Diagnostics of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) nucleocapsid antigen using chicken immunoglobulin Y.

The goal of this study was to develop a quantitative detection system for severe acute respiratory syndrome-associated coronavirus (SARS-CoV), targeting the nucleocapsid protein (NP), to determine the presence and degree of infection in suspected individuals. Because the NP is the viral protein shed during infection and its template mRNA is the most abundant subgenomic RNA, it is a suitable candidate for developing antibodies for diagnostic applications. In this study, we have prepared full-length SARS-CoV NP expressed in Escherichia coli and purified. Full-length NP was used for the preparation of mouse monoclonal antibody and chicken polyclonal IgY antibodies for the development of heterosandwich ELISA for early diagnostics of SARS-suspected individuals. The sensitivity of the developed heterosandwich ELISA can detect the viral antigen at 18.5 pg/mL of recombinant NP. This study describes ultrasensitive ELISA using 19B6 monoclonal antibody as the capture antibody and IgY as the detecting antibody against the most abundant SARS-CoV NP antigens. One of the most important findings was the use of inexpensive polyclonal IgY antibody to increase the sensitivity of the detection system for SARS-CoV at the picogram level. Furthermore, the immunoassay of SARS-CoV NP antigen developed could be an effective and sensitive method of diagnosing SARS-suspected individuals during a future SARS-CoV outbreak.

Maternal dietary N-3 fatty acids alter the spleen fatty acid composition and bovine serum albumin-induced wing web swelling in broilers.

Effects of maternal dietary polyunsaturated fatty acids (PUFA) on the spleen fatty acid composition and BSA-induced wing web swellings were investigated in broilers. One hundred twenty broiler breeder hens 26 wk of age were randomly assigned to diets containing mainly wheat, corn, soy meal, barley, oat and 5% (wt/wt) added sunflower oil, fish oil, or a mix of sunflower and fish oils (1:1). After 2 wk on the experimental diets, birds were inseminated, eggs were collected and incubated. Progeny chicks were then fed identical diets for 6 wk. The maternal dietary oils affected (P < 0.05) n-6 and n-3 PUFA in the spleens of hatching chicks. After 2 wk, n-6 PUFA did not differ among the groups; n-3 PUFA, docosapentaenoic, and docosahexaenoic (DHA) acids were higher (P < 0.05) in the spleens of broilers from hens fed 2.5 or 5% fish oil. After 4 wk, broilers from hens fed 5% fish oil still had higher levels of DHA (P < 0.05) in their spleens than those from hens fed 5% sunflower oil. The BSA-induced wing web swelling response was suppressed (P < 0.05) by n-3 PUFA in breeder hens. Broilers from hens fed high levels of n-3 PUFA had lower (P < 0.05) wing web swelling reactions to BSA at 2 wk (2.5% fish oil) and 4 wk (2.5 and 5% fish oil). In conclusion, n-3 PUFA in breeder hen diets suppressed the BSA-induced wing web swellings of the hens, increased the spleen n-3 fatty acids (especially DHA), and decreased BSA-induced wing web swellings of progeny up to 4 wk of age.

In vitro studies of chicken egg yolk antibody (IgY) against Salmonella enteritidis and Salmonella typhimurium.

Chicken egg yolk antibody (IgY) raised against Salmonella enteritidis or Salmonella typhimurium was found in highly specific activity levels by ELISA. S. enteritidis- and S. typhimurium-specific IgY powder, prepared by freeze-drying the egg yolk water-soluble fraction, contained 15.5 and 10.0% of specific IgY, respectively. Anti-S. enteritidis IgY cross-reacted 55.3% with S. typhimurium. The cross-reactivity of anti-S. typhimurium IgY with S. enteritidis was 42.4%. Salmonella-specific IgY was demonstrated to inhibit Salmonella growth in liquid medium. The growth rate of S. enteritidis incubated with S. enteritidis-specific IgY was fourfold less than that of the control group during a 4-to-6-h incubation. Cell counts of S. typhimurium incubated with S. typhimurium-specific IgY were reduced by 1.6 log cfu/mL in comparison to that of the control group after 6 h of incubation. The specific binding activity of IgY was further evaluated by using immunofluorescence and immunoelectron microscopy. It was found that Salmonella-specific IgY could bind to the antigens expressed on the Salmonella surface, resulting in structural alterations of the bacterial surface.

Isolation, characterization and localization of glycosaminoglycans in growing antlers of wapiti (Cervus elaphus).

Glycosaminoglycans were isolated from the four sections (tip, upper, middle and base) of the main beam of growing antlers of wapiti (Cervus elaphus) by papain digestion and DEAE-Sephacel chromatography. Chondroitin sulfate was the major glycosaminoglycan in each section of antler accounting for, on average, 88% of the total uronic acid. The yield of chondroitin sulfate liberated from the tissue was approximately 6-fold greater in the cartilaginous (tip and upper) sections than in the bony (middle and base) sections. This was consistent with the higher intensity of glycosaminoglycan staining with either Alcian Blue or Safranin-O. The majority (average 88%) of chondroitin sulfate was precipitated with 40 and 50% ethanol. The average molecular size of chondroitin sulfate determined by gel chromatography on Sephacryl S-300 tended to be greater in the 40% ethanol than in the 50% ethanol fraction. In either fraction, the molecular size of chondroitin sulfate was smaller in cartilaginous tissues than in osseous tissues of growing antler. In addition to chondroitin sulfate, the antler contained small amounts of hyaluronic acid, dermatan sulfate and keratan sulfate. The immunohistochemical study showed wide distribution of chondroitin sulfate, decorin, and keratan sulfate throughout the antler. On the other hand, keratan sulfate was more prominent in the cartilaginous sections than in the bony sections where the anti-keratan sulfate monoclonal antibody staining was seen in the osteoid tissue only.

