Treatment with 2 commercial antibiotics reduced clinical and systemic signs of pneumonia and the abundance of pathogenic bacteria in the upper respiratory tract of preweaning dairy calves.

The aim of this study was to evaluate the effect of therapeutically administered tildipirosin or florfenicol + flunixin meglumine for the treatment of bovine respiratory disease (BRD) accompanied by fever in calves before weaning compared with diseased and untreated animals. As specific objectives, we evaluated the composition of the bacterial microbiota of the upper respiratory tract (URT) and blood and health parameters of the animals. Preweaning Holstein female calves diagnosed with naturally acquired pneumonia were randomly assigned to one of the following experimental groups on the day of diagnosis (d 0): (1) TLD (n = 36): single subcutaneous injection with 4 mg/kg tildipirosin; (2) FLF (n = 33): single subcutaneous injection with an antimicrobial (40 mg/kg florfenicol) combined with a nonsteroidal anti-inflammatory drug (2.2 mg/kg flunixin meglumine); and (3) NEG (n = 35): no treatment within the first 5 d following enrollment. The NEG treatment group was closely monitored for 5 d, and calves were removed from the study following a standardized late treatment protocol, when necessary, to minimize health concerns. Healthy untreated calves (CTR; n = 31) were also selected for the study and used as controls. Blood samples used for biochemical analysis and nasopharyngeal swabs used for evaluation of URT microbiota were collected daily from d 0 until d 5 and then weekly until weaning. Next-generation sequencing of the 16S rRNA gene was used to assess the URT microbiota at the phylum and genus levels. Clinical signs associated with pneumonia and otitis media were assessed daily, as was the need for antibiotic interventions. Calves in the TLD and FLF groups had faster recovery from fever within the first 5 d after enrollment. In addition, antibiotic-treated calves reached the same serum haptoglobin levels as healthy calves on d 2 after diagnosis, whereas calves in the NEG group had higher haptoglobin levels than the CTR group until at least d 5 after BRD diagnosis. Calves in the TLD and FLF groups had a lower risk of treatment for pneumonia (FLF = 22.8%; TLD = 27.7%) from d 5 to weaning than calves in the NEG group (54.7%). Furthermore, FLF treatment had a significantly lower risk of nasal discharge, otitis media, and treatment failure compared with the NEG group, but did not differ from the TLD group. Differences in the composition of the URT microbiota were found between groups, and the genus Mycoplasma was the most abundant in samples collected from the URT of calves with and without pneumonia. Both drugs were effective in reducing the mean relative abundance (MRA) of important genera associated with pneumonia (Mannheimia and Pasteurella), although an increase in Mycoplasma MRA was observed for tildipirosin-treated calves. In conclusion, both drugs were effective in reducing the inflammatory signs of pneumonia and the need for antimicrobial treatment after enrollment compared with no treatment. In addition, both TLD and FLF were effective in reducing the MRA of important bacterial genera associated with pneumonia; however, TLD treatment was associated with increased Mycoplasma MRA compared with healthy and untreated calves.

Intrauterine infusion of a pathogenic bacterial cocktail is associated with the development of clinical metritis in postpartum multiparous Holstein cows.

Metritis is a uterine disorder common in dairy cattle caused by bacterial infection, with greater incidence in the early postpartum period. The disease causes delayed uterine involution, with a fetid, watery, red-brown discharge, with animals presenting different clinical signs including fever, dullness, inappetence, decreased milk yield, and dehydration. We developed an in vivo model of clinical metritis in Holstein multiparous cows using a pathogenic cocktail of Escherichia coli, Fusobacterium necrophorum, and Trueperella pyogenes. A total of 36 multiparous cows were randomly allocated to 1 of 3 treatment groups of 12 animals each. Cows assigned to the control group received an intrauterine administration of sterile saline solution, those in the low-dose group received a bacterial inoculum containing 106 cfu of Escherichia coli, Trueperella pyogenes, and Fusobacterium necrophorum; and those in the high-dose group received 109 cfu of these same organisms, all within 24 h of parturition. Clinical signs; milk yield; dry matter intake; serum concentration of acute phase proteins, metabolites, and cytokines; and bacterial counts in vaginal discharge were measured from parturition until 14 d in milk. Based on clinical signs, cows in the low-dose group had a greater incidence of metritis (83.3%) compared with the control (9%) or high-dose (25%) group. The low-dose group also had lesser dry matter intake compared with controls and an acute phase protein profile that typified metritis. The low-dose group presented greater relative abundance of bacteria from the genus Fusobacterium in the vaginal discharge compared with control cows, and the high-dose group was similar to the control group. Unexpectedly, intrauterine infusion of cows with a lower dose of bacteria was more effective than a higher dose, indicating that the bacterial load may affect metritis incidence in a nonlinear manner. These findings demonstrate that clinical metritis can be experimentally induced in postpartum multiparous Holstein cows, creating a relevant model for the study of uterine diseases.

