PESQUISA VETERINÁRIA BRASILEIRA

Abstract in English:

ABSTRACT.- Brandão L.N.S., Pitchenin L.C., Maruyama F.H., Chitarra C.S., Silva G.F.R., Klein C., Nakazato L. & Dutra V. 2014. [Standardization of unmodified gold nanoparticle (AuNPs) for detection of Actinobacillus pleuropneumoniae in swine lungs.] Padronização da técnica de nanopartícula de ouro não modificada (AuNPs) para detecção de Actinobacillus pleuropneumoniae em pulmões de suínos. Pesquisa Veterinária Brasileira 34(7):621-625. Laboratório de Microbiologia e Biologia Molecular, Universidade Federal do Mato Grosso, Av. Fernando Corrêa da Costa2367, Bairro Boa Esperança, Cuiabá, MT 78060-900, Brazil. E-mail: valdutra@ufmt.br Based on diagnostic tests for the detection of nucleic acids without amplification through the use of gold nanoparticles (AuNPs) have been described for various diseases. This study aimed to develop a technique of unmodified AuNPs to detect Actinobacillus pleuropneumoniae (App). We used 70 lung samples from pigs, 17 with and 53 without characteristic lesions of pneumonia, to detect App. The primer used was based on ApxIV gene. The AuNPs test had a sensitivity of 93.8% and specificity of 84.6% when compared with PCR detection. The results showed good agreement between AuNPs and PCR testing, and the technique can be used as an alternative to conventional tests, since it is quick and easy, and does not require implementation infrastructure and skilled labor.

• Pneumonia gold nanoparticle Actinobacillus pleuropneumoniae pneumonia suína nanopartícula de ouro

Abstract in Portuguese:

RESUMO.- Brandão L.N.S., Pitchenin L.C., Maruyama F.H., Chitarra C.S., Silva G.F.R., Klein C., Nakazato L. & Dutra V. 2014. [Standardization of unmodified gold nanoparticle (AuNPs) for detection of Actinobacillus pleuropneumoniae in swine lungs.] Padronização da técnica de nanopartícula de ouro não modificada (AuNPs) para detecção de Actinobacillus pleuropneumoniae em pulmões de suínos. Pesquisa Veterinária Brasileira 34(7):621-625. Laboratório de Microbiologia e Biologia Molecular, Universidade Federal do Mato Grosso, Av. Fernando Corrêa da Costa2367, Bairro Boa Esperança, Cuiabá, MT 78060-900, Brazil. E-mail: valdutra@ufmt.br Testes diagnósticos baseados na detecção de ácidos nucleicos sem amplificação prévia através da utilização de nanopartículas de ouro (AuNPs) têm sido descritos para várias enfermidades. Este trabalho teve como objetivo desenvolver uma técnica de AuNPs não modificada para detecção de Actinobacillus pleuropneumoniae (App). Utilizaram-se 70 amostras de pulmão de suínos, 17 sem lesão e 53 com lesões características de pneumonia, objetivando a detecção de App. O oligonucleotídeo utilizado foi baseado no gene ApxIV. O teste de AuNPs apresentou sensibilidade de 93,8% e especificidade de 84,6% quando comparado com a detecção pela PCR. Os resultados mostraram boa concordância entre os testes de AuNPs e a PCR, sendo que a técnica pode ser utilizada como alternativa aos testes convencionais, já que é de fácil e rápida execução e não exige infraestrutura e mão de obra especializada.

Abstract in English:

ABSTRACT.- Ji H.W., Li H.T., Zhu L., Zhang H., Wang Y., Zuo Z.C., Guo W.Z. & Xu Z.W. 2012. Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Actinobacillus pleuropneumoniae based the dsbE-like gene. Pesquisa Veterinária Brasileira 32(8):757-760. Key Laboratory of Animal Biotechnology, Center of Sichuan Province, College of Veterinary Medicine, Sichuan Agricultural University, Ya’an, Sichuan, 625014, P.R. China. E-mail: abtcxzw@126.com This paper reports on the development and validation of a loop-mediated isothermal amplification assay (LAMP) for the rapid and specific detection of Actinobacillus pleuropneumoniae (A. pleuropneumoniae). A set of six primers were designed derived from the dsbE-like gene of A.pleuropneumoniae and validate the assay using 9 A. pleuropneumoniae reference/field strains, 132 clinical isolates and 9 other pathogens. The results indicated that positive reactions were confirmed for all A. pleuropneumoniae strains and specimens by LAMP at 63°C for 60 min and no cross-reactivity were observed from other non-A.pleuropneumoniae including Haemophilus parasuis, Escherichia coli, Pasteurella multocida, Bordetella bronchiseptica, Streptococcus suis, Salmonella enterica, Staphylococcus, porcine reproductive and respiratory syndrome virus (PRRSV), and Pseudorabies virus. The detection limit of the conventional PCR was 102 CFU per PCR test tube, while that of the LAMP was 5 copies per tube. Therefore, the sensitivity of LAMP was higher than that of PCR. Moreover, the LAMP assay provided a rapid yet simple test of A. pleuropneumoniae suitable for laboratory diagnosis and pen-side detection due to ease of operation and the requirement of only a regular water bath or heat block for the reaction.

