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.

Optical fiber thermo-refractometer.

This work presents the implementation of a thermo-refractometer, which integrates the measurement of both refractive index and temperature in a single optical fiber structure. To this purpose, a lossy mode resonance (LMR)-based refractometer is obtained by means of the deposition of a titanium dioxide (TiO2) thin film onto a side-polished (D-shaped) single mode fiber. Measurement and subsequent temperature compensation are achieved by means of a fiber Bragg grating (FBG) inscribed in the core of the D-shaped region. The LMR wavelength shift is monitored in transmission while the FBG (FBG peak at 1533 nm) displacement is observed in reflection. The LMR is sensitive to both the surrounding refractive index (SRI), with a sensitivity of 3725.2 nm/RIU in the 1.3324-1.3479 range, and the temperature (- 0.186 nm/°C); while the FBG is only affected by the temperature (32.6 pm/°C in the 25°C - 45°C range). With these values, it is possible to recover the SRI and temperature variations from the wavelength shifts of the LMR and the FBG, constituting a thermo-refractometer, where it is suppressed the effect of the temperature over the refractometer operation, which could cause errors in the fourth or even third decimal of the measured SRI value.

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.

Nickel, manganese and copper removal by a mixed consortium of sulfate reducing bacteria at a high COD/sulfate ratio.

The use of sulfate-reducing bacteria (SRB) in passive treatments of acidic effluents containing heavy metals has become an attractive alternative biotechnology. Treatment efficiency may be linked with the effluent conditions (pH and metal concentration) and also to the amount and nature of the organic substrate. Variations on organic substrate and sulfate ratios clearly interfere with the biological removal of this ion by mixed cultures of SRB. This study aimed to cultivate a mixed culture of SRB using different lactate concentrations at pH 7.0 in the presence of Ni, Mn and Cu. The highest sulfate removal efficiency obtained was 98 %, at a COD/sulfate ratio of 2.0. The organic acid analyses indicated an acetate accumulation as a consequence of lactate degradation. Different concentrations of metals were added to the system at neutral pH conditions. Cell proliferation and sulfate consumption in the presence of nickel (4, 20 and 50 mg l(-1)), manganese (1.5, 10 and 25 mg l(-1)) and copper (1.5, 10 and 25 mg l(-1)) were measured. The presence of metals interfered in the sulfate biological removal however the concentration of sulfide produced was high enough to remove over 90 % of the metals in the environment. The molecular characterization of the bacterial consortium based on dsrB gene sequencing indicated the presence of Desulfovibrio desulfuricans, Desulfomonas pigra and Desulfobulbus sp. The results here presented indicate that this SRB culture may be employed for mine effluent bioremediation due to its potential for removing sulfate and metals, simultaneously.

Measurement of composite shrinkage using a fibre optic Bragg grating sensor.

Fibre Bragg grating is used to determine resin-based composite shrinkage. Two composite resins (Freedom from SDI and Z100 from 3M) were tested to determine the polymerization contraction behaviour. Each sample of resin was prepared with an embedded fibre Bragg grating. A LED activation unit with wavelength from 430 nm to 470 nm (Dabi Atlante) was used for resin polymerization. The wavelength position of the peak in the optical reflection spectra of the sensor was measured. The wavelength shift was related to the shrinkage deformation of the samples. Temperature and strain evolution during the curing phase of the material was monitored. The shrinkage in the longitudinal direction was 0.15 +/- 0.02% for resin Z100 (3M) and 0.06+/-0.01% for Freedom (SDI); two-thirds of shrinkage occurred after the first 50 s of illumination.

Effect of antibodies to intercellular adhesion molecule type 1 on the protection of distant organs during reperfusion syndrome in rats

We investigated kidney and lung alterations caused by intercellular adhesion molecule type 1 (ICAM-1) blockade after ischemia and reperfusion of hind limb skeletal muscles. Rats were submitted to ligature of the infrarenal aorta for 6 h. The animals were randomized into three groups of 6 rats each: group I, sacrificed after ischemia; group II, reperfusion for 24 h, and group III, reperfusion for 24 h after receiving monoclonal anti-ICAM-1 antibodies. At the end of the experiment, blood samples were collected for creatinine, lactate dehydrogenase, creatine phosphokinase, potassium, pH and leukocyte counts. Samples were taken from the muscles of the hind limbs and from the kidneys and lungs for histological analysis and measurement of the neutrophil infiltrate by myeloperoxidase staining. The groups did not differ significantly with regard to the laboratory tests. There were no major histological alterations in the kidneys. An intense neutrophil infiltrate in the lungs, similar in all groups, was detected. Myeloperoxidase determination showed that after reperfusion there was significantly less retention of polymorphonuclear neutrophils in the muscles (352 ± 70 vs 1451 ± 235 I 10² neutrophils/mg; P<0.01) and in the kidneys (526 ± 89 vs 852 ± 73 I 10² neutrophils/mg; P<0.01) of the animals that received anti-ICAM-1 before perfusion compared to the group that did not. The use of anti-ICAM-1 antibodies in this experimental model minimized neutrophil influx, thus reducing the inflammatory process, in the muscles and kidneys after ischemia and reperfusion of the hind limbs

Effect of antibodies to intercellular adhesion molecule type 1 on the protection of distant organs during reperfusion syndrome in rats.

We investigated kidney and lung alterations caused by intercellular adhesion molecule type 1 (ICAM-1) blockade after ischemia and reperfusion of hind limb skeletal muscles. Rats were submitted to ligature of the infrarenal aorta for 6 h. The animals were randomized into three groups of 6 rats each: group I, sacrificed after ischemia; group II, reperfusion for 24 h, and group III, reperfusion for 24 h after receiving monoclonal anti-ICAM-1 antibodies. At the end of the experiment, blood samples were collected for creatinine, lactate dehydrogenase, creatine phosphokinase, potassium, pH and leukocyte counts. Samples were taken from the muscles of the hind limbs and from the kidneys and lungs for histological analysis and measurement of the neutrophil infiltrate by myeloperoxidase staining. The groups did not differ significantly with regard to the laboratory tests. There were no major histological alterations in the kidneys. An intense neutrophil infiltrate in the lungs, similar in all groups, was detected. Myeloperoxidase determination showed that after reperfusion there was significantly less retention of polymorphonuclear neutrophils in the muscles (352 +/- 70 vs 1451 +/- 235 x 10(2) neutrophils/mg; P<0.01) and in the kidneys (526 +/- 89 vs 852 +/- 73 10(2) neutrophils/mg; P<0.01) of the animals that received anti-ICAM-1 before perfusion compared to the group that did not. The use of anti-ICAM-1 antibodies in this experimental model minimized neutrophil influx, thus reducing the inflammatory process, in the muscles and kidneys after ischemia and reperfusion of the hind limbs.