Comparison of effects of high-pressure processing and heat treatment on immunoactivity of bovine milk immunoglobulin G in enriched soymilk under equivalent microbial inactivation levels

Immunoglobulin-rich foods may provide health benefits to consumers. To extend the refrigerated shelf life of functional foods enriched with bovine immunoglobulin G (IgG), nonthermal alternatives such as high-pressure processing (HPP) may offer advantages to thermal processing for microbial reduction. To evaluate the effects of HPP on the immunoactivity of bovine IgG, a soymilk product enriched with milk protein concentrates, derived from dairy cows that were hyperimmunized with 26 human pathogens, was subjected to HPP or heat treatment. To achieve a 5 log reduction in inoculated Escherichia coli 8739, the HPP or heat treatment requirements were 345 MPa for 4 min at 30 degrees C or for 20 s at 70 degrees C, respectively. To achieve a 5 log reduction in natural flora in the enriched soymilk, the HPP or heat treatments needed were 552 MPa for 4 min at 30 degrees C or for 120 s at 78.2 degrees C, respectively. At equivalent levels for a 5 log reduction in E. coli, HPP and heat treatment caused 25% and no detectable loss in bovine IgG activity, respectively. However, at equivalent levels for a 5 log reduction in natural flora, HPP and heat resulted in 65 and 85% loss of bovine IgG activity, respectively. Results of combined pressure-thermal kinetic studies of bovine milk IgG activity were provided to determine the optimal process conditions to preserve product function.     

Analysis of the reactivity of anti-bovine CD8 monoclonal antibodies with cloned T cell lines and mouse L-cells transfected with bovine CD8

Mouse L-cells transfected with bovine CD8 and two Theileria parva-infected cloned T cell lines expressing bovine CD8 were used to screen the panel of ten monoclonal antibodies (mAbs) submitted to the workshop. Eight of the ten mAbs reacted with the transfectant and both the cloned T cell lines. However, two mAbs CC58 and BAT82A did not recognise the transfectant and only reacted with one of the T cell lines. Further biochemical studies indicated that the eight mAbs react with both homo- and heterodimeric forms of bovine CD8 whilst the two mAbs CC58 and BAT82A react with only heterodimeric forms. These data suggest that bovine DC8 is encoded by two genes as is the case in mouse and man.     

Accuracy of the withdrawal reflex for localization of the site of cervical disk herniation in dogs: 35 cases (2004-2007)

OBJECTIVE: To evaluate the accuracy of neurologic examination versus magnetic resonance imaging (MRI) in localization of cervical disk herniation and evaluate the usefulness of withdrawal reflex testing in dogs. DESIGN: Retrospective case series. ANIMALS: 35 client-owned dogs with a single-level cervical disk herniation as determined via MRI. PROCEDURES: 1 of 2 board-certified neurologists performed a complete neurologic examination in each dog. Clinical signs of a cervical lesion included evidence of neck pain and tetraparesis. The withdrawal reflex was used for neuroanatomic localization (C1-C5 or C6-T2). Agreement between results of neurologic and MRI examinations was determined. RESULTS: Agreement between neurologic and MRI diagnoses was 65.8%. In 11 dogs in which the lesion was clinically localized to the C6-T2 segment on the basis of a decreased withdrawal reflex in the forelimbs, MRI revealed an isolated C1-C5 disk lesion. In 1 dog, in which the lesion was suspected to be at the C1-C5 level, MRI revealed a C6-T2 lesion. Cranial cervical lesions were significantly associated with an incorrect neurologic diagnosis regarding site of the lesion. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that the withdrawal reflex in dogs with cervical disk herniation is not reliable for determining the affected site and that a decreased withdrawal reflex does not always indicate a lesion from C6 to T2.     

Effects of diet restriction on life span and age-related changes in dogs

OBJECTIVE: To evaluate the effects of 25% diet restriction on life span of dogs and on markers of aging. DESIGN: Paired feeding study. ANIMALS: 48 Labrador Retrievers. PROCEDURES: Dogs were paired, and 1 dog in each pair was fed 25% less food than its pair-mate from 8 weeks of age until death. Serum biochemical analyses were performed, body condition was scored, and body composition was measured annually until 12 years of age. Age at onset of chronic disease and median (age when 50% of the dogs were deceased) and maximum (age when 90% of the dogs were deceased) life spans were evaluated. RESULTS: Compared with control dogs, food-restricted dogs weighed less and had lower body fat content and lower serum triglycerides, triiodothyronine, insulin, and glucose concentrations. Median life span was significantly longer for dogs in which food was restricted. The onset of clinical signs of chronic disease generally was delayed for food-restricted dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that 25% restriction in food intake increased median life span and delayed the onset of signs of chronic disease in these dogs.     

