Bovine Abortions Revisited—Enhancing Abortion Diagnostics by 16S rDNA Amplicon Sequencing and Fluorescence in situ Hybridization

Abstract

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.

Antimicrobial Susceptibility of Bacterial Causes of Abortions and Metritis in Buffaloes (Bubalus bubalis) in Bareilly Region, Uttar Pradesh, India

“…However, many of the similar types of bacteria as identified in this study are reported to cause of abortions, as Salmonella enterica serovars including S. Dublin in cattle [23][24][25], Aeromonas species in equids [26] and bovines [27], Enterobacter agglomerans in equids [28,29], Moelerella wisconsensis in bovines [30,31], Xenorhabdus bovienii and X. poinarii in bovines [30], K. pneumoniae in cattle [27]. However, K. varians identified as cause of abortion and or me- [11,12,27,30,31,34,35] but some like K. varians and V. alginolyticus have rarely been associated with bovine infections but reported in human as opportunistic pathogens. Similar types of bacteria have been reported in vaginal tract of apparently healthy sows [35] and a closely related animal, mithun (Bos frontalis), to buffaloes [34] indicating that the bacteria identified in the present study might be opportunistic pathogens of reproductive tract.…”

Coxiella burnetii and Co-Infections with Other Major Pathogens Causing Abortion in Small Ruminant Flocks in the Iberian Peninsula

“…A panel of commercial qPRC and RT-qPCR assays were used for a rapid diagnosis of these pathogens. These molecular methods have been reported to provide highly sensitive, specific and rapid tools for the specific detection of different abortifacient infectious agents in various clinical samples, with multi-screening qPCR approaches allowing the simultaneous detection of the main abortive agents [ 45 , 46 , 47 ]. It is generally accepted that both standard PCR and qPCR provides diagnostic information on pathogens existing in samples [ 48 ].…”

Unconventional Sites for Diagnosis of Leptospirosis in Bovine Anicteric Fetuses

“…genus, which infects humans, domestic and wild animals [ 1 ]. In bovines, leptospirosis plays an important role as one of the main reproductive diseases, leading to important economic hazards [ 2 , 3 ]. Leptospires can colonize the genital tract and kidneys of bovines and its transmission occurs from direct contact with infected animals or a contaminated environment [ 4 , 5 ].…”

“…Since different pathogens can lead to abortions in cattle, the proper diagnosis is crucial to implement control measures. The necropsy of aborted fetuses followed by molecular analysis of its tissues and placenta is highly encouraged for the diagnosis of animal leptospirosis [ 3 , 22 , 23 ]. Detecting bacterial pathogens in fetuses is particularly challenging due to autolysis, which can happen even before the abortion is expelled, interfering with the bacterial load in the organs [ 12 , 13 , 23 ].…”