Systemic availability of bovine immunoglobulin G and chicken immunoglobulin Y after feeding colostrum and whole egg powder to newborn calves

In connection with a study on the prophylaxis of infectious diarrhea with specific egg yolk antibodies, the systemic availability of colostral bovine immunoglobulin G (bIgG) and chicken immunoglobulin Y (IgY) after feeding egg powder was investigated on 26 newborn calves from 23 different farms. Blood was sampled daily and at the same day time from these calves in the first 14 days of life. During the feeding of colostrum, the mean bIgG concentration was highest at day 1 post natum with a value of 9.3 mg/ml serum. Thereafter, the mean bIgG level was reduced continuously to a significant lower concentration of 4.9 mg/ml serum at day 12 post natum and remained nearly constant at 5.2 mg/ml till to the end of the observation period. Total protein concentrations in the serum did not change and plateaued at a mean value of 56.2 mg/ml (SD 11.2). The number of colostrum meals had no significant effect on the mean bIgG concentrations during that period. The individual variation of bIgG concentrations was very high on every day of the sampling period. The mean coefficient of variation was at 52.1 % (SD 5.7). After having described the individual bIgG concentration curves mathematically with a regression curve, two groups with significantly different bIgG elimination constants (k) could be obtained. Thus in one group (n = 10) with k-values of < -0.02 a mean half time of serum bIgG of 24.3 days (SD 4.6) was calculated. In the other group of calves (n = 16) with elimination constants of k > -0.02, a mean half time of 68.5 days (SD 36.7) could be calculated, possibly because these calves started earlier with their endogenous bIgG production. Additionally, to 18 of these calves 20 g egg powder with an IgY concentration of 15 mg/g was fed up to day 14. Calves had a maximal mean IgY concentration of 1.9 micrograms/ml serum if egg powder feeding started already during the first 12 hours of life. Starting at a later time resulted in a significant reduction of IgY levels. For example, the mean initial IgY concentration dropped to 0.035 micrograms/ml serum after having had the first egg powder application between 25 and 48 hours post natum. Using the individual IgY elimination constant derived from a regression analysis (r2 = 0.84) of the IgY concentration curve, a mean IgY half time of 5.0 days (SD 2.5) could be calculated. To prevent the absorption of heterologous antibodies and consecutively, also to prevent a possible systemic effect, egg powder for prophylactic purposes in newborn calves should be fed after the first 24, better 48 hour, post natum. Most important for the prophylactic effect of specific antibodies on infectious diarrhea is not their systemic but their high local intestinal availability.     

Serum immunoglobulin G concentration in goat kids fed colostrum or a colostrum substitute

To determine the suitability of a new colostrum substitute derived from goat serum and to determine the amount of colostral IgG needed to achieve serum IgG concentration > 800 mg/dl, twin kids from 14 does were fed colostrum or a colostrum substitute. The volume of colostrum or colostrum substitute fed was calculated so that half the kids in each group received IgG at a low dosage (1.5 g/kg of body weight) and the other half received IgG at a high dosage (3 g/kg). Kids were bottle fed the colostrum or colostrum substitute and then fed pooled goat's milk until 18 hours old, at which time they were allowed to nurse their dams. Does were milked manually every 2 hours after parturition until specific gravity of mammary secretions was < 1.02, the specific gravity of goat's milk. Serum IgG concentration of each kid was determined by means of single radial immunodiffusion at birth and 12, 18, and 24 hours and 7, 21, and 42 days after birth. Kids were weighed at each blood collection and monitored for illness daily. None of the kids had measurable serum IgG concentrations at birth. Mean serum IgG concentration was significantly higher in kids fed colostrum than in kids fed colostrum substitute at all times, except days 7 and 42 (P < 0.05). By 24 hours after birth, serum IgG concentration was > 800 mg/dl in all kids fed colostrum, in 4 of 7 kids fed the substitute at the higher dosage, and in 2 of 7 kids fed the substitute at the lower dosage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antigenic crossreactivity and lipopolysaccharide neutralization properties of bovine immunoglobulin G

