• 1.

    Walker D, Herrero-Herrero JI, Ruiz-Beltran R, Bulllon-Sopelana A, Ramos-Hidalgo A, 1987. The pathology of fatal Mediterranean spotted fever. Am J Clin Pathol 87: 669672.

    • Search Google Scholar
    • Export Citation
  • 2.

    Milagres BS, Padilha AF, Barcelos RM, Gomes GG, Montandon CE, Pena DC, Nieri FA, Silveira I, Pacheco R, Labruna MB, Bouyer DH, Freitas RN, Walker DH, Mafra CL, Galvão MA, 2010. Rickettsia in synanthropic and domestic animals and their hosts from two areas of low endemicity for Brazilian spotted fever in the eastern region of Minas Gerais, Brazil. Am J Trop Med Hyg 83: 13051307.

    • Search Google Scholar
    • Export Citation
  • 3.

    Rehacer J, Urvolgy J, 1978. Towards more standardized methods in ecological studies of Rickettsiae in relation to their vectors and vertebrate hosts. VII International Congress of Infectious and Parasitic Diseases, 18.

    • Search Google Scholar
    • Export Citation
  • 4.

    Walker DH, Liu QH, Yu XL, Li H, Taylor C, Feng HM, 1992. Antigenic diversity of Rickettsia conorii. Am J Trop Med Hyg 47: 7886.

  • 5.

    Forattini OP, 1992. Ecologia, Epidemiologia e Sociedade. São Paulo, Artes Médicas.

  • 6.

    Nagy T, 1993. Normas Operacionais de Centros de Controle de Zoonoses. Procedimentos para o Controle de Roedores. Brasilia: Coordenação Nacional de Controle de Zoonoses e Animais Peçonhentos. Brasília, 1993.

    • Search Google Scholar
    • Export Citation
  • 7.

    FUNASA, 2002. Manual de Controle de Roedores. Brasília: Fundação Nacional de Saúde.

  • 8.

    Pena DC, Mafra CL, Calic SB, Labruna MB, Milagres BS, Walker DH, Galvão MA, 2009. Serologic survey for antibodies to Rickettsia among domestic and wild animal populations in Brazil. Clin Microbiol Infect 15 (Suppl 2): 243244.

    • Search Google Scholar
    • Export Citation
  • 9.

    Ishikura M, Watanabe M, Nakayama T, Matsuura K, Morita O, Uchida T, 1992. Seroepidemiology of spotted fever group rickettsiae in small field rodents in Japan. Microbiol Immunol 36: 649653.

    • Search Google Scholar
    • Export Citation
  • 10.

    Ibrahim IN, Okabayashi T, Ristiyanto, Lestari EW, Yanase T, Muramatsu Y, Ueno H, Morita C, 1999. Serosurvey of wild rodents for Rickettsioses (spotted fever, murine typhus and Q fever) in Java Island, Indonesia. Eur J Epidemiol 15: 8993.

    • Search Google Scholar
    • Export Citation

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Spotted Fever Group Rickettsia in Small Rodents from Areas of Low Endemicity for Brazilian Spotted Fever in the Eastern Region of Minas Gerais State, Brazil

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  • Federal University of Viçosa, Viçosa, Brazil; Federal University of Ouro Preto, Ouro Preto, Brazil; University of São Paulo, São Paulo, Brazil; Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas

We investigated the humoral immune response against different species of Rickettsia in serum samples from small rodents collected in two areas of a silent focus for Brazilian spotted fever in the eastern region of Minas Gerais State, Brazil. Sera samples were analyzed by indirect immunofluorescence assay using antigens from Rickettsia species of the spotted fever, ancestral, and transition groups. Titers ≥ 1:64 were considered positive. In Santa Cruz do Escalvado, 94% (30 of 32) of the samples collected from Rattus rattus, 22% (5 of 23) from Nectomys squamipes, and 80% (4 of 5) from Akodon sp., reacted by indirect immunofluorescence assay with Rickettsia antigens of the spotted fever group. In the municipality of Pingo D'Água, 84% (26 of 31) of the samples collected from R. rattus, 86% (6 of 7) of the samples from Oryzomys subflavus, 86% (6 of 7) from N. squamipes, and 100% (1 of 1) from Bolomys sp. contained antibodies that reacted with rickettsial antigens of the spotted fever group. These results demonstrated the previous exposure of small rodents to spotted fever group Rickettsia, suggesting the participation of these animals in the natural history of these rickettsiae in this region.

