Temporal detection of equine herpesvirus infections of a cohort of mares and their foals

The objectives of this study were to estimate the prevalence of equine herpesviruses (EHV) 1-5 in the nasal secretions (NS) of a cohort of 12 mares and their foals from birth to 6 months of age, estimate the prevalence of EHV-1-5 infection of peripheral blood mononuclear cells (PBMC) of selected foals, and investigate phylogenetic relationships amongst the various strains of EHV-2 and 5. Virus-specific PCR assays were used to detect EHV-1-5 in NS and PBMC. A homologous portion of the glycoprotein B (gB) gene of the various strains of EHV-2 and 5 was sequenced and compared. EHV-2, 4, and 5 were all detected in NS from the horses, but only EHV-4 was associated with respiratory disease (P=0.005). EHV-2 and 5 infections were both common, but foals shed EHV-2 in their NS earlier in life than EHV-5 (P=0.01). Latent EHV-2 and 5 infections were detected in the PBMC of 75 and 88%, respectively, of the foals at approximately 6 months of age. The strains of EHV-2 shed in the NS of individual horses were more genetically heterogeneous than the strains of EHV-5 (95.5-99.3% versus 98.8-99.3% nucleotide identity, respectively). One-month-old foals typically shed strains of EHV-2 that were identical to those infecting their dams whereas older foals often shed virus strains that were different from those of their dams. Although herpesvirus infections were ubiquitous in this cohort of horses, there were distinct clinical consequences and clear epidemiological differences between infections with the different viruses.     

Spirulina maxima Derived Pectin Nanoparticles Enhance the Immunomodulation,Stress Tolerance,and Wound Healing in Zebrafish

In this study, Spirulina maxima derived pectin nanoparticles (SmPNPs) were synthesized and multiple biological effects were investigated using in vitro and in vivo models. SmPNPs were not toxic to Raw 264.7 cells and zebrafish embryos up to 1 mg/mL and 200 µg/mL, respectively. SmPNPs upregulated Il 10, Cat, Sod 2, Def 1, Def 2, and Muc 1 in Raw 264.7 cells and tlr2, tlr4b, tlr5b, il1β, tnfα, cxcl8a, cxcl18b, ccl34a.4, ccl34b.4, muc5.1, muc5.2, muc5.3, hamp, cstd, hsp70, cat, and sod1 in the larvae and adult zebrafish, suggesting immunomodulatory activity. Exposure of larvae to SmPNPs followed by challenge with pathogenic bacterium Aeromonas hydrophila resulted a two-fold reduction of reactive oxygen species, indicating reduced oxidative stress compared to that in the control group. The cumulative percent survival of larvae exposed to SmPNPs (50 µg/mL) and adults fed diet supplemented with SmPNPs (4%) was 53.3% and 76.7%, respectively. Topical application of SmPNPs on adult zebrafish showed a higher wound healing percentage (48.9%) compared to that in the vehicle treated group (38.8%). Upregulated wound healing markers (tgfβ1, timp2b, mmp9, tnfα, il1β, ccl34a.4, and ccl34b.4), enhanced wound closure, and restored pigmentation indicated wound healing properties of SmPNPs. Overall, results uncover the multiple bioactivities of SmPNPs, which could be a promising biocompatible candidate for broad range of aquatic and human therapies.     

Effect of black currant anthocyanins on the activation of endothelial nitric oxide synthase (eNOS) in vitro in human endothelial cells

