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.

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.

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 and activity. Cows were examined for diagnosis of metritis at 4 ± 1, 7 ± 1, and 9 ± 1 DIM. Cows diagnosed with metritis (n = 132), characterized by watery, fetid, reddish/brownish vaginal discharge (VD) were randomly allocated to one of 2 treatments: Control (CON; n = 62) - no treatment at the time of metritis diagnosis (d 0); 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 days in milk at metritis diagnosis) and behavior data (i.e., daily averages of rumination, activity generated by AHMD, and derived variables) to assess their association with metritis SC or TF. Variables with a P ≤ 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, sensitivity (Se) 87% and specificity (Sp) 71%. 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, Se of 93% and Sp of 82%. 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.

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.

Behavioral changes of metritic primiparous cows treated with chitosan microparticles or ceftiofur.

The main objective was to characterize behavioral changes in metritic primiparous cows treated with chitosan microparticles (CM) or ceftiofur (CEF). A secondary objective was to compare behavioral patterns of metritic cows with nonmetritic (NMET) cows. Nulliparous Holstein cows (n = 311) were fitted with a neck-mounted automated health-monitoring device (AHMD) from -21 to 60 d relative to calving. Cows diagnosed with metritis (d 0), characterized by watery, fetid, red-brownish uterine discharge within 21 d in milk were assigned randomly to CM (n = 45), intrauterine infusion of 24 g of CM dissolved in 40 mL of sterile distilled water on d 0, 2, and 4; CEF (n = 47), subcutaneous injection of 6.6 mg/kg ceftiofur crystalline-free acid on d 0 and 3; and control (CON; n = 39), no treatment. For comparison, NMET cows (n = 180) were matched with metritic cows according to age at calving and calving date. Postdiagnosis, there was an effect of treatment and an interaction between treatment and time on rumination and activity. The interaction showed that CM had lesser rumination than CEF from d 1 to 11, d 18, and d 20; CM had lesser rumination than CON from d 2 to 8; and CEF was not different from CON. The interaction showed that CM had lesser activity than CON on d 2, from d 6 to 11, and d 13 to 14; CM was not different from CEF; and CEF had lesser activity than CON on d 8, 9, 13, and 14. Prediagnosis, cows in CM, CEF, and CON had lesser rumination and activity than cows in NMET. Postdiagnosis, cows in CM, CEF, and CON had lesser rumination than NMET from d 0 to 2 and had lesser activity than NMET from d 0 to 5. In summary, CM decreased rumination and activity compared with CON, which indicates a negative systemic effect of CM. This may be associated with exacerbated inflammation in the uterus. Additionally, metritic cows had decreased rumination and activity prediagnosis, which may allow for the use of AHMD for metritis diagnosis.

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.

Validation of a fully automated chemiluminescent immunoassay for cattle serum and plasma progesterone measurement.

Introduction: Monitoring circulating progesterone concentration ([P4]) is an important component of basic and applied reproduction research and clinical settings. IMMULITE® 2000 XPi (Siemens Healthineers, Cary, NC) is a newly upgraded fully automated immunoassay system marketed for human use to measure concentrations of different measurands including P4. Objectives: Our objective was therefore to characterize the analytical performance of the IMMULITE® 2000 XPi P4 immunoassay (IPI) across the reportable range in serum and plasma of cattle. Methods: The IPI validation protocols included characterization of the method linearity, within-run, and between-run precision through calculation of the coefficient of variation (CV). The method accuracy was assessed through the calculation of the spiking-recovery (SR) bias across the reportable range (0.2-40.0 ng/mL). Passing-Bablok regression and Bland-Altman plots were used to determine the interlaboratory bias for two laboratories. Three types of observed total error (TEo) were calculated based on the considered type of bias, TEoSR (spiking-recovery), TEoRB (range-based bias), and TEoAB (average-based bias). Results: The IPI was linearly related to the true value (R 2 = 0.997) across the reportable range. The within-run and between-run precision (CV%) of the IPI for both serum and plasma [P4] of clinical relevance (1, 2, 5, and 10 ng/mL) were <5 and <10%, respectively. The TEo reported here for serum and plasma at [P4] of 1 and 5 ng/mL was ~20 and 25%, respectively. Of interest, the three types of TEo were relatively similar regardless of the considered bias. Conclusions: We concluded that the automated IPI provides a precise, accurate, reliable, and safe method for measuring [P4] in both serum and plasma of cattle. Consistent with the manufacturer's recommendations, the serum matrix is more accurate than plasma.

Bacterial communities from vagina of dairy healthy heifers and cows with impaired reproductive performance.

