High prevalence of porcine circovirus 2, porcine parvovirus, and pathogenic leptospires in mummified swine fetuses in Southern Brazil / Alta prevalência de parvovírus suíno, circovírus suíno 2 e leptospiras patogênicas em fetos suínos mumificados na região Sul do Brasil

ABSTRACT: Modern swine production employs a high degree of technology and is organized in various production stages, in which reproduction is one of the most significant. However, reproductive losses associated to fetal death are still high. Fetal losses, including fetal mummification, may occur at a rate below 1.5% in a sound herd. The causes of fetal death can be very diverse, and include various infectious agents. Therefore, investigating the causes of fetal losses is important in the decision-making process related to the control and prophylaxis of the herd. The goal of this study was to carry out a molecular survey, analyzing porcine circovirus 2 (PCV2), porcine parvovirus (PPV), and pathogenic Leptospira as potential agents related to fetal death in swine farms in Southern Brazil. We collected samples from three farms with a mummified index ≥ 2.5%. Fragments of brain, lung, kidney, liver, and heart were sampled for PCR analysis. Out of 100 samples examined, 87, 68, and 22 were positive for PCV2, PPV, and Leptospira spp., respectively. Moreover, we also identified coinfections with two and three pathogens in the same sample. Our findings contribute to the spreading of scientific knowledge related to infectious causes (PPV, PCV2, and pathogenic leptospires) of fetal losses in swine, as evidenced by the high frequencies of the investigated agents.

RESUMO: A produção moderna de suínos emprega um alto grau de tecnologia e é organizada em várias etapas de produção, nas quais a reprodução é uma das mais importantes. No entanto, as perdas reprodutivas associadas à morte fetal ainda são altas. Perdas fetais, incluindo mumificação, podem ocorrer em taxa abaixo de 1,5% em rebanhos sadios. As causas da morte fetal podem ser diversas, incluindo causas infecciosas. Portanto, a investigação de causas relacionadas à ocorrência de perdas fetais é importante no processo relacionado ao controle e profilaxia do rebanho. O objetivo deste estudo foi realizar um levantamento molecular, incluindo circovírus suíno 2 (PCV2), parvovírus suíno (PPV), e leptospiras patogênicas como potenciais agentes relacionados à morte fetal em suínos no sul do Brasil. Foram coletadas amostras de três granjas com índice de fetos mumificados ≥ 2,5%. Fragmentos de cérebro, pulmão, rim, fígado e coração foram amostrados para análise por PCR. Das 100 amostras examinadas, 87 foram positivas para PCV2, 68 foram positivas para PPV e 22 foram positivas para Leptospira spp. Além disso, também identificamos co-infecções com dois e três patógenos em uma mesma amostra. Os resultados encontrados contribuem para a disseminação do conhecimento científico relacionado às causas infecciosas (PCV2, PPV e Leptospiras patogênicas) relacionadas às perdas fetais em suínos, assim como evidenciam a alta frequência dos agentes pesquisados.

Structure analysis of capsid protein of Porcine circovirus type 2 from pigs with systemic disease

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.

Porcine circovirus type 2 ORF4 protein binds heavy chain ferritin.

Porcine circovirus type 2 (PCV2) is the primary infectious agent of PCV-associated disease (PCVAD) in swine. ORF4 protein is a newly identified viral protein of PCV2 and is involved in virus-induced apoptosis. However, the molecular mechanisms of ORF4 protein regulation of apoptosis remain unclear, especially given there is no information regarding any cellular partners of the ORF4 protein. Here, we have utilized the yeast two-hybrid assay and identified four host proteins (FHC, SNRPN, COX8A and Lamin C) interacting with the ORF4 protein. Specially, FHC was chosen for further characterization due to its important role in apoptosis. GST pull-down, subcellular co-location and co-immunoprecipitation assays confirmed that the PCV2 ORF4 protein indeed interacted with the heavy-chain ferritin, which is an interesting clue that will allow us to determine the role of the ORF4 protein in apoptosis.

