Biological Effects of Drug-Free Alginate Beads Cross-Linked by Copper Ions Prepared Using External Ionotropic Gelation.

External ionotropic gelation offers a unique possibility to entrap multivalent ions in a polymer structure. The aim of this experimental study was to prepare new drug-free sodium alginate (ALG) particles cross-linked by Cu2+ ions and to investigate their technological parameters (particle size, sphericity, surface topology, swelling capacity, copper content, release of Cu2+ ions, mucoadhesivity) and biological activity (cytotoxicity and efficiency against the most common vaginal pathogens-Herpes simplex, Escherichia coli, Candida albicans) with respect to potential vaginal administration. Beads prepared from NaALG dispersions (3 or 4%) were cross-linked by Cu2+ ions (0.5 or 1.0 M CuCl2) using external ionotropic gelation. Prepared mucoadhesive beads with particle size over 1000 µm exhibited sufficient sphericity (all ˃0.89) and copper content (214.8-249.07 g/kg), which increased with concentration of polymer and hardening solution. Dissolution behaviour was characterized by extended burst effect, followed by 2 h of copper release. The efficiency of all samples against the most common vaginal pathogens was observed at cytotoxic Cu2+ concentrations. Anti-HSV activity was demonstrated at a Cu2+ concentration of 546 mg/L. Antibacterial activity of beads (expressed as minimum inhibition concentration, MIC) was influenced mainly by the rate of Cu2+ release which was controlled by the extent of swelling capacity. Lower MIC values were found for E. coli in comparison with C. albicans. Sample ALG-3_1.0 exhibited the fastest copper release and was proved to be the most effective against both bacteria. This could be a result of its lower polymer concentration in combination with smaller particle size and thus larger surface area.

Evaluation of the use of recombinant Bhlp29.7 in immunoblotting with pig serum as a means to identify herds infected with Brachyspira hyodysenteriae.

AIMS: Aim of the study is to evaluate the use of recombinant Bhlp29.7 in immunoblotting with sera as a means to detect pig herds infected with Brachyspira hyodysenteriae. METHODS AND RESULTS: Sera samples from 789 sows and rectal swabs from 838 pigs of various categories on 22 farms of different size (median 450 animals), production type and history of swine dysentery (SD) were examined. Sera from 378 sows from farms with previous SD history were examined via immunoblotting. Specific antibodies were detected in 79 of these (20.9%). Examination of 411 serum samples from sows and gilts taken on 11 farms without previous history of SD detected specific antibodies in 13 sows and gilts (3.2%). These 13, however, had come from farms where the presence of B. hyodysenteriae was confirmed or SD status was not known. Seroprevalence in herds with previous SD history ranged from 2.5 to 35.7%. B. hyodysenteriae was confirmed on six (27.3%) of 22 monitored farms. CONCLUSIONS: Immunoblotting using recombinant antigen Bhlp29.7 in conjunction with culturing B. hyodysenteriae proved to be a valuable tool for detecting swine herds latently infected with B. hyodysenteriae. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of immunoblotting with recombinant Bhlp29.7 should prove to be a useful adjunct to detecting herds with SD, and hence, it will assist in controlling this important disease.

In vitro neutralization of equid herpesvirus 1 mediated by recombinant antibodies.

A phage antibody display library of single chain fragment variables (scFv) was applied to develop anti-equid herpesvirus-1 (EHV-1) glycoprotein D (gD) neutralizing antibodies. To enrich for specific scFvs, the phage antibody library was panned against epitope derived from the N-terminal part of EHV-1 gD. Unique clones were differentiated by BstNI fingerprinting and further characterized by sequencing and immunoreactivity. The neutralizing effect of each clone was assessed by plaque reduction assay. Three clones with neutralizing effect were isolated.

Isolation of Lawsonia intracellularis specific single-chain Fv antibody fragments from phage display library.

Single-chain antibodies (scFv) exhibiting specific binding to Lawsonia intracellularis were isolated from a phagemid library expressing scFvs molecules on the surface of filamentous bacteriophages. For scFv selection whole bacterial cells were used and individual clones were tested in ELISA test. The total of seven unique clones with different fingerprint profiles was isolated. All clones were able to bind specifically in immunofluorescence assay. This is the first report of species specific recombinant antibodies against L. intracellularis.

The levels of PCV2 specific antibodies and viremia in pigs.

