Identification of the capsule type of Pasteurella multocida isolates from cases of fowl cholera by multiplex PCR and comparison with phenotypic methods

Furian, Thales Quedi; Borges, Karen Apellanis; Pilatti, RM; Almeida, Carlos Podalírio Borges de; Nascimento, Vladimir Pinheiro do; Salle, Carlos Tadeu Pippi; Moraes, HL de S

doi:10.1590/1516-635x160231-36

Cited by 14 publications

(15 citation statements)

References 22 publications

(32 reference statements)

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“…An aliquot of BHI after an overnight incubation (1 mL) was selected for DNA extraction using the commercial kit NucleoSpin ® Tissue (MachereyNagel ® , Düren, Germany). Initially, a PCR protocol for species-specific amplification of the kmt gene was performed, as described by Townsend et al 15 Fifteen genes associated with virulence ( ompH , oma87 , sodA , sodC , hgbA , hgbB , exBD-tonB , nanB , nanH , ptfA , pfhA , toxA ), including the genes for capsule molecular typing of serogroup A ( hyaD-hyaC ), B ( bcbD ) and D ( dcbF ), were surveyed according to the multiplex PCR protocols standardized by Furian et al 16 , 17 An additional seven genes ( hsf-1 , pmHAS , fur , psl , ompA , plpB , tadD ) were surveyed according to protocols described by Ewers et al 4 and Tang et al 6 with modifications. Reference strains of P. multocida (ATCC 15742, ATCC 12945 and ATCC 12946) were selected as positive controls.…”

Section: Methodsmentioning

confidence: 99%

Virulence genes and antimicrobial resistance of Pasteurella multocida isolated from poultry and swine

Furian

Borges

Laviniki

et al. 2016

Brazilian Journal of Microbiology

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Pasteurella multocida causes atrophic rhinitis in swine and fowl cholera in birds, and is a secondary agent in respiratory syndromes. Pathogenesis and virulence factors involved are still poorly understood. The aim of this study was to detect 22 virulence-associated genes by PCR, including capsular serogroups A, B and D genes and to evaluate the antimicrobial susceptibility of P. multocida strains from poultry and swine. ompH, oma87, plpB, psl, exbD-tonB, fur, hgbA, nanB, sodA, sodC, ptfA were detected in more than 90% of the strains of both hosts. 91% and 92% of avian and swine strains, respectively, were classified in serogroup A. toxA and hsf-1 showed a significant association to serogroup D; pmHAS and pfhA to serogroup A. Gentamicin and amoxicillin were the most effective drugs with susceptibility higher than 97%; however, 76.79% of poultry strains and 85% of swine strains were resistant to sulphonamides. Furthermore, 19.64% and 36.58% of avian and swine strains, respectively, were multi-resistant. Virulence genes studied were not specific to a host and may be the result of horizontal transmission throughout evolution. High multidrug resistance demonstrates the need for responsible use of antimicrobials in animals intended for human consumption, in addition to antimicrobial susceptibility testing to P. multocida.

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Section: Methodsmentioning

confidence: 99%

Virulence genes and antimicrobial resistance of Pasteurella multocida isolated from poultry and swine

Furian

Borges

Laviniki

et al. 2016

Brazilian Journal of Microbiology

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“…Also, PCR is used to accurate, rapid detection of toxigenic P. Multocida from swabs and tissues (Carol et al, 1996). Multiplex PCR is an alternative to comparative phenotypic tests for the capsular typing of P.multocida , for simultaneous and rapid detection of genes that provides a greater capacity for strain typing (Furian et al, 2014). Therefore, Pasteurellosis is considered an important disease in camels due to its higher economic losses.…”

Section: Introductionmentioning

confidence: 99%

Pasteurella multocida in camels: incidence, capsular and virulence genes characterization

Tawab

Hofy

Al-Jeddawy

et al. 2016

Benha Veterinary Medical Journal

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Pasteurella multocida is the main cause of hemorrhagic septicemia in camels. This study deals with the isolation and molecular examination of hemorrhagic septicemia in camels from May 2014 to March 2016 from 30 camel nasal swabs in Marsa Matruh and 120 camel lungs (70 slaughtered in Basateen abattoir in Giza Governorate and 50 slaughtered in Al-Shohada abattoir at Al-Menofia Governorate). All collected samples were subjected to clinical, postmortem examination as well as for bacteriological and molecular examination. Totally P. multocida was isolated from the examined samples with percentage of 5(3.3%). While the percentage of the isolation rate from 120 camel lungs was 5(4.2%). In contrast, all 30 nasal swabs were negative. In the pathogenicity test, all P.multocida isolates were highly pathogenic. Pasteurella multocida isolates were identified by PCR and 23 S RNA gene was amplified at 1432bp. Three out of five isolates were identified as P.multocida type B with amplification at 760bp while other two isolates identified as P.multocida type A and amplified at 1044bp. Also, PCR showed that toxA gene was amplified in all isolates and giving product of 864bp but ptfA gene was not detected. As conclusion, P.multocida in camels can be diagnosed with different methods such as confirmatory biochemical and molecular assays.

