Turkey Embryo Lethality Assay of Streptococcus gallolyticus Field Isolates from Poultry

Since 2020, <i>Streptococcus gallolyticus</i> infection has emerged as one of the leading causes of disease in commercial turkey poults, causing fatal bacterial sepsis without any prior clinical signs except in unusual cases involving central nervous system signs. In this study, eight <i>S. gallolyticus</i> strains-<i>S. gallolyticus</i> subsp. <i>pasteurianus</i> (<i>n</i> = 7) and <i>S. gallolyticus</i> subsp. <i>gallolyticus</i> (<i>n</i> = 1)-were used in a turkey embryo lethality assay that were isolated from outbreaks involving commercial poults in the U.S. Midwest. API 20 STREP V8.0 and API RAPID ID 32 STREP V4.0 were used to identify these <i>S. gallolyticus</i> strains to subspecies level and obtain biochemical profile codes. Whole-genome sequencing revealed eight genetically distinct strains with more than 1283 single-nucleotide polymorphism differences to each other. Additionally, three of these strains contained a potential virulence factor similar to Acb protein, a putative collagen adhesin that promotes high-affinity binding to host-cell-immobilized collagen, suggesting increased advantage for intestinal tissue colonization and bloodstream translocation. Each strain was inoculated at a high (10⁶ CFU/ml) and low dose (10³ CFU/ml) through the air cell of eggs obtained from Nicholas breeding line into the allantoic cavity on day 15 of embryogenesis. Eggs were candled daily for 7 days to determine embryo mortality, thus assessing virulence potential of the strains. The cumulative embryo mortality was highest in the neurological <i>S. gallolyticus</i> subsp. <i>pasteurianus</i> strain in both high (10⁶ CFU/ml) and low (10³ CFU/ml) doses and <i>S. gallolyticus</i> subsp. <i>pasteurianus</i> strain 4 in the high dose (10⁶ CFU/ml). Most embryos that died were inoculated with the high dose of any strain on one day postinoculation. Dead embryos infected with the high dose on days 1 and 2 postinoculation showed generalized superficial and internal petechial hemorrhages and congestion of organs, as well as aggregates of Gram-positive coccoid bacteria in blood vessels and organs, consistent with bacterial sepsis. Only one of the three strains containing the Acb-like protein, the neurological <i>S. gallolyticus</i> subsp. <i>pasteurianus</i> strain, caused increased mortality, suggesting that Acb alone is not responsible for enhanced virulence in this embryo model. Rather, it is possible that additional novel virulence factors, such as L-arabinose metabolism discovered in the neurological <i>S. gallolyticus</i> subsp. <i>pasteurianus</i> strain, may enhance virulence fitness.