PESQUISA VETERINÁRIA BRASILEIRA

Abstract in English:

ABSTRACT.- Rosa G.N., Domingues H.G., Santos M.M.A.B., Felippe P.A.N., Spilki F.R. & Arns C.W. 2012. [Molecular detection and phylogenetic analysis of the gene H from canine distemper virus isolates circulating at the municipality of Campinas, São Paulo.] Detecção molecular e análise filogenética do gene H de amostras do vírus da cinomose canina em circulação no município de Campinas, São Paulo. Pesquisa Veterinária Brasileira 32(1):72-77. Laboratório de Microbiologia Molecular, Instituto de Ciências da Saúde, Universidade Feevale, Rodovia RS-239 2755, Novo Hamburgo, RS 93352-000, Brazil. E-mail: fernandors@feevale.br Canine distemper virus (CDV), a Morbillivirus of the family Paramyxoviridae, is the etiological agent of neurological and systemic disease in dogs. The laboratory diagnosis of infection requires viral isolation or detection of genetic material of the virus in secretions or tissues of dogs with clinical suspicion of the disease. The genetic diversity among isolates of CDV can be assessed by sequencing and phylogenetic analysis of the gene that encodes the viral hemagglutinin (H gene), and there is currently a special interest in comparing the strains currently circulating in the field with the genogroup America-1, which comprises strains present in vaccines available in the market. In this study, the molecular detection of CDV gene H was performed from biological samples harvested ante-and post-mortem from 15 dogs with clinical signs suggestive of canine distemper in the metropolitan region of Campinas, São Paulo. Ten of the 15 dogs examined had at least one positive organ under molecular detection and the obtained amplicons were sequenced and further analyzed by molecular phylogenetic analysis. Similarly to what has already been reported on previous studies regarding the diversity of the gene H in other countries, the phylogenetic reconstruction obtained for the samples of cases of distemper from Campinas region showed they were grouped with the North American, European and Japanese newly described samples, a genetic group distinguished from classical samples of CDV, named America-1, which encompasses the vaccine strains Snyder Hill, Onderstepoort and Lederle.

• Canine distemper virus Vírus da cinomose

Abstract in Portuguese:

RESUMO.- Rosa G.N., Domingues H.G., Santos M.M.A.B., Felippe P.A.N., Spilki F.R. & Arns C.W. 2012. [Molecular detection and phylogenetic analysis of the gene H from canine distemper virus isolates circulating at the municipality of Campinas, São Paulo.] Detecção molecular e análise filogenética do gene H de amostras do vírus da cinomose canina em circulação no município de Campinas, São Paulo. Pesquisa Veterinária Brasileira 32(1):72-77. Laboratório de Microbiologia Molecular, Instituto de Ciências da Saúde, Universidade Feevale, Rodovia RS-239 2755, Novo Hamburgo, RS 93352-000, Brazil. E-mail: fernandors@feevale.br O vírus da cinomose canina (CDV), um Morbillivirus da família Paramyxoviridae, é o agente etiológico de doença neurológica e sistêmica em cães. O diagnóstico laboratorial da infecção requer o isolamento viral ou detecção do material genético do vírus em secreções ou tecidos de cães com suspeita clínica da doença. A diversidade genética entre os isolados de CDV pode ser aferida pelo sequenciamento e filogenia molecular do gene que codifica a hemaglutinina viral (gene H), havendo atualmente um especial interesse em comparar as amostras circulantes a campo com o genogrupo América-1, que abrange as cepas presentes nas vacinas disponíveis no mercado. No presente estudo, foi realizada a detecção molecular do gene H de CDV a partir de amostras biológicas colhidas ante- e post-mortem de 15 cães com sinais clínicos sugestivos de cinomose na região metropolitana de Campinas, São Paulo. Dez dos 15 cães analisados tiveram ao menos um órgão positivo na detecção molecular e os amplicons obtidos foram submetidos ao sequenciamento nucleotídico seguido de análise filogenética molecular. De forma semelhante ao que já foi reportado para estudo analisando a diversidade do gene H em outros países, a reconstrução filogenética obtida para as amostras de casos de cinomose da região de Campinas demonstrou as mesmas foram agrupadas junto a amostras norte-americanas, europeias e japonesas recentes, em um grupo genético distinto do grupo de amostras clássicas de CDV, nomeado America-1, o qual engloba as estirpes vacinais Snyder Hill, Onderstepoort e Lederle.

