Biochemical screening of highly toxic aromatic contaminants in river sediment and comparison of sensitivity of biological model systems.

Fractions containing polycyclic aromatic hydrocarbons (PAHs), polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and polychlorinated biphenyls (PCBs) were extracted from river sediments by various extraction methods. The amount of individual pollutants was determined analytically and data compared with biological assays. These were based on the induction of cytochrome P450 1A1 (CYPIA1) after treatment with sediment fractions in two different biological model systems, a mouse hepatoma cell line Hepa-1 and a chick embryo. In the hepatoma cell culture Hepa-1 significant correlations with analytical results were found for fractions containing PCDD/Fs and planar and mono-ortho-chlorinated PCBs. However for PAH fraction an undesirable decrease of P450 1A1 induction was observed in higher concentrations of this fraction. This decrease was not observed in the chick embryo liver microsomes and biological responses towards the PAH fractions correlated with analytical data. Comparative investigations demonstrated that the chicken embryo hepatic microsomes were more sensitive for PAHs, and the hepatoma cell line Hepa-1 for PCDD/Fs and planar and mono-ortho-chlorinated PCBs.

[Culture and control of cells producing bovine leukemia virus]. / Kultivace a kontrola bunek produkujících bovinní leukemický virus.

In the field surveys of the occurrence of enzootic bovine leucosis caused by the bovine leucosis virus (BLV), the identification of positive animals is based on the detection of specific antiviral antibodies by serological methods. The reliability of these tests (particularly their sensitivity and specificity) depends on the quality of the virus antigen. The preparation of the antigen is based on the cultivation of BLV virus in cultures of the FLS cell line. A modified procedure of preparing the BLV antigen in the FLS cell culture is described, along with the control of its production by the immunoperoxidase test.

[Use of the tetrazolium salt reduction test in the detection of phagocytic activity in pigs]. / Pouzití testu redukce tetrazoliové soli k prukazu fagocytární aktivity u prasat.

One of the indicators of non-specific, cell-mediated immunity can be the ability of phagocytic system to react to the presence of antigenic impulses and stimulators. The phagocytic activity induced in this way in vitro can be evaluated quantitatively by help of different methods. In this study the method of the reduction of colourless tetrazolium salt (INT) to the red formazane was used. The optimum conditions for carrying out this test in isolated peripheral pig leucocytes were determined. It was stated that 3-4 ml samples of pig blood, from which 6.10(6) leucocytes necessary for the test can be isolated, were sufficient for the examination of phagocytic activity. Further, 45 minutes were determined as the optimum time of incubation It was found that the INT test can be reproduced by 24 hours after blood sampling provided the blood is kept at the temperature of +4 degrees C. The INT test was used for examining 38 pig blood samples before and after myostress; this stress has a significant effect both on a decrease of phagocytic activity and on the ability of leucocytes to migrate in the LMI test. The INT test was further used for examining pigs before the administration of the first dose of the inactivated vaccine against the Aujeszky's disease virus and two days after; the application of the vaccine significantly increased the phagocytic activity.

[Cell-mediated immunity in calves immunized against or infected with the bovine rhinotracheitis virus]. / Bunkami zprostredkovaná imunita u telat imunizovaných nebo infikovaných virem infekcní bovinní rhinotracheitidy.

Simple and expeditious methods--leucocyte adherence inhibition LAI and leucocyte migration inhibition LMI--are described in the present paper; these methods enable to investigate cell-mediated immunity of animals. The two tests, along with serological and virological methods, were used to study changes in the immunity of calves after experimental infection by IBRV and after immunization by inactivated oil vaccine against IBRV. The results have indicated that the changes in cell-mediated immunity after experimental infection and vaccination of calves assumed courses independent of the changes in humoral reaction. Total cell-mediated immunity reaction started earlier in experimentally infected calves than in vaccinated ones. In some vaccinated calves a modified course of cellular immunity reactions was observed, characterized also by elimination of IBR virus on the nasal mucous membrane after challenge infection. The LAI and LMI test can be recommended for immunological monitoring in clinical laboratories and in research.

[Detection of cytotoxic antibodies in the serum of cattle with leukemia]. / Prukaz cytotoxických protilátek v séru leukózního skotu.

