The agglutination test of coracidia of pseudophyllidean cestodes and its use in cestodological research

Ability of coracidia to agglutination in homologous antiserum was established. The method of reaction of a direct agglutination of coracidia (RDAC) in the culture of alive larvae was described. When comparing the RDAC results in homologous and heterologous variants a dependence of the agglutination pattern on the degree of antiserum homology was established. On the example of RDAC of the cestodes Diphyllobothrium dendriticum and D. ditremum in polyvalent and species-specific antisera to D. dendriticum possibility was shown of RDAC usage in the studies on specific taxonomy of pseudophyllid cestodes.     

Effects of volume, culture media and type of culture dish on in vitro development of hamster 1-cell embryos

We examined effects of medium volume and two different culture media (HECM-3 and HECM-4) on in vitro development of hamster embryos. Groups of 5 to 8 1-cell embryos were cultured for 72 h in either < or =100 or > or =100 microl volumes. In the first experiment, embryos were cultured in Petri dishes with 2, 5, 20, 50 or 100 microl of medium using the two media (2 x 5 factorial experiment). Optimal volumes for morula and blastocyst development were 100 microl of HECM-3 and > or =50 microl of HECM-4; in HECM-4, > or =20 microl volumes were suitable whereas in HECM-3 < or = 50 microl volumes were unsuitable. In the second experiment, embryos were cultured in 100, 200, 500 and 1000 microl of HECM-3 and HECM-4 using organ culture dishes. Controls were 100 microl drops in Petri dishes. In organ culture dishes, blastocyst development was < or =6% in HECM-3 and 33-41% in HECM-4, and suitable volumes for development to at least morulae were > or =200 microl of HECM-3, and > or =100 microl of HECM-4. In both experiments development to morula and blastocyst stages with 100 microl volume in Petri dishes was significantly higher with HECM-4 (96 and 85% in Experiment 1 and 2, respectively) than that with HECM-3 (52 and 40% in Experiment 1 and 2, respectively; P < 0.05). These results indicate that attention should be paid to both type and volume of medium and interaction with type of culture dish for optimizing development of embryos in vitro.     

Adjusting the number of spermatozoa in mini-straws by determination of the operative volume of bovine semen

The importance of the number of sperm per insemination on fertility has been well demonstrated in cattle. This number is usually calculated from the concentration in the extended semen and a theoretical value for the operative volume of semen delivered during insemination. The objective of this experiment was to investigate the usefulness of the measurement of the delivered volume of semen when estimating sperm numbers from frozen-thawed mini-straws, by comparing the results obtained with an analytical balance to the theoretical volume. The density of semen extended with Biociphos Plus and Triladyl was determined to be 1.033 g/ml using the gamma sphere method. This value was used to convert semen weight into operative volume. The effect of semen temperature at the time of weighing (37 degrees C versus 20 degrees C) was investigated on six semen batches, two technicians measuring the operative volume of 50 straws for each combination of temperature and semen batch (a total of 1200 weighings). The temperature effect was found to be insignificant, which allowed warm semen to be weighed before motility was assessed during routine quality control. The operative volume was then measured in straws routinely produced at 17 bovine Al centers (12-105 semen batches per center, mostly three straws from each batch, a total of 1912 measurements). The observed volumes were normally distributed around 198.7 microl, 98% measuring between 180 and 210 microl. The operative volume was significantly different among centers (from 192 to 205 microl, P < 0.0001) and among batches within centers (P < 0.0001). The S.D. among straws within batches was 3.4 microl. Some centers showed high variability in straw volume whereas others were more consistent. Determination of the operative volume of frozen-thawed mini-straws by weighing the delivered contents is an accurate method for estimation of the number of sperm per dose.     

Development of a sensitive enzymeimmunoassay (EIA) for FSH determination in bovine plasma.