Effect of treatment of pneumonia and otitis media with tildipirosin or florfenicol + flunixin meglumine on health and upper respiratory tract microbiota of preweaned Holstein dairy heifers.

The objective of this randomized clinical study was to compare the effect of 2 antimicrobial interventions, tildipirosin or florfenicol + flunixin meglumine, used for treatment of pneumonia and extralabel treatment for otitis on health parameters and upper respiratory tract (URT) microbiota of preweaned Holstein calves. Housed preweaned Holstein heifers diagnosed with either otitis or pneumonia were assigned into 1 of 2 treatment groups, receiving a single subcutaneous injection of either 4 mg/kg of tildipirosin (TLD; n = 444) or 40 mg/kg of florfenicol combined with 2.2 mg/kg of a nonsteroidal anti-inflammatory, flunixin meglumine (FLF; n = 442). Calves were enrolled and treated on the day of diagnosis of the first case of pneumonia or otitis. If a calf had a recurrent case, the opposite drug was administered, respecting an interval of 5 d between drug injections. Blood samples for leukocyte counts were collected at 0, 2, 4, and 6 d after treatment, and rectal temperature was measured daily during the 5 d after treatment. Ear scores were observed from calves with otitis. Additionally, swabs of the URT were collected from a subset of 20 calves in each treatment group at d 0, 3, 6, 9, and 11 following enrollment for analysis of URT microbiota through next-generation sequencing of the 16S rRNA gene and quantitative PCR. Swabs were also collected from a comparative group of 20 healthy calves that did not receive any drug. No differences were observed between groups for recurrence risk of either pneumonia (TLD = 32.4%; FLF = 29.7%) or otitis (TLD = 72.7%; FLF = 73.6%). Similarly, no differences were observed for the total number of treatments for pneumonia (TLD = 1.45; FLF = 1.42) or otitis (TLD = 2.96; FLF = 3.07). On the other hand, both drugs reduced rectal temperature, ear scores, and leukocyte counts, with FLF calves having a greater reduction in rectal temperature within 4 d after treatment. Both TLD and FLF reduced the total bacterial load when compared with healthy untreated calves, but no differences were observed between treatment groups. Furthermore, compared with the untreated group, treated calves had lower mean relative abundances (MRA) of the genera Mannheimia, Moraxella, and Pasteurella within 11, 9, and 3 d after treatment, respectively; however, no significant differences were observed between TLD and FLF. On the other hand, MRA of Mycoplasma was not decreased by both treatments compared to untreated animals, and a higher MRA was observed in the TLD group during 11 d after treatment in comparison to FLF and untreated calves. Based on this data, we concluded that both drugs used in the study were effective in reducing rectal temperature, ear scores, leukocyte counts, and MRA of the genera Mannheimia, Pasteurella, and Moraxella in the URT, and calves treated with FLF had a greater reduction in rectal temperature.

Immunization with a novel recombinant protein (YidR) reduced the risk of clinical mastitis caused by Klebsiella spp. and decreased milk losses and culling risk after Escherichia coli infections.

