Abstract
Dairy cow mastitis is an important disease in the dairy industry. Different microbial species have been identified as causative agents in mastitis, and are traditionally diagnosed by bacterial culture. The objective of this study was to use metagenomic pyrosequencing of bacterial 16S rRNA genes to investigate bacterial DNA diversity in milk samples of mastitic and healthy dairy cows and compare the results with those obtained by classical bacterial culture. One hundred and thirty-six milk samples were collected from cows showing signs of mastitis and used for microbiological culture. Additionally, 20 milk samples were collected from healthy quarters. Bacterial DNA was isolated from the same milk samples and the 16S rRNA genes were individually amplified and pyrosequenced. Discriminant analysis showed that the groups of samples that were most clearly different from the rest and thus easily discriminated were the normal milk samples from healthy cows and those characterised by culture as Trueperella pyogenes and Streptococcus spp. The mastitis pathogens identified by culture were generally among the most frequent organisms detected by pyrosequencing, and in some cases (Escherichia coli, Klebsiella spp. and Streptococcus uberis mastitis) the single most prevalent microorganism. Trueperella pyogenes sequences were the second most prevalent sequences in mastitis cases diagnosed as Trueperella pyogenes by culture, Streptococcus dysgalactiae sequences were the second most prevalent sequences in mastitis cases diagnosed as Streptococcus dysgalactiae by culture, and Staphyloccocus aureus sequences were the third most prevalent in mastitis cases diagnosed as Staphylococcus aureus by culture. In samples that were aerobic culture negative, pyrosequencing identified DNA of bacteria that are known to cause mastitis, DNA of bacteria that are known pathogens but have so far not been associated with mastitis, and DNA of bacteria that are currently not known to be pathogens. A possible role of anaerobic pathogens in bovine mastitis is also suggested.
Highlights
Dairy cow mastitis is arguably the most important disease for the dairy industry worldwide, causing economic losses due to reduced milk production, discarded milk, premature culling, and antibiotic usage
Diagnosis of Mastitis Aetiology by Bacterial Culture Based on bacterial culture of milk samples from 136 mastitic dairy cows, cases were diagnosed as infected with Streptococcus uberis, as Escherichia coli, 11 as Klebsiella spp., 17 as Staphylococcus aureus, 20 as Staphylococcus spp., 17 as Streptococcus dysgalactiae, 2 as Streptococcus spp., 3 as Trueperella pyogenes, and 33 samples were characterized as culture negative
The same 156 milk samples were subjected to metagenomic pyrosequencing of bacterial 16S rRNA genes
Summary
Dairy cow mastitis is arguably the most important disease for the dairy industry worldwide, causing economic losses due to reduced milk production, discarded milk, premature culling, and antibiotic usage. Limitations of classical bacterial culture such as a delay of 24–48 hours to obtain results, or the fact that in approximately 25% of milk samples from clinical mastitis cases bacteria are not detected in conventional culture [2] have spurred investigations of cultureindependent, molecular techniques for mastitis diagnosis. Methods such as real-time PCR [3], multiplex PCR (mPCR) [4], denaturing gradient gel electrophoresis (DGGE) PCR [5], and PCRsingle-strand conformation polymorphism (SSCP) [6] are being used to identify bacterial DNA in milk samples. Apart from addressing the above mentioned limitation in classical culture, molecular techniques have the potential to offer some insight in the microbial communities present in milk [7]
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