Research Article
 

Molecular Characterization of Pasteurella multocida Isolated from Rabbit in Egypt



S. Ahmed, M.S. Abdel-Salam, S.S. Hafez and W. Nemr
 
Facebook Twitter Digg Reddit Linkedin StumbleUpon E-mail
ABSTRACT

Objective: The study aimed to molecular characterization of Pasteurella multocida local strain using Enterobacterial Repetitive Intergenic Consensus (ERIC), 13 virulence genes and the outer membrane protein (OmpH) gene. Also, development of diagnostic markers by multiplex PCR from investigated genes. Methodology: The ERIC-PCR was used for the detection of the finger print of Pasteurella multocida local strain isolated from rabbits. Polymerase Chain Reaction (PCR) with specific primers of the virulence genes and OmpH gene was used for molecular characterization of Pasteurella multocida local strain. The multiplex-PCR protocol was used to develop diagnostic genes markers. Results: Pasteurella multocida ERIC pattern was identified as seven DNA bands ranging from less than 100-850 bp. The results determined the molecular sizes of toxA, pfhA, soda, nanB, nanH, fimA, hsf-1, hsf-2, hgbA, hgbB, ptfA, oma87, tbpA and OmpH genes. Four multiplex-PCR were configured from investigated genes. Multiplex 1 amplified both of hgbB and oma87 genes. Multiplex 2 included fimA and ptfA genes. Multiplex 3 used nanB and hgbB genes. Multiplex 4 amplified nanB and ptfA genes. Conclusion: Molecular characterization of P. multocida local strain using Enterobacterial Repetitive Intergenic Consensus (ERIC), 13 virulence genes and the outer membrane protein (OmpH) gene provided data information that could be used as diagnostic tool, epidemiological markers and in vaccine development.

Services
Related Articles in ASCI
Search in Google Scholar
View Citation
Report Citation

 
  How to cite this article:

S. Ahmed, M.S. Abdel-Salam, S.S. Hafez and W. Nemr, 2016. Molecular Characterization of Pasteurella multocida Isolated from Rabbit in Egypt. American Journal of Biochemistry and Molecular Biology, 6: 53-59.

DOI: 10.3923/ajbmb.2016.53.59

URL: https://scialert.net/abstract/?doi=ajbmb.2016.53.59
 
Received: February 13, 2016; Accepted: March 02, 2016; Published: March 15, 2016



INTRODUCTION

Pasteurella multocida is a common bacterial disease contributes to heavy losses in the animal production as well as a hazardous threat to human health. It is associated with haemorrhagic septicaemia in cattle and buffaloes, pneumonic pasteurellosis in sheep and goats, fowl cholera in poultry, snuffles in rabbits and cellulitis and septicemia in humans1.

Advanced molecular biology techniques become a very useful approach for detecting microbes universally. Stahel et al.2 proved that the REP-PCR typing represents a suitable tool for genetic characterization of rabbit Pasteurellaceae isolates while the biochemical analysis showed high heterogeneity and in some cases provided unclear results.

The wide host of P. multocida and different courses of infection lead to the need for extensive studies concerning the prevalence and distribution of capsule and virulence genes. The distribution of virulence genes of P. multocida in different animals has been studied by Ewers et al.3 Their study recorded a high prevalence of the toxin gene among the strains from small ruminants.

Development of the multiplex-PCR protocol depends on the molecular characterization of P. multocida strains has been successfully used as a useful tool for rapid and simultaneous detection of virulence genes4,5. The significance of the molecular characterization of P. multocida strains confirmed by Gautier et al.6, Sellyei et al.7, Prabhakar et al.8, Ferreira et al.9 and Verma et al.10 studies. They clarified that the epidemiological data together with molecular characteristics of individual strains could help to design and implement adequate preventive and intervention strategies.

In Egypt, Pasteurella multocida is a common pathogen in rabbits causing series outbreaks and considerable economic losses in rabbit industry11-13. The study aimed: (1) To molecular characterization of P. multocida isolated from rabbit using Enterobacterial Repetitive Intergenic Consensus (ERIC) and specific virulence genes of P. multocida and (2) To develop a P. multocida diagnostic tool by a multiplex-PCR.

