New application of indirect fluorescent antibody (IFA) technique using latex particles coupled with verotoxin 2 from Escherichia coli O157:H7 in order to determine colostral antibody titers in immunized dairy cows.

A simple and novel assay method for determining colostral and serum against soluble verotxin 2 (VT2) titers by indirect fluorescent antibody (IFA) assay using latex sensitized with VT2 was devised. The latex particles did not auto-fluoresce, and non specific reactions disappeared after washing with phosphate buffered saline containing 3 M Nacl. The highest titer measured by neutralizing test was observed at 1 day after delivery. The highest titer for each immunoglobulin class measured by enzyme-linked immunosorbent assay (ELISA) or IFA using latex sensitized with VT2 was also observed at 1 day after delivery. The changes in titer measured by each method showed similar patterns. Furthermore, the titers for IgG antibody were higher than those for IgM or IgA antibodies. Thus, the titers of bovine immune colostral antibody and each immunoglobulin class could be measured by IFA using latex sensitized with VT2.

Determination of antibody titers against soluble antigen by indirect fluorescent antibody (IFA) assay using latex particles coupled with soluble antigen: a preliminary study.

A method for indirect fluorescent antibody (IFA) assay for soluble antigen has been developed using latex particles (latex) as a carrier for soluble antigen. Two types of latex having grain sizes of 1.0 and 6.0 µm were used for IFA assay and were evaluated in this study. Bovine IgG and rabbit anti-bovine IgG antibody were used as soluble antigen and as primary antibody, respectively. Bovine IgG-sensitized latex on slide glass was not washed off after immersion for 45 min. IFA using latex appears to have high specificity, as no reactions between fluorescein isothiocyanate (FITC)-conjugated antibody and protein-sensitized latex were observed, and the latex did not show auto-fluorescence or non-specific reactions. Latex with a grain size of 6.0 µm was appropriate as a carrier of soluble antigen, as the amount of soluble antigen necessary for sensitization was approximately one-eighth that necessary for the small-diameter latex (1.0 µm), and large-diameter latex is easier to distinguish under the fluorescence microscope. IFA assay using soluble antigen-sensitized latex is suitable for more widespread use in the future.

Comparison of efficacies of bovine immune colostral antibody and each immunoglobulin class against verotoxin 2, flagellum and somatic cells of Escherichia coli O157:H7 in mice.

PURPOSE: The efficacy of bovine immune colostral (colostral) antibodies against verotoxin (VT) 2, flagellum and somatic cells of Escherichia coli (E. coli) O157:H7 in mice was determined. METHODS: Three major immunoglobulin (Ig) classes were isolated from the colostral antibody against VT2 by affinity chromatography and were used for estimation. Mice inoculated with VT2 were administered each Ig class from the colostral antibody, colostral antibody (colostral whey containing antibody) or serum antibody against VT2 at 1 hour after VT2 inoculation. RESULTS: All control mice (20/20) died after administration of sterilized saline instead of the colostral antibody. The survival rate was 93.3% (14/15) after administration of S-IgA or IgM antibody, or colostral antibody. Survival rates for IgG antibody and serum antibody administration were 80% (12/15) and 60% (9/15), respectively. Serum concentrations of VT2, which was absorbed from the small intestine in mice after administration of VT2 and colostral antibody, were measured by fluorescence enzyme immunoassay (FEIA). Serum concentrations of VT2 after administration of colostral antibody were lower than those after administration of sterilized saline. Mice inoculated with VT2-producing E. coli 157:H7 were administered anti-flagellum or anti-somatic colostral antibodies. Survival rates for E. coli O157:H7-infected mice administered the anti-flagellum and anti-somatic colostral antibodies were 52.4% (11/21) and 22.2% (4/18), respectively. Furthermore, survival rates increased to 89.5% (17/19) with combined administration of anti-flagellum and anti-VT2 colostral antibodies. CONCLUSION: These results suggest that colostral antibodies against VT2, flagellum and somatic cells are effective against E. coli O157:H7 infection.

Relationship between production of acute-phase proteins and strength of inflammatory stimulation in rats.

The relationship between intensity of inflammatory stimulation and production of α(2)-macroglobulin (α2M) and α(1)-acid glycoprotein (AAG) in rats was investigated. Sprague-Dawley rats were injected with turpentine oil at doses of 0.05, 0.2 or 0.4 mL/rat. Serum levels of α2M, interleukin (IL)-6 and cytokine-induced neutrophil chemoattractant-1 (CINC-1) were measured by enzyme-linked immunosorbent assay, and AAG was measured by single radial immunodiffusion. Peak serum levels of α2M and AAG in rats injected at 0.05 mL/rat were significantly lower than those at 0.2 or 0.4 mL/rat. However, no significant differences were observed for peak serum levels of these acute-phase proteins between 0.2 and 0.4 mL/rat. Furthermore, peak serum levels of IL-6 and CINC-1 in rats injected at 0.05 mL/rat were significantly lower than those at 0.2 or 0.4 mL/rat. Thus, the production of these acute-phase proteins has upper limits, even under increased strength of inflammatory stimulation in rats injected with turpentine oil.