Abstract in English:

ABSTRACT.- Domingues H.G., Spilki F.R. & Arns C.W. 2011. [Molecular detection and phylogenetic analysis of bovine respiratory syncytial virus (BRSV) in swabs and lung tissues of adult cattle.] Detecção molecular e análise filogenética de vírus respiratório sincicial bovino (BRSV) em swabs e tecido pulmonar de bovinos adultos. Pesquisa Veterinária Brasileira 31(11):961-966. Laboratório de Microbiologia Molecular, Instituto de Ciências da Saúde, Universidade Feevale, Rodovia RS-239, 2755, Novo Hamburgo, RS 93352-000, Brazil. E-mail: fernandors@feevale.br Bovine respiratory syncytial viruses virus (BRSV) is one of the etiologic agents of pneumonia in young cattle. Few studies have been made aiming detection of the virus in samples collected from adult animals, especially those asymptomatic bovines. However, it is assumed that infections in these groups may occur mostly asymptomatic and this would be an important mechanism for maintaining of BRSV in herds. In this study, the goal was to conduct an analysis of the occurrence of asymptomatic infections by BRSV in lung samples (n=68) and nasal swabs (209) taken from adult animals collected in abattoirs from Southern and Southeastern Brazil respectively, to detect via polymerase chain reaction the occurrence of infected animals in populations of adult cattle. The samples that resulted positive (6) on RT-PCR were subsequently subjected to cutting with restriction enzymes and sequencing for genetic characterization (2 samples). All samples belongs to subgroup B of BRSV, which is reported as the one circulating in Brazil. The results obtained demonstrate that BRSV may be present in samples taken from adult animals, which is in agreement the hypothesis that infections in adults run in a sub-clinical way that may be of importance as a maintenance mechanism of the virus in bovine herds.

• BRSV bovine respiratory syncytial virus vírus respiratório sincicial bovino

Abstract in Portuguese:

RESUMO.- Domingues H.G., Spilki F.R. & Arns C.W. 2011. [Molecular detection and phylogenetic analysis of bovine respiratory syncytial virus (BRSV) in swabs and lung tissues of adult cattle.] Detecção molecular e análise filogenética de vírus respiratório sincicial bovino (BRSV) em swabs e tecido pulmonar de bovinos adultos. Pesquisa Veterinária Brasileira 31(11):961-966. Laboratório de Microbiologia Molecular, Instituto de Ciências da Saúde, Universidade Feevale, Rodovia RS-239, 2755, Novo Hamburgo, RS 93352-000, Brazil. E-mail: fernandors@feevale.br O vírus respiratório sincicial bovino (BRSV) é um dos agentes etiológicos de pneumonias em bovinos jovens. Poucos estudos foram realizados visando à detecção do agente em amostras coletadas de animais adultos, e em especial de bovinos assintomáticos. No entanto, presume-se que as infecções ocorridas nestes grupos possam ocorrer em sua maioria de forma assintomática e este seria um mecanismo importante para manutenção do BRSV nos rebanhos. No presente estudo, o objetivo foi realizar uma análise da prevalência de infecções assintomáticas pelo BRSV em pulmões (n=68) e swabs nasais (209) coletados de bovinos adultos coletadas em frigoríficos da região Sul e Sudeste respectivamente, no sentido de detectar por intermédio de reação da polimerase em cadeia qual a taxa de animais infectados em populações de animais adultos onde não ocorram sinais clínicos da infecção. As amostras positivas à RT-PCR (6) foram posteriormente submetidas ao corte com enzimas de restrição (REA) e sequenciamento para caracterização genética do gene F (2 das amostras). Todas as amostras se enquadram no subgrupo B de BRSV, o grupo circulante no Brasil conforme estudos anteriores. Os resultados obtidos demonstram que o BRSV pode estar presente em amostras obtidas de animais sadios, reforçando a hipótese de que infecções subclínicas fazem parte do mecanismo de manutenção do vírus nos rebanhos.

