RESUMO
Ochratoxin A (OTA), frequently existing in the food and feeds, could induce immunotoxicity. Porcine circovirus type 2 (PCV2), as a primary causative agent of porcine circovirus-associated disease, also could induce immunosuppression. However, it is still unknown whether PCV2 infection impacts OTA-induced immunotoxicity. The pigs and porcine alveolar macrophages (PAMs) were used as the model in the present experiment. The results in vivo indicated that PCV2 infection exacerbated OTA-induced immunotoxicity, NF-κB p65 phosphorylation, and TLR4 and MyD88 mRNA and protein expression in spleen. The results in vitro showed that OTA at 7.0 and 9.0 µM decreased cell viability and increased LDH release of PAMs without PCV2 infection. However, with PCV2 infection, OTA at 5.0, 7.0 and 9.0 µM significantly decreased cell viability and increased LDH release compared with absence of PCV2 infection. In addition, OTA at 5.0 and 7.0 µM significantly increased Annexin V/PI-positive rate, apoptosis of nuclear, γ-H2AX foci, IL-1α and TNF-α expression in PAMs with PCV2 infection compared with absence of PCV2 infection. In addition, PCV2 infection enhanced OTA-induced TLR4 and MyD88 mRNA and protein expression and NF-κB p65 phosphorylation. Knockdown of TLR4 alleviated the exacerbating effects of PCV2 infection on OTA-induced cytotoxicity, apoptosis and DNA damage in PAMs. These results indicated that PCV2 infection aggravated OTA-induced immunotoxicity and reduced the dose of OTA-induced immunotoxicity via TLR4/NF-κB p65 signaling pathway, which could provide basis for establishing limits for OTA.
Assuntos
Circovirus , Ocratoxinas , Animais , Macrófagos Alveolares , Ocratoxinas/toxicidade , Transdução de Sinais , SuínosRESUMO
BACKGROUND: Porcine circovirus type 2 (PCV2)-associated diseases are a major problem for the swine industry worldwide. In addition to vaccines, the availability of antiviral polymers provides an efficient and safe option for reducing the impact of these diseases. By virtue of their molecular weight and repetitious structure, polymers possess properties not found in small-molecule drugs. In this perspective, we focus on chitosan, a ubiquitous biopolymer, that adjusts the molecular weight and sulfated-mediated functionality can act as an efficient antiviral polymer by mimicking PCV2-cell receptor interactions. METHODS: Sulfated chitosan (Chi-S) polymers of two molecular weights were synthesized and characterized by FTIR, SEM-EDS and elemental analysis. The Chi-S solutions were tested against PCV2 infection in PK15 cells in vitro and antiviral activity was evaluated by measuring the PCV2 DNA copy number, TCID50 and capsid protein expression, upon application of different molecular weights, sulfate functionalization, and concentrations of polymer. In addition, to explore the mode of action of the Chi-S against PCV2 infection, experiments were designed to elucidate whether the antiviral activity of the Chi-S would be influenced by when it was added to the cells, relative to the time and stage of viral infection. RESULTS: Chi-S significantly reduced genomic copies, TCID50 titers and capsid protein of PCV2, showing specific antiviral effects depending on its molecular weight, concentration, and chemical functionalization. Assays designed to explore the mode of action of the low molecular weight Chi-S revealed that it exerted antiviral activity through impeding viral attachment and penetration into cells. CONCLUSIONS: These findings help better understanding the interactions of PCV2 and porcine cells and reinforce the idea that sulfated polymers, such as Chi-S, represent a promising candidates for use in antiviral therapies against PCV2-associated diseases. Further studies in swine are warranted.
Assuntos
Quitosana , Infecções por Circoviridae , Circovirus , Doenças dos Suínos , Animais , Antivirais/metabolismo , Antivirais/farmacologia , Proteínas do Capsídeo/genética , Quitosana/metabolismo , Quitosana/farmacologia , Infecções por Circoviridae/prevenção & controle , Circovirus/genética , Peso Molecular , Sulfatos/metabolismo , Suínos , Replicação Viral/genéticaRESUMO
Porcine circovirus type 2 (PCV2) and protoparvovirus 1 (PPV) were detected as single infection (6/131) and (11/131) respectively, or co-infection (6/131) in fetuses and stillborn piglets from normal deliveries in a farm without reproductive problems. Twenty in twenty-three positive samples were over 70 days of gestation, which is when the fetus becomes immunocompetent, and the presence of a NADL-2 PPV strain suggests fetal immune system impairment. Phylogenetic analysis of sequences obtained showed that 8/9 sequences are related to cluster 13 and the remaining is grouped into cluster 11 sequences. An increase in variability in ORF2 sequences in Argentina was observed. It is not clear whether the detection of fetuses positive to PPV and PCV2 is of epidemiological importance in a subclinically affected farm. However, the results of this study showed that currently used vaccines and vaccine protocols do not fully protect against PPV or PCV2 fetus infection.
