Seroprevalence to Rabies Virus in Wildlife in Brazil

Wildlife has emerged as an important source of human rabies in Brazil, which is currently transmitted mainly by hematophagous bats, Desmodus rotundus (Ministry of Health, Brazil 2020). In São Paulo state, the most populated and developed state of Brazil, detection of rabies virus (RABV) in wildlife has been largely restricted to bats. However, in 2016, RABV was isolated from a fox (Cerdocyon thous) from São Paulo state (Favoretto et al. 2016), indicating that other wild mammals can be affected in this region. Thus, our study sought to investigate RABV exposure in wildlife other than bats from São Paulo state.

Serum samples from 638 free-ranging wild mammals comprising 24 species were analyzed (Table 1). These were opportunistic samples that had been obtained during previous studies on zoonotic diseases (Paiz et al. 2015; Fornazari et al. 2018; Batista et al. 2019) and remained stored at –80 C. The sampled population was heterogeneous, with different animals according to locations and year. Animals were surveyed between 2008 and 2017, with most samples (n=345) obtained between 2012 and 2013. Animals were from 46 municipalities, mainly in the central region of São Paulo state, with the majority of the samples coming from Botucatu municipality (Supplementary Material Figure). Some samples from Botucatu were from animals captured using nonlethal tomahawk traps. Captured animals were anesthetized in the field by intramuscular injection of ketamine hydrochloride plus midazolam, using species-specific dosages. Venous blood (1–5 mL) was collected, and the animals were released after complete recovery from the effects of the anesthesia (approximately 8 hr after the procedures). The other samples came from the Center for Medicine and Wildlife Research, São Paulo State University, Botucatu, which is responsible for receiving and caring for rescued animals, regardless of their origin and condition. Animals from the Center for Medicine and Wildlife Research were sampled during veterinary care. More details on the sampling methods were described previously (Fornazari et al. 2018).

Detection of RABV-neutralizing antibodies (rVNAs) was considered as evidence of exposure, and was assessed through the rapid fluorescent focus inhibition test using the protocol described by Smith et al. (1996) with minor modifications. A standard serum (National Institute for Biological Standards and Control 1997) containing 30 IU/mL was diluted to 0.5 IU/mL and was used as reference in the tests. Serum samples were inactivated at 56 C for 30 min. Six dilutions were prepared for each serum at a 1:5 ratio (from 1:2.5 to 1:3,125) in 96-well microplates, at a final volume of 50 µL per well, using Eagle's minimal essential medium with Earle's balanced salt solution (Vitrocell, Campinas, Brazil), containing 10% fetal bovine serum (Vitrocell). A Challenge Virus Standard RABV strain (CVS-132-11A, Institut Pasteur, São Paulo, Brazil) solution with 100 FFD₅₀ (fluorescence focus dose 50%) was added to the microplates, and incubated for 90 min at 37 C with 5% CO₂. Next, 100 µL of a BHK-21 (American Type Culture Collection, Manassas, Virginia, USA) cell suspension (2.5×10⁵ cells/mL) was added, followed by 20 hr incubation at 37 C with 5% CO₂. Cells were fixed in acetone (80%) at –20 C. An anti-RABV conjugate (Caporale et al. 2009) produced at the Institut Pasteur (São Paulo, Brazil) was added, and the microplates were incubated in a humid chamber at 37 C for 1 hr. The microplates were washed three times with phosphate buffered saline (prepared in-house), followed by another three washes with distilled water. The results were visualized using an inverted fluorescent microscope with 200× magnification. In each well, 18 microscopic fields were evaluated for the presence of fluorescent foci, and the titration was calculated through the Spearman-Kärber method (Aubert 1996) using the standard serum as reference. The minimum level of detection of the tests was considered 0.01 IU/ mL. Human sera from the Institut Pasteur were used as positive and negative controls in all assays, and were certified by the French Agency for Food, Environmental and Occupational Health Safety—Nancy Laboratory for rabies and wildlife. Because the rapid fluorescent focus inhibition test has been validated for humans and domestic species, not for wildlife, rather than the standard 0.1 IU/mL cutoff point, we adopted a more conservative interpretation and considered samples as positive with rVNA ≥0.2 IU/mL, as recommended by Berentsen et al. (2013).

We found 11/638 mammals positive (1.7%; 95% confidence intervals of 0.7–2.7) with titers ranging from 0.22 to 0.71 IU/mL. Seropositive animals (Tables 1, 2 and Supplementary Figure) were from two municipalities and included six species: 5/312 (1.6%) opossums (Didelphis albiventris), 2/104 (1.9%) coatis (Nasua nasua), 1/33 (3.0%) gray brockets (Mazama gouazoubira), 1/18 (5.6%) porcupines (Sphiggurus villosus), 1/3 (33.3%) capybaras (Hydrochoerus hydrochaeris), and the single tested otter (Lontra longicaudis). Based on clinical signs or laboratory testing, rabies was not suspected in any of the seropositive animals (Table 2).

Vaccination of free-ranging wildlife is not performed in Brazil, so this cannot be the basis for these positive data. Rather, as in many other studies, our results indicate natural exposure to RABV. Abortive infection, in which RABV is cleared by the immune system before the onset of clinical signs (Gold et al. 2020), is the most probable explanation for the presence of rVNAs. Contact with RABV could occur through bites of hematophagous bats, which exhibit a generalist feeding behavior and bite a diversity of species (Galetti et al. 2016; Bohmann et al. 2018). Alternatively, oral exposure to RABV might occur through contact with secretions or tissues of rabid animals. For example, production of rVNAs has been observed in red foxes (Vulpes vulpes) and striped skunks (Mephitis mephitis) after experimental feeding with infected mice (Ramsden and Johnston 1975).

Two previous studies in Brazil support our seroprevalence findings, with detection of rVNAs in several mammal species from São Paulo state (Almeida et al. 2001; Araujo et al. 2014), demonstrating the frequency and diversity of animals other than bats with contact to RABV in this state. Opossums and coatis were the most representative species in our study, both with seroprevalence values close to the 1.7% overall seroprevalence, indicating a low level of exposure to RABV. These results are compatible with multiple seroprevalence studies worldwide with different rates of rVNAs (Gold et al. 2020). In summary, we detected low frequency of wildlife with rVNAs in São Paulo state, indicating natural exposure to RABV. As our study was opportunistic and limited in scope, more focused studies are needed to elucidate the origins and outcomes of such exposures.

We thank the São Paulo Research Foundation (FAPESP 2012/02927-1 and 2012/05285-0) for financial support.

Supplementary material for this article is online at http://dx.doi.org/10.7589/JWD-D-21-00065.