Serological evidence of exposure to tick-borne agents in opossums ( Didelphis spp . ) in the state of São Paulo , Brazil

This work involved a serological investigation of tick-borne pathogens in opossums in eight municipalities of the state of São Paulo, Brazil. Serum samples from 109 opossums (91 Didelphis aurita and 18 Didelphis albiventris) were tested to detect antibodies to Rickettsia rickettsii (Taiaçu strain, 1:64 cut-off) and Ehrlichia canis (São Paulo strain, 1:40 cut-off), by indirect immunofluorescence assay (IFA); and against Borrelia burgdorferi (strain G39/40) by enzyme-linked immunosorbent assay (ELISA). The presence of antibodies to anti-R. rickettsii, anti-E. canis and anti-B. burgdorferi was detected in 32 (29.35%), 16 (14.67%) and 30 (27.52%) opossums, respectively. Opossum endpoint titers ranged from 64 to 1,024 for R. rickettsii, from 40 to 160 for E. canis, and from 400 to >51,200 for B. burgdorferi. These serological results suggest that opossums have been exposed to Rickettsia spp., Ehrlichia spp., and B. burgdorferi-related agents in the state of São Paulo. Our study underscores the need for further research about these agents in this study area, in view of the occurrence of Spotted Fever and Baggio-Yoshinari Syndrome disease in humans in the state of São Paulo, Brazil.


Introduction
Worldwide tick-borne diseases have been highlighted in both human and veterinary medicine.Various tick species act as vectors of several pathogens, and many wild animals act as reservoirs for these agents.Therefore, rickettsiosis, ehrlichiosis, and borreliosis have been reported around the world to cause illness in animals and humans (CASTELLAW et al., 2011).
Ticks are considered vectors of a large number of infectious agents (HOOGSTRAAL, 1967).Only a few of the approximately 825 species of ticks described worldwide are directly relevant to public health, because of the possibility of these ticks parasitizing humans (OLIVER, 1989).However several other species that have never been described in humans may play an important, albeit indirect role in public health because they contribute to the enzootic maintenance of infectious agents in nature (HOOGSTRAAL, 1967).In this context, the genera Rickettsia, Ehrlichia and Borrelia are important tick borne agents that cause disease in humans and animals (DUMLER et al., 2001;KRUPKA et al., 2007;PAROLA et al., 2013).
In Brazil, opossums are infested with different species of ticks and can often act as amplifier hosts of some pathogens, such as R. rickettsii (HORTA et al., 2009), which is the world's most pathogenic species of Rickettsia and is responsible for causing Brazilian Spotted Fever (BSF) (PAROLA et al., 2013).These animals are abundant in all areas endemic for BSF, where they present high antibody titers to R. rickettsii (HORTA et al., 2007) and are usually infested with ticks of the A. cajennense complex, the most important vector in South America (LABRUNA, 2009).
As for the genus Ehrlichia, there are few studies about the presence of antibodies in opossums.Animals seropositive to Ehrlichia chaffeensis have been reported in the United States (CASTELLAW et al., 2011).On the other hand, Lyme disease, a common zoonosis in the northern hemisphere (United States, Europe and Asia), is considered an infectious disease caused by spirochetes of the Borrelia burgdorferi sensu lato complex which is transmitted by ticks of the Ixodes ricinus complex (STEERE, 2001).In the case of borreliosis, this disease can be caused by a number of species of worldwide distribution, such as Borrelia burgdorferi (United States and Europe); Borrelia garinii and Borrelia afzelii (Europe), and Borrelia japonica (Asia) (STEERE, 2001;FELSENFELD, 1965;BARANTON et al., 1992).These species were isolated from Ixodes sp.ticks and another spirochete, called Borrelia lonestari, found in the United States, was isolated from Amblyomma americanum (BARBOUR et al., 1996).In Brazil, the disease is known as Baggio-Yoshinari Syndrome (BYS) or Lyme-like disease, and the first cases similar to this disease in the country were described in 1992 in siblings, who developed erythema migrans, flu-like symptoms and arthritis after being bitten by ticks.However, B. burgdorferi has so far not been isolated in this country.Specimens of the genera Ixodes and Amblyomma naturally infected with unidentified spirochetes have been found in areas where Lyme-like disease has been reported (YOSHINARI et al., 2010).
This work involved a serological evaluation of tick-borne agents (Ehrlichia spp., Rickettsia spp.and Borrelia spp.) in blood collected from opossums (Didelphis spp.) in the state of São Paulo, Brazil.

