Enzymatic activity of albumin shown by coelenterazine chemiluminescence.

Bioluminescence, the emission of light from live organisms, occurs in 18 phyla and is the major communication system in the deep sea. It has appeared independently many times during evolution but its origins remain unknown. Coelenterazine bioluminescence discovered in luminous jellyfish is the most common chemistry causing bioluminescence in the sea, occurring in seven phyla. Sequence similarities between coelenterazine luciferases and photoproteins from different phyla are poor (often < 5%). The aim of this study was to examine albumin that binds organic substances as a coelenterazine luciferase to test the hypothesis that the evolutionary origin of a bioluminescent protein was the result of the formation of a solvent cage containing just a few key amino acids. The results show for the first time that bovine and human albumin catalysed coelenterazine chemiluminescence consistent with a mono-oxygenase, whereas gelatin and haemoglobin, an oxygen carrier, had very weak activity. Insulin also catalysed coelenterazine chemiluminescence and was increased by Zn(2+). Albumin chemiluminescence was heat denaturable, exhibited saturable substrate characteristics and was inhibited by cations that bound these proteins and by drugs that bind to human albumin drug site I. Molecular modelling confirmed the coelenterazine binding site and identified four basic amino acids: lys195, arg222, his242 and arg257, potentially important in binding and catalysis similar to naturally occurring coelenterazine bioluminescent proteins. These results support the 'solvent cage' hypothesis for the evolutionary origin of enzymatic coelenterazine bioluminescent proteins. They also have important consequences in diseases such as diabetes, gut disorders and food intolerance where a mono-oxygenase could affect cell surface proteins.

Comparison of red clover and ryegrass silage for dry cows and influence on subsequent lactation performance.

The objective of this study was to investigate the use of red clover (RC) silage as a forage for dry dairy cows, primarily relative to its impact on tissue mobilization and repletion during the transition period and performance during the first 10 wk of lactation. Forty multiparous lactating Holstein-Friesian dairy cows were divided into 2 paired groups at 70 d before predicted calving dates; a subset (n = 8) of the cows were used for N and P balance measurements twice during the study. From the start of the experiment until 4 wk before predicted calving date all cows were offered ad libitum access to a ryegrass (RG) silage with no concentrate. At 4 wk before predicted calving date, one group of cows remained on the same diet, and the other group was changed to a diet of ad libitum access to RC silage. There was no difference in feed intakes, but CP intake was higher in cows fed RC silage, whereas ME intake was higher in cows fed RG silage. Cows fed RG silage gained more weight over the last 4 wk of the dry period (DP) than those fed RC silage, but there was no treatment effect on BCS. During the DP fecal N excretion was higher for cows fed RC silage, and there were no treatment differences in urine N excretion or overall N balance. At birth, calves from cows fed the RC silage were heavier. After calving, all cows were offered the same diet of ad libitum access to the same RG silage and a standard lactation concentrate. During the first 10 wk of lactation there was no difference in feed intake between the 2 previous treatment groups, and feed intake reached a maximum at approximately 4 wk of lactation. Cows on the RG treatment during the DP gained more longissimus dorsi muscle depth during the DP and retained it during early lactation. Mobilization of this muscle occurred before calving, indicating repartitioning of amino acids to other body tissues. There were no carryover effects of DP treatment on apparent partitioning of N from diet to milk, urine, or feces at wk 3 of lactation. Feeding RC silage during the DP had almost no impact on subsequent performance of dairy cows in early lactation, probably because the 2 silages were nutritionally very similar.

Effects of dietary sulphur sources on concentrations of hydrogen sulphide in the rumen head-space gas of dairy cows.

