RESUMO
Equid alphaherpesvirus 3 (EHV3) is the etiological agent of equine coital exanthema (ECE), which is a venereal, highly contagious disease, characterized by the formation of papules, vesicles, pustules and ulcers on the external genitalia of mares and stallions. EHV3 remains in a latent state after a successful infection and there are latently infected animals in which the virus is reactivated and generally re-excreted subclinically. There are no available vaccines for this condition and prevention is based on the clinical examination of mares prior to mating, which allows to segregate those showing clinical signs. As this approach does not eliminate the risk of contagion in stallions from subclinically infected mares, there is a need for a specific EHV3 treatment. Nowadays, there exist various antiviral compounds of proven effectiveness for other alphaherpesviruses affecting humans and animals. The aim of the present study was to compare the efficacy of three antiviral compounds, acyclovir, ganciclovir and cidofovir against EHV3 in vitro, and to assess their efficacy against six EHV3 Argentinian field isolates. To determine the efficacy of these compounds in vitro, three parameters were analyzed: reduction of plaque number, reduction of plaque size and reduction of viral production. Additionally, the effectiveness of the three compounds at an optimum concentration previously determined in this study was investigated for the EHV3 field isolates. Based on our results, ganciclovir was the most potent antiviral compound to reduce EHV3 replication in vitro and may thus be a valuable candidate for treatment and prevention of ECE in mares and stallions.
Assuntos
Aciclovir/farmacologia , Antivirais/farmacologia , Cidofovir/farmacologia , Ganciclovir/farmacologia , Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 3/efeitos dos fármacos , Doenças dos Cavalos/virologia , Animais , Células Cultivadas , Feminino , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 3/isolamento & purificação , CavalosRESUMO
HoBi-like pestiviruses (also known as bovine viral diarrhea virus 3) have been sporadically reported from naturally infected cattle in Brazil, Asia, and Europe. Although HoBi-like viruses seem to be endemic in Brazilian cattle and buffalo, they have not been studied in the other countries of South America to our knowledge. Herein we report serologic results of buffalo from 12 large farms in Argentina located near the Brazilian border. These buffalo were not vaccinated against pestiviruses. Our results indicate that HoBi-like virus may be circulating in the northeastern region of Argentina given that half of the analyzed animals showed high levels of neutralizing antibodies against the pestivirus. The HoBi-like seropositive animals were also checked for neutralizing antibodies against BVDV-1a, BVDV-1b, and BVDV-2, and in most cases these animals had low levels or no detectable antibodies against these other pestiviruses. Our study suggests a need for continued pestivirus surveillance in Argentinean cattle and buffalo.
Assuntos
Búfalos , Infecções por Pestivirus/veterinária , Pestivirus/isolamento & purificação , Animais , Argentina/epidemiologia , Feminino , Masculino , Infecções por Pestivirus/epidemiologia , Infecções por Pestivirus/virologia , Prevalência , Estudos SoroepidemiológicosRESUMO
In this study, we evaluated the immunogenicity and efficacy of mucosal delivery of a recombinant modified vaccinia Ankara virus (MVA) expressing the secreted version of bovine herpesvirus type 1 (BoHV-1) glycoprotein D (MVA-gDs) without addition of adjuvant in two animal models. First, we demonstrated the capability of MVA-gDs of inducing both local and systemic anti-gD humoral immune response after intranasal immunization of mice. Then, we confirmed that two doses of MVA-gDs administered intranasally to rabbits induced systemic anti-gD antibodies and conferred protection against BoHV-1 challenge. Our results show the potential of using MVA as a vector for the rational design of veterinary vaccines capable of inducing specific and protective immune responses both at local and systemic level.
