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Mannheimia haemolytica suşlarının farklı besiyerlerinde üreme ve lökotoksin oluşturma özelliklerinin incelenmesi

Year 2020, Volume: 5 Issue: 2, 33 - 42, 31.08.2020
https://doi.org/10.31797/vetbio.692422

Abstract

Mannheimia haemolytica hayvanlarda pnömoniye sebebiyet veren en etkili bakterilerden biridir. Etkenin en önemli virülans faktörü olan lökotoksine karşı gelişen antikorlar, hastalığa karşı dirençte önemli bir rol oynarlar. Bu nedenle aşılama çalışmalarında uygun miktarda lökotoksin üretecek suşun ve besiyerinin seçimi önemlidir. Bu amaçla, çalışmada 4 ayrı besiyeri ve 4 ayrı suş test edildi. Test edilen besiyerlerinde canlılık sayımı bakımından RPMI besiyeri diğer test edilen besiyerlerinden önemli derecede farklı bulundu (P<0.001). Öbür taraftan BHIB, BHIB+ %5 serum ve BHIB+%1 yeast extract besiyerleri canlılık sayımı açısından birbirlerinden istatistiki olarakfarklı bulunmadı (P>0.05). Ancak, test edilen suşların tümünün 24 saatlik kültürleri en fazla canlılık sayımını BHIB + %5 at serumu içeren besiyerinde gösterdiler. Suşlar arasında bakteriyel sayıma ilişkin istatistiki olarak önemli bir fark saptanmadı (P>0.05). RPMI besiyeri üretilen lökotoksin açısından diğer besiyerlerine göre istatistiki olarak önemli derecede farklı bulundu (P<0.001). Tüm test suşları RPMI besiyerinde diğer 3 besiyerine göre çok daha fazla miktarda lökotoksin üretti. Alınan sonuçlara göre, MH05 suşu en fazla miktarda lökotoksin üretti ve bunu MH06 suşu izledi. MH04 ve MH03 suşları arasında bu yönden anlamlı bir farklılık saptanmadı. Test edilen kültür besiyerlerinde üreyen mikroorganizma sayısı ile üretilen lökotoksin arasında negatif bir korelasyon olabileceği düşünüldü. Sonuç olarak, açlık ve kısıtlı demir varlığı gibi stress faktörlerinin Mannheimia haemolytica’nın daha fazla lökotoksin ve muhtemelen diğer virülans faktörleri üretmesine yol açtığı kanısına varıldı. Mannheimia haemolytica is one of the most effective bacteria causing pneumonia in animals. Since antibodies against leukotoxin, which is the most important virulence factor of the agent, play an important role in resistance to disease, selection of strain and medium that produce leukotoxin is most important in vaccination studies. For this purpose, 4 different strains were tested in 4 different culture media tested in the study. Based on the viability counts in tested media, RPMI media was found significantly different from the rest of media (P<0.001). On the other hand, BHIB, BHIB+5% serum and BHIB+%1 Yeast extract media were not found statistically different from each others (P>0.05) for the viability counts. However, the viability counts measured after 24 hours of culture of tested strains were found to be highest in medium containing BHIB + 5% horse serum. There were no significant differences among strains related to bacterial counts in tested media (P>0.05). RPMI media was significantly different from the rest of media regarding to amount of produced leukotoxin in direct ELISA (P<0.001). All the strains produced more leukotoxins in RPMI media compared to other 3 tested media. According to our results, the strain MH05 produced the highest amount of leukotoxin followed by MH06. There was no significant differences between MH03 and MH04 strains in this regard. It was thought that there could be negative correlation between viability count of the bacteria and the produced leukotoxin amount in tested media. As conclusion, it was assumed that stress factors like starvation or iron restriction might cause Mannheimia haemolytica to produce more leukotoxin and possibly other virulence factors.

Supporting Institution

Harran Üniversitesi Bilimsel Araştırma Projeleri Koordinatörlüğü

Project Number

16107

Thanks

Bu çalışma, Harran Üniversitesi Bilimsel Araştırma Projeleri Koordinatörlüğü tarafından 16107 proje numarası ile Yüksek lisans tezi olarak desteklenmiştir. Destekleri için kuruma teşekkür ederiz.