Three change-over design experiments investigated the origin of hydrogen sulphide in the rumen head-space gas of dairy cows, comparing the effects of single iso-S additions of methionine, cysteine and sodium sulphate, as well as the effects of single meals of fresh ryegrass or white clover. The concentration of hydrogen sulphide in rumen gas declined close to zero within 4 h after withdrawal of the previous feed. Sulphur sources were then given to cows and concentrations of hydrogen sulphide recorded in rumen head-space gas at 30-min intervals. Cysteine addition (8 g) led to a rapid (within 30 min) and a large (490 and 957 p.p.m. respectively in two experiments) increase in hydrogen sulphide concentration. Concentrations were significantly less following methionine addition. Increasing levels of cysteine addition led to significant increases in hydrogen sulphide concentrations ( P < 0.001 for the linear effect), although peak hydrogen sulphide was delayed and concentrations remained higher for longer with the highest (12 g) addition of cysteine ( P < 0.01 for the 'cysteine level' × 'time' interaction). The increase in concentration of hydrogen sulphide from sodium sulphate was smaller (230 p.p.m.) and slower (2 h) than for cysteine. Despite the much higher intake of cystine for white clover in comparison with perennial ryegrass ( P < 0.001), there was almost no increase in hydrogen sulphide concentration in rumen head-space gas from cows fed white clover. It seems likely that this is associated with the use of sulphur to produce thiocyanate to detoxify the hydrogen cyanide from cyanogenic white clover.

Effects of dairy cow diet forage proportion on duodenal nutrient supply and urinary purine derivative excretion.

Four mature Holstein-Friesian dairy cows were used in a 4 x 4 Latin square change-over design experiment made up of four 4-wk periods to investigate the relationship between microbial protein flow to the duodenum and excretion of purine derivatives (PD) in the urine. Four dietary treatments based on ad libitum access to ryegrass silage were offered, with a standard dairy concentrate included at different forage:concentrate (F:C) ratios, calculated on a dry matter basis: 80:20, 65:35, 50:50, and 35:65. Feed intakes increased as the proportion of concentrate in the diet increased, despite a concurrent decrease in silage intake. Increased feed intake led to increased nutrient flow to the duodenum. Milk yields increased as the diet F:C ratio decreased, with cows offered the 35:65 diet yielding nearly 8 kg/d more milk than cows offered the 80:20 diet; the concentrations of milk fat decreased and milk protein increased with a decreasing F:C ratio. Purine derivative excretion in the urine increased with an increasing proportion of concentrate in the diet, and there was a strong linear relationship between total PD excretion (allantoin and uric acid) and microbial N flow to the duodenum: microbial N (g/d) = 19.9 + 0.689 x total PD (mmol/d); R = 0.887. This strengthens the case for using PD excretion as a noninvasive marker of microbial protein flow from the rumen in dairy cows.

Tannerella forsythia-induced alveolar bone loss in mice involves leucine-rich-repeat BspA protein.

Tannerella forsythia (formerly Bacteroides forsythus) is one of the periodontal pathogens recently implicated in the development of periodontal disease. The cell-surface-associated, as well as the secreted, leucine-rich-repeat protein (BspA) of this bacterium have been suggested to play roles in bacterial adherence, and also in inflammation, by triggering release of pro-inflammatory cytokines from monocytes and chemokines from osteoblasts, leading to inflammation and bone resorption. In this study, we sought to determine the pathogenic potential of T. forsythia and the in vivo role of the BspA protein in pathogenesis in the mouse model of infection-induced alveolar bone loss. The results showed alveolar bone loss in mice infected with the T. forsythia wild-type strain, whereas the BspA mutant was impaired. In conclusion, evidence is presented in support of T. forsythia as an important organism involved in inducing alveolar bone loss, and the BspA protein is an important virulence factor of this bacterium.

Comparison of grass and legume silages for milk production. 2. In vivo and in sacco evaluations of rumen function.

