Ischemic myelomalacia and closed spinal dysraphism in multiple finishing swine.

Ischemic myelomalacia secondary to fibrocartilaginous emboli (FCE) is an idiopathic disease in humans and animals. On the other hand, congenital spinal cord malformations result from neural tube defects in fetal development (ie, spinal dysraphism), with structural anomalies referred to collectively as myelodysplasia. Spinal dysraphisms are frequently accompanied by skin and vertebral abnormalities because of the embryogenic relationship. In this observational case study, we report the pathologic findings of 13, 18- to 24-weeks-old pigs from a large conventional operation that presented with acute paraparesis. Ischemic myelomalacia secondary to FCE was observed in 5 of 13 examined pigs. Congenital spinal cord malformations located between the caudal thoracic and sacral spinal cord were identified in 7 pigs, with structural abnormalities that ranged from diplomyelia/split cord malformation to segmental spinal dysgenesis (myelodysplasia) to caudal agenesis. Concurrent myelomalacia and congenital spinal cord malformations in the same or different sites were noted in 2 pigs. No spinal lesion was observed in 3 pigs. Although gross vertebral abnormalities were not observed herein, intervertebral instability due to minor defects in the articular facets, as well as other unidentified factors, is suspected to contribute high incidence of FCE. It is likely that these congenital malformations were previously underdiagnosed or are possibly new conditions associated with continuous inbreeding and genetic improvement in the modern swine industry.

Monitoring Farmed Fish Welfare by Measurement of Cortisol as a Stress Marker in Fish Feces by Liquid Chromatography Coupled with Tandem Mass Spectrometry.

The aquaculture industry has become a sustainable source of food for humans. Remaining challenges include disease issues and ethical concerns for the discomfort and stress of farmed fish. There is a need for reliable biomarkers to monitor welfare in fish, and the stress hormone cortisol has been suggested as a good candidate. This study presents a novel method for measurement of cortisol in fish feces based on enzymatic hydrolysis, liquid−liquid extraction, derivatization, and finally instrumental analysis by liquid chromatography coupled with tandem mass spectrometry. Hydrolysis and extraction conditions were optimized. Cortisol appeared to be mostly conjugated to sulfate and less conjugated to glucuronic acid in the studied samples of feces from farmed Atlantic salmon. The method was suitable for quantification of cortisol after enzymatic deconjugation by either combined glucuronidase and sulfatase activity, or by glucuronidase activity alone. The limit of detection was 0.15 ng/g, the limit of quantification was 0.34 ng/g, and the method was linear (R2 > 0.997) up to 380 ng/g, for measurement of cortisol in wet feces. Method repeatability and intermediate precision were acceptable, both with a coefficient of variation (CV) of 11%. Stress level was high in fish released into seawater, and significantly reduced after eight days.

Replication and Transmission of the Novel Bovine Influenza D Virus in a Guinea Pig Model.

UNLABELLED: Influenza D virus (FLUDV) is a novel influenza virus that infects cattle and swine. The goal of this study was to investigate the replication and transmission of bovine FLUDV in guinea pigs. Following direct intranasal inoculation of animals, the virus was detected in nasal washes of infected animals during the first 7 days postinfection. High viral titers were obtained from nasal turbinates and lung tissues of directly inoculated animals. Further, bovine FLUDV was able to transmit from the infected guinea pigs to sentinel animals by means of contact and not by aerosol dissemination under the experimental conditions tested in this study. Despite exhibiting no clinical signs, infected guinea pigs developed seroconversion and the viral antigen was detected in lungs of animals by immunohistochemistry. The observation that bovine FLUDV replicated in the respiratory tract of guinea pigs was similar to observations described previously in studies of gnotobiotic calves and pigs experimentally infected with bovine FLUDV but different from those described previously in experimental infections in ferrets and swine with a swine FLUDV, which supported virus replication only in the upper respiratory tract and not in the lower respiratory tract, including lung. Our study established that guinea pigs could be used as an animal model for studying this newly emerging influenza virus. IMPORTANCE: Influenza D virus (FLUDV) is a novel emerging pathogen with bovine as its primary host. The epidemiology and pathogenicity of the virus are not yet known. FLUDV also spreads to swine, and the presence of FLUDV-specific antibodies in humans could indicate that there is a potential for zoonosis. Our results showed that bovine FLUDV replicated in the nasal turbinate and lungs of guinea pigs at high titers and was also able to transmit from an infected animal to sentinel animals by contact. The fact that bovine FLUDV replicated productively in both the upper and lower respiratory tracts of guinea pigs, similarly to virus infection in its native host, demonstrates that guinea pigs would be a suitable model host to study the replication and transmission potential of bovine FLUDV.

