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.

Diverse ß-lactam antibiotic-resistant bacteria and microbial community in milk from mastitic cows.

Intramammary bacterial infection, the most common cause of mastitis, is the most costly disease in dairy cattle in the US and reason for antibiotic usage. Ceftiofur, a third-generation cephalosporin, is generally used to treat such disease, but it has a high treatment failure rate. Though the reason is not known clearly, it is hypothesized that multiple factors are associated with the treatment failure. In this study, we analyzed 169 milk samples from cows with mastitis in two independent dairy farms (Farm A and B) in which 19.4% (Farm A) and 14.3% (Farm B) of the antibiotic treated cows were not cured. The prevalence of cephalosporin-resistant bacteria (CRB) in milk was 72.0% and 42.1% in Farm A and B, respectively. Nineteen and nine bacterial genera were identified in Farm A and B respectively, with the most abundant genus being Staphylococcus (27.1%; Farm A) and Bacillus (63.5%; Farm B). However, no strong relationship between the treatment failure rate and the CRB prevalence was observed. Furthermore, the metagenomic analysis showed no significant differences in the α- and ß-diversities of microbiota in milk samples from cured and uncured cows, suggesting that antibiotic-resistant bacteria were not the sole reason for the antibiotic treatment failure. KEY POINTS: • The mastitic milk samples had high prevalence of cephalosporin-resistant bacteria (CRB). • The CRB identified belong to diversified species. • Antibiotic treatment failure was not solely caused by the abundance of CRB.

Effects of adding an automated monitoring device to the health screening of postpartum Holstein cows on survival and productive and reproductive performances.

Automated monitoring devices (AMD) have become more affordable, and consequently more popular among dairy producers. We hypothesized that the addition of AMD-generated health alerts to a health-screening program improves survival, milk production, and reproductive success. In addition, we hypothesized that cows diagnosed with clinical disease that have AMD alerts are at greater risk of culling, lower milk production, and decreased risk of pregnancy than cows without AMD alerts. Holstein cows (nulliparous = 282, parous = 328) were enrolled at -60 ± 3 d (d 0 = calving), when they were fitted with an AMD and assigned randomly to 1 of 2 health-screening strategies: (1) control: AMD alerts not provided to farm personnel; and (2) automated device: AMD alerts provided to farm personnel. Twice daily, study personnel determined which cows had AMD alerts (health index ≤79, rumination <200 min/d, or difference between current rumination and the average of the 3 preceding days <0) and provided the information to farm personnel. Farm personnel examined cows at 3, 5, and 9 d in milk (DIM) and when daily milk yield decreased ≥25% on consecutive days. We detected no differences between health-screening strategies regarding morbidity (control = 49.7 ± 3.3%, automated device = 52.8 ± 3.2%), but the interaction between health-screening strategy and parity tended to be associated with the number of clinical diseases per cow (primiparous: control = 0.46 ± 0.06, automated device = 0.65 ± 0.07 cases/cow; multiparous: 0.88 ± 0.08, automated device = 0.86 ± 0.08 cases/cow). Cows enrolled in the automated device strategy were more likely to be treated with supportive therapy (64.4 ± 3.1 vs. 55.0 ± 3.2%), whereas primiparous cows in the automated device strategy were more likely to be treated with anti-inflammatory drugs than those in the control strategy (41.6 ± 4.7 vs. 23.8 ± 4.0%). Health-screening strategy did not affect survival or total milk yield up to 22 wk postpartum, but cows in the automated device strategy had reduced risk of pregnancy after the first 2 services (54.5 ± 3.0 vs. 46.2 ± 3.2%). Cows diagnosed with a clinical disease without AMD alerts had reduced risk of removal from the herd by 150 DIM (5.7 ± 2.0 vs. 19.0 ± 3.3%), greater risk of pregnancy after the first 2 services (49.6 ± 4.5 vs. 33.6 ± 3.9%), and greater milk by 22 wk postpartum (6.7 ± 0.2 vs. 5.3 ± 0.2 × 103 kg) than cows diagnosed with a clinical disease that had an AMD alert. Adding AMD-generated health alerts to the health screening of postpartum cows in a herd with an existing screening program did not improve survival, milk yield, or reproductive success. In addition, AMD alerts in cows diagnosed with a clinical disease may be indicative of the future success of such cows.

Effect of Chitosan Microparticles on the Uterine Microbiome of Dairy Cows with Metritis.

