ANALYSIS OF THE UTERINE MICROBIOME IN COWS WITH RETENTIO SECUNDINARUM AND THE DYNAMICS OF ITS CHANGES DURING THERAPY

Abstract

Retentio secundinarum (RS) in cows is defined as the failure to expel the placenta 24 hours
after calving. This complication is the cause of significant economic losses due to
reproductive disorders including: uterine inflammation, prolonged postpartum downtime,
reduced mating rates, prolonged inter-partum period and the development of secondary
metabolic diseases. These losses add to the cost of treating the animals. The microorganisms
most commonly found in RS cases were Escherichia coli, Fusobacterium necrophorum,
Clostridium spp. and Trueperella pyogenes, the presence of which in these cases justified the
domaic and general use of antibiotics, including third-generation cephalosporins. Their
widespread use in the current social context is unjustified. Other antibiotics have been
suggested for the treatment of RS. There is now a growing interest in other alternative
treatments. Nowadays, precise genetic tools are available to assess the type of
microorganisms. The aim of this study was to determine the uterine microbiome of cows with
RS. Metagenomic analysis of the popuation of batteries and archaeons was carried out on the
basis of the hypo-ergodic V3-V4 region of the S rRNA gene. Specific primer sequences 34IF
and 785R (16S analysis) were used to amplify the selected region and prepare the library.
PCR was performed using Q5 Hot Start High-Fidelity 2X Master Mix, according to the
manufacturer’s recommendations. Sequencing was performed on a MiSeq, paired-end (PE),
2×300 nt, using Illumina’s v3 kit. Automatic pre-analysis of the data was performed on a
Miseq sequencer using Miseq Reporter (MSR) v 2.6 software. The analysis consisted of two
steps: (1) automatic demultiplexing of samples and (2) generation of fastq files containing
raw reads. Based on the evaluation of the preliminary results of the NGS sequencing analysis,
a predominance of the bacteria Fusobacterium necrophorum, Coxiella burnetii, Esscherichia
coli, Streprococcus pluranimalium and Clostridium perfringens was observed in the test
specimens immediately after placenta detachment. It is planned to continue the study to
confirm the composition of the uterine microbiome in a larger number of animals in order to
develop a microbiome typical of RS cases and also to observe it after antibiotic and eubiotic
therapy.