Introduction

Rickettsial diseases are of wordwide importance.1 Generally, the primary focus involves mainly small sylvan mammals of class Rodentia as hosts for vector arthropods that transmit infections of domestic and synanthropic animals, which may play a role in the maintenance of Rickettsia in nature.2 Experimental studies with some small rodents have demonstrated that they are susceptible to rickettsial infection and develop rickettsemia at levels high enough to infect their ectoparasites during blood feeding.3 Those mammals have a limited dispersion and short lifespan and thus can serve as a good indicator of the circulation of rickettsial agents in a particular area by natural environmental dispersion.

In Minas Gerais State, Brazil, the cited municipalities are located at the biome Mata Atlantica, one of the last of a kind of rain forest in the southwest region of Brazil. Pingo D'Água is located on Rio Doce Valley. This municipality was selected with the aim of investigating the presence of Rickettsia organisms because of recent cases of Brazilian spotted fever (BSF) in that region. Santa Cruz do Escalvado, located in the Piranga Valley, had a history of occurrence of BSF cases, but this municipality has been considered as a silent focus of the disease during the past 20 years. In the beginning of the last decade, this municipality passed through several modifications in its natural landscape because of the building of a small hydroelectric power plant. We evaluated the current situation to understand the possibility of Rickettsia spread and the potential risk for a new BSF outbreak. In this investigation, we determined the occurrence of antibodies against different species of Rickettsia in serum samples of small sylvan rodents captured in these two municipalities.

Materials and Methods

Locations.

Santa Cruz do Escalvado occupies an area of 258.34 km,2 with an estimated population of 5,321 inhabitants, and 80% of those living in the rural area dedicated especially to farming activities (Brazilian Institute of Geography and Statistics, 2009). Rural dwellings in Minas Gerais countryside are characteristically made of wattle and daub, which favors the periodomestic presence of vectors of diseases and small rodent reservoirs of such diseases. In Pingo D'Água, which has an area of 67 km2 and an estimated population of 4,420, 90% of the inhabitants reside in an urban area.

Animals.

Rodents were captured during a period of two years (2005–2007) in a three-month frequency. Approximately 100 live-traps were placed near dwellings, including garages, storage facilities, corn fields, bamboo fields, landfills, and around dwellings. The rodents were anesthetized with halothane and then blood samples were collected.

Serum sample collection.

Serum samples were analyzed by indirect immunofluorescence assay (IFA), according to the protocol described by Walker and others4 using Rickettsia antigens of the spotted fever group (R. rickettsii, R. parkeri, R. rhipicephali, and R. amblyommii), an ancestral group (R. bellii), and a transitional group (R. felis).4 Serum samples that reacted at the screening dilution of 1:64 were then tested in serial two-fold dilutions to determine the endpoint titer. As a positive control serum, we used serum collected from a laboratory-reared water rat (Nectomys squamipes) 21 days after it was experimentally inoculated with R. rickettsii strain Taiaçu. In all reactions, we used a fluorescein isothiocyanate–labeled goat anti-rat IgG (Sigma, St. Louis, MO) as secondary antibody.

Results

In Santa Cruz do Escalvado, 62 rodents were trapped, of which 23 were identified as N. squamipes, 2 as Oryzomys subflavus, 32 as Rattus rattus, and five as Akodon sp. Of these 62 rodents, 39 (63%) had antibody titers ≥ 1:64 by IFA against R. rickettsii, R. parkeri, R. rhipicephali, and R. amblyommii antigens (Table 1). The highest titer (1:16,384) was found in serum of R. rattus using R. rickettsii antigen. None of the serum samples contained antibodies against R. felis or R. bellii antigens.

Table 1

Serum sample activity of rodents against spotted fever group Rickettsia antigens by indirect immunofluorescence assay, Santa Cruz do Escalvado, Minas Gerais, Brazil

Rodent speciesNo.Rickettsia rickettsii, No. (%)R. parkeri, No. (%)R. amblyommii, No. (%)R. rhipicephali, No. (%)
Rattus rattus3230 (94)30 (94)28 (88)23 (77)
Nectomys squamipes235 (22)4 (17)3 (13)3 (13)
Oryzomys subflavus20000
Akodon sp.54 (80)4 (80)1 (20)1 (20)
Total6239 (63%)38 (62%)32 (52%)27 (44%)

In Pingo D'Água, 46 rodents were trapped and 31 identified as R. rattus, 7 as O. subflavus, 7 as N. squamipes, and 1 as Bolomys sp. Of these 46 rodents, 39 (85%) contained antibodies against R. rickettsii, R. parkeri, R. rhipicephali, and R. amblyommii antigen, with titers ranging from 1:64 to 1:4,096. The highest titers were obtained against R. rickettsii. Two serum samples tested against R. bellii antigens showed positive results, and all showed negative results against R. felis antigen (Table 2).

