Testing the Induction of Metritis in Healthy Postpartum Primiparous Cows Challenged with a Cocktail of Bacteria.

Metritis is a postpartum uterine disease with greater incidence in primiparous than in multiparous cows. In primiparous cows, the impact on production and health is lessened, presumably due to a superior immune response. Here, we tested whether an in vivo model of clinical metritis induction developed for postpartum multiparous Holstein cows would produce similar results in primiparous cows. Thirty-six cows were randomly assigned to one of three groups and received intrauterine infusion within 24 h of parturition. The controls were infused with sterile saline; the low-dose group received a bacterial cocktail containing 103 cfu of Escherichia coli, Trueperella pyogenes, and Fusobacterium necrophorum; and the high-dose group were infused with 106 cfu of the same cocktail. Production, health traits, and the vaginal discharge culture were assessed daily, from enrollment until 14 d in milk. Clinical metritis occurred in 64% of high-dose cows, 33% of the controls, and 42% of low-dose cows, with no significant difference of incidence between groups. However, when accounting by time, high-dose cows had a 2.7 times greater hazard of metritis compared with the controls. The bacterial challenge affected milk production and dry matter intake tended to decrease. In the high-dose group, a greater growth of F. necrophorum in the selective medium was also observed, suggesting an association with metritis. Therefore, this study suggests intrauterine inoculation with 106 cfu of this bacterial cocktail elicits physical and clinical outcomes consistent with clinical metritis.

Short communication: Analysis of the nasal microbiota in newly received feedlot heifers according to subsequent incidence of bovine respiratory disease.

This study compared the relative abundance of bacteria in the nasal cavity of high-risk beef heifers at feedlot arrival according to subsequent incidence of bovine respiratory disease (BRD). Angus-influenced heifers (n = 76) were transported for 1,100 km (11 h) to the feedlot (day -1). At feedlot arrival (day 0), heifers were weighed [shrunk body weight (BW) = 234 ± 15 kg] and a nasal cavity swab collected for microbiota analysis. Heifers were ranked by arrival BW and allocated into 6 pens on day 1 where they remained until day 55. Heifers were evaluated daily for BRD signs (days 0 to 55), and a final shrunk BW was recorded on day 56 (16-h feed and water deprivation). Heifers were classified according to number of antimicrobial treatments for BRD received (0, 1, or ≥2), or according to time of the first incidence of BRD signs (no incidence [NOBRD], early incidence [EARLY; 4.1 ± 0.1 d, ranging from 3 to 6 d], or late incidence [LATE; 18.5 ± 9.6 d, ranging from 10 to 28 d]). Average daily gain decreased linearly (P = 0.04) according to number of BRD treatments, and was less (P = 0.04) in LATE and tended (P = 0.08) to be less in EARLY compared with NOBRD. The abundance of the Tenericutes phylum increased linearly (P < 0.01), while the abundance of other phyla (e.g., Firmicutes and Bacteroidetes) decreased linearly (P ≤ 0.05) and phyla diversity tended to decrease linearly (P = 0.10) according to number of BRD treatments. Heifers classified as EARLY had greater (P = 0.01) abundance of Tenericutes compared with NOBRD, whereas Tenericutes abundance in LATE heifers was intermediate and did not differ (P = 0.22) compared with EARLY and NOBRD. The abundance of Mycoplasma genus increased linearly (P < 0.01) while the abundance of other genera (e.g., Corynebacterium and Blautia) and genera diversity decreased linearly (P ≤ 0.03) according to number of BRD treatments. Heifers classified as EARLY had greater (P = 0.01) abundance of Mycoplasma and reduced (P = 0.01) genera diversity compared with NOBRD, and values noted in LATE heifers for these variables were intermediate and not different (P ≥ 0.27) compared with EARLY and NOBRD. Hence, heifers that developed BRD during the experiment had altered nasal microbiota at arrival compared with heifers that remained healthy, particularly increased prevalence of Tenericutes and Mycoplasma. Such differences in nasal microbiota were heightened in heifers that developed BRD shortly after arrival, or that required multiple antimicrobial treatments.