The objective of this study was to evaluate the effect of chitosan microparticles on the uterine microbiome of cows with metritis. Dairy cows with metritis (n = 89) were assigned to 1 of 3 treatments: chitosan microparticles (n = 21), in which the cows received an intrauterine infusion of chitosan microparticles at metritis diagnosis (day 0), day 2, and day 4; ceftiofur (n = 25), in which the cows received a subcutaneous injection of ceftiofur on day 0 and day 3; and no intrauterine or subcutaneous treatment (n = 23). Nonmetritic cows (n = 20) were healthy cows matched with cows with metritis by the number of days postpartum at metritis diagnosis. Uterine swab samples collected on days 0, 3, 6, 9, and 12 were used for 16S rRNA gene sequencing and 16S RNA gene copy number quantification by quantitative PCR. Principal-coordinate analysis showed that the microbiome of the ceftiofur-treated and metritic untreated groups progressed toward that of the nonmetritic group by day 3, whereas that of the chitosan microparticle-treated group remained unchanged. The differences on day 3 were mainly due to a greater relative abundance of Fusobacteria, particularly Fusobacterium, in the chitosan microparticle-treated group than in the ceftiofur-treated and metritic untreated groups. Furthermore, the microbiome of the ceftiofur-treated group became similar to that of the nonmetritic group by day 9, whereas the microbiome of the chitosan microparticle-treated and metritic untreated groups became similar to that of the nonmetritic group only by day 12. The total bacterial 16S rRNA gene counts in the chitosan microparticle-treated group were greater than those in the metritic untreated controls on days 6 and 9, whereas the ceftiofur treatment group was the only group in which the total bacterial 16S rRNA gene count became similar to that in the nonmetritic group by day 12. In summary, chitosan microparticles slowed the progression of the uterine microbiome toward a healthy state, whereas ceftiofur hastened the progression toward a healthy state.IMPORTANCE Third-generation cephalosporins, such as ceftiofur, are commonly used to treat metritis in dairy cows. Chitosan microparticles has been shown to have a broad spectrum of activity in vitro and to be effective against uterine pathogens in vivo; therefore, they have been hailed as a possible alternative to traditional antibiotics. Nonetheless, in the present study, we saw that chitosan microparticle treatment slowed the progression of the uterine microbiome of cows with metritis toward a healthy state, whereas ceftiofur treatment hastened the progression toward a healthy state. Given the lack of an effective alternative to traditional antibiotics and an increased concern about antimicrobial resistance, a greater effort should be devoted to the prevention of metritis in dairy cows.

Genomic Diversity, Virulence, and Antimicrobial Resistance of Klebsiella pneumoniae Strains from Cows and Humans.

Klebsiella pneumoniae is a leading cause of severe infections in humans and dairy cows, and these infections are rapidly becoming untreatable due to the emergence of multidrug-resistant (MDR) strains. However, little is known about the relationship between bovine and human K. pneumoniae isolates at the genome population level. Here, we investigated the genomic structures, pangenomic profiles, virulence determinants, and resistomes of 308 K. pneumoniae isolates from humans and dairy cows, including 96 newly sequenced cow isolates. We identified 177 functional protein families that were significantly different across human and bovine isolates; genes expressing proteins related to metal ion (iron, zinc, and calcium) metabolism were significantly more prevalent among the bovine isolates. Siderophore systems were found to be prevalent in both the bovine and the human isolates. In addition, we found that the Klebsiella ferric uptake operon kfuABC was significantly more prevalent in clinical mastitis cases than in healthy cows. Furthermore, on two dairy farms, we identified a unique IncN-type plasmid, pC5, coharboring blaCTX-M-1 and mph(A) genes, which confer resistance to cephalosporins and macrolides, respectively. We provide here the complete annotated sequence of this plasmid.IMPORTANCE We demonstrate here the genetic diversity of K. pneumoniae isolates from dairy cows and the mixed phylogenetic lineages between bovine and human isolates. The ferric uptake operon kfuABC genes were more prevalent in strains from clinical mastitis cows. Furthermore, we report the emergence of an IncN-type plasmid carrying the blaCTX-M-1 and mph(A) genes among dairy farms in the United States. Our study evaluated the genomic diversity of the bovine and human isolates, and the findings uncovered different profiles of virulence determinants among bovine and human K. pneumoniae isolates at the genome population level.

Symposium review: The uterine microbiome associated with the development of uterine disease in dairy cows.

