PESQUISA VETERINÁRIA BRASILEIRA

Abstract in English:

ABSTRACT.- Casagrande R.A., Machado G., Guerra P.R., Castro L.A., Spanamberg A., Silva S.C., Cardoso M.R.I. & Driemeier D. 2017. [Pathological and bacteriological characterization on broilers totally condemned due to colibacillosis under the control of the Federal Inspection Service.] Caracterização anatomopatológica e bacteriológica em frangos de corte condenados totalmente por colibacilose sob Serviço de Inspeção Federal. Pesquisa Veterinária Brasileira 37(9):949-957. Setor de Patologia Veterinária, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9090, Bairro Agronomia, Porto Alegre, RS 91540-000, Brazil. E-mail: davetpat@ufrgs.br Colibacillosis is the main infectious cause of total carcass condemnation in broilers in southern Brazil. This study aims to determine the degree of agreement between the total carcass condemnation for colibacillosis in broilers slaughtered in establishments under Federal Inspection Service (SIF) with the pathological and bacteriological diagnosis. The study was conducted with 45 broilers totally condemned by colibacillosis (case) and theirs 45 respective controls (chickens without lesions). All broilers condemned had gross lesions and the controls had not. The Kappa-Cohen’s test showed that these two variables had almost perfect agreement. Broilers condemned showed lesions in liver (27/45); liver and air sacs (11/45); liver and heart (2/45); liver, heart and air sacs (2/45); liver, air sacs and oviduct (1/45); liver, air sacs, heart and subcutaneous (1/45); and liver, air sacs, oviduct and spleen (1/45). There is almost perfect agreement between carcass condemnation and liver damage. Histologically, in 41 cases and 12 controls were observed lesions, the most frequent diagnoses were random necrotizing hepatitis, fibrinous-heterophilic bronchitis, acute pericarditis and lymphoplasmacytic tracheitis. In hepatitis cases was isolated Escherichia coli, Enterococcus sp. and Streptococcus sp. (10/38) and in bronchitis or bronchopneumonia E. coli and coagulase positive Staphylococcus (9/14). The polymerse chain reaction (PCR) and immunohistochemistry (IHC) tests for Mycoplasma gallisepticum and M. synoviae were negative. In cases of total carcass condemnation by Colibacillosis the liver was the main organ affected. Therefore, the condemnation criteria should be revised, suggesting conviction for hepatitis in these cases, because other bacteria can cause hepatitis, as demonstrated in this study.

• Broiler colibacilosis condemnation poutry frangos de corte colibacilose condenação ave

Abstract in Portuguese:

RESUMO.- Casagrande R.A., Machado G., Guerra P.R., Castro L.A., Spanamberg A., Silva S.C., Cardoso M.R.I. & Driemeier D. 2017. [Pathological and bacteriological characterization on broilers totally condemned due to colibacillosis under the control of the Federal Inspection Service.] Caracterização anatomopatológica e bacteriológica em frangos de corte condenados totalmente por colibacilose sob Serviço de Inspeção Federal. Pesquisa Veterinária Brasileira 37(9):949-957. Setor de Patologia Veterinária, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9090, Bairro Agronomia, Porto Alegre, RS 91540-000, Brazil. E-mail: davetpat@ufrgs.br A colibacilose é a principal causa infecciosa de condenação total de carcaça em frangos de corte no sul do Brasil. Esse trabalho tem por objetivo determinar o grau de concordância entre a condenação total por colibacilose de frangos de corte abatidos em estabelecimento sob Serviço de Inspeção Federal (SIF) com o diagnóstico anatomopatológico e bacteriológico. O estudo foi realizado com 45 frangos de corte condenados totalmente por colibacilose (caso) e seus respectivos 45 controles (frangos sem lesões). Em todos os frangos condenados pelo SIF havia lesões macroscópicas e, nos controles não se observou. Através do teste Kappa-Cohen´s essas duas variáveis apresentaram concordância quase perfeita. As aves condenadas apresentaram lesões em fígado (27/45); em fígado e sacos aéreos (11/45); em fígado e coração (2/45); fígado, sacos aéreos e coração (2/45); fígado, sacos aéreos e oviduto (1/45); fígado, sacos aéreos, coração e tecido subcutâneo (1/45); e fígado, sacos aéreos, oviduto e baço (1/45). Observou-se concordância quase perfeita entre condenação e lesão hepática. Histologicamente, em 41 casos e 12 controles observaram-se lesões, sendo os mais frequentes hepatite necrosante aleatória, bronquite fibrino-heterofílica, pericardite aguda e traqueíte linfoplasmocitária. Nas aves com hepatite identificou-se E. coli, Enterococcus sp. e Streptococcus sp. (10/38) e, nas aves com bronquite ou broncopneumonia isolou-se Escherichia coli e Staphylococcus coagulase positiva (9/14). O PCR em tempo real e a imuno-histoquímica para Mycoplasma gallisepticum e M. synoviae foram negativos. Nos casos de condenação total por colibacilose o fígado foi o principal órgão acometido, portanto, o critério de condenação deveria ser revisto, sugerindo condenação por hepatite nesses casos, já que outras bactérias podem causar hepatite, como foi demonstrado nesse estudo.