Polyphenols are known to induce vasodilatory function via activation of the redox-sensitive phosphatidylinositol-3 (PI3)/protein kinase B (Akt) pathway. Black currant fruits have appreciable amounts of polyphenolic compounds including cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside, delphinidin-3-O-glucoside, and delphinidin-3-O-rutinoside. It was hypothesized that black currant fruit extracts would cause activation of endothelial nitric oxide synthase (eNOS) through activation of redox-sensitive PI3 kinase/Akt signaling pathway. To test this hypothesis, human umbilical vein endothelial cells (HUVECs) were treated with different concentrations/times of black currant juice concentrates (Ben Gairn and Ben Hope) and the activation of Akt and eNOS was measured using immunoblotting. Vitamin C is also known to activate Akt and eNOS in in vitro models, and black currants are rich in vitamin C. Therefore, the effect of black currant extracts with and without coexisting vitamin C was investigated, using SPE columns to eliminate vitamin C content. The individual (and combined) effects of the major anthocyanins present in black currant juice samples with and without vitamin C were investigated and compared to the effects of the whole extract. Black currant juice samples (1 μL/mL) significantly increased the phosphorylation of Akt (p-Akt) and eNOS (p-eNOS) (P < 0.05). Activation of Akt and eNOS was abolished by incubation with wortmannin, a PI3K inhibitor, supporting the involvement of PI3K/Akt. Vitamin C alone significantly increased the p-Akt and p-eNOS (P < 0.05); however, removal of vitamin C from black currant did not significantly affect p-Akt and p-eNOS compared to black currant with vitamin C. Assessment of individual anthocyanins also showed significant effects on p-Akt and p-eNOS. In summary, in the present study data suggested that black currant concentrates, Ben Gairn and Ben Hope, activated eNOS via Akt/PI3 kinase pathway in vitro in HUVECs and that the effect was not dependent on vitamin C.     

Perchlorate, nitrate, thiocyanate, and iodide levels in chicken feed, water, and eggs from three farms

Perchlorate is an inhibitor of iodide uptake that is found widely in the environment. Given the potential for perchlorate accumulation during egg formation and the widespread consumption of eggs, it is important to examine eggs as a source of exposure to perchlorate and other potential inhibitors of iodide uptake (nitrate and thiocyanate). This study was conducted to determine potential human exposure to perchlorate from eggs produced by chicken flocks consuming differing amounts of perchlorate. The mean concentrations of perchlorate (7.16 ( 1.99 microg/kg of dry weight), nitrate (2820 ( 2100 microg/kg of dry weight), thiocyanate (574 +/- 433 microg/kg of dry weight), and iodide (2980 ( 1490 microg/kg of dry weight) in eggs (n = 180) from 15 chicken houses on 3 U.S. farms were determined. Chickens secreted into eggs an average of 23% of the perchlorate ingested from feed and water. Perchlorate levels in eggs were positively correlated with perchlorate intake (p < 0.001). Increased intake of perchlorate, nitrate, and thiocyanate was associated with decreased iodide levels in eggs, possibly indicating a competitive transport mechanism, such as sodium-iodide symporter. It was estimated that egg consumption contributes minimal perchlorate (approximately 0.040 microg) compared to the average total intake of approximately 10.5 microg for U.S. adults. Additionally, it was found that egg consumption was not associated with increased perchlorate exposure in 2820 individuals from the National Health and Nutrition Examination Survey (p value for the difference of least-squares means, pDiff = 0.225). From these findings it was concluded that, although chickens secrete perchlorate in eggs, eggs do not appear to be a significant source of perchlorate exposure for adults in the United States.     

Vitamin K2 is a mitochondrial electron carrier that rescues pink1 deficiency

Human UBIAD1 localizes to mitochondria and converts vitamin K(1) to vitamin K(2). Vitamin K(2) is best known as a cofactor in blood coagulation, but in bacteria it is a membrane-bound electron carrier. Whether vitamin K(2) exerts a similar carrier function in eukaryotic cells is unknown. We identified Drosophila UBIAD1/Heix as a modifier of pink1, a gene mutated in Parkinson's disease that affects mitochondrial function. We found that vitamin K(2) was necessary and sufficient to transfer electrons in Drosophila mitochondria. Heix mutants showed severe mitochondrial defects that were rescued by vitamin K(2), and, similar to ubiquinone, vitamin K(2) transferred electrons in Drosophila mitochondria, resulting in more efficient adenosine triphosphate (ATP) production. Thus, mitochondrial dysfunction was rescued by vitamin K(2) that serves as a mitochondrial electron carrier, helping to maintain normal ATP production.     