Until 2010, our knowledge of the uterine microbiome in cows that developed uterine disease relied almost exclusively on culture-dependent studies and mostly included cows with clinical endometritis (i.e., with purulent uterine discharge). Those studies consistently found a strong positive correlation between Trueperella pyogenes and clinical endometritis, whereas other pathogens such as Escherichia coli, Fusobacterium necrophorum, Prevotella melaninogenica, and Bacteroides spp. were also commonly cocultured. In contrast, Streptococcus spp., Staphylococcus spp., and Bacillus spp. were usually isolated from healthy cows. Starting in 2010, culture-independent studies using PCR explored the microbiome of cows with metritis and clinical endometritis, and observed that E. coli was a pioneer pathogen that predisposed cows to infection with F. necrophorum, which was strongly associated with metritis, and to infection with T. pyogenes, which was strongly associated with clinical endometritis. Starting in 2011, culture-independent studies using metagenomic sequencing expanded our knowledge of the uterine microbiome. It has been shown that cows have bacteria in the uterus even before calving, they have an established uterine microbiome within 20 min of calving, and that the microbiome structure is identical between cows that develop metritis and healthy cows until 2 d postpartum, after which the bacterial structure of cows that developed metritis deviates in favor of greater relative abundance of Bacteroidetes and Fusobacteria and lesser relative abundance of Proteobacteria and Tenericutes. The shift in the uterine microbiome in cows that develop metritis is characterized by a loss of heterogeneity and a decrease in bacterial richness. At the genus level, Bacteroides, Porphyromonas, and Fusobacterium have the strongest association with metritis. At the species level, we observed that Bacteroides pyogenes, Porphyromonas levii, and Helcococcus ovis were potential emerging uterine pathogens. Finally, we have shown that the hematogenous route is a viable route of uterine infection with uterine pathogens. Herein, we propose that metritis is associated with a dysbiosis of the uterine microbiota characterized by decreased richness, and an increase in Bacteroidetes and Fusobacteria, particularly Bacteroides, Porphyromonas, and Fusobacterium.

Epidemiologic and economic analyses of pregnancy loss attributable to mastitis in primiparous Holstein cows.

The main objective of the study reported here was to examine the association between pregnancy loss (PL) and previous exposure to clinical or subclinical mastitis before breeding or during gestation in primiparous Holstein cows. A secondary objective was to estimate the cost of clinical mastitis during gestation, including that of PL attributable to mastitis in study cows. A total of 687 primiparous Holstein cows from 1 dairy farm were included in a matched case-control study. Study cows were declared pregnant via ultrasound on d 33 after timed artificial insemination (TAI). Case cows (n = 78) were those diagnosed as nonpregnant by rectal palpation on d 47 or 75 after TAI. Control cows were those confirmed as pregnant by rectal palpation on d 47 and 75 after TAI. Case cows were matched with eligible controls according to year of calving and calving-to-conception interval ±3 d. Cows were assigned to 1 of 3 groups: (1) cows not affected with clinical or subclinical mastitis; (2) cows affected with subclinical mastitis (Dairy Herd Improvement Association somatic cell score >4.5); and (3) cows affected with clinical mastitis during 2 exposure periods, 1 to 42 d before breeding or during gestation (1 to PL diagnosis day for case cows, and 1 to 75 d for control cows). Conditional logistic regression was used to model the odds of PL as a function of previous exposure to mastitis in study cows. Mastitis before breeding was not associated with PL. The odds of PL were 2.21 times greater in cows affected with clinical mastitis during gestation (95% confidence interval = 1.01, 4.83), compared with cows without mastitis, after controlling for breeding type and lameness. The cost of clinical mastitis during gestation was $149, which includes the cost ($27) of PL attributable to mastitis. In conclusion, this study provides evidence that clinical mastitis during gestation can cause PL in primiparous dairy cows leading to economic losses.

Evidence that mastitis can cause pregnancy loss in dairy cows: A systematic review of observational studies.