References

  • Adamu J.Y. (2007). Mannheimia haemolytica: Phylogeny and genetic analysis of its majör virulence factors. Israel Journal of Veterinary Medicine, 61, 85-96.
  • Clinkenbeard K.D., Mosier D.A., Confer A.W.(1989). Transmembrane pore size and role of cell swelling in cytotoxicity caused by Pasteurella haemolytica leukotoxin. Infect Immun , 57(2), 420-425.
  • Davies R.L., Parton R., Coote J.G., Alison Gibbs H., Freer J.H.(1992). Outer-membrane protein and lipopolysaccharide variation in Pasteurella haemolytica serotype A1 under different growth conditions. Journal of General Microbiology, 138, 908-922.
  • Davies R.L., Arkinsaw S., Selander R.K.(1997). Evolutionary genetics of Pasteurella haemolytica isolates recovered from cattle and sheep. Infect Immun, 65, 3585–3593.
  • Davies R.L., Baillie S.(2003). Cytotoxic activity of Mannheimia haemolytic strains in relation to diversity of the leukotoxi structural gene lktA. Vet Microbiol , 92, 263-279.
  • Du Preez J.C., Van Rensburg E., Kilian S.G.(2008). Kinetics of growth and leutoxin production by Mannheimia haemolytica in continious culture. J. Ind. Microbiol. Biotechnol, 35, 611-618. Jeyaseelan S., Sreevatsan S., Maheswaran S.K.(2002). Role of Mannheimia haemolytica leukotoxin in the pathogenesis of bovine pneumonic pasteurellosis. Anim. Health Res. Rev, 3, 69- 82.
  • Odendaal M.W., Ellis C.E.(1999). The production and evaluation of Pasteurella haemolytica leukotoxin in the supernatant of submerged cultures in fermenters. Onderstepoort Journal of Veterinary Research , 66, 265-272.
  • Oppermann T., Busse N., Czermak P.(2017). Mannheimia haemolytica growth and leukotoxin production for vaccine manufacturing-A bioprocess review. Electronic Journal of Biotechnology, 28, 95-100.
  • Panciera R.J., Corsvet R.E. (1984).Bovine Pneumonic Pasteurellosis: Model for Pasteurella haemolytica and Pasteurella multocida induced pneumonia in cattle. Am J Vet Res, 45(12), 2532-3537. Retzer M.D., Yu R., Zhang Y.(1998). Discrimination between apo and iron-loaded forms of transferrin by transferrin binding protein B and its Nterminal subfragment. Microb Pathogenesis , 25(4), 175-180.
  • Rice J.A., Carrasco- Medina L., Hodgins D.C., Shewen P.E.(2008). Mannheimia haemolytica and bovine respiratory disease. Anim. Health. Res. Rev, 8, 117- 128.
  • Saadati M., Gibbs H.A., Parton R., Coote J.G.(1997). Characterisation of the leukotoxin produced by different strains of Pasteurella haemolytica. J Med Microbiol , 46, 276-284.
  • Shewen P.E., Wilkie B.N.(1985). Evidence for the Pasteurella haemolytica cytotoxin as a product of actively growing bacteria. Am J Vet Res, 46(5), 1212-1214.
  • Shewen P.E., Wilkie B.N.(1988). Vaccination of calves with leukotoxic culture supernatant from Pasteurella haemolytica. Can. J. Vet. Res, 52, 30-36.
  • Singh K., Ritchey J.W., and Confer A.W.(2011). Mannheimia haemolytica: Bacterial-Host Interactions in Bovine Pneumonia. Veterinary Pathology 2011, 48(2): 338-348.
  • Smith H. Pathogenicity and the microbe in vivo. Journal of General Microbiology 1990, 136: 377-383
  • Stevens PK, Czuprynski CJ. Pasteuralla haemolytica Leukotoxin Induces Bovine Leukocytes To Undergo Morphologic Changes Consistent with Apoptosis In Vitro. Infect. Immun 1996, 64: 2687-2694.
  • Winn W, Allen S, Janda W, Koneman E, Procop G, Schreckenberger P, Woods G. Koneman’s Color Atlas and Textbook of Diagnostic Microbiology. (6. Edition). Lippincott Williams & Wilkins. Chepter 8. 2006, 459-467.
  • Urban-Chmiel R, Wernicki A, Puchalski A, Mikucki P. Evaluation of Mannheimia haemolytica leukotoxinprepared in nonsupplemented and BSA and FBS supplemented RPMI 1640 medium. Pol J Vet Sci 2004, 7: 1-8.
  • Zechinon L, Fett T, Desmecht D. How Mannheimia haemolytica defeats host defence through a kiss of death mechanism. Vet. Res 2005, 36: 133-156.

