Archives of Virology

, Volume 163, Issue 9, pp 2369–2376 | Cite as

Analysis of rabies diagnosis in dogs and cats in the state of São Paulo, Brazil

  • Juliana Galera CastilhoEmail author
  • Samira Maria Achkar
  • Rafael de Novaes Oliveira
  • Enio Mori
  • Pedro CarnieliJr
  • Carla Isabel Macedo
Original Article


The genetic lineage of rabies virus (RABV) associated with dogs has not been found in the state of São Paulo since 1998, and all cases of rabies in domestic animals reported since then have involved the RABV lineage that circulates in bats. As there has been a change in the rabies transmission cycle in cats and dogs, we decided to analyze the tests used to diagnose rabies in these animals in the 15-year period from 2002 to 2016 in the state of São Paulo. During this period, 85,508 central nervous system (CNS) samples from dogs and cats were submitted to the Rabies Diagnosis Section at the Pasteur Institute of São Paulo for testing. All of the samples were tested by the fluorescent antibody test (FAT) and at least one of the following three tests: mouse inoculation test (MIT), rabies tissue culture infection test (RTCIT) and reverse transcription polymerase chain reaction (RT-PCR). Of all the samples tested, twenty were positive in at least one of these assays. Four other positive samples were identified at other institutions in the state of São Paulo. Of the twenty samples that tested positive at the Pasteur Institute of São Paulo, nine were tested by FAT, and the results were subsequently confirmed by other techniques; five gave inconclusive results, and therefore, other techniques had to be used as soon as possible in case the samples were positive; and six were negative by FAT and positive by one or more of the following tests: RTCIT, MIT and RT-PCR. Genetic typing of isolates from eighteen samples identified them as the lineage circulating in bats. In light of this finding, which indicates that genetic lineages associated with bats are circulating in domestic animals in the state of São Paulo, when the results of FAT carried out with samples from aggressive cats and dogs are inconclusive, complementary tests should be used. Decomposing samples and samples for which FAT was inconclusive should be tested using molecular techniques so that a definitive result can be obtained quickly and timely post-exposure prophylaxis can be administered to exposed individuals.


Compliance with ethical standards


This study was funded by Instituto Pasteur.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

This study is a retrospective analysis of the diagnosis of rabies in which viral isolation in mice is one of the techniques recommended by the World Health Organization. The Pasteur Institute is the reference center for the diagnosis of rabies in Brazil, and all samples that are referred for diagnosis are subjected to these techniques that were analyzed in the study. Therefore, both the animal samples received and the laboratory animals used are part of the laboratory diagnosis routine for rabies. Thus, since this is not a study linked to a research project but an analysis of the diagnostic tests, there is no opinion of the ethics committee of the institution. The laboratory animals used routinely for the diagnosis of rabies are maintained according to federal legislation.

This study did not include human samples, so there was no need for an ethics committee opinion.