The objective of this study was to conduct a systematic review to identify and assess evidence and knowledge gaps in published observational studies that have investigated the relationship between mastitis and pregnancy loss (PL) in dairy cows. PubMed and ScienceDirect were used to search pertinent peer-reviewed research reports of interest. Screening of research reports was conducted at 3 levels: titles, abstracts, and full-text articles. The search identified 651 records for initial screening. The final screening process identified 8 qualified articles for review after removing 10 duplicate records, 582 titles, 31 abstracts, and 20 full-text articles. Two studies produced strong epidemiologic evidence indicating that (1) exposure to clinical mastitis during early gestation (first 45 d of gestation) is associated with subsequent PL during the following 90 d; and (2) subclinical mastitis 1 to 30 d before artificial insemination (AI) is associated with subsequent PL at 35 to 41 d of gestation. An additional study showed that exposure to clinical mastitis during early lactation in combination with low body condition can increase the risk of PL in dairy cows; however, the interaction effect between clinical mastitis and low body condition on PL was considered weak. Four other studies produced inconclusive evidence indicating that mastitis is a predisposing factor for PL in dairy cows, as the exposure risk period for mastitis overlapped with the follow-up period for diagnosis of PL in dairy cows. Finally, one study failed to identify a relationship between mastitis and PL in dairy cows. Further research is needed to (1) support the hypothesis that mastitis in combination with low body condition score (or other exposure factors) can increase the risk of PL, (2) compare the effect of clinical versus subclinical mastitis on PL, (3) compare the effect of mastitis before breeding and during gestation on PL, and (4) compare the effect of mastitis on PL in dairy cows during different lactations.

Using Doppler ultrasonography on day 34 of pregnancy to predict pregnancy loss in lactating dairy cattle.

The objective of this experiment was to determine whether uterine or ovarian vascular dynamics could be used to identify cows at risk for pregnancy loss. Our hypothesis was that cows that subsequently lose their pregnancy will have decreased corpus luteal (CL) perfusion, or an increased resistance index (RI; reduced blood flow), or both, at d 34 of pregnancy. Day 34 was chosen because it is a common time for dairy cattle to be checked for pregnancy. This experiment was performed in 2 replicates from November 2011 to April 2012 (n = 69) and from November 2012 to April 2013 (n = 53). Cows were bred via timed artificial insemination using Ovsynch-56 and checked for pregnancy on d 32 after artificial insemination. At d 34, cows confirmed pregnant were examined via transrectal Doppler ultrasonography. Blood samples collected via coccygeal vein were used to measure circulating plasma progesterone concentrations. Diameter of the corpus luteum and crown-rump length were measured. Color power Doppler ultrasonography was used to determine vascular perfusion to the CL, and RI was measured for the uterine arteries just after branching from the umbilical artery. Records were later examined to identify pregnancy status of cows after reconfirmation. Abortion rate did not differ between replicates (11.6% in replicate 1, 9.4% in replicate 2). Mean crown-rump length of embryos that were carried to term was greater on d 34 than that in cows that aborted (14.23 ± 0.27 vs. 13.21 ± 0.53 mm). Circulating progesterone concentration at d 34 was greater for cows that carried pregnancies to term than for those that aborted (9.1 ± 0.7 vs. 7.5 ± 1.0 ng/mL). The final logistic regression model consisted of crown-rump length, progesterone concentration, and RI of the uterine artery contralateral to pregnancy. Decreased crown-rump length and progesterone concentration tended to be associated with increased odds ratio for pregnancy loss, whereas CL perfusion and uterine blood flow were not associated with increased odds ratio of pregnancy loss. In conclusion, examining CL perfusion and RI of the uterine arteries on d 34 of pregnancy does not offer a method to identify lactating Dairy cattle at risk for pregnancy loss after d 34.

Effects of intrauterine infusion of Escherichia coli lipopolysaccharide on uterine mRNA gene expression and peripheral polymorphonuclear leukocytes in Jersey cows diagnosed with purulent vaginal discharge.

