An injectable subunit vaccine containing Elongation Factor Tu and Heat Shock Protein 70 partially protects American bison from Mycoplasma bovis infection.

Mycoplasma bovis (M. bovis) is the etiologic agent of high mortality epizootics of chronic respiratory disease in American bison (Bison bison). Despite the severity of the disease, no efficacious commercial vaccines have been licensed for the prevention of M. bovis infection in bison. Elongation factor thermal unstable (EFTu) and Heat Shock Protein 70 (Hsp70, DnaK) are highly conserved, constitutively expressed proteins that have previously been shown to provide protection against M. bovis infection in cattle. To assess the suitability of EFTu and Hsp70 as vaccine antigens in bison, the immune response to and protection conferred by an injectable, adjuvanted subunit vaccine comprised of recombinantly expressed EFTu and Hsp70 was evaluated. Vaccinates developed robust antibody and cellular immune responses against both EFTu and Hsp70 antigens. To assess vaccine efficacy, unvaccinated control and vaccinated bison were experimentally challenged with bovine herpes virus-1 (BHV-1) 4 days prior to intranasal infection with M. bovis. Vaccinated bison displayed reductions in joint infection, lung bacterial loads, and lung lesions compared to unvaccinated controls. Together, these results showed that this subunit vaccine reduced clinical disease and bacterial dissemination from the lungs in M. bovis challenged bison and support the further development of protein subunit vaccines against M. bovis for use in bison.

Optimizing the Integration of Microwave Processing and Enzymatic Extraction to Produce Polyphenol-Rich Extracts from Olive Pomace.

The integration of green technologies such as microwave- and enzyme-assisted extraction (MEAE) has been shown to improve the extraction efficiency of bioactive compounds while reducing processing time and costs. MEAE using tannase alone (MEAE-Tan), or in combination with cellulase and pectinase (MEAE-Tan-Cel-Pec), was optimized to produce enriched phenolic and antioxidant extracts from olive pomace. The individual and integrated impact of enzyme concentration, temperature, and pomace/water ratio were determined using a central composite rotatable design. Optimal extraction conditions for MEAE-Tan (60 °C, 15 min, 2.34% of enzyme (w/w), and 1:15 pomace/water ratio) and MEAE-Tan-Cel-Pec (46 °C, 15 min, 2% of enzymes (w/w), in the proportion of 1:1:1, and 1:20 pomace/water ratio) resulted in extracts containing 7110.6 and 2938.25 mg GAE/kg, respectively. The antioxidant activity of the extracts was correlated with phenolic acid release, which was enzyme-dependent, as determined with HPLC-DAD analysis. Enzyme selection had a significant impact on the phenolic profile of extracts, with tannase releasing high concentrations of chlorogenic acid and the combined use of enzymes releasing high concentrations of hydroxytyrosol and chlorogenic and ferulic acids. The novelty of this study relies on the integration and optimization of two green technologies (microwave- and enzyme-assisted extraction) to improve the extraction efficiency of bioactive phenolics from olive pomace while reducing processing time and costs. While these techniques have been evaluated isolated, the benefits of using both processing strategies simultaneously remain largely unexplored. This study demonstrates the effectiveness of the integration and processing optimization of two environmentally friendly technologies as a promising alternative to treat agro-industrial byproducts.

Effects of concurrent administration of modified live viral vaccines with RB51 on immune responses to RB51.