  1. 1.
    King AMK, Adans MJ, Carstens EB, Lefkowitz EJ (2012) Genus Lyssavirus. In: King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ (eds) International Committee on Taxonomy of Viruses, 9th edn. Elsevier Inc., San Diego, pp 696–699Google Scholar
  2. 2.
    Fisher CR, Streicker DG, Schnell MJ (2018) The spread and evolution of rabies virus: conquering new frontiers. Nat Rev Microbiol. PubMedCrossRefGoogle Scholar
  3. 3.
    Acha PN, Szyfres B (2003) Zoonoses y enfermeddes transmisibles comunes al hombre y a los animales, vol 2, 3rd edn. Organization Panamericana de la Salud, WashingtonGoogle Scholar
  4. 4.
    Schneider MC, Romijn PC, Uieda W et al (2009) Rabies transmitted by vampire bats to humans: an emerging zoonotic disease in Latin America? Rev Panam Salud Publica 25:260–269CrossRefPubMedGoogle Scholar
  5. 5.
    Rocha SM, de Oliveira SV, Heinemann MB, Gonçalves VSP (2017) Epidemiological profile of wild rabies in Brazil (2002–2012). Transbound Emerg Dis 64:624–633. CrossRefPubMedGoogle Scholar
  6. 6.
  7. 7.
    Kotait I, Favoretto SR, Carrieri ML, Takaoka N (2001) Raiva humana causada pela variante 3—Desmodus rotundus—no estado de São Paulo. In: Institute P (ed) Seminário Internacional: Morcegos Como Transmissores da Raiva, São Paulo, pp 70–71Google Scholar
  8. 8.
    Diaz AM, Papo S, Rodriguez A, Smith JS (1994) Antigenic analysis of rabies-virus isolates from Latin America and the Caribbean. Zentralblatt für Veterinärmedizin R B J Vet Med 41:153–160Google Scholar
  9. 9.
    Castilho JG, de Souza DN, Oliveira RN et al (2017) The epidemiological importance of bats in the transmission of rabies to dogs and cats in the state of São Paulo, Brazil, between 2005 and 2014. Zoonoses Public Health 64:423–430. CrossRefPubMedGoogle Scholar
  10. 10.
    Dean DJ, Abelseth MK, Atanasiu P (1996) The fluorescent antibody test. In: Meslin FX, Kaplan MM, Koprowski H (eds) Laboratory techniques in rabies, 4th edn. World Health Organization, Geneva, pp 88–93Google Scholar
  11. 11.
    Koprowski H (1996) The mouse inoculation test. In: Meslin FX, Kaplan MM, Koprowski H (eds) Laboratory techniques in rabies, 4th edn. World Health Organization, Geneva, pp 80–86Google Scholar
  12. 12.
    Castilho JG, Iamamoto K, Yoshitaka J et al (2007) Padronização e aplicação da técnica de isolamento do vírus da raiva em células de neuroblastoma de camundongo (N2A). Bol Epidemiológico Paul (BEPA) 4:12–18Google Scholar
  13. 13.
    Orciari LA, Niezgoda M, Hanlon CA et al (2001) Rapid clearance of SAG-2 rabies virus from dogs after oral vaccination. Vaccine 19:4511–4518CrossRefPubMedGoogle Scholar
  14. 14.
    Takaoka N (2000) Alteração no perfil epidemiológico da raiva no Estado de São Paulo. In: Pasteur I (ed) Seminário Internacional de Raiva, São Paulo, pp 23–24Google Scholar
  15. 15.
    Albas A, Ferrari CI, da Silva LH et al (1999) Influence of canine brain decomposition on laboratory diagnosis of rabies. Rev Soc Bras Med Trop 32:19–22CrossRefPubMedGoogle Scholar
  16. 16.
    McElhinney LM, Marston DA, Brookes SM, Fooks AR (2014) Effects of carcase decomposition on rabies virus infectivity and detection. J Virol Methods 207:110–113. CrossRefPubMedGoogle Scholar
  17. 17.
    Trimarchi CV, Davis Nadin SA (2007) Diagnostic Evaluation. In: Jackson AC, Wunner W (eds) Rabies, 2nd edn. Academic Press, San Diego, pp 411–469CrossRefGoogle Scholar
  18. 18.
    David D, Yakobson B, Rotenberg D et al (2002) Rabies virus detection by RT-PCR in decomposed naturally infected brains. Vet Microbiol 87:111–118CrossRefPubMedGoogle Scholar
  19. 19.
    Oliveira R, Takaoka N, Brandao P et al (2006) Postmortem confirmation of human rabies source [7]. Emerg Infect Dis 12:867–869CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Bingham J, van der Merwe M (2002) Distribution of rabies antigen in infected brain material: determining the reliability of different regions of the brain for the rabies fluorescent antibody test. J Virol Methods 101:85–94CrossRefPubMedGoogle Scholar
  21. 21.
    Tierkel E, Atanasiu P (1996) Rapid microscopic examination for Negri bodies and preparation of specimens for biological test. In: Meslin FX, Kaplan MM, Koprowski H (eds) Laboratory techniques in rabies, 4th edn. World Health Organization, Geneva, pp 55–65Google Scholar
  22. 22.
    Charlton KM, Casey GA, Campbell JB (1983) Experimental rabies in skunks: mechanisms of infection of the salivary glands. Can J Comp Med Rev Can Med Comp 47:363–369Google Scholar
  23. 23.
    CDC—Center for Disease Control and Prevention (2006) Protocol for postmortem diagnosis of rabies in animals by direct fluorescent antibody testing: a minimum standard for rabies diagnosis in the United States. Accessed 22 Aug 2017
  24. 24.
    Fooks AR, Johnson N, Freuling CM et al (2009) Emerging technologies for the detection of rabies virus: challenges and hopes in the 21st century. PLoS Negl Trop Dis 3:e530. CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Tordo N, Sacramento D, Bourhy H (1996) The polymerase chain reaction (PCR) technique for diagnosis, typing and epidemiological studies. In: Meslin FX, Kaplan MM, Koprowski H (eds) Laboratory techniques in rabies, 4th edn. World Health Organization, Geneva, pp 157–170Google Scholar
