Evaluation of novel multiplex qPCR assays for diagnosis of pathogens associated with the bovine respiratory disease complex.

Bovine respiratory disease complex is the most common disease requiring the use of antimicrobials in industrial calf production worldwide. Pathogenic bacteria (Mannheimia haemolytica (Mh), Pasteurella multocida (Pm), Histophilus somni (Hs), and Mycoplasma bovis) and a range of viruses (bovine respiratory syncytial virus, bovine coronavirus, bovine parainfluenza virus type 3, bovine viral diarrhea virus and bovine herpesvirus type 1) are associated with this complex. As most of these pathogens can be present in healthy and diseased calves, simple detection of their presence in diseased calves carries low predictive value. In other multi-agent diseases of livestock, quantification of pathogens has added substantially to the predictive value of microbiological diagnosis. The aim of this study was to evaluate the ability of two recently developed quantitative PCR (qPCR) kits (Pneumo4B and Pneumo4V) to detect and quantify these bacterial and viral pathogens, respectively. Test efficiencies of the qPCR assays, based on nucleic acid dilution series of target bacteria and viruses, were 93-106% and 91-104%, respectively, with assay detection limits of 10-50 copies of nucleic acids. All 44 strains of target bacteria were correctly identified, with no false positive reactions in 135strains of non-target bacterial species. Based on standard curves of log10 CFU versus cycle threshold (Ct) values, quantification was possible over a 5-log range of bacteria. In 92 tracheal aspirate samples, the kappa values for agreement between Pneumo4B and bacterial culture were 0.64-0.84 for Mh, Pm and Hs. In an additional 84 tracheal aspirates, agreement between Pneumo4B or Pneumo 4V and certified diagnostic qPCR assays was moderate (0.57) for M. bovis and high (0.71-0.90) for viral pathogens. Thus Pneumo4 kits specifically detected and quantified the relevant pathogens.

Presence and localization of bacteria in the bovine endometrium postpartum using fluorescence in situ hybridization.

The aim of this study was to investigate bacterial invasiveness of the bovine endometrium during the postpartum period. Fluorescence in situ hybridization was applied to endometrial biopsies using probes for Fusobacterium necrophorum, Porphyromonas levii, Trueperella pyogenes, Escherichia coli and a probe for bacteria in general (the overall domain Bacteria) to determine their tissue localization. Holstein cows were sampled at three time points postpartum (T1: 4-12 days postpartum, T2: 24-32 days postpartum and T3: 46-54 days postpartum). At T1, cows were clinically scored as having a uterine infection based on presence of a brownish, fetid vaginal discharge or as normal if having normal lochia. An endometrial biopsy was taken from all cows at T1 (n = 57). Endometrial biopsies were taken from the same cows at T2 and T3 if allowed by the size of the cervical canal and if the cow had not been inseminated. Fifty and 39 biopsies were obtained at T2 and T3, respectively. The biopsies were evaluated for inflammation and for presence and localization of bacteria. When analyzed by the probe for the entire domain Bacteria, bacteria were found in most biopsies irrespectively of time (T1: 79.0%, T2: 82.0%, T3: 89.7%). Fusobacterium necrophorum and Porphyromonas levii were often present in the endometrium at T1 (61.1% and 47.8%, respectively), but the prevalence decreased significantly over time. Trueperella pyogenes and Escherichia coli were less prevalent at T1 (8.8% and 10.5%, respectively) and their prevalence also decreased significantly over time. Fusobacterium necrophorum and Porphyromonas levii were often co-localized intraepithelially or in the lamina propria. Trueperella pyogenes and Escherichia coli were located only on the endometrial surface. Due to the high prevalence of tissue invasiveness, these findings emphasize the importance of Fusobacterium necrophorum and Porphyromonas levii in postpartum uterine disease of cattle and indicate that tissue invasiveness is an important aspect of the pathogenesis.

Intranasal deferoxamine improves performance in radial arm water maze, stabilizes HIF-1α, and phosphorylates GSK3ß in P301L tau transgenic mice.

Deferoxamine (DFO), a metal chelator, has been previously reported to slow the loss of spatial memory in a mouse model of amyloid accumulation when delivered intranasally (IN). In this study, we determined whether IN DFO also has beneficial effects in the P301L mouse, which accumulates hyperphosphorylated tau. Mice were intranasally treated three times per week with either 10% DFO (2.4 mg) or saline for 5 months, and a battery of behavioral tests were conducted before tissue collection and biochemical analyses of brain tissue with Western blot and ELISA. Wild-type (WT) mice statistically outperformed transgenic (TG) saline mice in the radial arm water maze, while performance of TG-DFO mice was not different than WT mice, suggesting improved performance in the radial arm water maze. Other behavioral changes were not evident. Beneficial changes in brain biochemistry were evident in DFO-treated mice for several proteins. The TG mice had significantly less pGSK3ß and HIF-1α, with more interleukin-1ß and total protein oxidation than wild-type controls, and for each protein, DFO treatment significantly reduced these differences. There was not a significant decrease in phosphorylated tau in brain tissue of DFO-treated mice at the sites we measured. These data suggest that IN DFO is a potential treatment not only for Alzheimer's disease, but also for other neurodegenerative diseases and psychiatric disorders in which GSK3ß and HIF-1α play a prominent role.

Separate and simultaneous enzymatic hydrolysis and fermentation of wheat hemicellulose with recombinant xylose utilizing Saccharomyces cerevisiae.

Fermentations with three different xylose-utilizing recombinant Saccharomyces cerevisiae strains (F12, CR4, and CB4) were performed using two different wheat hemicellulose substrates, unfermented starch free fibers, and an industrial ethanol fermentation residue, vinasse. With CR4 and F12, the maximum ethanol concentrations obtained were 4.3 and 4 g/L, respectively, but F12 converted xylose 15% faster than CR4 during the first 24 h. The comparison of separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) with F12 showed that the highest, maximum ethanol concentrations were obtained with SSF In general, the volumetric ethanol productivity was initially, highest in the SHF, but the overall volumetric ethanol productivity ended up being maximal in the SSF, at 0.013 and 0.010 g/Lh, with starch free fibers and vinasse, respectively.