The objectives of the current experiment were to investigate the effects of intrauterine treatment of cows with purulent vaginal discharge (PVD) using lipopolysaccharide (LPS) from Escherichia coli on uterine mRNA expression of genes related to inflammatory responses, peripheral polymorphonuclear leukocyte (PMN) function, hematological parameters, and blood concentrations of cortisol, haptoglobin, and progesterone (P4). Jersey cows (n = 3,084) were examined for PVD at 35 ± 6 d postpartum using the Metricheck device (Simcro, Hamilton, New Zealand). At examination, 310 cows had PVD (10.1%), but to ensure proper collection and processing of samples, 267 cows were used in this experiment. Cows were balanced for lactation number, body condition score, and milk yield, and randomly assigned to the control treatment [intrauterine infusion of 20 mL of phosphate-buffered saline (PBS); n = 87] or to receive intrauterine infusion of 20 mL of PBS containing 150 µg (LPS150; n = 91) or 300 µg (LPS300; n = 89) of E. coli LPS. Uteri were biopsied in a subgroup of cows at 6 h after infusion and in another subgroup of cows at 24 h after infusion. Peripheral PMN expression of adhesion molecules (L-selectin and MAC-1) and phagocytosis and oxidative burst were evaluated at 0, 2, and 6 h after infusion. Blood sampled 0, 2, 6, 24, 48, and 168 h after infusion was used for complete hemogram and to determine concentrations of cortisol, haptoglobin, and P4. Treatment did not affect uterine mRNA expression of adhesion molecules [endothelial leukocyte adhesion molecule (E-selectin), intracellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1)], cytokines (tumor necrosis factor-α, IL-1ß, IL-6, IL-8, and IL-10), and toll-like receptor-4. Treatment did not affect PMN expression of L-selectin, but intensity of expression of MAC-1 was higher for LPS150 cows than PBS cows, and tended to be higher in LPS150 than LPS300 cows. Furthermore, a greater percentage of PMN from LPS300 cows were positive for phagocytosis and oxidative burst compared with PBS and LPS150 cows. No effects were observed of treatment on hematological parameters and concentrations of cortisol, haptoglobin, and P4. These observations suggest that intrauterine infusion of E. coli LPS moderately stimulates peripheral PMN function, but further research is needed to better understand the immunomodulatory effects of LPS in the uterus of cows with PVD.

Uterine Microbiota Progression from Calving until Establishment of Metritis in Dairy Cows.

The objective of this study was to evaluate the progression of the uterine microbiota from calving until establishment of metritis. Uterine swabs (n = 72) collected at 0, 2, and 6 ± 2 days postpartum (dpp) from 12 metritic and 12 healthy cows were used for metagenomic sequencing of the 16S rRNA gene on the Illumina MiSeq platform. A heat map showed that uterine microbiota was established at calving. The microbiota changed rapidly from 0 to 6 ± 2 dpp, with a decrease in the abundance of Proteobacteria and an increase in the abundance of Bacteroidetes and Fusobacteria, which were dominant in metritic cows. Uterine microbiota composition was shared; however, metritic and healthy cows could be discriminated using relative abundance of bacterial genera at 0, 2, and 6 ± 2 dpp. Bacteroides was the main genus associated with metritis because it was the only genus that showed significantly greater abundance in cows with metritis. As the abundance of Bacteroides organisms increased, the uterine discharge score, a measure of uterine health, worsened. Fusobacterium was also an important genus associated with metritis because Fusobacterium abundance increased as Bacteroides abundance increased and the uterine discharge score worsened as the abundance increased. The correlation with uterine discharge score and the correlation with Bacteroides or Fusobacterium showed that other bacteria, such as Helcoccocus, Filifactor, and Porphyromonas, were also associated with metritis. There were also bacteria associated with uterine health, such as "Candidatus Blochmannia," Escherichia, Sneathia, and Pedobacter.

Utility of inline milk fat and protein ratio to diagnose subclinical ketosis and to assign propylene glycol treatment in lactating dairy cows.

The objective was to identify a fat-to-protein ratio (FPR) cut-off to diagnose subclinical ketosis (SCK) and to evaluate the effect of propylene glycol (PPG) treatment of cows with high FPR. The optimized cut-off was > 1.42; sensitivity (Se) = 92%; specificity (Sp) = 65%. A cut-off > 1.5 was selected for the PPG trial for balanced Se-Sp. Fat-to-protein ratio cut-offs > 1.25, 1.35, 1.50, 1.60, and 1.70 resulted in Se-Sp of 100% to 49%, 96% to 59%, 75% to 78%, 33% to 90%, and 8% to 96%, respectively. The proportions of cows with FPR > 1.25, 1.35, 1.42, 1.50, 1.60, and 1.70 were 60%, 50%, 44%, 30%, 14%, and 6%, respectively. Incidences of clinical ketosis and milk yield were similar between cows that received 400 mL of PPG (n = 34) and control cows (n = 38). Prevalence of SCK at enrollment was 29.2%; therefore, FPR > 1.5 is not indicated for treatment. Lower cut-offs should be used for screening.

