Use of molecular biology tools for rapid identification and characterization of  Pasteurella  spp

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Abstract

Aim:

This study aimed to create rapid characterization and genotyping of Pasteurella multocida (PM) protocol using modern molecular biology techniques.

Materials and Methods:

Thirty bacterial isolates were characterized by capsular and somatic identification using conventional procedure followed by multiplex polymerase chain reaction (PCR), restriction endonucleases analysis (REA), and finally confirmed by sequence analysis. Two local vaccine strains and two field isolates were identified as PM Type A and B.

Results:

A total of 30 isolates were found positive for PM either morphologically and biochemically; however, multiplex PCR technique identified only 22 isolates as Pasteurella species using universal primers while 8 isolates were found negative for PM. 12 of 22 isolates (54%) were characterized at the same reaction into PM Type A, five isolates (23%) were Type B and the rest five isolates (23%) of tested isolates were negative for Types A, B, and D. Hemorrhagic septicemia Type B: 2 or B: 5 could be identified somatically within PM capsular serogroup B using PCR technique. Somatic characterization of PM was done using REA that could identify all PM Type A into A:1 and all PM Type B into B: 2. These protocols were verified for its accuracy and reliability by sequence analysis of two vaccine strains of PM Type A and B that were characterized previously by biochemical and serological methods as well as two selected isolates from the 22 positive isolates representing PM Type A and B.

Conclusion:

PCR and REA could confirm the identity of PM and provide a rapid and reliable characterization in comparison with biochemical analysis and conventional serotyping that may take up to 2 weeks. Hence, they can reduce the time needed for polyvalent vaccine production and when the reference antisera are unavailable. Moreover, the identity of Omp-H for vaccine and field strains may provide better data to control Pasteurellosis in Egypt.

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Most cited references 33

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Development of PCR assays for species- and type-specific identification of Pasteurella multocida isolates.

Chong-Kil Lee, J. Frost, Julie S. Townsend … (1998)

Genomic subtractive hybridization of closely related Pasteurella multocida isolates has generated clones useful in distinguishing hemorrhagic septicemia-causing type B strains from other P. multocida serotypes. Oligonucleotide primers designed during the sequencing of these clones have proved valuable in the development of PCR assays for rapid species- and type-specific detection of P. multocida and of type B:2 in particular. This study demonstrated that the primer pair designed from the sequence of the clone 6b (KTT72 and KTSP61) specifically amplified a DNA fragment from types B:2, B:5, and B:2,5 P. multocida and that the primers KMT1T7 and KMT1SP6 produced an amplification product unique to all P. multocida isolates analyzed. It was also shown that PCR amplification performed directly on bacterial colonies or cultures represents an extremely rapid, sensitive method of P. multocida identification.

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Typing methods used in the molecular epidemiology of microbial pathogens: a how-to guide.

Reza Ranjbar, Ali Karami, Shohreh Farshad … (2014)

Microbial typing is often employed to determine the source and routes of infections, confirm or rule out outbreaks, trace cross-transmission of healthcare-associated pathogens, recognize virulent strains and evaluate the effectiveness of control measures. Conventional microbial typing methods have occasionally been useful in describing the epidemiology of infectious diseases. However, these methods are generally considered too variable, labour intensive and time-consuming to be of practical value in epidemiological investigations. Moreover, these approaches have proved to be insufficiently discriminatory and poorly reproducible. DNA-based typing methods rely on the analysis of the genetic material of a microorganism. In recent years, several methods have been introduced and developed for investigation of the molecular epidemiology of microbial pathogens. Each of them has advantages and limitations that make them useful in some studies and restrictive in others. The choice of a molecular typing method therefore will depend on the skill level and resources of the laboratory and the aim and scale of the investigation. This study reviews the most popular DNA-based molecular typing methods used in the epidemiology of bacterial pathogens together with their advantages and limitations.