Assuntos
Infecções por Circoviridae/veterinária , Circovirus/isolamento & purificação , Parvovirus Suíno/isolamento & purificação , Doenças dos Suínos/fisiopatologia , Animais , Infecções por Circoviridae/fisiopatologia , SuínosRESUMO
Porcine Circovirus Type 2 (PCV2) is one of the most important pathogens in pigs around the world. PCV2 is a non-enveloped virus and its capsid is formed by a single protein known as open reading frame 2 (ORF2). The aim of this study was to evaluate the antigenicity and immunogenicity of genetically-encoded protein nanoparticles (NPs) containing ORF2 from PCV2 fused to the first 110 amino acids of the N-terminus of polyhedrin from the insect virus Autographa californica nucleopolyhedrovirus (PH(1â¯-1â¯1â¯0)). Our group has previously described that some polyhedrin fragments self-aggregate forming polyhedra-like particles. We identified a self-aggregating signal within the first 110 amino acids from polyhedrin (PH(1â¯-1â¯1â¯0)). Fusing the ORF2 from PCV2 to the carboxyl terminus from PH(1â¯-1â¯1â¯0) results in the formation of NPs which incorporate the antigen of interest. Using this system we synthesized NPs containing PH(1â¯-1â¯1â¯0) fused to ORF2 (PH(1â¯-1â¯1â¯0)PCV2) and purify them to immunize pigs and evaluate the humoral immune response generated by these NPs comparing them to a commercially available vaccine. Pigs immunized with PH(1â¯-1â¯1â¯0)PCV2 NPs produced antibodies against ORF2 from PCV2 as indicated by western blot and ELISA analysis. Antibodies obtained with PH(1â¯-1â¯1â¯0)PCV2 NPs were comparable to those obtained using a commercial PCV2 vaccine. These antibodies neutralized the infection of a recombinant PCV2 expressing the green fluorescent protein (GFP). These results together suggest that the self-aggregating peptide PH(1â¯-1â¯1â¯0) can be used for the synthesis of subunit vaccines against PCV2.
Assuntos
Infecções por Circoviridae/veterinária , Circovirus/genética , Circovirus/imunologia , Nanopartículas , Fases de Leitura Aberta/imunologia , Síndrome Definhante Multissistêmico de Suínos Desmamados/prevenção & controle , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Feminino , Imunização , Masculino , Fases de Leitura Aberta/genética , Suínos , Vacinas de Subunidades Antigênicas , Vacinas Virais/química , Vacinas Virais/genéticaRESUMO
Background: Porcine Circovirus type 2 (PCV2) infections are distributed worldwide and cause Porcine Circovirus Associated Disease (PCVAD). To minimize the impact of PCV2 infection on swine health and production, different vaccination schemes have been used since 2006. However, the association between vaccination schemes, virus load and disease under field conditions are not completely understood. Therefore, the objective of this study was to compare the effect of two different PCV2 vaccination schemes on the humoral response and PCV2 load in pigs after weaning under field conditions. Methods: Two commercial pig farms (Farm A and B), endemically infected with PCV2, which were using two different PCV2 subunit vaccinations schemes for sow, gilts and piglets, were selected. We designed a longitudinal study and measured IgG levels by ELISA and virus load by quantitative PCR in pigs after weaning. Forty 3-week old piglets were randomly selected at weaning and followed for 20 weeks. IgG levels and virus loads were compared within and between farms and considered statistically different if the non-parametric Kruskal Wallis test p value was lower than 0.05. Results: We found that low virus loads were maintained in pigs from both farms regardless of the vaccination scheme used (p>0.05). However, there was significant difference in the mean IgG levels observed over time (p<0.05), suggesting that different humoral immune response are not necessarily associated with different virus loads observed over time. Conclusions: These results are important because they can help to prevent PCV2 infections using different vaccination schemes to minimize the effect of PCVAD on swine health and production.
Assuntos
Circovirus/imunologia , Circovirus/fisiologia , Imunidade Humoral , Vacinação , Carga Viral , Animais , Imunoglobulina G/imunologia , SuínosRESUMO
Porcine circovirus type 2 (PCV2), family Circoviridae, genus Circovirus infection in domestic pig has been associated with several pathological conditions being the most important of them the postweaning multisystemic wasting syndrome. Many studies have demonstrated the existence of three PCV2 genotypes (a, b, and c) and recently PCV3. Until now, these genotypes or subgenotypes have not been described in Mexico. We found genetic changes in ORF2 from nine strains of PCV2 obtained from samples of Jalisco, Veracruz, Estado de México, Hidalgo and Sonora states of Mexico. Our results shown the presence of two genotypes (PCV2a and PCV2b) as well as, the presence and differences between the reported subgenotypes. The subgenotype PCV2b (1A/1B, 1A) has a higher prevalence (87.5%) in comparison with PCV2a (2C) (12.5%).
RESUMO
Abstract Economic losses with high mortality rate associated with Porcine circovirus type 2 (PCV2) is reported worldwide. PCV2 commercial vaccine was introduced in 2006 in U.S. and in 2008 in Brazil. Although PCV2 vaccines have been widely used, cases of PCV2 systemic disease have been reported in the last years. Eleven nursery or fattening pigs suffering from PCV2 systemic disease were selected from eight PCV2-vaccinated farms with historical records of PCV2 systemic disease in Southern Brazil. PCV2 genomes were amplified and sequenced from lymph node samples of selected pigs. The comparison among the ORF2 amino acid sequences of PCV2 isolates revealed three amino acid substitutions in the positions F57I, N178S and A190T, respectively. Using molecular modeling, a structural model for the capsid protein of PCV2 was built. Afterwards, the mutated residues positions were identified in the model. The structural analysis of the mutated residues showed that the external residue 190 is close to an important predicted region for antibodies recognition. Therefore, changes in the viral protein conformation might lead to an inefficient antibody binding and this could be a relevant mechanism underlying the recent vaccine failures observed in swine farms in Brazil.
