Optimization and Testing of a Commercial Viability PCR Protocol to Detect Escherichia coli in Whole Blood.

Bacteremia, specifically if progressed to sepsis, poses a time-sensitive threat to human and animal health. Escherichia coli is a main causative agent of sepsis in humans. The objective was to evaluate a propidium monoazide (PMA)-based viability PCR (vPCR) protocol to detect and quantify live E. coli from whole blood. We optimized the protocol by adding a eukaryotic-specific lysis step prior to PMA exposure, then used spiking experiments to determine the lower limit of detection (LOD) and linear range of quantification. We also compared the vPCR quantification method to standard colony count of spiked inoculum. Lastly, we calculated percent viability in spiked samples containing 50% live cells or 0% live cells. The LOD was 102 CFU/mL for samples containing live cells only and samples with mixed live and heat-killed cells. The linear range of quantification was 102 CFU/mL to 108 CFU/mL (R2 of 0.997) in samples containing only live cells and 103 CFU/mL to 108 CFU/mL (R2 of 0.998) in samples containing live plus heat-killed cells. A Bland-Altman analysis showed that vPCR quantification overestimates compared to standard plate count of the spiked inoculum, with an average bias of 1.85 Log10 CFU/mL across the linear range when only live cells were present in the sample and 1.98 Log10 CFU/mL when live plus heat-killed cells were present. Lastly, percent viability calculations showed an average 89.5% viable cells for samples containing 50% live cells and an average 19.3% for samples containing 0% live cells. In summary, this optimized protocol can detect and quantify viable E. coli in blood in the presence of heat-killed cells. Additionally, the data presented here provide the groundwork for further development of vPCR to detect and quantify live bacteria in blood in clinical settings.

Application of behavior data to predictive exploratory models of metritis self-cure and treatment failure in dairy cows.

The objective was to evaluate the performance of exploratory models containing routinely available on-farm data, behavior data, and the combination of both to predict metritis self-cure (SC) and treatment failure (TF). Holstein cows (n = 1,061) were fitted with a collar-mounted automated-health monitoring device (AHMD) from -21 ± 3 to 60 ± 3 d relative to calving to monitor rumination time and activity. Cows were examined for diagnosis of metritis at 4 ± 1, 7 ± 1, and 9 ± 1 d in milk (DIM). Cows diagnosed with metritis (n = 132), characterized by watery, fetid, reddish/brownish vaginal discharge (VD), were randomly allocated to 1 of 2 treatments: control (CON; n = 62), no treatment at the time of metritis diagnosis (d 0); or ceftiofur (CEF; n = 70), subcutaneous injection of 6.6 mg/kg of ceftiofur crystalline-free acid on d 0 and 3 relative to diagnosis. Cure was determined 12 d after diagnosis and was considered when VD became mucoid and not fetid. Cows in CON were used to determine SC, and cows in CEF were used to determine TF. Univariable analyses were performed using farm-collected data (parity, calving season, calving-related disorders, body condition score, rectal temperature, and DIM at metritis diagnosis) and behavior data (i.e., daily averages of rumination time, activity generated by AHMD, and derived variables) to assess their association with metritis SC or TF. Variables with P-values ≤0.20 were included in the multivariable logistic regression exploratory models. To predict SC, the area under the curve (AUC) for the exploratory model containing only data routinely available on-farm was 0.75. The final exploratory model to predict SC combining routinely available on-farm data and behavior data increased the AUC to 0.87, with sensitivity (Se) of 89% and specificity (Sp) of 77%. To predict TF, the AUC for the exploratory model containing only data routinely available on-farm was 0.90. The final exploratory model combining routinely available on-farm data and behavior data increased the AUC to 0.93, with Se of 93% and Sp of 87%. Cross-validation analysis revealed that generalizability of the exploratory models was poor, which indicates that the findings are applicable to the conditions of the present exploratory study. In summary, the addition of behavior data contributed to increasing the prediction of SC and TF. Developing and validating accurate prediction models for SC could lead to a reduction in antimicrobial use, whereas accurate prediction of cows that would have TF may allow for better management decisions.

