Seroprevalence of IgG1 and IgG4 class antibodies against Mycobacterium avium subsp. paratuberculosis in Japanese population.

Mycobacterium avium subsp. paratuberculosis (MAP) is the established causative agent of Johne's disease in cattle and other ruminants, and it has also been speculated to be a putative etiological agent of several human autoimmune diseases. It is acknowledged that dairy products deriving from infected animals play a role (could be vehicles) in exposing humans to MAP. MAP could stimulate the human immune system by means of their complex antigen (in the case of lipids, multivalent antigens) and may modulate it, acting as adjuvant molecules such as Freund's complete adjuvant. The immune system might be abnormally stimulated by the constant presence of MAP antigens (for example, in the dairy products), and this might be particularly relevant in genetically predisposed individuals. However, there is limited understanding about the current human exposure to MAP. The present study analyzed the antibody recognition profile of MAP lipophilic antigens in a cohort of 126 healthy Japanese. We measured the serum levels of total immunoglobulin G (IgG) and subclasses targeting MAP surface antigens through ethanol vortex indirect enzyme-linked immunosorbent assay (EVELISA) by using serum absorbed with Mycobacterium phlei. Elevated IgG (especially IgG1 and IgG4) responses were observed in 14% of the sera. To assess the specificity of EVELISA, the same samples were analyzed by means of a commercially available Johnelisa II kit. It was noteworthy that a high degree of correlation was observed when comparing the two methodologies (rs=0.7, p<0.0001). Moreover, in order to investigate the specificity of the binding, inhibition assay experiments were carried out also searching for antibodies against Bacillus Calmette-Guérin antigens, but no cross-reaction was observed. The result obtained represents the first evidence implying that the Japanese population is exposed to MAP, and additionally the existence of a foodborne chain of exposure that transmits MAP antigens to humans.

Elimination Half-Lives of Acute Phase Proteins in Rats and Beagle Dogs During Acute Inflammation.

The half-lives of typical acute phase proteins in rats and beagle dogs during acute inflammation were investigated. Acute inflammation was induced by injection of turpentine oil in rats and administration of indomethacin in beagle dogs. Serum concentrations of α2-macroglobulin (α2M) and C-reactive protein (CRP) were measured by enzyme-linked immunosorbent assay and α1-acid glycoprotein (AAG) was measured by single radial immunodiffusion. Half-life was calculated as 0.693/elimination rate constant (K). The mean half-lives in the terminal elimination phase of α2M and AAG were 68.1 and 164.8 h, respectively. The half-life of AAG was significantly longer than that of α2M. Mean half-lives in the terminal elimination phase of CRP and AAG were 161.9 and 304.4 h, respectively. The half-life of AAG was significantly longer than that of CRP in beagle dogs. No significant differences in the half-life of AAG were observed between rats and beagle dogs. Furthermore, serum concentrations in the terminal elimination phase could be simulated with the K data acquired in this study.

Inhibition of verotoxin (VT) 2 absorption into systemic blood from intestine by repeated administration of bovine immune colostral antibody against VT2 in mice.

BACKGROUND/PURPOSE: Whether absorption of verotoxin (VT) 2 from the intestine in mice is inhibited by administration bovine immune colostral antibody against VT2 was investigated. METHODS: Three-week-old mice were administered VT2 solution at 477.8 ng/mL or 955.6 ng/mL, and bovine immune colostral antibody against VT2 was then administered three times. Whey without antibody against VT2 was administered to control mice. Serum levels of VT2 were measured by fluorescence enzyme immunoassay. RESULTS: Serum levels of VT2 in mice administered VT2 solution at 477.8 ng/mL and bovine immune colostral antibody against VT2 scarcely changed. By contrast, serum levels of VT2 in control mice increased and peaked 12 hours after administration. Peak values were 15.4 ± 5.04 ng/mL. Furthermore, serum levels of VT2 at 12 hours and 16 hours in control mice were significantly higher than in mice administered bovine colostral antibody against VT2. Serum levels of VT2 in mice administered antibody at 955.6 ng/mL showed no significant differences between repeated administration of bovine immune colostral antibody and controls. CONCLUSION: These results suggest that absorption of VT2 from the intestine was inhibited by repeated administration of bovine immune colostral antibody against VT2 at early stages of Escherichia coli O157:H7 infection, whereas VT2 in the intestine remained at low levels.