Two experiments were conducted to investigate the basis for higher voluntary intakes and increased alpha-linolenic acid content in milk from cows offered clover silages. Six cows with rumen and duodenal cannulae were used in a four-period changeover-design experiment. Cows received 8 kg/d of dairy concentrate and had ad libitum access to one of six silage treatments: grass, red clover, white clover, alfalfa, and 50/50 (dry matter basis) mixtures of grass with red clover or white clover. The rumen fermentability of grass, red clover, white clover, and grass/red clover silages was also evaluated in a nylon bag study. Legume silages led to increased dry matter intake and milk production in comparison with grass silage. There was no significant effect of legume silages on rumen pH and volatile fatty acid concentrations, but a significant increase in rumen ammonia concentration with the legume silages, reflecting their higher protein content. The inclusion of white clover or alfalfa silage, but not red clover silage, in diets led to an increase in molar proportions of isobutyric, iso-valeric, and n-valeric acids in comparison with diets based on grass silage. Rumen fill was significantly lower, and rumen passage rates were significantly higher for cows offered alfalfa or white clover silages. However, the markedly different particle size distribution of rumen contents with these feeds suggests very different mechanisms for the high intake characteristics: high rates of particle breakdown and passage with alfalfa, and high rates of fermentation and passage with white clover. Microbial energetic efficiency (grams microbial N per kilogram organic matter apparently digested in the rumen) was highest for cows offered alfalfa silage, intermediate for clover silage, and lowest for cows offered grass silage. These differences reflect the higher rumen outflow rates for legume silages in comparison with grass silage. However, the effect of these differences on N-use efficiency (feed to milk) was probably quite small in comparison with effects of N intake. Although the biohydrogenation of alpha-linolenic acid was still high for red clover silage (86.1% compared with 94.3% for grass silage), there was a 240% increase in the proportion of alpha-linolenic acid passing through the rumen. This explains the increased recovery of alpha-linolenic acid from feed into milk with diets based on red clover silage.

Effects of level of concentrate feeding during the second gestation of Holstein-Friesian dairy cows. 2. Nitrogen balance and plasma metabolites.

We offered 43 Holstein-Freisian dairy heifers that calved for the first time at either 2 or 3 yr of age ad libitum access to ryegrass silage and a standard concentrate allowance of either 2 or 7 kg/d from the middle to the end of their first lactation. All animals were given the same relatively poor quality dry period diet of a mixture of ryegrass silage and barley straw (63:37 dry matter basis) from 6 wk before their predicted second-calving date. Following their second calving, all animals received access to the same ration of ad libitum grass silage and concentrates at a rate of 8 kg/d to 120 d of lactation and 5 kg/d thereafter until the end of the experimental recording at about 150 d of lactation. Nitrogen balance was significantly higher at the end of the first lactation for animals that were given the higher concentrate allowance and tended to be higher for older animals. There was no effect of age or residual effect of concentrate allowance on N balance during the dry period or during the second lactation. Labile body protein reserves, as estimated by the depth of the muscle Longissimus dorsi (which was significantly correlated with body condition score), were similar for all animals during the dry period, but younger animals previously offered the lower concentrate allowance did not lose L. dorsi depth early in the second lactation as did other animals. Arterial plasma concentrations of amino acids Phe, Trp, and Leu were significantly higher in younger animals at wk 8 of the second lactation, and Gly was significantly lower, although mammary blood flow, arteriovenous differences, and rates of uptake of the AA measured were unaffected by treatment. It is concluded that differences in second-lactation milk yield were not mediated through the availability of labile body protein or the supply of nutrients to the mammary gland.

Assessment of rumen processes by selected-ion-flow-tube mass spectrometric analysis of rumen gases.

