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.

Orientações para o diagnóstico de influenza em suínos / Guidelines for diagnosis of swine influenza

Este trabalho descreve a colheita adequada de amostras, as técnicas/procedimentos disponíveis para o diagnóstico de influenza A em suínos, assim como os resultados e suas respectivas interpretações, para auxiliar médicos veterinários de campo na identificação dessa doença. Em suínos vivos, as amostras adequadas são: secreção nasal, fluido oral e sangue (soro). Para suínos mortos, colher preferencialmente amostras de pulmão com consolidação cranioventral. Secreção nasal e fragmentos de pulmão refrigerado são utilizados para detectar partícula viral viável (isolamento viral - IV) ou ácido nucleico viral (RT-PCR convencional e RT-PCR em tempo real). As amostras não devem ser congeladas, pois o vírus é inativado a -20°C. A caracterização molecular dos isolados é feita pela análise filogenética obtida pelo sequenciamento de DNA. O soro é utilizado para a detecção de anticorpos (Acs) por meio do teste da inibição da hemaglutinação e ELISA. O fluido oral pode ser utilizado para detecção de anticorpo (ELISA) ou de vírus. Fragmentos de pulmão fixados em formol a 10% são examinados microscopicamente para identificar pneumonia broncointersticial e para detecção de antígeno viral pela imuno-histoquímica (IHQ). Para o sucesso do diagnóstico, as amostras devem ser colhidas de suínos que estão preferencialmente na fase aguda da doença, para aumentar as chances de detecção viral. As melhores opções para o diagnóstico de influenza A em suínos vivos são RT-PCR e isolamento viral de amostras de swab nasal ou fluido oral. Pulmão para análise por RT-PCR, isolamento viral ou IHQ é a amostra de escolha em suínos mortos. Testes sorológicos têm valor diagnóstico limitado e são utilizados apenas para determinar o estado imune do rebanho, não indicando doença clínica, pois os Acs são detectados 7-10 dias pós-infecção (fase subaguda). O diagnóstico de influenza é importante para avaliar o envolvimento desse agente no complexo de doença respiratória suína. Além disso, o isolamento do vírus influenza é essencial para o monitoramento dos principais subtipos circulantes em uma determinada região ou país, assim como para a detecção de novos rearranjos virais, já que influenza é considerada uma zoonose.

This article is intended to describe the adequate sample collection, the laboratory procedures/techniques, the expected results and their interpretation for diagnosis of influenza infection in swine, serving as a support for field veterinarians. In live pigs, the samples to be taken are nasal secretions, oral fluids and blood. For dead pigs, preference should be given to samples of cranioventral lung consolidation. Nasal discharge and chilled lung fragments are used for detection of virus (virus isolation - VI) or viral nucleic acids (conventional RT-PCR and real-time RT-PCR). Samples should not be frozen, because the virus is inactivated at -20°C. Molecular characterization of isolates is performed by phylogenetic analysis of gene sequences obtained by DNA sequencing. Serum is used for the detection of antibodies using hemagglutination inhibition (HI) test and ELISA. Oral fluid may be used for either antibody (ELISA) or viral detection. Fragments of lung fixed in 10% formaldehyde are used for histopathological analysis to identify bronchointerstitial pneumonia, and for immunohistochemistry (IHC) for antigens. For a successful diagnosis, sampling should be preferably performed in the acute phase of the disease to improve chances of virus detection. The best options to perform the diagnosis of influenza A in a swine herd are RT-PCR and VI from nasal swabs or oral fluid in live pigs and/or lung tissue for RT-PCR, VI or IHC in dead pigs. Serological tests are of very limited diagnostic value and are useful only to determine the immune status of the herd, not indicating clinical disease, because antibodies are detected after 7-10 days post infection (subacute phase). The diagnosis of influenza is important to evaluate the involvement of this agent in the complex of respiratory diseases in pigs. Furthermore, the isolation of influenza virus is essential for monitoring the main subtypes circulating in a given region or country, as well as for the detection of potential new viral reassortants, because influenza is considered a zoonosis.