Vaginal microenvironment plays a significant role in bovine fertility since its resident microorganisms interact with the host mucosa and constitutes the first barrier against ascending pathogens in the reproductive tract. In this study, the vaginal microbiome of healthy heifers (H) and cows with impaired reproductive performance, metritis complex (MT) or repeat breeders (RB), was assessed using a 16S rRNA gene sequencing approach. Analysis revealed that even though a vaginal microbiological guild (Firmicutes, Bacteroidetes, Proteobacteria, Tenericutes, Fusobacteria and Actinobacteria) was shared among healthy heifers and cows with uterine disease; further analysis at genus level showed significant differences depending on the reproductive health status. The relative abundances of recognized uterine pathogens such as Bacteroidetes, Fusobacterium and Helcococcus were higher in MT when compared with H and RB; therefore, their presence in vagina can be considered as a risk factor for fertility. The present study describes for the first time, the composition of native bacterial communities in the vagina of cows undergoing the repeat breeding syndrome (RBS), and reports an association between this disease and the presence of Porphyromonas and unassigned genera of the Pasteurellaceae family. In addition, this work highlights the bacteria associated with a healthy vagina: genera from the families Lachnospiraceae, Rikenellaceae and the genera Acinetobacter, Bacillus, Oscillospira, CF231 and 5-7NS. Results highlighted herein, signify the potential of the evaluation of the bovine vaginal microbiome to future design therapeutic interventions to improve pregnancy rates however, further research is needed to elucidate the balance of bacterial species resulting in an optimal reproductive health.

Investigating the Use of Dry Matter Intake and Energy Balance Prepartum as Predictors of Digestive Disorders Postpartum.

One objective was to evaluate the association of dry matter intake as a percentage of body weight (DMI%BW) and energy balance (EB) prepartum and postpartum, and energy-corrected milk (ECM) postpatum with digestive disorders postpartum. For this, ANOVA was used, and DMI%BW, EB, and ECM were the outcome variables, and left displaced abomasum (LDA), indigestion, and other digestive disorders (ODDZ) were the explanatory variables. The main objective was to evaluate prepartum DMI%BW and EB as predictors of digestive disorders. For this, logistic regression was used, and LDA, indigestion, and ODDZ were the outcome variables and DMI%BW and EB were the explanatory variables. Data from 689 cows from 11 experiments were compiled. Left displaced abomasum was not associated with prepartum DMI%BW or EB. Postpartum data were normalized to the day of the event (day 0). Cows that developed LDA had lesser postpartum DMI%BW on days -24, -23, -12, -7 to 0 and from days 1 to 8, 10 to 12, and 14 and 16, lesser postpartum EB from days -7 to -5, -3 to 0, and 12, and lesser postpartum energy-corrected milk on days -19, -2, -1, 0, 7, 9, 10, 15, and 17 relative to diagnosis than cows without LDA. Cows that developed indigestion had lesser prepartum DMI%BW and EB than cows without indigestion, and lesser postpartum DMI%BW on days -24, -1, 0, 1, and 2, and greater DMI%BW on day 26, lesser ECM on days -24, -2, -1, 0, 1, and 2 relative to diagnosis. Postpartum EB was not associated with indigestion postpartum. Cows that developed ODDZ had lesser prepartum DMI%BW on day -8 and from days -5 to -2, lesser prepartum EB on day -8 and from days -5 to -2, and lesser postpartum DMI%BW than cows without ODDZ. Each 0.1 percentage point decrease in the average DMI%BW and each Mcal decrease in the average EB in the last 3 days prepartum increased the odds of having indigestion by 9% each. Cutoffs for DMI%BW and EB during the last 3 days prepartum to predict indigestion were established and were ≤1.3%/day and ≤0.68 Mcal/day, respectively. In summary, measures of prepartum DMI%BW and EB were associated with indigestion and ODDZ postpartum and were predictors of indigestion postpartum, although the effect sizes were small.

Molecular characterization of carbapenem-resistant and virulent plasmids in Klebsiella pneumoniae from patients with bloodstream infections in China.

Bloodstream infections (BSIs) caused by carbapenem-resistant Klebsiella pneumoniae (CRKP) are potentially life-threatening and an urgent threat to public health. The present study aims to clarify the characteristics of carbapenemase-encoding and virulent plasmids, and their interactions with the host bacterium. A total of 425 Kp isolates were collected from the blood of BSI patients from nine Chinese hospitals, between 2005 and 2019. Integrated epidemiological and genomic data showed that ST11 and ST307 Kp isolates were associated with nosocomial outbreak and transmission. Comparative analysis of 147 Kp genomes and 39 completely assembled chromosomes revealed extensive interruption of acrR by ISKpn26 in all Kp carbapenemase-2 (KPC-2)-producing ST11 Kp isolates, leading to activation of the AcrAB-Tolc multidrug efflux pump and a subsequent reduction in susceptibility to the last-resort antibiotic tigecycline and six other antibiotics. We described 29 KPC-2 plasmids showing diverse structures, two virulence plasmids in two KPC-2-producing Kp, and two novel multidrug-resistant (MDR)-virulent plasmids. This study revealed a multifactorial impact of KPC-2 plasmid on Kp, which may be associated with nosocomial dissemination of MDR isolates.

