Amplicon sequencing of bacterial microbiota in abortion material from cattle

Abstract

Abortions in cattle have a significant economic impact on animal husbandry and require prompt diagnosis for surveillance of epizootic infectious agents. Since most abortions are not epizootic but sporadic with often undetected etiologies, this study examined the bacterial community present in the placenta (PL, n = 32) and fetal abomasal content (AC, n = 49) in 64 cases of bovine abortion by next generation sequencing (NGS) of the 16S rRNA gene. The PL and AC from three fetuses of dams that died from non-infectious reasons were included as controls. All samples were analyzed by bacterial culture, and 17 were examined by histopathology. We observed 922 OTUs overall and 267 taxa at the genus level. No detectable bacterial DNA was present in the control samples. The microbial profiles of the PL and AC differed significantly, both in their composition (PERMANOVA), species richness and Chao-1 (Mann–Whitney test). In both organs, Pseudomonas was the most abundant genus. The combination of NGS and culture identified opportunistic pathogens of interest in placentas with lesions, such as Vibrio metschnikovii, Streptococcus uberis, Lactococcus lactis and Escherichia coli. In placentas with lesions where culturing was unsuccessful, Pseudomonas and unidentified Aeromonadaceae were identified by NGS displaying high number of reads. Three cases with multiple possible etiologies and placentas presenting lesions were detected by NGS. Amplicon sequencing has the potential to uncover unknown etiological agents. These new insights on cattle abortion extend our focus to previously understudied opportunistic abortive bacteria.

Review of Diagnostic Procedures and Approaches to Infectious Causes of Reproductive Failures of Cattle in Australia and New Zealand

“…Individual or multiplex PCR analyses can be performed on fresh fetal tissues. If bacteria are also isolated from the placenta or cultured from fetal stomach contents, liver or lung, this provides a highly probable positive diagnosis ( 203 ). Even more so if lesions consistent with bacterial infection are present, and other causes of abortion have been ruled out.…”

Limited added value of fungal ITS amplicon sequencing in the study of bovine abortion

“…The macroscopic findings for 52 cases and corresponding histological findings for 40 placentas are summarized in Table 5 and Supplementary Table 2, together with the results of comprehensive microbiological analyses. The results of six cases previously reported in Vidal et al. (2017b) are included.…”

“…Maternal serum was also received for 63 cases. Samples were obtained according to Vidal et al. (2017b) .…”

“…Extraction of total genomic DNA was performed for all samples of PL (n = 72) and AC (n = 63) submitted and from healthy fetuses [PL (n = 3), AC (n = 3) and AF (n = 3)] according to Vidal et al. (2017a ; 2017b) . DNA extraction included two extraction control tubes containing only reagents.…”

“…Recently, 16S rRNA gene amplicon sequencing was found to be useful for identifying a wide spectrum of pathogens from bovine abortion material, providing insight into new species and polymicrobial infections ( Vidal et al., 2017b ). Due to the considerable limitations in detecting mycobiota by culture methods, the number of studies investigating fungal microbiota using amplicon sequencing of the ribosomal internal transcribed spacer (ITS) region, the universal DNA barcode marker for fungi, is increasing ( Schoch et al., 2012 ; Zoll et al., 2016 ).…”