The primary objective of this study was to evaluate the protective efficacy of a novel recombinant subunit vaccine containing the protein YidR (rYidR) against clinical mastitis (CM) caused by Klebsiella spp. and Escherichia coli. Given that E. coli infection is known to cause metritis, we also evaluated the effect of rYidR vaccination on the incidence of metritis and conception at the first artificial insemination. Retained placenta and abortion incidence, milk production and composition, and serological responses to specific antigens were also evaluated. In total, 3,107 cows were blocked by parity and randomly allocated into 1 of 3 treatment groups: experimental recombinant subunit vaccine containing the YidR protein (rYidR); commercial vaccine composed of Klebsiella pneumoniae siderophore receptors and porin protein (Kleb-SRP; KlebVax, Epitopix, Willmar, MN); and sterile water adjuvanted with aluminum hydroxide (20%; placebo). Vaccinations were performed at the dry-off for cows, and at 223 ± 3 d of pregnancy for pre-fresh heifers. A second administration was given at 21 ± 3 d after the first injection. Vaccination with rYidR significantly reduced the incidence of CM caused by Klebsiella spp. (3.2%) when compared with the placebo (5.1%) group. No difference was observed on risk of Klebsiella CM between Kleb-SRP (5.9%) and placebo groups. Cows in the rYidR group that experienced E. coli CM had a lower risk of death or culling (12.5%) compared with the Kleb-SRP (27.6%) and placebo groups (27.8%). Furthermore, among cows that developed E. coli CM, rYidR-immunized cows produced more milk than did cows in the placebo and Kleb-SRP groups. Regardless of CM occurrence, rYidR-immunized cows tended to have higher milk production up to the eighth month of lactation than cows in the other groups. No significant effect of treatment was observed on the overall incidence of abortion and metritis; however, the risk of retained placenta tended to be lower for the rYidR group (4.7%) compared with the placebo group (6.7%). In addition, primiparous cows in the rYidR group had the highest conception risk at the first artificial insemination (48.3%) compared with the placebo (39.5%) group, and no significant difference was observed when the Kleb-SRP (40.1%) group was compared with the placebo group. Generally, higher antibody serum titers (IgM and IgG) were observed for the immunized groups compared with the placebo. In conclusion, the rYidR vaccine reduced the risk of CM caused by Klebsiella spp. and the mortality or culling of cows with E. coli infections. Other benefits of the novel vaccine include maintenance of milk production after CM caused by E. coli, and higher conception risk at the first service in primiparous cows compared with cows in the placebo and Kleb-SRP groups.

Negatively controlled, randomized clinical trial comparing different antimicrobial interventions for treatment of clinical mastitis caused by gram-positive pathogens.

The general objective of this study was to evaluate the effect of 3 intramammary antibiotic interventions using 2 commercially available antibiotics with narrow- or broad-spectrum activity on cure rates of clinical mastitis (CM) caused by gram-positive bacteria. We also compared the efficacy of treatment protocols, including a negative control, on outcomes at the cow and mammary quarter level. Before the onset of the study, 5,987 animals more than 12 mo old were randomly preassigned to 1 of 4 protocols in the event of gram-positive CM (except for Staphylococcus aureus and Trueperella pyogenes) during lactation: 3 infusions with 62.5 mg of amoxicillin performed 12 h apart (AMOX-L); 5 infusions once a day with 62.5 mg of amoxicillin (AMOX-EL); 5 infusions once a day with 125 mg of ceftiofur hydrochloride (CEFT-L); or negative control, no treatment performed until 5 d after diagnosis (NEG-CTR). Randomization was performed to preassign 90% of cows to one of the antibiotic protocols (30% in each group) and 10% to the negative control. A total of 696 quarter cases of CM met the inclusion criteria and were evaluated in the study. Quarter-level outcomes were assessed based on 5 milk samples collected up to 14 ± 3 d following enrollment (i.e., first day of treatment), whereas variables at the cow level [composite somatic cell count (SCC), milk production, and survival in the herd] were assessed up to 90 d after CM diagnosis. Streptococcus uberis, followed by Streptococcus dysgalactiae, were the main causes of gram-positive CM. Overall, clinical cure was higher for CEFT-L than for AMOX-EL, and no difference was observed between CEFT-L and AMOX-L. Likewise, no significant differences were detected on overall bacteriological cure, although some treatment effects were observed at the species level. Compared with antibiotic-treated groups, quarters assigned to NEG-CTR had higher counts of colony-forming units (cfu), 16S rRNA gene copy numbers, and Streptococcus relative abundance (RA) until d 5 after enrollment. Quarters treated with AMOX-L had higher cfu counts on d 5, 8, and 14 after enrollment compared with the other antibiotic protocols. In addition, the RA of Streptococcus spp. was higher on d 14 after enrollment for AMOX-treated quarters compared with the CEFT-L group. Linear score of SCC was higher for AMOX-treated cows than for CEFT-L in the first test day after CM. However, cows assigned to AMOX-L had higher milk production than those submitted to the AMOX-EL and CEFT-L protocols. In conclusion, the 2-d protocol with 3 intramammary infusions of amoxicillin (narrow-spectrum antimicrobial) had similar overall clinical and bacteriological cures as 5 administrations (once a day) with ceftiofur hydrochloride (wide spectrum). No significant difference was observed on CM recurrence and cow survival. However, quarters treated with 5-d protocols were more effective at reducing milk cfu counts than quarters in the AMOX-L protocol. In addition, lower Streptococcus spp. RA was observed in ceftiofur-treated quarters compared with the amoxicillin protocols at d 14 after CM diagnosis. Based on results of microbiome and bacterial load (quantitative PCR and cfu count) up to 5 d after CM diagnosis, antibiotic use remains an indispensable strategy for treatment of CM caused by gram-positive bacteria.