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.

Ceftiofur reduced Fusobacterium leading to uterine microbiota alteration in dairy cows with metritis.

BACKGROUND: Metritis is an inflammatory uterine disease found in ~ 20% of dairy cows after parturition and associated with uterine microbiota with high abundance of Fusobacterium, Bacteroides, and Porphyromonas. Ceftiofur is a common treatment, but the effect on uterine microbiota is poorly understood. Herein, we investigated the short-term impact of ceftiofur on uterine microbiota structure and function in cows with metritis. Eight cows received ceftiofur (CEF) and 10 remained untreated (CON). Uterine swabs were collected for PCR and metagenomic analysis at diagnosis before treatment (5 ± 1 DPP) and 2 days after diagnosis/treatment (7 ± 1 DPP) from the same individuals. Seven CEF and 9 CON passed quality control and were used for 16S rRNA gene sequencing. RESULTS: Ceftiofur treatment resulted in uterine microbiota alteration, which was attributed to a decrease in relative abundance of Fusobacterium and in gene contents involved in lipopolysaccharide biosynthesis, whereas uterine microbiota diversity and genes involved in pantothenate and coenzyme A biosynthesis increased. Ceftiofur treatment also reduced rectal temperature and tended to reduce total bacteria in the uterus. However, other uterine pathogens such as Bacteroides and Porphyromonas remained unchanged in CEF. The blaCTX-M gene was detected in 37.5% of metritic cows tested but was not affected by CEF. We found that ß-hydroxybutyric acid, pyruvic acid, and L-glutamine were preferentially utilized by Fusobacterium necrophorum according to metabolic activity with 95 carbon sources. CONCLUSIONS: Ceftiofur treatment leads to alterations in the uterine microbiota that were mainly characterized by reductions in Fusobacterium and genes involved in LPS biosynthesis, which may be associated with a decrease in rectal temperature. The increase in pantothenate and coenzyme A biosynthesis indicates microbial response to metabolic stress caused by ceftiofur. Preference of Fusobacterium for ß-hydroxybutyric acid may help to explain why this strain becomes dominant in the uterine microbiota of cows with metritis, and it also may provide a means for development of new therapies for the control of metritis in dairy cows.

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.

Combined effect of mastitis and parity on pregnancy loss in lactating Holstein cows.

The main objective of this study was to examine the combined effect of mastitis and parity on pregnancy loss (PL) in lactating Holstein cows. A secondary objective was to estimate the cost of mastitis including that of PL attributable to mastitis. A total of 1,774 lactation periods from 1,047 Holstein cows with different parities from one dairy farm were included in a matched case-control study. All study cows were diagnosed pregnant by transrectal ultrasonography on day 33 after timed artificial insemination (TAI). Case cows (n = 222 lactations) were those later diagnosed non-pregnant by transrectal palpation on day 47 or 75 after TAI. Control cows (n = 1,552 lactations) were those confirmed pregnant by transrectal palpation on day 75 after TAI. Case cows were matched with eligible controls according to year of calving and calving-to-conception interval (CCI) ± 3 days. Cows with different parities were classified as exposed to subclinical mastitis (somatic cell score (SCS) > 4.5 in at least one Dairy Herd Improvement Association (DHIA) test day) or clinical mastitis (with or without evidence of subclinical mastitis) during two exposure periods: 1-42 days before breeding or 1-75 days during gestation (1 to PL diagnosis day in case cows, or 1-75 day in control cows). Conditional logistic regression was used to model the odds of PL as a function of previous exposure to mastitis in different parities. Cost of PL attributable to mastitis ($/case) among cows with mastitis was estimated based on attributable risk calculated in the epidemiologic analysis. We observed a higher than expected combined effect between exposure to mastitis (subclinical or clinical) before breeding and parity 3 or ≥ 4, and during gestation and parity ≥ 4 on PL. The cost of PL attributable to mastitis was highest ($196/case) in cows in parity ≥ 4 affected with clinical mastitis during gestation. Overall, study results indicate the impact of mastitis on PL is higher in older cows (parity ≥ 3). Dairy farmers and attending veterinarians can consider the combined effect of mastitis and parity when evaluating causes for PL and strategies for optimizing reproductive performance in dairy cows.

Draft Genome Sequences of Bacteroides pyogenes Strains Isolated from the Uterus of Holstein Dairy Cows with Metritis.

Bacteroides pyogenes is found in the human and animal gut and is implicated in the pathogenesis of metritis in cows. We report the draft genome sequences of four Bacteroides pyogenes isolates obtained from the uterus of metritic cows. This will increase the understanding of its pathogenicity, antimicrobial resistance, and differentiation across hosts.