Bovine respiratory disease (BRD) is the most common disease in feedlot cattle and costs the US cattle industry more than $2 billion annually. Such economical losses include mortality, wasted feed resources, pharmaceutical inputs, and decreased performance of morbid cattle. Hence, research to understand the etiology of BRD is critical to lessen the incidence and productive impacts of this disease in feedlot systems. The upper respiratory tract is home to a plethora of bacteria associated with BRD in cattle, whereas the composition and stress-related imbalances in this microbiota can lead to the disease. Based on this rationale, this experiment evaluated the microbiota composition in the nasal cavity of newly receiving feedlot heifers and contrasted with subsequent prevalence of BRD. In general, heifers that develop BRD had altered nasal microbiota at the time of feedlot arrival compared with heifers that remained healthy. Such differences in microbiota were heightened in heifers that developed BRD shortly after arrival, or heifers that required multiple antimicrobial treatments upon disease occurrence.

Characterization of rumen microbiome and metabolome from oro-esophageal tubing and rumen cannula in Holstein dairy cows.

Less invasive rumen sampling methods, such as oro-esophageal tubing, became widely popular for exploring the rumen microbiome and metabolome. However, it remains unclear if such methods represent well the rumen contents from the rumen cannula technique. Herein, we characterized the microbiome and metabolome in the rumen content collected by an oro-esophageal tube and by rumen cannula in ten multiparous lactating Holstein cows. The 16S rRNA gene was amplified and sequenced using the Illumina MiSeq platform. Untargeted metabolome was characterized using gas chromatography of a time-of-flight mass spectrometer. Bacteroidetes, Firmicutes, and Proteobacteria were the top three most abundant phyla representing ~ 90% of all samples. Although the pH of oro-esophageal samples was greater than rumen cannula, we found no difference in alpha and beta-diversity among their microbiomes. The overall metabolome of oro-esophageal samples was slightly different from rumen cannula samples yet more closely related to the rumen cannula content as a whole, including its fluid and particulate fractions. Enrichment pathway analysis revealed a few differences between sampling methods, such as when evaluating unsaturated fatty acid pathways in the rumen. The results of the current study suggest that oro-esophageal sampling can be a proxy to screen the 16S rRNA rumen microbiome compared to the rumen cannula technique. The variation introduced by the 16S rRNA methodology may be mitigated by oro-esophageal sampling and the possibility of increasing experimental units for a more consistent representation of the overall microbial population. Studies should consider an under or over-representation of metabolites and specific metabolic pathways depending on the sampling method.

Virulence factors, antimicrobial resistance and phylogeny of bovine mastitis-associated Streptococcus dysgalactiae.

We carried out a thorough genetic evaluation of Streptococcus dysgalactiae isolated from clinical bovine mastitis cases and performed a phylogenetic analysis to represent the evolutionary relationship between S. dysgalactiae sequences. A total of 35 S. dysgalactiae strains were isolated from cases of clinical mastitis identified at a large commercial dairy farm located near Ithaca, New York. Whole-genome sequencing identified twenty-six antibiotic resistance genes, four of which were acquired genes, in addition to fifty virulence genes. Multi-locus sequence typing detected three new sequence types (STs). We conclude that a high proportion of this microorganism carries multiple virulence determinants and resistance genes, and that this indicates its potential to cause mastitis. Eight different STs were identified, of which ST453 (n = 17) was the most prevalent and ST714, ST715, ST716 were novel STs.

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.

Evaluation of Recombinant Bovine Interleukin-8 (rbIL-8) as a Treatment for Chronic Intramammary Infection in Dairy Cows.

Mastitis is one of the main contributors to antimicrobial resistance in livestock, so alternative therapies are being investigated to address it. The present study assessed the capability of recombinant bovine interleukin-8 (rbIL-8) to improve neutrophil function in the mammary gland and resolve chronic high somatic cell count (SCC) in Holstein cows. Multiparous cows (n = 8) with more than 300,000 SCC per mL were allocated to one of two intramammary infusions: saline (10 mL of saline solution) or rbIL-8 (1.57 mg/mL of recombinant bovine IL-8 diluted in 9 mL of saline). In addition, there was an untreated control group (n = 2, SCC < 300,000 SCC/mL). Milk samples were collected post-treatment at 0, 4, 8, 12, 24, 48, and 144 h to quantify milk SCC, haptoglobin, and IgG concentrations. Neutrophil's phagocytosis in milk and blood was evaluated via flow cytometry at 0, 24, and 48 h. The log of SCC did not differ between the infused groups (p = 0.369). Neutrophils presented a similar log of cells with high fluorescence for propidium-iodide (PI) and dihydrorhodamine (DHR) in milk (p = 0.412) and blood samples (p = 0.766) in both infused groups. Intramammary infusion of 1.57 mg/mL of rbIL-8 did not improve neutrophils response and failed to resolve chronic high SCC.