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“…However, in that study, unlike our study, the strains classified as untypable were negative for both the hyaluronidase and acriflavine tests. Similarly, it was found that 11 of 54 (20.37%) strains isolated during a fowl cholera outbreak in Brazil were untypable using these phenotypic tests [7].…”

Section: Discussionmentioning

confidence: 96%

Comparison of Phenotypic and Genotypic Identification Methods of Pasteurella multocida Serotypes Isolated from Pigs

Amaral

Rebelatto

Klein

et al. 2016

Acta Scientiae. Vet.

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Background: Pasteurella multocida serotypes A and D are commonly associated with pneumonia and pleuritis in pigs. Different phenotypic techniques, such as hyaluronidase and acriflavine tests, and genotyping techniques, such as PCR, are used to distinguish between these serotypes. The objective of this study was to compare the capsular identification methods of type A and type D P. multocida isolated from pigs using both phenotypic (hyaluronidase and acriflavine tests) and genotypic (multiplex PCR) techniques.Materials, Methods & Results: A total of 44 lyophilized P. multocida isolates, obtained between 1981 and 1997 from pig farms at Rio Grande do Sul State, Brazil, were analyzed. The isolates were reactivated in Brain Heart Infusion (BHI) broth and cultured in BHI broth and blood agar supplemented with 5% sheep blood. Colony identity was further confirmed by evaluating colony morphology in blood agar and confirming the absence of growth on MacConkey agar. Bacteria in Tryptone Soy Agar (TSA) were used for the Triple Sugar Iron (TSI), Sulfide-Indole-Motility (SIM), and nitrate, glucose, lactose, sucrose and mannitol fermentation tests. For hyaluronidase test, P. multocida colonies were streaked transversally across the entire plate, approximately 3-5mm apart, in order to observe their lines of growth. Following this, a hyaluronidase producing strain of Staphylococcus aureus was heavily streaked at right angles to the P. multocida lines and the plates were incubated at 37°C for 24 h. Type A isolates were then identified as those with smaller colonies in the region adjacent to the Staphylococcus aureus streak (negative satellitism). For acriflavine test, the isolates were inoculated into tubes containing 2 mL of BHI, incubated at 37°C for 18-24 h, centrifuged (500 g for 15 min) and 1.5 mL of the supernatant was discarded. A 1:1000 solution of acriflavine neutral (0.5 mL) was then added to the residual broth containing bacteria and kept at room temperature. Solutions of acriflavine were freshly prepared each week and stored protected from light. Type D strains were identified by the appearance of a heavy flocculent precipitate within 5 min. DNA extraction by heat shock was performed prior to multiplex PCR for the detection of capsular genes hyaD-hyaC (capsular typing A) and dcbF (capsular typing D). Test of symmetry and a weighted kappa coefficient were used to evaluate correlations and to assess agreement of the results between the identification methods, respectively. Phenotypic tests showed that two isolates were type D (4.55%), 40 were type A (90.9%) and two (4.55%) were untypable isolates (4.55%) while PCR showed that 38 isolates were type A (86.36%) and six were type D (13.64%). The correlation analysis between the phenotypic and genotypic tests showed that 90.9% of the strains were identified as belonging to the same serotype by both tests and the weighted kappa coefficient (K = 0.633) indicates a substantial agreement between the two tests.Discussion: There was a disagreement between the phenotypic and genotypic results in four of the isolates (9.09%). The phenotypically untypable isolates were classified as type D by multiplex PCR. Nonetheless, we conclude that PCR testing is a more reliable method to differentiate between P. multocida serotypes A and D.

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Identification of the capsule type of Pasteurella multocida isolates from cases of fowl cholera by multiplex PCR and comparison with phenotypic methods

Cited by 14 publications

References 22 publications

Virulence genes and antimicrobial resistance of Pasteurella multocida isolated from poultry and swine

Virulence genes and antimicrobial resistance of Pasteurella multocida isolated from poultry and swine

Pasteurella multocida in camels: incidence, capsular and virulence genes characterization

Comparison of Phenotypic and Genotypic Identification Methods of Pasteurella multocida Serotypes Isolated from Pigs

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