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

RESUMO

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.

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RESUMO

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

RESUMO

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

RESUMO

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

RESUMO

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

RESUMO

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

RESUMO

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

RESUMO

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

RESUMO

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 SOlmonella 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.

RESUMO

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

Assuntos

RESUMO

Bovine rotaviruses isolated from beef and dairy herds in Argentina were serotyped by the immunoperoxidase focus reduction assay as previously described (G. Gerna, M. Battaglia, G. Milenesi, N. Passarani, E. Percivalle, and E. Cattaneo, Infect. Immun. 43:722-729, 1984). Three strains from beef herds were related to the UK and NCDV bovine rotavirus strains defined as serotype 6 (Y. Hoshino, R. G. Wyatt, H. B. Greenberg, J. Flores, and A. Z. Kapikian, J. Infect. Dis. 149:694-702, 1984). Two other strains from dairy herds were classified as bovine viruses related to the bovine B223 strain reported by Woode and co-workers (G. N. Woode, N. E. Kelso, T. F. Simpson, S. K. Gaul, L. E. Evans, and L. Babiuk, J. Clin. Microbiol. 18:358-364, 1983) in the United States. A serotyping antibody-capture enzyme-linked immunoassay to detect serotype 6 rotavirus using a serotype 6-specific monoclonal antibody was developed and evaluated for strain characterization. Characterization of 72 group A rotavirus-positive fecal samples from beef herds and 43 fecal samples from dairy herds showed a predominance of serotype 6 rotavirus in beef herds but both serotype 6 and non-serotype 6 rotaviruses in dairy herds. Analysis of genomic double-stranded RNA by polyacrylamide gel electrophoresis showed that when outbreaks were caused by one serotype only a single electropherotype was present in all samples.

Assuntos

RESUMO

We have assessed the potency of an inactivated oil-adjuvanted rotavirus vaccine in beef herds in Argentina. Two different vaccine trials were conducted. In a small-scale experimental trial, involving 21 pregnant cows (13 vaccinated and eight unvaccinated controls), a significant increase in neutralizing antibody titres against different serotypes of bovine rotaviruses was found in both the colostrum and serum of vaccinated cows compared with that of unvaccinated controls. Seven days after birth, half of the calves born to vaccinated dams or to control cows were challenged with live virulent virus whereas the other half of both groups were left in contact with the infected calves in order to mimic a natural field challenge. Although no statistically significant differences in the rate of protection were observed among the different groups of animals, a larger number of vaccinated calves were protected in comparison with their controls, particularly where animals in contact with infected calves were concerned. Secondly, a large-scale field trial was carried out in 17 beef herds involving a total of 4066 vaccinated pregnant cows. In 11 farms morbidity and mortality in calves from vaccinated cows were compared with historical data from the previous 3 years at the same locations. In the other six herds, control groups were used to compare data of the same year: 1540 cows were vaccinated and 2700 were left as controls. Taking into account the previous and current incidence of diarrhoea, morbidity and mortality were significantly reduced in 16 of the 17 beef herds tested. Vaccine effectiveness was also evident in farms where other enteropathogens such as cryptosporidium and coronaviruses were present, together with rotavirus.

Assuntos

RESUMO

Fecal samples from 156 diarrheic piglets were collected from several herds located in two main breeding areas of Argentina. Rotaviruses were detected in 60 samples (38.4%) by polyacrylamide gel electrophoresis and in 55 samples by a group A-specific enzyme-linked immunosorbent assay (ELISA). All samples which were positive by polyacrylamide gel electrophoresis and negative by ELISA had elicited atypical electropherotypes resembling those of group B or C. ELISA-positive samples showing genome rearrangements were also detected (R.C. Bellinzoni, N.M. Mattion, O.R. Burrone, S.A. González, J.L. La Torre, and E.A. Scodeller, J. Clin. Microbiol. 25:952-954, 1987; N.M. Mattion, S.A. González, O.R. Burrone, R.C. Bellinzoni, J.L. La Torre, and E.A. Scodeller, J. Gen. Virol. 69:695-698, 1988). By subgrouping with monoclonal antibodies, it was found that of 32 positive samples, 13 belonged to subgroup I, 2 belonged to subgroup II, 2 samples had both specificities, and 15 samples were neither subgroup I nor subgroup II (non-I/II). In addition, 10 samples were adapted to grow in tissue culture, cloned, and serotyped by means of neutralization assays. Two samples were classified as serotype 5, and none of them were classified as serotype 4. The other strains showed only a one-way relationship with serotype 5 and can be tentatively classified as new porcine serotypes. Two samples with rearranged genomes had a one-way relationship with antiserum to human strain 69M, which displays a supershort electropherotype and was classified as a new human serotype (S. Matsuno, A. Hasegawa, A. Mukoyama, and S. Inouye, J. Virol. 54:623-624, 1985). At one farm, similar rearranged strains were detected during three successive years. Serotype changes were found between the isolates of the first and the second year, suggesting that a high degree of antigenic variability went on during continuous circulation of these strains in the field.