We investigated a possible mechanism by which immunization against core and lipid A determinants of lipopolysaccharide reduced clinical cases of mastitis and symptoms commonly associated with heterologous Gram-negative IMI. The IgG fraction of sera from cows immunized with either Escherichia coli J5 bacterin, E. coli J5 lipopolysaccharide conjugate vaccine, or unimmunized controls was purified by precipitation with caprylic acid and ammonium sulfate. The degree of IgG crossreactivity with Gram-negative bacteria that were isolated from clinical quarters was greater than that with Gram-positive isolates of Staphylococcus aureus. The highest magnitude of crossreactivity was against smooth strain E. coli isolates, followed by heterologous species of Enterobacter, Serratia, and Klebsiella isolates. Serum IgG from cows immunized with conjugate was highly crossreactive to E. coli J5, E. coli O111:B4, Serratia marcescens, Klebsiella pneumoniae, and Salmonella typhimurium lipopolysaccharides. The magnitude of antibody crossreactivity with lipopolysaccharides coincided with the ability of IgG to suppress the mitogenic effect of lipopolysaccharides on bovine lymphocytes.     

Prophylactic effect of bovine anti-Cryptosporidium hyperimmune colostrum immunoglobulin in healthy volunteers challenged with Cryptosporidium parvum

Bovine hyperimmune anti-Cryptosporidium colostrum immunoglobulin (BACI) decreases the intensity of Cryptosporidium parvum infection in vitro. We investigated the prophylactic effect of BACI in healthy adults challenged with C. parvum. After we established an oocyst dose that resulted in 100% infection in four volunteers (baseline group), 16 volunteers were randomized to receive (1) BACI prior to C. parvum challenge (BACI group) and a nonfat milk placebo 30 minutes later, (2) BACI prior to and 30 minutes after challenge (reinforced BACI group), or (3) nonfat milk placebo prior to and 30 minutes after challenge. Subjects received BACI (10 g) or nonfat milk placebo three times a day for a total of 5 days and were followed for clinical symptoms and oocyst excretion for 30 days. A trend toward less diarrhea (P = .08) was observed for subjects receiving BACI in comparison with occurrences in placebo recipients. Subjects receiving BACI or nonfat milk placebo had a 100-fold reduction in oocyst excretion as compared with excretion in the baseline group.     

Thermal inactivation of Mycobacterium paratuberculosis in milk

Thermal inactivation of Mycobacterium paratuberculosis, a suspected human pathogen, was determined in ultrahigh-temperature whole milk. Three strains of M. paratuberculosis were examined for survival at temperatures from 55 to 75 degrees C using a submerged glass capillary tube method. Clumped and declumped suspensions of the cultures were used to determine the rate of heat inactivation and survival at pasteurization temperatures. Methods for declumping M. paratuberculosis included the use of glass beads, vortexing, and passing the cells through a 26-gauge needle. The latter procedure was found to be superior over other methods and did not affect the viability of cells. Capillary tubes filled with milk containing 4 x 10(6) to 3 x 10(7) CFU/ml were heated at temperatures ranging from 55 to 75 degrees C. At 55 degrees C, minimal thermal inactivation was observed for clumped and declumped cells. At 58 degrees C, thermal inactivation ranging from 0.3 to 0.7 log reduction was observed for both clumped and declumped suspensions. D values at 60 degrees C ranged from 8.6 to 11 min and 8.2 to 14.1 min for clumped and declumped cells, respectively. At 63 degrees C, the D values ranged from 2.7 to 2.9 and 1.6 to 2.5 min for clumped and declumped cells, respectively. Survival of M. paratuberculosis at initial levels ranging from 44 to 10(5) CFU/ml at pasteurization treatment (63 degrees C for 30 min and 72 degrees C for 15 s) was also determined. No survivors were observed after incubating plates for up to 4 months on Middlebrook 7H11 agar and up to 2 months on Herrold's egg yolk medium. The sensitivity of the plating method was 1 CFU/250 microliters. These results demonstrate that low levels of M. paratuberculosis, as might be found in raw milk, will not survive pasteurization treatments.     