Whole-genome sequencing reveals high genetic diversity of Streptococcus uberis isolated from cows with mastitis.

BACKGROUND: Bovine mastitis is an important cause of economic loss in dairy farms. Streptococcus uberis is among the most frequently isolated bacterial species isolated from cows with mastitis. The aim of this study was to perform an in-depth genetic assessment of S. uberis strains isolated from bovine clinical mastitis (CM) and to perform a phylogenetic analysis to represent the evolutionary relationship among S. uberis sequences. RESULTS: A total of 159 isolates was genetically characterized using whole genome sequencing. According to the virulence determinants, all strains harbored the hasC, leuS, perR, purH, and purN virulence genes. Thirty-four resistance genes were identified in at least one strain. In terms of acquired genes, we observed that 152 (95.6 %) strains had a resistance gene to lincosamine (lnuD), 48 (30.2 %) to tetracycline (tetM), 4 (2.51 %) to tobramicine (ant6), and 1 to lincosamide (lsa(E)). MLST detected the Sequence Type (ST)797 (n = 23), while 85.5 % of the strains did not match to known STs. CONCLUSIONS: Then, eleven distinct ST were identified after we submitted the new alleles to assign new STs. The other prevalent STs observed were ST1215 (n = 58), ST1219 (n = 35), and ST1213 (n = 15). And it was not possible to identify the MLST of four strains. Phylogenetic lineages indicated a high genomic diversity of S. uberis in our collection, confirming that most strains isolated from bovine mastitis have different reservoirs, typical of environmental pathogens.

Components of the Gut Microbiome That Influence Bone Tissue-Level Strength.

Modifications to the constituents of the gut microbiome influence bone density and tissue-level strength, but the specific microbial components that influence tissue-level strength in bone are not known. Here, we selectively modify constituents of the gut microbiota using narrow-spectrum antibiotics to identify components of the microbiome associated with changes in bone mechanical and material properties. Male C57BL/6J mice (4 weeks) were divided into seven groups (n = 7-10/group) and had taxa within the gut microbiome removed through dosing with: (i) ampicillin; (ii) neomycin; (iii) vancomycin; (iv) metronidazole; (v) a cocktail of all four antibiotics together (with zero-calorie sweetener to ensure intake); (vi) zero-calorie sweetener only; or (vii) no additive (untreated) for 12 weeks. Individual antibiotics remove only some taxa from the gut, while the cocktail of all four removes almost all microbes. After accounting for differences in geometry, whole bone strength was reduced in animals with gut microbiome modified by neomycin (-28%, p = 0.002) and was increased in the group in which the gut microbiome was altered by sweetener alone (+39%, p < 0.001). Analysis of the fecal microbiota detected seven lower-ranked taxa differentially abundant in animals with impaired tissue-level strength and 14 differentially abundant taxa associated with increased tissue-level strength. Histological and serum markers of bone turnover and trabecular bone volume per tissue volume (BV/TV) did not differ among groups. These findings demonstrate that modifications to the taxonomic components of the gut microbiome have the potential to decrease or increase tissue-level strength of bone independent of bone quantity and without noticeable changes in bone turnover. © 2021 American Society for Bone and Mineral Research (ASBMR).

Preliminary functional analysis of the subgingival microbiota of cats with periodontitis and feline chronic gingivostomatitis.

The subgingival microbial communities of domestic cats remain incompletely characterized and it is unknown whether their functional profiles are associated with disease. In this study, we used a shotgun metagenomic approach to explore the functional potential of subgingival microbial communities in client-owned cats, comparing findings between periodontally healthy cats and cats with naturally occurring chronic periodontitis, aggressive periodontitis, and feline chronic gingivostomatitis. Subgingival samples were subjected to shotgun sequencing and the metagenomic datasets were analyzed using the MG-RAST metagenomic analysis server and STAMP v2.1.3 (Statistical Analysis of Metagenomic Profiles) software. The microbial composition was also described to better understand the predicted features of the communities. The Respiration category in the level 1 Subsystems database varied significantly among groups. In this category, the abundance of V-Type ATP-synthase and Biogenesis of cytochrome c oxidases were significantly enriched in the diseased and in the healthy groups, respectively. Both features have been previously described in periodontal studies in people and are in consonance with the microbial composition of feline subgingival sites. In addition, the narH (nitrate reductase) gene frequency, identified using the KEGG Orthology database, was significantly increased in the healthy group. The results of this study provide preliminary functional insights of the microbial communities associated with periodontitis in domestic cats and suggest that the ATP-synthase and nitrate-nitrite-NO pathways may represent appropriate targets for the treatment of this common disease.