Unusual outcome of in utero infection and subsequent postnatal super-infection with different PCV2b strains / 中国病毒学·英文版

VC2002, isolated from postweaning multisystemic wasting syndrome (PMWS)-affected pig, is a mixture of two porcine circovirus genotype 2b (PCV2b) viruses, K2 and K39. Preliminary experiments disclosed short-term adverse effects of K39, but not K2, on porcine foetuses. These findings led to the hypothesis that infection of immuno-incompetent foetuses with K2 confers a status of immunotolerance, and postnatal super-infection with K39 triggers PMWS. To explore this hypothesis, nine 55-day-old foetuses were inoculated in utero (three with K2-104.3TCID50, three with K39-104.3TCID50 and three with medium), and foeto-pathogenicity examined. At 21 days post-inoculation (dpi), K2 did not induce pathology, whereas pathological effects of K39 were evident. Twenty-four 45-day-old foetuses were subsequently inoculated to examine the long-term effect of K2, including six with K2-high dose-104.3TCID50, six with K2-low dose-102.3TCID50 and 12 mock-inoculated controls. Both doses resulted in ifve mummiifed foetuses and one live-born piglet each (69dpi). K2 was recovered from all mummies. K2 and K2-speciifc antibodies were not detected in serum of the two live-born piglets at birth, indicating full control of K2 infection. The K2-low dose-infected piglet was immunostimulated at day 2, but not the K2-high dose-infected piglet. Both non-stimulated and stimulated K2-infected piglets were super-inoculated with K39 at day 6 or 8 (taken as 0 days post super-inoculation). Low viral replication was observed in the non-stimulated K2-K39 piglet (up to 103.3 TCID50/g;identiifed as K39). In contrast, viral replication was extremely high in the stimulated K2-K39 piglet (up to 105.6TCID50/g) and identiifed as K2, indicating that K2 infection is controlled during foetal life, but emerges after birth upon immunostimulation. However, none of the piglets showed any signs of PMWS.

Expresión de citocinas en cerdos co-infectados con circovirus porcino tipo 2 y el virus del síndrome respiratorio y reproductivo porcino / Cytokine expression in growing pigs co-infected with porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus

The aim of this work was to quantify porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), as well as cytokine mRNA expression (IL-6, IL-12, TNF-α, and IFN-γ) in superficial inguinal lymph nodes (SILN) of pigs from postweaning multisystemic wasting syndrome (PMWS)-affected and healthy farms. Genetic characterization of detected PCV2 sequences was also carried out. Based on the clinical outcome and the quantification of PCV2 nucleic acid by in situ hybridization and real time PCR, pigs were grouped into three categories: 1) PMWS, pigs with signs of wasting and high viral load in SILN (n = 4); 2) wasted-non-PMWS, pigs with signs of wasting and low to intermediate viral loads in SILN (n = 3); and 3) healthy, pigs with no clinical signs and low viral loads (n = 3). PRRSV was detected in three PMWS affected and two-wasted-non-PMWS pigs. The genetic analysis of PCV2 sequences revealed the presence of two genotypes PCV2a and PCV2b in PMWS-affected farms. Cytokine mRNA expression revealed that PMWS affected pigs had low expression of IFN-γ, whereas wasted-non-PMWS pigs showed higher amounts of IFN-γ. These results suggest that an imbalance in cytokines could be involved in the pathogenesis of PMWS.

Los objetivos de este trabajo fueron la cuantificación de circovirus porcino tipo 2 (PCV2) de virus del síndrome respiratorio y reproductivo porcino (PRRSV), así como la expresión de citocinas (IL-6, IL-12, TNF-α, e IFN-γ) en nódulos linfáticos inguinales superficiales (NLIS) de cerdos afectados por el síndrome de desmedro posdestete (postweaning multisystemic wasting syndrome, PMWS) y en cerdos sanos. Con base en el diagnóstico clínico y a la cuantificación de PCV2 por hibridación in situ y qRT-PCR, los cerdos se distribuyeron en tres grupos: 1) PMWS, cerdos con signos de desmedro y con cargas virales altas en NLIS (n = 4); 2) con desmedro sin PMWS, cerdos con signos de desmedro y cargas virales de intermedias a bajas en NLIS (n = 3); y 3) cerdos sanos, sin signos clínicos y con cargas virales bajas (n = 3). El PRRSV fue detectado en tres de los cerdos afectados por el PMWS y en dos cerdos con desmedro, pero sin el síndrome. Se caracterizaron las secuencias nucleotídicas del ORF2 de los PCV2 encontrados y los análisis genéticos revelaron la presencia de 2 genotipos PCV2a y PCV2b en las granjas afectadas con el PMWS. El perfil de expresión de citocinas mostró una baja expresión de IFN-γ en los cerdos con PMWS, mientras que los cerdos con desmedro sin PMWS mostraron valores elevados de esta citocina. Estos resultados sugieren que existe un desbalance en la producción de citocinas que puede estar implicado en la patogénesis de la enfermedad.