Brucella abortus is a gram negative, zoonotic pathogen that can cause abortions and stillbirths in the cattle industry and has contributed to significant economic losses to cow-calf producers. Cell mediated immunity (CMI) is an important component of the immune response associated with protection against Brucella abortus and other intracellular pathogens. Brucellosis and viral modified live vaccines (vMLV) are licensed individually but may be used concurrently under field conditions. Peripheral blood mononuclear cells (PBMC) from non-vaccinated cattle and cattle vaccinated with either Brucella abortus strain RB51, a vMLV or both RB51 and a vMLV vaccine were isolated. The frequency of CD4+, CD8+ and γδ+ T cell populations within PBMC, and the frequency of interferon gamma (IFN-γ) production within these cell types was characterized via flow-cytometry. The goal of this study was to characterize immune responses to RB51 vaccination and determine the effect of concurrent vaccine administration. Although immune responses were greatest in PBMC from cattle vaccinated with only RB51, cattle vaccinated with both RB51 and vMLV demonstrated measurable T cell responses associated with protective immunity. Data suggests a lack of significant biological differences between the groups in protective immune responses. Collectively, our data demonstrated a lack of vaccine interference following concurrent administration of vMLV and RB51. Although concurrent administration of individually licensed vaccines may influence immune responses and contribute to vaccine interference, potential vaccine combinations should be evaluated for biological effects.

Response to Bovine Viral Diarrhea Virus in Heifers Vaccinated with a Combination of Multivalent Modified Live and Inactivated Viral Vaccines.

Bovine viral vaccines contain both live or inactivated/killed formulations, but few studies have evaluated the impact of vaccinating with either live or killed antigens and re-vaccinating with the reciprocal. Commercial dairy heifers were utilized for the study and randomly assigned to three treatment groups. Treatment groups received a commercially available modified-live viral (MLV) vaccine containing BVDV and were revaccinated with a commercially available killed viral (KV) vaccine containing BVDV, another group received the same KV vaccine and was revaccinated with the same MLV vaccine, and yet another group served as negative controls and did not receive any viral vaccines. Heifers in KV/MLV had higher virus neutralizing titers (VNT) at the end of the vaccination period than heifers in MLV/KV and control groups. The frequency of IFN-γ mRNA positive CD4+, CD8+, and CD335+ populations, as well as increased mean fluorescent intensity of CD25+ cells was increased for the MLV/KV heifers as compared to KV/MLV and controls. The data from this study would suggest that differences in initial antigen presentation such as live versus killed could augment CMI and humoral responses and could be useful in determining vaccination programs for optimizing protective responses, which is critical for promoting lifetime immunity.

Experimental Senecavirus A Infection of Bovine Cell Lines and Colostrum-Deprived Calves.

Senecavirus A (SVA) is a causative agent for vesicular disease in swine, which is clinically indistinguishable from other vesicular diseases of swine including foot-and-mouth disease (FMD). Recently, it was reported that buffalo in Guangdong, China were experiencing clinical symptoms similar to FMD including mouth ulcers and lameness tested positive for SVA. The objective of this study was to determine the susceptibility of cattle (Bos taurus) to SVA infection. Initial in vitro work using the PrimeFlow assay demonstrated that bovine cell lines and peripheral blood mononuclear cells from cattle were susceptible to SVA infection. Subsequently, six colostrum-deprived Holstein calves were challenged with SVA intranasally. No vesicular lesions were observed after challenge. Serum, oral, nasal, and rectal swabs tested for SVA nucleic acid did not support significant viral replication and there was no evidence of seroconversion. Therefore, demonstrating cattle from this study were not susceptible to experimental SVA infection.

Influence of species of negative control sera on results of a brucellosis fluorescence polarization assay.