Sequencing and characterization of Helcococcus ovis: a comprehensive comparative genomic analysis of virulence.

BACKGROUND: Helcococcus ovis (H. ovis) is an emerging bacterial pathogen that commonly causes opportunistic respiratory, mammary, and uterine infections across mammalian hosts. This study applied long- and short-read whole genome sequencing technologies to identify virulence factors in five H. ovis isolates with low, medium, and high virulence phenotypes. RESULTS: The resulting assemblies contained one circular chromosome ranging from 1,744,566 to 1,850,083 bp in length and had a mean GC content of 27.6%. Phylogenetic and nucleotide identity analyses found low virulence strain KG38 to be part of a clade that forms an outgroup apart from the rest of the H. ovis taxon. Assembling the first complete genomes of the species revealed major genomic rearrangements in KG38. One to six prophage regions were identified in each genome. A novel pathogenicity island was found exclusively in the two high virulence strains (KG37 and KG104), along with two hypothetical transmembrane proteins designated as putative VFs. Finally, three zinc ABC transporters and three Type-II/IV secretion systems were identified as possible virulence determinants in this species. The low virulence strain KG38 has fewer intact paralogs of these operons in its genome compared to the higher virulence isolates, which strongly suggests a role in virulence. This strain is also missing four putative virulence factors (VFs) found in other isolates associated with adherence (collagen adhesin precursor), immune evasion (choline-binding protein A and a PspA-like hypothetical protein) and cell wall synthesis (glycerol-3-phosphate cytidylyltransferase). CONCLUSIONS: In this study, we assembled reference-quality complete genomes for five H. ovis strains to identify putative virulence factors. Phylogenetic analyses of H. ovis isolates revealed the presence of a clade representing a potentially novel species within the genus Helcococcus. A novel pathogenicity island and two hypothetical transmembrane proteins were found exclusively in high-virulence strains. The identification of Zinc ABC transporters and Type-II/IV secretion systems as possible virulence determinants, along with the differences in operon content between the low and high virulence isolates, strongly suggests they also play a role in the bacterium's pathogenicity. Taken together, these findings are a valuable first step toward deciphering the pathogenesis of H. ovis infections.

Targeted reproductive management for lactating Holstein cows: Reducing the reliance on exogenous reproductive hormones.

Adoption of automated monitoring devices (AMD) affords the opportunity to tailor reproductive management according to the cow's needs. We hypothesized that a targeted reproductive management (TRM) would reduce the use of reproductive hormones while increasing the percentage of cows pregnant 305 d in milk (DIM). Holstein cows from 2 herds (n = 1,930) were fitted with an AMD at 251.0 ± 0.4 d of gestation. Early-postpartum estrus characteristics (EPEC; intense estrus = heat index ≥70; 0 = minimum, 100 = maximum) of multiparous cows were evaluated at 40 (herd 1) or 41 (herd 2) DIM and EPEC of primiparous cows were evaluated at 54 (herd 1) or 55 (herd 2) DIM. Control cows received the first artificial insemination at fixed time (TAI; primiparous, herd 1 = 82 and herd 2 = 83 DIM; multiparous, herd 1 = 68 and herd 2 = 69 DIM) following the Double-Ovsynch (DOV) protocol. Cows enrolled in the TRM treatment were managed as follows: (1) cows with at least one intense estrus were inseminated upon AMD detected estrus for 42 d and, if not inseminated, were enrolled in the DOV protocol; and (2) cows without an intense estrus were enrolled in the DOV protocol at the same time as cows in the control treatment. Control cows were re-inseminated based on visual or patch aided detection of estrus, whereas TRM cows were re-inseminated as described for control cows with the aid of the AMD. Cows received a GnRH injection 27 ± 3 d after insemination and, if diagnosed as nonpregnant, completed the 5-d Cosynch protocol and received TAI 35 ± 3 d after insemination. Among cows in the TRM treatment, 55.8 and 42.9% of primiparous and multiparous cows, respectively, received the first insemination in spontaneous estrus. The interaction between treatment and parity affected pregnancy 67 d after the first AI (primiparous: control = 37.6%, TRM = 27.4%; multiparous: control = 41.0%, TRM = 44.7%). The TRM treatment increased re-insemination in estrus (control = 48.3%, TRM = 70.5%). Pregnancy 67 d after re-inseminations tended to be affected by the interaction between treatment and EPEC (no intense estrus: control = 25.3%, TRM = 32.0%; intense estrus: control = 32.9%, TRM = 32.2%). The interaction between treatment and EPEC affected pregnancy by 305 DIM (no intense estrus: control = 80.8%, TRM = 88.2%; intense estrus: control = 87.1%, TRM = 86.1%). Treatment did not affect the number of reproductive hormone treatments among cows that had not had an intense estrus (control = 10.5 ± 0.3, TRM = 9.1 ± 0.2 treatments/cow), but cows in the TRM treatment that had an intense estrus received fewer reproductive hormone treatments than cows in the control treatment (2.0 ± 0.1 vs. 9.6 ± 0.2 treatments/cow). Selecting multiparous cows for first AI in estrus based on EPEC reduced the use of reproductive hormones without impairing the likelihood of pregnancy to first AI. The use of AMD for re-insemination expedited the establishment of pregnancy among cows that did not display an intense estrus early postpartum.