Retrospective study on Porcine circovirus-2 by nested pcr and real time pcr in archived tissues from 1978 in Brazil

Porcine circovirus-2 (PCV-2) infection is currently considered an important disease of swine. The pathogenic agent was first described in Brazil in 2000. This study detected the PCV-2 DNA in four Brazilian pig tissues collected between 1978 and 1979. This observation is the oldest description of this virus in Brazil.

Rectal stenosis in pigs associated with Salmonella Typhimurium and porcine circovirus type 2 (PCV2) infection / Estenose retal em suínos associada à enterite causada por Salmonella Typhimurium em animais infectados pelo circovírus suíno tipo 2 (PCV2)

Rectal stricture is an acquired annular fibrous constriction of the rectum that results from a variety of chronic necrotizing enteric diseases. In pigs, it is in most cases a sequel of Salmonella infection. Porcine circovirus type 2 (PCV2) is a known pathogen causing immunosuppression in pigs worldwide. PCV2 infected pigs may be predisposed to salmonellosis. In this report, rectal stenosis was observed in 160 pigs from a herd that experienced an outbreak of enteric salmonellosis over a 4-month period. Distension of the abdominal wall and diarrhea were the main clinical signs observed. Five animals were analyzed showing annular cicatrization of the rectal wall 5.0-7.0 cm anterior to the anorectal junction and Salmonella-positive immunostaining in the large intestine. Salmonella Typhimurium was isolated from fragments of the large intestine. Porcine circovirus type 2 antigen was observed in the mesenteric lymph-node in 4 pigs and in the large intestine in 3 pigs.

Estenose retal é uma constrição anular fibrosa do reto que pode ser decorrente de qualquer doença entérica crônica necrotizante. Em suínos, é em muitos casos uma seqüela de infecção por Salmonella. Circovírus suíno tipo 2 é um patógeno bem conhecido que causa imunodepressão em suínos e apresenta distribuição mundial. No presente trabalho, estenose retal foi observada em 160 suínos em um rebanho que teve um surto de salmonelose entérica durante 4 meses. Distensão da parede abdominal e diarreia foram os principais sinais clínicos observados. Foram analisados cinco suínos que demonstraram cicatrização anular da parede do reto 5,0 a 7,0 cm anterior a junção anoretal e imuno-histoquímica positiva para Salmonella spp. no intestino grosso. Antígeno de Circovírus suíno tipo 2 foi observado no linfonodo mesentérico de quatro suínos e no intestino grosso de três.

Quantification and genotyping of PCV2 DNA in the tissues of PCV2-infected conventional pigs with different clinical signs / 대한수의학회지

This study was focused on the genotyping and quantification of Porcine circovirus type 2 (PCV2) in thirty PCV2-positive pigs with different clinical symptoms (PCV2-infected without wasting, PCV2-infected with wasting, PCV2-infected with wasting and lymphoid depletion). The quantity of PCV2 DNA in diverse tissues was significantly differed among these groups. (One-way ANOVA test, p < 0.001) Interestingly, PCV2-DNA load in tissues of PCV2-infected pigs without wasting and PCV2-infected pigs with wasting and lymphoid depletion were not significantly differed (p = 0.38), while they were all significantly higher when compared with PCV2-infected pigs with wasting-only. PCV2 DNA quantity in tissues was significantly higher in PCV2a and 2b co-infected pigs compared to the PCV2b only-infected pigs (Wilcoxon test, p = 0.039). The PCV2a and 2b co-infected pigs had increased wasting and lymphoid depletion rate but it was not statistically significant. Therefore, this cross-sectional study suggested that PCV2 DNA load in tissues was diverse by clinical and histological findings. Furthermore, co-infection of PCV2a and 2b affected to the PCV2 DNA load in tissues with increased rate of wasting and lymphoid depletion.

Epidemiological characteristics of pulmonary pneumocystosis and concurrent infections in pigs in Jeju Island, Korea

Epidemiological characteristics of swine pulmonary Pneumocystis (P.) carinii and concurrent infections were surveyed on Jeju Island, Korea, within a designated period in 172 pigs submitted from 54 farms to the Department of Veterinary Medicine, Jeju National University. The submitted cases were evaluated by histopathology, immunohistochemistry, PCR/RT-PCR, and bacteriology. P. carinii infection was confirmed in 39 (22.7%) of the 172 pigs. Histopathologically, the lungs had moderate to severe lymphohistioctyic interstitial pneumonia with variable numbers of fungal organisms within lesions. Furthermore, porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV-2) co-infection was a common phenomenon (12.8%, 20.5%, and 48.7% were positive for PRRS, PCV-2, or both, respectively, as determined by PCR/RT-PCR). Infection was much more concentrated during winter (December to March) and 53.8% of the infected pigs were 7- to 8-weeks old. In addition, three pigs showed co-infection with bacteria such as Pasteurella multocida and Streptococcus suis. The results of the present study suggest that the secondary P. carinii infection is common following primary viral infection in swine in Korea. They further suggest that co-infection of P. carinii might be enhanced by the virulence of primary pathogens or might have synergistic effects in the pigs with chronic wasting diseases.