A highly sensitive enzymeimmunoassay (EIA) procedure for FSH determination in bovine plasma on microtiterplates using the biotin-streptavidin amplification system and the second antibody coating was developed. Biotin was coupled to FSH and used to bridge between streptavidin-peroxidase and the immobilized antiserum in the competitive assay. The EIA was carried out directly in 50 microl of bovine plasma and compared with an established radioimmunoassay (RIA) employing 100 microl plasma. Same FSH standards and FSH specific antiserum were used in both procedures. FSH standards prepared in hormone free plasma were used. The sensitivity of the EIA procedure was 6.25 pg/well FSH which corresponded to 125 pg/ml plasma; the 50% relative binding sensitivity was seen at 200 pg/well. In comparison to RIA, the EIA was at least four times more sensitive besides requiring 6 times less FSH specific antiserum. Plasma volumes for the EIA ranging from 12.5 to 50 microl did not influence the shape of the standard curve even though a slight drop in the OD450 was seen with higher plasma volumes. When both EIA and RIA methods were used to measure FSH in cows, the levels were detectable only by the EIA procedure. The assay detects high and low plasma FSH levels within the physiological variation as well as changes in plasma FSH after stimulation with a GnRH analog. In conclusion, in addition to being non-radioactive and low cost in nature, the method offers several advantages over the conventional FSH RIA procedure; these are (a) higher sensitivity, (b) less labour and time saving, (c) more economical use of precious FSH antiserum and (d) long shelf-life of the biotinyl-FSH label (in contrast to the short half life of iodinated FSH in RIA).     

Specific activity of colored variants of N. meningitidis colonies

The character of fluorescence of the colonies formed by serogroup A meningococci (229 strains) in oblique light and their activity in the agglutination test with group specific S and RD antisera to the meningococcal dissociant of the same serogroup were studied. Orange and orange-green colonies were found to have pronounced group-specific activity, and faint gray colonies changed the character of their agglutination with the group-specific antiserum up to the loss of agglutinability; and simultaneously their capacity for agglutination with the antiserum to the dissociant was revealed. The study of the character of fluorescence and the group-specific activity of meningococcal colonies belonging to other serogroups provided similar results.     

Nitric oxide of the supraoptic nucleus influences the salivary secretion, sodium renal excretion, urinary volume and arterial blood pressure induced by pilocarpine

Male Holtzman rats weighting 200-250 g were anesthetized with zoletil 50 mg/Kg (tiletamine chloridrate 125.0 mg and zolazepan chloridrate 125.0 mg) into quadriceps muscle and stainless steel cannulas were implanted into their supraoptic nucleus (SON). We investigated the effects of the injection into the supraoptic nucleus (SON) of FK 409, a nitric oxide donor, and NW-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor (NOS), on the salivary secretion, arterial blood pressure, sodium excretion and urinary volume induced by pilocarpine, which was injected into SON. The drugs were injected in 0.5 microl volume over 30-60 s. Controls was injected with a similar volume of 0.15 M NaCl. FK 409 and L-NAME were injected at doses of 20 microg/0.5 microl and 40 microg/0.5 microl respectively. The amount of saliva secretion was studied over a five-minute period after injection of pilocarpine into SON. Injection of pilocarpine (10, 20, 40, 80, 160 microg/microl) into SON produced a dose-dependent increase in salivary secretion. L-NAME was injected into SON prior to the injection of pilocarpine into SON, producing an increase in salivary secretion due to the effect of pilocarpine. FK 409 injected into SON attenuating the increase in salivary secretion induced by pilocarpine. Mean arterial pressure (MAP) increase after injections of pilocarpine into the SON. L-NAME injected into the SON prior to injection of pilocarpine into SON increased the MAP. FK 409 injected into the SON prior to pilocarpine attenuated the effect of pilocarpine on MAP. Pilocarpine (0.5 micromol/0.5 microl) injected into the SON induced an increase in sodium and urinary excretion. L-NAME injected prior to pilocarpine into the SON increased the urinary sodium excretion and urinary volume induced by pilocarpine. FK 409 injected prior to pilocarpine into the SON decreased the sodium excretion and urinary volume induced by pilocarpine. All these roles of pilocarpine depend on the release of nitric oxide into the SON. In summary the present results show: a) SON is involved in pilocarpine-induced salivation; b) that mechanism involves increase in MAP, sodium excretion and urinary volume.     

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.     

Reactions of antibodies against ferredoxin, ferredoxin-NADP+ reductase and plastocyanin with spinach chloroplasts

Purified antisera against ferredoxin, ferredoxin-NADP+ reductase and plastocyanin agglutinated osmotically shocked and washed spinach chloroplasts, prepared according to standard procedures. The monomeric antibody (immunoglobulin G fraction) of the reductase antiserum agglutinated chloroplasts specifically and directly, indicating that protruding structures (for example, the coupling factor) do not act as steric hindrances as has been suggested. With ferredoxin antiserum, the presence of a pentameric antibody (immunoglobulin M fraction) was obligatory to observe a positive agglutination reaction. Immunoglobulin G only inhibited ferredoxin-dependent reactions, like NADP+-photoreduction, but did not cause agglutination. Ferredoxin seems to be located in depressions of the membrane, possibly caused by a partial release of this protein in shocked chloroplasts. Similar results were obtained with purified immunoglobulins from a plastocyanin antiserum. Again the immunoglobulin G fraction inhibited electron transport reactions catalyzed by plastocyanin, whereas immunoglobulin M showed a positive agglutination, but had no influence on electron transport. It is concluded that ferredoxin, ferredoxin-NADP+ reductase and plastocyanin are peripheral electron transport components, located at the outer thylakoid membrane.     