Assuntos

RESUMO

Some properties of Triatoma virus (TrV), a picorna-like virus recently isolated from Triatoma infestans, have been studied. Electron microscopic observations of purified viral preparations showed the presence of non-enveloped viral particles 30 nm in diameter. The sedimentation coefficient of virus particles was about 165S and the buoyant density in CsCl was 1.39 g/ml. The viral genome was composed of one single-stranded RNA molecule with an Mr of 3 x 10(6). Three major polypeptides with Mr values of 39K, 37K and 33K and a minor one of about 45K were found in the virus particle. TrV particles contain about 35% RNA and 65% protein by weight. These data support the classification of this virus in the family Picornaviridae.

Assuntos

RESUMO

Two aphthovirus intertypic recombinants between the virulent strain A Venceslau and guanidine-resistant attenuated mutants of either strain C3 Resende or O1 Campos were obtained in an attempt to establish the region(s) of the viral genome responsible for attenuation in cattle. Recombinants that inherited the 3' half of the genome from either attenuated parent and the 5' half from the virulent strain were selected and analyzed with respect to their ability to grow in cells of bovine origin and for their virulence in cattle. The results obtained support our previous conclusion, derived from studies with homotypic recombinants between attenuated aphthovirus type O1 and its original virulent strain, that the host range restriction phenotype for fetal bovine kidney cells of the attenuated strain is inherited from the 3' half of the genome. For the intertypic recombinants, however, this restriction is enhanced, presumably by the presence of a heterologous 5' half of the genomic region. In addition, we demonstrate that the results in vitro correlate with those of virulence tests in cattle.

Assuntos

RESUMO

The evolution in the field is described for foot-and-mouth disease viruses belonging to serotype C3 in Argentina between 1981 and 1986. During 1981 and 1982 only three isolations of this serotype took place, which showed minor serological and biochemical variations from the prototype strain C3 Resende-Brasil/55. At the beginning of 1983 an outbreak was detected in a restricted geographical region caused by strains which had important serological and biochemical differences from the prototype strain. However, revaccination of the animals with the available commercial vaccine was sufficient to control the disease. During 1984 and up to 1986, a new outbreak of the same serotype took place in an extended geographical region of Argentina. During this outbreak, two field strains were isolated, studied and later included in the vaccine. One of them, strain C84, was a serological variant of the prototype strain and its inclusion in the vaccine had only a limited effect in controlling the disease. Inclusion of the other representative strain, C85, together with virus C84 finally reduced the number of outbreaks to the usual number reported.

Assuntos

RESUMO

We have recently reported the isolation of two group A swine rotaviruses each lacking normal genomic RNA segment 11 and showing instead one extra segment that migrated abnormally on gel electrophoresis. Hybridization studies performed with segment-specific probes and with a purified abnormal RNA segment showed that the extra bands had sequence homology to normal segment 11. Analysis of protein profiles of normal and rearranged strains showed that the gene product of segment 11 had no apparent change in its relative electrophoretic migration, suggesting that the rearranged genes remained functional.

Assuntos

RESUMO

A sample of aphthovirus type C3 strain Resende carrying two polyribocytidilic acid [poly(C)] tracts was cloned in tissue culture. One clone with a poly(C)-rich tract of about 145 nucleotides long (clone 3B) and another with a poly(C)-rich tract of about 230 nucleotides long (clone 12) and a mixture of both were injected intralingually into three steers. Samples from all three animals were recovered during the acute phase of the disease, from the blood and from the feet, and at various days after inoculation from the oesophageal-pharyngeal (OP) fluids. Analysis of the viral RNAs of the positive samples by means of RNase T1 maps on one- and two-dimensional gels showed (1) changes in the electrophoretic mobility of the poly(C)-rich tracts of viruses recovered from the OP fluids at various times after infection; (2) selection of virus populations with poly(C)-rich tracts of increased size; (3) later on, changes in the patterns of oligonucleotides of persistent viruses. These variations may lead to the production of new strains with altered biological properties that may contribute to the maintenance and spread of these viruses in the field.

Assuntos

RESUMO

In Argentina the presence of rotavirus was investigated in a chicken flock experiencing periodic episodes of diarrhoea during the winter of 1986. All the samples analysed were negative by the enzyme-linked immunosorbent assay (ELISA). However, when samples were observed by electron microscopy, particles which were indistinguishable from standard rotaviruses were detected in some samples. Ten of the 36 samples were positive after polyacrylamide gel electrophoresis (PAGE) analysis, all of them showing the same electropherotype. Based on these results these viruses were classified as rotavirus-like or atypical rotaviruses.

Assuntos