Assessment of correlation between in vitro CD3+ T cell susceptibility to EAV infection and clinical outcome following experimental infection

In a recent study, we demonstrated that the virulent Bucyrus strain (VBS) of EAV could infect in vitro a small population of CD3(+) T lymphocytes from some but not all horses. Furthermore, we have shown that a common haplotype is associated with this in vitro CD3(+) T cell susceptibility/resistance phenotype to EAV infection. In this study, we investigated whether the differences in the susceptibility or resistance of CD3(+) T cells in vitro correlate with the outcome and severity of clinical signs in vivo. Thus, horses were divided into two groups based on their CD3(+) T cell susceptible or resistant phenotype. Following experimental inoculation with the recombinant VBS of EAV, horses were assessed for presence and severity of clinical signs, duration and magnitude of virus shedding, as well as production of proinflammatory and immunomodulatory cytokines in peripheral blood mononuclear cells using real-time quantitative RT-PCR. The data showed that there was a significant difference between the two groups of horses in terms of cytokine mRNA expression and evidence of increased clinical signs in horses possessing the in vitro CD3(+) T cell resistant phenotype. This is the first study to provide direct evidence for a correlation between variation in host genotype and phenotypic differences in terms of the extent of viral replication, presence and severity of clinical signs and cytokine gene expression caused by infection with virulent EAV.     

Differentiation of strains of equine arteritis virus of differing virulence to horses by growth in equine endothelial cells

OBJECTIVE: To compare growth characteristics of strains of equine arteritis virus (EAV) of differing virulence to horses in rabbit kidney (RK)-13 cells and equine endothelial cells (EECs) cultured from the pulmonary artery of a foal. SAMPLE POPULATION: 13 strains of EAV, including 11 field isolates of differing virulence to horses; the highly virulent, horse-adapted Bucyrus strain; and the modified-live virus (MLV) vaccine derived from it. PROCEDURE: The growth characteristics of the 13 strains were compared in EECs and RK-13 cells. Viral nucleoprotein expression, cytopathogenicity, and plaque size were compared to determine whether growth characteristics of the 13 strains were predictive of their virulence to horses. RESULTS: Cytopathogenicity, viral nucleoprotein expression, and plaque size induced by all 13 viruses were similar in RK-13 cells, whereas virulent strains of EAV caused significantly larger plaques in EECs than did the avirulent strains of EAV. Paradoxically, the highly attenuated MLV vaccine and 1 field isolate of EAV caused plaques in EECs that were larger than those caused by any of the other viruses, and sequence analysis confirmed the field isolate of EAV to be indistinguishable from the MLV vaccine. CONCLUSIONS AND CLINICAL RELEVANCE: With the notable exception of the MLV vaccine, growth of the various strains of EAV in EECs was predictive of their individual virulence to horses. Thus, EECs provide a relevant and useful model to further characterize determinants of virulence and attenuation amongst strains of EAV.     

Isolation of equine herpesvirus-5 from blood mononuclear cells of a gelding.

Horses are commonly infected by herpesviruses, but isolation of equine herpesvirus-5 (EHV-5) has only infrequently been reported. We describe the isolation and characterization of a strain of EHV-5 from the blood mononuclear cells of a healthy adult horse in California. The virus was initially identified by EHV-5 specific polymerase chain reaction (PCR), and it caused lytic infection of cultured rabbit kidney cells only after repeated serial passage. Virions with characteristic herpesvirus morphology were readily demonstrated in cell culture lysate by transmission electron microscopy. A portion of the glycoprotein B gene of this strain of EHV-5 had 99% identity to the published EHV-5 sequence, and it was clearly distinguishable from other EHV (1-4) by virus-specific PCR assays. Prevalence of EHV-5 infection in a group of young racehorses was estimated at 64% using the EHV-5 specific PCR on nasopharyngeal secretions.