We evaluated serologic responses of cattle, bison, elk, and swine representing negative control, early vaccination (4-8 wk), late vaccination (21-28 wk) or booster vaccination, early after-experimental challenge (2-4 wk), and late after-experimental challenge (8-21 wk), in a brucellosis fluorescence polarization assay (FPA; n = 10 sera per species per treatment) using negative control sera from cattle, bison, elk, and swine (n = 5 per species). Sera from cattle shedding Brucella abortus strain RB51 in milk were also evaluated against the 20 negative control sera. The species of negative control sera used in the FPA could increase (p < 0.05) delta millipolarization (mP; delta mP = sample mP - negative control mP) results. In general, the species of negative control sera did not alter the interpretation of FPA results in control, vaccinated, or infected animals. Even after repeated RB51 vaccinations in bison, cattle, or elk, or in cattle shedding RB51 in milk, serologic results from the FPA remained negative. Species differences in FPA results were noted; elk developed robust humoral responses very quickly after infection that resulted in strong positive FPA results. In cattle and bison, humoral responses appeared to develop over a longer period of time, and greater delta mP values were detected at later times after infection. Sensitivity of the FPA for detecting infected animals was greatest for elk in early challenge samples and bison in late challenge samples. Our data suggest that species of origin of negative control sera does not influence interpretation of the FPA in natural hosts of Brucella abortus.

Case Report: Fading Elk Syndrome in a Herd of Captive Elk (Cervus elaphus) in the North American Midwest.

Fading elk syndrome, or chronic ill-thrift of elk, is a disease associated with abomasal parasitism with Ostertagia species, of which elk appear to be particularly susceptible. While this syndrome has been extensively reported to affect wapiti-type red deer hybrids farmed in New Zealand since the mid 1980's, there is only a single report of this disease in North America. Here, we report a case of fading elk syndrome in a herd of 34 elk (Cervus elaphus) in Ames, Iowa, at the National Animal Disease Center. Analysis of complete blood counts were unremarkable, but blood chemistry demonstrated a severe hypoalbuminemia. Fecal floatations were also unremarkable, and non-diagnostic. Histological examination of tissues collected at necropsy revealed proliferative abomasitis and nematodes consistent with Ostertagia spp. Anthelmintic treatment consisting of a combination of pour-on Cydectin® and injectable Noromectin Plus®, at double the recommended dose for cattle, showed positive results, as all remaining animals in the herd recovered. The work presented here is the first report of naturally-acquired disease in a herd of captive elk used for research and sheds light on this seldomly-reported disease in North America.

Assessing Parents' Communication of Weight and Weight Management from Clinic to Home.

Background: Family-oriented therapies are the gold standard of childhood obesity treatment, yet little is known about if or how information gathered by one parent from a health care provider is translated to the home. We assessed how families of children and adolescents with overweight and obesity communicate weight-related information received from their provider to family members not present at the visit. Methods: Parents and children (9-18 years old, N = 112) completed the McMaster's Family Assessment Device Communication Subscale (FADc) and investigator-derived questions describing weight-related communication practices with family members. We used descriptive statistics to describe communication practices and separate logistic regression models to assess associations of communication practices with parent-reported FADc, child BMI z-score, child sex, parent BMI, household income, and site. Results: Most parents discuss with other family members: their child's weight (60.4%) or weight management discussions with the child's provider (57.9%). Median parent FADc score was 2.0 (IQR 0.5). The most common facilitator to weight-related conversations was understanding what the provider said (95.1%). Higher FADc score (worse communication) was associated with whether parents ask other family members' opinions about weight information received from their child's provider [odds ratio 0.22 (95% confidence interval 0.05-0.99)]. Higher income was associated with many healthy communication practices. Conclusions: Slightly more than half of parents discuss with family members what their provider said regarding their child's weight. More effort must be placed on aiding parents in relaying information from the provider to other family members in the home to encourage family lifestyle changes and alleviate childhood obesity.

Analysis of Microsatellites (SSRs) in Processed Olives as a Means of Cultivar Traceability and Authentication.

Select cultivars of table olives have more desirable traits and a higher economic value. There are suspected issues with cultivar mislabeling and traceability in the supply chain. Here, we describe a method to identify cultivars by genotyping of processed olives. DNA was extracted from leaves and California-style olives of seven commonly packed cultivars. Processed olive fruits yielded relatively low DNA concentrations (0.04-0.86 µg/g), and extracts had more impurities compared with leaves. From 15 candidate SSRs, five markers showing the highest number of unique allele combinations and discriminatory power were selected. These SSRs were successfully amplified and analyzed in all cultivars of olives except one. When directly comparing any two cultivars, different allele combinations were typically present for at least four of the five SSRs. Microsatellite analysis shows potential as a simple yet robust diagnostic tool. The method can be expanded to include other cultivars, styles of table olives, and potentially other processed plant-based foods.