Development of a Loop-Mediated Isothermal Amplification Assay for Porcine Circovirus Type 2 / 中国病毒学·英文版

In this study,the loop-mediated isothermal amplification(LAMP)method was used to develop a rapid and simple detection system for porcine circovirus type 2(PCV2).According to the PCV2 sequences published in GenBank,multiple LAMP primers were designed targeting conserved sequences of PCV2.Using the DNA extracted from PCV2 isolates HUN-09 and SD-09 as the template,LAMP reactions in a PCV2 LAMP system was performed,the amplification products were detected by adding SYBR Green I and could be observed directly by the naked eye.The results showed highly-efficient and specific amplification in 30 min at 63℃ with a LAMP real-time turbidimeter.Furthermore,PCV2 DNA templates,with a detection limit of 5.5×10-5ng of nucleic acid,indicated that this assay was highly sensitive.The results obtained with the naked eye after SYBR Green I staining were consistent with those detected by the real-time turbidimeter,showing the potential simplicity of interpretation of the assay results.The LAMP assay appeared to have greater accuracy than PCR and virus isolation for the analysis of 18 clinical samples.In addition it offers higher specificity and sensitivity,shorter reaction times and simpler procedures than the currently available methods of PCV2 detection.It is therefore a promising tool for the effective and efficient detection of PCV2.

Porcine circovirus-2 load in the gastrointestinal tract, mesenteric lymph node, tonsil, and feces of wasted pigs with and without diarrhea / Carga viral de circovírus suíno-2 no trato gastrintestinal, linfonodo mesentérico, tonsila e fezes de animais refugo com e sem diarreia

Relata-se a quantificação de PCV-2, por meio de PCR em tempo real, no trato gastrintestinal, linfonodo mesentérico, tonsilas e fezes de nove suínos refugo da fase de crescimento, provenientes de rebanhos com histórico de diarreia não responsiva ao tratamento com antibioticoterapia. Com base nos resultados histopatológicos e imunoistoquímicos, os animais foram classificados como afetados por PCV-2 (n=5) e não afetados (n=4). Foi observada diarreia em todos os animais do grupo afetado por PCV-2. Em todas as amostras testadas foi detectado PCV-2, no entanto, a carga viral observada nos tecidos, bem como a excreção nas fezes foi significantemente maior (P<0.01) nos animais com diarreia. A maior concentração de PCV-2 foi observada nos linfonodos mesentéricos. A carga viral observada em tecido fresco e nas mesmas amostras emblocadas em parafina foi semelhante, sugerindo que o PCR em tempo real pode ser uma ferramenta útil em estudos retrospectivos da infecção por PCV-2.

The cap gene of porcine circovirus type 2 (PCV2) evolves by positive selection in vitro / O gene cap de circovírus suíno tipo 2 (PCV2) evolui por seleção positiva in vitro

This article reports the genetic divergence of PCV2 after passages in VERO and SK-RST cell lines. Fisher’s exact test indicated a trend for positive selection on the cap gene. These results allow insights on the development of PCV2 vaccines and the evolution of the genus Circovirus.

Este artigo relata a divergência genética de PCV2 após passagens em células das linhagens VERO e SK-RST. O teste exato de Fisher indicou tendência para seleção positiva no gene cap. Estes resultados permitem inferências relativas ao desenvolvimento de vacinas contra PCV2 e sobre a evolução do gênero Circovirus.