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Characterisation of bovine strains of Pasteurella multocida and comparison with isolates of avian, ovine and porcine origin.

Roslyn MacCorquodale, Cavan S Reilly, Claire L. Davies (2004)

One hundred and fifty-three bovine Pasteurella multocida strains recovered primarily from cases of pneumonia and mastitis in England and Wales over an 11-year period were characterised by capsular PCR typing, comparison of outer membrane protein (OMP) profiles, and multilocus sequence analysis. All of the strains were of capsular type A with the exception of a single capsular type F isolate. Thirteen distinct OMP profiles (OMP-types) were identified based mainly on molecular mass heterogeneity of the heat-modifiable (OmpA) and porin (OmpH) proteins. However, 85% of the isolates were represented by just five OMP-types and 39% of the strains were of a single OMP-type. Multilocus sequence analysis revealed a limited degree of genetic diversity among bovine P. multocida isolates; strains of the same OMP-type have identical genetic backgrounds and represent distinct clones. Analysis of OMP variation was more discriminating than multilocus sequence analysis because strains of different OMP-types had the same, or similar, genetic backgrounds. The association of a small number of clones with the majority of cases of bovine pneumonia suggests that these clones have an increased capacity to cause disease compared to less frequently recovered clones. Molecular mass heterogeneity of OmpA and OmpH, in strains of the same or similar genetic background, suggests that these proteins are subject to diversifying selection within the host and might play important roles in host-pathogen interactions. Comparison of the OMP profiles of bovine isolates with those of avian, ovine and porcine strains showed that a high proportion of the respiratory tract infections in each of these species are caused by different strains of P. multocida. However, the presence of small numbers of closely related strains in more than one host species suggests that transmission of bacteria between different host species is also a factor in the population biology of P. multocida.

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Author and article information

Journal

Journal ID (nlm-ta): Vet World

Journal ID (iso-abbrev): Vet World

Title: Veterinary World

Publisher: Veterinary World (India )

ISSN (Print): 0972-8988

ISSN (Electronic): 2231-0916

Publication date (Print): July 2018

Publication date (Electronic): 29 July 2018

Volume: 11

Issue: 7

Pages: 1006-1014

Affiliations

[1 ]Genetic Engineering Research Department, Veterinary Serum and Vaccine Research Institute, Cairo Egypt

[2 ]Aerobic Bacterial Vaccine Research Department, Veterinary Serum and Vaccine Research Institute, Cairo Egypt

[3 ]Anaerobic Bacterial Vaccine Research Department, Veterinary Serum and Vaccine Research Institute, Cairo Egypt

[4 ]Department of Microbiology and Immunology, Veterinary Division, National Research Center, Dokki, Egypt

Author notes

Corresponding author: Dalia Ahmed Mohammed. Abd El-Moaty, e-mail: dody.ahmed@ 123456gmail.com Co-authors: AMA: ashraf_vir@ 123456hotmail.co.uk , ESAZ: dremeyzaki@ 123456yahoo.com , EFE: elham1832@ 123456hotmail.com , NAE: nsebaie@ 123456gmail.com , HAF: Ahmed_essam2022@ 123456hotmail.com , AAS: ayman_samy@ 123456hotmail.com

Article

DOI: 10.14202/vetworld.2018.1006-1014

PMC ID: 6097567

PubMed ID: 30147273

SO-VID: b700d2d7-7d5e-41a1-a028-bcbcd26e49a0

License:

Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Use of molecular biology tools for rapid identification and characterization of Pasteurella spp

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Abstract

Aim:

Materials and Methods:

Results:

Conclusion:

Related collections

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Most cited references 33

Development of PCR assays for species- and type-specific identification of Pasteurella multocida isolates.

Typing methods used in the molecular epidemiology of microbial pathogens: a how-to guide.

Characterisation of bovine strains of Pasteurella multocida and comparison with isolates of avian, ovine and porcine origin.

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