Abstract in English:

ABSTRACT.- Almeida P.R., Andrade C.P., Almeida L.L., Oliveira L.G.S., Castro L.A., Zlotowski P., Silva S.C. & Driemeier D. 2012. Nested-PCR for the detection of Mycoplasma hyopneumoniae in bronchial alveolar swabs, frozen tissues and formalin-fixed paraffin-embedded swine lung samples: Comparative evaluation with immunohistochemical findings and histological features. Pesquisa Veterinária Brasileira 32(8):715-720. Setor de Patologia Veterinária, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9090, Porto Alegre, RS 91540-000, Brazil. E-mail: davetpat@ufrgs.br. The diagnosis of Mycoplasma hyopneumoniae infection is often performed through histopathology, immunohistochemistry (IHC) and polymerase chain reaction (PCR) or a combination of these techniques. PCR can be performed on samples using several conservation methods, including swabs, frozen tissue or formalin-fixed and paraffin-embedded (FFPE) tissue. However, the formalin fixation process often inhibits DNA amplification. To evaluate whether M. hyopneumoniae DNA could be recovered from FFPE tissues, 15 lungs with cranioventral consolidation lesions were collected in a slaughterhouse from swine bred in herds with respiratory disease. Bronchial swabs and fresh lung tissue were collected, and a fragment of the corresponding lung section was placed in neutral buffered formalin for 48 hours. A PCR assay was performed to compare FFPE tissue samples with samples that were only refrigerated (bronchial swabs) or frozen (tissue pieces). M. hyopneumoniae was detected by PCR in all 15 samples of the swab and frozen tissue, while it was detected in only 11 of the 15 FFPE samples. Histological features of M. hyopneumoniae infection were presented in 11 cases and 7 of these samples stained positive in IHC. Concordance between the histological features and detection results was observed in 13 of the FFPE tissue samples. PCR was the most sensitive technique. Comparison of different sample conservation methods indicated that it is possible to detect M. hyopneumoniae from FFPE tissue. It is important to conduct further research using archived material because the efficiency of PCR could be compromised under these conditions.

• Mycoplasma hyopneumoniae

Abstract in Portuguese:

RESUMO.- Almeida P.R., Andrade C.P., Almeida L.L., Oliveira L.G.S., Castro L.A., Zlotowski P., Silva S.C. & Driemeier D. 2012. Nested-PCR for the detection of Mycoplasma hyopneumoniae in bronchial alveolar swabs, frozen tissues and formalin-fixed paraffin-embedded swine lung samples: Comparative evaluation with immunohistochemical findings and histological features. Pesquisa Veterinária Brasileira 32(8):715-720. Setor de Patologia Veterinária, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9090, Porto Alegre, RS 91540-000, Brazil. E-mail: davetpat@ufrgs.br. O diagnóstico de infecção por Mycoplasma hyopneumoniae é frequentemente realizado através de histopatologia, imuno-histoquímica (IHQ) e reação em cadeia da polimerase (PCR), ou uma combinação dessas técnicas. PCR pode ser realizada a partir de amostras submetidas a vários métodos de conservação, incluindo swabs, tecido refrigerado ou congelado, ou ainda tecido fixado em formalina e embebido em parafina (FFEP). Entretanto, o processo de fixação em formalina pode inibir a amplificação de DNA. Para avaliar se DNA de M. hyopneumoniae poderia ser recuperado de tecido FFEP, 15 pulmões com lesões de consolidação crânio-ventral de suínos oriundos de rebanhos com problemas respiratórios foram selecionados no abatedouro. Swabs bronquiais e pulmão fresco foram colhidos, e um fragmento da mesma porção de pulmão foi colocado por 48 horas em solução de formalina tamponada e posteriormente processado e embebido em parafina. PCR foi realizada comparando amostras de tecido fixado em formalina com amostras que passaram somente por refrigeração (swab bronquial) ou foram congeladas (fragmentos de tecido). A detecção de M. hyopneumoniae ocorreu em todas as 15 amostras de swabs e tecido congelado enquanto em amostras de tecido FFEP, o agente foi detectado somente em 11 das 15 amostras. Características histológicas de infecção por M. hyopneumoniae ocorreram em 11 casos e 7 destas amostras obtiveram marcação imuno-histoquímica positiva. Concordância entre histologia e detecção a partir de tecido FFEP foi observada em 13 casos. Dentre as técnicas analisadas, a PCR foi a mais sensível. A comparação de diferentes métodos de conservação de amostras indica que é possível detectar M. hyopneumoniae a partir de tecido FFEP, fato importante para pesquisa utilizando material arquivado, porém a eficácia do teste de PCR pode ficar comprometida sob essas condições.

Abstract in English:

ABSTRACT.- Andrade C.P. de, Almeida L.L., Castro L.A., Leal J.S., Silva S.C. & Driemeier D. 2011. Single nucleotide polymorphisms at 15 codons of the prion protein gene from a scrapie-affected herd of Suffolk sheep in Brazil. Pesquisa Veterinária Brasileira 31(10):893-898. Setor de Patologia Veterinária, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9090, Porto Alegre, RS 91540-000, Brazil. E-mail: davetpat@ufrgs.br Scrapie is a transmissible spongiform encephalopathy of sheeps and goats, associated with the deposition of a isoform of the prion protein (PrPsc). This isoform presents an altered conformation that leads to aggregation in the host’s central nervous and lymphoreticular systems. Predisposition to the prion agent infection can be influenced by specific genotypes related to mutations in amino acids of the PrPsc gene. The most characterized mutations occur at codons 136, 154 and 171, with genotypes VRQ being the most susceptible and ARR the most resistant. In this study we have analyzed polymorphisms in 15 different codons of the PrPsc gene in sheeps from a Suffolk herd from Brazil affected by an outbreak of classical scrapie. Amplicons from the PrPsc gene, encompassing the most relevant altered codons in the protein, were sequenced in order to determine each animal’s genotype. We have found polymorphisms at 3 of the 15 analyzed codons (136, 143 and 171). The most variable codon was 171, where all described alleles were identified. A rare polymorphism was found at the 143 codon in 4% of the samples analyzed, which has been described as increasing scrapie resistance in otherwise susceptible animals. No other polymorphisms were detected in the remaining 12 analyzed codons, all of them corresponding to the wild-type prion protein. Regarding the risk degree of developing scrapie, most of the animals (96%) had genotypes corresponding to risk groups 1 to 3 (very low to moderate), with only 4% in the higher risks group. Our data is discussed in relation to preventive measures involving genotyping and positive selection to control the disease.