  26. 26.
    Araujo DB, Langoni H, Almeida MF et al (2008) Heminested reverse Transcriptase polymerase chain reaction (hnRT-PCR) as a tool for rabies virus detection in stores and decomposed samples. BMC Res Notes 1:17CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Whitby JE, Johnstone P, Sillero-Zubiri C (1997) Rabies virus in the decomposed brain of an Ethiopian wolf detected by nested reverse transcription-polymerase chain reaction. J Wildl Dis 33:912–915CrossRefPubMedGoogle Scholar
  28. 28.
    Rupprecht CE, Hanlon CA, Hemachudha T (2002) Rabies re-examined. Lancet Infect Dis 2:327–343CrossRefPubMedGoogle Scholar
  29. 29.
    Shuangshoti S, Thepa N, Phukpattaranont P et al (2013) Reduced viral burden in paralytic compared to furious canine rabies is associated with prominent inflammation at the brainstem level. BMC Vet Res 9:31. CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Brandão PE, Carnieli Junior P, Castilho JG, et al (2004) Rabies virus versus frugivorous and vampire bats: evolutive and public health insights. In: Abstracts book of the XV International Conference Rabies in the Americas (XV RITA). organizer committee, Santo Domingo, pp 41–42Google Scholar
  31. 31.
    Albas A, de Souza EAN, Lourenço RA et al (2009) Antigen profile of rabies virus isolated from different species of non-hematophagous bats in the region of Presidente Prudente, State of São Paulo. Rev Soc Bras Med Trop 42:15–17CrossRefPubMedGoogle Scholar
  32. 32.
    Fahl WO, Carnieli P, Castilho JG et al (2012) Desmodus rotundus and Artibeus spp. bats might present distinct rabies virus lineages. Braz J Infect Dis 16:545–551. CrossRefPubMedGoogle Scholar
  33. 33.
    Kobayashi Y, Sato G, Shoji Y et al (2005) Molecular epidemiological analysis of bat rabies viruses in Brazil. J Vet Med Sci 67:647–652CrossRefPubMedGoogle Scholar
  34. 34.
    Shoji Y, Kobayashi Y, Sato G et al (2004) Genetic characterization of rabies viruses isolated from frugivorous bat (Artibeus spp.) in Brazil. J Vet Med Sci 66:1271–1273CrossRefPubMedGoogle Scholar
  35. 35.
    Gomes MN (2008) Padrões espaciais da raiva bovina e seus determinantes no Estado de São Paulo entre 1992 e 2003. Instituto Nacional de Pesquisas Espaciais (INPE) São José do CamposGoogle Scholar
  36. 36.
    Sazima I (1978) Aspectos Do Comportamento Alimentar Do Morcegos Hematófago, Desmodus Rotundus. Bol Zool Univ São Paulo 3:97–120Google Scholar
  37. 37.
    Lucca T, Rodrigues RCA, Nitsche A, Von Zuben AP (2017) Ações de Vigilância e controle da raiva frente a caso positivo em felino no município de Campinas, São Paulo, Brasil. Bol Epidemiológico Paul 14:33–41Google Scholar
  38. 38.
    Threlfall CG, Law B, Banks PB (2012) Influence of landscape structure and human modifications on insect biomass and bat foraging activity in an urban landscape. PLoS One 7:e38800. CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Queiroz LH, Favoretto SR, Cunha EMS et al (2012) Rabies in southeast Brazil: a change in the epidemiological pattern. Arch Virol 157:93–105. CrossRefPubMedGoogle Scholar
  40. 40.
    Morikawa VM, Ribeiro J, Biondo AW et al (2012) Cat infected by a variant of bat rabies virus in a 29-year disease-free urban area of southern Brazil. Rev Soc Bras Med Trop 45:255–256CrossRefPubMedGoogle Scholar
  41. 41.
    Albas A, de Campos AC, Araujo DB et al (2011) Molecular characterization of rabies virus isolated from non-haematophagous bats in Brazil. Rev Soc Bras Med Trop 44:678–683CrossRefPubMedGoogle Scholar
  42. 42.
    Ministério da Saúde—Brasil (2012) Nota Técnica n. 19 – CGDT/DEVEP/SVS/MS. Diretrizes da vigilância em saúde para atuação diante de casos de raiva em morcegos em áreas urbanasGoogle Scholar
  43. 43.
    Pereira MG (1995) Doenças infecciosas. In: Koogan Guanabara (ed) Epidemiologia: teoria e prática, 1st. Guanabara Koogan S.A, Rio de Janeirol, pp 419–448Google Scholar
  44. 44.
    Schneider MC (1990) Estudo de avaliação sobre área de risco para a raiva no Brasil. Escola Nacional de Saúde Pública da Fundação Oswaldo CruzGoogle Scholar
  45. 45.
    Martins CM (2015) Avaliação e proposta de reestruturação do sistema de vigilância da raiva humana, canina e felina no Estado de São Paulo. Faculdade de Medicina Veterinaria da Universidade de São PauloGoogle Scholar
  46. 46.
    Woods M, Mcdonald RA, Harris S (2003) Predation of wildlife by domestic cats Felis catus in Great Britain. Mamm Rev 33:174–188. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  • Juliana Galera Castilho
    • 1
    Email author
  • Samira Maria Achkar
    • 1
  • Rafael de Novaes Oliveira
    • 1
  • Enio Mori
    • 1
  • Pedro CarnieliJr
    • 1
  • Carla Isabel Macedo
    • 1
  1. 1.Pasteur InstituteSão PauloBrazil

Personalised recommendations