Table 2

Serum sample activity of rodents against spotted fever group Rickettsia antigens by indirect immunofluorescence assay, Pingo D'Água, Minas Gerais, Brazil

Rodent speciesNo.Rickettsia rickettsii, No. (%)R. parkeri, No. (%)R. amblyommii, No. (%)R. rhipicephali, No. (%)R. belli, No. (%)
Rattus rattus3126 (84)24 (77)18 (58)15 (48)1 (2)
Nectomys squamipes76 (86)6 (86)5 (71)3 (13)0
Oryzomys subflavus76 (86)6 (86)3 (43)3 (43)0
Bolomys sp.11 (100)1 (100)1 (100)1 (100)1 (100)
Total4639 (85)38 (83)32 (70)27 (59)2 (4)

The species R. rickettsii showed a higher percentage of positive serologic results. In Santa Cruz do Escalvado, Minas Gerais, of the 39 rodent serum samples positive for R. rickettsii, 11 (28.2%) sera had titers four times higher for R. rickettsii antigens than for other species of rickettsial antigens. In Pingo D'Água, of the 39 rodent sera positive for R. rickettsii, 9 (23.07%) had titers four times higher for R. rickettsii antigens than for other of species of rickettsial antigens. These results show this species or another closely related species stimulated the immune response, and that there was no serologic cross-reactivity. For the remaining rodent serum samples positive for rickettsial antigens, the species that triggered the immune response was not known, and cross-reactivity with other nonpathogenic species might have occurred.

Discussion

The results obtained indicated exposure of small sylvan rodents to spotted fever group rickettsial organisms, suggesting the participation of these animals in the maintenance of ecologic cycle of rickettsiae in these areas in Brazil. The presence of antibodies in small rodents, such as R. rattus, N. squamipes, Bolomys sp., O. subflavus, and Akodon sp., indicates advantageous conditions for rickettsial development and reproduction in those animals. These animals are losing their natural predators because of alterations in the ecologic balance caused by human activities in the studied regions. Also, availability of food, such as grains found in storage structures, or the replacement of native vegetation by corn and sugar cane fields can be responsible for maintaining these species in nature.

The presence of these rodent species in the study region is consistent with the synanthropic behavior of small sylvan rodents, which are well adapted to the residential environment.57 The percentage of seroreactivity detected in R. rattus samples was high, which is an important result, because R. rattus is the most synanthropic species among those collected. Because these rodents live near humans, they maintain their location under constant risk of infection by pathogens maintained the zoonotic cycles by other small mammals and their ectoparasite vectors. In addition, the fact that this mammalian species has a large range of distribution increases the possibility of contacts between domestic and sylvan animals, and dispersion of their ticks or other ectoparasites into the environment, which leads to infection of domestic animals and humans. Rickettsial vectors (ticks and fleas) and small rodents can serve as hosts in amplifying cycles.8

Laboratory studies have demonstrated that some small rodents species are competent hosts of rickettsiae and develop rickettsemia levels capable of infecting their ectoparasites, despite the short period of rickettsemia.3,9 There have been reports showing R. rattus to contain antibodies against rickettsial antigens810 Furthermore, our study is the this first to identify a prevalent immune response in the other mammalian species by IFA using specific rickettsial antigens. Further studies are necessary to better understand the actual importance of these animals in the sylvan and domestic cycles of BSF, as well their interaction with their arthropods vectors.

ACKNOWLEDGMENTS

Bruno S. Milagres has been awarded a PhD degree, and Amanda F. Padilha has been awarded a Master's degree from the Federal University of Ouro Preto for their investigation of rickettsiosis under the direction of Dr. Márcio A. M. Galvão.

  • 1.

    Walker D, Herrero-Herrero JI, Ruiz-Beltran R, Bulllon-Sopelana A, Ramos-Hidalgo A, 1987. The pathology of fatal Mediterranean spotted fever. Am J Clin Pathol 87: 669672.

    • Search Google Scholar
    • Export Citation
  • 2.