An ELISA Based on a Truncated Soluble ORF2 Protein for the Detection of PCV2 Antibodies in Domestic Pigs / 中国病毒学·英文版

Postweaning multisystemie wasting syndrome (PMWS) is an important swine disease that is closely associated with porcine circovirus type 2 (PCV2). The capsid protein (Cap protein) is a major structural protein that has at least three immunoreactive regions, and it can be a suitable candidate antigen for detecting the specific antibodies of a PCV2 infection. In the present study, an indirect enzyme-linked immunosorbent assay (TcELISA)based on a truncated soluble Cap protein produced in Escherichia coli (E.coli) was established and validated for the diagnostic PCV2 antibodies in swine. The TcELISA was validated by comparison with an indirect immunofluorescence assay (IIFA). The diagnostic sensitivity (DSN), specificity (DSP), and accuracy of the TcELISA were 88.6%, 90.7% and 89.4%, respectively. The agreement rate was 89.38% between results obtained with TcELISA and IIFA on 113 field sera. A cross-reactivity assay showed that the method was PCV2-specific by comparison with other sera of viral disease. Therefore ,the TcELISA will be helpful for the development of a reliable serology diagnostic test for large scale detection of PCV2 antibodies and for the evaluation of vaccine against PCV2 in swine.

Necrotic enterocolitis in pigs naturally infected by porcine circovirus type 2 / Enterocolite necrótica em suínos naturalmente infectados por circovírus suíno tipo 2

Samples of intestine with necrotic enteritis from 63 pigs naturally infected with porcine circovirus type 2 (PCV2) were studied. Colon was the main target of PCV2 associated necrotic enteritis in 60 cases. Immunohistological investigations were carried out to detect the presence of PCV2 in necrotic lesions and to identify the type of cells infected by the virus. Crypt epithelial cells had positive labelling for PCV2 in 17 cases. Depletion of goblet cells occurred in 10 cases. In 24 necrotic enteritis cases, co-infection of PCV2 and Salmonella was identified. An increased rate of apoptosis in the crypt epithelial cells of the large intestine from PCV2 of naturally infected pigs was observed. Immunohistochemical findings confirmed the presence of PCV2 within cells from necrotic intestinal tissue, suggesting that PCV2 may play a role in the development of those lesions. Diagnosis of necrotic enteritis associated with PCV2 should be based on the detection of PCV2 antigen or DNA in the necrotizing lesions. However, bacteriological examination should be performed to rule out the presence of bacterial agents, since co-infections are likely to occur in PCV2 affected pigs.

Foram selecionadas amostras intestinais com enterite necrótica de 63 suínos naturalmente infectados pelo circovírus suíno tipo 2 (PCV2). Enterite necrótica associada com PCV2 ocorreu principalmente no cólon, em 60 casos. Análise imuno-histoquímica foi realizada para identificar a presença de PCV2 em lesões necróticas e o tipo de células infectadas pelo vírus. Células epiteliais das criptas apresentaram marcação positiva para PCV2 em 17 casos. Depleção de células caliciformes ocorreu em 10 casos. Em 24 casos de enterite necrótica, observou-se co-infecção por PCV2 e Salmonella. Foi observado um aumento no índice de apoptose nas células das criptas do intestino grosso de suínos naturalmente infectados com PCV2. Os achados imuno-histoquímicos e histopatológicos sugerem que a infecção por PCV2 das células do tecido intestinal pode ocasionar enterite necrótica. O diagnóstico de enterite necrótica associada com PCV2 deve ser baseado na detecção do antígeno ou do DNA viral nas lesões necróticas. Contudo, análise bacteriológica deve ser realizada para descartar a presença de agente bacteriano, já que co-infecções são comuns.

Development and evaluation of a recombinant DNA vaccine candidate expressing porcine circovirus 2 structural protein: [revision] / Desenvolvimento e avaliação de um candidato à vacina de DNA recombinante expressando a proteína estrutural do circovírus suíno: [revisão]

Porcine circovirus 2 (PCV2) is generally associated with the porcine circovirosis syndrome, which is considered an important disease of swine and has potentially serious economic impact on the swine industry worldwide. This article describes the construction of a recombinant plasmid expressing the PCV2 structural protein and the evaluation of cellular and humoral immune responses produced by this recombinant vaccine in BALB/c mice. The vaccine candidate was obtained and analyzed in vivo, in an effort to determine the ability to induce a specific immune response in mice. DNA was extracted from a Brazilian PCV2 isolate and the gene coding for Cap protein was amplified by PCR and inserted into an expression plasmid. Groups of BALB/c mice were inoculated intra-muscularly and intradermally in a 15-day interval, with 100 µg and 50 µg of the vaccine construct, respectively. Another group was inoculated intramuscularly with 100 µg of empty plasmid, corresponding to the control group. Seroconversion and cellular response in BALB/c mice were compared and used for vaccine evaluation. Seroconversion was analyzed by ELISA. After a series of 3 immunizations the spleen cells of the immunized animals were used to perform lymphocyte proliferation assays. Seroconversion to PCV2 was detected by ELISA in the animals inoculated with the vaccine construct when compared with control groups. Lymphocyte proliferation assays showed a stronger cell proliferation in the inoculated animals compared with the control group. Thus, the vaccine candidate construct demonstrated to be able to induce both humoral and cellular responses in inoculated mice.