Optimization of a 12-hour TaqMan PCR-based method for detection of Salmonella bacteria in meat

We developed a 12-h Salmonella detection method, based on 8 h of preenrichment, followed by automated DNA extraction and a sensitive real-time PCR. The method was optimized to obtain the highest possible yield of cells and DNA. The growth of different Salmonella strains in various preenrichment media and the effects of adding growth-promoting and selective reagents were explored, taking into account their PCR compatibility. The effects of (i) analyzing larger volumes (1 to 5 ml) from preenriched samples and introducing wash steps prior to DNA extraction, (ii) regulating the amount of paramagnetic particles (increasing it from 60 to 90 microl) in the DNA extraction, (iii) eluting the DNA in reduced volumes (25 or 50 microl rather than 100 microl), and (iv) increasing the PCR template volume (from 5 to 20 microl) were investigated. After 8 h of preenrichment, buffered peptone water yielded the highest number of salmonellae. When analyzing minced meat samples, positive effects of increasing the initial sampling volume from 1 to 5 ml and increasing the amount of paramagnetic particles to 90 microl were observed. However, washing the pellet and eluting the DNA in reduced volumes (25 and 50 microl) had no positive effects and resulted in decreased reproducibility. Increasing the amount of PCR template DNA from 5 to 20 mul improved the threshold cycle value by approximately 2. The improved 12-h PCR method was successfully compared to a reference culture method with 100 minced meat and poultry samples, with a relative accuracy of 99%, a relative sensitivity of 98%, and a relative specificity of 100%.     

Context-dependent foraging decisions in rufous hummingbirds

A core assumption implicit in economic models of animal choice is that subjects assign absolute utilities to options that are independent of the type and number of alternatives available. Humans sometimes appear to violate this assumption and employ relative, as opposed to absolute, currencies when making choices. Recent evidence suggests that animals too might sometimes employ relative choice mechanisms. We tested this idea by measuring the foraging preferences of rufous hummingbirds (Selasphorus rufus) faced with choices analogous to those in which human use of relative currencies is evident. The birds experienced three treatments: a binary choice between two artificial flower types designated concentration (20 microl, 40% sucrose solution) and volume (40 microl, 20%), and two trinary treatments in which a third decoy option (either concentration decoy: 10 microl, 30% or volume decoy: 30 microl, 10%) was added to the set. The birds' preferences differed significantly across the three treatments. In the trinary treatments, the effect of the decoy options was to increase the preference for the option that dominated the decoy. These results are similar to those reported in the human choice literature, and are compatible with the hummingbirds using a relative evaluation mechanism in decision making.     

Serological method for identification of Vibrio parahaemolyticus from marine samples

Use of agglutination with antiserum against lateral flagella (H-agglutination) for the identification of Vibrio parahaemolyticus was studied. Sucrose-negative bacteria were isolated from seawater, and their characterization was carried out by traditional biological tests and slide agglutination with antiserum specific to lateral flagella of V. parahaemolyticus. Of 135 strains isolated, 78 were identified as V. parahaemolyticus by biological tests and were agglutinated with the above serum. Fifty-five strains did not agglutinate with the serum, and their biological characteristics were different from those of V. parahaemolyticus. Two strains also differed from V. parahaemolyticus in some biological characteristics but agglutinated with the antiserum. All clinically isolated V. parahaemolyticus strains also agglutinated with the above serum. These results suggest that our serological method is useful for the identification of V. parahaemolyticus, especially for samples in which there are many organisms related to V. parahaemolyticus, because many biological tests can be omitted.     