Effect of metaphylactic administration of tildipirosin on the incidence of pneumonia and otitis and on the upper respiratory tract and fecal microbiome of preweaning Holstein calves.

The objectives of this study were to evaluate the effect of the metaphylactic use of a semi-synthetic long-acting macrolide (tildipirosin) on the prevention of pneumonia and otitis in preweaning Holstein calves, as well as its effects on the microbiome of their upper respiratory tract (URT) and feces. Newborn healthy Holstein heifers, collectively housed, were randomly allocated to 1 of 2 treatment groups: treatment (TRT; n = 932) or control (CTR; n = 927). Calves in the TRT group received a single subcutaneous injection of 4 mg/kg tildipirosin (Zuprevo, Merck Animal Health) at 7 ± 7 d of life. Calves in the CTR group received no drug injection. All enrolled calves were evaluated from 1 to 63 ± 3 d of life (weaning age) and monitored daily for any adverse health events during this period. Daily physical examination was performed to diagnose pneumonia and otitis, and body weight was measured weekly in all animals. From a randomly selected subset of 217 calves, blood samples for biochemical variables analysis and swabs were collected weekly from the URT and rectum for analysis of the nasal and fecal microbiome, respectively, via next-generation sequencing of the 16S rRNA gene. Total bacterial load was evaluated using quantitative PCR. In addition, another subset of 26 calves was randomly selected and fecal swabs were collected in a more intensive sampling to investigate the short-term effect of tildipirosin administration on the fecal microbiome. We performed general mixed linear models and logistic regression to analyze continuous and binary outcomes, respectively. Tildipirosin metaphylaxis reduced the incidence of otitis (CTR = 47.03%; TRT = 37.55%) and tended to reduce the incidence of pneumonia (CTR = 20.71%; TRT = 17.38%) and the overall mortality risk (CTR = 6.69%; TRT = 4.94%). We observed no significant differences between groups for mortality due to pneumonia (CTR = 0.86%; TRT = 0.97%) or mortality due to otitis (CTR = 2.05%; TRT = 1.39%). Calves in the TRT group had a higher average daily gain than calves in the CTR group. Furthermore, metaphylaxis had no significant effects on the total bacterial load, genus, or phylum analysis of the fecal microbiome from the 2 subset groups. However, for the URT microbiota, we observed a significant decrease in total bacterial load for the TRT group compared to the CTR group 1 week after metaphylactic injection. Tildipirosin metaphylaxis decreased the mean relative abundance of the genera Mannheimia, Moraxella, and Pasteurella but significantly increased the mean relative abundance of Mycoplasma. Although tildipirosin had no positive effect on Mycoplasma, it reduced the mean relative abundance of important pathogenic bacteria in the URT and had positive effects for the control of otitis. The metaphylactic use of tildipirosin can be a suitable strategy for the control of otitis on farms with a high prevalence of this disease.

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.

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.

Development and evaluation of a new recombinant protein vaccine (YidR) against Klebsiella pneumoniae infection.

Vaccination is a promising approach to prevent Klebsiella infection; however, the high heterogeneity of strains is a limiting factor. The best antigenic target for an anti-Klebsiella vaccine should be expressed by all or most of strains. We previously found YidR protein to be highly conserved among K. pneumoniae strains independently of antigen serotype. Therefore, in the present study, we developed a recombinant YidR protein vaccine and evaluated its protective efficacy against lethal challenge with K. pneumoniae in a mouse model. The yidR gene was cloned in Escherichia coli for recombinant expression. The lethal dose (LD100) of K. pneumoniae was determined and lethal challenge was carried out after immunization with recombinant purified YidR. After immunization, the concentration of total serum IgG was significantly higher in YidR-immunized mice than in non-immunized mice, indicating strong induction of antibodies. Mice were challenged with LD100 of K. pneumoniae, and significantly lower murine sepsis and higher body weight were observed in YidR-immunized mice compared to unvaccinated controls. Moreover, ∼90% of YidR-immunized mice survived beyond 10 days of observation, whereas none of the control mice survived past 48 h. The protective effect of YidR recombinant protein vaccine was demonstrated and YidR may be a promising vaccine candidate to prevent klebsiellosis.

Bovine Endometritis and the Inflammatory Peripheral Cholinergic System.