Assuntos
Animais , Circovirus/química , Proteínas do Capsídeo/química , Conformação Proteica , Suínos , Doenças dos Suínos/virologia , Brasil , Modelos Moleculares , Circovirus/isolamento & purificação , Circovirus/genética , Infecções por Circoviridae/veterinária , Infecções por Circoviridae/virologia , Substituição de Aminoácidos , Proteínas do Capsídeo/genéticaRESUMO
Economic losses with high mortality rate associated with Porcine circovirus type 2 (PCV2) is reported worldwide. PCV2 commercial vaccine was introduced in 2006 in U.S. and in 2008 in Brazil. Although PCV2 vaccines have been widely used, cases of PCV2 systemic disease have been reported in the last years. Eleven nursery or fattening pigs suffering from PCV2 systemic disease were selected from eight PCV2-vaccinated farms with historical records of PCV2 systemic disease in Southern Brazil. PCV2 genomes were amplified and sequenced from lymph node samples of selected pigs. The comparison among the ORF2 amino acid sequences of PCV2 isolates revealed three amino acid substitutions in the positions F57I, N178S and A190T, respectively. Using molecular modeling, a structural model for the capsid protein of PCV2 was built. Afterwards, the mutated residues positions were identified in the model. The structural analysis of the mutated residues showed that the external residue 190 is close to an important predicted region for antibodies recognition. Therefore, changes in the viral protein conformation might lead to an inefficient antibody binding and this could be a relevant mechanism underlying the recent vaccine failures observed in swine farms in Brazil.(AU)
Assuntos
Animais , Circovirus/ultraestrutura , Capsídeo/ultraestrutura , Suínos/virologia , Epitopos , Vacinas ViraisRESUMO
Economic losses with high mortality rate associated with Porcine circovirus type 2 (PCV2) is reported worldwide. PCV2 commercial vaccine was introduced in 2006 in U.S. and in 2008 in Brazil. Although PCV2 vaccines have been widely used, cases of PCV2 systemic disease have been reported in the last years. Eleven nursery or fattening pigs suffering from PCV2 systemic disease were selected from eight PCV2-vaccinated farms with historical records of PCV2 systemic disease in Southern Brazil. PCV2 genomes were amplified and sequenced from lymph node samples of selected pigs. The comparison among the ORF2 amino acid sequences of PCV2 isolates revealed three amino acid substitutions in the positions F57I, N178S and A190T, respectively. Using molecular modeling, a structural model for the capsid protein of PCV2 was built. Afterwards, the mutated residues positions were identified in the model. The structural analysis of the mutated residues showed that the external residue 190 is close to an important predicted region for antibodies recognition. Therefore, changes in the viral protein conformation might lead to an inefficient antibody binding and this could be a relevant mechanism underlying the recent vaccine failures observed in swine farms in Brazil.
Assuntos
Proteínas do Capsídeo/química , Circovirus/química , Substituição de Aminoácidos , Animais , Brasil , Proteínas do Capsídeo/genética , Infecções por Circoviridae/veterinária , Infecções por Circoviridae/virologia , Circovirus/genética , Circovirus/isolamento & purificação , Modelos Moleculares , Conformação Proteica , Suínos , Doenças dos Suínos/virologiaRESUMO
Abstract Economic losses with high mortality rate associated with Porcine circovirus type 2 (PCV2) is reported worldwide. PCV2 commercial vaccine was introduced in 2006 in U.S. and in 2008 in Brazil. Although PCV2 vaccines have been widely used, cases of PCV2 systemic disease have been reported in the last years. Eleven nursery or fattening pigs suffering from PCV2 systemic disease were selected from eight PCV2-vaccinated farms with historical records of PCV2 systemic disease in Southern Brazil. PCV2 genomes were amplified and sequenced from lymph node samples of selected pigs. The comparison among the ORF2 amino acid sequences of PCV2 isolates revealed three amino acid substitutions in the positions F57I, N178S and A190T, respectively. Using molecular modeling, a structural model for the capsid protein of PCV2 was built. Afterwards, the mutated residues positions were identified in the model. The structural analysis of the mutated residues showed that the external residue 190 is close to an important predicted region for antibodies recognition. Therefore, changes in the viral protein conformation might lead to an inefficient antibody binding and this could be a relevant mechanism underlying the recent vaccine failures observed in swine farms in Brazil.