Utilité du ratio de matière grasse et de protéine sur la ligne de traite pour diagnostiquer une cétose subclinique et assigner le traitement au propylèneglycol chez les vaches laitières en lactation. L'objectif consistait à identifier un seuil du ratio de matière grasse et de protéine (RMGP) pour diagnostiquer une cétose subclinique et évaluer l'effet du traitement au propylèneglycol (PPG) chez les vaches présentant un RMGP élevé. Le seuil optimisé était de > 1,42; la sensibilité (Se) = 92 %; la spécificité (Sp) = 65 %. Un seuil de > 1,5 a été choisi pour l'essai au PPG pour des Se-Sp équilibrées. Des seuils de ratios de matière grasse et protéine de > 1,25, 1,35, 1,50, 1,60 et 1,70 ont produit des Se-Sp de 100 % et 49 %, de 96 % et 59 %, 75 % et 78 %, de 33 % et 90 % et de 8 % et 96 %, respectivement. Les proportions des vaches avec un RMGP de > 1,25, 1,35, 1,42, 1,50, 1,60 et 1,70 étaient de 60 %, 50 %, 44 %, 30 %, 14 % et 6 %, respectivement. L'incidence de cétose clinique et la production de lait étaient semblables entre les vaches qui avaient reçu 400 mL de PPG (n = 34) et les vaches témoins (n = 38). La prévalence de cétose subclinique au recrutement était de 29,2 %; par conséquent, un RMGP de > 1,5 n'est pas indiqué pour le traitement. Des seuils inférieurs devraient être utilisés pour le dépistage.(Traduit par Isabelle Vallières).

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.

Pangenomic and biochemical analyses of Helcococcus ovis reveal widespread tetracycline resistance and a novel bacterial species, Helcococcus bovis.

Helcococcus ovis (H. ovis) is an opportunistic bacterial pathogen of a wide range of animal hosts including domestic ruminants, swine, avians, and humans. In this study, we sequenced the genomes of 35 Helcococcus sp. clinical isolates from the uterus of dairy cows and explored their antimicrobial resistance and biochemical phenotypes in vitro. Phylogenetic and average nucleotide identity analyses classified four Helcococcus isolates within a cryptic clade representing an undescribed species, for which we propose the name Helcococcus bovis sp. nov. By establishing this new species clade, we also resolve the longstanding question of the classification of the Tongji strain responsible for a confirmed human conjunctival infection. This strain did not neatly fit into H. ovis and is instead a member of H. bovis. We applied whole genome comparative analyses to explore the pangenome, resistome, virulome, and taxonomic diversity of the remaining 31 H. ovis isolates. An overwhelming 97% of H. ovis strains (30 out of 31) harbor mobile tetracycline resistance genes and displayed significantly increased minimum inhibitory concentrations of tetracyclines in vitro. The high prevalence of mobile tetracycline resistance genes makes H. ovis a significant antimicrobial resistance gene reservoir in our food chain. Finally, the phylogenetic distribution of co-occurring high-virulence determinant genes of H. ovis across unlinked and distant loci highlights an instance of convergent gene loss in the species. In summary, this study showed that mobile genetic element-mediated tetracycline resistance is widespread in H. ovis, and that there is evidence of co-occurring virulence factors across clades suggesting convergent gene loss in the species. Finally, we introduced a novel Helcococcus species closely related to H. ovis, called H. bovis sp. nov., which has been reported to cause infection in humans.

Integrating uterine microbiome and metabolome to advance the understanding of the uterine environment in dairy cows with metritis.