• Spongiform encephalopathy Prion protein scrapie encefalopatia espongiforme proteína priônica

Abstract in Portuguese:

RESUMO.- Andrade C.P. de, Almeida L.L., Castro L.A., Leal J.S., Silva S.C. & Driemeier D. 2011. Single nucleotide polymorphisms at 15 codons of the prion protein gene from a scrapie-affected herd of Suffolk sheep in Brazil. [Polimorfismos de nucleotídeos únicos em 15 códons do gene da proteína priônica em um rebanho Suffolk afetado com scrapie no Brasil.] Pesquisa Veterinária Brasileira 31(10):893-898. Setor de Patologia Veterinária, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9090, Porto Alegre, RS 91540-000, Brazil. E-mail: davetpat@ufrgs.br Scrapie é uma encefalopatia espongiforme transmissível de ovinos e caprinos, associado a deposição da isoforma da proteína priônica (PrPsc). Essa isoforma apresenta uma alteração conformacional que leva ao acúmulo da proteína no sistema nervoso central e linforeticular do hospedeiro. A predisposição a infecção pelo agente priônico pode ser influenciado por genótipos específicos relacionados a mutações na sequência de aminoácidos do gene PrPsc. As principais mutações caracterizadas ocorrem nos códons 136, 154 e 171, sendo o genótipo VRQ o mais suscetível e o genótipo ARR o mais resistente. Nesse estudo nós analisamos os polimorfismos de 15 códons diferentes da gene PrPsc em ovinos de um rebanho da raça Suffolk no Brasil afetado com scrapie clássico. Os amplicons do gene da PrPsc, que contem os códons mais frequentemente encontrados foram sequenciados para determinar o genótipo de cada animal. Nós encontramos 3 polimorfismos do 15 códons analisados (136, 143 e 171). O códon que mais teve variações foi o códon 171, onde todos os alelos foram identificados. Um polimorfismo raro foi encontrado no códon 143, em 4% das amostras analisadas, o qual tem sido descrito por aumentar a resistência a scrapie em animais suscetíveis. Nenhum outro polimorfismo foi detectado nos 12 códons restantes, todos então, correspondendo à proteína priônica selvagem. De acordo com a grau de risco a desenvolver scrapie, a maioria dos animais (96%) tiveram genótipo correspondentes aos grupos de risco 1 a 3 (muito baixo a moderado), e somente 4% no grupo de risco alto. Nossos dados discutem a relação das medidas de prevenção envolvendo a genotipagem e a seleção positiva para o controle da doença.

Abstract in English:

ABSTRACT.- Silva Júnior A., Castro L.A., Chiarelli Neto O., Silva F.M.F., Vidigal P.M.P., Moraes M.P. & Almeida M.R. 2009. Development and evaluation of a recombinant DNA vaccine candidate expressing porcine circovirus 2 structural protein. Pesquisa Veterinária Brasileira 29(1):76-82. Laboratório de Infectologia Molecular Animal, Instituto de Biotecnologia Aplicada à Agropecuária, Universidade Federal de Viçosa, Av. PH Rolfs s/n, Campus Universitário, Viçosa, MG 36570-000, Brazil. E-mail: marcia@ufv.br 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.

• PCV2 DNA vaccine porcine circovirosis

Abstract in Portuguese:

ABSTRACT.- Silva Júnior A., Castro L.A., Chiarelli Neto O., Silva F.M.F., Vidigal P.M.P., Moraes M.P. & Almeida M.R. 2009. Development and evaluation of a recombinant DNA vaccine candidate expressing porcine circovirus 2 structural protein. Pesquisa Veterinária Brasileira 29(1):76-82. Laboratório de Infectologia Molecular Animal, Instituto de Biotecnologia Aplicada à Agropecuária, Universidade Federal de Viçosa, Av. PH Rolfs s/n, Campus Universitário, Viçosa, MG 36570-000, Brazil. E-mail: marcia@ufv.br 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.