Effects of a colostrum replacement product derived from serum on immunoglobulin G absorption by calves

Calves are born hypogammaglobulinemic and rely on immunoglobulin (Ig) from colostrum to obtain passive immunity. Previous research has indicated that colostrum supplements derived from milk are less effective than is maternal colostrum in providing adequate IgG to neonatal calves. Our objective was to determine the absorption of IgG by newborn calves fed a USDA food-grade colostrum supplement derived from bovine serum or fed pooled maternal colostrum. Holstein calves (n = 20; 10 bulls) were removed from the dam within 1 h of birth and were housed in individual stalls for the 24-h study. Calves were fed 2 L of colostrum or colostrum replacer at 1.5 and 13.5 h (+/- 0.1 h). Calves were blocked by colostrum pool, and replacer was fed to provide equal intakes of IgG within blocks. Jugular blood was collected at 1 and 24 h (+/- 0.1 h) for analysis of IgG by radial immunodiffusion. At 24 h, calves were injected with 1.5 ml of Evans blue dye to estimate plasma volume. Mean plasma IgG at 24 h of age was 7.3 +/- 0.4 g/L and was affected by an interaction of block and treatment. Apparent efficiency of IgG absorption of 24 h was reduced when 750 g of the colostrum replacement product were fed but was increased when 266 g of colostrum replacement product were fed. Mean plasma volume was unaffected by treatment and was 3.5 +/- 0.2 L or 9.1% of BW. These data indicate that efficiency of IgG absorption from the colostrum replacement product may be affected by amount of material fed. Proteins other than IgG in the colostrum replacement product might have reduced the efficiency of IgG absorption.     

Development of an automated turbidimetric immunoassay for quantification of bovine serum immunoglobulin G

OBJECTIVE: To develop an automated turbidimetric immunoassay (TIA) for measurement of bovine IgG. SAMPLE POPULATION: 24 bovine serum samples. PROCEDURE: IgG concentration was determined by use of the TIA, and results were compared with those of the radial immunodiffusion (RID) method. Variables were determined, using commercially available reagents and a clinical biochemical analyzer. For the TIA, polyclonal goat anti-bovine IgG (Fc specific) serum, bovine IgG calibrator serum, and polyethylene glycol reaction buffer were used. Sample concentrations were determined by the instrument, using the linear regression method of least squares. The accuracy of this assay was validated by referencing to a purified bovine IgG standard and by recovery of control standards. Parallelism was documented by assay linearity and serial sample dilution linearity. Interference resulting from hemolyzed samples was examined. RESULTS: The TIA method correlated positively (r = 0.9957) and significantly (P < 0.05) with the RID method, yielding a regression equation with slope of 0.78708 and y-intercept of 1.02102. Bias attributable to hemolysis was not observed. CONCLUSIONS: The TIA method is automated, accurate, and precise for bovine serum IgG quantification. CLINICAL RELEVANCE: This assay provides sample results in approximately 10 minutes and may be used as an alternative to the manual RID method.     

Survival of T. pallidum under microaerobic conditions in cell and tissue cultures

The survival of pathogenic T. pallidum was studied under microaerobic conditions (partial pressure of oxygen 7 mm Hg) with and without tissue cell cultures. As between one third and half of the germs were motile even after 96 hours of incubation, treponemes do not seem to be strictly anerobic as has been generally believed so far. Flocks containing enormous numbers of treponemes were observed in the nutrient medium. For their formation, human serum, testicular components from rabbits and modified Nelson's survival medium were essential. Although the problem of cultivation of T. pallidum remains to be solved the formation of flocks indicates a possible transient multiplication of treponemes in vitro.     

Dynamics of submerged growth of Mycobacterium tuberculosis under aerobic and microaerophilic conditions