Recombinant protein subunit vaccine reduces puerperal metritis incidence and modulates the genital tract microbiome.

The objective of this study was to evaluate the efficacy of 3 vaccine formulations containing proteins (FimH, leukotoxin, and pyolysin), inactivated whole cells (Escherichia coli, Fusobacterium necrophorum, and Trueperella pyogenes), or both, in the prevention of postpartum uterine diseases. A randomized clinical trial was conducted at a commercial dairy farm; 800 heifers were assigned into 1 of 4 different treatment groups: control, vaccine 1 (bacterin and subunit proteins), vaccine 2 (bacterin), and vaccine 3 (recombinant subunit proteins), and each heifer received a subcutaneous injection of its respective treatment at 240 ± 3 and 270 ± 3 d of gestation. Vaccination significantly reduced the incidence of puerperal metritis when compared with control (9.1% vs. 14.9%, respectively; odds ratio 0.51). Additionally, vaccine 3 was found to reduce the incidence of puerperal metritis when compared with the control (8.0% vs. 14.9%, respectively; odds ratio 0.46). Reproduction was improved for metritic cows that were vaccinated, and the effect was stronger for cows that were treated with vaccine 3. In general, vaccination decreased the total vaginal bacterial load and decreased the vaginal load of F. necrophorum by 9 d in milk. Vaccination reduced the prevalence of puerperal metritis in the first lactation of dairy cows, leading to less metritic disease and improved reproduction.

Dynamics of the microbiota found in the vaginas of dairy cows during the transition period: Associations with uterine diseases and reproductive outcome.

We investigated the microbiota found in the vaginas of Holstein dairy cows during the transition period and described the differences in bacterial composition and total bacterial load (TBL) associated with disease and fertility. Vaginal swabs were collected at -7, 0, 3, and 7 d relative to parturition from 111 dairy cows housed on a commercial dairy farm near Ithaca, New York. Microbiota were characterized by next-generation DNA sequencing of the bacterial 16S rRNA gene, and TBL was determined by real-time quantitative PCR. We applied repeated-measures ANOVA to evaluate the associations of uterine disease and related risk factors with the microbiota and TBL. We estimated phylum-specific bacterial load by multiplying the TBL by the relative abundance of each phylum observed in the metagenomics results. We confirmed the validity of this approach for estimating bacterial load by enumerating the number of bacteria in an artificial sample mixed in vitro and in clinical and healthy vaginal samples. Phyla associated with uterine disease and related risk factors were Proteobacteria, Fusobacteria, and Bacteroidetes. Cows with retained placenta and healthy cows had similar TBL at the day of parturition, but at d 7 postpartum, cows with retained placenta showed a significantly higher TBL, mainly driven by higher estimated loads of Fusobacteria and Bacteroidetes. Cows diagnosed with metritis had a significantly higher estimated load of Proteobacteria at d -7 and at calving and higher estimated loads of Fusobacteria in the postpartum samples. Additionally, the estimated load of Bacteroidetes at d 7 postpartum was higher for cows diagnosed with endometritis at 35 days in milk. Higher estimated loads of Fusobacteria and Bacteroidetes were also evident in cows with postpartum fever, in primiparous cows, in cows with assisted parturition, and in cows that gave birth to twins. Our findings demonstrated that microbiota composition and TBL were associated with known periparturient risk factors of uterine diseases and reproductive failure, including parity, assisted parturition, and retained fetal membranes.