Endometritis is an inflammation of the endometrium associated with bacterial infection. The pathogenesis of endometritis in cows is still not completely understood. The combined analysis of the markers of inflammation and oxidative stress has contributed to a better understanding of disease mechanisms, but is still unexplored in uterine disorders. Moreover, research provides evidence about an important role of the vagus nerve in regulating the innate immune function through the cholinergic anti-inflammatory pathway in response to bacterial infections. This new pathway has demonstrated a critical role in controlling the inflammatory system. The aim of this study was to evaluate the activity of cholinesterase in total blood, lymphocytes, and serum of dairy cows with clinical and subclinical endometritis. Sixty-one Holstein cows, between 30 and 45 days in milk, were classified into 3 groups of animals: presenting clinical endometritis (n = 22), subclinical endometritis (n = 17), and healthy (n = 22). Mean leukocyte counts did not differ among groups, but the neutrophil number was significantly higher in cows with clinical endometritis than those in healthy animals. Also, serum concentration of interleukin-1beta (pg/mL) was significantly higher in cows with endometritis. The activity of acetylcholinesterase in blood and lymphocytes increased in both groups with endometritis. Animals with endometritis presented an increase in lipid peroxidation, but the antioxidant enzyme activity (catalase levels) was higher in endometritis groups than in normal cows. In conclusion, the inflammatory process of clinical and subclinical endometritis leads to systemic lipid peroxidation despite the compensatory increase of the antioxidant enzyme. These data also provide evidence of an important role of the cholinergic pathway in regulating dairy cows with clinical and subclinical endometritis.

Endodontic Microbiome of Fractured Non-vital Teeth in Dogs Determined by 16S rRNA Gene Sequencing.

Dental fractures resulting in pulp exposure will lead to an endodontic infection with microbes from the oral cavity. However, data on the endodontic microbial composition in veterinary dentistry is lacking. The aim of this study was to examine the microbiome of naturally occurring primary endodontic infections in client-owned dogs. The endodontic microbiome of 10 non-vital teeth with exposed pulp cavities was assessed using a 16S rRNA gene sequencing approach. The results were compared to the microbiome of the subgingival plaque of the same teeth. Analysis revealed an abundant mixed microflora of a comparable richness and diversity and with mostly the same phyla obtained from sulcal and endodontic samples. However, further analysis revealed significant differences between sulcal and endodontic samples in the relative abundance of the most abundant phyla and genera, with the relative abundance of Bacteriodetes being significantly higher in endodontic samples. Although each sample presented a particular profile regarding the genera identified, Bacteroides was the most abundant genus in the endodontic samples. Snowella was also significantly more abundant in endodontic samples, while Porphyromonas and Fusobacterium were significantly more abundant in sulcal samples. We confirmed that the microbiome of the diseased endodontic system is comparably abundant with microorganisms to the healthy subgingival plaque indicating that previous culture-based studies of primary endodontic infections in dogs underestimated the richness and diversity of the endodontic microbiota.

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.

The subgingival microbial community of feline periodontitis and gingivostomatitis: characterization and comparison between diseased and healthy cats.

Periodontitis is a common and important health problem in domestic cats. The subgingival microbiota of cats diagnosed with chronic periodontitis (CP), aggressive periodontitis (AP), and feline chronic gingivostomatitis (FCGS) are not well characterized. Thus, the aim of the present study was to characterize and compare the periodontal microbiota of periodontally healthy cats versus cats diagnosed with CP, AP, and FCGS by using next-generation sequencing. In total, 44 domestic cats were enrolled, and 139 subgingival samples were subjected to 16S rRNA gene sequencing to investigate the microbiota composition of each periodontal group evaluated. Our results identified several key genera previously described in periodontal disease (e.g. Treponema and Filifactor) and in the oral microbiota (e.g. Moraxella and Capnocytophaga) of healthy cats. Phylogenetic beta diversity analysis showed that the microbiota of periodontally healthy cats were distinguishable from diseased cats. Even though most of the genera known to be associated with periodontal disease were also identified in healthy cats, they were present at significantly lower relative abundance. Remarkably, alpha diversity was found to be higher in the disease groups compared to healthy animals. These results suggest a pathological mechanism involving opportunistic behavior. Our findings corroborate those in the current literature regarding the complexity of the subgingival microbiota of the domestic cat and reveal both differences and similarities among periodontally healthy and diseased cats.

The microbial metagenome and bone tissue composition in mice with microbiome-induced reductions in bone strength.