In a set of 55 serum samples obtained from the herd of bulls heavily infested with bovine leukosis we tested the applicability of cytotoxic test for the diagnostics of bovine leukosis we tested the applicability of cytotoxic test for the diagnostics of bovine leukosis and compared the results with immunodiffusion test in agar. The two tests show approximately the same sensitivity because in each examined sample the results of cytotoxic test corresponded at least in two out of three serum dilutions (1:2, 1:5 and 1:7) to the immunodiffusion test. It has been demonstrated that as regards the cytotoxic test, the final serum dilution of 1:5 was satisfying. At the same time it has been checked that the bovine foetal serum, required for the growth of cell cultures, could be replaced by the bovine serum treated with the 40% solution of polyethylene glycol (PEG) 6000 without any influencing the results of cytotoxic test.

[Detection of bovine leukemia virus antibodies using the cytotoxicity test in comparison with other serologic methods]. / Prükaz protilátek proti viru bovinní leukózy cytotoxickým testem ve srovnání s jinými sérologickými metodami.

In ninety-five serum samples taken in a herd of five-year to seven-year cattle that was heavily infected by bovine leukosis virus, the four serological assays were used for demonstration of the antibodies to bovine leukosis virus; cytotoxic test, immunodiffusion test in agar-agar, immunoenzymatic test and serum neutralizing test. The serum neutralizing test was found to be the most sensitive: further seven positive reagents were diagnosed in comparison with immunoenzymatic test; cytotoxic and immunodiffusion tests in agar-agar have the lowest sensitivity and the results of these tests are almost identical. It was found out in forty titrated samples that serum neutralizing test was by as much as 20 times more sensitive than immunoenzymatic test, the latter being about 50 times more sensitive than cytotoxic and immunodiffusion tests.

[Immunoenzyme detection of enzootic bovine leukemia viruses (BLV) in cell cultures]. / Imunoenzymatický prukaz viru enzootické leukózy skotu (BLV) v bunecných kulturách.

There is a description of the method of immunoenzymic test used for detection of enzootic bovine leukosis virus (BLV) on the cells of foetal lamb spleen (FLS) permanently producing BLV and on the lymphocytes of infected animals. In both cases (in FLS cells and lymphocytes) the presence of BLV was demonstrated by the immunoperoxidase test. Using the defined serums from the animals after experimental and natural infection, BLV was detected by the above-mentioned test in FLS cells in all cases of the use of positive serums with anti-BLV antibodies. The specificity of the antibodies was also demonstrated in this way. The presence of BLV was analogically detected in the lymphocytes of the infected animals where the infection time was precisely defined. The method of immunoperoxidase test on lymphocytes is recommended in indicated cases for the demonstration of the presence of BLV in infected animals.

[Detection of enzootic bovine leukemia virus using the syncytium test]. / Prükaz viru enzootické leukózy skotu (BLV) syncytiálním testem (SyT).

BLV detection by the syncytial test was performed in 27 heifers experimentally and naturally infected by the enzootic bovine leukosis virus (BLV). The presence of BLV was demonstrated in 94.7% of the animals. The bovine foetal spleen cells (FBS) were found to be suitable for the syncytial test. Positive animals not reacting to infection by the production of anti-BLV antibodies were identified during the syncytial-test investigation. The importance of this finding for the programme of controlling enzootic bovine leukosis on farms is discussed. As suggested by the results, temporary occurrence of anti-BLV antibodies followed by their disappearance can be observed together with a negative result of the syncytial test in some circumstances. The discussion deals with the problems of the determination of anti-BLV antibodies in milk, and/or milk secretion, by the ELISA method.

[Experimental and natural infection with the enzootic leukosis virus of cattle]. / Experimentální a prirozená infekce virem enzootické leukózy skotu (BLV).

A trial was performed with heifers at the age of six to seven months. The animals were experimentally infected with the lymphocytes of a virus-productive donor. Infection was produced in all the nine cases, as demonstrated by means of the positive syncytial test. As indicated by the results of the trial, the antibodies to the enzootic bovine leucosis virus (BLV) were produced soon after experimental infection. A high sensitivity of the serum-neutralization test and the ELISA method was demonstrated in this connection: by these methods, the antibodies were identified already two to three weeks after experimental infection whereas by the immunodiffusion test they could be detected only after five weeks. Twenty-four animals were exposed to natural contact infection. Within 270 days of the trial, the disease after contact was recorded only in one heifer out of the four that were in close contact with the experimentally infected animals. In this case, as compared with experimental infection, the antibodies were produced much later--after 85 to 93 days. Leucosis was recorded in none of the remaining animals. The reasons why such a favourable result was obtained were the thorough disinfection of the stables after blood collections and the strict observance of the aseptic conditions. The results of experimental infection in three cows were identical with those obtained in young cattle. In the experimentally infected dairy cows, antibodies in milk were determined by the ELISA method. As found, in milk the antibodies to BLV appear two to three weeks later than they do in serum. The ELISA method of BLV antibody detection can be used for the identification of infected animals in herds where enzootic bovine leucosis occurs.

