Presence of anti-Leptospira spp. antibodies in captive yellow-spotted river turtles (Podocnemis unifilis) in the Eastern Amazon

ABSTRACT: We describe anti-Leptospira spp. agglutinin in yellow-spotted river turtles (Podocnemis unifilis)in the Amazon region. Ninety-eight serum samples from individuals housed at the Bosque Rodrigues Alves Zoobotanical Garden of Amazonia, Belém, PA, Brazil, were subject to the microscopic agglutination test (MAT) using 19 different Leptospira spp. antigen serogroups. Thirty-four of the 98 samples (35%) were reactive, with titers ranging from 100 to 3200, and eight 8 reactive samples (23.5%) co-agglutinated under two or more serovars.The most common serogroup was Hebdomadis (26.9%, 7/26), followed by Semaranga (23%, 6/26), Shermani (19.2%, 5/26), Djasiman (11.5%, 3/26), and Australis (7.7%, 2/26); Bataviae, Javanica, and Sejroewere represented by a single sample each (3.9%). The presence of turtles reactive to anti-Leptospira spp. antibodies implies exposure to the pathogen.

are typically asymptomatic carriers, which may hinder infection diagnosis and treatment. Nevertheless, few serologic investigations have been performed in captivity including zoos, research centers, and breeding centers (SILVA et al., 2010;EBANI, 2017).
The studied specimens inhabited artificial lakes alongside other turtle species. These lakes communicate with tanks containing other animals such as the spectacled caiman (Caiman crocodilus). Water is only replaced when the enclosures are completely drained for cleaning, and the 98 P. unifilis examined in the present study were captured manually during these full-drain periods, with the exception of those in one tank that could not be emptied; here, a net was used instead. All P. unifilis were healthy, without clinical signs of infection.
Blood samples (1-2 mL) were collected aseptically from dorsal tail veins of physically restrained turtles using 3-mL syringes and 25 × 7 needles, immediately stored in tubes without anticoagulant, and sent to the Laboratory of Zoonoses and Public Health, Institute of Veterinary Medicine, Federal University of Pará (IMV/UFPA), Brazil. Samples were centrifuged to extract the serum, and serum samples were transferred to Eppendorf micro tubes and stored at -20 ºC for serological tests.
The presence of anti-Leptospira spp. antibodies was assessed using the microscopic agglutination test (MAT) (FAINE, 1999), and been used 31 standard strains. The seeds derived from these strains used in the present research were four months old and grown in culture medium at our laboratory, allowing retrieving 19 serogroups: Australis, Autumnalis, Ballum, Bataviae, Canicola, Celedoni, Cynopteri, Djasiman, Grippotyphosa, Hebdomadis, Icterohaemorrhagiae, Javanica, Pamana, Pomona, Pyrogenes, Sejroe, Shermani, Andamana, and Seramanga. Samples with ≥50% agglutination compared to the control sample were considered reactive and were sent for titration with the cutoff point set at 100, considering this value as the dilution.
Agglutinins were detected for eight serogroups, with Hebdomadis being the most frequent. This serogroup was not identified for P. unifilis in the Amazon, as this species is typically reported in the northern Amazon region. However, a frequent Hebdomadis serogroup has been described for other wild animal species and cattle (MINEIRO et al., 2007;LENHARO et al., 2012;RODRIGUES et al., 2012). Due to the lack of serologic studies in P. unifilis, we compared our findings with that obtained for other turtle species, including Geochelones spp., red-eared tortoise, Amazonian tortoise, and water tiger.  Reactivity to Semaranga (serovar Patoc), the second most common serogroup in our study, has been reported incaptive Geochelones spp. And Trachemys scripta (pond slider) (ESTEVES et al., 2005;SILVA et al., 2010).Despite originatingfrom the non-pathogenic and saprophytic Leptospira biflexa, detecting Semaranga has important implications for anthropozoonosis because its presence may stimulate an immune response and trigger crossreactions with other pathogenic serovars (MYERS, 1976;PAZ et al., 2015). This might be the reason why Semaranga and Bataviae serogroups had the highest titers (3200). Bataviae has also been detected in Trachemys dorbigni (D'Orbigny's slider) (SILVA et al., 2009). Because the water at the enclosures where P. unifilis live are freely accessible to other species, it may become a reservoirof Leptospira spp. Mammals, in particular, might be sources of infection for P. unifilis. Thus, serological tests for the diagnosis of serovars belonging to the serogroups identified in the present study have been conducted in rodents; PAIXÃO et al. (2014) detected samples of rats captured in a wild animal preservation reactive to the serogroups identified for P. unifilis, while MESQUITA et al. (2018) detected samples of Didelphis marsupialis (common opossum), a common mammal in the region, reactive to the icterohaemorragiae and Nupezo serogroups, which are different from the ones detected in this study.
In addition to regular serologic tests on animals to better understand the infection risk, hygienic and sanitary measures are necessary in captive environments. These include regular sanitization of enclosures, removal of synanthropic animals, analysis of water quality, and implementation of a quarantine period before the introduction of a new specimen (PÉREZ-FLORES et al., 2016;ESTEVES-MACHADO et al., 2010;RATAJ et al., 2011;RODRIGUES et al., 2016;DETTUZZO, et al., 2017;EBANI, 2017). Such measures can minimize pathogen incidence in zoos and other facilities were animals are held captive.
In conclusion, this study successfully detected anti-Leptospira spp. antibodies in P. unifilis, an Amazonian turtle species held in captivity, providing direct evidence that this population has been exposed to the pathogen.