    Milagres BS, Padilha AF, Barcelos RM, Gomes GG, Montandon CE, Pena DC, Nieri FA, Silveira I, Pacheco R, Labruna MB, Bouyer DH, Freitas RN, Walker DH, Mafra CL, Galvão MA, 2010. Rickettsia in synanthropic and domestic animals and their hosts from two areas of low endemicity for Brazilian spotted fever in the eastern region of Minas Gerais, Brazil. Am J Trop Med Hyg 83: 13051307.

    • Search Google Scholar
    • Export Citation
  • 3.

    Rehacer J, Urvolgy J, 1978. Towards more standardized methods in ecological studies of Rickettsiae in relation to their vectors and vertebrate hosts. VII International Congress of Infectious and Parasitic Diseases, 18.

    • Search Google Scholar
    • Export Citation
  • 4.

    Walker DH, Liu QH, Yu XL, Li H, Taylor C, Feng HM, 1992. Antigenic diversity of Rickettsia conorii. Am J Trop Med Hyg 47: 7886.

  • 5.

    Forattini OP, 1992. Ecologia, Epidemiologia e Sociedade. São Paulo, Artes Médicas.

  • 6.

    Nagy T, 1993. Normas Operacionais de Centros de Controle de Zoonoses. Procedimentos para o Controle de Roedores. Brasilia: Coordenação Nacional de Controle de Zoonoses e Animais Peçonhentos. Brasília, 1993.

    • Search Google Scholar
    • Export Citation
  • 7.

    FUNASA, 2002. Manual de Controle de Roedores. Brasília: Fundação Nacional de Saúde.

  • 8.

    Pena DC, Mafra CL, Calic SB, Labruna MB, Milagres BS, Walker DH, Galvão MA, 2009. Serologic survey for antibodies to Rickettsia among domestic and wild animal populations in Brazil. Clin Microbiol Infect 15 (Suppl 2): 243244.

    • Search Google Scholar
    • Export Citation
  • 9.

    Ishikura M, Watanabe M, Nakayama T, Matsuura K, Morita O, Uchida T, 1992. Seroepidemiology of spotted fever group rickettsiae in small field rodents in Japan. Microbiol Immunol 36: 649653.

    • Search Google Scholar
    • Export Citation
  • 10.

    Ibrahim IN, Okabayashi T, Ristiyanto, Lestari EW, Yanase T, Muramatsu Y, Ueno H, Morita C, 1999. Serosurvey of wild rodents for Rickettsioses (spotted fever, murine typhus and Q fever) in Java Island, Indonesia. Eur J Epidemiol 15: 8993.

    • Search Google Scholar
    • Export Citation

Author Notes

* Address correspondence to Bruno S. Milagres, School of Pharmacy and Medicine, Federal University of Ouro Preto, Ouro Preto, Minas Gerais State, 35420-000. E-mail: bsmilagres@gmail.com

Financial support: This study was supported by Fundação de Amparo à Pesquisa do Estado de Minas Gerais/Brazil (APQ-01602-09).

Authors' addresses: Bruno S. Milagres and Márcio A. M. Galvão, School of Pharmacy, Department of Medicine, Ouro Preto Federal University, Ouro Preto, Minas Gerais, Brazil, E-mails: bsmilagres@gmail.com and galvaomarcio@oi.com.br. Amanda F. Padilha, Department of Biological Sciences, Federal University of Ouro Preto, Ouro Preto, Minas Gerais, Brazil, E-mail: amandaep12@gmail.com. Carlos E. Montandon and Cláudio L. Mafra, Department of Biochemistry, Federal University of Viçosa, Viçosa, Minas Gerais, Brazil, E-mails: em.carlos@yahoo.com.br and mafra@ufv.br. Renata N. Freitas, Department of Nutrition, Federal University of Ouro Preto, Ouro Preto, Minas Gerais, Brazil, E-mail: rfreitasmail@gmail.com. Richard Pacheco, Department of Basic Sciences and Animal Production, Federal University of Mato Grosso, Cuiabá, Mato Grosso, Brazil, E-mail: pachecorichard@yahoo.com.br. David H. Walker, Department of Pathology, University of Texas Medical Branch, Galveston, TX, E-mail: dwalker@utmb.edu. Marcelo B. Labruna, Department of Preventive Veterninary Medicine, University of Sao Paulo, Sao Paulo, Brazil, E-mail: labruna@usp.br.

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