Investigations on growth and leukotoxin production of mannheimia haemolytica strains growing in different culture media

Year 2020, Volume: 5 Issue: 2, 33 - 42, 31.08.2020
https://doi.org/10.31797/vetbio.692422

Abstract

Mannheimia haemolytica is one of the most effective bacteria causing pneumonia in animals. Since antibodies against leukotoxin, which is the most important virulence factor of the agent, play an important role in resistance to disease, selection of strain and medium that produce leukotoxin is most important in vaccination studies. For this purpose, 4 different strains were tested in 4 different culture media tested in the study. Based on the viability counts in tested media, RPMI media was found significantly different from the rest of media (P<0.001). On the other hand, BHIB, BHIB+5% serum and BHIB+%1 Yeast extract media were not found statistically different from each other’s (P>0.05) for the viability counts. However, the viability counts measured after 24 hours of culture of tested strains were found to be highest in medium containing BHIB + 5% horse serum. There were no significant differences among strains related to bacterial counts in tested media (P>0.05). RPMI media was significantly different from the rest of media regarding to amount of produced leukotoxin in direct ELISA (P<0.001). All the strains produced more leukotoxins in RPMI media compared to other 3 tested media.
According to our results, the strain MH05 produced the highest amount of leukotoxin followed by MH06. There was no significant difference between MH03 and MH04 strains in this regard. It was thought that there could be negative correlation between viability count of the bacteria and the produced leukotoxin amount in tested media. As conclusion, it was assumed that stress factors like starvation or iron restriction might cause Mannheimia haemolytica to produce more leukotoxin and possibly other virulence factors.