Porcine circovirus 2 quantification by real-time PCR and determination of virus specific antibodies using a peptide based ELISA test was performed on a total of 400 serum samples. Samples for antibody measurement and virus quantification were obtained from a conventional pig farm with a clinical form of post-weaning multisystemic wasting syndrome. Samples were taken during the post-weaning period. Seven litters of weaned piglets (6-25 weeks) were systematically sampled at 2-3 week intervals. Porcine circovirus 2 specific antibodies were detected using the peptide based ELISA test. IgM antibodies were first detected at week 8 and reached their highest levels at week 12; IgG antibody appeared at week 10 and peaked at week 16 with an average titer at 1:3500. Although, the viral load peaked at week 10 (7x10(7) genomes copy/ml of sera), viral infection persisted through to adult age (10(5) genomes copy/ml of sera).

Recombinant single chain Fv antibodies specific for glycoprotein D of equid herpesvirus 1.

Single chain Fv (scFv) molecules generated by phage-display technology represent a new and efficient tool in the research and diagnostics of infectious diseases. The recombinant glycoprotein D of Equid herpesvirus 1 was successfully expressed in E. coli cells. The protein was produced predominantly in soluble fraction and was then purified on a nickel-agarose column. The scFv antibodies against the glycoprotein were selected and several clones of glycoprotein D-specific scFv-antibodies were identified; t of them was expressed as a soluble scFv molecule, purified by immobilized metal-affinity chromatography and used as reagent in an immunofluorescence test.

Detection of feline immunodeficiency provirus by seminested polymerase chain reaction.

Specific primers for the detection of the FIV provirus in peripheral blood mononuclear cells (PBMC) by seminested PCR (snPCR) were developed. Forty cats (patients from veterinary hospitals) were investigated for the presence of FIV serum antibodies by immunoblot and for the presence of provirus in PBMC by snPCR. Seventeen of the 40 examined samples (42.5%) showed the presence of antibodies against FIV. The total number of animals that were found positive in snPCR was 19 (47.5%). Fourteen of the seropositive animals (35%) were positive by snPCR whereas three seropositive animals (7.5%) turned out to be snPCR negative. Of twenty-three animals that were negative by immunoblot, five (12.5%) were found to be positive by snPCR.

Isolation and partial characterization of ovine lentivirus in Czech Republic.

The first isolation and partial characterization of ovine lentivirus in Czech Republic is described. The virus was isolated in a tissue culture system derived from plexus choroideus from sheep. The new isolate was compared with prototypic K1514 Maedi-Visna strain; these two viruses shared antigenic determinants as determined by serological testing. Both viral strains reacted in a PCR reaction with primers situated in the gag and pol gene. Based on similarities of growth characteristics, antigenic determinants and primer binding sites it can be concluded that the isolate OPM is an ovine lentivirus and is at least partly related to the prototypic Maedi-Visna strain K1514.

The production and application of single-chain antibody fragments.

This review discusses methods for the single-chain antibody fragment ($cFv) generation and scFv expression systems, and describes potential applications of scFv in the therapy of viral diseases and cancer, with emphasis on intracellularly expressed scFvs (intrabodies), application of scFvs in detection and diagnostics, and their use in proteomics.

Isolation and partial characterization of equine herpesvirus type 1 in Czechia.

Equine herpesvirus type 1 was determined as the etiological cause of an abortion storm in Czechia in 2003 after the virus strain was isolated from aborted fetus and identified by serological means and by PCR technique. Cloning and sequencing of the glycoprotein D confirmed the identity of the isolates and showed molecular relationships to known EHV-1 strains. Comparison of glycoprotein D sequences with corresponding sequence of EHV-1 reference strains (Kentucky-A and Ab1) revealed high nucleotide homology. The Czech isolate of EHV-1 virus does not differ significantly from the Ab1 strain regarding the glycoprotein D gene and does not bear the frameshift in the 3' terminus which occurs in the Kentucky-A strain.

Recombinant single-chain Fv antibodies that recognize the p25 protein of the Maedi-Visna virus.