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.

Comparison of α2-macroglobulin synthesis by juvenile vs. mature rats after identical inflammatory stimulation.

Synthesis of α2-macoglobulin (α2M) by 3-week-old juvenile rats was compared to that of mature 7- and 11-week-old rats. Serum concentrations of α2M, interleukin (IL)-6- and cytokine-induced neutrophil chemoattractant (CINC)-1 were measured by enzyme-linked immunosorbent assay. The area under the concentration vs. time curve (AUC) for α2M was significantly different among the three groups. The synthesis of α2M increased in an age-dependent manner. No significant difference was observed for the AUC of IL-6, but that of CINC-1 in 3-week-old rats was significantly lower than that in 7- or 11-week-old rats. These results suggest that synthesis of α2M was increased in mature compared to juvenile rats, possibly due to differences in liver function. The maximum concentration of CINC-1 in 3-week-old rats was observed 6 h after turpentine oil injection. The serum concentrations of IL-6 and CINC-1 increased more quickly in juvenile rats than in mature rats after inflammatory stimulation.

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.

Impairment of α(2)-macroglobulin synthesis in experimental hepatopathic rats treated with turpentine oil.

The aim of this study was to investigate the synthesis of α(2)-macroglobulin (α2M) in hepatopathic rats injected with turpentine oil to induce acute inflammation. Hepatopathy was induced by oral administration of acetaminophen at a dose of 1 g/kg daily for 2 weeks or a 25% solution of carbon tetrachloride (CCl(4)) at 2 ml/kg body weight three times per week for 7 weeks. Acute inflammation was induced by intramuscular injection of turpentine oil at a dose of 1.0 ml/kg body weight. Serum concentrations of α2M were measured by enzyme-linked immunosorbent assay. Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and total protein differed significantly between acetaminophen or CCl(4)-induced hepatopathic rats and acetaminophen control (AA-control) or CCl(4) control (CC-control) rats. Furthermore, pathological examination confirmed hepatopathy in rat livers. Peak serum concentrations and area under the time-concentration curve for α2M showed significant differences between hepatopathic rats and AA-control or CC-control rats. Thus, serum concentrations of α2M did not increase when compared with nontreated rats.

Mycobacterium avium subsp. paratuberculosis lipophilic antigen causes Crohn's disease-type necrotizing colitis in Mice.

BACKGROUND: A 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced murine colitis model was developed to investigate the pathogenesis and to evaluate a method of treating human Crohn's disease. This experimental model rapidly induces colitis similar to human Crohn's disease lesion in a reproducible manner. However, natural exposure of the human digestive tract to TNBS is unrealistic. A novel animal model based on realistic data is eagerly anticipated in future research on pathogenesis of CD. METHOD: We evaluated the potency of Map antigen molecules in an effort to develop a novel colitis model using a more realistic source than TNBS. We prepared the Map antigen by ethanol extraction and developed a mouse model in a manner similar to that of the well-known TNBS-induced colitis in mice. In the experiment, seven days after subcutaneous (SC) injection of the antigen into normal C57BL/6 mice, the same antigen in 50% ethanol was injected into the colon by the transanal route with a fine cannula. RESULTS: On the fifth day after the transanal injection, histopathological examination revealed full-thickness necrotizing colitis with erosion and ulcers; severe infiltration with neutrophils, lymphocytes, macrophages, and perforation. However, no change was detected with each single Map-antigen injection. CONCLUSION: The present results provide a novel animal model for research on CD and may be the key to clarifying the relationship between CD and Map. This is the first evidence that mycobacterium antigen induces necrotizing colitis.