When Mycobacterium tuberculosis is grown in detergent-containing medium under continous agitation, multiplication is known to follow a logarithmic mode. When the cultures are not continuously shaken, but only agitated a few times a week to resuspend the bacilli and permit turbidity to be measured, the net increase suggests an arithmetic growth mode. It is shown here that a single pulse of aeration of an unshaken submerged culture of M. tuberculosis causes an almost instantaneous acceleration of growth, followed rapidly by a cessation of growth. Whether or not the bacilli will subsequently resume growth depends on the bacillary population density of the cuture at the time of application of the pulse of aeration. If the bacilli are permitted to grow in the depths of Dubos Tween Albumin broth without any agitation, they exhibit net arithmetic growth and attain a maximal population density greater than is seen in cultures exposed to occasional pulses of aeration. By the use of isotopically labeled cells, it has been shown that replication occurs ar a logarithmic rate amoung the small proportion of the bacilli that remain suspended in nonagitated cultures. This replication is balanced by settling of cells, resulting in a net appearance of arithmetic multiplication. The cells that have settled into the sediment replicate at a very slow rate, if at all, but do retain their viability for 4 weeks or longer. This suggests a possible analogy to quiescent tubercle bacilli in vivo.     

Microstructural evolution of austenite under conditions simulating thin slab casting and hot direct rolling

Abstract

The hot direct rolling (HDR) of thin slabs introduces some new microstructural phenomena with respect to conventional hot rolling of steels. This paper aims to investigate the microstructural changes of as cast austenite under these conditions. Current laboratory techniques for HDR simulation require a freshly cast slab for every experiment and a perfect link between casting and hot deformation. The present work adopted a new approach; the C–Mn steel is substituted by austenitic Fe–30Ni alloy, Conventional reheating before rolling replaces the direct link. The experimental ingot casting of Fe–30Ni alloy resulted in a solidification structure in good agreement with that of thin slabs of C–Mn steels. From metallographic observations, a mixed softening process and a strong grain refinement and homogenisation characterise the microstructural changes during HDR simulation. The microstructural behaviour and the grain refinement measured for the Fe–30Ni alloy is closely comparable with that predicted for C–Mn steels for the same conditions. The steel substitution appears to constitute a suitable and advantageous experimental approach for HDR simulation.     

Diagnosis of paratuberculosis in dairy cattle, using enzyme-linked immunosorbent assay for detection of antibodies against Mycobacterium paratuberculosis in milk

An ELISA containing lipoarabinomannan (LAM) antigen was used to detect antibodies in milk and serum for diagnosis of Mycobacterium paratuberculosis infection in dairy cattle. In experiment 1, milk and serum samples were obtained from 25 cows, and subjected to LAM ELISA testing immediately, and after 1 year of storage at -70 C. Milk samples, with and without a commonly used chemical preservative, were tested. There was no significant difference in LAM ELISA results between fresh and frozen samples or between preserved and unpreserved milk samples. In experiment 2, milk samples were collected daily from 30 cows over a 14-day period. The day-to-day coefficient of variation was 0.19 for milk LAM ELISA and was 0.15 for serum LAM ELISA, with no statistically significant time effect detected. In experiment 3, single milk, serum, and fecal samples were obtained from 764 cows. The fecal samples were cultured for M paratuberculosis to identify infected cows, and the serum and milk samples were subjected to LAM ELISA testing. Results were compared, using the area under the receiver operating characteristic curves. The milk LAM ELISA had specificity (+/- 95% confidence limits) of 87 +/- 8.1% when the cutoff was set at 50% sensitivity, and specificity of 83 +/- 9.1% when sensitivity was set at 60%. The area under the receiver operating characteristic curve was 0.85 +/- 0.03 for the milk ELISA and 0.75 +/- 0.02 for the serum ELISA. In this population of cattle, the milk LAM ELISA had comparable accuracy to serum LAM ELISA, although the milk LAM ELISA was slightly less reproducible (higher coefficient of variation).     

Molecular dynamics simulation of glycoprotein-glycans of immunoglobulin G and immunoglobulin M

In this paper, the authors introduce a workflow model. The development of computer network technology enables us to share the distributed data in real time. It is a considerable significance in the practical application of network capabilities not only to office work but also to the medical environment. In order to construct a well-connected, managed post (environment, scene), a model is needed to design the workflow. Here we propose a workflow model to cope with the scene of unforeseen events that we usually encounter in daily clinical activities. We give careful consideration to the ability of this model to manage dynamic changes within the workflow and describe its application to a medical scene (triage) and then carry out simulations based on this model. The authors are able to demonstrate the validity of this model through this simulation.     