The effects of interleukin-6 and cytokine-induced neutrophil chemoattractant-1 on α2-macroglobulin production in rats.

We investigated the effects of interleukin-6 (IL-6) and cytokine-induced neutrophil chemoattractant-1 (CINC-1) on α(2)-macroglobulin (α2M) production in rats. IL-6-rich and CINC-1-rich fractions were separated from serum obtained from rats 12 h after injection with turpentine oil using gel-chromatography. Sexual dimorphism was observed in the peak levels of α2M after injection of IL-6-, CINC-1-, or a mixture of IL-6-and-CINC-1-rich fractions. No significant differences in α2M levels were observed in males after injection with IL-6- or CINC-1-rich fractions and those injected with normal serum obtained from healthy rats (control). In contrast, serum levels of α2M, 6 to 120 h after injection of a mixture of IL-6- and CINC-1-rich fractions were significantly higher than in control rats. These results suggest that IL-6 and CINC-1 contribute to α2M production in rats only when IL-6 and CINC-1 act synergistically.

Carbonic anhydorase isoenzyme I (CA-I) concentration in feces and urine as a temporary marker of occult blood in beagle dogs.

This study was undertaken to investigate whether the concentration of carbonic anhydorase isoenzyme I (CA-I) in canine feces and urine is useful as a temporary marker of occult blood. Concentrations of CA-I were measured by enzyme-linked immunosorbent assay (ELISA). Fecal CA-I concentrations in 113 healthy beagle dogs (50 male and 63 female) of various ages ranged from 4.3 to 16.7 ng/g feces (mean; 7.0 +/- 2.9 ng/g feces). One milliliter of blood from 3 healthy beagle dogs was found to contain 1,047, 1,062 and 1,150 microg CA-I. The fecal CA-I concentrations of dogs receiving intragastric infusions of autologous blood (10 ml) were very low. However, the fecal CA-I concentrations of dogs receiving infusion of autologous blood (5 ml) into the ascending colon were very high. Detection of fecal CA-I would be useful for identifying dogs with hemorrhaging of the large intestine. Of 55 urinary samples collected from healthy beagle dogs by catheter, chemical tests for occult blood were negative in 44, but CA-I concentrations ranged from 1.8 to 12.6 ng/ml (mean; 6.9 +/- 5.4 ng/ml) by ELISA. The CA-I concentrations of the other 11 samples, which tested positive for occult blood on chemical testing, ranged from 41.2 to 525.0 ng/ml by ELISA. Although CA-I is not a specific marker of erythrocytes, CA-I may be used to detect occult blood in canine feces and urine until a specific immunological test kit using antibody for Hb is developed.

Neutralizing activity of bovine colostral antibody against verotoxin derived from enterohemorrhagic Escherichia coli O157:H7 in mice.

The neutralization efficacy of bovine colostral antibody against verotoxin (VT) 1 and 2 was investigated. Cows were immunized with VT1 or VT2 fourteen times at 7-day intervals. A colostral antibody exhibiting high titers was obtained from immunized cows. Survival rates were evaluated in mice administered VT1 or VT2, and those infected with Escherichia coli (E. coli) O157:H7 producing VT1 or VT2. Survival rates after VT1 administration were 100% in the single-administration group, 90% in the repeat-administration group, and 78.6% in the control group. Survival rates after VT2 were 75.0% in the single-administration group, and 100% in the repeat-administration group. All mice in the control group died. Colostral antibody and fosfomycin (FOM) in the colostral antibody group and FOM and skim milk in the control group were administered three times per day for 5 days to mice infected with E. coli O157:H7 producing VT1 or VT2. Survival rates after inoculation with E. coli O157:H7 producing VT1 were 80.0% in the colostral antibody group, and 63.6% in the control group. Survival rates after inoculation with E. coli O157:H7 producing VT2 were 83.3% in the colostral antibody group, and 20.0% in the control group. The survival rate in mice without treatment following inoculation with E. coli O157:H7 producing VT2 was 88.2%. The survival rates in mice infected with E. coli O157:H7 strains producing VT1 or VT2 improved after administration of this colostral antibody, which exhibited neutralization efficacy against VT.