Comparison of mouse and chick embryo liver and hepatoma cell lines as model systems used for enzymological estimation of toxicity potentials of organic pollutants.

Cytochromes P450-dependent monooxygenase activities were determined and compared in mouse liver microsomes and in hepatoma cell homogenates after exposure to prototype inducers of individual P450 enzymes. In vivo inductions of levels of mouse hepatic monooxygenase activities have been found as effective biochemical markers of toxicity potentials of a series of classes of xenobiotics (CYP1A induction for toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin, coplanar polychlorinated biphenyls, polycyclic aromatic hydrocarbons and related pollutants; CYP2E induction for dialkylnitrosamines and organic solvents, e.g. acetone and ethanol; CYP2B and CYP3A induction for phenobarbital- and dexamethasone-type of xenobiotics). A specific induction of CYP1A-dependent O-dealkylase activities by TCDD was found in Hepa-1 and Hep G2 cell cultures, but no in vitro induction of other P450 enzymes was found after the treatment with phenobarbital, acetone or dexamethasone. Therefore, mouse liver is a suitable in vivo system for the testing of inducing effects of xenobiotics on all relevant P450 forms, while hepatoma cell cultures are usable only for the bioassay of TCDD-like toxicity.

[Radioimmunologic detection of antibodies to bovine leukemia virus]. / Radioimunologický prukaz protilátek proti viru leukózy skotu.

The radioimmunologic assay (RIA) was elaborated for a demonstration of serum antibodies to bovine leukosis virus. The procedure makes use of the viral antigen bond to the fixed phase of a polystyrene carrier. The method was compared with the ELISA method and pseudoneutralizing and immunodiffusion tests. High congruence of the results of the RIA and ELISA methods was achieved, making 95%. The RIA method is more sensitive than the immunodiffusion test.

Monoclonal antibody for the demonstration by ELISA of antibodies to protein p24 of enzootic bovine leukosis virus in individual and pooled blood serum and milk samples.

A sensitive and specific ELISA for the demonstration of antibodies to the protein p24 of enzootic bovine leukosis virus (EBVL) using a 'capture' monoclonal antibody to this protein (MAb p24) was developed. The method is sensitive enough to detect the international reference serum E4/10 in pooled blood serum samples collected from up to 50 cows, or, if a 10-fold concentrate of milk whey is tested, in samples of bulk milk collected from up to 400 cows. The application of MAb p24 has considerably increased not only the sensitivity, but also the specificity of ELISA. Moreover it is possible to differentiate reliably between positive and 'false positive' reagents by testing a suspicious sample in a pair of wells of which one is coated with MAb p24 alone and the other with the complex MAb p24 + EBLV antigen and the subsequent calculation of 'specific absorbance'. This method, showing the highest sensitivity of detection of antibodies to EBLV p24 described so far, can become an effective tool on the sanitation of infected herds as well as in checks of the EBL-free status. A diagnostic kit suitable for commercial manufacture has been devised.

Monoclonal antibodies to rabbit haemorrhagic disease virus and their use in the diagnosis of infection.

Hybridomas producing monoclonal antibodies (MAbs) to rabbit haemorrhagic disease virus (RHDV) were prepared. Using Western blot (WB) analysis, the MAbs obtained were divided into two groups, one reacting with the major structural proteins of Mr 61K and 38K, and the other giving negative reactions. Both groups of MAbs, however, reacted specifically with RHDV in ELISA and by immunoperoxidase (IP) and immunofluorescence (IF) tests with infected cells. As demonstrated by WB using RHDV-specific MAbs and a MAb to feline calicivirus (FCV) strain F9, the major structural (capsid) proteins of RHDV and FCV have very similar sizes (Mr61K and 38K compared to 62K to 64K and 40K respectively). No cross-reactions of MAbs with proteins of the other virus were observed in WB analysis, ELISA, IP tests or IF. The high specificity and sensitivity of RHDV-specific MAbs make them suitable for the routine IP and IF diagnosis of RHDV in liver cells of rabbits dying after natural or experimental infections.