Release of Shiga toxin by membrane vesicles in Shigella dysenteriae serotype 1 strains and in vitro effects of antimicrobials on toxin production and release

Effects of various antimicrobials on in vitro Shiga toxin production and release by Shigella dysenteriae serotype 1 was investigated in this study with particular reference to the role of outer membrane vesicles in toxin release by the organism. Five antimicrobials, namely nalidixic acid, ciprofloxacin, norfloxacin, fosfomycin and mitomycin C, were chosen for the study and the toxin titre was measured by the reverse passive latex agglutination (RPLA) method using an available kit. Only mitomycin C was found to induce production of Shiga toxin in the bacteria and its release by outer membrane vesicles. The highest titre of toxin was obtained in vesicle fraction suggesting that the vesicles play an important role in the release of Shiga toxin from periplasmic space by the organism.     

Immune parameters in carpet shell clams naturally infected with Perkinsus atlanticus

Defence parameters of non-infected clams (Ruditapes decussatus) and clams heavily infected with Perkinsus atlanticus were assessed. Cellular (haemocyte density and phagocytic activity) and humoral (lysozyme and anti-bacterial activities, protein concentration and agglutination titre) parameters were measured in clams collected in an area enzootic for P. atlanticus. The infection intensity of each clam was assessed, and the immune parameters measured in the most infected clams were compared with those measured in the non-infected ones. Only the serum anti-bacterial activity and the agglutination titre were significantly different between infected and non-infected clams. The phagocytic rate, haemocyte density, lysozyme concentration and protein concentration were not statistically different but they showed the same trend in the two trials performed. Phagocytic rate, haemocyte concentration and anti-bacterial activity were higher in non-infected clams, while they had lower lysozyme concentration, serum protein concentration and agglutination titre than infected clams. Although infected and healthy clams were not different for every parameter measured, probably due to the high variability among individuals, P. atlanticus seems to affect the clam immune system, at least in advanced stages of the infection.     

Evaluation of the enzyme-linked immunosorbent assay for the serodiagnosis of tularemia

The usefulness of the ELISA using as antigen prepared in our laboratory supernatant obtained after centrifugation of sonicated F. tularensis cell suspension was compared with the tube agglutination test with commercial available antigen. Paired serum specimens obtained from 6 patients with ulceroglandular syndrome of tularemia were tested in both tests. The cut-off limit of serum antibodies was set at mean antibody titre determined in the sera of 115 blood donors exceeded by three standard deviations. Antibodies to F. tularensis in diagnostically significant titre were detected in all 12 serum samples by both tests. However the titres obtained in ELISA were several times higher than in tube agglutination test. In the second serum sample the level of IgA and IgM was lower but the level of IgG higher than in the first sample. We could not observe any difference in the level of antibodies between paired serum specimens in tube agglutination test.     

Effects of Carbohydrates in Growth Medium on Agglutination of Several Species of Salmonella with Polyvalent H Antiserum

The effect of various carbohydrates in the growth medium on agglutination of salmonellae with polyvalent H antiserum was studied. There appeared to be a relationship between fermentation of the carbohydrate by the organism and resultant agglutination with the antiserum. It is recommended that the tube test for flagellar antigens be allowed to remain in a water bath for 2 hr before the final observation is made. Sorbitol, dulcitol, mannose, maltose, rhamnose, or trehalose, when included in the growth medium for Salmonella, yielded high percentages of positive agglutinations with all conditions of the experiment.     

Estudio comparativo de las reacciones serologicas cuantitativas con un antigeno metabolico de Aspergillus fumigatus