MATERIALS AND METHODS

Samples: The samples were collected by sterile swabs from external nares of healthy and suspected infected rabbits. Each swab was plated on tryptic soy agar (Oxoid), supplemented with 5% sheep blood14. The isolates were subjected to further identification using Gram staining and standard biochemical procedures according to MacFaddin15.

Pasteurella multocida DNA extraction: Bacterial genomic DNA was extracted according to Ozbey et al.16.

Molecular characterization of P. multocida genes: Polymerase chain reaction with specific primers for Enterobacterial Repetitive Intergenic Consensus (ERIC), 13 virulence genes and the outer membrane protein (OmpH) gene was used.

The primers sequences of enterobacterial repetitive intergenic consensus was according to Amonsin et al.17. The PCR program was denaturation at 95°C, 10 min, then 30 cycles of denaturation at 94°C for 1 min, annealing temperature at 52°C for 1 min, extension at 65°C for 8 min and a final extension at 65°C for 16 min.

The specific primers of 13 genes studied were obtained from available scientific research articles (Table 1). The OmpH primers were deduced in this study from the GenBank sequencing data [F: GCG TTT CAT TCA AAG CAT CTC, R: TTT AGA TTG TGC GTA GTC AAC]. The optimized PCR program was denaturation at 95°C: 5 min, 35 cycles of denaturation at 94°C: 45 sec, annealing temperature (Table 1) 30 sec, 72°C: 30 sec, extension at 72°C for 7 min.

Multiplex-PCR was developed (Table 2) from investigated genes in this study. The optimized PCR program was denaturation at 95°C: 5 min, 30 cycles of denaturation at 94°C: 30 sec, annealing 56°C, 30 sec, 72°C: 30 sec, extension at 72°C for 7 min. For each amplification reaction, a mixture of reagents (master mix) and primers (total volume 25 μL) were prepared.

The PCR products were electrophoresed on 1% agarose gel (Invitrogen UltrapureTM Agrose®-Carlsbad, USA) together with a 100 bp DNA ladder (Promega Corporation, France) for molecular weight estimation. The amplified products were visualized in an ultraviolet light transilluminator, photographed and analyze.

RESULTS

The result of P. multocida ERIC electrophoresis patterns showed seven DNA bands there sizes were less than 100, 100, 175, 280, 390, 710 and 850 bp (Fig. 1).

Thirteen virulence genes (toxA, pfhA, sodaA, nanB, nanH, fimA, hsf-1, hsf-2, hgbA, hgbB, ptfA, oma87 and tbpA) and the outer membrane protein (OmpH) gene were identified. The fragments size of the successful PCR amplified products of the tested genes are recorded in Table 1 and Fig. 2-7.

Four multiplex-PCR were configured from investigated genes. Multiplex 1 amplified both o f hgbB and oma87 genes, multiplex 2 included fimA and ptfA genes, multiplex 3 used nanB and hgbB genes and multiplex 4 amplified nanB and ptfA genes. The developed multiplex-PCR fragments are shown in Fig. 8-11.

Image for - Molecular Characterization of Pasteurella multocida Isolated from Rabbit in Egypt
Fig. 1:Agarose gel electrophoreses Lane 1: A 100 bp ladder, Lane 2-4: Enterobacterial Repetitive Intergenic Consensus (ERIC)

Image for - Molecular Characterization of Pasteurella multocida Isolated from Rabbit in Egypt
Fig. 2:
Agarose gel electrophoreses of DNA fragments obtained with specific primers Lane 1: A 100 bp ladder, Lane 2: phfA, Lane 3: ptfA, Lane 4: fimA and Lane 5: hsf-2 genes

Image for - Molecular Characterization of Pasteurella multocida Isolated from Rabbit in Egypt
Fig. 3:
Agarose gel electrophoreses of DNA fragments obtained with specific primers Lane 1: A 100 bp ladder, Lane 2: nanH, Lane 3: hsf-1 and Lane 4: tbpA genes

Image for - Molecular Characterization of Pasteurella multocida Isolated from Rabbit in Egypt
Fig. 4:
Agarose gel electrophoreses of DNA fragments obtained with specific primers Lane 1: A 100 bp ladder, Lane 2: hgbA, Lane 3: nanB and Lane 4: oma87 genes

Image for - Molecular Characterization of Pasteurella multocida Isolated from Rabbit in Egypt
Fig. 5:
Agarose gel electrophoreses of DNA fragments obtained with specific primers Lane 1: A 100 bp ladder, Lane 2: hgbB and Lane 3: toxA genes

