RESUMO
Our proposal was to develop a vaccine based on total Leishmania antigens (TLA) adjuvanted with polyinosinic-polycytidylic acid [Poly(I:C)] able to induce a Th1 response which can provide protection against Leishmania infection. Mice were vaccinated with two doses of TLA-Poly(I:C) administered by subcutaneous route at 3-week interval. Humoral and cellular immune responses induced by the immunization were measured. The protective efficacy of the vaccine was evaluated by challenging mice with infective promastigotes of Leishmania (Leishmania) amazonensis into the footpad. Mice vaccinated with TLA-Poly(I:C) showed a high anti-Leishmania IgG titre, as well as increased IgG1 and IgG2a subclass titres compared with mice vaccinated with the TLA alone. The high IgG2a indicated a Th1 bias response induced by the TLA-Poly(I:C) immunization. Accordingly, the cellular immune response elicited by the formulation was characterized by an increased production of IFN-γ and no significant production of IL-4. The TLA-Poly(I:C) immunization elicited good protection, which was associated with decreased footpad swelling, a lower parasite load and a reduced histopathological alteration in the footpad. Our findings demonstrate a promising vaccine against cutaneous leishmaniasis that is relatively economic and easy to develop and which should be taken into account for preventing leishmaniasis in developing countries.
Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Leishmania/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Cutânea/prevenção & controle , Poli I-C/imunologia , Células Th1/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antiprotozoários/imunologia , Feminino , Imunidade Celular , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Interferon gama/biossíntese , Interleucina-4/biossíntese , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Poli I-C/administração & dosagem , VacinaçãoRESUMO
A simple and reliable indirect enzyme-linked immunosorbent assay for detection of antibodies directed against a major bovine viral diarrhea virus (BVDV) immunogen, the E2 glycoprotein (tE2-ELISA), has been developed using the recombinant C-terminal truncated E2 glycoprotein (tE2) expressed in a Drosophila melanogaster system. This strategy demonstrated that tE2 is secreted efficiently in the supernatant, no purification steps are necessary, it is easy to produce and carries out the post translational modifications necessary to preserve its native conformation. Preliminary analysis of 183 cattle serum samples using tE2-ELISA showed a 98% specificity and a 100% sensitivity compared with the standard homologous BVDV virus neutralization test. The results also showed that the tE2 is immunoreactive because the conformation and antigenicity of the original E2 are maintained to a large extent. To our knowledge this is the first study report of the recombinant tE2 of BVDV expressed in D. melanogaster system as an antigen for ELISA.
Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Vírus da Diarreia Viral Bovina/imunologia , Ensaio de Imunoadsorção Enzimática , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos Virais/biossíntese , Antígenos Virais/isolamento & purificação , Bovinos , Vírus da Diarreia Viral Bovina/genética , Drosophila melanogaster , Testes de Neutralização , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Sensibilidade e Especificidade , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/isolamento & purificaçãoRESUMO
Triatoma virus (TrV) is the only virus described to date that infects triatomines, and has previously been considered to be a member of the family Picornaviridae on the basis of physico-chemical properties. The genome of TrV was sequenced completely (9010 nt). Analysis of the sequence revealed the presence of two large open reading frames (ORFs). The predicted amino acid sequence of ORF1 (nt 549-5936) showed significant similarity to the non-structural proteins of several animal and plant RNA viruses. This ORF product contains sequence motifs characteristic of RNA-dependent RNA polymerases (RdRp), cysteine proteases and RNA helicases. ORF1 is preceded by 548 nucleotides of non-coding RNA and the two ORFs are separated by 172 nucleotides of non-coding RNA. Direct N terminus sequence analysis of two capsid proteins showed that ORF2 (nt 6109-8715) encodes the structural proteins of TrV. The predicted amino acid sequence of ORF2 is very similar to the corresponding regions of Drosophila C virus, Plautia stali intestine virus, Rhopalosiphum padi virus and Himetobi P virus and to a partial sequence from the 3' end of the cricket paralysis virus genome. All of these viruses have a novel genome organization and it has been proposed that they are not members of the Picornaviridae, as previously thought, but belong to a new virus family. On the basis of similarities of genome organization, we propose that TrV also belongs to this new virus family.