Summary The following quantitative serologic reactions: agar-gel immunodiffusion, complement-fixation, opposite electrophoresis and latex particle agglutination tests have been performed in 38 sera from mycologically proved pulmonary aspergillosis cases. A metabolic antigen from a strain ofAspergillus fumigatus according toAjello et al technic modified by us, has been employed. Sera from 120 subjects suffering from non-mycotic lung conditions, as well as 10 sera from histoplasmosis cases, 10 sera from S. A. blastomycosis and 2 sera from patients with lung aspergillosis produced byA. niger, gave negative results with the above mentioned seroligic reactions.One hundred per cent of positive results were obtained with the complement-fixation test (titre ranging from 1/20 to 1/1280), agar-gel immunodiffusion test (titre up to 1/64) and the opposite immunoelectrophoresis (titre ranging from 1/2 to 1/256). Twenty five per cent negative and 4 non-specific results were registered with the latex particle agglutination test.A correlation of the number of serum precipition bands obtained by the electrophoresis technic with the titre of the quantitative serologic reactions, as well as a correlation of the titre of the circulating antibodies with the severity of the clinical form of aspergillosis seems to be present.Electrophoretic motility of the specific antibody performed in 10 sera showed results like the IgM in 1 instance and an intermediate position between IgA and IgG in 9 samples.     

Revision of the antigenic structure of genus Listeria

O-antigenic structure of genus Listeria was studied, using antisera (obtained from rabbits) against different O-antigens of reference strains of each serovar. The titres of sera were determined by agglutination using antigens of the same reference strains as well. Some differences from the actual scheme were found: serum antifactor-IX gave a lower titre than expected against antigens 4ab and 6b, while the titre observed against antigen 4b was higher than the expected in this case. Serum antifactor-VIII presented a higher titre than could be expected against antigen 6b. The strains of serovars 4d and 4e used in this experience were impossible to distinguish, and could have been classified in the same serovar. We could not obtain serum antifactor-XI from serovar 6b after several trials. From these differences we propose some modifications of the current antigenic scheme of genus Listeria.     

Purification and chemical characterization of antigens from sheep erythrocytes

A mixture of glycoproteins and glycolipids was solubilized from sheep erythrocytes membranes under the effect of high ionic strength (2 M NaCl, 0.5 M Tris). Several antigenic fractions could be purified from the mixture using gel filtration on Sephadex G-200 and block electrophoresis on Pevikon C870; two fractions were found to raise antibodies in a primary reaction and these antibodies effectively sensitized erythrocytes to lysis by complement. The majority of other fractions elicited a weaker primary reaction which was detectable by both agglutination and haemolysis. The fraction, migrating fastest towards the cathode, elicited after immunization a formation of antibodies that could be detected almost exclusively by haemagglutination. The fraction, which elicited in the primary reaction a high titre of haemolytic antibodies, is composed of 72% proteins, 11% lipids and 15% saccharides.     

Evaluation of a serological test for tuberculosis.

The agglutination test of Nicholls was found to be ineffective in diagnosing active tuberculosis. A positive result (titre of 1/125 or more) was found in the serum of 74 (70%) out of 105 patients with newly diagnosed, smear-positive pulmonary tuberculosis; 61 (62%) out of 98 healthy family contacts; and 19 (63%) out of 30 patients with non-tuberculous conditions. These findings were not due to faulty technique since the results obtained at Hammersmith were similar to those obtained by Nicholls''s laboratory in the same serum samples. Twenty-seven of the tuberculous patients who had a negative result before treatment were retested two months after the start of chemotherapy but showed no evidence of a rising titre.     

Kinetics of Pseudomonas fluorescens growth under different conditions of culturing

The dynamics of Pseudomonas fluorescens VKM-1472 growth was studied under the conditions of batch and continuous cultivation, and the behaviour of the organism was investigated upon nutrition shifts and starvation. At D greater than 0.375 h-1, just as in the batch culture with a substrate excess, the strain realised excessive metabolism [15]: the yield in terms of the substrate fell down (38% for the batch culture and 40% for the continuous culture), the titre of viable cells decreased to a considerable extent, and maintenance expenditures increased (3 times for qgluc and 7.5 times for qO2). When the culture was incubated under the oligotrophous conditions, the biomass yield decreased fourfold within 8 days and the titre of viable cells dropped down twofold within the same period of time. However, the organism was still capable of oxidising a wide range of substrates (17 substrates were studied). As soon as a substrate was added, it was oxidised at a high rate and changes in the macromolecular composition were characteristic of an "up-jump" in the growth rate [11]. It is possible that the growth and behaviour of this organism are associated with its ecological niche occupied in natural systems.     