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.

Kinetics of {alpha}2-macroglobulin and {alpha}1-acid glycoprotein in rats subjected to repeated acute inflammatory stimulation.

The kinetics of alpha(2)-macroglobulin (alpha2M) and alpha(1)-acid glycoprotein (AAG) in rats repeatedly stimulated with intramuscular injections of turpentine oil at doses 0.05 and 0.4 mL/rat were investigated. Mean serum levels of alpha2M peaked at 48 h after the first turpentine oil injection, reaching 1.74 and 2.36 mg/mL at 0.05 and 0.4 mL/rat, respectively. AAG peaks were also observed at 48 h after injection, and the mean values were 2.02 and 2.53 mg/mL, respectively. These peak values of alpha2M and AAG differed significantly between the 0.05 and 0.4 mL/rat injection groups. Mean serum levels of interleukin-6 (IL-6) at 0.05 mL/rat were 52.61 pg/mL at 12 h, 48.86 pg/mL at 36 h and 81.93 pg/mL at 84 h after the first injection. Mean IL-6 serum levels at 0.4 mL/rat were 215.24 pg/mL at 12 h, 56.33 pg/mL at 36 h and 39.25 pg/mL at 84 h after the first injection. Mean serum levels of cytokine-induced chemoattractant-1 (CINC-1) at a dose of 0.05 mL/rat were 5.70 ng/mL at 12 h, 5.58 ng/mL at 36 h and 4.58 ng/mL at 84 h after the first injection. Mean serum levels of CINC-1 after injection at 0.4 mL/rat were 11.57 ng/mL at 12 h, 4.68 ng/mL at 36 h and 4.42 ng/mL at 84 h. Serum levels of IL-6 differed significantly at 12, 24, 72 and 84 h, while those of CINC-1 differed significantly at 12, 24, 48 and 96 h between the 0.05 and 0.4 mL/rat injection groups. Differences in peak serum levels in the 0.05 and 0.4 mL/rat groups were attributed to differences in the production of IL-6 and CINC-1, which are thought to contribute to alpha2M and AAG production.

Bovine colostral antibody against verotoxin 2 derived from Escherichia coli O157:H7: resistance to proteases and effects in beagle dogs.

A bovine colostral antibody against verotoxin (VT) 2 of Escherichia coli O157:H7 was administered orally to beagle dogs. The antibody remained in the dogs' small intestine for at least 2 h, whereas little serum antibody remained 1.5 h after administration. Furthermore, the antibody activity of secretory IgA did not change until 2 h after administration; however, the activity of IgG and IgM antibodies decreased by approximately 60% and 40% at 2 h after administration, respectively. Seven beagle dogs inoculated with Escherichia coli O157:H7 producing VT2 were administered bovine colostral antibody or bovine colostral whey without antibody. With administration of bovine colostral whey without antibody, the amount of VT2 in feces decreased gradually after administration and increased again at 5 d after inoculation, whereas bovine colostral antibody significantly reduced the amount of VT2 in feces on the day after administration. In addition, 9 beagle dogs were given bovine colostral antibody, bovine plasma antibody, or saline. The amount of VT2 in feces again decreased significantly more rapidly after administration of bovine colostral antibody than after administration of bovine plasma antibody or saline.

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.

Alpha 1-acid glycoprotein (AAG) levels in healthy and pregnant beagle dogs.

Serum alpha 1-acid glycoprotein (AAG) levels were measured in healthy beagles of various ages (66 male and 74 female) by turbidimetric immunoassay (TIA), and then separately--in pregnant beagles--by single radial immunodiffusion (SRID). The first experiment revealed that serum AAG levels ranged from 40 to 960 microg/ml (mean of 322 +/- 202 microg/ml) in male dogs, and from 47 to 833 microg/ml--in female dogs (mean of 316 +/- 199 microg/ml), without any significant sex- or age-related variation. The second experiment, however, revealed that serum AAG levels increased in all pregnant beagles and peaked in the middle of gestation at 250-1,000 microg/ml (mean of 634 +/- 246 microg/ml). In 7 of 8 dogs the AAG levels peaked about 45 days after ovulation. Despite a high value of 1,210-1,360 microg/ml being observed for serum AAG levels in 3 pregnant beagles inoculated with Staphylococcus aureus, its levels in umbilical cord blood were below the detection limit of SRID (40 microg/ml).