Assuntos
Portadores de Fármacos , Infecções por Herpesviridae/prevenção & controle , Herpesvirus Bovino 1/imunologia , Vacinas contra Herpesvirus/imunologia , Vaccinia virus/genética , Proteínas Virais/imunologia , Administração Intranasal , Animais , Anticorpos Antivirais/sangue , Modelos Animais de Doenças , Feminino , Infecções por Herpesviridae/imunologia , Vacinas contra Herpesvirus/administração & dosagem , Vacinas contra Herpesvirus/genética , Camundongos Endogâmicos BALB C , Coelhos , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Proteínas Virais/genéticaRESUMO
Herpesviruses have mainly co-evolved with their hosts for millions of years. However, bovine herpesvirus 1 (BoHV1) and related ruminant alphaherpesviruses have been reported to cross the species barrier. Bubaline herpesvirus 1 (BuHV1) is an alphaherpesvirus closely related to BoHV1 and BoHV5. According to the serological cross-relationships between ruminant alphaherpesviruses, several surveys have studied the occurrence of BoHV1-related virus infection in wild and domestic ruminant species. Recent studies in Argentina showed an increase in serological prevalence against BoHV1 related viruses in water buffaloes (Bubalus bubalis) population. The aim of this study was to investigate the presence of related ruminant alphaherpesvirus in the Argentinean water buffalo population. BuHV1 was successfully isolated from 5 out of 225 buffaloes analyzed. One isolate was obtained from nasal secretions, and the others were from vaginal swabs. The buffaloes belonged to four different farms located in northeastern Argentina. The isolates were characterized as alphaherpesvirus by direct immunofluorescence using FITC-anti-BoHV1 IgG. Restriction analysis performed with BamHI and BstEII on the complete genome showed differences between the isolates and those from BoHV1 and BoHV5 subtypes. Phylogenetic analysis on both UL27 and US6 showed similarity in tree topology. While three of the isolates grouped together with sequences of BoHV5, two other isolates clustered separately. Genetic analysis of eight concatenated sequences from all isolates and references strains showed high nucleotide sequence identity between BuHV1 and BoHV5. While three of the isolates clustered together with the BoHV5 reference strain, the last two isolates were closely related to an Australian BuHV1 strain. To our knowledge, this is the first report on the isolation and molecular characterization of BuHV1 in South America. Phylogenetic analysis suggested that two different BuHV1 lineages circulate in the Argentinean water buffalo population.
Assuntos
Alphaherpesvirinae/isolamento & purificação , Búfalos/virologia , Infecções por Herpesviridae/veterinária , Alphaherpesvirinae/classificação , Alphaherpesvirinae/genética , Animais , Argentina , Infecções por Herpesviridae/virologia , Dados de Sequência Molecular , FilogeniaRESUMO
BACKGROUND: Parainfluenza virus type 3 (PIV3) was isolated from dairy buffaloes (Bubalus bubalis) naturally affected with respiratory and reproductive clinical conditions. RESULTS: Examination of nasal and vaginal swabs collected from 12 diseased buffaloes led to the isolation of three paramyxovirus isolates from two animals. Antigenic, morphological and biological characteristics of these three isolates were essentially similar to those of members of the Paramyxoviridae family. Antigenic analysis by direct immunofluorescence and cross neutralization test placed these isolates together with bovine parainfluenza virus type 3 (BPIV3). Nucleotide and amino acid phylogenetic analysis of partial matrix gene sequences of the buffalo isolates and six field BPIV3 isolates from bovines in Argentina were studied. Buffalo isolates were similar to genotype B (BPIV3b) while the six BPIV3 isolates were similar to genotypes A (BPIV3a) and C (BPIV3c). CONCLUSIONS: This is the first characterization of BPIV3 in water buffalo.According to the samples analyzed, in Argentina, the genotype B was found in buffalo and the genotypes A and C were found in cattle.