This work investigated the potential to use measurement of the concentration of certain gases in the rumen headspace to gain information about rumen processes and as a potential diagnostic tool. We used new equipment (selected-ion-flow-tube mass spectrometer) that allows rapid and precise analysis of many of the gases present in a sample. Samples of rumen headspace gas and corresponding samples of rumen liquor were taken from three lactating cows, prepared with rumen fistulae, at intervals after receiving their morning feed allocation (grass silage and concentrates). Hydrogen sulfide, methyl sulfide, and dimethyl sulfide, were the predominant gases that were measured in the rumen headspace by this technique. The concentrations of these sulfur compounds declined over the interval after feeding, mirroring ammonia concentrations measured in rumen liquor, reflecting their common dependence on the fermentation of sulfur amino acids. Ammonia concentrations in rumen headspace gas varied in the opposite direction to the concentration of ammonia in rumen liquor and likely depend more on the pH of rumen liquor. Consideration of the pKa of ammonia suggests that ammonia concentrations in rumen gas will be very low below pH 6, representing a useful diagnostic for subacute ruminal acidosis. Low concentrations of volatile fatty acids were detected in rumen gas. The molar proportions of volatile fatty acids were similar in gas and liquor samples, with rumen gas containing slightly less acetic acid and disproportionately more valeric and caproic acids.

Oral immunization with recombinant Streptococcus gordonii expressing porphyromonas gingivalis FimA domains.

Porphyromonas gingivalis, a gram-negative anaerobe, is implicated in the etiology of adult periodontitis. P. gingivalis fimbriae are one of several critical surface virulence factors involved in both bacterial adherence and inflammation. P. gingivalis fimbrillin (FimA), the major subunit protein of fimbriae, is considered an important antigen for vaccine development against P. gingivalis-associated periodontitis. We have previously shown that biologically active domains of P. gingivalis fimbrillin can be expressed on the surface of the human commensal bacterium Streptococcus gordonii. In this study, we examined the effects of oral coimmunization of germfree rats with two S. gordonii recombinants expressing N (residues 55 to 145)- and C (residues 226 to 337)-terminal epitopes of P. gingivalis FimA to elicit FimA-specific immune responses. The effectiveness of immunization in protecting against alveolar bone loss following P. gingivalis infection was also evaluated. The results of this study show that the oral delivery of P. gingivalis FimA epitopes via S. gordonii vectors resulted in the induction of FimA-specific serum (immunoglobulin G [IgG] and IgA) and salivary (IgA) antibody responses and that the immune responses were protective against subsequent P. gingivalis-induced alveolar bone loss. These results support the potential usefulness of the S. gordonii vectors expressing P. gingivalis fimbrillin as a mucosal vaccine against adult periodontitis.

Interlaboratory study into the proficiency of serum cholinesterase activity measurement.

The measurement of cholinesterase activity is an important function of a clinical laboratory. Participation in appropriate quality assurance schemes is essential in ensuring a high analytical standard. Samples of human serum were distributed to thirty-five laboratories for the measurement of cholinesterase activity. Because of methodological differences between the participants, findings of each laboratory were compared either by the use of duplicate samples or by analysis of six mixtures of two samples, one having a high and one a low activity. Of 4,964 distributed samples 95% were analysed and findings reported in 596 reports. Thirty-four percent of all reports were considered very good (less than 5% within-run error) and 38% less than satisfactory (within-run error over 10%). Access to a proficiency programme such as this enables laboratories to evaluate the quality of their analytical service.

Effects of altering energy and protein supply to dairy cows during the dry period. 1. Intake, body condition, and milk production.

We used 48 Holstein-Friesian cows to investigate the effects of altering energy and protein supply to dry cows. Cows were fed one of three diets for 6 wk prior to parturition: (a) a 60:40 (DM basis) mixture of grass silage with barley straw ad libitum; (b) grass silage ad libitum; or (c) 0.5 kg/d of prairie meal with grass silage ad libitum. The standard lactation diet was a flat-rate allocation of concentrates and grass silage ad libitum. We evaluated dry-period diets using four dry fistulated cows; rumen pH remained high (mean = 6.6) and ammonia concentrations followed N intake. The inclusion of straw reduced apparent ruminal digestion of OM, N, and NDF as well as microbial protein yield, though microbial yield per unit of OM apparently digested in the rumen remained unchanged. Voluntary intake of forage was reduced by the inclusion of straw, while the inclusion of prairie meal had little effect. The decline in intake as calving approached was lower with the silage and straw mix diet. There were large differences in the BW change over the final 5 wk of the dry period, although the opposite effect was seen in early lactation, and differences in BW and body condition score were small by lactation wk 22. Despite the substantial differences in nutrient supply and effects on body reserves, there was little effect of dry-period diet on subsequent performance. Lower forage intakes and yields of protein and lactose were confined to the first month of lactation for cows previously offered straw.