• Actinobacillus pleuropneumoniae lamp

Abstract in Portuguese:

RESUMO.- Ji H.W., Li H.T., Zhu L., Zhang H., Wang Y., Zuo Z.C., Guo W.Z. & Xu Z.W. 2012. Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Actinobacillus pleuropneumoniae based the dsbE-like gene. Pesquisa Veterinária Brasileira 32(8):757-760. Key Laboratory of Animal Biotechnology, Center of Sichuan Province, College of Veterinary Medicine, Sichuan Agricultural University, Ya’an, Sichuan, 625014, P.R. China. E-mail: abtcxzw@126.com This paper reports on the development and validation of a loop-mediated isothermal amplification assay (LAMP) for the rapid and specific detection of Actinobacillus pleuropneumoniae (A. pleuropneumoniae). A set of six primers were designed derived from the dsbE-like gene of A.pleuropneumoniae and validate the assay using 9 A. pleuropneumoniae reference/field strains, 132 clinical isolates and 9 other pathogens. The results indicated that positive reactions were confirmed for all A. pleuropneumoniae strains and specimens by LAMP at 63°C for 60 min and no cross-reactivity were observed from other non-A.pleuropneumoniae including Haemophilus parasuis, Escherichia coli, Pasteurella multocida, Bordetella bronchiseptica, Streptococcus suis, Salmonella enterica, Staphylococcus, porcine reproductive and respiratory syndrome virus (PRRSV), and Pseudorabies virus. The detection limit of the conventional PCR was 102 CFU per PCR test tube, while that of the LAMP was 5 copies per tube. Therefore, the sensitivity of LAMP was higher than that of PCR. Moreover, the LAMP assay provided a rapid yet simple test of A. pleuropneumoniae suitable for laboratory diagnosis and pen-side detection due to ease of operation and the requirement of only a regular water bath or heat block for the reaction.

Abstract in English:

Thirty-three strains of Haemophilus (Actinobacillus) pleuropneumoniae biovar 1 were isolated from swine lungs with acute or chronic lesions of swine pleuropneumonia, with one exception, which was isolated from the nasal cavity of a pig from a problem herd. These strains originated from 14 herds, ten located in the State of Santa Catarina, three in Rio Grande do Sul and one in São Paulo. Serotyping was done by rapid slide agglutination (RSA), immunofluorescence (IF), counterimmunoelectrophoresis (CIE) and immunodiffusion (ID). Twenty-two (66%) of the 33 strains could be serotyped, 19 belonged to serotype 5 and three to serotype 3. All serotype 3 strains together with those not possible to be serotyped originated from swine of the sarne herd, Santa Catarina, one in which serotype 5 was also isolated. The IF and CIE were the most sensitive tests, while the ID was the least sensitive. Three strains could not be serotyped by the RSA because they auto-agglutinated.

• Swine pleuropneumonia serotypes Haemophilus pleuropneumoniae Actinobacillus pleuropneumoniae Brazil

Abstract in Portuguese:

Isolaram-se 33 amostras de Haemophilus (Actinobacillus) pleuropneumoniae, biovar 1, sendo que 32 originaram-se de pulmões de suínos com lesões macroscópicas agudas ou crônicas de Pleuropneumonia Suína (PPS) e uma amostra da cavidade nasal de um suíno de granja problema. Estas amostras foram coletadas em 14 propriedades, sendo dez localizadas no Estado de Santa Catarina, três no Rio Grande do Sul e uma em São Paulo. Para a sorotipagem utilizaram-se as técnicas de soroaglutinação rápida (SAR), imunofluorescência (IF), imunoeletroosmoforese (IEOF) e imunodifusão (ID). Das 33 amostras, 22 (66%) foram sorotipadas e, entre estas, 19 pertenciam ao sorotipo 5 e três ao sorotipo 3. As amostras do sorotipo 3 e aquelas não sorotipadas originaram-se de suínos de uma mesma propriedade do Estado de Santa Catarina, onde também isolou-se o sorotipo 5. Os testes de IF e IEOF foram os mais sensíveis e o de ID apresentou a menor sensibilidade. Três amostras não puderam ser sorotipadas pelo teste de SAR porque auto-aglutinavam.

• Pleuropneumonia suína sorotipos Haemophilus pleuropneumoniae Actinobacillus pleuropneumoniae Brasil