Enhanced Antiviral Activity of Human Surfactant Protein D by Site-Specific Engineering of the Carbohydrate Recognition Domain.

Innate immunity is critical in the early containment of influenza A virus (IAV) infection and surfactant protein D (SP-D) plays a crucial role in innate defense against IAV in the lungs. Multivalent lectin-mediated interactions of SP-D with IAVs result in viral aggregation, reduced epithelial infection, and enhanced IAV clearance by phagocytic cells. Previous studies showed that porcine SP-D (pSP-D) exhibits distinct antiviral activity against IAV as compared to human SP-D (hSP-D), mainly due to key residues in the lectin domain of pSP-D that contribute to its profound neutralizing activity. These observations provided the basis for the design of a full-length recombinant mutant form of hSP-D, designated as "improved SP-D" (iSP-D). Inspired by pSP-D, the lectin domain of iSP-D has 5 amino acids replaced (Asp324Asn, Asp330Asn, Val251Glu, Lys287Gln, Glu289Lys) and 3 amino acids inserted (326Gly-Ser-Ser). Characterization of iSP-D revealed no major differences in protein assembly and saccharide binding selectivity as compared to hSP-D. However, hemagglutination inhibition measurements showed that iSP-D expressed strongly enhanced activity compared to hSP-D against 31 different IAV strains tested, including (pandemic) IAVs that were resistant for neutralization by hSP-D. Furthermore, iSP-D showed increased viral aggregation and enhanced protection of MDCK cells against infection by IAV. Importantly, prophylactic or therapeutic application of iSP-D decreased weight loss and reduced viral lung titers in a murine model of IAV infection using a clinical isolate of H1N1pdm09 virus. These studies demonstrate the potential of iSP-D as a novel human-based antiviral inhalation drug that may provide immediate protection against or recovery from respiratory (pandemic) IAV infections in humans.

Effects of a feed additive blend on broilers challenged with heat stress.

We evaluated a blend of medium-chain fatty acids (MCFA), organic acids, and a polyphenol antioxidant on gut integrity. Eighty Ross Broilers were exposed to 20-22°C (control - normothermic) or to 35-39.5°C (heat stress) for eight hours a day for a period of 1 or 5 days. Birds were fed a standard diet, or a diet supplemented with the test blend. Thereafter, birds were euthanized, and intestinal sections were excised for morphological, morphometric and gene expression analyses. Blood samples were collected for glucose-6-phosphate dehydrogenase (G6PD), glutathione peroxidase (GSH-Px) activity and trolox equivalent antioxidant capacity (TEAC) determination. Heart and liver tissues were used to quantify the expression of heat shock proteins 60 and 70 (HSP60 and HSP70, respectively) and inhibitor of kappa light chain gene enhancer in B cells alpha (IKBA). The jejunum was the most sensitive intestinal section, where heat stress modulated the expression of HSP70, of the inflammatory markers IKBA, interleukin 8 (IL-8), interferon gamma (IFNγ), and toll-like receptor 4 (TLR4). Moreover, expression of tight junctions (CLDN1, ZO1 and ZO2) and nutrient transporters (PEPT1 and EAAT3) was modulated especially in the jejunum. In conclusion, the feed additive blend protected intestines during heat stress from the decrease in villus height and crypt depth, and from the increase in villus width. Especially in the jejunum, heat stress played an important role by modulating oxidative stress and inflammation, impairing gut integrity and nutrient transport, and such deleterious effects were alleviated by the feed additive blend. RESEARCH HIGHLIGHTS Jejunum is the most sensitive intestinal segment during heat stress. Heat stress affects the expression of tight junctions and nutrient transporters. Feed management helps to alleviate the disturbances caused by heat stress. A blend of MCFA, organic acids and a polyphenol protects broilers under heat stress.

Quantitative histo-morphometric analysis of heat-stress-related damage in the small intestines of broiler chickens.

The aim of the current research was to present a methodological approach allowing reproducible morphometric and morphological (Chiu/Park scale) analyses of the alterations in the intestines of broilers exposed to heat stress. Ross broilers were exposed over four consecutive days to a high-temperature regime in controlled climate rooms, with a day temperature of 39°C (±1°C) and a night temperature of 25°C (±1°C), respectively. A control group was kept at an ambient temperature of 25°C (±1°C) during the entire experimental period. At the end of the exposure period, the birds were sacrificed and specimens were taken of the duodenum, jejunum and ileum for histology. Blood was collected for oxidative stress analysis. Histo-morphological and morphometric analyses of the intestines indicated that the duodenum and jejunum showed more damage than the ileum. The major alterations in the control intestines were limited to the villus tips, while heat stress led to villus denudation and crypt damage. When compared with morphologically normal villi, heat-stress-associated alterations were also observed in villus height (decreased), villus breadth at base (increased) and epithelial cell area (decreased). Birds exposed to heat stress presented with an increase in glutathione peroxidase activity and a decreased antioxidant capacity. It can be concluded that the chosen model allows a reproducible quantification of heat stress effects, which is suitable for the evaluation of dietary intervention strategies to combat heat stress conditions.

Microbiological Conditions and Keeping Quality of Veal Tongues as Affected by Lactic Acid Decontamination and Vacuum Packaging.

The effect of a lactic acid decontamination treatment on the microbiological condition and keeping quality of veal calf tongues was assessed. Thirty tongues were collected 45 min post mortem. Ten were washed with tap water in a centrifuge, 10 were treated with 2.0% (v/v) L-lactic acid instead of water, and 10 tongues received no treatment and served as control samples. Immediately following these treatments all tongues were vacuum-packaged, chilled 2 h in ice-water and stored at 3±1 °C and 85±5 % ERH. At 0, 14, and 28 d postmortem samples were taken for bacteriological, histobacterioscopic and sensory examination. The histobacterioscopic examination showed that the initial microflora appeared to be predominantly located under and between the papillae of the tongue surface. Centrifugation with water only did not significantly affect the bacteriological condition of tongues, although the overall appearance improved. Decontamination with lactic acid decreased mesophilic aerobic colony counts from 5.6 to 2.7 log10 CFU/cm2. After 14 d of storage the so-called "delayed" effect of lactic acid was still observed. At that time aerobic colony counts and Enterobacteriaceae counts of controls were 6.5 and 2.8 log10 CFU/cm2, while these counts of the lactic acid treated group were 4.0 and <1.3, respectively. Results of the bacteriological examinations were substantiated by the histobacterioscopic findings. Centrifugation with lactic acid detached superficial cells from the stratified squamous epithelium. Decontamination of tongues by centrifugation with lactic acid before vacuum packaging will increase storage life and safeguard public health.