Phenethyl isothiocyanate upregulates death receptors 4 and 5 and inhibits proliferation in human cancer stem-like cells.

BACKGROUND: The cytokine TRAIL (tumor necrotic factor-related apoptosis-inducing ligand) selectively induces apoptosis in cancer cells, but cancer stem cells (CSCs) that contribute to cancer-recurrence are frequently TRAIL-resistant. Here we examined hitherto unknown effects of the dietary anti-carcinogenic compound phenethyl isothiocyanate (PEITC) on attenuation of proliferation and tumorigenicity and on up regulation of death receptors and apoptosis in human cervical CSC. METHODS: Cancer stem-like cells were enriched from human cervical HeLa cell line by sphere-culture method and were characterized by CSC-specific markers' analyses (flow cytometry) and Hoechst staining. Cell proliferation assays, immunoblotting, and flow cytometry were used to assess anti-proliferative as well as pro-apoptotic effects of PEITC exposure in HeLa CSCs (hCSCs). Xenotransplantation study in a non-obese diabetic, severe combined immunodeficient (NOD/SCID) mouse model, histopathology, and ELISA techniques were further utilized to validate our results in vivo. RESULTS: PEITC attenuated proliferation of CD44(high/+)/CD24(low/-), stem-like, sphere-forming subpopulations of hCSCs in a concentration- and time-dependent manner that was comparable to the CSC antagonist salinomycin. PEITC exposure-associated up-regulation of cPARP (apoptosis-associated cleaved poly [ADP-ribose] polymerase) levels and induction of DR4 and DR5 (death receptor 4 and 5) of TRAIL signaling were observed. Xenotransplantation of hCSCs into mice resulted in greater tumorigenicity than HeLa cells, which was diminished along with serum hVEGF-A (human vascular endothelial growth factor A) levels in the PEITC-pretreated hCSC group. Lung metastasis was observed only in the hCSC-injected group that did not receive PEITC-pretreatment. CONCLUSIONS: The anti-proliferative effects of PEITC in hCSCs may at least partially result from up regulation of DR4 and possibly DR5 of TRAIL-mediated apoptotic pathways. PEITC may offer a novel approach for improving therapeutic outcomes in cancer patients.

Natural infection with highly pathogenic avian influenza virus H5N1 in domestic pigeons (Columba livia) in Egypt.

The highly pathogenic avian influenza virus (HPAIV) subtype H5N1 threatens animal and human health worldwide. Susceptibility of pigeons to HPAIV (H5N1) and their role in avian influenza virus transmission to domestic birds and humans remain questionable. In this study, an outbreak in domestic pigeons (1 to 18 months old) with 50% mortality was investigated. Pigeons exhibited nervous manifestations and greenish diarrhoea. Necropsy of the naturally infected pigeons revealed congestion of the internal organs, particularly the lungs and brain. The HPAIV subtype H5N1 designated A/Pigeon/Egypt/SHAH-5803/2011 was isolated from a 40-day-old pigeon. Sequencing of the haemagglutinin gene showed it to be closely related to viruses in group 2.2.1/C. Intravenous inoculation of the isolate in chickens induced 100% mortality within 2 days post inoculation and the intravenous pathogenicity index was 2.7. Virus pathogenicity and transmissibility was determined experimentally in 6-week-old domestic pigeons. Thirty per cent of pigeons inoculated oronasally with 10(6) median embryo infective dose showed congested beak, conjunctivitis, depression, and greenish diarrhoea. A mortality rate of 10% was recorded preceded by severe neurologic signs consisting of torticollis, incoordination, tremors, and wing paralysis. Pathological examination revealed a friable brain tissue and congested meningeal blood vessels. The lungs appeared oedematous and severely haemorrhagic. Subepicardial and petechial haemorrhages on the coronary fat were observed. Both infected and contact pigeons shed virus via the oropharynx and cloaca. To our knowledge, this is the first description and characterization of HPAIV in naturally infected pigeons in Egypt. Our findings reveal that pigeons can indeed be susceptible to H5N1 HPAIVs and could be a source of infection to other birds and humans.