RESUMO
Porcine Circovirus type 2 (PCV2) is a worldwide distributed pathogen and one of the most economically relevant swine infections. Four genotypes have been recognized and it is well known that PCV2a, PCV2b and PCV2d have a global distribution. However, the information about recombinant strains circulation and their influence in driving PCV2 evolution is a poorly studied area. In Uruguay, PCV2 associated symptoms began to be frequently observed in pigs from different farms since 2010. The main purpose of this study was to thoroughly investigate the molecular epidemiology of PCV2 in nationwide swine herds and free-living wild boars during the period 2010-2014, providing an extensive viral sequence dataset. Surprisingly, the findings revealed a predominance of recombinant strains circulation, evidencing for the first time in the field that PCV2 recombination can lead to the emergence of strains able to compete and potentially displace parental ones. In addition, the circulation of the genotypes PCV2d (29%), PCV2b (10.5%) and PCV2a (7.9%) were also observed. Since 2013, a high circulation of PCV2d was identified in the country and probably reflected the recent global scenario of the emergence of this genotype. In addition, fluctuations in the frequency of PCV2 infection in the period evaluated may suggest a limitation of biosecurity strategies implemented in Uruguay for the disease control, including the instability of vaccination practices. On the other hand, the sustained PCV2 infection observed in wild boar population and the similarity among circulating viral strains from these animals and domestic pigs, suggested that wild animals could serve as permanent reservoir of the disease. Altogether, this work put forward that many factors play a role in PCV2 heterogeneity including rapid viral spread and evolution, recombination, wide movement within national boundaries and multiples introduction events resulting of international trade. Continuous monitoring of viral epidemiology is needed to better understand the PCV2 population dynamics in Uruguay and the development of appropriate strategies are required for disease control.
Assuntos
Proteínas do Capsídeo/genética , Infecções por Circoviridae/epidemiologia , Circovirus/genética , Sus scrofa/genética , Doenças dos Suínos/epidemiologia , Suínos/genética , Animais , Infecções por Circoviridae/diagnóstico , Infecções por Circoviridae/genética , Infecções por Circoviridae/virologia , Circovirus/classificação , DNA Viral/genética , Genoma , Genótipo , Epidemiologia Molecular , Filogenia , Recombinação Genética , Análise de Sequência de DNA , Sus scrofa/virologia , Suínos/virologia , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/genética , Doenças dos Suínos/virologia , Fatores de Tempo , Uruguai/epidemiologiaRESUMO
The objective of this study was to evaluate the seroprevalence and identify the strains of swine influenza virus (SwIV), as well as the seroprevalence of porcine parvovirus (PPV), transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine respiratory coronavirus (PRCV), porcine circovirus type 2 (PCV-2), and classical swine fever virus (CSFV) in pigs in Trinidad and Tobago (T&T). Blood samples (309) were randomly collected from pigs at farms throughout T&T. Serum samples were tested for the presence of antibodies to the aforementioned viruses using commercial ELISA kits, and the circulating strains of SwIV were identified by the hemagglutination inhibition test (HIT). Antibodies against SwIV were detected in 114 out of the 309 samples (37%). Out of a total of 26 farms, 14 tested positive for SwIV antibodies. HI testing revealed high titers against the A/sw/Minnesota/593/99 H3N2 strain and the pH1N1 2009 pandemic strain. Antibodies against PPV were detected in 87 out of the 309 samples (28%), with 11 out of 26 farms testing positive for PPV antibodies. Antibodies against PCV-2 were detected in 205 out of the 309 samples tested (66%), with 25 out of the 26 farms testing positive for PCV-2 antibodies. No antibodies were detected in any of the tested pigs to PRRSV, TGEV, PRCV, or CSFV.
Assuntos
Orthomyxoviridae/isolamento & purificação , Doenças dos Suínos/epidemiologia , Viroses/veterinária , Animais , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/veterinária , Infecções por Orthomyxoviridae/virologia , Prevalência , Estudos Soroepidemiológicos , Suínos , Doenças dos Suínos/virologia , Trinidad e Tobago/epidemiologia , Viroses/epidemiologia , Viroses/virologiaRESUMO
Few studies have described enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against porcine circovirus type 2 (PCV2) based on antigens produced in cell culture. Furthermore, few articles have described viral purification techniques for members of the family Circoviridae. This occurs because circoviruses are difficult to isolate, noncytopathogenic, and produce low viral titres in cell culture. Thus, for overcoming these difficulties in the cultivation of PCV2, this study aimed to develop a double-antibody sandwich ELISA based on the cell culture antigen PCV2b for the quantification of anti-PCV2 antibodies. A 20% and 50% discontinuous sucrose cushion was used for viral purification, which enabled the separation of cell culture proteins in the 20% sucrose cushion and a greater viral concentration in the 50% sucrose cushion. Following isopycnic centrifugation, PCV2 was concentrated in the band with density values from 1.330 to 1.395g/cm3. Viral purification was assessed using SDS-PAGE, indirect ELISA and electron microscopy. The standardised ELISA revealed a strong linear correlation (r= 0.826, p < 0.001) when compared with a commercial ELISA kit. The assay exhibited low variability (inter-assay coefficient of variation of 4.24% and intra-assay of 1.80%) and excellent analytical specificity conferred by the capture antibody produced in rabbit. Thus, this ELISA is a rapid, specific and convenient method for the detection of antibodies against PCV2 in studies of experimental and natural infection, and in monitoring the response to vaccination on commercial farms.(AU)