The microbiome of bulk tank milk: Characterization and associations with somatic cell count and bacterial count.

Numerous studies have evaluated associations between bacterial groups and milk quality parameters. However, to our knowledge, no research has been published that has analyzed associations between the microbiome and quality parameters of bulk tank milk (BTM). Thus, the aims of this study were to identify the core microbiome of BTM and to examine associations between the microbiome and milk quality parameters. Four hundred seventy-two BTM samples from 19 different dairy farms located in New York State were analyzed by next-generation sequencing and quantitative PCR of the 16S rRNA gene to assess the milk microbiome and measure total bacterial load, respectively. Flow cytometry was used to determine bacterial and somatic cell counts. Heatmaps were constructed and simple linear regressions and response screening analysis were performed. To facilitate data analysis and interpretation of the results, we dichotomized the BTM samples into high (HSCC, >200,000) and low somatic cell count (LSCC, ≤200,000) and into high (HSPC, >3.6) and low log10 SPC (LSPC, ≤3.6). Spoilage-causing, spore-forming, and pathogenic bacteria of importance to the dairy industry were identified in the core microbiome. In addition, the taxa Thermoanaerobacterium and 5-7N15 were identified in the core microbiome; to our knowledge, these genera have not been previously identified in milk samples. Several bacterial genera were encountered in significantly higher relative abundances in the HSCC group when compared with the LSCC group, including Corynebacterium, Streptococcus, Lactobacillus, Coxiella, Arthrobacter, and Lactococcus. Additionally, several bacterial taxa were found in significantly higher relative abundances in the HSPC groups versus the LSPC groups: Acinetobacter, Enterobacteriaceae, Corynebacterium, and Streptococcus. In addition, Streptococcus was highly correlated with HSPC, and this genus was the second most abundant bacterial taxon detected in samples classified as HSCC. Bacterial diversity (Shannon index) was negatively correlated with bacterial load, suggesting that the microbiomes of high-bacterial load BTM samples are dominated by smaller groups of bacterial taxa. In conclusion, the associations described corroborated current knowledge about pathogens and spoilage bacteria in relationship to milk quality, and also indicated that other bacterial taxa should be a focus of further investigations.

The Lactococcus genus as a potential emerging mastitis pathogen group: A report on an outbreak investigation.

The bacterium Lactococcus lactis is widely used in food production and in medical applications, and is considered safe for human and animal use. However, studies have also linked Lactococcus bacteria to infection. For example, certain variants of Lactococcus species have been associated with bovine mastitis (e.g., Lactococcus lactis and Lactococcus garvieae). In this study, we investigated an outbreak of bovine mastitis thought to be associated with Lactococcus bacteria by using microbiological and molecular techniques. We used bacterial isolation, next-generation sequencing, DNA fingerprinting, and other methods to test our hypothesis that Lactococcus microbes were the primary pathogen causing the mastitis outbreak. Twenty-eight Lactococcus isolates were obtained from mastitic milk of 28 dairy cows. The isolates were identified as L. lactis (27 isolates) and L. garvieae (1 isolate). Phylogenetic analysis based on 16S rDNA gene sequence comparison indicated similarity among the L. lactis isolates as well as between the isolates and reference sequences. The DNA fingerprinting analysis based on random amplified polymorphic DNA results of the 27 L. lactis isolates identified different random amplified polymorphic DNA profiles, which suggests they originated from multiple sources. Microbiome analysis determined Lactococcus to be the dominant genus in the majority of the mastitic milk samples, whereas it was found in low relative abundance in healthy milk samples. The Lactococcus genus was detected in all environmental samples tested, and sampling of bulk tank milk corroborated that Lactococcus was not abundant in healthy milk from the same dairy herd. In summary, our findings suggest that Lactococcus bacteria are a potential etiological agent in the mastitis outbreak studied. Further studies should be conducted to understand the importance of Lactococcus, especially L. lactis, as pathogenic microbes in veterinary medicine and food safety.