Bordetella pertussis agglutinogens in cultivation dynamics

Bordetella pertussis growth phases during homogenous batch dynamic cultivation in the liquid medium as well as during the static cultivation on the solid medium were established. The maximal activity of agglutination reaction with antisera to B. pertussis agglutinogens 1, 2, and 3 was detected in bacterial culture at the end of exponential phase of growth. The activity of agglutination reaction decreased when cultures in stationary and death phases were used. During transition from exponential to death phase level of antibodies to agglutinogen 2 decreased by4 - 32 times. 2 - 4-fold decrease of antibodies level was observed when antiserum to agglutinogen 3 was used. Activity of agglutination reaction with antiserum to agglutinogen 1 was high and did not depend from phase of growth. When polyvalent antiserum to B. pertussis was used 4-fold decrease of antibody titers was observed in parallel with change of growth phases. Sera from rabbits immunized with B. pertussis cultures from the middle of exponential growth phase, the end of this phase, and begin of the death phase had high (maximal) level of agglutinating antibodies (6400), which was detected on 101 day after immunization with the former culture and on 31 day after immunization with either of the two latter cultures. To the end of experiment (292 day) titers decreased to 800, 3200, and 1600 respectively. These findings confirm an advisability of use of exponential growth culture for immunization of rabbits in order to obtain highly active diagnostic antisera to B. pertussis.     

Immunological Methods for the Detection of a Coryneform Isolated from Ratoon Stunted Sugarcane

A coryneform bacterium, isolated from ratoon stunted sugarcane, has been obtained in pure culture. Its identification by immunological means was investigated. Optimum conditions for the light microscopic observation of bacterial agglutination by specific antiserum, immunofluorescence and the microcapillary haemagglutination of IgG-sensitized erythrocytes by the bacterium were established. All three assay procedures are simple and rapid. By comparison, more samples can be handled at any one time bythe microcapillary haemagglutination method. Based on bacterial agglutination, the antiserum is specific for the coryneform isolated from ratoon-stunted sugarcane.     

A bacterial-induced lectin which triggers hemocyte coagulation in Manduca sexta

An inducible hemagglutinin termed M13, was purified from M. sexta hemolymph. M13 is a glucose-specific lectin which in addition to erythrocyte agglutination, can activate dedifferentiation of various hemocytes into a filamentous coagulation network. When lectin activity was inhibited with glucose or antiserum, neither erythrocyte agglutination or hemocyte coagulation occurred. When M13 was boiled or trypsin treated, hemocyte activation was lost, but erythrocyte agglutination remained. Hence M13 activity appears to be bimodal, possessing both a lectin activity and a hemocyte-coagulating activity.     

Serological method for identification of Vibrio parahaemolyticus from marine samples.

Use of agglutination with antiserum against lateral flagella (H-agglutination) for the identification of Vibrio parahaemolyticus was studied. Sucrose-negative bacteria were isolated from seawater, and their characterization was carried out by traditional biological tests and slide agglutination with antiserum specific to lateral flagella of V. parahaemolyticus. Of 135 strains isolated, 78 were identified as V. parahaemolyticus by biological tests and were agglutinated with the above serum. Fifty-five strains did not agglutinate with the serum, and their biological characteristics were different from those of V. parahaemolyticus. Two strains also differed from V. parahaemolyticus in some biological characteristics but agglutinated with the antiserum. All clinically isolated V. parahaemolyticus strains also agglutinated with the above serum. These results suggest that our serological method is useful for the identification of V. parahaemolyticus, especially for samples in which there are many organisms related to V. parahaemolyticus, because many biological tests can be omitted.     

南方鲇血型的初步鉴定

本研究旨在通过观察南方鲇血清与其红细胞的交叉反应以鉴定南方鲇的血型.实验结果表明:南方鲇的血清与同种其他个体的红细胞进行交叉反应时均未出现凝集现象,这表明南方鲇可能不存在血型或南方鲇具备血型但血清中相应的凝集素含量不足.以南方鲇的红细胞为抗原免疫日本种大耳白兔制备的抗血清与南方鲇的红细胞进行交叉反应,出现了不同程度的凝集反应,这表明南方鲇存在血型.据上述两个实验结果可以推断,南方鲇可能存在4种血型,分别命名为NA、NB、NAB和NO型;同时也证实,在鉴定南方鲇血型的研究中,通过制备抗血清与红细胞进行交叉反应的方法更为可靠.     

Isolation and concentration of Salmonellae with an immunoaffinity column

A method for rapidly and selectively isolating Salmonellae from buffer solutions and concentrating the bacteria by a factor of approximately 500 was developed. Anti-Salmonellae antibody was covalently linked to 40 microm polyacrylamide beads to prepare a solid phase with affinity for the bacteria. The beads were packed into 1-mm diameter glass tubes to form a column 20 microl in volume. Buffer containing Salmonellae at concentrations ranging from 10(2) to 10(6)/ml was pumped through the column to trap and concentrate the bacteria. At a flow rate of 50 microl/min, more than 95% of the bacteria introduced to the column were captured, while at 800 microl/min capture dropped to 32%. Specificity was high, with no detectable capture of Escherichia coli at a concentration of 10(5)/ml. Capture of more than 90% of Salmonellae in a 5-ml sample was achieved in 40 min by re-circulating the sample through the column at a flow rate of 500 microl/.     