An evaluation of contaminated complete feed as a vehicle for porcine epidemic diarrhea virus infection of naïve pigs following consumption via natural feeding behavior: proof of concept.

BACKGROUND: Since its initial detection in May 2013, porcine epidemic diarrhea virus (PEDV) has spread rapidly throughout the US swine industry. Initially, contaminated feed was proposed as a risk factor for PEDV; however, data were not available to support this theory. Here we provide proof of concept of this risk by describing a novel means for recovering PEDV-contaminated complete feed material from commercial swine sites and conducting an in vivo experiment to prove its infectivity. RESULTS: For on-farm detection of PEDV RNA in feed, paint rollers were used to collect material from at-risk feed bins from 3 clinically affected breeding herds. This material was tested by PCR and determined to be positive for PEDV-RNA (Ct = 19.50-22.20 range). To test infectivity, this material was pooled (Ct = 20.65) and a Treatment group of 3-week old PEDV-naïve piglets were allowed to consume it via natural feeding behavior. For the purpose of a Positive control, piglets were allowed to ingest feed spiked with stock PEDV (Ct = 18.23) while the negative control group received PEDV-free feed. Clinical signs of PEDV infection (vomiting and diarrhea) and viral shedding were observed in both the Positive control and Treatment group' post-consumption with virus and microscopic lesions detected in intestinal samples No evidence of infection was observed in the Negative controls. CONCLUSIONS: These data provide proof of concept that contaminated complete feed can serve as a vehicle for PEDV infection of naïve pigs using natural feeding behavior.

Heat-labile- and heat-stable-toxoid fusions (LTR192G-STaP13F) of human enterotoxigenic Escherichia coli elicit neutralizing antitoxin antibodies.

Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and animals. Adhesins and enterotoxins, including heat-labile (LT) and heat-stable (STa) toxins, are the key virulence factors. Antigenic adhesin and LT antigens have been used in developing vaccines against ETEC diarrhea. However, STa has not been included because of its poor immunogenicity and potent toxicity. Our recent study showed that porcine-type STa toxoids became immunogenic and elicited neutralizing anti-STa antibodies after being genetically fused to a full-length porcine-type LT toxoid, LT(R192G) (W. Zhang et al., Infect. Immun. 78:316-325, 2010). In this study, we mutated human-type LT and STa genes, which are highly homologous to porcine-type toxin genes, for a full-length LT toxoid (LT(R192)) and a full-length STa toxoid (STa(P13F)) and genetically fused them to produce LT192-STa13 toxoid fusions. Mice immunized with LT192-STa13 fusion antigens developed anti-LT and anti-STa IgG (in serum and feces) and IgA antibodies (in feces). Moreover, secretory IgA antibodies from immunized mice were shown to neutralize STa and cholera toxins in T-84 cells. In addition, we fused the STa13 toxoid at the N terminus and C terminus, between the A1 and A2 peptides, and between the A and B subunits of LT192 to obtain different fusions in order to explore strategies for enhancing STa immunogenicity. This study demonstrated that human-type LT192-STa13 fusions induce neutralizing antitoxin antibodies and provided important information for developing toxoid vaccines against human ETEC diarrhea.

Protective Efficacy of an Orf Virus-Vector Encoding the Hemagglutinin and the Nucleoprotein of Influenza A Virus in Swine.

