The Animal Health Centre (AHC) is the provincial veterinary diagnostic laboratory for British Columbia and is accredited by the American Association of Veterinary Laboratory Diagnosticians (AAVLD). Case accessions included a range of tissue samples and whole carcasses obtained from local producers, veterinarians, and the general public. Bovine fetuses presented for necropsy underwent a standardized protocol, during which body weight and crown-to-rump length are recorded. All organ systems were examined internally and externally, and tissue samples were collected for routine bacteriology, histopathology, and other ancillary testing as deemed appropriate by a veterinary pathologist. Samples for bacteriology included the abomasal content, placenta, and lung tissues. These tissues were processed using conventional techniques, which included initial surface searing, followed by inoculation onto blood agar and MacConkey agar plates (Oxoid, ON). The samples were then incubated aerobically for up to 48 h. For any abortion cases, selective Salmonella culture was performed on Hektoen and XLT4 agar plates (Oxoid, ON), followed by selective enrichment in selenite broth. The colonies that grew on the agar plates were identified using biochemical tests and MALDI-TOF mass spectrometry (Bruker, ON), and the results were recorded in the proprietary veterinary information management system, VADDS&Vetstar developed by Advanced Technology Corp (Ramsey, NJ). For this study, S. pluranimalium isolates were archived at -20C for further analysis. The subsamples of the isolates were stored at -80C. Additional tissue samples were systematically collected for histopathological analysis, processed with an automated processor, embedded in paraffin, and sectioned at 5 um. These sections were stained with hematoxylin and eosin using an automated stainer, cover-slipped, and then reviewed by board-certified veterinary pathologists. Microscopic lesions were graded on a scale from 0 (no apparent lesions) to 4 (severe lesions). A summary case report was prepared. Based on the histopathology results, ancillary diagnostic studies were conducted to screen for Neospora caninum, bovine viral diarrhea, Chlamydophila abortus, and Ureoplasma diversum (Hewinson et al., 1997), along with radial immunodiffusion testing for bovine IgG and IgM in select cases.

To determine the number of S. pluranimalium isolates recovered at the AHC, a database search covering the period from 2008 to 2024 was performed using the parameters of bovine, fetus, and abortion.

Bacterial isolates were recovered from freezer stocks by initial culturing on tryptic soy agar plates supplemented with 5% defibrinated sheep’s blood and incubating overnight at 37°C. A single colony was picked and inoculated into tryptic soy broth supplemented with 5% defibrinated sheep’s blood, and the culture was shaken overnight at 37°C. DNA extractions were performed using Qiagen’s DNeasy Blood & Tissue kit (Toronto, ON), following the supplemental steps for the pretreatment of Gram-positive bacteria. The extracted DNA was then assessed for purity and concentration using gel electrophoresis, Nanodrop, and Qubit.

The samples were then prepared for sequencing on MinION, Oxford Nanopore Technologies (ONT) (Oxford, UK), using their Native Barcoding Kit (EXP-NBD114). This process involved repairing the DNA and performing end-preparation using the NEBNext FFPE DNA Repair Mix and the NEBNext Ultra II End Repair/dA-Tailing Module reagents (New England Biolabs, NEB; Whitby, ON). The samples were purified using AMPure XP beads, and the native barcodes were then ligated onto the fragments using Blunt/TA Ligase Master Mix (NEB). The samples were purified using AMPure XP beads, quantified, and then pooled in equimolar amounts. The ONT adapters were ligated to the barcoded pool using T4 Ligase (NEB) before being loaded onto an R10.3 flow cell, following ONT specifications. The samples were sequenced for 72 h and base-called using the MinKNOW (v22.03.6) high-accuracy base-calling model.

