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2012 | 15 | 2 |
Tytuł artykułu

Application of real-time PCR for detection of Lawsonia intracellularis and Brachyspira hyodysenteriage in fecal samples from pigs

Treść / Zawartość
Warianty tytułu
Języki publikacji
EN
Abstrakty
EN
The aim of the study was to develop and validate real-time PCR method for the quantification of Lawsonia intracellularis and Brachyspira hyodysenteriae in porcine feces. Before the optimization process was performed two different extraction methods were compared to select the more efficient one. Based on the results achieved at this stage the boiling procedure was rejected and a commercially available silica-membrane based method was chosen for further analysis. The primers and the Taqman probe for B. hyodysenteriae and L. intracellularis were based on the sequence of NADH oxidase gene and 16S rDNA gene, respectively. The detection limit of the real-time PCR for suspension of feces inoculated with B. hyodysenteriae and L. intracellularis was determined to be 1.5x103 CFU/ml and 6.5x101 CFU/ml, respectively. The results of this study demonstrate that our real-time PCR is able to detect low number of B. hyodysenteriae and L. intracellularis cells which is satisfying in routine diagnosis of swine dysentery and proliferative enteropathy. Therefore, it is possible to identify both subclinically infected pigs and those representing an acute form of mentioned diseases. In summary, the quantitative real-time PCR is useful for routine diagnosis of L. intracellularis and B. hyodysenteriae. Compared to conventional PCR, the new validated quantification method based on real-time PCR is fast and with reduced risk of laboratory contamination. The novel technique is specific and even more sensitive than the previously used one. Furthermore, the new real-time PCR enables quick detection and quantification of both pathogens in fecal samples, which helps to estimate the health status of a pig herd.
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-
Rocznik
Tom
15
Numer
2
Opis fizyczny
p.267-273,fig.,ref.
Twórcy
autor
  • Department of Swine Diseases, National Veterinary Research Institute, Partyzantow 57, 24-100 Pulawy, Poland
autor
autor
autor
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Bibliografia
  • Akase S, Uchitani Y, Sohmura Y, Tatsuta K, Sadamasu K, Adachi Y (2009) Application of real time PCR for diagnosis of Swine Dysentery. J Vet Med Sci 71: 359-362.
  • Elder RO, Duhamel GE, Schafer RW, Mathiesen MR, Ramanathan M (1994) Rapid detection of Serpulina hyodysenteriae in diagnostic specimens by PCR. J Clin Microbiol 32: 1497-1502.
  • Freschi CR, Carvalho LF de Oliveira CJ (2005) Comparison of DNA-extraction methods and selective enrichment broths on the detection of Salmonella typhimurium in swine feces by polymerase chain reaction (PCR). Braz J Microbiol 36: 363-367.
  • Hampson DJ, Fellström C, Thomson JR (2006) Swine dysentery. In: Straw BE, Zimmerman JJ, D’Allaire S, Taylor DJ (eds) Diseases of Swine. 9th ed., Wiley-Blackwell Publishing, USA, pp 785-805.
  • Jacobson M, Aspan A, Königsson MH, Segerstad CH, Wallgren P, Fellström C, Jensen-Waern M, Gunnarson A (2004) Routine diagnostics of Lawsonia intracellularis performed by PCR, serological and post mortem examination, with special emphasis on sample preparation methods for PCR. Vet Microbiol 102: 189-201.
  • Jensen TK, Möller K, Leser TD, Jorsal SE (1997) Comparison of histology, immunohistochemistry and polymerase chain reaction for detection of Lawsonia intracellularis in natural porcine proliferative enteropathy. Eur J Vet Pathol 3: 115-123.
  • Kunkle RA, Kinyon JM (1988) Improved selective medium for the isolation of Treponema hyodysenteriae. J Clin Microbiol 26: 2357-2360.
  • Lindecrona RH, Jensen TK, Andersen PH, Möller K (2002) Application of a 5’ nuclease assay for detection of Lawsonia intracellularis in fecal samples from pigs. J Clin Microbiol 40: 984-987.
  • McOrist S, Gebhart CJ (2006) Proliferative enteropathies. In: Straw BE, Zimmerman JJ, D’Allaire S, Taylor DJ (eds) Diseases of Swine. 9th ed., Wiley-Blackwell Publishing, USA, pp 727-737.
  • Nathues H, Holthaus K, Beilage E (2009) Quantification of Lawsonia intracellularis in porcine faeces by real-time PCR. J Appl Microbiol 107: 2009-2016.
  • Neef NA, Lysons RJ, Trott DJ, Hampson DJ, Jones PW, Morgan JH (1994) Pathogenicity of porcine intestinal spirochetes in gnobiotic pigs. Infect Immun 62: 2395-2403.
  • Nelson EA, Palombo EA, Knowles SR (2010) Comparison of methods for the extraction of bacterial DNA from human faecal samples for analysis by real-time PCR. In: Méndez-Vilas A (ed) Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology, Formatex, Microbiology Series No 2 Vol. 1, C/ Zurbarhn 1, 2o – Oficina 106002 Badajoz, Spain, pp 1479-1485.
  • Osek J (2001) Multiplex polymerase chain reaction assay for identification of enterotoxigenic Escherichia coli strains. J Vet Diagn Invest 13: 308-311.
  • Smith SH, McOrist S (1997) Development of persistent intestinal infection and excretion of Lawsonia intracellularis by piglets. Res Vet Sci 62: 6-10.
  • Song Y, Liu C, Finegold SM (2004) Real-time PCR quantitation of clostridia in feces of autistic children. Appl Environ Microbiol 70: 6459-6465.
  • Szynkiewicz ZM, Binek M (1986) Evaluation of selective media for primary isolation of Treponema hyodysenteriae and Treponema innocens. Comp Immunol Microbiol Infect Dis 9: 71-77.
  • Yeh JY, Kim TJ, Park SY, Song CS, Yoon YD, Kim SK, Lee JB, Choi IS (2006) Isolation of Lawsonia intracellularis in Korea and reproduction of proliferative enteropathy in pigs and hamsters. J Vet Med Sci 68: 499-501.
  • Żmudzki J, Osek J, Stankevicius A, Pejsak Z (2004) Rapid detection of Brachyspira hyodysenteriae and Lawsonia intracellularis in swine faecal and mucosal specimens by multiplex PCR. Bull Vet Inst Pulawy 48: 207-214.
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Bibliografia
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