Assuntos
Vírus de RNA/classificação , Vírus de RNA/genética , Triatoma/virologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Genoma Viral , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Feces from Triatoma infestans (Klug) infected with TrV showed a large number of well-preserved viral particles when examined by electron microscopy. No viral particles were observed in suspensions of feces uninfected insects. Fecal suspensions inoculated parenterally into uninfected triatomines killed the insects within 36 h, showing that infective TrV is present in the feces of infected insects. It also is demonstrated that T. infestans becomes infected with TrV while feeding on contaminated chickens, and all the chickens used to feed a colony of triatomines infected with TrV showed high anti-TrV titer in their sera, although no TrV replication could be demonstrated in chickens. Oral infection of T. infestans by contaminated feces probably contributes to virus dispersal in nature. This observation provides the rationale for the potential use of TrV as a biological control agent.
Assuntos
Vírus de Insetos , Picornaviridae , Triatoma/virologia , Animais , Anticorpos Antivirais/imunologia , Galinhas , Fezes/virologia , Vírus de Insetos/fisiologia , Picornaviridae/fisiologiaRESUMO
In a survey of wild populations of Triatoma infestans (Klug) in Argentina, 10% were infected with Triatoma virus (TrV). The virus also was detected in a laboratory colony 18 mo after being established, with infection rates up to 100%. Mortality rate was 97.6% in nymphs and the molting process was inhibited, thereby increasing development time. Because the virus was detected in colony nymphs. TrV may be transmitted vertically. However, the higher infection rate in the colony compared with natural populations also indicates other route(s) of transmission.
Assuntos
Vírus de Insetos/patogenicidade , Picornaviridae/patogenicidade , Triatoma/virologia , Animais , Cobaias , Vírus de Insetos/isolamento & purificação , Muda , Ninfa , Picornaviridae/isolamento & purificação , Coelhos , Fatores de Tempo , Triatoma/crescimento & desenvolvimentoRESUMO
A set of antisera specific for each viral polypeptide of foot-and-mouth disease virus was used to provide a full comparison of polypeptides of two strains attenuated for cattle with respect to their parental virulent strains. Both attenuated strains, belonging to serotypes O1 Campos and C3 Resende, were obtained through serial passages of the corresponding virulent strains in chicken embryos. Although mutations were scattered throughout the genome, both attenuated strains showed an electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of viral polypeptide 3A faster than that of their respective wild-type strains. To determine the nature of this alteration, the nucleotide sequences of the genomic region encoding this polypeptide were determined. Comparative sequence analysis of wild-type and attenuated strains revealed 57 and 60 nucleotide deletions in the attenuated strains O1 Campos and C3 Resende, respectively. These studies, in conjunction with our previous analysis of recombinant viruses between wild-type and attenuated strains, which concluded that the major determinants of attenuation are located in the 3' half of the viral genome, strongly suggest that the alteration in polypeptide 3A of the attenuated strains is important for their reduced virulence in cattle.