Swine influenza is a highly contagious respiratory disease of pigs caused by influenza A viruses (IAV-S). IAV-S causes significant economic losses to the swine industry and poses challenges to public health given its zoonotic potential. Thus effective IAV-S vaccines are needed and highly desirable and would benefit both animal and human health. Here, we developed two recombinant orf viruses, expressing the hemagglutinin (HA) gene (OV-HA) or the HA and the nucleoprotein (NP) genes of IAV-S (OV-HA-NP). The immunogenicity and protective efficacy of these two recombinant viruses were evaluated in pigs. Both OV-HA and OV-HA-NP recombinants elicited robust virus neutralizing antibody response in pigs, with higher levels of neutralizing antibodies (NA) being detected in OV-HA-NP-immunized animals pre-challenge infection. Although both recombinant viruses elicited IAV-S-specific T-cell responses, the frequency of IAV-S-specific proliferating CD8+ T cells upon re-stimulation was higher in OV-HA-NP-immunized animals than in the OV-HA group. Importantly, IgG1/IgG2 isotype ELISAs revealed that immunization with OV-HA induced Th2-biased immune responses, whereas immunization with OV-HA-NP virus resulted in a Th1-biased immune response. While pigs immunized with either OV-HA or OV-HA-NP were protected when compared to non-immunized controls, immunization with OV-HA-NP resulted in incremental protection against challenge infection as evidenced by a reduced secondary antibody response (NA and HI antibodies) following IAV-S challenge and reduced virus shedding in nasal secretions (lower viral RNA loads and frequency of animals shedding viral RNA and infectious virus), when compared to animals in the OV-HA group. Interestingly, broader cross neutralization activity was also observed in serum of OV-HA-NP-immunized animals against a panel of contemporary IAV-S isolates representing the major genetic clades circulating in swine. This study demonstrates the potential of ORFV-based vector for control of swine influenza virus in swine.

Small-Scale Dynamic Aurora.

Small-scale dynamic auroras have spatial scales of a few km or less, and temporal scales of a few seconds or less, which visualize the complex interplay among charged particles, Alfvén waves, and plasma instabilities working in the magnetosphere-ionosphere coupled regions. We summarize the observed properties of flickering auroras, vortex motions, and filamentary structures. We also summarize the development of fundamental theories, such as dispersive Alfvén waves (DAWs), plasma instabilities in the auroral acceleration region, ionospheric feedback instabilities (IFI), and the ionospheric Alfvén resonator (IAR). SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11214-021-00796-w.

Genetic fusions of heat-labile (LT) and heat-stable (ST) toxoids of porcine enterotoxigenic Escherichia coli elicit neutralizing anti-LT and anti-STa antibodies.

Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and farm animals. E. coli fimbriae, or colonization factor antigens (CFAs), and enterotoxins, including heat-labile enterotoxins (LT) and heat-stable enterotoxins (ST), are the key virulence factors in ETEC diarrhea. Unlike fimbriae or LT, STa has not often been included as an antigen in development of vaccines against ETEC diarrhea because of its poor immunogenicity. STa becomes immunogenic only after being coupled with a strongly immunogenic carrier protein. However, native or shorter STa antigens either had to retain toxic activity in order to become antigenic or elicited anti-STa antibodies that were not sufficiently protective. In this study, we genetically mutated the porcine LT (pLT) gene for a pLT(192(R-->G)) toxoid and the porcine STa (pSTa) gene for three full-length pSTa toxoids [STa(11(N-->K)), STa(12(P-->F)), and STa(13(A-->Q))] and used the full-length pLT(192) as an adjuvant to carry the pSTa toxoid for pLT(192):pSTa-toxoid fusion antigens. Rabbits immunized with pLT(192):pSTa(12) or pLT(192):pSTa(13) fusion protein developed high titers of anti-LT and anti-STa antibodies. Furthermore, rabbit antiserum and antifecal antibodies were able to neutralize purified cholera toxin (CT) and STa toxin. In addition, preliminary data suggested that suckling piglets born by a sow immunized with the pLT(192):pSTa(13) fusion antigen were protected when challenged with an STa-positive ETEC strain. This study demonstrated that pSTa toxoids are antigenic when fused with a pLT toxoid and that the elicited anti-LT and anti-STa antibodies were protective. This fusion strategy could provide instructive information to develop effective toxoid vaccines against ETEC-associated diarrhea in animals and humans.