The genetic components of microbial species that inhabit the body are known collectively as the microbiome. Modifications to the microbiome have been implicated in disease processes throughout the body and have recently been shown to influence bone. Prior work has associated changes in the microbial taxonomy (phyla, class, species, etc.) in the gut with bone phenotypes but has provided limited information regarding mechanisms. With the goal of achieving a more mechanistic understanding of the effects of the microbiome on bone, we perform a metagenomic analysis of the gut microbiome that provides information on the functional capacity of the microbes (all microbial genes present) rather than only characterizing the microbial taxa. Male C57Bl/6 mice were subjected to disruption of the gut microbiota (ΔMicrobiome) using oral antibiotics (from 4 to 16 weeks of age) or remained untreated (n = 6-7/group). Disruption of the gut microbiome in this manner has been shown to lead to reductions in tissue mechanical properties and whole bone strength in adulthood with only minor changes in bone geometry and density. ΔMicrobiome led to modifications in the abundance of microbial genes responsible for the synthesis of the bacterial cell wall and capsule; bacterially synthesized carbohydrates; and bacterially synthesized vitamins (B and K) (p < 0.01). Follow up analysis focused on vitamin K, a factor that has previously been associated with bone health. The vitamin K content of the cecum, liver and kidneys was primarily microbe-derived forms of vitamin K (menaquinones) and was decreased by 32-66% in ∆Microbiome mice compared to untreated animals (p < 0.01). Bone mineral crystallinity determined using Raman spectroscopy was decreased in ∆Microbiome mice (p = 0.01). This study illustrates the use of metagenomic analysis to link the microbiome to bone phenotypes and provides preliminary findings implicating microbially synthesized vitamin-K as a regulator of bone matrix quality.

The Bos taurus maternal microbiome: Role in determining the progeny early-life upper respiratory tract microbiome and health.

Natural transference of maternal microbes to the neonate, especially at birth via the vaginal canal, has recently been recognized in humans and cows; however, its microbial influence on calf health has not yet been documented. We compared the bacterial communities in vaginal and fecal samples from 81 pregnant dairy cows versus those in nasopharyngeal and fecal samples collected at 3, 14 and 35 days of life from their respective progeny. The microbiota of the calf upper respiratory tract (URT), regardless of calf age, was found to be highly similar to the maternal vaginal microbiota. Calf fecal microbiota clustered closely to the maternal fecal microbiota, progressing toward an adult-like state over the first 35 days when relative abundances of taxa were considered. Sixty-four, 65 and 87% of the detected OTUs were shared between cow and calf fecal microbiota at days 3, 14 and 35 respectively, whereas 73, 76 and 87% were shared between maternal vaginal microbiome and calf URT microbiota at days 3, 14 and 35, respectively. Bacteroidetes, Ruminococcus, Clostridium, and Blautia were the top four genera identified in maternal and calf fecal samples. Mannheimia, Moraxella, Bacteroides, Streptococcus and Pseudomonas were the top five genera identified in maternal vaginal and calf URT samples. Mannheimia was relatively more abundant in the vaginal microbiota of cows whose progeny were diagnosed with respiratory and middle ear disease. Our results indicate that maternal vaginal microbiota potentially influences the initial bacterial colonization of the calf URT, and that might have an important impact on the health of the calf respiratory tract and middle ear.

Hair and saliva analysis fails to accurately identify atopic dogs or differentiate real and fake samples.

BACKGROUND: The availability of direct-to-consumer medical testing for human and veterinary health conditions has increased in recent years. For allergies, several companies market proprietary hair and saliva tests directly to pet owners. These tests have not been validated and there is limited regulatory oversight for such tests in veterinary medicine. HYPOTHESIS/OBJECTIVES: To examine the accuracy and reproducibility of a commercial direct-to-consumer hair and saliva allergen test. ANIMALS: Seven healthy animals (six dogs, one cat); six animals (five dogs, one cat) with atopic dermatitis; 11 samples of synthetic fur and sterile saline. METHODS AND MATERIALS: Duplicate animal hair and saliva, and 11 synthetic fur and saline samples were collected (total samples 35) and submitted to the company for analysis, yielding 12,075 outcomes for statistical analysis. RESULTS: Positive test results were provided by the direct-to-consumer pet allergy for all submitted samples, including synthetic fur and saline. The test results for healthy and atopic animal samples were no different from each other or from synthetic fur and saline samples. Reproducibility for paired samples was not different from random chance. The results for real animals correlated strongly with results for synthetic fur and saline samples (r = 0.71, P < 0.05). CONCLUSIONS AND CLINICAL IMPORTANCE: The direct-to-consumer hair and saliva test for pet allergies examined in this study performed no better than chance and the results were not reproducible.