Há poucos relatos na literatura de métodos de ELISA (Enzyme-linked immunosorbent assay), para a detecção de anticorpos contra o circovírus suíno tipo 2 (PCV2), baseados em antígenos produzidos em cultivo celular, bem como uma escassez de trabalhos descrevendo técnicas de purificação viral para os membros da família Circoviridae. Isso ocorre, pois os circovírus são de difícil isolamento, não causam efeito citopático e produzem um baixo título viral em cultivo celular. Assim, para superar essas dificuldades encontradas no cultivo do PCV2, este estudo objetivou desenvolver um sandwich ELISA com duplo anticorpo, baseado no antígeno de PCV2 produzido em cultivo celular, para a quantificação de anticorpos anti-PCV2. Um colchão de sacarose descontínuo a 20% e 50% foi utilizado para a purificação viral, o qual possibilitou a separação das proteínas oriundas do cultivo celular no colchão de sacarose a 20% e uma maior concentração viral no colchão de sacarose a 50%. Com a ultracentrifugação isopícnica, o PCV2 ficou mais concentrado na banda com valores de densidade de 1,330 a 1,395g/cm3. A purificação viral foi avaliada pelas técnicas de SDS-PAGE, ELISA indireto e microscopia eletrônica. Assim, o método de ELISA padronizado revelou uma forte correlação linear (r = 0,826, p < 0,001) quando comparado com um kit de ELISA comercial. O ensaio demonstrou baixa variabilidade (coeficientes de variação inter-teste de 4,24% e intra-teste de 1,80%) e uma excelente especificidade analítica conferida pelo anticorpo de captura produzido em coelho. Portanto, o método de ELISA demonstrou ser rápido, específico e conveniente para a detecção de anticorpos contra o PCV2 em estudos de infecção natural e experimental, além da monitoria da resposta à vacinação contra o PCV2 em granjas comerciais.(AU)
Assuntos
Ensaio de Imunoadsorção Enzimática , Circovirus , Sacarose , Anticorpos , Centrifugação IsopícnicaRESUMO
Few studies have described enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against porcine circovirus type 2 (PCV2) based on antigens produced in cell culture. Furthermore, few articles have described viral purification techniques for members of the family Circoviridae. This occurs because circoviruses are difficult to isolate, noncytopathogenic, and produce low viral titres in cell culture. Thus, for overcoming these difficulties in the cultivation of PCV2, this study aimed to develop a double-antibody sandwich ELISA based on the cell culture antigen PCV2b for the quantification of anti-PCV2 antibodies. A 20% and 50% discontinuous sucrose cushion was used for viral purification, which enabled the separation of cell culture proteins in the 20% sucrose cushion and a greater viral concentration in the 50% sucrose cushion. Following isopycnic centrifugation, PCV2 was concentrated in the band with density values from 1.330 to 1.395g/cm3. Viral purification was assessed using SDS-PAGE, indirect ELISA and electron microscopy. The standardised ELISA revealed a strong linear correlation (r= 0.826, p<0.001) when compared with a commercial ELISA kit. The assay exhibited low variability (inter-assay coefficient of variation of 4.24% and intra-assay of 1.80%) and excellent analytical specificity conferred by the capture antibody produced in rabbit. Thus, this ELISA is a rapid, specific and convenient method for the detection of antibodies against PCV2 in studies of experimental and natural infection, and in monitoring the response to vaccination on commercial farms.(AU)
Há poucos relatos na literatura de métodos de ELISA (Enzyme-linked immunosorbent assay), para a detecção de anticorpos contra o circovírus suíno tipo 2 (PCV2), baseados em antígenos produzidos em cultivo celular, bem como uma escassez de trabalhos descrevendo técnicas de purificação viral para os membros da família Circoviridae. Isso ocorre, pois os circovírus são de difícil isolamento, não causam efeito citopático e produzem um baixo título viral em cultivo celular. Assim, para superar essas dificuldades encontradas no cultivo do PCV2, este estudo objetivou desenvolver um sandwich ELISA com duplo anticorpo, baseado no antígeno de PCV2 produzido em cultivo celular, para a quantificação de anticorpos anti-PCV2. Um colchão de sacarose descontínuo a 20% e 50% foi utilizado para a purificação viral, o qual possibilitou a separação das proteínas oriundas do cultivo celular no colchão de sacarose a 20% e uma maior concentração viral no colchão de sacarose a 50%. Com a ultracentrifugação isopícnica, o PCV2 ficou mais concentrado na banda com valores de densidade de 1,330 a 1,395g/cm3. A purificação viral foi avaliada pelas técnicas de SDS-PAGE, ELISA indireto e microscopia eletrônica. Assim, o método de ELISA padronizado revelou uma forte correlação linear (r = 0,826, p <0,001) quando comparado com um kit de ELISA comercial. O ensaio demonstrou baixa variabilidade (coeficientes de variação inter-teste de 4,24% e intra-teste de 1,80%) e uma excelente especificidade analítica conferida pelo anticorpo de captura produzido em coelho. Portanto, o método de ELISA demonstrou ser rápido, específico e conveniente para a detecção de anticorpos contra o PCV2 em estudos de infecção natural e experimental, além da monitoria da resposta à vacinação contra o PCV2 em granjas comerciais.(AU)
Assuntos
Anticorpos , Circovirus , Ensaio de Imunoadsorção Enzimática , Sacarose , Centrifugação IsopícnicaRESUMO
Background: porcine circovirus type 2 (PCV2) is associated with reproductive disease in newly populated herds and in replacement breeding stock from new sources and is almost exclusively reported in gilts. Objective: the main purpose of this study was to assess the dynamics of porcine circovirus type 2 infection and neutralizing antibodies in subclinically infected gilts and the effect on their piglets. Methods: the study was conducted with 40 gilts selected at random from four breeding herds. Blood samples, nasal and vaginal swabs were obtained from the gilts at arrival, acclimatization, farrowing, and one day after farrowing. Colostrum samples were collected immediately after parturition and one day after farrowing. Blood, nasal swab, or tissue samples were collected from four piglets prior to suckling. All serums were analyzed by virus neutralization test (VNT) to establish the presence of antibodies. All samples were subjected to SYBER Green real-time PCR assay to detect PCV2 DNA. Results: high levels of viremia and viral load of PCV2 in nasal and vaginal swabs were found in healthy gilts at arriving, confirming the introduction of infected animals into the farms. In addition, most gilts were positive for PCV2 DNA in serum, nasal and vaginal swabs at farrowing. PCV2 shedding was also observed in nasal and vaginal fluids and colostrum even in presence of serum neutralizing antibodies (NA). Subclinically infected dams had detectable viremia, developed anti-PCV2 antibodies, and there was PCV2 DNA in tissue samples of their born alive and healthy piglets. PCV2a and PCV2b genotypes were confirmed in PCV2 subclinical infection in both dams and piglets in utero. Conclusion: replacement gilts can be infected with PCV2 before entering the farm and continuous exposure seems to occur horizontally in acclimatization and gestation units or before farrowing. Exposure and infection during gestation may result in infected but apparently healthy piglets.