Melatonin reduces apoptotic cells,SOD2 and HSPB1 and improves the in vitro production and quality of bovine blastocysts

Effects of adding different concentrations of melatonin (10^?7, 10^?9 and 10^?11 M) to maturation (Experiment 1; Control, IVM  + 10^?7, IVM  + 10^?9, IVM  + 10^?11) and culture media (Experiment 2; Control, IVC  + 10^?7, IVC  + 10^?9, IVC  + 10^?11) were evaluated on in vitro bovine embryonic development. The optimal concentration of melatonin (10^?9 M) from Experiments 1–2 was tested in both maturation and/or culture media of Experiment 3 (Control, IVM  + 10^?9, IVC  + 10^?9, IVM /IVC  + 10^?9). In Experiment 1, maturated oocytes from Control and IVM  + 10^?9 treatments showed increased glutathione content, mitochondrial membrane potential and percentage of Grade I blastocysts (40.6% and 43%, respectively). In Experiment 2, an increase in the percentage of Grade I blastocysts was detected in IVC  + 10^?7 (43.5%; 56.7%) and IVC  + 10^?9 (47.4%; 57.4%). Moreover, a lower number and percentage of apoptotic cells in blastocysts were observed in the IVC  + 10^?9 group compared to Control (3.8 ± 0.6; 3.6% versus 6.1 ± 0.6; 5.3%). In Experiment 3, the IVC  + 10^?9 treatment increased percentage of Grade I blastocysts with a lower number of apoptotic cells compared to IVM /IVC  + 10^?9 group (52.6%; 3.0 ± 0.5 versus 46.0%; 5.4 ± 1.0). The IVC  + 10^?9 treatment also had a higher mRNA expression of antioxidant gene (SOD 2) compared to the Control, as well as the heat shock protein (HSPB 1) compared to the IVM  + 10^?9. Reactive oxygen species production was greater in the IVM /IVC  + 10^?9 treatment group. In conclusion, the 10^?9 M concentration of melatonin and the in vitro production phase in which it is used directly affected embryonic development and quality.     

Eel acetylcholinesterase inhibition studies with heteroarylphosphinates

The inhibition of eel acetylcholinesterase by the 4-nitrophenyl esters of 2-furyl(methyl)-, methyl(2-thienyl)-, di-2-furyl-, and di-2-thienylphosphinic acid (I, II, III, and IV, respectively) was investigated at pH 6.90 in 0.067 M phosphate buffer (25.0°C) using stopped-flow instrumentation and automated data processing. Our evaluation of the dissociation constant, K_d, the unimolecular bonding rate constant, k₂, and the bimolecular reaction constant, k_i, are the first reported values for these constants for alkyl/heteroaryl and diheteroaryl esters of phosphinic acids. The largest k_i value (19,330 M^?1 sec^?1) was observed for the reaction of I with the enzyme. The order for the remaining three is II > IV > III. There is no direct relationship between the hydrolysis rates of the esters and their anticholinesterase activities on eel acetylcholinesterase. Likewise, there is no direct relationship between their anticholinesterase activities and the LD₅₀ values in rats.     

Biofilm bacteria: formation and comparative susceptibility to antibiotics

The Calgary Biofilm Device (CBD) was used to form bacterial biofilms of selected veterinary gram-negative and gram-positive pathogenic bacteria from cattle, sheep, pigs, chicken, and turkeys. The minimum inhibitory concentration (MIC) and minimum biofilm eradication concentration (MBEC) of ampicillin, ceftiofur, cloxacillin, oxytetracycline, penicillin G, streptomycin, tetracycline, enrofloxacin, erythromycin, gentamicin, tilmicosin, and trimethoprim-sulfadoxine for gram-positive and -negative bacteria were determined. Bacterial biofilms were readily formed on the CBD under selected conditions. The biofilms consisted of microcolonies encased in extracellular polysaccharide material. Biofilms composed of Arcanobacterium (Actinomyces) pyogenes, Staphylococcus aureus, Staphylococcus hyicus, Streptococcus agalactiae, Corynebacterium renale, or Corynebacterium pseudotuberculosis were not killed by the antibiotics tested but as planktonic bacteria they were sensitive at low concentrations. Biofilm and planktonic Streptococcus dysgalactiae and Streptococcus suis were sensitive to penicillin, ceftiofur, cloxacillin, ampicillin, and oxytetracycline. Planktonic Escherichia coli were sensitive to enrofloxacin, gentamicin, oxytetracycline and trimethoprim/ sulfadoxine. Enrofloxacin and gentamicin were the most effective antibiotics against E. coli growing as a biofilm. Salmonella spp. and Pseudomonas aeruginosa isolates growing as planktonic populations were sensitive to enrofloxacin, gentamicin, ampicillin, oxytetracycline, and trimethoprim/sulfadoxine, but as a biofilm, these bacteria were only sensitive to enrofloxacin. Planktonic and biofilm Pasteurella multocida and Mannheimia haemolytica had similar antibiotic sensitivity profiles and were sensitive to most of the antibiotics tested. The CBD provides a valuable new technology that can be used to select antibiotics that are able to kill bacteria growing as biofilms.     