Abstract in English:

ABSTRACT.- Ståhl K., Benito A., Felmer R., Zuñiga J., Reinhardt G., Rivera H., Baule C. & Moreno-López J. 2009. Genetic diversity of bovine viral diarrhoea virus (BVDV) from Peru and Chile. Pesquisa Veterinária Brasileira 29(1):41-44. Joint Virology Research and Development Division, National Veterinary Institute and Swedish University of Agricultural Sciences Uppsala, Sweden. E-mail: Karl.Stahl@bvf.slu.se Twenty-five BVDV strains, detected in serum from persistently infected cattle from Peru (n=15) and Chile (n=10) were genetically characterized. The phylogenetic analysis based on the 5’ UTR showed that all 25 strains belonged to genotype 1. Twenty-three of the strains could further be subdivided into subtype 1b, and two out of ten Chilean strains into subtype 1a. In conclusion, in total 23 out of 25 strains analyzed were of genotype 1, subtype 1b. This is the predominant BVDV subtype in many countries all over the world, including USA. The close homology with previously described strains reflects the influence of livestock trade on the diversity of BVDV circulating within and between countries and continents. Peru and Chile have imported large numbers of cattle from USA and Europe, mostly with insufficient or lacking health documentation.

• Bovine viral diarrhoea virus BVDV diversity pestivirus phylogenetic analysis

Abstract in Portuguese:

ABSTRACT.- Ståhl K., Benito A., Felmer R., Zuñiga J., Reinhardt G., Rivera H., Baule C. & Moreno-López J. 2009. Genetic diversity of bovine viral diarrhoea virus (BVDV) from Peru and Chile. Pesquisa Veterinária Brasileira 29(1):41-44. Joint Virology Research and Development Division, National Veterinary Institute and Swedish University of Agricultural Sciences Uppsala, Sweden. E-mail: Karl.Stahl@bvf.slu.se Twenty-five BVDV strains, detected in serum from persistently infected cattle from Peru (n=15) and Chile (n=10) were genetically characterized. The phylogenetic analysis based on the 5’ UTR showed that all 25 strains belonged to genotype 1. Twenty-three of the strains could further be subdivided into subtype 1b, and two out of ten Chilean strains into subtype 1a. In conclusion, in total 23 out of 25 strains analyzed were of genotype 1, subtype 1b. This is the predominant BVDV subtype in many countries all over the world, including USA. The close homology with previously described strains reflects the influence of livestock trade on the diversity of BVDV circulating within and between countries and continents. Peru and Chile have imported large numbers of cattle from USA and Europe, mostly with insufficient or lacking health documentation.

Abstract in English:

ABSTRACT.- Campos T.A., Lago J.C., Nakazato G., Stehling E.G., Brocchi M., Castro A.F.P. & Silveira W.D. 2008. Occurrence of virulence-related sequences and phylogenetic analysis of commensal and pathogenic avian Escherichia coli strains (APEC). Pesquisa Veterinária Brasileira 28(10):533-540. Departamento de Microbiologia e Immunologia, Instituto de Biologia, Unicamp, Cidade Universitrária Zeferino Vaz s/n, Campinas, SP 13081-862, Brazil. E-mail: wds@unicamp.br The presence of iron uptake (irp-2, fyuA, sitA, fepC, iucA), adhesion (iha, lpfAO157/O141, lpfAO157/O154, efa, toxB) and invasion (inv, ial-related DNA sequences and assignment to the four main Escherichia coli phylogenetic groups (A, B1, B2 e D) were determined in 30 commensal E. coli strains isolated from healthy chickens and in 49 APEC strains isolated from chickens presenting clinical signs of septicemia (n=24) swollen head syndrome (n=14) and omphalitis (n=11) by PCR. None of the strains presented DNA sequences related to the inv, ial, efa, and toxB genes. DNA sequences related to lpfAO157/O154, iucA, fepC, and irp-2 genes were significantly found among pathogenic strains, where iucA gene was associated with septicemia and swollen head syndrome and fepC and irp-2 genes were associated with swollen head syndrome strains. Phylogenetic typing showed that commensal and omphalitis strains belonged mainly to phylogenetic Group A and swollen head syndrome to phylogenetic Group D. Septicemic strains were assigned in phylogenetic Groups A and D. These data could suggest that clonal lineage of septicemic APEC strains have a multiple ancestor origin; one from a pathogenic bacteria ancestor and other from a non-pathogenic ancestor that evolved by the acquisition of virulence related sequences through horizontal gene transfer. Swollen head syndrome may constitute a pathogenic clonal group. By the other side, omphalitis strains probably constitute a non-pathogenic clonal group, and could cause omphalitis as an opportunistic infection. The sharing of virulence related sequences by human pathogenic E. coli and APEC strains could indicate that APEC strains could be a source of virulence genes to human strains and could represent a zoonotic risk.

