Development of a murine ocular posterior segment explant culture for the study of intravitreous vector delivery

The objective of this study was to develop a murine retinal/choroidal/scleral explant culture system to facilitate the intravitreous delivery of vectors. Posterior segment explants from adult mice of 2 different age groups (4 wk and 15 wk) were cultured in serum-free medium for variable time periods. Tissue viability was assessed by gross morphology, cell survival quantification, activated caspase-3 expression, and immunohistochemistry. To model ocular gene therapy, explants were exposed to varying transducing units of a lentiviral vector expressing the gene for green fluorescent protein for 48 h. Explant retinal cells remained viable for approximately 1 wk, although the ganglion cell layer developed apoptosis between 4 and 7 d. Following vector infusion into the posterior segment cups, viral transduction was noted in multiple retinal layers in both age groups. An age of donor mouse influence was noted and older mice did not transduce as well as younger mice. This explant offers an easily managed posterior segment ocular culture with minimum disturbance of the tissue, and may be useful for investigating methods of enhancing retinal gene therapy under controlled conditions.     

Staphylococcus spp., Streptococcus canis,and Arcanobacterium phocae of healthy Canadian farmed mink and mink with pododermatitis

Pododermatitis is a disease of concern for mink breeders in Canada and worldwide, as it causes discomfort and lowers the breeding rates on farms affected by the disease. Unfortunately, the etiology and pathogenesis of pododermatitis are still unknown. In this study, we compared Staphylococcus spp. and Streptococcus canis isolates from healthy mink with isolates from animals with pododermatitis on 2 farms in Ontario. Almost all hemolytic Staphylococcus spp. isolated were shown to be Staphylococcus delphini Group A by 16S ribosomal ribonucleic acid (rRNA) sequence analysis and polymerase chain reaction (PCR). Pulsed-field gel electrophoresis (PFGE) did not reveal any S. delphini or S. canis clonal lineages specifically associated with pododermatitis, which suggests that these bacteria do not act as primary pathogens, but does not dismiss their potential roles as opportunistic pathogens. While S. delphini and S. canis were the most prevalent bacterial pathogens in mink pododermatitis, they were also present in samples from healthy mink. Arcanobacterium phocae is occasionally isolated from pododermatitis cases, but is difficult to recover with conventional culture methods due to its slow growth. A quantitative real-time PCR was developed for the detection of A. phocae and was tested on 138 samples of footpad tissues from 14 farms. The bacterium was detected only in pododermatitis-endemic farms in Canada and was at higher concentrations in tissues from infected footpads than in healthy tissues. This finding suggests that A. phocae is involved in the pathogenesis of pododermatitis.     

Glycoprofiling of bifidobacterial consumption of human milk oligosaccharides demonstrates strain specific, preferential consumption of small chain glycans secreted in early human lactation

The molecular basis by which human breast milk supports the development of a protective intestinal microbiome in infants is unknown. After lactose and lipids, human milk oligosaccharides (HMOs) are quantitatively the third largest and most diverse component of breast milk. In this work, glycomic profiling of HMO consumption by bifidobacteria using Fourier transform ion cyclotron resonance mass spectrometry reveals that one species, Bifidobacterium longum biovar infantis ATCC 15697, an isolate from the infant gut, preferentially consumes small mass oligosaccharides, representing 63.9% of the total HMOs available. These HMOs were detected in human breast milk at the onset and constantly through the first month of lactation by use of high performance liquid chromatography-chip time-of-flight mass spectrometry. Further characterization revealed that strain ATCC 15697 possesses both fucosidase and sialidase activities not present in the other tested strains. This work provides evidence that these small mass HMOs are selectively metabolized by select bifidobacterial strains and represent a potential new class of bioactive molecules functioning as prebiotics to facilitate a protective gut colonization in breast-fed newborns.     