Table 1:Optimized annealing temperatures and the amplified DNAs size of fourteen specific genes of P. multocida
Image for - Molecular Characterization of Pasteurella multocida Isolated from Rabbit in Egypt

Image for - Molecular Characterization of Pasteurella multocida Isolated from Rabbit in Egypt
Fig. 6:Agarose gel electrophoreses of DNA obtained with specific primers Lane1: 100 bp ladder and Lane 2: sodA

Image for - Molecular Characterization of Pasteurella multocida Isolated from Rabbit in Egypt
Fig. 7:Agarose gel electrophoreses of DNA fragments obtained with specific primers Lane 1: A 100 bp ladder and Lane 2-3: OmpH gene

Image for - Molecular Characterization of Pasteurella multocida Isolated from Rabbit in Egypt
Fig. 8:
Agarose gel electrophoreses of DNA fragments obtained with specific primers Lane 1: A 100 bp ladder and Lane 2: hgbB and oma87 genes as multiplex 1

Image for - Molecular Characterization of Pasteurella multocida Isolated from Rabbit in Egypt
Fig. 9:
Agarose gel electrophoreses of DNA fragments obtained with specific primers Lane 1: A 100 bp ladder and Lane 2: fimA and ptfA genes as multiplex 2

Image for - Molecular Characterization of Pasteurella multocida Isolated from Rabbit in Egypt
Fig. 10:
Agarose gel electrophoreses of DNA fragments obtained with specific primers Lane 1: nanB and hgbB genes as multiplex 3 and Lane 2: A 100 bp ladder

Image for - Molecular Characterization of Pasteurella multocida Isolated from Rabbit in Egypt
Fig. 11:
Agarose gel electrophoreses of DNA fragments obtained with specific primers Lane 1: A 100 bp ladder and Lane 2: nanB and ptfA genes as multiplex 4

Table 2:Multiplex-PCR for P. multocida diagnoses
Image for - Molecular Characterization of Pasteurella multocida Isolated from Rabbit in Egypt

DISCUSSION

The ERIC typing has been identified as a tool for Pasteurella genetic characterization and it used extensively for bacterial differentiation, rapid epidemiological analysis and determination of outbreak-related strains16,19. In this study, to generate DNA fingerprint for P. multocida the ERIC marker was used. The obtained result was different from previously published studies19,2 used ERIC-PCR for characterization of P. multocida local strain, that difference could be related to the specificity of the P. multocida strains.

The investigated genes in this study play an important role in epidemiological and pathogenesis effect of P. multocida3,20,18,7,8. The previous study by Shayegh et al.20, Prabhakar et al.8 and Verma et al.10 recommended using tbpA, phfA and ptfA genes as an epidemiological marker. The fimA, hsf-2, hgbA, nanB and oma87 genes of P. multocida recognized as virulence factors responsible for the pathogenicity in the target host21,22,5. The hgbB and toxA genes are virulence genes used for detection of the P. multocida pathogenicity and the multiplex-PCR methodology for disease diagnosis5. The studies related to the sodA sequences showed higher divergence than the corresponding 16S rRNA genes which makes it as a potent target to differentiate related species9,10. The results of this study revealed that the amplified PCR products of tested genes of P. multocida isolated from Egyptian rabbits showed differences in genes fragments sizes amplifications compared to several previous studies which used the same genes loci isolated from different animals (cattle, buffalo, pig and sheep)3,13.

The OmpH gene is reported as surface-exposed conserved immunodominant porin23. In this study, the specific primers for OmpH gene was developed using the GenBank sequencing data. Development of these primers aimed to obtain specific primers from the conservative gene of P. multocida. Accordingly, these primers could be considered as a diagnostic tool reference for the P. multocida local strain.

Multiplex-PCR is a rapid and reliable technique to determine multiple genes of microorganisms for diseases diagnosis, reducing the amount of reagents used and time required4,15. The results of tested genes were used to develop a multiplex-PCR for diseases diagnosis induced by P. multocida local strain (Table 2). Optimization of the multiplex-PCR protocols developed in this study depends on the same primers annealing conditions without interfering with one another in the multiplex-PCR and to the different in DNA bands produced. The results confirmed the significance study of molecular characterization of P. multocida local strain.

