Faculty of Veterinary Medicine University of Zagreb
Abstract
Metagenomic and traditional microbial culture based analyses of milk samples from cows harbouring subclinical mastitis pathogens were carried out to identify the microbial community structure of milk. A total of 77 Triple cross (TP), Kankrej and Gir lactating cows and 301 quarters were screened for subclinical mastitis. A total of 106 isolates belonging to five different microbial genera were recovered from 91 quarters of 41 cows, including 15 quarters having mixed bacterial infections by cultural examination. Pyrosequencing readings obtained from the breed wise pooled DNA of subclinical mastitis milk samples were analyzed using the SEED subsystem database of Meta Genome Rapid Annotation with Subsystem Technology (MG-RAST). Among the five genera, Staphylococcus, Streptococcus, Micrococcus, Bacillus and Escherichia, detected in the subclinical mastitis milk samples by culture based methods, four genera, Staphylococcus, Streptococcus, Bacillus and Escherichia, were identified in the corresponding pyrosequencing data, while Micrococcus was not found. In contrast, the pyrosequencing yielded 28 bacterial species, of which only two species, S. aureus and E. coli, were identified by the cultural method. S. agalactiae, the third species identified by cultural method, was not found in the pyrosequencing data. Metagenomic analysis additionally identified 19 genera and 26 species in comparison with the routine cultural methods. Many of the fastidious / anaerobic bacterial organisms, which are difficult to cultivate by routine methods, were identified by metagenomic analyses.Radi identifikacije mikrobne zajednice u mlijeku provedena je metagenomska i uobičajena kulturelna pretraga uzoraka mlijeka krava sa supkliničkim mastitisom. Ukupno je 77 trostruko križanih Kankrej i Gir mliječnih krava i 301 četvrt vimena bilo pretraženo na supklinički mastitis. Izdvojeno je bilo 106 izolata svrstanih u pet različitih rodova iz 91 četvrti od 41 krave uključujući i 15 četvrti kod kojih je kulturelnom pretragom bila ustanovljena mješovita bakterijska infekcija. Sljedovi mješavine DNA izdvojeni iz uzoraka mlijeka kod supkliničkog mastitisa očitani pirosekvenciranjem bili su analizirani po podsustavu SEED baze podataka „Meta Genome Rapid Annotation with Subsystem Technology (MG-RAST)“. Iz pretraženih uzoraka mlijeka bilo je izdvojeno pet rodova: Staphylococcus, Streptococcus, Micrococcus, Bacillus i Escherichia. Četiri su bila dokazana postupkom pirosekvenciranja: Staphylococcus, Streptococcus, Bacillus i Escherichia, dok Micrococcus nije bio dokazan. S druge strane, pirosekvenciranjem je bilo dokazano 28 bakterijskih vrsta, od kojih su samo dvije, S. aureus i E. coli, bile dokazane klasičnom kulturelnom pretragom. S. agalactiae, treća vrsta identificirana kulturelnom pretragom nije bila dokazana postupkom pirosekvenciranja. Metagenomskom analizom dodatno je bilo dokazano 19 rodova i 26 vrsta u usporedbi s rutinskom kulturelnom pretragom. Mnoge anaerobne bakterije, koje je vrlo teško uzgojiti rutinskim metodama, bile su identificirane metagenomskom analizom
