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Development of a lateral flow test for the rapid detection of Avibacterium paragallinarum in chickens suspected of having infectious coryza.

ABSTRACT: BACKGROUND:Infectious coryza (IC) is an acute respiratory disease of growing chickens and layers caused by Avibacterium paragallinarum. The development of tools that allow rapid pathogen detection is necessary in order to avoid disease dissemination and economic losses in poultry. An Av. paragallinarum-specific Ma-4 epitope of the TonB-dependent transporter (TBDT) was selected using bioinformatic tools in order to immunize a BalbC mouse and to produce monoclonal antibodies to be used in a lateral flow test (LFT) developed for Av. paragallinarum detection in chicken nasal mucus samples. RESULTS:The 1G7G8 monoclonal antibody was able to detect TBDT in Av. paragallinarum cultures (serogroups: A, B and C) by Western blot and indirect ELISA assay. Consequently, we developed a self-pairing prototype LFT. The limit of detection of the prototype LFT using Av. paragallinarum cultures was 1?×?104 colony-forming units (CFU)/mL. Thirty-five nasal mucus samples from chickens suspected of having infectious coryza were evaluated for the LFT detection capacity and compared with bacterial isolation (B.I) and polymerase chain reaction (PCR). Comparative indicators such as sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive values (NPV) and the kappa index (K) were obtained. The values were 100.0% Se, 50% Sp, 65.4% PPV, 100% NPV, and 0.49?K and 83.9% Se, 100% Sp, 100% PPV, 44.4% NPV, and 0.54?K for the comparison of the LFT with B.I and PCR, respectively. Additionally, the LFT allowed the detection of Av. paragallinarum from coinfection cases of Av. paragallinarum with Gallibacterium anatis. CONCLUSIONS:The results indicate that the self-pairing prototype LFT is suitable for the detection of TBDT in Av. paragallinarum cultures as well as in field samples such as nasal mucus from Av. paragallinarum-infected chickens. Therefore, this prototype LFT could be considered a rapid and promising tool to be used in farm conditions for Av. paragallinarum diagnosis.

SUBMITTER: Morales Ruiz S

PROVIDER: S-EPMC6300026 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Development of a lateral flow test for the rapid detection of Avibacterium paragallinarum in chickens suspected of having infectious coryza.

Morales Ruiz Sandra S Bendezu Jorge J Choque Guevara Ricardo R Montesinos Ricardo R Requena David D Choque Moreau Luz L Montalván Ávalos Ángela Á Fernández-Díaz Manolo M

BMC veterinary research 20181219 1

<h4>Background</h4>Infectious coryza (IC) is an acute respiratory disease of growing chickens and layers caused by Avibacterium paragallinarum. The development of tools that allow rapid pathogen detection is necessary in order to avoid disease dissemination and economic losses in poultry. An Av. paragallinarum-specific Ma-4 epitope of the TonB-dependent transporter (TBDT) was selected using bioinformatic tools in order to immunize a BalbC mouse and to produce monoclonal antibodies to be used in ...[more]

PMID: 30567563

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Project description:Avibacterium paragallinarum is the pathogen involved in infectious coryza (IC), an acute infectious upper respiratory disease in chickens. The prevalence of IC has increased in China in recent years. There is a lack of reliable and effective procedures for gene manipulation, which has limited the research on the bacterial genetics and pathogenesis of A. paragallinarum. Natural transformation has been developed as a method of gene manipulation in Pasteurellaceae by the introduction of foreign genes or DNA fragments into bacterial cells, but there has been no report on natural transformation in A. paragallinarum. In this study, we analyzed the existence of homologous genetic factors and competence proteins underlying natural transformation in A. paragallinarum and established a method for transformation in it. Through bioinformatics analysis, we identified 16 homologs of Haemophilus influenzae competence proteins in A. paragallinarum. We found that the uptake signal sequence (USS) was overrepresented in the genome of A. paragallinarum (1,537 to 1,641 copies of the core sequence ACCGCACTT). We then constructed a plasmid, pEA-KU, that carries the USS and a plasmid, pEA-K, without the USS. These plasmids can be transferred via natural transformation into naturally competent strains of A. paragallinarum. Significantly, the plasmid that carries USS showed a higher transformation efficiency. In summary, our results demonstrate that A. paragallinarum has the ability to undergo natural transformation. These findings should prove to be a valuable tool for gene manipulation in A. paragallinarum. IMPORTANCE Natural transformation is an important mechanism for bacteria to acquire exogenous DNA molecules during the process of evolution. Additionally, it can also be used as a method to introduce foreign genes into bacteria under laboratory conditions. Natural transformation does not require equipment such as an electroporation apparatus. It is easy to perform and is similar to gene transfer under natural conditions. However, there have been no reports on natural transformation in Avibacterium paragallinarum. In this study, we analyzed the presence of homologous genetic factors and competence proteins underlying natural transformation in A. paragallinarum. Our results indicate that natural competence could be induced in A. paragallinarum serovars A, B, and C. Furthermore, the method that we established to transform plasmids into naturally competent A. paragallinarum strains was stable and efficient.

Dataset Information

Development of a lateral flow test for the rapid detection of Avibacterium paragallinarum in chickens suspected of having infectious coryza.

Publications

Development of a lateral flow test for the rapid detection of Avibacterium paragallinarum in chickens suspected of having infectious coryza.

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