Antecedentes: el circovirus porcino tipo 2 (PCV2) es asociado con casos de falla reproductivas en granjas recién pobladas, en granjas de cría para cerdas jóvenes y casi exclusivamente en cerdas de reemplazo. Los signos clínicos descritos son: incremento en los abortos durante la segunda y tercera etapa de la gestación, fetos momificados, mortinatos y el nacimiento de lechones débiles no viables. Objetivo: el principal propósito de este estudio fue evaluar la dinámica de la infección por el circovirus porcino tipo 2 y títulos de anticuerpos neutralizantes en las cerdas de reemplazo subclinicamente infectadas y el efecto en su camada. Métodos: este estudio se realizó con 40 cerdas de reemplazo seleccionadas al azar en cuatro granjas porcinas de cría. De cada animal se colectaron muestras de sangre, hisopados nasales y vaginales al ingresar a la explotación, durante la cuarentena, en el momento del parto y un día post-parto. Igualmente, se colectaron muestras de calostro al terminar el parto y un día post-parto. De cuatro lechones neonatos, se colectaron muestras de sangre, hisopado nasal y tejidos antes de consumir calostro. Todos los sueros fueron analizados mediante la técnica de sero-neutralización para detectar anticuerpos anti-PCV2 y todas las muestras se analizaron por una técnica SYBER Green en tiempo real para detectar el ADN viral. Resultados: la detección de un alto nivel de viremia y la demostración de la eliminación viral en hisopados nasales y vaginales permitió demostrar la introducción a las granjas de cerdas de reemplazo infectadas, aparentemente sanas. Igualmente, el suero y los hisopados nasales y vaginales fueron positivos por PCR SYBER Green en la mayoría de las hembras al parto. Se demostró eliminación viral en fluidos nasales, vaginales y en calostro en presencia de anticuerpos séricos neutralizantes. La infección de las cerdas se manifestó en viremia, en el desarrollo de anticuerpos frente al PCV2 y en la presencia del ADN viral en los tejidos de lechones neonatos aparentemente sanos. Los genotipos PCV2a y PCV2b fueron detectados en la infección in utero. Conclusiones: las cerdas de reemplazo pueden estar infectadas con el PCV2 antes de ingresar a las explotaciones de cría o pueden infectarse por transmisión horizontal durante la cuarentena y gestación. La exposición e infección viral de las cerdas durante la gestación puede resultar en infección subclínica de los lechones neonatos.
Antecedentes: o circovírus suíno tipo 2 (PCV2) está associado a casos de falha reprodutiva em granjas recém-assentadas e fazendas de criação de marrãs e afeta principalmente a porcas nulíparas. Clinicamente observam-se aumento dos fetos abortados no segundo e terceiro estágios da gravidez, fetos mumificados, natimortos e nascimento de leitões inviáveis. Objetivo: o principal objetivo deste estudo foi avaliar a dinâmica da infeção pelo circovirus porcino tipo 2 e os títulos de anticorpos neutralizantes em porcas nulíparas com infecção subclínica e o efeito em sua leitegada. Métodos: este estudo foi realizado com 40 porcas em quatro granjas selecionadas aleatoriamente. Foram coletados de cada animal amostras de sangue, esfregaços nasais e vaginais ao entrar na fazenda, durante a quarentena, no parto e um dia pós-parto. Também foram coletadas amostras de colostro no parto e um dia pós-parto. Amostras de sangue, esfregaços nasal e tecido foram tomadas de quatro leitões antes de consumir colostro. As amostras foram analisadas pelo PCR Sybr Green para detectar e quantificar o PCV2. A detecção de anticorpos contra o vírus do PCV2 em soro foi realizada pelo teste de soroneutralização e todas as amostras foram analisadas através da técnica de SYBR Green PCR para a detecção do ADN viral. Resultados: a detecção de um nível elevado de viremia e a demonstração da excreção viral em esfregaços nasais e vaginais nas fêmeas permitiram demonstrar a introdução de porcas nulíparas aparentemente saudávels. Igualmente, o soro e as secreções vaginais e nasais foram positivos por PCR SYBR Green em tempo real na maioria das porcas no parto. Observou-se excreção viral em secreções vaginais e nasais e colostro na presença de anticorpos neutralizantes. A infecção das porcas manifestou-se no desenvolvimento de anticorpos neutralizantes e detecção de infecção fetal em leitões recém-nascidos aparentemente saudáveis , confirmando a transmissão vertical do PCV2. Os genótipos PCV2a e PCV2b foram detectados na infecção in utero. Conclusões: as porcas nulíparas podem estar infectadas com PCV2 antes de entrar nas granjas de criação e pode ser infectadas por transmissão horizontal durante a quarentena e gravidez. Exposição e infecção viral durante a gestação pode resultar em infecção subclínica de leitões recém-nascidos.