Assuntos
Búfalos , Vírus da Parainfluenza 3 Bovina/isolamento & purificação , Infecções por Respirovirus/veterinária , Animais , Argentina/epidemiologia , Sequência de Bases , Bovinos , Feminino , Genótipo , Dados de Sequência Molecular , Vírus da Parainfluenza 3 Bovina/classificação , Vírus da Parainfluenza 3 Bovina/genética , Filogenia , RNA Viral/genética , Infecções por Respirovirus/epidemiologia , Infecções por Respirovirus/virologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Infections by Babesia bovis limit cattle production and cause important economic losses in tropical and subtropical areas around the world. Monitoring of calf sera can be used to detect unprotected cattle herds and to decide on strategic control measures, as well as for epidemiological studies. Merozoite surface antigen 2c (MSA-2c) is an immunodominant surface protein expressed in B. bovis merozoites and sporozoites and contains B-cell epitopes that are conserved among geographic isolates. A monoclonal antibody against recombinant MSA-2c (rMSA-2c) was previously shown to inhibit the binding of anti-B. bovis antibodies to a parasite B-cell epitope in a competitive enzyme-linked immunosorbent assay (cELISA) format. In the work at hand, the parameters of this cELISA were reevaluated and adjusted when necessary, and a cutoff value was determined by receiver operator characteristic (ROC) curve analysis of a total of 357 bovine sera of known reactivity, as assessed by indirect immunofluorescence assay (IFAT). The established rMSA-2c cELISA demonstrated a specificity of 98% and a sensitivity of 96.2%. An additional set of 303 field bovine sera from regions where ticks are endemic and tick-free regions of Argentina was tested by both rMSA-2c cELISA and IFAT, and the results were shown to be in very good agreement (kappa index, 0.8325). The performance shown by rMSA-2c cELISA in the detection of B. bovis-specific antibodies and its suitability for standardization and large-scale production, as well as the possibility of its application in most veterinary diagnostic laboratories, make the assay a powerful tool for the surveillance of herd immunity as a strategic measure for the control of bovine babesiosis.
Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários , Babesiose/veterinária , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/parasitologia , Técnicas de Laboratório Clínico/métodos , Proteínas de Membrana , Proteínas de Protozoários , Medicina Veterinária/métodos , Animais , Anticorpos Monoclonais , Antígenos de Protozoários/imunologia , Argentina , Babesiose/diagnóstico , Babesiose/parasitologia , Bovinos , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas de Membrana/imunologia , Proteínas de Protozoários/imunologia , Sensibilidade e EspecificidadeRESUMO
Bovine herpesvirus-1 (BoHV-1) infection is distributed worldwide and the development of new tools to fight against this pathogen has become extremely important. In this work a recombinant modified vaccinia virus Ankara (MVA) vector expressing the secreted version of glycoprotein D, MVA-gDs, was obtained and evaluated as a candidate vaccine. First, the correct expression, antigenicity, and N-glycosylation of glycoprotein D were confirmed by molecular techniques. Then MVA-gDs was used as parenteral immunogen in BALB/C mice in which a specific anti-gD humoral immune response was induced and maintained for 7 mo. Two doses of MVA-gDs supplemented with cholera toxin delivered by intranasal immunization induced IgA anti-gD humoral immune responses in nasal and bronchopulmonary washes, as well as IgG anti-gD antibodies in serum samples. In order to evaluate the protection conferred by MVA-gDs immunization, a rabbit BoHV-1 challenge assay was performed. A shorter viral excretion period and a reduction in the number of animals shedding BoHV-1 was observed in the group immunized with recombinant MVA-gDs. In conclusion our data encourage further studies to evaluate MVA-gDs, alone or combined with other immunogens, as a candidate vaccine for BoHV-1.