Abstract in English:

Abstract.- Pescador C.A., Bandarra P.M., Castro L.A., Antoniassi N.A.B., Ravazollo, A.P., Sonne L. Cruz C.E.F. & Driemeier D. 2007. Co-infection by porcine circovirus type 2 and porcine parvovirus in aborted fetuses and stillborn piglets in southern Brazil. Pesquisa Veterinária Brasileira 27(10):425-429. Departamento de Patologia Clínica Veterinária, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9090, Porto Alegre, RS 91540-000, Brazil. E-mail: davetpat@ufrgs.br Porcine circovirus types 1 and 2 (PCV1, PCV2) and porcine parvovirus (PPV) are widespread in pig populations around the world. Nevertheless, only PCV2 has been associated with different clinical syndromes, thus representing a major problem to the pig industry. The association of cases of swine abortions and stillborns with PCV1 and PCV2 and PPV was studied retrospectively (2005-2007). Additional pathogens were also investigated in lesioned fetuses. The studied litters included stillborn piglets and several mummified fetuses of varied sizes. Ventricular dilatation, myocardial pale areas, and mesocolic edema were the gross lesions. Escherichia coli was detected as co-infecting with PCV2 the cases in which mesocolic edema was seen. Microscopic lesions included non-suppurative myocarditis, myocardial necrosis and fibrosis, mineralization foci and intranuclear inclusion bodies in cardiomyocytes, and interstitial mononuclear pneumonia. Samples from 7 (5.78 per cent) of 121 aborted fetuses and stillborn piglets had lesions consistent with a viral cause and showed both positive anti-PCV2 immunostaining as well as PCV2-PCR. In samples from 3 (2.47 per cent) of these 7 fetuses, co-infection with PPV was confirmed by Nested-PCR. Both viruses were detected in fetuses at different stages of gestation. Viral antigens of PCV2 were detected by immunohistochemistry mainly in macrophages and myocytes. PCV1 individually was not detected in any of these affected fetuses, but it was associated with PCV2 and/or PPV in some of them. These findings indicate that PCV2 alone or in association with PPV should be kept in mind when investigating causes of infectious abortion in pigs in Brazil.

• Abortion PCV2 PCV1 PPV stillborn swine Brazil

Abstract in Portuguese:

Abstract.- Pescador C.A., Bandarra P.M., Castro L.A., Antoniassi N.A.B., Ravazollo, A.P., Sonne L. Cruz C.E.F. & Driemeier D. 2007. Co-infection by porcine circovirus type 2 and porcine parvovirus in aborted fetuses and stillborn piglets in southern Brazil. Pesquisa Veterinária Brasileira 27(10):425-429. Departamento de Patologia Clínica Veterinária, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9090, Porto Alegre, RS 91540-000, Brazil. E-mail: davetpat@ufrgs.br Porcine circovirus types 1 and 2 (PCV1, PCV2) and porcine parvovirus (PPV) are widespread in pig populations around the world. Nevertheless, only PCV2 has been associated with different clinical syndromes, thus representing a major problem to the pig industry. The association of cases of swine abortions and stillborns with PCV1 and PCV2 and PPV was studied retrospectively (2005-2007). Additional pathogens were also investigated in lesioned fetuses. The studied litters included stillborn piglets and several mummified fetuses of varied sizes. Ventricular dilatation, myocardial pale areas, and mesocolic edema were the gross lesions. Escherichia coli was detected as co-infecting with PCV2 the cases in which mesocolic edema was seen. Microscopic lesions included non-suppurative myocarditis, myocardial necrosis and fibrosis, mineralization foci and intranuclear inclusion bodies in cardiomyocytes, and interstitial mononuclear pneumonia. Samples from 7 (5.78 per cent) of 121 aborted fetuses and stillborn piglets had lesions consistent with a viral cause and showed both positive anti-PCV2 immunostaining as well as PCV2-PCR. In samples from 3 (2.47 per cent) of these 7 fetuses, co-infection with PPV was confirmed by Nested-PCR. Both viruses were detected in fetuses at different stages of gestation. Viral antigens of PCV2 were detected by immunohistochemistry mainly in macrophages and myocytes. PCV1 individually was not detected in any of these affected fetuses, but it was associated with PCV2 and/or PPV in some of them. These findings indicate that PCV2 alone or in association with PPV should be kept in mind when investigating causes of infectious abortion in pigs in Brazil.