Single chain Fv (scFv) antibodies (generated by phage display technology, molecules representing new and efficient tools in the research and diagnostics of infectious diseases) against the capsid protein (p25) of Maedi-Visna virus were selected. Several clones of p25 specific scFv antibodies were identified; one of them was expressed as a soluble scFv molecule, purified by immobilized metal-affinity chromatography and further characterized by sequencing and determination of the kinetic equilibrium association constant. Sequence analysis showed that the rearranged VL and VH domains of the analyzed scFv clone used sequences from the VL3 family (germline DPL16/VL3.1) and VH1 family (germline VH20), respectively. The kinetic equilibrium association constant was determined as KA = 1.12 +/- 0.52 L/mumol.

[The role of small terrestrial mammals in the epidemiology of rabies]. / Uloha drobných zemních savcu v epidemiologii vztekliny.

The examination should help to elucidate the possibility of virus occurrence in free living small terrestrial rodents. The examination was oriented on the following: 1. Animals living in natural surroundings. 2. Rodents examined after injuring man. The examination was carried out by means of the direct immunofluorescent test and, partially, also by biological assay on suckling laboratory mice. In the first part of experiment, more than 10,000 small terrestrial mammals were entrapped, belonging to 16 species. In the second part of experiment, 1,969 rodents, belonging to 12 species, were examined after injuring humans. In these cases, a biological test was also carried out to demonstrate the occurrence of the virus. In neither of the above mentioned experiment the occurrence of rabies was proved.

[Suppressive effects of ionizing radiation on immunoproductive cells in laboratory mice]. / Supresivní úcinek ionizujícího zárení na imunoproduktivní bunky laboratorních mysí.

The very sensitive Jerne method of detecting the plaque-forming cells was used in the study of the immunosuppressive effect of ionizing radiation on laboratory mice. A significant immunosuppression was obtained after irradiation of the individual groups of mice with 3, 4, 5 and 6 Gy. Successive regeneration of the immunity system occurred after 42 hours only in the groups of mice irradiated with 3 and 4 Gy. mice; ionizing radiation; immunoproductive cells; Jerne method.

[Preparation of precipitation antigens for the diagnosis of Maedi-Visna]. / Príprava precipitacního antigenu k diagnostice Maedi-Visna.

In the present paper antigen preparation is described to be used for diagnostics of Maedi-Visna disease by immunodiffusion test (IDT). The antigen was prepared by cultivation of the Maedi-Visna virus, OLV strain, in cell culture of the synovial membrane of carpal joint and plexus choroides of lamb. Cell cultures were used for 10-25 subcultures. The antigen was concentrated by polyethylene glycol application. The characteristics of the prepared diagnostic antigen were evaluated by its comparison with a reference, commercial preparation. Sufficient sensitivity and specificity of the prepared antigen was demonstrated by testing in a sample of 100 ovine serums.

[Detection of parvoviruses in dogs using the hemagglutination test]. / Prukaz parvoviru psu hemaglutinacním testem.

The haemagglutination activity of the causal agent of canine parvovirosis is described. Out of the tested erythrocytes of pig, monkey, cat, horse, cattle, sheep, rabbit and guinea-pig, a positive reaction was recorded only in the erythrocytes of pig and monkey at the temperature of 4 degrees C. As a result of the examination of 20 faeces samples of hospitalized dogs by the method of haemagglutination reaction, a positive reaction typical of canine parvovirosis was obtained in 17 cases. The specificity of the reaction was confirmed by antiserum against the virus of panleucopenia of cat in the haemagglutination-inhibition test.

[Pathology and differential diagnosis of Maedi-Visna disease]. / Patologie a diferenciální diagnostika onemocnení Maedi-Visna.

The intensity and distribution of typical lesions in organs and tissues were determined in 38 sheep with serological positivity to the Maedi-Visna disease. The lesions in the lungs, mammary gland, CNS and joints were evaluated according to the number of marks. The lungs, mammary gland and CNS were affected most frequently. Concurrent incidence of pulmonary adenomatosis was determined in 57% of the examined cases. Differential diagnostics of typical M-V virus induced lesions and pulmonary adenomatosis is discussed in this paper. Identification of eight cases of meningoencephalitis, which is considered as the initial stage of Visna form, is a priority observation in the Czech Republic. With respect to distribution of lentivirus carriers, the authors emphasize the need of detailed pathomorphological examination of culled sheep.

[Pathologic changes in the organs of sheep serologically positive for Maedi-Visna virus antibodies]. / Výskyt patologických zmen v orgánech ovcí sérologicky pozitivních na protilátky proti viru Maedi-Visna.