Rapid detection and identification of Erwinia carotovora subsp. atroseptica by a conjugated Staphylococcus aureus slide agglutination test

A. MCLEOD AND M.C.M. PEROMBELON. 1992. A conjugated Staphylococcus aureus slide agglutination test was used to detect and identify the potato blackleg pathogen, Erwinia carotovora subsp. atroseptica. Agglutination was obtained with > 10⁸ cfu/ml of the homologous strain with a polyclonal antiserum (171) against E.c. atroseptica serogroup I which is the predominant E.c. atroseptica serogroup on potatoes in Scotland. The titre of antiserum 171 against live cells of E.c. atroseptica groups I and XXII was 2000 whereas that of other serogroups was considerably less; only 1 and 4 out of 22 serogroups of E. carotovora subsp. carotovora reacted at 1:1500 and 1:1000 antiserum dilutions, respectively and one of the three less common other E.c. atroseptica serogroups reacted at 1:1000. When tested against 24 different bacterial species including E. chrysanthemi and saprophytic bacteria present in potato tuber rots, negative results were obtained with 1:1000 antiserum dilution. The titre against heat-treated (1 h, 70°C) cells of E.c. atroseptica serogroups I and XXII was1700–2000 whereas it was < 10 against other bacteria including E.c. carotovora. Detection of E.c. atroseptica serogroups I and XXII in diseased potato tissues was achieved directly by the slide agglutination test, but lower antiserum dilutions (1:700–1000) were needed. Still lower antiserum dilutions were needed with heat-treated test material for E.c. atroseptica identification.     

Use of ISO-NOP200 for measurement of NO in the gas phase under controlled humidity conditions.

A technique has been developed to measure nitric oxide (NO) in the gas phase using the ISO-NOP200 NO-specific probe, which was designed to only measure NO in solution. It was found that probe output was responsive to the relative humidity (RH) of the atmosphere. Increasing sensitivity of probe output to NO was observed with increasing RH but the time to achieve a stable output was also increased. The recommended method to give high sensitivity but an acceptable time between analyses was to hold the probe at a constant temperature (20 degrees C) in a sealed 20 ml glass vial containing 4 ml of a saturated solution of NaCl, which provides a constant RH of 75%. NO standards and samples were injected directly into the vial and provided good baseline stability and a limit of detection of 0.18 microl/L in the vial. The limit of detection of the analytical sample will depend on the volume of gas injected into the vial. Up to 4 ml could be injected without disturbing probe stability and this equates to a detection limit of 0.75 microl/L NO. However, analysis of the internal atmosphere of banana fruit could only consistently extract 1 ml of gas, which gave a detection limit of 3 microl/L NO.     

Left ventricular volume measurement in mice by conductance catheter: evaluation and optimization of calibration

The conductance catheter (CC) allows thorough evaluation of cardiac function because it simultaneously provides measurements of pressure and volume. Calibration of the volume signal remains challenging. With different calibration techniques, in vivo left ventricular volumes (V(CC)) were measured in mice (n = 52) with a Millar CC (SPR-839) and compared with MRI-derived volumes (V(MRI)). Significant correlations between V(CC) and V(MRI) [end-diastolic volume (EDV): R(2) = 0.85, P < 0.01; end-systolic volume (ESV): R(2) = 0.88, P < 0.01] were found when injection of hypertonic saline in the pulmonary artery was used to calibrate for parallel conductance and volume conversion was done by individual cylinder calibration. However, a significant underestimation was observed [EDV = -17.3 microl (-22.7 to -11.9 microl); ESV = -8.8 microl (-12.5 to -5.1 microl)]. Intravenous injection of the hypertonic saline bolus was inferior to injection into the pulmonary artery as a calibration method. Calibration with an independent measurement of stroke volume decreased the agreement with V(MRI). Correction for an increase in blood conductivity during the in vivo experiments improved estimation of EDV. The dual-frequency method for estimation of parallel conductance failed to produce V(CC) that correlated with V(MRI). We conclude that selection of the calibration procedure for the CC has significant implications for the accuracy and precision of volume estimation and pressure-volume loop-derived variables like myocardial contractility. Although V(CC) may be underestimated compared with MRI, optimized calibration techniques enable reliable volume estimation with the CC in mice.