Identification and characterization of a novel extracellular ferric reductase from Mycobacterium paratuberculosis

Diaspirin cross-linked hemoglobin (DCLHb) is an intramolecularly cross-linked hemoglobin-based oxygen carrier being developed as a therapy for acute blood loss. We report here the absence of immunogenicity of DCLHb in patients enrolled in phase II and III clinical trials of DCLHb. Two very sensitive immunoassays, an enzyme-linked immunosorbent assay (ELISA) and a Western blot assay, were developed and validated for this assessment. The DCLHb-antibodies used in these assays were raised in monkeys, had similar affinities for DCLHb and native human hemoglobin (SFHb), and showed cross-reactivity for subunits of DCLHb and SFHb on the Western blot, suggesting that these antibodies were elicited as a xenogenic response to the protein. In the ELISA, the optical density of a patient sample exposed to DCLHb-coated wells was compared with that of the patient sample exposed to carbonate buffer-coated wells; an optical density ratio of 1.4 was established for discriminating between a positive (reactive) or negative DCLHb antibody response. To date, all of the more than 300 patient specimens (preinfusion and postinfusion) from clinical trials have exhibited a ratio of less than 1.4, confirming the lack of preexisting antibodies to DCLHb and clearly showing the absence of DCLHb antibodies after exposure to this new biologic entity. There has been no requirement for use of the confirmatory Western blot assay. Taken together, the results from this study indicate DCLHb is not immunogenic in humans at doses evaluated clinically.     

Reduction in heat-induced gastrointestinal hyperpermeability in rats by bovine colostrum and goat milk powders

Male Sprague-Dawley rats were assigned to one of three dietary groups [standard diet (Cont; n = 8), standard diet plus bovine colostrum powder (BColost 1.7 g/kg; n = 8), or goat milk powder (GMilk 1.7 g/kg; n = 8)] to determine the ability of these supplements to reduce gastrointestinal hyperpermeability induced by heat. Raising core body temperature of rats to 41.5 degrees C increased transfer of (51)Cr-EDTA from gut into blood 34-fold relative to the ambient temperature value (P < 0.05) in the Cont group of rats, indicative of increased gastrointestinal permeability. Significantly less (P < 0.01) (51)Cr-EDTA was transferred into the blood of rats in either the BColost (27% of Cont) or GMilk group (10% of Cont) after heating, showing that prior supplementation with either bovine colostrum or goat milk powder significantly reduced the impact of heat stress on gastrointestinal permeability. The changes in the BColost group were not significantly different than those of the GMilk group. The potential mechanism of the protective effect of bovine colostrum and goat milk powders may involve modulation of tight junction permeability, because both powders were able to maintain transepithelial resistance in Madin Darby canine kidney cells challenged with EGTA compared with cells maintained in media only. The results show that bovine colostrum powder can partially alleviate the effects of hyperthermia on gastrointestinal permeability in the intact animal. Moreover, goat milk powder was equally as effective as bovine colostrum powder, and both may be of benefit in other situations where gastrointestinal barrier function is compromised.     

Survival of fish-virulent strains of Photobacterium damselae subsp. damselae in seawater under starvation conditions

A bacterial complementation assay has been developed for the rapid screening of a large number of compounds to identify those that inhibit an enzyme target for structure-based inhibitor design. The target enzyme is the hypoxanthine phosphoribosyltransferase (HPRT). This enzyme has been proposed as a potential target for inhibitors that may be developed into drugs for the treatment of diseases caused by several parasites. The screening assay utilizes genetically deficient bacteria complemented by active, recombinant enzyme grown in selective medium in microtiter plates. By comparing absorbance measurements of bacteria grown in the presence and absence of test compounds, the effect of the compounds on bacterial growth can be rapidly assayed. IC50 values for inhibition of bacterial growth are a reflection of the ability of the compounds to bind and/or inhibit the recombinant enzyme. We have tested this bacterial complementation screening assay using recombinant HPRT from the parasites. Plasmodium falciparum and Trypanosoma cruzi, as well as the human enzyme. The results of these studies demonstrate that a screening assay using bacterial complement selection can be used to identify compounds that target enzymes and can become an important part of structure-based drug design efforts.