Immunodominant epitopes in nsp2 of porcine reproductive and respiratory syndrome virus are dispensable for replication, but play an important role in modulation of the host immune response.

Non-structural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) is the largest protein of this virus. In addition to its crucial role in virus replication, recent studies have indicated its involvement in modulating host immunity. In this study, each of the six identified immunodominant nsp2 B-cell epitopes (ES2-ES7) was deleted using a type I PRRSV cDNA infectious clone. Deletion of ES3, ES4 or ES7 allowed the generation of viable virus. In comparison with the parental virus, the DeltaES3 mutant showed increased cytolytic activity and more vigorous growth kinetics, whilst the DeltaES4 and DeltaES7 mutants displayed decreased cytolytic activity and slower growth kinetics in MARC-145 cells. These nsp2 mutants were characterized further in a nursery pig disease model. The results showed that the DeltaES4 and DeltaES7 mutants exhibited attenuated phenotypes, whereas the DeltaES3 mutant produced a higher peak viral load in pigs. The antibody response reached similar levels, as measured by IDEXX ELISA at 21 days post-infection, and slightly higher levels of mean virus neutralizing titres were observed from pigs infected by the DeltaES4 and DeltaES7 mutants. The expression of innate and T-helper 1 cytokines was measured in peripheral blood mononuclear cells or virus-infected macrophages. The results consistently showed that interleukin-1beta and tumour necrosis factor alpha expression levels were downregulated in cells that were stimulated (or infected) with the DeltaES3 mutant compared with parental virus and the other nsp2 deletion mutants. These results suggest that certain regions in nsp2 are non-essential for PRRSV replication but may play an important role in modulation of host immunity in vivo.

Dietary soya saponins increase gut permeability and play a key role in the onset of soyabean-induced enteritis in Atlantic salmon ( Salmo salar L.).

Saponins are naturally occurring amphiphilic molecules and have been associated with many biological activities. The aim of the present study was to investigate whether soya saponins trigger the onset of soyabean-induced enteritis in Atlantic salmon (Salmo salar L.), and to examine if dietary soya saponins increase the epithelial permeability of the distal intestine in Atlantic salmon. Seven experimental diets containing different levels of soya saponins were fed to seawater-adapted Atlantic salmon for 53 d. The diets included a fishmeal-based control diet, two fishmeal-based diets with different levels of added soya saponins, one diet containing 25% lupin kernel meal, two diets based on 25% lupin kernel meal with different levels of added soya saponins, and one diet containing 25% defatted soyabean meal. The effect on intestinal morphology, intestinal epithelial permeability and faecal DM content was examined. Fish fed 25% defatted soyabean meal displayed severe enteritis, whereas fish fed 25% lupin kernel meal had normal intestinal morphology. The combination of soya saponins and fishmeal did not induce morphological changes but fish fed soya saponins in combination with lupin kernel meal displayed significant enteritis. Increased epithelial permeability was observed in fish fed 25% defatted soyabean meal and in fish fed soya saponin concentrate independent of the protein source in the feed. The study demonstrates that soya saponins, in combination with one or several unidentified components present in legumes, induce an inflammatory reaction in the distal intestine of Atlantic salmon. Soya saponins increase the intestinal epithelial permeability but do not, per se, induce enteritis.

Co-infection of highly pathogenic avian influenza and duck hepatitis viruses in Egyptian backyard and commercial ducks.