Acrylamide Content of Experimental Flatbreads Prepared from Potato, Quinoa, and Wheat Flours with Added Fruit and Vegetable Peels and Mushroom Powders.

Flatbreads are a major food consumed worldwide. To mitigate an undesirable safety aspect of flatbreads that might be associated with the potentially-toxic compound acrylamide, we recently developed recipes using a variety of grains that resulted in the production of low-acrylamide flatbreads. To further enhance the functionality of flatbreads, we have developed, in this work, new experimental flatbreads using potato, quinoa, and wheat flours supplemented with peel powders prepared from commercial nonorganic and organic fruits and vegetables (apples, cherry tomatoes, melons, oranges, pepino melons, sweet potato yams), potato peels, and mushroom powders (Lion's Mane, Hericium erinaceus; Reishi, Ganoderma lucidum; and Turkey Tail, Trametes versicolor). These additives have all been reported to contain beneficial compositional and health properties. The results of fortification of the baked flatbreads showed either no effect or increases in acrylamide content by unknown mechanisms. Since the additives did not increase the acrylamide content of the quinoa flour flatbreads for the most part, such supplemented quinoa flatbreads have the potential to serve as a nutritional, gluten-free, low-acrylamide, and health-promoting functional food. Mushroom powder-fortified wheat flatbreads with relatively low acrylamide content may also have health benefits.

Comparative Study of Four Analytical Methods for the Routine Determination of Acrylamide in Black Ripe Olives.

Reducing acrylamide in foods is an important scientific and regulatory goal. Black ripe olives contain significant levels of acrylamide. However, unlike cereal and potato products, there are no standardized methods or certified reference materials for olives and no harmonization between laboratories performing routine analyses. The industry has observed inconsistencies between laboratories using different analytical methods. To narrow the cause of this variability, acrylamide was extracted from olives using a single protocol and analyzed with the most commonly used routine methods: liquid chromatography with ultraviolet detection (LC-UV) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) without bromination and LC-UV and gas chromatography-mass spectrometry (GC-MS) following bromination. This design specifically evaluated the effects of sample derivatization and detector on acrylamide measurements, which has not been documented for any food. Accuracy, precision, sensitivity, linearity, and reproducibility were assessed, and 10 commercial olive samples were analyzed. LC-MS/MS demonstrated the best overall performance. Although derivatization decreased precision and reproducibility, all detection methods had comparable accuracy and calculated acrylamide values. The results suggest that harmonization of extraction protocols across laboratories is the most important area of future study. This study provides the framework for a standardized method for acrylamide analysis in olives. Reliable measurements are essential for aiding the decision making of the industry and regulatory agencies.

Acrylamide Content of Experimental and Commercial Flatbreads.