Determination of serum C-reactive protein (CRP) in healthy beagle dogs of various ages and pregnant beagle dogs.

Serum C-reactive protein (CRP) concentrations in healthy beagle dogs of various ages and in pregnant beagles were measured by enzyme-linked immunosorbent assay (ELISA). Serum CRP concentrations were 1.5-16.0 microg/ml (mean 7.9 +/- 3.4 microg/ml) in male, and 1.8-18.9 microg/ml (mean 8.3 +/- 4.0 microg/ml) in female dogs. No significant sex-related differences were observed in the values. Further, there were no significant age-related differences either. Serum CRP concentrations increased during pregnancy. The concentration of serum CRP in pregnant dogs peaked at 70.2-90.4 microg/ml (mean 77.5 +/- 7.1 microg/ml) 30 or 45 days after ovulation, demonstrating two characteristic features of CRP concentration change in pregnant dogs.

Analysis of 16S rRNA gene sequences of Ehrlichia canis, Anaplasma platys, and Wolbachia species from canine blood in Japan.

In the present study, three canine blood samples from Japan, that were suspected to be ehrlichia positive were examined. After sequencing the 16S rRNA genes, each dog was found to be infected either with Ehrlichia canis (Kagoshima 1), Anaplasma platys (Okinawa 1), or Wolbachia sp. (Okinawa 2). Phylogenic analysis was performed on these sequences. The nearly entire 16S rRNA sequence of Kagoshima 1 was found to be most similar to the sequences from Oklahoma and Venezuela E. canis strains (1 base pair difference out of 1,387, 99.9% sequence identity). The 16S rRNA gene sequence of Okinawa 1 showed the closest DNA identity to the French strain of A. platys (1 base deletion out of 1,385 bp, 99.6% sequence identity). The 16S rRNA gene sequence of Okinawa 2 illustrated the closest DNA identity to that of a Wolbachia sp. from Dirofilaria immitis (98.9% sequence similarity). These data imply a low diversity within E. canis strains and within A. platys strains, including those strains reported in this study. This is also the first demonstration of Wolbachia DNA in dog blood, suggesting the involvement of Wolbachia sp. in canine febrile illnesses.

Transplacental transport of alpha 2-macroglobulin (alpha 2M) and induction of alpha 2M in maternal and neonatal rats with acute inflammation.

The aims of this study were to investigate transplacental transport of alpha 2-macroglobulin (alpha 2M) in rats in rats and to examine the degree of alpha 2M induction in maternal and neonatal rats with acute inflammation. Serum was collected from healthy pregnant CD (IGS) rats, neonates of the pregnant rats and their cord blood. Additional serum samples were obtained from pregnant rats inoculated with an inflammatory agent, turpentine oil, their neonates and cord blood, and neonates inoculated with turpentine oil. The serum levels of alpha 2M were measured by means of an enzyme-linked immunosorbent assay. The average serum levels of alpha 2M in healthy neonates and cord blood were about 380 micrograms/ml. Serum a2M level in neonates inoculated with turpentine oil averaged about 580 micrograms/ml. Serum alpha 2M levels in maternal rats inoculated with turpentine oil, neonates from those rats and their cord blood were elevated, the values being 2,000 micrograms/ml or higher. It was demonstrated that induction of alpha 2M in neonatal rats was lower than in maternal rats when inoculated with turpentine oil. These results suggest that alpha 2M is transplacentally transported from maternal rats to fetal ones.