Indução de partos em suínos: uso de cloprostenol associado com ocitocina ou carbetocina / Farrowing induction in swine: use of cloprostenol associated with oxytocin or carbetocin

O presente estudo teve como objetivo avaliar o efeito da aplicação de um análogo sintético da PGF2 (cloprostenol sodico), associado à ocitocina ou carbetocina, sobre a eficiência da indução ao parto em suínos. O experimento I foi realizado com 284 fêmeas, distribuídas em quatro tratamentos: cloprostenol; cloprostenol e 0,100mg de carbetocina; cloprostenol e 10UI de ocitocina; solução salina (NaCl 0,9 por cento). No experimento II, foram utilizadas 276 fêmeas, distribuídas em quatro tratamentos: cloprostenol; cloprostenol e 0,10mg de carbetocina; cloprostenol e 0,05mg de carbetocina; cloprostenol e 10UI de ocitocina. A indução do parto foi realizada aos 113 dias de gestação, pela aplicação de 0,175mg de cloprostenol, via submucosa vulvar. A carbetocina ou a ocitocina foram aplicadas 24h após a aplicação de cloprostenol, pela via intramuscular. O intervalo indução-parto foi menor (P<0,05) nos grupos cujo parto foi induzido com cloprostenol, em comparação ao grupo sem indução do parto. A aplicação de carbetocina ou ocitocina resultou em maior concentração dos partos (P<0,05) até 26 e 28h após a administração de cloprostenol. A utilização de carbetocina resultou em menor (P<0,05) duração do parto. No experimento I, o uso de ocitocina ou de carbetocina resultou em maior natimortalidade (P<0,05) do que no grupo sem indução do parto. Além disso, a natimortalidade foi maior (P<0,05) com o uso de carbetocina do que na indução somente com cloprostenol. No experimento II, não houve diferença na natimortalidade (P>0,05) entre os tratamentos. A utilização de ocitócitos, em associação com cloprostenol, resulta em partos antecipados e mais sincronizados. O uso associado de cloprostenol e carbetocina reduz o tempo de parto e 99 por cento ou mais dos partos ocorrem em até quatro horas após a aplicação de carbetocina, independentemente da dose utilizada.

The aim of the present study was to evaluate the effect of a synthetic analogue of PGF2 (sodium cloprostenol) associated to carbetocin or oxytocin on the efficiency of farrowing induction in swine. In Experiment I, 284 females were distributed in four treatments: - cloprostenol; - cloprostenol and 0.10mg of carbetocin; - cloprostenol and 10UI of oxytocin; and saline solution. In Experiment II, 276 females were distributed in four treatments: cloprostenol; cloprostenol and 0.10mg of carbetocin; cloprostenol and 0.05mg of carbetocin; and cloprostenol and 10UI of oxytocin. Farrowing induction was performed at 113 days of gestation using an injection of 0.175mg cloprostenol by vulvar submucosal route. Carbetocin or oxytocin was administered 24h after cloprostenol, by intramuscular route. The interval induction-farrowing was shorter (P<0.05) in groups with cloprostenol compared to the group without farrowing induction. Cumulative farrowing, within 26h and 28h after cloprostenol administration, was higher (P<0.05) when carbetocin or oxytocin was used. The use of carbetocin resulted in a shorter (P<0.05) farrowing length. In experiment I, higher stillbirth rate (P<0.05) was observed with the use of carbetocin or oxytocin compared to group without farrowing induction. Furthermore, stillbirth rate was higher (P<0.05) with cloprostenol + carbetocin than with cloprostenol alone. In experiment II, there was no difference (P>0.05) in stillbirth rate among treatments. The use of oxytocic drugs, in association with cloprostenol, results in anticipated and more synchronized farrowings. Following the use of carbetocin in association with cloprostenol, occurs a reduction in farrowing length and 99 percent or more of farrowings take place within 4h after carbetocin administration, regardless of the dose used.