Project Number

16107

References

  • Adamu J.Y. (2007). Mannheimia haemolytica: Phylogeny and genetic analysis of its majör virulence factors. Israel Journal of Veterinary Medicine, 61, 85-96.
  • Clinkenbeard K.D., Mosier D.A., Confer A.W.(1989). Transmembrane pore size and role of cell swelling in cytotoxicity caused by Pasteurella haemolytica leukotoxin. Infect Immun , 57(2), 420-425.
  • Davies R.L., Parton R., Coote J.G., Alison Gibbs H., Freer J.H.(1992). Outer-membrane protein and lipopolysaccharide variation in Pasteurella haemolytica serotype A1 under different growth conditions. Journal of General Microbiology, 138, 908-922.
  • Davies R.L., Arkinsaw S., Selander R.K.(1997). Evolutionary genetics of Pasteurella haemolytica isolates recovered from cattle and sheep. Infect Immun, 65, 3585–3593.
  • Davies R.L., Baillie S.(2003). Cytotoxic activity of Mannheimia haemolytic strains in relation to diversity of the leukotoxi structural gene lktA. Vet Microbiol , 92, 263-279.
  • Du Preez J.C., Van Rensburg E., Kilian S.G.(2008). Kinetics of growth and leutoxin production by Mannheimia haemolytica in continious culture. J. Ind. Microbiol. Biotechnol, 35, 611-618. Jeyaseelan S., Sreevatsan S., Maheswaran S.K.(2002). Role of Mannheimia haemolytica leukotoxin in the pathogenesis of bovine pneumonic pasteurellosis. Anim. Health Res. Rev, 3, 69- 82.
  • Odendaal M.W., Ellis C.E.(1999). The production and evaluation of Pasteurella haemolytica leukotoxin in the supernatant of submerged cultures in fermenters. Onderstepoort Journal of Veterinary Research , 66, 265-272.
  • Oppermann T., Busse N., Czermak P.(2017). Mannheimia haemolytica growth and leukotoxin production for vaccine manufacturing-A bioprocess review. Electronic Journal of Biotechnology, 28, 95-100.
  • Panciera R.J., Corsvet R.E. (1984).Bovine Pneumonic Pasteurellosis: Model for Pasteurella haemolytica and Pasteurella multocida induced pneumonia in cattle. Am J Vet Res, 45(12), 2532-3537. Retzer M.D., Yu R., Zhang Y.(1998). Discrimination between apo and iron-loaded forms of transferrin by transferrin binding protein B and its Nterminal subfragment. Microb Pathogenesis , 25(4), 175-180.
  • Rice J.A., Carrasco- Medina L., Hodgins D.C., Shewen P.E.(2008). Mannheimia haemolytica and bovine respiratory disease. Anim. Health. Res. Rev, 8, 117- 128.
  • Saadati M., Gibbs H.A., Parton R., Coote J.G.(1997). Characterisation of the leukotoxin produced by different strains of Pasteurella haemolytica. J Med Microbiol , 46, 276-284.
  • Shewen P.E., Wilkie B.N.(1985). Evidence for the Pasteurella haemolytica cytotoxin as a product of actively growing bacteria. Am J Vet Res, 46(5), 1212-1214.
  • Shewen P.E., Wilkie B.N.(1988). Vaccination of calves with leukotoxic culture supernatant from Pasteurella haemolytica. Can. J. Vet. Res, 52, 30-36.
  • Singh K., Ritchey J.W., and Confer A.W.(2011). Mannheimia haemolytica: Bacterial-Host Interactions in Bovine Pneumonia. Veterinary Pathology 2011, 48(2): 338-348.
  • Smith H. Pathogenicity and the microbe in vivo. Journal of General Microbiology 1990, 136: 377-383
  • Stevens PK, Czuprynski CJ. Pasteuralla haemolytica Leukotoxin Induces Bovine Leukocytes To Undergo Morphologic Changes Consistent with Apoptosis In Vitro. Infect. Immun 1996, 64: 2687-2694.
  • Winn W, Allen S, Janda W, Koneman E, Procop G, Schreckenberger P, Woods G. Koneman’s Color Atlas and Textbook of Diagnostic Microbiology. (6. Edition). Lippincott Williams & Wilkins. Chepter 8. 2006, 459-467.
  • Urban-Chmiel R, Wernicki A, Puchalski A, Mikucki P. Evaluation of Mannheimia haemolytica leukotoxinprepared in nonsupplemented and BSA and FBS supplemented RPMI 1640 medium. Pol J Vet Sci 2004, 7: 1-8.
  • Zechinon L, Fett T, Desmecht D. How Mannheimia haemolytica defeats host defence through a kiss of death mechanism. Vet. Res 2005, 36: 133-156.
There are 19 citations in total.

Details

Primary Language Turkish
Subjects Veterinary Surgery
Journal Section Research Articles
Authors

Mehmet Çelik 0000-0002-3541-9544

Sevil Erdenliğ Gürbilek 0000-0002-0377-2650

Project Number 16107
Publication Date August 31, 2020
Submission Date February 21, 2020
Acceptance Date June 4, 2020
Published in Issue Year 2020 Volume: 5 Issue: 2

Cite

APA Çelik, M., & Erdenliğ Gürbilek, S. (2020). Mannheimia haemolytica suşlarının farklı besiyerlerinde üreme ve lökotoksin oluşturma özelliklerinin incelenmesi. Journal of Advances in VetBio Science and Techniques, 5(2), 33-42. https://doi.org/10.31797/vetbio.692422

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