Acrylamide, formed in baked and fried plant-based foods, is reported to induce numerous adverse effects in cells, animals, and humans. Examples from the literature show that processed potato- and cereal-based products are two major food types that seem to contribute the highest amounts of acrylamide to the diet worldwide. To meet both the demand for gluten-free products and the interest in alternative grains, we previously developed recipes for flatbreads using a variety of different grains. In this study, we determined the acrylamide content of 15 experimental flatbreads made from a variety of flours and 21 commercial flatbreads. The application of a validated, highly sensitive HPLC/MS method revealed that flatbreads made with the following flours baked at 195.5 °C for 2 min had very low (<10 µg/kg) levels of acrylamide: brown rice, buckwheat, cornmeal, millet, oat, and quinoa. The acrylamide levels of the following flatbreads were 14 to 59 µg/kg: rye, sorghum, soy, wheat, commercial pita, pita crackers, pizza, naan, and lavash. Wheat-based matzo breads, which are rapidly baked to a crisp texture at high heat (∼400 °C), contained 101 to 504 µg/kg acrylamide. Potato-based products were some of the highest of the products tested, ranging from 153 (potato pancakes) to 2,070 (potato-containing gluten-free matzos) µg/kg acrylamide. Except for the potato-containing products, the flatbreads made in this study were lower in acrylamide content (<3 to 21.3 µg/kg) than any of the commercial products tested. Of these experimental flatbreads, wheat- and sorghum-based products were the highest. Flatbreads from alternative grains can result in gluten-free products with high nutritional value and less acrylamide. PRACTICAL APPLICATION: Acrylamide formation is dependent on both the composition of the food product and the method of cooking. Flatbreads have the potential to be high in acrylamide due to cooking methods which lead to the development of desirable browning products. Flatbreads developed in this study using alternative and ancient grains were mostly lower in acrylamide content than their wheat counterpart, suggesting that they can serve as a low-acrylamide, gluten-free functional food.

Phenolics and Antioxidant Capacity of Pitted Olive Pomace Affected by Three Drying Technologies.

This study investigated drum-drying's ability to produce dried food-grade olive pomace as a potential food ingredient that is more nutritionally dense than its freeze-dried and hot-air dried counterparts. The pits and skin were removed from fresh olive pomace, and the remaining pulp was dried to <5% moisture through freeze-drying, hot-air drying, and drum-drying at two rotational speeds. The drying treatments had no significant (P ≤ 0.05) effect on the olive pomace's fat or dietary fiber contents but did increase the L* , a* , and b* color parameter values. Although all the drying treatments significantly (P ≤ 0.05) decreased the fresh olive pomace's antioxidant capacity, drum-drying preserved the olive pomace's antioxidant capacity significantly (P ≤ 0.05) better than freeze-drying and hot-air drying. The drum-dried samples had concentrations of caffeic acid and verbascoside that were significantly (P ≤ 0.05) higher than the other dried pomace samples and were not significantly (P ≤ 0.05) different from the fresh pomace. The drum-dried olive pomace contained concentrations of hydroxytyrosol, tyrosol, vanillic acid, luteolin-7-glucoside, and rutin that were not significantly (P ≤ 0.05) different from the dried sample with the highest concentration of each respective phenolic compound. No oleuropein was found in the fresh or dried olive pomace. The results of this study show that drum-drying is an energy efficient method for converting olive pomace into a stable food-grade supplement that preserves its high phenolic, antioxidant, and dietary fiber contents to potentially benefit human health when incorporated into food or supplement products. PRACTICAL APPLICATION: Pitting and drying converts the olive pomace into a stable form that is free of physical hazards and could be incorporated into food products to increase their nutritional quality through olive pomaces' high fiber, antioxidant, and phenolic contents. Drum-drying allows food-grade olive pomace to retain higher amounts of beneficial soluble phenolics and a higher antioxidant capacity than conventional drying methods, thus furthering olive pomace's potential valorization as a food ingredient.

ß-Glucosidase Discovery and Design for the Degradation of Oleuropein.

Current lye processing for debittering California black table olives produces large amounts of caustic wastewater and destroys many of the beneficial phenolic compounds in the fruit. Herein, we propose using enzyme treatment in place of lye, potentially reducing the amount and causticity of wastewater produced. By specifically targeting the bitterness-causing compound, oleuropein, retention of other beneficial phenolics may be possible. A ß-glucosidase from Streptomyces sp. was identified from a screen of 22 glycosyl hydrolases to completely degrade oleuropein in 24 h. Computational modeling was performed on this enzyme, and mutation C181A was found to improve the rate of catalysis by 3.2-fold. This mutant was tested in the context of the olive fruit and leaf extract. Degradation was observed in the olive leaf extract but not in the fruit matrix, suggesting that enzyme fruit penetration is a limiting factor. This work discovers and begins the refinement process for an enzyme that has the catalytic properties for debittering olives and provides direction for future engineering efforts required to make a product with commercial value.