• Avian colibacillosis Escherichia coli APEC virulence related-genes pathogenic clones

Abstract in Portuguese:

ABSTRACT.- Campos T.A., Lago J.C., Nakazato G., Stehling E.G., Brocchi M., Castro A.F.P. & Silveira W.D. 2008. Occurrence of virulence-related sequences and phylogenetic analysis of commensal and pathogenic avian Escherichia coli strains (APEC). Pesquisa Veterinária Brasileira 28(10):533-540. Departamento de Microbiologia e Immunologia, Instituto de Biologia, Unicamp, Cidade Universitrária Zeferino Vaz s/n, Campinas, SP 13081-862, Brazil. E-mail: wds@unicamp.br The presence of iron uptake (irp-2, fyuA, sitA, fepC, iucA), adhesion (iha, lpfAO157/O141, lpfAO157/O154, efa, toxB) and invasion (inv, ial-related DNA sequences and assignment to the four main Escherichia coli phylogenetic groups (A, B1, B2 e D) were determined in 30 commensal E. coli strains isolated from healthy chickens and in 49 APEC strains isolated from chickens presenting clinical signs of septicemia (n=24) swollen head syndrome (n=14) and omphalitis (n=11) by PCR. None of the strains presented DNA sequences related to the inv, ial, efa, and toxB genes. DNA sequences related to lpfAO157/O154, iucA, fepC, and irp-2 genes were significantly found among pathogenic strains, where iucA gene was associated with septicemia and swollen head syndrome and fepC and irp-2 genes were associated with swollen head syndrome strains. Phylogenetic typing showed that commensal and omphalitis strains belonged mainly to phylogenetic Group A and swollen head syndrome to phylogenetic Group D. Septicemic strains were assigned in phylogenetic Groups A and D. These data could suggest that clonal lineage of septicemic APEC strains have a multiple ancestor origin; one from a pathogenic bacteria ancestor and other from a non-pathogenic ancestor that evolved by the acquisition of virulence related sequences through horizontal gene transfer. Swollen head syndrome may constitute a pathogenic clonal group. By the other side, omphalitis strains probably constitute a non-pathogenic clonal group, and could cause omphalitis as an opportunistic infection. The sharing of virulence related sequences by human pathogenic E. coli and APEC strains could indicate that APEC strains could be a source of virulence genes to human strains and could represent a zoonotic risk.

Abstract in English:

Abstract.- Barry A.F, Alfieri A.F. & Alfieri A.A. 2008. Detection and phylogenetic analysis of porcine enteric calicivirus, genetically related to the Cowden strain of sapovirus genogroup III, in Brazilian swine herds. Pesquisa Veterinária Brasileira 28(1):82-86. Laboratório de Virologia Animal, Departamento de Medicina Veterinária Preventiva, Centro de Ciências Agrárias, Universidade Estadual de Londrina, Campus Universitário, Londrina, PR 86051-990, Brazil. E-mail: alinebarry@uol.com.br Sapovirus of the Caliciviridae family is an important agent of acute gastroenteritis in children and piglets. The Sapovirus genus is divided into seven genogroups (G), and strains from the GIII, GVI and GVII are associated with infections in swine. Despite the high prevalence in some countries, there are no studies related to the presence of porcine enteric sapovirus infections in piglets in Brazil. In the present study, 18 fecal specimens from piglets up to 28 days were examined to determine the presence of sapovirus genome by RT-PCR assay, using primers designed to amplify a 331 bp segment of the RNA polymerase gene. In 44.4% (8/18) of fecal samples, an amplified DNA fragment was obtained. One of these fragments was sequenced and submitted to molecular and phylogenetic analysis. This analysis revealed high similarity, with nucleotides (87%) and amino acids (97.8%), to the Cowden strain, the GIII prototype of porcine enteric calicivirus. This is the first description of sapovirus in Brazilian swine herds.