O circovírus suíno 2 (PCV2) é geralmente associado à síndrome da circovirose suína, que é considerada uma importante doença de suínos e possui um sério impacto econômico na suinocultura mundial. Este trabalho descreve a construção de um plasmídeo recombinante que expressa a proteína estrutural do PCV2 e a avaliação das respostas imune humoral e celular por meio de vacinação em camundongos BALB/c. O candidato vacinal foi submetido a análises in vivo, determinando a capacidade de induzir resposta imune específica em camundongos. O DNA de um isolado brasileiro de PCV2 foi extraído e o gene que codifica para a proteína do capsídeo foi amplificado por PCR e inserido num plasmídeo de expressão. Grupos de camundongos BALB/c foram inoculados por via intramuscular e intradérmica a cada 15 dias, com 100µg e 50µg da construção vacinal, respectivamente. Outro grupo foi inoculado com 100µg do plasmídeo original, correspondente ao grupo controle. A soroconversão e a resposta celular dos grupos de camundongos BALB/c vacinados foram comparados como parâmetros de avaliação vacinal. A soroconversão foi avaliada por um teste de ELISA. Após 3 imunizações, as células esplênicas dos animais imunizados foram utilizadas nos ensaios de linfoproliferação. A soroconversão para o PCV2 foi detectada por ELISA nos animais inoculados com a construção vacinal quando comparados com o grupo controle. Nos ensaios de linfoproliferação foi observada uma grande proliferação celular nos animais inoculados comparados ao grupo controle. Portanto, o candidato vacinal demonstrou ser capaz de induzir tanto uma resposta humoral e celular nos camundongos inoculados.

Obtaining of union expression virus-like particle of PPV VP2 and PCV2 ORF2 and its immunogenicity / 中国兽医学报

The amplified VP2 gene (PPV strain SC-1) and PCV20RF2 gene were inserted into the eukaryotic expression vector pPI-2.EGFP.Then the recombinant plasmid named pPI-2.EGFP.VP2.ORF2 was obtained.Mediated by liposome,the recombinant plasmid was transfected into Veto cells and expressed.Using immunofluorescence assay,the fluorescence of expression products were first detected at 20 h after transfection and peaked 36 h.Under electronmicroscope,virus-like particles (VLPs) can be observed in the transfected cells.To confirm the obtained VLPs to be recombinant particles,piglets were immunized using purified VLPs.The dynamic variation of blood T lymphocytes and serum antibody level of PPV and PCV2 were measured.The results showed that the ratio of CD3+,CD4+ T lymphocyte in peripheral blood lymphocyte of immunized piglets raised in a certain degree,the number of CD8+ T lymphocytes fell at 7-14 d after immunization,and then raised.Relatively high level of PP-V,PCV2 specific antibody could be detected.This indicated that the expression of recombinant plasmid pPI-2.EGFP.VP2.ORF2 was successful,the virus-like particles were formed and showed favourable immunogenicity.

Effects on humoral immune response to classical swine fever vaccine after experimental infection of porcine circovirus type 2 / 中国兽医学报

The levels of the antibody response against,classic swine fever (CSF) vaccine were measured by blocking ELISA at different time for pigs vaccinated with CSF vaccine (CSFV group,n= 3),pigs inoculated with porcine eircovirus type 2 (PCV2) and with CSF vaccine two weeks later (when PCV2 viremia was detected by PCR,PCV2/CSFV group,n=3),or inoculated with both CSF vaccine and PCV2 at the same time (CSFV/PCV2 group,n=3),respectively.And distribution of genome or antibodies specific to PCV2 of the PCV2 infection group,PCV2/CSFV group and CSFV/PCV2 group were also detected by indirect ELISA or PCR,respectively.The results showed that experimental infection of PCV2 was successful.In pigs inoculated with both PCV2 and CSF vaccine,the antibody response to CSF vaccine was significantly lower than that of animals treated with CSF vaccine vaccination alone at 52 days postinoculation (DPI).The average titers of antibodies against CSFV in animals of CSFV group were obviously higher than those of the PCV2/CSFV or CSFV/PCV2 group at 49 and 52DPI,respectively.The positive rate of antibodies against CSFV in pigs of CSFV group was 100% in comparison with that of animals inoculated with PCV2 and the vaccine (only 67%).The results suggested that the infection of PCV2 could suppress the antibody response to classical swine fever vaccine.