Childhood Stroke and Vision: A Review of the Literature.

OBJECTIVE: Here we review the current literature regarding visual outcome after perinatal and childhood stroke. BACKGROUND: Visual deficits following stroke in adults are common and have been previously reviewed. Less is known about visual deficits following stroke in neonates and older children. Most of the literature regarding this subject has focused on preterm infants, or on other types of brain injury. This review summarizes the types of visual deficits seen in term infants following perinatal stroke and children following childhood stroke and predictors of outcome. This review suggests areas for future research. METHODS: We performed Ovid MEDLINE searches regarding visual testing in children, vision after childhood stroke, neuroplasticity of vision, treatment of visual impairment after stroke, and driving safety concerns after stroke. RESULTS: Visual field defects were the most commonly reported visual deficits after perinatal and childhood stroke. There is a significant lack of literature on this subject, and most is in the form of case reports and case series. Children can experience significant visual morbidity after stroke, and have the potential to show some recovery, but guidelines on assessment and treatment of this population are lacking. CONCLUSIONS: There were limitations to this study, given the small amount of literature available. Although stroke in children can result in severe visual deficits, most children regain at least a portion of their vision. However, more research is needed regarding visual assessment of this population, long-term visual outcomes, specific predictors of recovery, and treatment options.

Orbital Apex Metastasis from Adenoid Cystic Carcinoma: Acute Loss of Vision and Subsequent Recovery with the Radiation.

Orbital apex metastasis from adenoid cystic carcinoma (ACC) is rare. We present a patient with known metastatic ACC presented with a rapidly declining vision with visual acuity oculus dexter (OD) equal to counting fingers at two feet. On imaging, she was found to have a right orbital apex tumor causing compressive optic neuropathy. She received the intensity modulated radiation therapy (IMRT). After completion of the therapy, she had regained essentially a full vision with visual acuity OD of 20/30 without corrective lenses. The treatment rationale and pertinent literature are discussed in this article.

Molecular characterization of a partitivirus from Ophiostoma himal-ulmi.

The complete nucleotide sequences of two double-stranded (ds) RNA molecules, S1 (1,744 bp) and S2 (1,567 bp), isolated from an isolate HP62 of the Himalayan Dutch elm disease fungus, Ophiostoma himal-ulmi, were determined. RNA S1 had the potential to encode a protein, P1, of 539 amino acids (62.7 kDa), which contained sequence motifs characteristic of RNA-dependent RNA polymerases (RdRps). A database search showed that P1 was closely related to RdRps of members of the genus Partitivirus in the family Partitiviridae. RNA S2 had the potential to encode a protein, P2, of 430 amino acids (46.3 kDa), which was related to capsid proteins of members of the genus Partitivirus. Virus particles isolated from isolate HP62 were shown to be isometric with a diameter of 30 nm, and to contain dsRNAs S1 and S2 and a single capsid protein of 46 kDa. N-terminal sequencing of tryptic peptides derived from the capsid protein proved unequivocally that it is encoded by RNA S2 and corresponds to protein P2. It is concluded that O. himal-ulmi isolate HP62 contains a new member of the genus Partitivirus, which is designated Ophiostoma partitivirus 1. A phylogenetic tree of RdRps of members of the family Partitiviridae showed that there are least two RdRp lineages of viruses currently classified in the genus Partitivirus. One of these lineages contained viruses with fungal hosts and viruses with plant hosts, raising the possibility of horizontal transmission of partitiviruses between plants and fungi. The partitivirus RdRp and capsid proteins appear to have evolved in parallel with the capsid proteins evolving much faster than the RdRps.