RESUMO
Since its discovery, Porcine circovirus type 2 has emerged as one of the most relevant swine infectious diseases, causing relevant economic losses for the pig industry. While four genotypes were identified, only three (PCV2a, PCV2b and PCV2d) are currently circulating and display a worldwide distribution. Another genotype, PCV2c, has been described only once in Danish archive samples collected between 1980 and 1990. In addition to commercial pigs, PCV2 has been demonstrated to infect wild boars and other wild species, which can potentially serve as a reservoir for domestic populations. In this study, eight sequences obtained from feral pigs in the Pantanal region (Mato Grosso do Sul State, Brazil) were compared with reference sequences and other Brazilian sequences, and the results revealed remarkable genetic diversity, with all four genotypes currently recognised being detected (PCV2a, PCV2b, PCV2c and PCV2d). This finding represents a remarkable discovery, as it is the first detection of PCV2c since 1990 and the first-ever detection of PCV2c in live animals. The peculiar population history and ecological scenario of feral pigs in the Pantanal coupled with the complex, and still only partially known relationship of feral pigs with other PCV2 susceptible species (i.e., domestic pigs, wild boars and peccaries), open exciting questions concerning PCV2 origin and evolution. Overall, the results of the present study led us to form the following hypothesis: the PCV2 strains found in feral pigs may be the last descent of the strains that circulated among European pigs in the past, or they may have infected these feral pigs more recently through a bridge species.
Assuntos
Circovirus/genética , Evolução Molecular , Variação Genética , Filogenia , Suínos/virologia , Animais , Sequência de Bases , Brasil , Genótipo , Dados de Sequência Molecular , Análise de Sequência de DNA , Áreas AlagadasRESUMO
Porcine circovirus 2 (PCV2) is a common virus in pig population and is associated with the postweaning multisystemic wasting disease (PMWS). In this study, it was developed and evaluated the single-tube nested PCR (STNPCR) method for the detection of PCV2 DNA. PCV2 reference controls and swine tissue samples were used, and primers were selected for targeting specific regions of the viral genome. In comparison of the methods, STNPCR was 10 times more sensitive than conventional PCR and showed the same sensitivity to nested PCR (NPCR), but with reduction in the risk of cross-contamination. In clinical application, 55 tissue samples were analysed by conventional PCR and resulted in 67% (37/55) of positive reactions, while the NPCR and STNPCR were able to identify the presence of viral DNA in 100% (55/55) of the samples. The high sensitivity combined with the elimination of cross-contamination makes the STNPCR method suitable for the epidemiological studies of PCV2 and can aid in the diagnosis of PMWS.
Assuntos
Circovirus/isolamento & purificação , Reação em Cadeia da Polimerase/veterinária , Síndrome Definhante Multissistêmico de Suínos Desmamados/diagnóstico , Síndrome Definhante Multissistêmico de Suínos Desmamados/virologia , Animais , Sequência de Bases , Circovirus/genética , Biologia Computacional , Primers do DNA/genética , DNA Viral/genética , Feminino , Genoma Viral/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Análise de Sequência de DNA/veterinária , SuínosRESUMO
Porcine Circovirus Type 2 (PCV2) is a worldwide distributed virus and is considered an important emerging pathogen related to several distinct disease syndromes in pigs. Genomic structure consists of three major open reading frames (ORFs). ORF1 (rep gene) encodes replication-related proteins, ORF2 (cap gene) encodes the capsid protein and ORF3 encodes a protein putatively involved in virus-induced apoptosis. Based on cap gene sequences, PCV2 strains are classified into two main genotypes, PCV2a with five clusters (2A-2E) and PCV2b with three clusters (1A-1C). According to previous theoretical studies, PCV2 strains can eventually undergo intra and inter-genotype recombination, mainly within the rep gene. Ever since, several evidences of recombination in the field have been reported and confirmed this hypothesis. In South America, data regarding molecular characterization of PCV2 strains is still scant. Genotyping studies in the region have concluded that PCV2b is the predominant circulating genotype in the region and till now, no recombinant strains have ever been reported. In this work we thoroughly characterized at the molecular level Uruguayan PCV2 strains by extensive sequence data analysis. Moreover, recombination software tools were applied to explore and characterize eventual occurrence of natural recombination events. Two recombinant PCV2 strains were detected in this study, as a consequence of an inter-genotype recombination event between PCV2b-1A and PCV2a-2D, as the major and minor parent, respectively. According to recombination software analysis, in both cases the event occurred within the ORF1. Herein, extensive viral sequence dataset is provided, including the characterization of the first PCV2 recombinant strains ever reported in South America. Additionally, our results suggested a multi-centered source of PCV2 infection in Uruguay, which probably involved Brazilian and European origins.