Highly pathogenic avian influenza (HPAI) H5N1 virus poses a major challenge to the poultry industry and human health in Egypt. Twenty one households and eight duck farms in Sharkia Province, Egypt were investigated for the presence of avian influenza virus (AIV) and/or duck hepatitis virus 1 (DHV-1). Mortality rates among the investigated farms and yards were, 18.9% (69/365) of native ducks, 60.9% (25/41) of Pekin ducks, 60.2% (6306/10473) of Muscovy ducks and 44.9% (1353/3015) of Mallard ducks. The RT-PCR revealed the circulation of HPAI-H5N1 virus (81/104) among the examined birds with a high percentage in Muscovy (83.7%) and Pekin (83.4%) ducks. Interestingly, co-infection of HPAI and DHV-1 viruses in three ducklings with age of 4-19 days was detected. Severe neurological signs with high mortality were observed in ducklings as early as 4 days of age. Influenza virus antigen was detected in the neurons and glial cells of the brain, hepatocytes, and the intestinal submucosal plexus. Although, genetic characterization of H5N1 isolates revealed HPAIV of clade 2.2.1.2, such increased mortalities and neurological signs regardless of the duck age might imply the natural selection of HPAI in ducks. Crucial monitoring of the disease situation in ducks is essential for the implementation of an effective prevention and control program.

New science in plain sight: Citizen scientists lead to the discovery of optical structure in the upper atmosphere.

A glowing ribbon of purple light running east-west in the night sky has recently been observed by citizen scientists. This narrow, subauroral, visible structure, distinct from the traditional auroral oval, was largely undocumented in the scientific literature and little was known about its formation. Amateur photo sequences showed colors distinctly different from common types of aurora and occasionally indicated magnetic field-aligned substructures. Observations from the Swarm satellite as it crossed the arc have revealed an unusual level of electron temperature enhancement and density depletion, along with a strong westward ion flow, indicating that a pronounced subauroral ion drift (SAID) is associated with this structure. These early results suggest the arc is an optical manifestation of SAID, presenting new opportunities for investigation of the dynamic SAID signatures from the ground. On the basis of the measured ion properties and original citizen science name, we propose to identify this arc as a Strong Thermal Emission Velocity Enhancement (STEVE).

Phylogenetic Analysis and Characterization of a Sporadic Isolate of Equine Influenza A H3N8 from an Unvaccinated Horse in 2015.

Equine influenza, caused by the H3N8 subtype, is a highly contagious respiratory disease affecting equid populations worldwide and has led to serious epidemics and transboundary pandemics. This study describes the phylogenetic characterization and replication kinetics of recently-isolated H3N8 virus from a nasal swab obtained from a sporadic case of natural infection in an unvaccinated horse from Montana, USA. The nasal swab tested positive for equine influenza by Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR). Further, the whole genome sequencing of the virus confirmed that it was the H3N8 subtype and was designated as A/equine/Montana/9564-1/2015 (H3N8). A BLASTn search revealed that the polymerase basic protein 1 (PB1), polymerase acidic (PA), hemagglutinin (HA), nucleoprotein (NP), and matrix (M) segments of this H3N8 isolate shared the highest percentage identity to A/equine/Tennessee/29A/2014 (H3N8) and the polymerase basic protein 2 (PB2), neuraminidase (NA), and non-structural protein (NS) segments to A/equine/Malaysia/M201/2015 (H3N8). Phylogenetic characterization of individual gene segments, using currently available H3N8 viral genomes, of both equine and canine origin, further established that A/equine/Montana/9564-1/2015 belonged to the Florida Clade 1 viruses. Interestingly, replication kinetics of this H3N8 virus, using airway derived primary cells from multiple species, such as equine, swine, bovine, and human lung epithelial cells, demonstrated appreciable titers, when compared to Madin-Darby canine kidney epithelial cells. These findings indicate the broad host spectrum of this virus isolate and suggest the potential for cross-species transmissibility.

Saponin-containing subfractions of soybean molasses induce enteritis in the distal intestine of Atlantic salmon.

