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

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.

Diagnostic studies of abortion in Danish cattle 2015-2017.

BACKGROUND: Abortion is a major source of economic losses in cattle breeding. Abortion occurs due to a wide range of causes, but infections are the most frequently diagnosed. However, establishing an aetiological diagnosis remains challenging due to the large variety of bacteria, protozoa, viruses, and fungi that have been associated with abortion in cattle. Economic restraints limit the range of diagnostic methods available for routine diagnostics, and decomposition of the conceptus or lack of proper fetal and/or maternal samples further restrict the diagnostic success. In this study, we report recent diagnostic findings from bovine abortions in Denmark, a country that has a large dairy sector and is free from most infectious agents causing epizootic abortion in cattle. The aims of the study were: (i) to identify infectious causes of bovine abortion in Denmark, (ii) to categorise the diagnostic findings based on the level of diagnostic certainty, and (iii) to assess the diagnostic rate. Due to economic restraints, only a limited panel of routine diagnostic methods were available. Placentas and/or fetuses from mid- to late-term abortions and stillbirths (n = 162) were submitted to the Danish National Veterinary Institute between January 2015 and June 2017. The aborted materials were examined macroscopically, histologically, and by bacterial culture. Maternal blood samples were tested for bovine viral diarrhoea virus (BVDV) antibodies. RESULTS: The likely aetiology of the abortion was diagnosed in 52 cases, resulting in a diagnostic rate of 33%. The most common cause was protozoal infection (19%) followed by infection with Trueperella pyogenes (3%), Staphylococcus aureus (2%), and non-haemolytic Escherichia coli (2%). Lesions in fetuses with a protozoal infection were consistent with neosporosis. In many cases (38%), inflammatory changes were found in the placenta and/or fetal organs but no specific aetiology was identified. Neither infection with Brucella spp. nor maternal BVDV antibodies were detected. The majority of submitting herds (92%) were each represented by fewer than three abortion cases over the study period. CONCLUSIONS: Protozoal infection, most likely neosporosis, was the most commonly diagnosed cause of abortion and the only one associated with potential epizootic abortion events. Despite using a reduced number of diagnostic methods in comparison to other abortion studies, the diagnostic rate of this study was within the range reported in an earlier Danish study, as well as in recent international studies. The low number of submitted cases per herd and the sparse anamnestic information provided at submission hampered conclusions on the potential epizootic character of the abortion events in question.

Fate of ptaquiloside-A bracken fern toxin-In cattle.

Ptaquiloside is a natural toxin present in bracken ferns (Pteridium sp.). Cattle ingesting bracken may develop bladder tumours and excrete genotoxins in meat and milk. However, the fate of ptaquiloside in cattle and the link between ptaquiloside and cattle carcinogenesis is unresolved. Here, we present the toxicokinetic profile of ptaquiloside in plasma and urine after intravenous administration of ptaquiloside and after oral administration of bracken. Administered intravenously ptaquiloside, revealed a volume of distribution of 1.3 L kg-1 with a mean residence-time of 4 hours. A large fraction of ptaquiloside was converted to non-toxic pterosin B in the blood stream. Both ptaquiloside and pterosin B were excreted in urine (up to 41% of the dose). Oral administration of ptaquiloside via bracken extract or dried ferns did not result in observations of ptaquiloside in body fluids, indicating deglycosolidation in the rumen. Pterosin B was detected in both plasma and urine after oral administration. Hence, transport of carcinogenic ptaquiloside metabolites over the rumen membrane is indicated. Pterosin B recovered from urine counted for 7% of the dose given intravenously. Heifers exposed to bracken for 7 days (2 mg ptaquiloside kg-1) developed preneoplastic lesions in the urinary bladder most likely caused by genotoxic ptaquiloside metabolites.