartments are completely drained for cleaning. On one such occasion, in August 2015, 74 P. expansa (23 males, 48 females and three juveniles of undetermined sex) were captured manually for blood sampling. About 1-2 mL of blood were collected by venipuncture into the caudal vertebrae or occipital sinus using 3-mL syringes and tubes without anticoagulant. The injection site was cleaned with 2% chlorhexidine prior to injection. The samples were transported to the Zoonosis and Public Health Laboratory of the Institute of Veterinary Medicine of Universidade Federal do Pará (IMV−UFPA), where they were centrifuged to separate coagulated blood from serum.
The serum was placed in 1.5 ml Eppendorf® tubes, identified and stored at -20 ° C until analysis.
Of the 74 serum samples analyzed, 40.5% (30/74) were positive for Leptospira spp., with titrations between 100 to 3.200 for one or more serogroups, while no samples showed coagglutination with two or more serogroups (Table 1). Two samples were positive for the Djasiman serogroup, with titrations between 100 and 200, and only one sample was positive for the Celledoni and Bataviae serogroups, with titrations of 400 and 100, respectively.
Rhinoclemmys punctularia turtles kept in the same tanks as the P. expansa in our study showed a prevalence of 54.8% (17/31) of animals reactive to Leptospira spp. (Rocha et al. 2019), a high frequency similar to that found in P. expansa. Although the animals were kept in the same environment, and blood collection was performed on the same day, only antigen-antibody reactions for the Celledoni serogroup occurred in both species. This suggests that the diversity of infecting strains and the pathophysiology of Leptospira spp. is variable among reptile species. The presence of anti-Leptospira spp. antibodies in aquatic turtles has been related to prolonged exposure to Leptospira present in water (Andrews et al. 1965), contact with captive infected individuals (Grimm et al. 2015), to the presence of rodents in turtle farms (Lindtner-Knific et al. 2013) or carnivorous habits, such as ingestion of small vertebrates that may have been exposed to bacteria (Glosser et al. 1974).
Only four serogroups were detected in P. expansa in our study, yet the presence of different serogroups in a single species indicates that they circulate in the habitat and that the host species plays a role in the life cycle of the agent (Oliveira et al. 2013). Although the source of infection of the turtles is unknown, the aquatic environment was the likely transmission route, since the tanks in which turtles are kept are not cleaned regularly, which may result in the persistence of bacteria in the water and explain the high titration rate of antibodies (Andrews et al. 1965).
The detected serogroups were initially isolated from humans (Kmety and Dikken 1993), and later in domestic and wild animals (Rossetti et al. 2005;Betance et al. 2017;Rajeev et al. 2017;Fornazari et al. 2018), indicating that these serovars are not species-specific, making studies of their epidemiology more complicated, as transmission networks can be very complex. Little is known about infectious serotypes, their respective wild reservoirs, and their correlation between urban animals and wildlife (Grimm et al. 2015). The extent of the Amazon region, and the complexity of its habitats and biodiversity, can lead to regionally differentiated epidemiological cycles, so that turtles are possibly exposed to different serovars throughout their extensive distribution range (Ferrara et al. 2017).
We provide the first record of the presence of antibodies in P. expansa to the Hebdomadis, Djasiman, Celledoni and Bataviae serogroups. Our findings demonstrate that P. expansa in captive conditions were exposed to different Leptospira spp. serogroups, and that it is important to isolate environmental Leptospira and to test wild animals kept in captivity in the Amazon region for exposure to Leptospira.