In vitro oncospheral agglutination given by immune sera from mice infected, and rabbits injected, with eggs of Hymenolepis nana.

Oncospheral agglutination given by sera immunized with Hymenolepis nana eggs is described as a new way of assessing H. nana infection. All sera of mice which possessed acquired protective immunity against reinfection by H. nana eggs had the potency to induce oncospheral agglutination in vitro. Only oncospheres, which had been hatched, agglutinated, no agglutination occurred in sera from uninfected mice. Oncospheral agglutination was carried out by mixing 0-1 ml of serial two-fold dilutions of serum and 0-1 ml of Hanks' balanced salt solution containing about 600 hatched oncospheres. Titre of agglutinins was indicated as a reciprocal of the final dilution capable of giving agglutination clusters made of three or more oncospheres. Agglutinins developed within 14 days after a primary infection with 500 shell-free eggs. There was no rapid increase of agglutinins within 4 days following a secondary infection. The titre increase coincided with the increase in dosages of eggs. Agglutinins were thought to be immunoglobulins, because the potency of the serum to agglutinate oncospheres was extinguished after absorption of globulins with rabbit anti-mouse globulin serum. Agglutinins were produced in rabbits by intravenous injections of shell-free eggs. The titres of the rabbit sera were much higher than those of mouse sera.     

Cultivation of lymphatic cells in protein-free medium and chemical study of the products

Rabbits immunized withBrucella suis within a period of one week formed antibodies in high titres. These antibodies were of a macroglobulin character only. Cells from the spleen and popliteal lymph nodes were cultivatedin vitro in protein-free medium, in which the only macromolecular substance was dextran or carbowax. Physico-chemical analysis of secretion products was not succesful, since under the given cultivation conditions the cells started to disintegrate from the outset and the medium contained nucleic acids as well as proteins. Some of these substances were present in particles of the size of different subcellular particles. After cultivation of spleen and lymph node cells, antibodies were detected by an agglutination test in media containing dextran and carbowax. When particles substantially larger than the antibody molecule were removed by ultracentrifugation, the agglutination antibody titre in the medium fell.     

Control of sperm concentration is necessary for standardization of sperm cryopreservation in aquatic species: evidence from sperm agglutination in oysters

A lack of standardization in sperm cryopreservation of aquatic organisms is one of the main reasons for inconsistency observed among various studies. In particular, there have been few attempts to standardize sperm concentration during procedural optimization. This study was intended to call attention to sperm concentration standardization through research of sperm agglutination in Pacific oysters Crassostrea gigas. Sperm agglutination after thawing is a relatively frequent phenomenon observed for various aquatic species, especially when sub-optimal cryopreservation protocols are used; however, no systematic attempts have been made to explain this phenomenon. The present study evaluated various factors affecting sperm agglutination of thawed samples from diploid and tetraploid Pacific oysters, and is the first detailed report addressing the sperm agglutination phenomenon of thawed samples from any aquatic organism. Agglutination of oyster sperm was classified into six levels with a scale ranging from 0 (homogenous suspension) to 5 (well-developed "noodles"). It was found that agglutination in thawed samples was mainly due to the lack of sufficient cryoprotectant for a specific sperm concentration. Interestingly, high levels of agglutination did not necessarily lead to low fertilization. On the contrary, some sperm cells appeared to gain protection from the formation of peripheral agglutination within 0.5-ml French straws. The exact mechanism of sperm agglutination remains unclear. However, morphological examination of cross sections of the noodles (agglutination level 5) indicated at least two forms of agglutination (formed with and without cryoprotectant) which could be used as a tool to understand the cryopreservation process within the micro-environment of the straw. Furthermore, the fact that the level of sperm agglutination was directly determined by sperm concentration, in addition to the type of cryoprotectant, cryoprotectant concentration, and cooling and thawing methods emphasized the importance of procedural standardization and systematic optimization and integration of protocols involving multiple factors.     

Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood cells are particularly useful targets as they are readily available and agglutination is observable using the naked eye. This technique is commonly used to determine the titre of an antibody (Ab), for blood grouping and viral quantification. In this video, the steps involved in preparing and performing a haemagglutination assay is demonstrated using antibodies specific to blood group A-antigens added to red blood cells (Revercells). The antiserum is serially diluted in a 96 well U-bottom microtitre tray, to which is added a suspension of Revercells. The samples are mixed and then incubated at 37°C for 60 minutes. After this time, the samples can then be easily scored for ve, +ve and intermediate (-/+) haemagglutination reactions. This approach allows for the reactivity and titre of a serum sample to be assessed using a rapid and simple technique. The video will cover the theory behind the assay, how the results are read and interpreted, how the titre is determined, how the assay can be modified and any issues associated with the use of this technique.Open in a separate windowClick here to view.^{(20M, flv)}     

Neospora caninum infections in bovine foetuses and dairy cows with abortions in Argentina

Antibodies to Neospora caninum were measured in bovine foetuses, dairy cows and beef cows in Argentina using the IFAT, the N. caninum agglutination test, and the recombinant NCDG1 and NCDG2 ELISA. Serum antibodies (IFAT titre 1:80) were found in 20 of 82 (24.4%) dairy cow foetuses and one of 22 (4.5%) beef cow foetuses. Microscopic lesions suggestive of neosporosis were seen in brains of seven of eight foetuses with IFAT titres of 1:80. Antibodies (IFAT) were found in 122 of 189 (64.5%) dairy cows that aborted. Serum antibody titres (IFAT) of 189 dairy cows that aborted were: < 1:25 (67 cows), 1:25 (four cows), 1:50 (16 cows), 1:200 (seven cows), 1:> or = 800 (95 cows). Of the 87 sera with IFAT titres of < or = 1:50, 57 had no antibodies in 1:40 dilution and 30 had titres of 1:40 in the N. caninum agglutination test. Thus, sera from at least 56 dairy cows which had aborted were seronegative both in the N. caninum agglutination test and the IFAT. The distribution of positive and negative sera was similar when measured by ELISA, except that, depending on cut-off titre, the ELISA indicated a greater number of seropositive cows that were negative by the IFAT and N. caninum agglutination test. These results suggest that transplacental transmission of N. caninum in dairy cows in Argentina is frequent.     

Effect of dietary beta-1,3 glucan on immune responses and disease resistance of healthy and aflatoxin B1-induced immunocompromised rohu (Labeo rohita Hamilton).

The immunostimulant beta-1,3 glucan was fed at 0.1% in feed for 7 days to healthy and aflatoxin B1 (AFB1)-induced immunocompromised fish, Labeo rohita (one of the major tropical carp species), in a 60 day trial. The effects of AFB1, glucan and their interactions on non-specific and specific immunity levels and disease resistance of fish were studied. A single intraperitoneal injection of AFB1 at 1.25 mg kg(-1) body weight) caused a significant (P < 0.05) reduction in non-specific immunity as measured through neutrophil phagocytic indices, serum bactericidal activity, and specific immunity as measured through bacterial agglutination titre against Edwardsiella tarda, as well as reduced protection against Aeromonas hydrophila challenge in comparison to control fish which were exposed neither to aflatoxin nor to glucan. Feeding of glucan to healthy fish raised the non-specific and specific immunity level and protection against bacterial infection compared with the control. Feeding of glucan to AFB1-induced immunocompromised fish for 7 days significantly raised the degree of resistance against A. hydrophila challenge and the non-specific immunity level in comparison to non-treated AFB1 exposed fish. Although feeding of glucan was able to increase specific immunity, all measured through haemagglutination titre against sheep red blood cells, and bacterial (E. tarda) agglutination titre in healthy fish in comparison to all other groups, no significant increase in specific immunity to the aflatoxin-exposed group was seen.     