Heterogeneity of Porphyromonas gingivalis strains in the induction of alveolar bone loss in mice.

These experiments examine alveolar bone loss in a model in which specific pathogen-free mice are exposed orally with Porphyromonas gingivalis. Alveolar bone loss was induced as a result of a specific infection with P. gingivalis, rather than other environmental antigens. Infection with live P. gingivalis was required, as significant bone loss did not follow gavage with formalin-killed P. gingivalis. The virulence of different strains of P. gingivalis was compared. Two laboratory strains of the bacteria (ATCC 53977 and W50) and a mutant strain lacking the 43-kDa fimbrillin (strain DPG3) induced bone loss. P. gingivalis 381, however, did not induce bone loss. There was a strong immunoglobulin G (IgG) antibody response to infection with each strain but a significant serum IgA response only to strain 381. These studies show that in mice with a background oral microflora bone loss is induced by a specific infection with P. gingivalis and that bacterial strain variation is important in determining whether alveolar bone loss will ensue.

Lesions in cattle exposed to Mycobacterium bovis-inoculated calves.

Nine calves were housed for periods ranging from 24 to 117 days in close contact with cattle inoculated intranasally with Mycobacterium bovis. These "in-contact" calves were examined immunologically and bacteriologically during the period of exposure, and pathologically and immunocytochemically post mortem. Three became infected by day 14, as indicated by the detection of M. bovis in nasal mucus. In-vitro interferon-gamma production and lymphocyte proliferation were detected after stimulation of peripheral blood with M. bovis antigens in the majority of in-contact animals by day 28; this provided support for the role of immunological mechanisms in pathogenesis. Tuberculous lesions were found in the submandibular and bronchomediastinal lymph nodes and in the lungs of the in-contact calves; in distribution and appearance the lesions resembled those observed in naturally occurring disease. The distribution of M. bovis antigen and the numbers of mycobacteria within pulmonary lesions are reported. 1999 Harcourt Publishers Ltd.

Serum antibody response to oral infection precedes but does not prevent Porphyromonas gingivalis-induced alveolar bone loss in mice.

The purpose of this study was to determine whether humoral immunity prevents bacterially induced alveolar bone loss. BALB/cByJ mice were orally infected with the human periodontopathic bacterium Porphyromonas gingivalis, and were compared with sham-infected mice. Specific serum antibody titers to P. gingivalis were measured by enzyme-linked immunosorbent assay. Alveolar bone levels were measured as the distance from the cementoenamel junction to the alveolar bone crest and bone loss was defined as a change in bone levels over time or between infected and sham-infected animals. The specific humoral response was predominantly of the IgG isotype, although low levels of specific serum IgA were also present. Antibody titers in the infected animals were significantly different from those in the sham-infected animals by 18 days and remained at maximal levels at 47 days. Bone loss became significant by 26 days and continued to progress at 47 days. Thus the serum antibody response to oral infection with P. gingivalis preceded detectable bone loss and remained elevated while bone loss increased. The presence of specific antibody did not prevent the onset or progression of bone loss.

Studies on the efficacy of intranasal vaccination for the prevention of experimentally induced parainfluenza type 3 virus pneumonia in calves.