Transmission of porcine circovirus 2 (PCV2) by semen and viral distribution in different piglet tissues

Porcine circovirus infections are caused by the porcine circovirus 2 (PCV2). Among six different clinical manifestations involving respiratory, enteric, nervous and reproductive signs, the postweaning multisystemic wasting syndrome (PMWS) is the most important and studied disease. However, reproductive failures associated with PCV2 have been increasingly reported. Some studies have shown the possible contamination of sows by semen of PCV2 positive boars. In order to investigate the transmission of PCV2 by contaminated semen and its ability to infect the sow and piglets, 20 PCV2 negative sows were inseminated, 10 with negative boar semen and 10 with previously nested-PCR tested positive boar semen. The sows were weekly monitored and blood samples were collected. Based on the results, 4 out 20 sows were selected (1 sow was PCR negative and inseminated with a negative semen, 2 sows were PCR negative and inseminated with a positive semen and 1 sow was PCR negative and inseminated with a positive semen, but became PCR positive around the 30 days of pregnancy). After weaning, 12 male piglets, 3 of each sow, were selected and maintained under isolation. In order to investigate which organs harbored the virus, the young pigs were necropsied around 9 months of age. Samples of serum collected monthly were tested by immunocitochemistry (ICC), and all 12 pigs serum converted. Samples of lymphoid, systemic and reproductive organs were analyzed by nested-PCR and immunohistochemistry (IHC). Evaluation of the samples by nested-PCR, revealed that several tissues were positive in 10 of 12 pigs, mainly the lymph nodes, bone marrow and spleen. Various samples were positive by IHC in 8 of 12 piglets, being the lymph nodes, tonsils and bulbourethral glands the most frequently positive. Thus, the results of testing different samples, in the 3 tests (ICC, nested-PCR and IHC) were complementary. These results show that PCV2 transmission through semen to the sows...

Identificação do circovírus suíno tipo 2 por reação em cadeia da polimerase e por imunoistoquímica em tecidos suínos arquivados desde 1988 no Brasil / Identification of porcine circovirus type 2 by polymerase chain reaction and immunohistochemistry on archived porcine tissues since 1988 in Brazil

A síndrome multissistêmica do definhamento dos suínos (SMDS) é uma doença de importância econômica causada pelo circovírus suíno tipo 2 (PCV2). Uma pesquisa retrospectiva foi realizada em amostras de órgãos de suínos fixados em blocos de parafina arquivados, que haviam sido submetidos à Embrapa Suínos e Aves entre 1985 e 1998 para o diagnóstico histopatológico. Vinte e cinco casos foram selecionados com base nas lesões histológicas características da SMDS, tais como linfoadenopatia, pneumonia intersticial, hepatite e nefrite intersticial. A presença de PCV2 nos cortes histológicos foi pesquisada por reação em cadeia da polimerase interna (nested-PCR), na qual utilizou-se primers específicos para a seqüência da ORF2 do PCV2 e também por imunoistoquímica, utilizando um anticorpo monoclonal específico para o capsídeo do PCV2. O DNA viral e os antígenos específicos do PCV2 foram detectados em amostras de tecidos de dois dos 25 casos analisados, sendo um desses datado de 1988. Esses resultados indicam que o PCV2 já estava presente no Brasil desde 1988.

Postweaning multisystemic wasting syndrome (PMWS) is of economical importance a disease caused by porcine circovirus type 2 (PCV2). A retrospective investigation was performed on paraffin-embedded organs samples from swine submitted to Embrapa Swine and Poultry Research Center between 1985 and 1998 for histopathologic diagnosis. A total of 25 cases were chosen from the archival collection of the Animal Health Laboratory at Embrapa Swine and Poultry based on characteristic pathological lesions of PMWS, such as lymphadenopathy, interstitial pneumonia, hepatitis and interstitial nephritis. The sections were investigated by nested-PCR (polymerase chain reaction) which used specific primers for the ORF2 sequence of the PCV2 and by immunohistochemistry using a monoclonal antibody specific for PCV2 capsid antigen. Virus specific DNA and antigen were detected in tissue samples of two out of 25 analyzed cases. The earliest positive sample originated from 1988. These results indicate that PCV2 is present in Brazil since 1988.