Assuntos
Aphthovirus/genética , Peptídeos/genética , Animais , Aphthovirus/imunologia , Aphthovirus/patogenicidade , Sequência de Bases , Bovinos , Linhagem Celular , Deleção Cromossômica , Genes Virais , Soros Imunes , Dados de Sequência Molecular , Peptídeos/análise , Vacinas Atenuadas , VirulênciaRESUMO
Rotavirus, Cryptosporidium sp, and Salmonella spp. were investigated in the faeces of 452 diarrhoeic calves from 36 beef and 33 dairy herds. Animals surveyed were from a few days of age up to approximately 1 month of life. Enterotoxigenic Escherichia coli (ETEC) was studied in 212 calves, aged 15 days or less. The animals were from the Provinces of Buenos Aires (59% of the calves), Córdoba (18%), Santa Fe (16%), Entre Ríos (5%) and La Pampa (2%). A minimum of 4 calves were sampled on each farm. In beef calves rotavirus was excreted by 45.1% of the animals. Cryptosporidium by 30.5% and Sàlmonella serovars Arechabaleta, Livingstone, Panama and Typhimurium by 1.9%. In dairy calves Cryptosporidium was excreted by 29.6%, rotavirus by 23% and Salmonella serovar Dublin by 1.6%, ETEC was not detected in any calf. Rotavirus was the most widespread agent, detected in 32 (88.9%) beef herds and excreted by more than 50% of the calves in half of these herds. In contrast, rotavirus was only detected in 19 (57.5%) dairy herds and was excreted by more than 50% of the calves in 6 of these herds. Crytosporidium oocysts were identified in 27 (75%) beef and in 23 (69.7%) dairy farms. Salmonellosis due to serovar Dublin was associated with diarrhoea in 2 dairy herds. Concurrent infection with two or three agents occurred in 36 (8%) calves and 38 (55.1%) farms; the combination rotavirus-Cryptosporidium was found in 32 (6.9%) calves an in 33 (47.8) farms.
Assuntos
Doenças dos Bovinos/microbiologia , Diarreia/veterinária , Animais , Argentina/epidemiologia , Bovinos , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/parasitologia , Criptosporidiose/epidemiologia , Diarreia/epidemiologia , Diarreia/microbiologia , Diarreia/parasitologia , Surtos de Doenças/veterinária , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/veterinária , Fezes/microbiologia , Fezes/parasitologia , Infecções por Rotavirus/epidemiologia , Infecções por Rotavirus/veterinária , Salmonelose Animal/epidemiologiaRESUMO
Two porcine rotavirus strains (CN86 and CC86) isolated during an epidemiological survey of diarrhoea in swine in Argentina were studied because of several unique characteristics. Both these strains were isolated and cloned from the same faecal sample and the electrophoretic migration of 10 of their 11 genomic dsRNA genomic segments in polyacrylamide gels was identical, but strain CC86 had a supershort electropherotype. We analysed biochemical, serological and biological properties of both viruses. In vitro translation of genome segment 11 RNAs showed that both viruses produced a polypeptide with an apparent Mr of 26K. No differences in any of the other virus-induced proteins made in infected MA104 cells were found on one- and two-dimensional gels for either strain. In addition, the serotype and the subgroup specificities of both viruses were identical (group A, subgroup I, serotype 5). These results suggest that the rearranged strain was probably generated from the standard one and that the coding capacity of the rearranged segment was conserved. Consistent with this hypothesis, primer extension analysis revealed that the supershort strain had a rearrangement involving partial duplication of genomic segment 11. Biological studies showed differences between these viruses. The rearranged strain (CC86) produced larger plaques in monolayers of MA104 cells and outgrew the standard strain (CN86) when cells were coinfected with both viruses at different relative concentrations and different m.o.i. The possibility that large plaque formation and efficient virus replication can be influenced by the products of genomic segment 11, in addition to segment 4, is discussed.