Raw reads had adaptors trimmed using Porechop v0.2.4 (Wick, 2018) and were quality-filtered using NanoFilt v2.8.0 (using the parameters -q 10, −l 300, −-headcrop 40) (De Coster et al., 2018). The filtered reads were assembled using Flye v2.9.1 (Kolmogorov et al., 2019) and polished using Medaka v1.11.1 (ONT, 2024). The genomes were annotated using Bakta v1.7 (Schwengers et al., 2021), and a Maximum likelihood phylogenetic tree was created using the tool autoMLST (accessed August 23, 2022) (Alanjary et al., 2019). Genome completeness was assessed using BUSCO v5.7.1 (Simão et al., 2015). The Virulence Factor Database (accessed May 10, 2024) (Liu et al., 2022) and Abricate v1.0.0 (Seemann, 2020) were used to search for the presence of virulence factors. The Comprehensive Antibiotic Resistance Database (CARD) v3.2.9 (McArthur et al., 2013), coupled with RGI v6.0.2, was used to examine antibiotic resistance genes (ARGs). Clusters of Orthologous Groups analysis was conducted using the EggNOG-mapper v2.1.9 (Cantalapiedra et al., 2021) and EggNOG Database v5.0.2 (Huerta-Cepas et al., 2018). Plasmids were identified using Mobsuite v3.0.3 (Robertson and Nash, 2018), and phage sequences were identified using Virsorter v2.2.3 (Guo et al., 2021).

Between 2008 and 2024, 382 fetuses were submitted to the AHC for diagnostic evaluation. Each case was reviewed individually, and the signalment, gross pathology, histopathology, bacteriology, and molecular results were tabulated and scored. Using MALDI-TOF mass spectrometry, S. pluranimalium was identified in 10 fetal samples, which then underwent further characterization. In these 10 samples, additional bacterial isolates were taxonomically identified at the species level or, if the species level could not be determined, at the genus level. Overall, 13 bacterial species and genera were isolated from the lung, stomach contents, and placenta (Supplementary Table 1). These included S. pluranimalium (n = 11), Streptococcus uberis (n = 2), the Glutamicibacter genus (n = 2), the Staphylococcus genus (n = 3), Enterococcus saccharolyticus (n = 1), the Psychrobacter genus (n = 1), the Arthrobacter genus (n = 3), Aerococcus viridans (n = 1), the Vibrio genus (n = 1), the Acinetobacter genus (n = 5), the Corynebacterium genus (n = 2), Enterococcus faecium (n = 1), and Escherichia coli (n = 5). In addition, sample BC-AHC-08 contained the fungi Aspergillus fumigatus and a species of Candida. The presence of three other pathogens—Neospora caninum (n = 8), Bovine Viral Diarrhea Virus (n = 1), and Ureoplasma diversum (n = 1)—was confirmed either through polymerase chain reaction or serology.

The histopathology results indicated the presence of several lesions in the aborted fetuses (Table 1), affecting various tissues including the brain, lungs, heart, muscles, kidneys, and eyes. Lesions in the eyes (n = 6) and placenta (n = 7) were the most common. Sample BC-AHC-03 exhibited the most extensive lesions, affecting six different tissues. No histopathological abnormalities were apparent in sample BC-AHC-05.

Among the 10 fetal samples with MALDI-TOF-confirmed S. pluranimalium, isolates from 9 were further characterized using molecular methods. Whole-genome sequencing was performed on one isolate from each sample, except for sample BC-AHC-09, from which two isolates were sequenced: BC-AHC-09_a isolated from the lung and BC-AHC-09_b isolated from the stomach. The BC strains had an average genome size of 2,313,582 base pairs (bp) and an average GC content of 38.59%, similar to the reference strain TH11417 (Pan et al., 2018), which has a genome size of 2,065,522 and a GC content of 38.65 (Table 2). The BC strains had an average of 15.2 contigs per assembly and an average genome completeness of 96.63%, as assigned by BUSCO.