The current work aimed at tracing the causative components for soybean-induced enteritis in Atlantic salmon (Salmo salar L.). Soybean molasses was subjected to phase separation using n-butanol. Three subfractions were obtained as follows: butanol phase, precipitate, and water phase. The biochemical composition of soybean molasses and the obtained subfractions were analyzed in detail: Protein, fat, and ash were quantified according to standard methods. Sucrose, raffinose, and stachyose were quantified using high-performance anion-exchange chromatography. Soyasaponins were quantified using reverse-phase high-performance liquid chromatography. Finally, sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to evaluate the size distribution of the proteins present in each fraction. Molasses and the different subfractions were thereafter fed to Atlantic salmon in two successive fish trials. The level of intestinal inflammation was evaluated by light microscopy using a semiquantitative scoring system. Histological assessments revealed that Atlantic salmon fed a combination of butanol phase and precipitate displayed significant enteritis. Atlantic salmon fed the water phase displayed normal intestinal morphology. The causative components for soybean-induced enteritis withstand butanol treatment and prolonged heating at 70 degrees C. Sucrose, raffinose, stachyose, nor soybean proteins larger than 10 kDa induce enteritis alone. Soyasaponins, or components that follow the same solubility pattern, trigger the inflammatory reaction. We therefore suggest that soybean-induced enteritis in Atlantic salmon is induced by soyasaponins alone or by soyasaponins in combination with other factors, e.g., antigenic soybean proteins or the intestinal microflora.

Soyasaponins resist extrusion cooking and are not degraded during gut passage in Atlantic salmon (Salmo salar L.).

The stability of soyasaponins in fish feed formulations was investigated. The level of soyasaponin Ab, Bb, Bc, Ba-2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (Ba-DDMP), Bb-DDMP, and Bc-DDMP was quantified in 15 samples of defatted soybean meal, two full fat soybean meals, and two soybean protein concentrates by reverse phase high-performance liquid chromatography. The total level of saponins in the 15 samples of commercial defatted soybean meal ranged from 4.8-6.8 micromol/g (5.1-7.0 g/kg). The two full fat meals contained 4.4 and 4.7 micromol/g whereas no saponins could be detected in the alcohol-extracted soybean protein concentrates. Fifteen batches of fish feed containing 20% defatted soybean meal were produced by twin-screw extrusion from the 15 different samples of defatted soybean meal. Extrusion did not reduce the total level of group B saponins, but the ratio between DDMP-conjugated group B saponins and non-DDMP-conjugated group B saponins was slightly reduced. A soybean-containing diet was fed to seawater adapted Atlantic salmon for 9 weeks. Yttrium oxide was included in the feed as an inert marker in order to estimate the disappearance of saponins during gut passage. High levels of intact non-DDMP-conjugated group B soyasaponins were found in feces whereas only low levels of DDMP-conjugated saponins could be detected. The overall disappearance of saponins was close to zero, and the concentration of intact saponins in dry feces reached levels several fold higher than dietary levels. The present work demonstrates that non-DDMP-conjugated group B soyasaponins resist extrusion cooking and remain intact during gut passage in Atlantic salmon. The latter is contrary to earlier findings in endothermic animals.

Effect of ripeness and postharvest storage on the phenolic profiles of Cherries (Prunus avium L.).

The phenolic compounds hydroxycinnamates, anthocyanins, flavonols, and flavan-3-ols of sweet cherry cultivars Burlat, Saco, Summit, and Van harvested in 2001 and 2002 were quantified by HPLC-DAD. Phenolics were analyzed at partially ripe and ripe stages and during storage at 15 +/- 5 degrees C (room temperature) and 1-2 degrees C (cool temperature). Neochlorogenic and p-coumaroylquinic acids were the main hydroxycinnamic acid derivatives, but chlorogenic acid was also identified in all cultivars. The 3-glucoside and 3-rutinoside of cyanidin were the major anthocyanins. Peonidin and pelargonidin 3-rutinosides were the minor anthocyanins, and peonidin 3-glucoside was also present in cvs. Burlat and Van. Epicatechin was the main monomeric flavan-3-ol with catechin present in smaller amounts in all cultivars. The flavonol rutin was also detected. Cultivar Saco contained the highest amounts of phenolics [227 mg/100 g of fresh weight (fw)] and cv. Van the lowest (124 mg/100 g of fw). Phenolic acid contents generally decreased with storage at 1-2 degrees C and increased with storage at 15 +/- 5 degrees C. Anthocyanin levels increased at both storage temperatures. In cv. Van the anthocyanins increased up to 5-fold during storage at 15 +/- 5 degrees C (from 47 to 230 mg/100 g of fw). Flavonol and flavan-3-ol contents remained quite constant. For all cultivars the levels of phenolic acids were higher in 2001 and the anthocyanin levels were higher in 2002, which suggest a significant influence of climatic conditions on these compounds.