Assuntos
Rearranjo Gênico , RNA de Cadeia Dupla/análise , RNA Viral/análise , Rotavirus/genética , Animais , Eletroforese em Gel de Poliacrilamida , Fezes/microbiologia , Genes Virais , Fenótipo , Biossíntese de Proteínas , Rotavirus/crescimento & desenvolvimento , Suínos , Ensaio de Placa ViralRESUMO
Rotavirus, Cryptosporidium sp. y Salmonella spp. fueron investigados en las heces de 452 terneros diarreicos provenientes de 36 rodeos de cría y 33 de tambo. Los animales muestreados tenían desde pocos días de vida hasta aproximadamente 1 mes de edad. Escherichia coli enterotoxigénica (ETEC) fue buscada en 212 terneros de 15 o menos días de edad. Los animales provenían de las Provincias de Buenos Aires (59%) de los terneros), Córdoba (18%), Santa Fe (16%), Entre Ríos (5%) y la Pampa (2%). Um mínimo de 4 terneros fue muestreado en cada establecimiento. En terneros de cría, rotavirus fue excretado por el 45,1% de los animales Cryptosporidium por el 30,5% y Salmonela serovariedades Arechabaleta, Livingstone, Panama y Typhimurium por el 1,9% (Cuadro 1). En terneros de tambo Cryptossporidium fue excretado per el 29,6%, rotavirus por el 23% y Salmonella serovariedad Dublin por el 1,6%. ETEC no fue detectado en ningún ternero. Rotavirus fue el agente más difundido, detectado en 32(88,9%) rodeos de cría (Cuadro 2) y excretado por más del 50% de los terneros en la mitad de esos rodeos. En contraste rotavirus fue solamente detectado en 19(57,5%) tambos y fue excretado por más del 50% de los terneros en 6 de esos rodeos. Se identificaron oocistos de Cryptosporidium en 27(75%) rodeos de cría y en 23(69,7%) tambos. La salmonelosis por la serovariedad Dublin se asoció con diarrea en 2 tambos. Infecciones concurrentes con dos o tres agentes ocurrieron en 36(8%) terneros y en 38(55,1%) establecimientos; la combinación rotavirus-Cryptosporidium se encontró en 31(6,9%) terneros y en 33(47,8) establecimientos (AU)
Assuntos
Estudo Comparativo , Animais , Bovinos , Diarreia/veterinária , Doenças dos Bovinos/microbiologia , Diarreia/epidemiologia , Diarreia/microbiologia , Diarreia/parasitologia , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/parasitologia , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/veterinária , Infecções por Rotavirus/epidemiologia , Infecções por Rotavirus/veterinária , Salmonelose Animal/epidemiologia , Fezes/microbiologia , Fezes/parasitologia , Criptosporidiose/epidemiologiaRESUMO
Rotavirus, Cryptosporidium sp. y Salmonella spp. fueron investigados en las heces de 452 terneros diarreicos provenientes de 36 rodeos de cría y 33 de tambo. Los animales muestreados tenían desde pocos días de vida hasta aproximadamente 1 mes de edad. Escherichia coli enterotoxigénica (ETEC) fue buscada en 212 terneros de 15 o menos días de edad. Los animales provenían de las Provincias de Buenos Aires (59%) de los terneros), Córdoba (18%), Santa Fe (16%), Entre Ríos (5%) y la Pampa (2%). Um mínimo de 4 terneros fue muestreado en cada establecimiento. En terneros de cría, rotavirus fue excretado por el 45,1% de los animales Cryptosporidium por el 30,5% y Salmonela serovariedades Arechabaleta, Livingstone, Panama y Typhimurium por el 1,9% (Cuadro 1). En terneros de tambo Cryptossporidium fue excretado per el 29,6%, rotavirus por el 23% y Salmonella serovariedad Dublin por el 1,6%. ETEC no fue detectado en ningún ternero. Rotavirus fue el agente más difundido, detectado en 32(88,9%) rodeos de cría (Cuadro 2) y excretado por más del 50% de los terneros en la mitad de esos rodeos. En contraste rotavirus fue solamente detectado en 19(57,5%) tambos y fue excretado por más del 50% de los terneros en 6 de esos rodeos. Se identificaron oocistos de Cryptosporidium en 27(75%) rodeos de cría y en 23(69,7%) tambos. La salmonelosis por la serovariedad Dublin se asoció con diarrea en 2 tambos. Infecciones concurrentes con dos o tres agentes ocurrieron en 36(8%) terneros y en 38(55,1%) establecimientos; la combinación rotavirus-Cryptosporidium se encontró en 31(6,9%) terneros y en 33(47,8) establecimientos