Assuntos
Portadores de Fármacos , Vacinas contra Herpesvirus/imunologia , Vaccinia virus/genética , Proteínas Virais/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/genética , Administração Intranasal , Animais , Anticorpos Antivirais/análise , Anticorpos Antivirais/sangue , Líquido da Lavagem Broncoalveolar/imunologia , Toxina da Cólera/administração & dosagem , Toxina da Cólera/genética , Modelos Animais de Doenças , Feminino , Vetores Genéticos , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/prevenção & controle , Vacinas contra Herpesvirus/administração & dosagem , Vacinas contra Herpesvirus/genética , Imunoglobulina A/análise , Imunoglobulina G/sangue , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mucosa Nasal/imunologia , Doenças dos Roedores/imunologia , Doenças dos Roedores/prevenção & controle , Fatores de Tempo , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Proteínas Virais/genética , Eliminação de Partículas ViraisRESUMO
Clostridium perfringens epsilon toxin (ETX) is responsible for a fatal enterotoxemia in different animal species, producing extensive renal damage, neurological disturbance and edema of lungs, heart and kidneys. However, there is no information about the susceptibility of humans to ETX. Here, we report that primary cultures of human renal tubular epithelial cells (HRTEC) exposed to ETX showed a marked swelling with subsequent large blebs surrounding most cells. The incubation of HRTEC with ETX produced a reduction of cell viability in a dose- and time-dependent manner. The CD(50) after 1-hour and 24-hour incubation were 3 µg/mL and 0.5 µg/mL, respectively. The pulse with ETX for 3 min was enough to produce a significant cytotoxic effect on HRTEC after 1-hour incubation. ETX binds to HRTEC forming a large complex of about 160 kDa similar to what was found in the Madin-Darby canine kidney (MDCK) cell line. The HRTEC could be a useful cell model to improve the understanding of the mechanisms involved on the cell damage mediated by ETX.
Assuntos
Toxinas Bacterianas/toxicidade , Células Epiteliais/efeitos dos fármacos , Túbulos Renais/efeitos dos fármacos , Toxinas Bacterianas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Ligação Proteica , Fatores de TempoRESUMO
The mce2 operon is one of the four mce operons present in Mycobacterium tuberculosis that encode exported proteins with a probable role in the virulence mechanisms of this bacterium. In the present study we demonstrated that Rv0586, which encodes a putative GntR-like regulator, is part of the mce2 operon. By using a promoter-lacZ fusion approach and bioinformatics tools, we found that Rv0586 represses the expression of Mce2 proteins and of a putative endonuclease IV, encoded by end (Rv0670) gene. For this reason, we have re-named the repressor protein Mce2R. By gel-shift experiments Mce2R binding was determined to be located within the mce2 promoter region. In addition, two FadR-like operator motifs were identified within the promoter regions of both the mce2 operon and the end gene. These motifs overlap putative -10 and -35 promoter boxes. M. tuberculosis carrying mce2 and end promoter-lacZ fusions were used to infect J774 macrophage-like cells. Expression of beta-galactosidase was induced after phagocytocis, suggesting that some cellular factor could be a key component of the molecular switch regulation expression of the mce2 operon. In conclusion, these results add novel evidence of the complex regulation of mce operon expression.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis/genética , Proteínas Repressoras/genética , Sítios de Ligação , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli/genética , Expressão Gênica , Genes Bacterianos , Humanos , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Regiões Operadoras Genéticas , Regiões Promotoras Genéticas , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transcrição Gênica , Virulência/genéticaRESUMO
BACKGROUND: mce3 is one of the four virulence-related mce operons of Mycobacterium tuberculosis. In a previous work we showed that the overexpression of Mce3R in Mycobacterium smegmatis and M. tuberculosis abolishes the expression of lacZ fused to the mce3 promoter, indicating that Mce3R represses mce3 transcription. RESULTS: We obtained a knockout mutant strain of M. tuberculosis H37Rv by inserting a hygromycin cassette into the mce3R gene. The mutation results in a significant increase in the expression of mce3 genes either in vitro or in a murine cell macrophages line as it was determined using promoter-lacZ fusions in M. tuberculosis. The abundance of mce1, mce2 and mce4 mRNAs was not affected by this mutation as it was demonstrated by quantitative RT-PCR. The mce3R promoter activity in the presence of Mce3R was significantly reduced compared with that in the absence of the regulator, during the in vitro culture of M. tuberculosis. CONCLUSION: Mce3R repress the transcription of mce3 operon and self regulates its own expression but does not affect the transcription of mce1, mce2 and mce4 operons of M. tuberculosis.