The efficacy of intranasal vaccination in preventing or limiting disease of the lower respiratory tract induced by parainfluenza 3 (PI3) virus was evaluated under experimental conditions, using a commercially available live vaccine containing a temperature-sensitive strain of PI3 virus. In a preliminary study four colostrum-deprived calves were vaccinated intranasally at one week and again at two months of age, and two similar calves were given an intranasal placebo. After the second vaccination serum antibodies to PI3 virus were detected in all four vaccinated calves, but not in the control animals. Seventeen days after the second vaccination all six calves were challenged with virulent PI3 virus, and they were killed six days later. The clinical scores and the extent of pulmonary consolidation were reduced in the vaccinated animals; PI3 virus was detected in the upper and lower respiratory tract of the control calves but in none of the vaccinated calves. In a larger scale study with 14 colostrum-fed calves, seven were vaccinated at one week and again at five weeks of age, and seven were given an intranasal placebo. Two weeks after the second vaccination all 14 calves were challenged with virulent PI3 virus. The clinical scores and lung consolidation were significantly reduced in the vaccinated calves in comparison with the controls. Six days after infection, 10 of the 14 calves were killed; PI3 virus was detectable in the nasal secretions of all seven control calves but in only one of the vaccinated animals, and PI3 viral antigen was detected in the lungs of the control calves but not in those of the vaccinated animals. One of the vaccinated calves had developed a severe clinical response after the challenge, but it had only minor lung consolidation when killed.

An explanation for the different inhibitory characteristics of human serum butyrylcholinesterase phenotypes deriving from inhibition of atypical heterozygotes.

The time course of inhibition of butyrylcholinesterase (EC 3.1.1.8) by the dimethylcarbamate Ro 02-0683 in sera taken from patients heterozygous for the usual (U), atypical (A), K or J variants was followed using propionylthiocholine as substrate. Data obtained were used to determine rate constants of inhibition together with the contribution made by each variant to total enzyme activity. The findings substantiate earlier reports that J and K mutations lead to quantitative changes in the concentration of usual enzyme in contrast to the qualitative changes of the atypical variant. The contribution of the atypical enzyme to the total activity in serum from UA, AK and AJ heterozygotes was respectively 17-20, 24-31 and 34-53%. The altered ratios of atypical to usual, K or J enzyme in UA, AK and AJ together with the constants on the usual enzyme alone, explain the differences in observed inhibitor numbers which enable these heterozygotes to be identified.

Catalytic parameters for the hydrolysis of butyrylthiocholine by human serum butyrylcholinesterase variants.

Catalysed hydrolysis of butyrylthiocholine (BTCh) by the usual (UU), fluoride-resistant (FS), AK, AJ and atypical (AA) human serum butyrylcholinesterase (EC 3.1.1.8) variants was measured in phosphate buffer pH 7.4 at 25 degrees C. pS-curves for all phenotypes were S-shaped; the activities rose to a plateau with increasing substrate concentration except at 100 mM where there was a small decrease. To obtain the catalytic constants, three equations were applied: Michaelis-Menten equation (Eq. 1), Hill equation (Eq. 2) and an equation which assumes simultaneous binding of the substrate to the catalytic site and to a peripheral site on the enzyme (Eq. 3). Over a range from 0.01 to 50 mM BTCh, the activity versus substrate concentration relationship deviated from Michaelis-Menten kinetics (Eq. 1) while data fitted well with Eqs. 2 and 3. The Michaelis-Menten equation was applied separately to two BTCh concentration ranges: the corresponding Km constants for the UU, FS, AK, AJ and AA phenotypes ranged from 0.1 to 0.2 mM (at 0.01-1.0 mM BTCh) and from 0.3 to 2.0 mM (at 1.0-50 mM BTCh). Hill coefficients (nH) calculated from Eq. 2 were similar for all phenotypes (nH approximately 0.5). The dissociation constants K1 and K2 calculated from Eq. 3 for two sites on the enzyme fell between 0.02 and 0.12 mM (K1) and 0.89 and 4.9 mM (K2) for the five phenotypes. Experimental data support the assumption that the phenotypes studied have two substrate binding sites.