Assuntos
Infecções por Circoviridae/virologia , Circovirus/genética , Animais , Infecções por Circoviridae/veterinária , Circovirus/classificação , Circovirus/isolamento & purificação , Análise por Conglomerados , DNA Viral/análise , DNA Viral/genética , Fases de Leitura Aberta/genética , Filogenia , Recombinação Genética/genética , Suínos , Uruguai , Virologia/métodosRESUMO
Background: Researches have suggested that mycotoxins could corroborate the pathogenesis of porcine viral diseases. Zearalenone (ZEA), one of the most import mycotoxicosis in pigs result significant reproductive disorders. The circovirosis syndromes in pigs have been associated with multifactorial or predisposing conditions. No previous data have correlated ZEA with viral syndromes in Sus scrofa. In this report are described anamnesis, clinical and histological findings with zearalenone ingestion and PCV-2 infection in a Brazilian livestock. Case: Feeding ration was mixed and produced at the farm with low quality corn that was stored in barn with cement floor. During ten months, 28 farrowing occurred. Fifteen of them were normal and produced 145 animals, mean of 9.66 piglets per litter. Thirteen sows presented reproductive disorders, and one died. Eight of them had the delayed farrowing and three aborted. These animals produced 57 mummified fetuses (10-16cm crown-rump length), 23 aborted fetuses (60-72 days of gestation), four stillborn, two teratogenic fetuses and only 16 normal piglets. Skin pustules, hemorrhagic spots and petechiae, as well as coughing were observed in 88% of the animals and four piglets died. Diarrhea outbreaks frequently occurred in this period. Twenty five of 34 gilts (75%) showed longer estrous cycle, estrus returning, vulvae volume and mammary glands en
Background: Researches have suggested that mycotoxins could corroborate the pathogenesis of porcine viral diseases. Zearalenone (ZEA), one of the most import mycotoxicosis in pigs result significant reproductive disorders. The circovirosis syndromes in pigs have been associated with multifactorial or predisposing conditions. No previous data have correlated ZEA with viral syndromes in Sus scrofa. In this report are described anamnesis, clinical and histological findings with zearalenone ingestion and PCV-2 infection in a Brazilian livestock. Case: Feeding ration was mixed and produced at the farm with low quality corn that was stored in barn with cement floor. During ten months, 28 farrowing occurred. Fifteen of them were normal and produced 145 animals, mean of 9.66 piglets per litter. Thirteen sows presented reproductive disorders, and one died. Eight of them had the delayed farrowing and three aborted. These animals produced 57 mummified fetuses (10-16cm crown-rump length), 23 aborted fetuses (60-72 days of gestation), four stillborn, two teratogenic fetuses and only 16 normal piglets. Skin pustules, hemorrhagic spots and petechiae, as well as coughing were observed in 88% of the animals and four piglets died. Diarrhea outbreaks frequently occurred in this period. Twenty five of 34 gilts (75%) showed longer estrous cycle, estrus returning, vulvae volume and mammary glands en
RESUMO
Background: Researches have suggested that mycotoxins could corroborate the pathogenesis of porcine viral diseases. Zearalenone (ZEA), one of the most import mycotoxicosis in pigs result significant reproductive disorders. The circovirosis syndromes in pigs have been associated with multifactorial or predisposing conditions. No previous data have correlated ZEA with viral syndromes in Sus scrofa. In this report are described anamnesis, clinical and histological findings with zearalenone ingestion and PCV-2 infection in a Brazilian livestock. Case: Feeding ration was mixed and produced at the farm with low quality corn that was stored in barn with cement floor. During ten months, 28 farrowing occurred. Fifteen of them were normal and produced 145 animals, mean of 9.66 piglets per litter. Thirteen sows presented reproductive disorders, and one died. Eight of them had the delayed farrowing and three aborted. These animals produced 57 mummified fetuses (10-16cm crown-rump length), 23 aborted fetuses (60-72 days of gestation), four stillborn, two teratogenic fetuses and only 16 normal piglets. Skin pustules, hemorrhagic spots and petechiae, as well as coughing were observed in 88% of the animals and four piglets died. Diarrhea outbreaks frequently occurred in this period. Twenty five of 34 gilts (75%) showed longer estrous cycle, estrus returning, vulvae volume and mammary glands en
Background: Researches have suggested that mycotoxins could corroborate the pathogenesis of porcine viral diseases. Zearalenone (ZEA), one of the most import mycotoxicosis in pigs result significant reproductive disorders. The circovirosis syndromes in pigs have been associated with multifactorial or predisposing conditions. No previous data have correlated ZEA with viral syndromes in Sus scrofa. In this report are described anamnesis, clinical and histological findings with zearalenone ingestion and PCV-2 infection in a Brazilian livestock. Case: Feeding ration was mixed and produced at the farm with low quality corn that was stored in barn with cement floor. During ten months, 28 farrowing occurred. Fifteen of them were normal and produced 145 animals, mean of 9.66 piglets per litter. Thirteen sows presented reproductive disorders, and one died. Eight of them had the delayed farrowing and three aborted. These animals produced 57 mummified fetuses (10-16cm crown-rump length), 23 aborted fetuses (60-72 days of gestation), four stillborn, two teratogenic fetuses and only 16 normal piglets. Skin pustules, hemorrhagic spots and petechiae, as well as coughing were observed in 88% of the animals and four piglets died. Diarrhea outbreaks frequently occurred in this period. Twenty five of 34 gilts (75%) showed longer estrous cycle, estrus returning, vulvae volume and mammary glands en