• Swine; piglets diarrhea calicivirus Sapovirus

Abstract in Portuguese:

Abstract.- Barry A.F, Alfieri A.F. & Alfieri A.A. 2008. Detection and phylogenetic analysis of porcine enteric calicivirus, genetically related to the Cowden strain of sapovirus genogroup III, in Brazilian swine herds. Pesquisa Veterinária Brasileira 28(1):82-86. Laboratório de Virologia Animal, Departamento de Medicina Veterinária Preventiva, Centro de Ciências Agrárias, Universidade Estadual de Londrina, Campus Universitário, Londrina, PR 86051-990, Brazil. E-mail: alinebarry@uol.com.br Sapovirus of the Caliciviridae family is an important agent of acute gastroenteritis in children and piglets. The Sapovirus genus is divided into seven genogroups (G), and strains from the GIII, GVI and GVII are associated with infections in swine. Despite the high prevalence in some countries, there are no studies related to the presence of porcine enteric sapovirus infections in piglets in Brazil. In the present study, 18 fecal specimens from piglets up to 28 days were examined to determine the presence of sapovirus genome by RT-PCR assay, using primers designed to amplify a 331 bp segment of the RNA polymerase gene. In 44.4% (8/18) of fecal samples, an amplified DNA fragment was obtained. One of these fragments was sequenced and submitted to molecular and phylogenetic analysis. This analysis revealed high similarity, with nucleotides (87%) and amino acids (97.8%), to the Cowden strain, the GIII prototype of porcine enteric calicivirus. This is the first description of sapovirus in Brazilian swine herds.

Abstract in English:

ABSTRACT.- Cortez A., Heinemann M.B., Castro A.M.M.G., Soares M.S, Pinto A.M.V., Alfieri A.A., Flores E.F., Leite R.C. & Richtzenhain L.J. 2006. Genetic characterization of Brazilian bovine viral diarrhea virus isolates by partial nucleotide sequencing of the 5’-UTR region. Pesquisa Veterinária Brasileira 26(4):211-216. Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87, Cidade Universitária, São Paulo, SP 05508-000, Brazil. E-mail: leonardo@usp.br Nineteen isolates of bovine viral diarrhea virus (BVDV) from Brazil were genetically characterized through partial nucleotide sequencing and analysis of the 5’UTR region. The isolates were grouped as BVDV-1 (11/19), BVDV-2 (6/19) or “atypical” pestivirus (2/19). Among the BVDV-1, eight isolates were classified as subgenotype BVDV-1a, whereas most (4 out of 6) BVDV-2 belonged to subgenotype 2b. Two isolates from aborted fetuses were not classified into any genetic group, being considered atypical BVDVs. Genetic diversity among Brazilian BVDV isolates may be responsible for vaccination and diag-nostic failure and therefore may influence the control strategies for BVDV infection in the country.

• BVDV-1 BVDV-2 Pestivirus phylogenetic analysis

Abstract in Portuguese:

ABSTRACT.- Cortez A., Heinemann M.B., Castro A.M.M.G., Soares M.S, Pinto A.M.V., Alfieri A.A., Flores E.F., Leite R.C. & Richtzenhain L.J. 2006. Genetic characterization of Brazilian bovine viral diarrhea virus isolates by partial nucleotide sequencing of the 5’-UTR region. Pesquisa Veterinária Brasileira 26(4):211-216. Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87, Cidade Universitária, São Paulo, SP 05508-000, Brazil. E-mail: leonardo@usp.br Nineteen isolates of bovine viral diarrhea virus (BVDV) from Brazil were genetically characterized through partial nucleotide sequencing and analysis of the 5’UTR region. The isolates were grouped as BVDV-1 (11/19), BVDV-2 (6/19) or “atypical” pestivirus (2/19). Among the BVDV-1, eight isolates were classified as subgenotype BVDV-1a, whereas most (4 out of 6) BVDV-2 belonged to subgenotype 2b. Two isolates from aborted fetuses were not classified into any genetic group, being considered atypical BVDVs. Genetic diversity among Brazilian BVDV isolates may be responsible for vaccination and diag-nostic failure and therefore may influence the control strategies for BVDV infection in the country.