Control of the disease outbreaks is dependent upon the system of monitoring, early detection and vaccination. Definitive diagnosis of bacterial or viral disease requires detection of the microorganism, antigen or genome in clinical material. Traditional microbiological methods for detection of pathogens can be slow, are not sensitive, may not distinguish infection from colonization and are influenced by previous antibiotic therapy24,25. As each bacterial strain has a specificity at the molecular level.

CONCLUSION

The molecular characterization of P. multocida local strain in rabbits investigated for the study using Enterobacterial Repetitive Intergenic Consensus (ERIC), 13 virulence genes and the outer membrane protein (OmpH) gene provided data information that could be used as diagnostic tool, epidemiological markers and in vaccine development.

ACKNOWLEDGMENT

This study was funded from the Science and Technology Development Fund, Egypt (STDF Technology Development Grant, project ID: 6025).

SIGNIFICANCE STATEMENT

Molecular characterization of the indigenous pathogen is required for diagnosis, epidemiological study, vaccine development and detection any shift or mutation of the microorganism genomic as the local vaccine strain should be prepared from the circulating strain in the region.

REFERENCES

  1. El Tayeb, A.B., T.Y. Morishita and E.J. Angrick, 2004. Evaluation of Pasteurella multocida isolated from rabbits by capsular typing, somatic serotyping and restriction endonuclease analysis. J. Vet. Diagnost. Invest., 16: 121-125.
    CrossRef  |  Direct Link  |  


  2. Stahel, A.B.J., R.K. Hoop, P. Kuhnert and B.M. Korczak, 2009. Phenotypic and genetic characterization of Pasteurella multocida and related isolates from rabbits in Switzerland. J. Vet. Diagnost. Invest., 21: 793-802.
    CrossRef  |  Direct Link  |  


  3. Ewers, C., A. Lubke-Becker, A. Bethe, S. Kiebling, M. Filter and L.H. Wieler, 2006. Virulence genotype of Pasteurella multocida strains isolated from different hosts with various disease status. Vet. Microbiol., 114: 304-317.
    CrossRef  |  PubMed  |  Direct Link  |  


  4. Atashpaz, S., J. Shayegh and M.S. Hejazi, 2009. Rapid virulence typing of Pasteurella multocida by multiplex PCR. Res. Vet. Sci., 87: 355-357.
    CrossRef  |  Direct Link  |  


  5. Furian, T.Q., K.A. Borges, S.L.S. Rocha, E.E. Rodrigues, V.P. do Nascimento, C.T.P. Salle and H.L.S. Moraes, 2013. Detection of virulence-associated genes of Pasteurella multocida isolated from cases of fowl cholera by multiplex-PCR. Vet. Bras., 33: 177-182.
    CrossRef  |  Direct Link  |  


  6. Gautier, A.L., D. Dubois, F.O. Escande, J.L. Avril, P. Trieu-Cuot and O. Gaillot, 2005. Rapid and accurate identification of human isolates of Pasteurella and related species by sequencing the sodA gene. J. Clin. Microbiol., 43: 2307-2314.
    CrossRef  |  Direct Link  |  


  7. Sellyei, B., K. Banyai and T. Magyar, 2010. Characterization of the ptfA gene of avian Pasteurella multocida strains by allele-specific polymerase chain reaction. J. Vet. Diagn. Invest., 22: 607-610.
    CrossRef  |  Direct Link  |  


  8. Prabhakar, P., A. Thangavelu, J.J. Kirubaharan, N.D.J. Chandran, S.M. Sakthivelan and M. Thangapandian, 2012. Outbreak of pasteurellosis in captive EMU birds and detection of virulence genes in P. multocida isolates. Tamilnadu J. Vet. Anim. Sci., 8: 299-305.
    Direct Link  |  


  9. Ferreira, T.S.P., M.R. Felizardo, D.D.S. de Gobbi, C.R. Gomes and P.H. de Lima Nogueira Filsner et al., 2012. Virulence genes and antimicrobial resistance profiles of Pasteurella multocida strains isolated from rabbits in Brazil. Sci. World J.
    CrossRef  |  Direct Link  |  