Enteritis associated with Lawsonia intracellularis and porcine circovirus type 2 infection in wild boars in Southern Brazil / Enterite associada com infecção por Lawsonia intracellularis e circovírus suíno tipo 2 em um rebanho de javalis no Sul do Brasil

In a commercial herd of 1250 wild boars, 56 animals were affected by anorexia, diarrhea, and wasting. Enlargement of mesenteric lymph nodes, thickening of ileal wall, and a white-yellowish ileal content were observed in two of the four animals necropsied. Microscopically, ileal crypts showed epithelial hyperplasia, moderate decrease in goblet cell density, lymphohistiocytic infiltrate in the lamina propria, and moderate depletion with histiocytic infiltrate in Peyer's patches. Curved rod-shaped organisms within the apical cytoplasm of enterocytes were observed in both Warthin-Starry and Lawsonia intracellularis-immunostained sections. PCV2 antigen was observed in ileal Peyer's patches of 3 wild pigs. Trichuris suis nematodes were found in the cecum of the four wild pigs. This report describes clinicopathological findings in cases of enteritis associated with Lawsonia intracellularis and PCV2 infection in a commercial herd of wild boars.

Neste trabalho, são relatados os achados clínico-patológicos de casos de enterite associados com infecção por Lawsonia intracellularis e PCV2 em um rebanho comercial com 1250 javalis, sendo que 56 foram afetados por anorexia, diarréia e emagrecimento progressivo. Em dois dos quatro animais necropsiados, foi observado aumento de linfonodos mesentéricos, espessamento da parede do íleo e conteúdo branco-amarelado no íleo. Microscopicamente, as criptas do íleo apresentavam hiperplasia epitelial, decréscimo moderado na densidade de células caliciformes, infiltrado linfo-histiocitário na lâmina própria e depleção moderada das placas de Peyer com infiltrado histiocitário. Bastonetes curvos no ápice citoplasmático dos enterócitos foram observados em preparações coradas com Warthin-Starry e pela imunoistoquímica contra Lawsonia intracellularis. Antígeno de PCV2 foi observado nas placas de Peyer do íleo de três animais e nematódeos Trichuris suis estavam presentes no ceco dos quatro javalis necropsiados.

Gastric ulcers in pigs affected with postweaning multisystemic wasting syndrome / Úlceras gástricas em suínos afetados pela síndrome multissistêmica do definhamento

Samples of gastric lymph nodes and the stomachs from 24 pigs selected from herds affected by postweaning multisystemic wasting syndrome (PMWS) and sudden death associated with gastric ulcers were studied. Pigs were selected on the basis of unthriftiness, decreased feed intake, and wasting. The stomachs were opened, inverted, and classified into 0-3 score according the severity of the gross lesions present in pars oesophagica (non-glandulargastric mucosa). Selected samples were processed for paraffin embedding and stained with hematoxylin and eosin. Immunohistochemistry using anti-PCV2 (porcine circovírus type 2) antibody, anti-Helicobacter pylori antibody and a wide-spectrum anti-cytokeratin antibody was performed. Gross changes in pars oesophagea were classified according to the severity of lesions as score 3, 2, and 1 in 8, 6, 5 stomachs respectivelly. Microscopically, hyperplastic lymphoid follicles, lymphohistiocytic inflammatory infiltrates and focci of necrosis in the gastric mucosa were common findings. Large amounts of PCV2 antigen were observed in the cytoplasm and nuclei from intralesional cells and debris from the gastric glandular mucosal zone; however, in the fundus, anti-PCV2 immunostaining was restricted to the surface mucosal cells and foveolar compartment. All gastric lymph nodes were positive for PCV2 antigen. Anti-H. pylori immunostaining was seen in eleven cases, mainly in the antrum, on the mucosal surface and foveolar compartment. The association of the anti-PCV2 immunostaining with the glandular mucus-producing cells suggests a role for PCV2 as an additional factor for the swine ulcer development.(AU)

