Evidence of Borrelia in wild and domestic mammals from the state of Minas Gerais , Brazil

The main of the study was to evaluate the presence of Borrelia burgdorferi infection in domestic and wild vertebrates and ectoparasites in endemic areas from the state of Minas Gerais, Brazil. A total of 445 serum samples were examined by ELISA, which used the Borrelia burgdorferi strain G39/40 U.S. source and 3,821 tick samples were tested by polymerase chain reaction (PCR). B. burgdorferi antibodies were found in 30 serum samples (6.74%); three in marsupials (7.69%), three in rodents (2.80%), nine in dogs (6.25%), and 15 in horses (9.68%). Nested-PCR performed in DNA samples obtained from collected ticks demonstrated negative results. Although attempts to amplify B. burgdorferi DNA from ticks had been not successful, the presence of seroreactive vertebrates suggests the possibility the Borrelia species circulating in these regions. Further research is required to provide information on the presence of Borrelia in Brazilian territory and its association with Baggio-Yoshinari syndrome.

Microorganisms of genus Borrelia are Gram-negative multi-flagellated bacteria, belonging to family Spirochetaceae (KRUPKA et al., 2007).Lyme disease (LD) is considered the most important emerging zoonosis in the United States of America and Europe (KRUPKA et al., 2007), causing skin, joint, neurological, cardiac and ocular symptoms.LD is caused by Borrelia burgdorferi sensu lato bacterium complex, which comprises a group of 12 distinct species; it is transmitted by ticks of Ixodes ricinus complex (JOPPERT, 1995;RANKA et al., 2004).
In Brazil, a similar disease, referred as Brazilian Lyme diseasesimile illness or Baggio-Yoshinari Syndrome (BYS) has been described, since last ending century (YOSHINARI et al., 2010;GOUVEIA et al., 2010).Mantovani et al. (2007) were unable to confirm whether this syndrome is caused by new Borrelia species or it is related to genetically modified Borrelia burgdorferi sensu lato microorganisms or other infectious agent.
Give that BYS is considered a zoonosis as LD, it is very important to verify the presence of the causing agents in wild and domestic animals and theirs ectoparasites.In this scenario, the goal of this research was to investigate the occurrence of antibodies against B. burgdorferi in domestic and wild animals and the presence of Borrelia DNA in ticks.
This study was realized in the municipalities of Santa Cruz do Escalvado (20°14'09" S 42°48'50" W), Pingo D'Água (19°43'37" S 42°24'28" W), and Caratinga (19°47'24" S 42°08'20" W).These municipalities are located in the Rio Doce Hydrographic Basin in the State of Minas Gerais, Brazil, being classified as endemic areas for tick-borne zoonosis, and characterized by intense anthropogenic destructive actions against the ecological system.In the municipalities of Santa Cruz do Escalvado and Pingo D'Água, blood samples from 445 animals were collected from the tail vein of rodents and marsupials, cephalic vein of dogs, and jugular vein of horses, as previously approved by Brazilian Institute of Environment and Natural Renewable Resources (IBAMA) and the Ethics Committee of the Federal University of Vicosa.These samples were stored at -20 °C until the time of use.After blood collection, the wild animals captured were morphologically identified (ROSSI et al., 2006;OLIVEIRA;BONVICINO, 2006) and returned to their natural habitat.
A total number of 3,821 ticks were collected from wild and domestic animals in the parasitic stage and from the ground in the municipalities of Santa Cruz do Escalvado, Pingo D'Água, and Caratinga.The ticks were taxonomically identified by external morphological characteristics according to Aragão and Fonseca (1961) and Linardi and Guimarães (2000); they were sorted into pools according to each mammalian parasitized, been maintained at 37 °C under biochemical oxygen demand (BOD) conditions for 48 hours.After that, the ectoparasites were separated in pools according to taxonomic identification and animal origin, followed by storage at -20 °C until DNA extraction.
To roll out possible cross reaction, but not covering all of them, serology for leptospirosis pre-exposure against 30 different serovars was performed in all serum samples using microscopic agglutination test (MAT), as previously described (POSTIC et al., 2000).In the analyzed samples, 20 (4.49%) showed reactivity against B. burgdorferi and Leptospira spp.antigens, being 211 animals (47.42%)Leptospira spp.reactive (Table 3).