Abstract in English:

ABSTRACT.- Callado A.K.C., Castro R.S. & Teixeira M.F.S. 2001. [Lentiviruses of small ruminants (CAEV and Maedi-Visna): a review and perspectives] Lentivírus de pequenos ruminantes (CAEV e Maedi-Visna): revisão e perspectivas. Pesquisa Veterinária Brasileira 21(3):87-97. Depto Medicina Veterinária, Universidade Federal Rural de Pernambuco, Rua Dom Manoel de Medeiros s/n, Dois Irmãos, Recife, PE 55171-000, Brazil. E-mail: callado@altavista.net Small ruminant lentiviruses (SRLV), whose prototypes are Caprine Arthritis-Encephalitis virus (CAEV) and Maedi-Visna virus, are the causative agents of slow progressive degenerative diseases of goats and sheep (infected animals), responsible for significant economic losses. These viruses cause persistent infections with long periods of incubation and induce inflammatory and degenerative lesions. The lesions are induced in target organs of the host such as joints, CNS, lungs and mammary glands dueto viral replication in cells of the monocyte/macrophage lineage which is the main target cell. Infections occur particularly in the young and are acquired through ingestion of virus in milk or colostrum from infected does or ewes. The induction of immune response is variable and does not protect against the infection. Diagnosis is primarily based on the presence of SRLV antibodies usually detected by agar gel immunodiffusion (AGID) or enzyme linked immunosorbent assays (ELISA). As no vaccine is available, most often employed schemes to prevent spread of SRLV are based on segregation or/and culling of positive animals associated with management practices, especially the offspring. The strategies of SRLV for dealing with the immune system make difficult to accomplish diagnosis of infection, control or prevention of the viral spread. This review shows aspects of SRLV based on their phylogenetic studies of fields isolates, clinical, and immunopathological features.

• Small ruminant lentiviruses caprine arthritis-encephalitis virus maedi-visna retroviruses phylogenetic analysis epidemiology goat sheep Lentivírus de pequenos ruminantes vírus da artrite-encefalite caprina retrovírus análises filogenéticas epidemiologia caprino ovino.

Abstract in Portuguese:

RESUMO.- Callado A.K.C., Castro R.S. & Teixeira M.F.S. 2001. [Lentiviruses of small ruminants (CAEV and Maedi-Visna): a review and perspectives] Lentivírus de pequenos ruminantes (CAEV e Maedi-Visna): revisão e perspectivas. Pesquisa Veterinária Brasileira 21(3):87-97. Depto Medicina Veterinária, Universidade Federal Rural de Pernambuco, Rua Dom Manoel de Medeiros s/n, Dois Irmãos, Recife, PE 55171-000, Brazil. E-mail: callado@altavista.net Os lentivírus de pequenos ruminantes (SRLV), cujos protótipos são os vírus da Artrite-Encefalite Caprina (CAEV) e Maedi-Visna, são patógenos amplamente distribuidos, os quais causam doenças degenerativas progressivas lentas em caprinos e ovinos, determinando importantes perdas econômicas. Estes vírus causam infecções persistentes com período de incubação longo e causam inflamatórias e degenerativas. As lesões são induzidas em tecidos específicos do hospedeiro como articulações, pulmões, CNS e glândulas mamárias devido à replicação virai em células da linhagem monocítico-fagocitária que são as principais células-alvo. A infecção ocorre principalmente durante os primeiros meses de vida, através da ingestão de vírus no leite ou colostro de cabras ou ovelhas infectadas. A indução da resposta imunológica é variável e não protege contra a infecção. O diagnóstico é baseado primariamente na detecção de anticorpos para SRLV, geralmente por imunodifusão em gel de agar (AGID) e enzyme linked immunosorbent assay (ELISA). O diagnóstico e separação ou descarte dos animais soropositivos associado ao uso de certas práticas de manejo, especialmente das crias, são os principais meios implementados para prevenir a disseminação de SRLV, uma vez que ainda não existe vacina contra o vírus. As estratégias adotadas pelos SRLV para enfrentar o sistema imune dificultam o diagnóstico da infecção, controle ou prevenção da disseminação de SRLV. Esta revisão apresenta alguns aspectos das lentivíroses de pequenos ruminantes baseadas em estudos filogenéticos de amostras isoladas, aspectos clínicos e imunopatológicos.