Amostras de linfonodos gástricos e os estômagos de 24 leitões selecionados de rebanhos afetados pela síndrome multissistêmica do definhamento suíno e mortes súbitas por úlceras gástricas foram estudados. Os animais foram seleciona dos por baixa performance, baixo consumo de alimento e desnutrição. Os estômagos foram abertos, invertidos e classificados, conforme a severidade das lesões presentes na pars oesophagica (porção não-glandular da mucosa gástrica). Amostras selecionadas foram processadas por método histológico convencional para coloração de hematoxilina-eosina. Testes imuno-histoquímicos utilizando anticorpos anti-PCV2 (circovírus suíno tipo 2), anti-Helicobacter pylori e anticitoqueratina de largo espectro foram feitos. As alterações macroscópicas na pars oesophagica foram classificadas como de escore 3, 2 e 1 respectivamente em 8, 6 e 5 estômagos. Microscopicamente, foram notados folículos linfóides hiperplásicos, infiltrados linfohistiocitários e focos de necrose na mucosa gástrica. Grandes quantidades de antígeno do PCV2 foram observadas no citoplasma, núcleo e restos necróticos de células intralesionais das glândulas gástricas nas regiões do antro e cárdia; entretanto, na região do fundo, a marcação anti-PCV2 foi restrita às células da superfície mucosa e fossetas gástricas. Todos os linfonodos gástricos foram positivos para PCV2. Coloração anti-H. pylori foi identificada em 11 casos, principalmente, na superfície mucosa e fossetas gástricas no antro. A associação de antígenos PCV2 com células produtoras de muco lesadas na zona glandular gástrica sugere o envolvimento de PCV2 como um fator adicional para o desenvolvimento da úlcera gástrica suína.(AU)

Associação entre otites bacterianas e infecção pelo circovírus suíno tipo 2 (PCV2) em suínos / Association between bacterial otitis and porcine circovirus 2 (PCV2) infection in pigs

Neste trabalho, foi estudada a ocorrência e a bacteriologia das otites purulentas em suínos apresentando três condições clínico-patológicas distintas: síndrome multissistêmica do definhamento dos suínos (SMDS), crescimento retardado (porém sem apresentação de sinais clínicos da SMDS) e crescimento normal. No total, foram examinados 385 animais com idade de 60-130 dias, sendo a confirmação do diagnóstico da SMDS obtida pelos sinais clínicos e achados patológicos e pela detecção de antígenos virais por imunoistoquímica. De 242 animais com a SMDS, 57 (23,5 por cento) apresentaram lesões purulentas no ouvido médio. Entre os 119 animais de baixo desenvolvimento, apenas 1 (0,7 por cento) apresentou a lesão. Não foram detectadas lesões macroscópicas no ouvido médio dos 24 animais com crescimento normal. Os agentes isolados com maior freqüência foram Arcanobacterium pyogenes (43 por cento), Streptococcus α-hemolíticos (37,2 por cento) e Pasteurella multocida (27,9 por cento) dos 86 ouvidos submetidos à bacteriologia. A alta ocorrência de lesões purulentas no ouvido médio de animais com a SMDS sugere que a infecção pelo PCV2 pode tornar o suíno mais suscetível às otites bacterianas. A ocorrência reduzida dessas lesões em suínos de baixo desenvolvimento, sem a manifestação clínica da SMDS, sugere que a otite média não representa uma causa importante de mau desempenho em leitões nas fases de crescimento e terminação. O envolvimento de A. pyogenes, Streptococcus α-hemolíticos e P. multocida na maioria das lesões apontam a importância desses organismos como agentes causais de otite média em suínos.(AU)

The occurrence and bacteriology of purulent otitis in groups of pigs affected by three different clinical conditions are described: postweaning multisystemic wasting syndrome (PMWS), depressed growth (attrition) and normal growth (control). A total of 385 animals, 60-130 days of age, were examined. Diagnosis of PMWS was confirmed by the presence of clinical signs and pathological findings compatible with the disease, and through detection of viral antigens in tissues by immunohistochemistry. From 242 pigs with PMWS, 57 (23.5 percent) showed purulent lesions in the middle ear. Among 119 pigs with attrition, only 1 (0.7 percent) presented the lesion. In 24 control pigs, middle ear lesions were not detected. The most frequently isolated agents from the lesions were Arcanobacterium pyogenes (43 percent), á-hemolytic Streptococci (37.2 percent) and Pasteurella multocida (27.9 percent) out of 86 materials. The frequent occurrence of purulent lesions in the middle ear of PMWS affected pigs suggests that PCV2 infection may increase the susceptibility to bacterial otitis. The low occurrence of this disease in piglets with attrition suggests that otitis does not represent a significant cause for depressed growth in pigs from growing and finishing age. The isolation of A. pyogenes, á-hemolytic Streptococci and P. multocida from most of the lesions emphasizes the importance of these organisms as causal agents of otitis media in swine.(AU)