Establishing Galleria mellonella as an invertebrate model for the emerging multi-host pathogen Helcococcus ovis.

Helcococcus ovis (H. ovis) can cause disease in a broad range of animal hosts, including humans, and has been described as an emerging bacterial pathogen in bovine metritis, mastitis, and endocarditis. In this study, we developed an infection model that showed H. ovis can proliferate in the hemolymph and induce dose-dependent mortality in the invertebrate model organism Galleria mellonella (G. mellonella). We applied the model and identified H. ovis isolates with attenuated virulence originating from the uterus of a healthy post-partum dairy cow (KG38) and hypervirulent isolates (KG37, KG106) originating from the uterus of cows with metritis. Medium virulence isolates were also isolated (KG36, KG104) from the uterus of cows with metritis. A major advantage of this model is that a clear differentiation in induced mortality between H. ovis isolates was detected in just 48 h, resulting in an effective infection model able to identify virulence differences between H. ovis isolates with a short turnaround time. Histopathology showed G. mellonella employs hemocyte-mediated immune responses to H. ovis infection, which are analogous to the innate immune response in cows. In summary, G. mellonella can be used as an invertebrate infection model for the emerging multi-host pathogen Helcococcus ovis.

Tracing the source and route of uterine colonization by exploring the genetic relationship of Escherichia coli isolated from the reproductive and gastrointestinal tract of dairy cows.

The source and route of bacterial colonization of the uterus are still not established. The objective was to investigate the source and route of bacterial colonization of the uterus by exploring the genetic relationship among E. coli strains isolated from the gastrointestinal and the reproductive tract of dairy cows pre- and postpartum. Secondarily, uterine health status (metritis vs. healthy) was evaluated. Cows (n = 34) had the rectoanal junction (RAJ), vulva, and vagina swabbed every three days starting six days before expected calving until nine days postpartum. The uterus was swabbed postpartum. A blood sample was collected at all time points, but cultures were negative. Whole-genome sequencing was performed on 44 isolates recovered from eight cows (four metritic and four healthy) with growth on selective E. coli media from the RAJ, vulva and/or vagina and uterus. Clonal isolates were found in the RAJ or the vulva prepartum and in the vulva, vagina or uterus postpartum. Clonal isolates were also found in the RAJ, the vulva, the vagina and the uterus postpartum. Clonal isolates were found in individual cows and different cows. Absence of clustering based on virulence factor genes and all genes indicate no strain specificity to body site or uterine health status. These findings indicate that the gastrointestinal tract is the likely source of bacteria that colonize the reproductive tract via ascending colonization of the uterus through the lower genital tract. Additionally, cow to cow transmission occurs, and strains are not specific to body site or to health status.