  10. Verma, S., M. Sharma, S. Katoch, L. Verma and S. Kumar et al., 2013. Profiling of virulence associated genes of Pasteurella multocida isolated from cattle. Vet. Res. Commun., 37: 83-89.
    CrossRef  |  Direct Link  |  


  11. Suelam, I.I.A. and L.K.A. Samie, 2011. Molecular diversity of Pasteurella multocida isolated from different rabbit outbreaks at Zagazig suburbs, Egypt. Global Vet., 6: 208-212.
    Direct Link  |  


  12. Nassar, S.A., A.H. Mohamed, H. Soufy and S.M. Nasr, 2013. Protective effect of egyptian propolis against rabbit pasteurellosis. Bio. Med. Res. Inter.
    CrossRef  |  Direct Link  |  


  13. Soliman, M.A, M.A.A. Abdel Rahman, M.S. Mohamed, O. Mehana and S.A. Nasef, 2016. Molecular, clinical and pathological studies on viral rabbit hemorrhagic disease. Alex. J. Vet. Sci., 48: 20-26.
    Direct Link  |  


  14. Mutters, R., W. Mannheim and M. Bisgaard, 1989. Taxonomy of the Group. In: Pasteurella and Pasteurellosis. Adlam, C. and J.M. Rutter (Eds.). Academic Press, London, UK., ISBN-13: 9780120442744, pp: 3-4


  15. MacFaddin, J.F., 2000. Biochemical Tests for Identification of Medical Bacteria. 3rd Edn., Lippincott Williams & Wilkins, Pennsylvania, United States, ISBN: 0-683-05318-3, Pages: 912
    Direct Link  |  


  16. Ozbey, G., A. Kilic, H.B. Ertas and A. Muz, 2004. Random amplified polymorphic DNA (RAPD) analysis of Pasteurella multocida and Mannheimia haemolytica strains isolated from cattle, sheep and goats. Vet. Med. Czech, 49: 65-69.
    Direct Link  |  


  17. Amonsin, A., J.F.X. Wellehan, L.L. Li, J. Laber and V. Kapur, 2002. DNA fingerprinting of Pasteurella multocida recovered from avian sources. J. Clin. Micro., 8: 3025-3031.
    CrossRef  |  Direct Link  |  


  18. Tang, X., Z. Zhao, J. Hu, B. Wu, X. Cai, Q. He and H. Chen 2009. Isolation, antimicrobial resistance, and virulence genes of Pasteurella multocida strains from swine in China. J. Clin. Microbiol 47: 951-958.
    CrossRef  |  PubMed  |  Direct Link  |  


  19. Leotta, G.A., I. Chinen, G.B. Vigo, M. Pecoraro and M. Rivas, 2006. Outbreaks of avian cholera in Hope Bay, Antarctica. J. Wildlife Dis., 42: 259-270.
    CrossRef  |  Direct Link  |  


  20. Shayegh, J., S. Atashpaz and M.S. Hejazi, 2008. Virulence genes profile and typing of ovine Pasteurella multocida. Asian J. Anim. Vet. Adv., 3: 206-213.
    CrossRef  |  Direct Link  |  


  21. Fuller, T.E., M.J. Kennedy and D.E. Lowery, 2000. Identification of Pasteurella multocida virulence genes in a septicemic mouse model using signature-tagged mutagenesis. Microbol. Pathog, 29: 25-38.
    CrossRef  |  PubMed  |  Direct Link  |  


  22. Harper, M., J.D. Boyce and B. Adler, 2006. Pasteurella multocida pathogenesis: 125 years after Pasteur. F.E.M.S. Microbiol. Lett., 265: 1-10.
    CrossRef  |  Direct Link  |  


  23. Dabo, S.M., J.D. Taylor and A.W. Confer, 2007. Pasteurella multocida and bovine respiratory disease. Anim. Health Res. Rev., 8: 129-150.
    CrossRef  |  Direct Link  |  


  24. Nolte, F.S., 2008. Molecular diagnostics for detection of bacterial and viral pathogens in community-acquired pneumonia. Clin. Infect. Dis., 47: S123-S126.
    CrossRef  |  Direct Link  |  


  25. Muldrew, K.L., 2009. Molecular diagnostics of infectious diseases. Curr. Opin. Pediatr., 21: 102-111.
    CrossRef  |  PubMed  |  Direct Link  |  


©  2022 Science Alert. All Rights Reserved