Changes in thyroid hormone levels in eggs and larvae and in iodide uptake by eggs of coho and chinook salmon,Oncorhynchus kisutsch andO. tschawytscha

Developmental profiles of thyroxin (T₄), triiodothyronine (T₃) and radioactive iodide uptake were established for eggs and T₄ and T₃ profiles were established for larvae (whole-body, yolk-only and body-only) of coho and chinook salmon. T₄ and T₃ were consistently present in all samples. In eggs, hormone levels remained fairly constant in all cohorst for at least the first three weeks of incubation, but then fluctuated in both directions in some sample groups. Large increases in T₄ (from 9 ng/g to 245 ng/g) were seen in 1985 chinook eggs 28 days after fertilization. Radioactive iodide uptake (which was used as a possible indicator of thyroxinogenesis) increased at least 10-fold in both 1986 coho and chinook eggs from 23–30 days after fertilization. T₄ (62 ng/g) and T₃ (393 ng/g) were found in the bodies of 28-day-old 1986 chinook embryos. In whole larvae, hormone levels varied depending upon the cohort studied. In general, initial body-only concentrations of both T₄ and T₃ decreased as body weight increased, but before yolksac resorption was completed, both thyroid hormone content and concentration increased (except for chinook T₃). T₄ and T₃ content in larval yolk stayed constant as yolksac size decreased, resulting in increased thyroid hormone concentration in the yolksac. All of these data suggest that the initial source of thyroid hormones in coho and chinook salmon eggs is maternal, but that by approximately 3–4 weeks after fertilization, the developing embryos begin to produce their own thyroid hormones. After hatching, increases in tissue T₄ and T₃ concentration coupled with constant T₄ and T₃ content in diminishing yolksacs suggest that larvae also produce their own thyroid hormones; yolksac content then may reflect both the original maternal hormones and the larva-producted hormones.     

IgE ELISA using antisera derived from epsilon chain antigenic peptides detects allergen-specific IgE in allergic horses

Equine disease with an allergic etiology is common. Environmental antigens most often implicated as allergens in horses include molds, dusty hay, grass pollen, hay dust mites, and insect saliva. Although intradermal testing with allergen is a useful diagnostic tool for some species, skin testing frequently produces false positive results in horses. Allergen deprivation as a diagnostic tool is often impossible and at best it is ineffective at diagnosing the specific allergic reactivity. Synthesis of IgE after exposure to allergen is the instigator of the allergic process. While IgE exerts its effect after binding strongly to mast cell Fc receptors, the presence of free IgE in the serum can be used to quantify and determine the allergen specificity of the allergic disease. A lack of widely available reagents for detection of equine IgE has limited this approach in horses. We have used the nucleotide sequence of equine IgE to prepare a peptide-based immunogen to elicit equine epsilon chain-specific antisera. Selection of peptides was based on antigenic attributes of the deduced amino acid sequence of the equine epsilon chain. Six peptides were selected for conjugation to carrier molecules and rabbit immunization. Of these, one peptide elicited antisera that was successfully used in enzyme linked immunosorbant assay (ELISA) to screen horse serum from 64 allergic horses for allergen-specific IgE. Twenty-four of the 64 horses showed positive reactivity to one or more of the following allergens: grass, grain mill dust, mosquito, and horsefly. This study demonstrates the usefulness of peptide-based immunogens for development of antisera to rare or difficult to purify antigens such as IgE. Resultant antisera has great usefulness in diagnostic assays for equine allergy and as a research tool.