Development of a pheromone trap monitoring system for orange wheat blossom midge, Sitodiplosis mosellana, in the UK

Field-trapping experiments with synthetic 2,7-nonadiyl dibutyrate, the female-produced sex pheromone of the orange wheat blossom midge, Sitodiplosis mosellana (Géhin), demonstrated that pheromone traps were highly attractive to males and caught very few non-target organisms. Different formulations of pheromone were tested to identify the optimum release rate and dispenser type for use in pheromone traps in the UK. Key findings were that racemic pheromone was as effective as enantiomerically pure (2S,7R)-2,7-nonadiyl dibutyrate, that release rates higher than 0.5 microg day(-1) were not necessary and that the optimal formulation was a 1 mg pheromone loading in a rubber septum. Pheromone traps gave a reliable indication of peak midge emergence, onset of flight and abundance of midges throughout the season. A strong correlation between maximum trap catch and crop infestation levels was obtained.     

Transepithelial ion exchange in smolting atlantic salmon (Salmo Salar L.)

Whole-body (but predominantly gill) Na⁺ exchange, gill Na⁺/K⁺/ATPase activity and seawater tolerance were examined in juvenile Atlantic salmon during the smolting period. Transepithelial net Na⁺ gain decreased steadily from late February showing a net loss in April and early May, returning to approximate equilibrium in mid-May. This seasonal net loss of Na⁺ to the environment occurred slightly after maximal gill epithelial Na⁺/K⁺/ATPase activity and preceded maximal seawater tolerance. The results are discussed in relation to changes in gill permeability and salt intake via the diet.     

Allozyme diversity in Australian rainbow trout, Oncorhynchus mykiss

Rainbow trout, Oncorhynchus mykiss (Walbaum), were first introduced into Australia over 100 years ago, and forms the basis of important recreational inland fisheries and an aquaculture industry in south‐eastern Australia. This paper investigates the genetic variation within and between samples of Australian rainbow trout using allozyme electrophoresis. The levels of genetic diversity within Australia do not show marked differences from those observed in hatchery and wild populations from throughout North America, New Zealand and South Africa, but there is evidence for the loss of some rare alleles during translocation from California to Australia via New Zealand. No appreciable difference in genetic diversity was apparent between hatchery and self‐sustaining wild populations of rainbow trout from mainland Australia. However, significant differences in allelic frequencies were observed, with consistent genetic differences between Victorian and New South Wales samples most likely reflecting state‐based hatchery and stocking policies.     

Protein metabolism during sexual maturation in female Atlantic salmon (Salmo salar L)

Body composition and fractional rates of protein synthesis (percentage of the protein mass synthesized per day) were determined in female Atlantic salmon returning to the River Tay, Scotland in July and in October after a 95 day period without food, during which time the animals became sexually mature. During the 95 day period of starvation/sexual maturation the ventricle and red muscle remained as a constant proportion of fresh weight whereas the liver, gill and ovary increased and the stomach and white muscle decreased. Fractional rates of protein synthesis increased markedly in the liver, stomach and ovary during the period of starvation/sexual maturation. In the gill, ventricle and white muscle fractional protein synthesis rates increased slightly or remained constant. From the estimated rates of protein loss or gain in the various tissues it is concluded that there is considerable protein turnover and repartitioning of amino acids during the period of starvation and sexual maturation. The absolute rate of protein synthesis rates in the ovary indicates that this tissue made the largest contribution to the energy and amino acid demands of the fish, whilst most of the amino acids required for maturation of the ovary were derived from white muscle, principally as the result of increased muscle protein degradation.     

Equine hyperimmune serum protects mice against Clostridium difficile spore challenge

Clostridium (C.) difficile is a common cause of nosocomial diarrhea in horses. Vancomycin and metronidazole have been used as standard treatments but are only moderately effective, which highlights the need for a novel alternative therapy. In the current study, we prepared antiserum of equine origin against both C. difficile toxins A and B as well as whole-cell bacteria. The toxin-neutralizing activities of the antibodies were evaluated in vitro and the prophylactic effects of in vivo passive immunotherapy were demonstrated using a conventional mouse model. The data demonstrated that immunized horses generated antibodies against both toxins A and B that possessed toxin-neutralizing activity. Additionally, mice treated with the antiserum lost less weight without any sign of illness and regained weight back to a normal range more rapidly compared to the control group when challenged orally with 10⁷ C. difficile spores 1 day after serum injection. These results indicate that intravenous delivery of hyperimmune serum can protect animals from C. difficile challenge in a dose-dependent manner. Hence, immunotherapy may be a promising prophylactic strategy for preventing C. difficile infection in horses.