In the present paper the results are presented of pathomorphological examination of 38 sacrificed sheep with positive reactions to the Maedi-Visna disease. Lungs, mammary gland, joints and other organs with macroscopical lesions were subjected to histopathological examination. The CNS was examined by the method of a complete series of frontal sections. Diagnostics of M-V typical lesions revealed them in 87% of the examined sheep, in 55.2% of examined lungs, in 58.6% of examined mammary glands, 27.3% examined CNS and 4.7% examined joints. In 57.1% of the cases, M-V lesions of the lungs were complicated by simultaneous incidence of pulmonary adenomatosis, chronic purulent or granulomatous pneumonia. No clinical symptoms were found in a majority of sheep, while the M-V typical lesions of organs were not determined in about fifty percent of sheep. The observed morphological lesions corresponded to initial or medium progressive affection. A proof of precipitation antibodies and identification of typical M-V lesions in the organs are needful for determination of M-V diagnosis. Incidence and character of lesions indicate the need of persistent implementation of virus-eradication treatments on sheep farms.

[Serological detection of parvovirus infection in dogs using the hemagglutination inhibition test]. / Sérologický prukaz parvovirové infekce psu hemaglutinacne inhibicním testem.

Throughout the year 1982 the parvovirus disease of dogs in the Czechoslovak Socialist Republic was proved by means of haemagglutination-inhibition test (HIT). 317 serum samples of dogs from different localities were examined. The highest spreading of this disease was detected in urban areas where the possibilities of direct and indirect infection were much higher. In 70% of the cases the titres of HI antibodies varied from 256 to 4096. The HIT method is suitable for serological screening of parvovirus disease of dogs.

[Antibodies to certain infections on large goat farms in the Czech Republic]. / Protilátky proti nekterým infekcím ve velkochovech koz v Ceské republice.

On five newly established large goat farms the incidence of antibodies to some chronic and latent infections (arthritis and encephalitis, Q-fever, caseous lymphadenitis and toxoplazmosis) was investigated in the first six months of the year 1994. Agar-gel immunodiffusion did not reveal any antibodies to arthritis and encephalitis of goats (CAE). Complement fixation test did not demonstrate any antibodies to Q-fever. Neither agar-gel immunodiffusion nor neutralization test confirmed any antibodies to caseous lymphadentitis. Complement fixation test (titer 1:8 and more) revealed antibodies to toxoplazmosis at 20.2% of the cases. No clinical symptoms of toxoplazmosis were observed on the investigated goat farms. It was recommended to take a preventive serological examination of goats against CAE, Q-fever and caseous lymphadenitis before they were housed on the given farms. By maintaining high zoohygienic parameters on the large goat farms it is possible to except a decrease in prevalence of antibodies to toxoplazmosis.

[Comparison of various antigens in the diagnosis of caprine arthritis-encephalitis virus using the ELISA test]. / Porovnání ruzných antigenu pro diagnostiku CAEV testem ELISA.

The most adequate means of diagnosing infections with caprine arthritis encephalitis (CAE) and Maedi-Visna (MV) viruses is the demonstration of antibodies to these viruses in milk or in blood serum. Various techniques and a range of different antigen preparations can be used for this purpose. We have evaluated two different whole virus antigen preparations derived from lamb synovial cells infected with CAE and MV viruses and recombinant antigen containing the capsid protein expressed in E. coli in ELISA test. The suitability of different detergents for solubilization of whole virus particles is shown in Tab. I. The detergents used were ether (50%, 10 min, 37 degrees C), octyl-beta-D glucopyranoside - OGP (2%, 2 h, 37 degrees C), and sodium dodecylsulphate SDS (0.125%, 10 min, 37 degrees C). Antigens prepared with SDS gave the best results and were then used in the following antigen comparative study. All antigens (whole virus and recombinant core protein) were used for the coating of ELISA plates and were evaluated on a panel of 130 sera. In general, whole virus antigens prepared from CAE- and MV-viruses gave identical results. The specificity achieved with recombinant antigen was superior but the sensitivity was lower compared with whole virus antigen. Immunoblotting served as the gold standard for discordant results. The other aim of our study was to compare antibody detection in blood serum and milk. Using whole virus antigen, antibodies could be detected with higher specificity and sensitivity in milk than in blood serum (Tab. II).(ABSTRACT TRUNCATED AT 250 WORDS)