Effects of egg and yolk weights on yolk antibody (IgY) production in laying chickens.

Twenty 35-wk-old chickens, including 10 Single Comb White Leghorn (SCWL) and 10 Rhode Island Red (RIR) hens, were used to examine the effects of egg and yolk weights on egg yolk antibody (IgY) production in the two strains of chickens immunized with BSA. The SCWL chickens had a greater (P < 0.01) percentage hen-day production and greater egg and yolk weights than did the RIR chickens. However, the anti-BSA antibody activities determined by ELISA in the serum and the egg yolk were similar (P > 0.05) between the SCWL and RIR chickens. Similarities between the two strains of hens were also observed in protein and total IgY contents (expressed as the percentage of wet weight of yolk) and the percentage of BSA-specific antibody in the total IgY. It was concluded that both the SCWL and RIR chickens immunized with BSA can produce egg yolk IgY containing similar proportions of BSA-specific antibodies. Therefore, the egg yolk weight and the percentage hen-day production, both of which are greater in the SCWL hens, are considered to be important factors for the efficient production of IgY.

Isolation and characterization of proteoglycans from growing antlers of wapiti (Cervus elaphus).

Proteoglycans were extracted with 4 M guanidine-HCl from the zone of maturing chondrocytes, the site of endochondral ossification of growing antlers of wapiti (Cervus elaphus). Proteoglycans were isolated by DEAE-Sephacel chromatography and separated by Sepharose CL-4B chromatography into three fractions. Fraction I contained a high molecular mass (> 1000 kDa) chondroitin sulfate proteoglycan capable of interacting with hyaluronic acid. Its amino acid composition resembled that of the cartilage proteoglycan, aggrecan. Fraction II contained proteoglycans with intermediate molecular weight which were recognized by monoclonal antibodies specific to chondroitin sulfate and keratan sulfate. Fraction III contained a low molecular mass (< 160 kDa) proteoglycan, decorin, with a glucuronate-rich glycosaminoglycan chain.

Immune responses in chickens against lipopolysaccharide of Escherichia coli and Salmonella typhimurium.

Immunization of chickens with whole bacteria results in the production of antibodies specific to lipopolysaccharide (LPS), a major constituent of the outer membrane of Gram-negative bacteria. However, there is relatively limited information available concerning immune response of purified LPS in this species. In the present study, immune responses were examined in serum and egg yolk from two groups of chickens injected with entire LPS from Escherichia coli and lipid A free LPS from Salmonella typhimurium. The results demonstrated that the increase of antibody activity occurs first in serum, and then in egg yolk with a lag in time of 1 to 3 wk in both groups of chickens. However, the time of elevated levels of antibodies activity was much shorter in chickens immunized with S. typhimurium LPS (< 1 wk) than in those immunized with E. coli LPS (4 wk). A lack of lipid A is the S. typhimurium antigen may be a factor related to this difference.

Effects of dietary alpha-linolenic acid and strain of hen on the fatty acid composition, storage stability, and flavor characteristics of chicken eggs.

A study was conducted to determine the effect of dietary alpha-linolenic acid on the fatty acid compositions of egg yolk lipids, tocopherols, and internal quality of raw eggs during storage and the sensory characteristics of hard-boiled eggs from six different laying hen strains. Laying hens (total 300 birds, 72 wk old) from six strains (Rhode Island Red, Barred Plymouth Rock, New Hampshire, Light Sussex, Brown Leghorn, and White Leghorn) were distributed in 12 floor pens (2 pens per strain, 25 birds per pen) with male roosters. One of the pens for each strain was fed with tallow-based control diet and another was assigned with 3% alpha-linolenic acid (LNA) enriched diet with 120 U of mixed tocopherol/kg diet for 3 mo. Ten eggs from each pen were collected every day after 2 wk with the experimental diets, and stored in a cold room at 4 C up to 4 wk. Total lipids, fatty acid compositions, Haugh units, and tocopherols of egg yolk were determined once a week during the 4-wk storage periods. Sensory studies were also conducted using the eggs stored for 2 wk at 4 C. Dietary LNA increased the amount of n-3 fatty acids (6.5%) in total lipid, and over 70% was C18:3n3, and the rest was C22:6n3 (20 to 25%) and C22:5n3 (5 to 10%). Only minor differences in fatty acids among strains were observed. The differences and the changes in tocopherols during storage periods by strain and diet appeared randomly and lacked consistency.(ABSTRACT TRUNCATED AT 250 WORDS)