BACKGROUND: Metritis is a prevalent uterine disease that affects the welfare, fertility, and survival of dairy cows. The uterine microbiome from cows that develop metritis and those that remain healthy do not differ from calving until 2 days postpartum, after which there is a dysbiosis of the uterine microbiome characterized by a shift towards opportunistic pathogens such as Fusobacteriota and Bacteroidota. Whether these opportunistic pathogens proliferate and overtake the uterine commensals could be determined by the type of substrates present in the uterus. The objective of this study was to integrate uterine microbiome and metabolome data to advance the understanding of the uterine environment in dairy cows that develop metritis. Holstein cows (n = 104) had uterine fluid collected at calving and at the day of metritis diagnosis. Cows with metritis (n = 52) were paired with cows without metritis (n = 52) based on days after calving. First, the uterine microbiome and metabolome were evaluated individually, and then integrated using network analyses. RESULTS: The uterine microbiome did not differ at calving but differed on the day of metritis diagnosis between cows with and without metritis. The uterine metabolome differed both at calving and on the day of metritis diagnosis between cows that did and did not develop metritis. Omics integration was performed between 6 significant bacteria genera and 153 significant metabolites on the day of metritis diagnosis. Integration was not performed at calving because there were no significant differences in the uterine microbiome. A total of 3 bacteria genera (i.e. Fusobacterium, Porphyromonas, and Bacteroides) were strongly correlated with 49 metabolites on the day of metritis diagnosis. Seven of the significant metabolites at calving were among the 49 metabolites strongly correlated with opportunistic pathogenic bacteria on the day of metritis diagnosis. The main metabolites have been associated with attenuation of biofilm formation by commensal bacteria, opportunistic pathogenic bacteria overgrowth, tissue damage and inflammation, immune evasion, and immune dysregulation. CONCLUSIONS: The data integration presented herein helps advance the understanding of the uterine environment in dairy cows with metritis. The identified metabolites may provide a competitive advantage to the main uterine pathogens Fusobacterium, Porphyromonas and Bacteroides, and may be promising targets for future interventions aiming to reduce opportunistic pathogenic bacteria growth in the uterus.

Shifts in uterine microbiome associated with pregnancy outcomes at first insemination and clinical cure in dairy cows with metritis.

Objectives were to assess differences in uterine microbiome associated with clinical cure and pregnancy outcomes in dairy cows treated for metritis. Cows with metritis (reddish-brownish, watery, and fetid vaginal discharge) were paired with cows without metritis based on parity and days postpartum. Uterine contents were collected through transcervical lavage at diagnosis, five days later following antimicrobial therapy (day 5), and at 40 days postpartum. Uterine microbiome was assessed by sequencing the V4 hypervariable region of the 16S rRNA gene. Although alpha-diversity based on Chao1, Shannon, and inverse Simpson indexes at diagnosis did not differ between cows with and without metritis, disease was associated with differences in beta-diversity. Prevalence of Porphyromonas, Bacteroides, and Veillonella was greater in cows with metritis. Streptococcus, Sphingomonas, and Ureaplasma were more prevalent in cows without metritis. Differences in beta-diversity between cows with and without metritis persisted on day 5. Uterine microbiome was not associated with clinical cure. Richness and alpha-diversity, but not beta-diversity, of uterine microbiome 40 days postpartum were associated with metritis and pregnancy. No relationship between uterine microbiome and pregnancy outcomes was observed. Results indicate that factors other than changes in intrauterine bacterial community underlie fertility loss and clinical cure in cows with metritis.

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.

Genome-wide association and genomic prediction for a reproductive index summarizing fertility outcomes in U.S. Holsteins.

Subfertility represents one major challenge to enhancing dairy production and efficiency. Herein, we use a reproductive index (RI) expressing the predicted probability of pregnancy following artificial insemination (AI) with Illumina 778K genotypes to perform single and multi-locus genome-wide association analyses (GWAA) on 2,448 geographically diverse U.S. Holstein cows and produce genomic heritability estimates. Moreover, we use genomic best linear unbiased prediction (GBLUP) to investigate the potential utility of the RI by performing genomic predictions with cross validation. Notably, genomic heritability estimates for the U.S. Holstein RI were moderate (h2 = 0.1654 ± 0.0317-0.2550 ± 0.0348), while single and multi-locus GWAA revealed overlapping quantitative trait loci (QTL) on BTA6 and BTA29, including the known QTL for the daughter pregnancy rate (DPR) and cow conception rate (CCR). Multi-locus GWAA revealed seven additional QTL, including one on BTA7 (60 Mb) which is adjacent to a known heifer conception rate (HCR) QTL (59 Mb). Positional candidate genes for the detected QTL included male and female fertility loci (i.e. spermatogenesis and oogenesis), meiotic and mitotic regulators, and genes associated with immune response, milk yield, enhanced pregnancy rates, and the reproductive longevity pathway. Based on the proportion of the phenotypic variance explained (PVE), all detected QTL (n = 13; P ≤ 5e - 05) were estimated to have moderate (1.0% < PVE ≤ 2.0%) or small effects (PVE ≤ 1.0%) on the predicted probability of pregnancy. Genomic prediction using GBLUP with cross validation (k = 3) produced mean predictive abilities (0.1692-0.2301) and mean genomic prediction accuracies (0.4119-0.4557) that were similar to bovine health and production traits previously investigated.