The results of our work support the evidence of Borrelia spp.infection in wild and domestic animals; it was lowest in rodents (2.80%) and highest in horses (9.68%).In the municipality of Santa Cruz do Escalvado the highest antibody titers were found, especially in serum samples collected from dogs and rodents.Similar results were observed by Alves et al. (2004) in dogs from the metropolitan region of Rio de Janeiro, Brazil, with titles ranging from 1:400 to 1:6,000.
Serological evidence of B. burgdorferi infection in dogs was observed in 6.25% animals; this frequency was similar to date previously reported in areas of risks to Lyme disease.In a study with 237 dogs from the risk area of Cotia, State of São Paulo, Joppert (1995) demonstrated by ELISA 33 animals (9.70%) showing IgG antibodies against B. burgdorferi.
In horses tested, 9.68% presented antibodies against B. burgdorferi, indicating the potential relevance of horses in the epidemiology of Borrelia spp. or non-identified BYS agent transmission.This result is similar to the results found in the municipalities of Três Rios and Vassouras in the State of Rio de Janeiro, Brazil (MADUREIRA et al., 2007).
In relation to wild animals, three of the 39 samples examined of marsupials (7.69%).showed antibodies against B. burgdorferi.
Barros-Battesti (1998) captured 62 marsupials and 72 rodents in a residential condominium located in the Atlantic Rain forest in the municipality of Itapevi, State of São Paulo, where the first cases of BYS in brothers were described in Brazil.This author observed under dark field microscopy the presence of Borrelialike spirochetes in blood of 13.00% marsupials and 36.40%wild rodents.Despite of low frequency of antibodies against B. burgdorferi verified in rodents (2.80%), these animals are considered major reservoirs and carriers of some Borrelia species (PAVLOVSKY, 1965;BARBOUR;HAYES, 1986).
By nested-PCR, using flgE primers, the DNA samples from 3,821 ticks collected in three municipalities showed negative results.Similar results were obtained by Ataliba (2006), who used nested-PCR to verify the presence of Borrelia DNA in a total of 349 A. cajennense adult ticks collected from BYS suspected areas.We propose some explanations to justify these PCR negative results: (1) The etiological agent of BYS is quite different from the B. burgdorferi described in LD, since it is probably found at its atypical morphology cystic presentation (MANTOVANI et al., 2007(MANTOVANI et al., , 2012;;MANTOVANI, 2010); (2) The BYS is caused by a new Borrelia species, or by a genetically modified B. burgdorferi strain, or other spirochete that was not yet described, that do not can be correctly identified when using B. burgdorferi Northern hemisphere primers (MANTOVANI et al., 2007(MANTOVANI et al., , 2012;;YOSHINARI et al., 2010); (3) As a rule, enormous amounts of DNA are necessary to obtain positive nested-PCR with the use of flgE primer in Brazil (MANTOVANI, 2010;MANTOVANI et al., 2012).
In conclusion, although the obtained results require further studies aiming the characterization of the etiologic agent in both samples of wild and domestic animals, the research demonstrated   by the first time in the State of Minas Gerais, Brazil, serological evidence of circulation of borrelia or the other related agent in wild and domestic animals in the municipalities of Santa Cruz do Escalvado and Pingo D'Água.Further research is required to provide information on the presence of Borrelia in Brazilian territory and its association with Baggio-Yoshinari syndrome.
against Borrelia using Borrelia burgdorferi of North American origin strain G39/40 performed by ELISA.n -number.

Table 1 .
Frequencies of antibodies against Borrelia burgdorferi (ELISA -IgG) in wild and domestic animals from the municipalities of Santa Cruz do Escalvado and Pingo D'Água, Minas Gerais State, Brazil.

Table 3 .
Serological response against Leptospira spp.and Borrelia burgdorferi in wild and domestic animals (n= 445) from the municipalities of Santa Cruz do Escalvado and Pingo D'Água, Minas Gerais State, Brazil.

Table 2 .
Anti-Borrelia antibody titers in serum samples from animals captured in the municipalities of Santa Cruz do Escalvado and Pingo D'Água, state of Minas Gerais, Brazil.