Materials and Methods
This study was conducted in eight municipalities (Barueri, Biritiba Mirim, Cotia, Osasco, Salesópolis, São Bernardo do Campo, São Paulo, and Sorocaba) in the state of São Paulo, Brazil (Figure 1).Free-living opossums were captured between 2003 and 2008.The opossums were anesthetized with 15-30 mg/kg The IFA was used to detect antibodies to Ehrlichia spp.and Rickettsia spp.Anti-Ehrlichia spp.antibodies were evaluated using the São Paulo strain of E. canis with a cut-off point at an initial dilution of 1:40 (AGUIAR et al., 2007).Anti-Rickettsia spp.antibodies were evaluated using the Taiaçu strain of R. rickettsii, with an initial dilution of 1:64 as previously described (PINTER & LABRUNA, 2006).Each slide contained serum previously shown to be non-reactive (negative control) and serum known to be reactive (positive control, obtained by experimentally infection of the agents in opossums).Anti-Borrelia antibodies were evaluated by ELISA using the North American strain G39/40 of B. burgdorferi as antigen (JOPPERT et al., 2001).

Discussion
This study detected the presence of antibodies against Rickettsia spp., Ehrlichia spp.and B. burgdorferi in free-living opossums in the state of São Paulo.In Brazil, serological studies in opossums infected experimentally with R. rickettsii, R. parkeri, R. bellii, and R. felis have shown that these animals have enough seroconversion to be positive by IFA (HORTA et al., 2009(HORTA et al., , 2010)).Studies on free-living opossums in the states of São Paulo and Minas Gerais detected reactivity rates of 59.5% (56/94) and 42.1% (16/38), respectively, against Rickettsia-specific antigen (HORTA et al., 2007;MILAGRES et al., 2010).More recently Silveira et al. (2015) found free living opossums seroreactive to Rickettsia spp.(R. parkeri, R. rickettsii, R. amblyommii and R. rhipicephali) from Paulicéia, state of São Paulo.Although our results indicated lower percentages than those found in another studies, we found evidence of rickettsial infection circulating among opossums in Brazil, an important synanthropic animal commonly present in areas endemic for BSF.Horta et al. (2009) had already demonstrated that R. rickettsii is able to infect opossums without clinical signs and to develop rickettsemia that can cause infection in guinea pigs and ticks.These results consistently demonstrate that opossums can serve as amplifier hosts for the horizontal transmission of this pathogen to A. cajennense ticks.Thus, it can be inferred that the participation of opossums in the epidemiology of the disease is very important for the maintenance of the agent in nature, not only in ticks but also in vertebrates hosts, including humans.
This study reports the presence of anti-Ehrlichia spp.antibodies in opossums.Evidence of the exposure of opossums to E. chaffeensis has been reported in the USA (CASTELLAW et al., 2011).Although research into Ehrlichia spp.infections in opossums are scanty, our results indicate the possibility that they occur due to the presence of vector ticks.The existence of cross-reactivity observed in the IFA among the species of the genus Ehrlichia (HARRUS & WANER, 2011) implies the possibility that an unrecognized ehrlichial agent is circulating among these animals.To date, only E. canis and an ehrlichial agent related to cattle and Rhipicephalus (Boophilus) microplus ticks have been isolated in Brazil (CABEZAS-CRUZ et al., 2012;AGUIAR et al., 2013AGUIAR et al., , 2014)).
Our results support the evidence of B. burgdorferi infection in opossums (27.52%).Similar results were observed in the state of Minas Gerais, where the presence of antibodies against B. burgdorferi was detected in 7.69% opossums (MONTANDON et al., 2014).Other studies in Brazil have reported humans and animals seropositive for B. burgdorferi (YOSHINARI et al., 2010;CORRADI et al., 2006;MADUREIRA et al., 2007;MONTANDON et al., 2014), demonstrating different prevalence rates in dispersed areas and showing that both domestic and wild animals are becoming infected with a closely related Borrelia spp., although the agents have never been isolated in Brazil.
Our findings reinforce the probable presence of these agents in opossums in eight municipalities in the state of São Paulo and emphasize the need for further research into these pathogens in this study area, in view of the occurrence of BSF and BYS in humans in the state of São Paulo.

Figure 1 .
Figure 1.Geographic locations of the 8 municipalities in which the opossums were caught.State of São Paulo, Brazil.

Table 1 .
Presence of anti-R.rickettsii, anti-E.canis, and anti-B.burgdorferi antibodies in opossums (D. albiventris and Didelphis aurita) from the state of São Paulo, Brazil.