A maximum likelihood phylogenetic tree was constructed based on the analysis of core, single-copy genes present in all strains (Figure 1). The phylogenetic tree was constructed using the 10 BC genomes and 6 other publicly available genomes from the NCBI as of July 2024. The six genomes from the NCBI include TH11417 (Pan et al., 2018), 14A0014 (Rodriguez Campos et al., 2018), Colony612 (Buathong et al., 2021), SP21-2 (Xu et al., 2023), SP28 (Zhu, 2023), and SUG2384 (Holman et al., 2024). The BC strains formed a distinct clade, with strain BC-AHC-06_a being the most divergent. Strains BC-AHC-09_a and BC-AHC-09_b were isolated from the same sample but were separated on the phylogenetic tree. The publicly available strain, 14A0014, which was also recovered from a bovine abortion sample, was the most genetically distinct compared to the BC strains.

Clusters of orthologous groups (COGs) is a method of phylogenetic classification that clusters each gene into a COG ID and assigns each COG ID to a functional category. The COG analysis, supported by Fisher’s exact test, revealed that the BC strains, except for strain BC-AHC-06_a, contained a significantly higher proportion of COG IDs associated with category X compared to other S. pluranimalium strains (p < 0.05) (Figure 2A). The BC strains, excluding BC-AHC-06_a, had an average of 12.5% of all COG IDs associated with category X. In comparison, strain BC-AHC-06_a had only 2.7% of its COG IDs associated with category X, which is similar to the non-BC strains that had an average of 3.2% of their COG IDs. With the exception of strain BC-AHC-06_a, many COG IDs associated with category X that were in high abundance were shared among all of the BC strains (Figure 2B).

Utilizing the tool Abricate and the Virulence Factor Database, seven different virulence factors (VFs) were identified in the BC strains, encompassing four virulence classes: immune modulation, stress survival, exotoxin, and adherence (Table 3). All BC strains contained the gene cluster rfbA, rfbB, and rfbC, with an 80–82% nucleotide identity. The VFs tig/ropA, plr/gapA, and eno were also found in all BC strains, with identities of approximately 82, 89, and 90%, respectively. Strain BC-AHC-06_a contained the SSU98_0978 virulence factor, with a nucleotide identity of 95%.

Utilizing the Resistance Gene Identifier tool and the Comprehensive Antibiotic Resistance Database, several genes conferring resistance to multiple antibiotics were identified using strict and perfect criteria (Figure 3). Antibiotic resistance genes (ARGs) conferring resistance to a number of antibiotics such as glycopeptides (vanY genes), lincosamides (lnuC), tetracycline (tet(M)), and macrolides (mreA) were detected (Table 2). Strains BC-AHC-08_a and BC-AHC-09_a contained the largest number of ARGs, with four each.

Using the tool MOB-suite, two novel plasmids were identified. The first was a 10.8 kb plasmid identified in strains BC-AHC-01_a and BC-AHC-02_a, and the second was a 5.4 kb plasmid identified in strains BC-AHC-03_a, BC-AHC-04_a, BC-AHC-08_a, and BC-AHC-09_b. Both plasmids showed the closest similarity to NSUI060 (Athey et al., 2016), a 5.6 kb Streptococcus suis plasmid. These S. pluranimalium plasmids did not contain any known virulence factors or antimicrobial resistance genes, had the MOBV relaxase type, and were classified as mobilizable.

Using VirSorter2, a machine learning tool that predicts phage sequences based on features such as the number of hallmark viral genes, the percentage of viral genes, and the mean GC content, we identified 41 putative prophages in the BC strains (Supplementary Table 2). Their lengths ranged from 1,024 bp to 295,435 bp. Each BC strain had an average of 291,212 bp of phage sequences. In comparison, the non-BC strains Colony612 and SP28 did not contain any phage sequences. The four other non-BC strains had an average of 147,501 bp of phage sequences per strain. Strains BC-AHC-03_a and BC-AHC-09_b contained two virulence factors, tig/ropA and plr/gapA, located on a phage sequence. The eno virulence factor was also located on a phage sequence in strain BC-AHC-06_a. Strain BC-AHC-08_a contained a phage sequence with three ARGs: the vanY gene in the vanB cluster, mreA, and lnuC. In addition, strain BC-AHC-09_a contained one phage sequence with one ARG, lnuC.