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Eklund, Madelene, 2017. Quantitative polymerase chain reaction method compared with traditional methods for Clostridium tyrobutyricum in grass-clover silage. Second cycle, A2E. Uppsala: SLU, Dept. of Animal Environment and Health

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Abstract

C. tyrobutyricum is the most common type of bacteria to contaminate milk and cause late blown cheese caused by high spore counts in silage. The purpose of this thesis was to test a qPCR method to quantify C. tyrobutyricum in silage, by investigating potential differences in the clostridia spore estimate between the qPCR method, the MPN method and the plate count method. Furthermore, relationships between silage fermentation characteristics and clostridia spore contents analysed with the different methods were evaluated. The silage that was used was from a sward containing 75% red clover and 25% grass that was harvested on the 4th of September 2011 (third harvest) at Skara in the southwest of Sweden. The silage fermentation parameters were analyzed and the C. tyrobutyricum count was analysed with Qauntitative Polymerase Chain Reaction (qPCR method), most probable number method (MPN-method) and plate count method. Relationships between clostridia spore counts from the traditional laboratory methods (the plate method and the MPN method) and the qPCR method were investigated by linear regression analysis using PROC REG in SAS Also, the least-square means of clostridia spore counts from the analytical methods were compared in the general linear model procedure of SAS (PROC GLM). Stepwise regressions on the relationships between silage fermentation characteristics and clostridia spore count from the different methods were performed. Comparisons of the least-square (LS) means of clostridia spore count from the different methods did not show any significant differences between the methods. The simple linear regressions showed that the MPN method performed at the laboratory of SLU, Skara, Sweden (MPN-S) and the plate method had moderate to strong correlations with the qPCR method, which indicates their reliability for estimating the Clostridium spore content in clover-grass silage. The MPN method performed at the LKS mbH, Germany (MPN-D) showed low correlations with the qPCR. The qPCR method showed very high correlation with the fermentation parameters compared to the MPN-D and the plate count methods. Generally, the qPCR method hade higher correlation with the fermentation parameters and smaller variations compared with MPN and plate-method. Conclusion of the study was no significant differences in Clostridia spore counts of red clover-grass silage between the qPCR, MPN-D, MPN-S and the plate methods when comparing the LS means of the Clostridia counts from the different methods. The simple linear regressions showed that the MPN-S and the plate method had moderate to strong correlations with the qPCR method. The MPN-D showed low correlations with the qPCR. The high specificity for the qPCR method decreases the risk of counting other lactate degraders than C. tyrobutyricum in the silage. The qPCR method showed very high correlation with the fermentation parameters compared to the MPN-D and the plate count methods. The MPN-S method showed promising relationships with the fermentation products in the silage.

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C. tyrobutyricum är den vanligaste bakterien till att kontaminera mjölk och orsaka sprängda ostar orsakat av höga sporhalter i ensilaget. Syftet med denna studie var att testa qPCR metoden för kvantifiering av C. tyrobutyricum i ensilage, genom att undersöka potentiella skillnader i den uppskattade mängden klostridiesporer mellan qPCR, MPN metod och platt metoden. Vidare undersöktes samband mellan ensilagets fermentationsparametrar och mängden klostridiesporer analyserade med de olika metoderna. Ensilaget som användes i undersökningen kommer från en vall som innehåller 75 % rödklöver och 25 % gräs som skördades den 4:e September 2011 (tredje skörden) i Skara i sydvästra Sverige. Ensilagets fermentationsparametrar analyserades och C. tyrobutyricum mängden analyserades med kvantitativ polymerasekedjereaktion (qPCR metoden), mest sannolika antalmetoden (MPN-metoden) och plattspridningsmetoden. Relationen mellan mängden klostridiesporer härrört ur de traditionella laboratoriemetoderna (plattmetoden och MPN-metoden) och qPCR-metoden analyserades i linjär regressionsmodell med PROC REG i SAS. Även jämförelser av medelvärden för klostridiespormängd mellan de olika metoderna utfördes i generell linjärmodell i SAS (PROC GLM). Stegvis regressionsanalys mellan fermentationsparametrar och klostridiespormängden från var och en av metoderna genomfördes. Det påvisades inga signifikanta skillnader i LS mean klostridiespormängd från de olika metoderna. De enkla linjära regressionerna visade att MPN-metoden som utförts vid laboratoriet i SLU, Skara, Sverige (MPN-S) och plattmetoden hade måttliga till starka korrelationer med qPCR-metoden, vilket indikerar deras tillförlitlighet för att uppskatta Klostridium sporinnehållet i klöver-gräs ensilage. MPN-metoden utförd vid LKS mbH, Tyskland (MPN-D) visade låga korrelationer med qPCR. QPCR-metoden visade mycket hög korrelation med fermentationsparametrarna jämfört med MPN-D- och platträkningsmetoden. Generellt hade qPCR-metoden högre korrelation med fermentationsparametrarna och mindre variationer jämfört med MPN- och plattmetoden. Slutsatsen av studien var, analysen där LS mean jämfördes mellan metoderna gav ingen signifikant skillnad i klostridium mängd mellan qPCR, MPN-S, MPN-D och plattspridnigsmetoden i klöver-gräs ensilage. Enkla linjära regressioner visade att MPN-S och Platt metoden hade starka korrelationer till qPCR metoden. MPN-D visar låg korrelation med qPCR Specificiteten hos qPCR metoden minskar risken att andra laktat fermenterare medräknas. qPCR- metoden visade stark korrelation med fermentationsparametrarna i jämförelse med MPN-D och plattspridningsmetoden. Även MPN-S visade korrelation med fermentationsparametrarna.

Main title:Quantitative polymerase chain reaction method compared with traditional methods for Clostridium tyrobutyricum in grass-clover silage
Authors:Eklund, Madelene
Supervisor:Nadeau, Elisabet
Examiner:Johansson, Birgitta
Series:Studentarbete / Sveriges lantbruksuniversitet, Institutionen för husdjurens miljö och hälsa
Volume/Sequential designation:719
Year of Publication:2017
Level and depth descriptor:Second cycle, A2E
Student's programme affiliation:VM003 Animal Science - Master's Programme 120 HEC
Supervising department:(VH) > Dept. of Animal Environment and Health
Keywords:C. tyrobutyricum, qPCR, silage
URN:NBN:urn:nbn:se:slu:epsilon-s-8247
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-s-8247
Subject. Use of subject categories until 2023-04-30.:Animal feeding
Language:English
Deposited On:25 Oct 2017 06:41
Metadata Last Modified:25 Oct 2017 06:41

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