Abortion in cattle causes significant economic losses for cattle farmers worldwide. The diversity of abortifacients makes abortion diagnostics a complex and challenging discipline that additionally is restrained by time and economy. Microbial culture has traditionally been an important method for the identification of bacterial and mycotic abortifacients. However, it comes with the inherent bias of favoring the easy-to-culture species, e.g., those that do not require cell culture, pre-enrichment, a variety of selective growth media, or different oxygen levels for in vitro growth. Molecular methods such as polymerase chain reaction (PCR) and next-generation sequencing have been established as alternatives to traditional microbial culturing methods in several diagnostic fields including abortion diagnostics. Fluorescence in situ hybridization (FISH), a bridging microscopy technique that combines molecular accuracy with culture independence, and spatial resolution of the pathogen-lesion relation, is also gaining influence in several diagnostic fields. In this study, real-time quantitative PCR (qPCR), 16S rDNA amplicon sequencing, and FISH were applied separately and in combination in order to (i) identify potentially abortifacient bacteria without the bias of culturability, (ii) increase the diagnostic rate using combined molecular methods, (iii) investigate the presence of the difficult-to-culture zoonotic agents Coxiella burnetii, Chlamydia spp., and Leptospira spp. in bovine abortions in Denmark. Tissues from 162 aborted or stillborn bovine fetuses and placentas submitted for routine diagnostics were screened for pathogenic bacteria using 16S rDNA amplicon sequencing. Lesion association of fungal elements, as well as of selection of bacterial abortifacients, was assessed using specific FISH assays. The presence of Chlamydia spp. and chlamydia-like organisms was assessed using qPCR. The study focused on bacterial and fungal abortifacients, because Danish cattle is free from most viral abortifacients. The 16S rDNA amplicon sequencing–guided FISH approach was suitable for enhancing abortion diagnostics, i.e., the diagnostic rate for cases with tissue lesions (n = 115) was increased from 46 to 53% when compared to routine diagnostic methods. Identification of Bacillus licheniformis, Escherichia coli, and Trueperella pyogenes accounted for the majority of additional cases with an established etiology. No evidence for emerging or epizootic bacterial pathogens was found. The difficult-to-culture abortifacients were either not detected or not identified as abortifacients.

The objective of the study was to identify the location of the present bacteria in the uterus and oviducts of cows with pyometra. Pyometra is one of the postpartum infectious diseases in cattle that can result in infertility and thereby affect reproduction performance. Reproductive tracts (n = 21) were collected at a slaughterhouse in Denmark and send to The University of Copenhagen for examination and sampling. The uteri were included in the study when the following criteria were met: the cow was more than 21 days postpartum, the uterus was distended with pus, the cervix was closed, and a corpus luteum was present in one or both ovaries. A full thickness uterine tissue sample from the previous pregnant horn and both oviducts were sampled and then fixed in formalin. The tissues were trimmed, processed by routine methods, embedded in paraffin, sectioned at 3 microns, and prepared for fluorescence in situ hybridization using a probe targeting the 16S ribosomal RNA of the domain bacteria (i.e. targeting all bacteria regardless of species). Using fluorescence microscopy, the presence of bacteria within or on the surface of the endometrium and in the oviducts were noted. The endometrial biopsies from all cows (n = 21) contained bacteria, while 75% (16/21) of the cows had bacteria in one or both oviducts. The bacteria were located on the luminal surface and in the lamina propria in 38.1% (8/21) of the uterine biopsies. In the remaining 62% of the uterine biopsies, the bacteria were only located above the basal membrane. Regarding the oviduct biopsies, the bacteria were located on the luminal surface and in lamina propria in 9.5% (2/21) of the biopsies, whereas the bacteria were located only above the basal membrane in 90.5% of the biopsies. In conclusion, 1) bacteria are present in the uteri and oviducts of cows with pyometra and 2) the bacteria are primarily located on the luminal epithelia surface above the basal membrane. Further analyses will investigate which specific species of bacteria that are located in the lamina propria of the uterine and oviduct biopsies.

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