Multiepitope fusion antigen induces broadly protective antibodies that prevent adherence of Escherichia coli strains expressing colonization factor antigen I (CFA/I), CFA/II, and CFA/IV.

Diarrhea is the second leading cause of death in children younger than 5 years and continues to be a major threat to global health. Enterotoxigenic Escherichia coli (ETEC) strains are the most common bacteria causing diarrhea in developing countries. ETEC strains are able to attach to host small intestinal epithelial cells by using bacterial colonization factor antigen (CFA) adhesins. This attachment helps to initiate the diarrheal disease. Vaccines that induce antiadhesin immunity to block adherence of ETEC strains that express immunologically heterogeneous CFA adhesins are expected to protect against ETEC diarrhea. In this study, we created a CFA multiepitope fusion antigen (MEFA) carrying representative epitopes of CFA/I, CFA/II (CS1, CS2, and CS3), and CFA/IV (CS4, CS5, and CS6), examined its immunogenicity in mice, and assessed the potential of this MEFA as an antiadhesin vaccine against ETEC. Mice intraperitoneally immunized with this CFA MEFA exhibited no adverse effects and developed immune responses to CFA/I, CFA/II, and CFA/IV adhesins. Moreover, after incubation with serum of the immunized mice, ETEC or E. coli strains expressing CFA/I, CFA/II, or CFA/IV adhesins were significantly inhibited in adherence to Caco-2 cells. Our results indicated this CFA MEFA elicited antibodies that not only cross-reacted to CFA/I, CFA/II and CFA/IV adhesins but also broadly inhibited adherence of E. coli strains expressing these seven adhesins and suggested that this CFA MEFA could be a candidate to induce broad-spectrum antiadhesin protection against ETEC diarrhea. Additionally, this antigen construction approach (creating an MEFA) may be generally used in vaccine development against heterogenic pathogens.

Toxicity and immunogenicity of Enterotoxigenic Escherichia coli heat-labile and heat-stable toxoid fusion 3xSTa(A14Q)-LT(S63K/R192G/L211A) in a murine model.

Diarrhea is the second leading cause of death to young children. Enterotoxigenic Escherichia coli (ETEC) are the most common bacteria causing diarrhea. Adhesins and enterotoxins are the virulence determinants in ETEC diarrhea. Adhesins mediate bacterial attachment and colonization, and enterotoxins including heat-labile (LT) and heat-stable type Ib toxin (STa) disrupt fluid homeostasis in host cells that leads to fluid hyper-secretion and diarrhea. Thus, adhesins and enterotoxins have been primarily targeted in ETEC vaccine development. A recent study reported toxoid fusions with STa toxoid (STa(P13F)) fused at the N- or C-terminus, or inside the A subunit of LT(R192G) elicited neutralizing antitoxin antibodies, and suggested application of toxoid fusions in ETEC vaccine development (Liu et al., Infect. Immun. 79:4002-4009, 2011). In this study, we generated a different STa toxoid (STa(A14Q)) and a triple-mutant LT toxoid (LT(S63K/R192G/L211A), tmLT), constructed a toxoid fusion (3xSTa(A14Q)-tmLT) that carried 3 copies of STa(A14Q) for further facilitation of anti-STa immunogenicity, and assessed antigen safety and immunogenicity in a murine model to explore its potential for ETEC vaccine development. Mice immunized with this fusion antigen showed no adverse effects, and developed antitoxin antibodies particularly through the IP route. Anti-LT antibodies were detected and were shown neutralizing against CT in vitro. Anti-STa antibodies were also detected in the immunized mice, and serum from the IP immunized mice neutralized STa toxin in vitro. Data from this study indicated that toxoid fusion 3xSTa(A14Q)-tmLT is safe and can induce neutralizing antitoxin antibodies, and provided helpful information for vaccine development against ETEC diarrhea.