Hemagglutinating activity of Mycoplasma salivarium and its attachment to sheep red blood cells

Mycoplasma salivarium (ATCC 23064) and 10 other strains isolated from human saliva agglutinated red blood cells of rabbits and human types A and O weakly, and those of sheep (SRBC) and human type B strongly. Glycoproteins on the surface of the organism cells and N-acetylneuraminic acid residues and some sugars on SRBC were suggested to be involved in agglutination of SRBC. Protein A-like activity was detected in the organism cells. The organism cells were also shown to attach to SRBC in PPLO broth (Difco) supplemented with 10% horse serum, and bivalent metal ions were suggested to be involved in the attachment. The organism cells attaching to SRBC activated complement through the alternative pathway and lyzed the SRBC.     

Rational method of obtaining sera with a high titre of virus-neutralizing antibodies. Report 2]

In addition to report I (ZHMEI, 1977, No. 1) a study was made of 9 more schemes of rabbit immunization with the poliomyelitis virus, type I, for the purpose of obtaining the neutralizing sera of high titre. Vitamins A and C were used in the experiments in the capacity of the activators of the organism reaction; Freund's adjuvant of different make was tested; different reimmunization periods and different amounts of the adjuvant were administered. Titration of rabbit sera in the process of immunization and reimmunization showed immunization into the lymph nodes with the subsequent single reimmunization in one month to be the most effective and economical method of obtaining high effective sera.     

Natural antibodies in Pseudomonas aeruginosa infections

Studying 347 sera of 49 patients suffering from Pseudomonas aeruginosa infection the mean titre value (MTV) calculated from the exponent of the binary logarithm of natural antibody (NA) titres, was found suitable to characterize the humoral immune status. As long as the organism is in equilibrium with the infection, the NA level rises. In septic shock, before death or at the development of a massive infection, the NA titre decreases rapidly. The decrease may be due partly to the permeability increasing effect of endotoxin and antigen-antibody reactions exerted on the capillaries. Consequently, in the most severe phase of sepsis, when bacteria enter the circulation less NA is available to fight against them. This might be a cause of the still very high lethality of septic shock due to Gram-negative bacteria. Finally, when applying the MTV one always has to consider that despite its advantages it gives less information than the Backhausz immunogram.     

Prevalence of antibodies to Francisella tularensis in forest workers from different regions of Poland

In the present study we evaluate the prevalence of antibodies to F. tularensis in 480 serum samples obtained from healthy forest workers from different regions of Poland. The investigations were performed using the tube agglutination test and ELISA. The cut-off limit of serum antibodies was set at mean antibody titre determined in the sera of 115 blood donors exceeded by three standard deviations. In none serum samples we detected antibodies to F. tularensis by tube agglutination test. Of the 480 tested sera IgA antibodies were detected by ELISA in 4.6%, antibodies IgG in 3.8% and antibodies IgM in 2.70% serum samples. The results of our study showed that antibodies to F. tularensis were slightly, but not statistically significant, more often diagnosed in healthy forest workers than healthy blood donors.     

Antibodies to Corynebacterium Pseudotuberculosis in Adult Goats from a Naturally Infected Herd

Serum samples taken in 3 successive years (1977, 1978 and 1979) from adult dairy goats (Norwegian breed) originating from 1 herd were examined for antibodies to Gorynebacterium pseudotuberculosis. Both bacterial agglutination test (BAT) and hemolysis inhibition test (HIT) were used. The proportion of seropositive goats increased 10–12 % during the investigation period. In 1979 all animals were seropositive to BAT and about 95 % had antihemolysins in their sera. Twenty-two of the 23 one-year old goats recruited to the herd in 1978 were seropositive. The average age-specific titres increased up to the age of 3 years, and subsequently decreased for goats aged 4–7 years. Caseous lymphadenitis is thus regarded as a chronic infection. The effect of age on the titre values was significant at the 5 % level in 1977 and 1978 when HIT was used and in 1978 when BAT was used. During the investigation period the same 36 and 40 goats were examined every year by BAT and HIT, respectively. Intermediate to high correlations between titre values for the same goats from year to year were found. Both BAT and HIT are suitable for sero-epidemiological investigations concerning infection with G. pseudotuberculosis in goats.