CD4(+) T cells and the proinflammatory cytokines gamma interferon and interleukin-6 contribute to alveolar bone loss in mice.

In this study, we used a mouse model to examine the role of the adaptive immune response in alveolar bone loss induced by oral infection with the human gram-negative anaerobic bacterium Porphyromonas gingivalis. Severe combined immunodeficient mice, which lack B and T lymphocytes, exhibited considerably less bone loss than did immunocompetent mice after oral infection, suggesting that lymphocytes contribute to this process. Bone loss after oral infection was decreased in mice deficient in major histocompatibility complex (MHC) class II-responsive CD4(+) T cells, but no change in bone loss was observed in mice deficient in MHC class I-responsive CD8(+) T cells or NK1(+) T cells. Mice lacking the cytokine gamma interferon or interleukin-6 also demonstrated decreased bone loss. These results suggest that the adaptive immune response, and in particular CD4(+) T cells and the proinflammatory cytokines that they secrete, are important effectors of bone loss consequent to P. gingivalis oral infection. The studies also reinforce the utility of the mouse oral infection model in dissecting the pathobiology of periodontal disease.

Immunostimulatory activity of LT-IIa, a type II heat-labile enterotoxin of Escherichia coli.

Certain bacterial molecules potentiate immune responses to parenterally administered antigens. One such molecule that has been intensely investigated is cholera toxin, a type I heat-labile enterotoxin produced by the Gram-negative bacterium Vibrio cholerae. Immunization with a mixture of a foreign antigen and cholera toxin enhances the immune response to the antigen. Similar adjuvant activity is associated with LT-I, a closely related type I heat-labile enterotoxin produced by Escherichia coli. The adjuvant activities of LT-IIa, a member of the type II heat-labile enterotoxins produced by E. coli, have not been described. LT-IIa and CT differ significantly in amino acid sequence of the B polypeptides and in receptor binding affinity. In this study, rats were subcutaneously immunized with fimbrillin, a protein isolated from the bacterium Porphyromonas gingivalis, and with fimbrillin in combination with LT-IIa, the prototypical type II enterotoxin. Previous studies documented that fimbrillin administered alone is a poor immunogen. Animals immunized with the mixture of fimbrillin and LT-IIa produced high titers of specific IgG antibody directed against fimbrillin. Anti-fimbrillin antibody titers in sera from animals receiving the combination of LT-IIa + fimbrillin were comparable to those obtained from sera of animals immunized with cholera toxin + fimbrillin. The results of these experiments demonstrate that LT-IIa exhibits an adjuvant activity that is equal to that of cholera toxin. Recombinant methods have been established for producing large amounts of LT-IIa, an advantage that will likely provide an economic impetus to consider incorporating the enterotoxin as an immunostimulatory agent in future vaccines.

Early lesion formation in cattle experimentally infected with Mycobacterium bovis.

Early lesion formation was examined in 13 calves inoculated intranasally with 2 x 10(7) colony-forming units of Mycobacterium bovis and killed either singly or in pairs at intervals of < or = 7 days from post-inoculation day (pid) 3 to pid 42. Immunological examinations were carried out before and after infection, and sequential necropsies were performed. M. bovis was recovered as early as pid 3, from the upper respiratory tract mucosae, retropharyngeal lymph nodes and caudal lung lobe. Gross tuberculous lesions were detected in both the upper respiratory tract mucosae and in the lungs of the calves killed from pid 14 onwards. Lesions were also present in the lymph nodes draining these areas. On histological examination, neutrophils appeared to play a key role in the earliest stages of lesion formation, and lesion mineralization was observed for the first time at pid 35. The contemporaneous development of lesions and cellular immunity, as demonstrated by in-vitro lymphocyte proliferation and interferon-gamma assay responses, provided further evidence of the role of immunopathogenic mechanisms in the development of bovine tuberculosis.