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.

Genetics of anti-EHV antibody responses in a horse population.

Individual variation in immune responses to herpesviruses was observed in various species. Here, associations between polymorphic molecular markers and life-long anti-EHV-1/4 antibody immune responses were analyzed in a model EHV-infected population of the Old Kladruber horses. Two-dimensional analysis including overall mean titers and titer dynamics expressed by differences between spring and autumn titers allowed identification of low-responders. 50 randomly selected microsatellites and nine single nucleotide polymorphisms in nine immunity-related candidate genes were genotyped. Due to differences (p<0.001) in antibody titers between two color varieties of Old Kladruber horses, separate association studies were performed in the two sub-populations by using the Fisher's exact test. In black horses, the interleukin 4 receptor and MxA protein coding genes, and the microsatellite TKY325 were associated with the responder status. In the grey population, the microsatellite TKY343 showed significant association with anti-EHV antibody responsiveness after Bonferroni corrections.

Expression and serological reactivity of Nsp7 protein of PRRS genotype I virus.

The aim of this work was to express recombinant nonstructural Nsp7 protein of European genotype of porcine reproductive and respiratory syndrome virus and to evaluate its diagnostic sensitivity and specificity in serological diagnostics of the disease. The gene coding for Nsp7 protein was expressed in Escherichia coli cells and purified by IMAC. Serological reactivity of purified protein was assessed on a panel of swine sera in indirect ELISA test. Serum samples originated from PRRS positive farms, herds free of PRRS infection and PRRS free herds vaccinated with an inactivated vaccine. Nsp7 antigen proved to be suitable for serological detection of PRRS specific antibodies, showing diagnostic sensitivity of 82.2% and specificity of 97.6% when compared with IDEXX ELISA test. The nonstructural protein proved to be suitable for use as an antigen for the differentiation of post-infection and post-vaccination antibodies in pigs vaccinated with the inactivated vaccine. But the low overall antibody response to N protein after this type of vaccination makes this concept rather theoretical.

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.

Prevalence and age distribution of porcine torque teno sus virus (TTSuV) in the Czech Republic.

Torque teno sus viruses (TTSuV) were detected in the pig population in the Czech Republic by a nested PCR technique. The prevalence of individual TTSuV was found to be 42.9% (TTSuV1) and 46.7% (TTSuV2). The prevalence for TTSuV 1 and TTSuV2 was determined to be 52.7% and 60.6% in piglets at weaning, 90.9% and 63.6% in gilts, and 75% and 87.5% in sows, respectively. Both virus species were detected in 12% of newborn piglets, which suggests possible transplacental transmission. Sequencing of several virus strains showed that the sequenced TTSuV strains belong to two different species of viruses. The average similarity on the nucleotide levels between these two species was 43.3%.

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.

The selection of single-chain Fv antibody fragments specific to Bhlp 29.7 protein of Brachyspira hyodysenteriae.

Single-chain antibodies (scFv) specific to Brachyspira hyodysenteriae were isolated from a phagemid library. Recombinant Bhlp 29.7 protein was used for scFv selection and individual clones were tested by ELISA and immunofluorescent test; four unique clones were isolated. One of selected clones was able to bind specifically B. hyodysenteriae in ELISA and immunofluorescence test. This is the first report of species-specific recombinant antibodies against B. hyodysenteriae.

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.

[Pharmacological aspects of Lyme borreliosis]. / Farmakologické aspekty lymeské borreliózy.

Clinical signs of Lyme boreliosis in humans are versatile and in their whole scope they finally affect the nervous system, heart, and joints. The therapeutic effect of antibiotics is maximal in the first acute stage of the disease when doxycycline and amoxiciline are administered. These antibiotics possess a comparable in vitro effect, tissue penetration, pharmacokinetics, and therapeutic effect. The treatment of disseminated infections in the second stage, such as neuroborreliosis, carditis, and iritis, is difficult and with relative success they are treated with large doses of penicillin G, or cefriaxon, and doxycycline. The treatment of the third stage of borreliosis aims at chronic inflammatory changes in the affected organs. Antibiotics, however, are successfully effective only in 50% of cases. Administration of antibiotics, such as tetracycline, cefuroxim, doxycycline, or large doses of penicillin is a long-term one, coming up to four weeks. A special therapeutic regimen is used in pregnant women and children.

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 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.

Detection of antibodies to ovine lentivirus using recombinant capsid and transmembrane proteins.

The coding sequences of the capsid protein p25 and transmembrane protein of Maedi-Visna virus were amplified using polymerase chain reaction and cloned into the plasmid expression vector pRSET-B. Both DNA constructs expressed proteins tagged with polyhistidine. The recombinant proteins were purified using Ni-NTA agarose and used in immunoblot to detect antibodies against Maedi-Visna virus. A total of 260 ovine serum specimens was analysed. The total number of p25-positive sera was 111 (42.7%). Higher sensitivity was achieved with rTM antigen, which detected antibodies in 118 (45.4%) sera. The combination of both recombinant proteins as antigens resulted in higher sensitivity of serological detection compared to whole virus antigen.

The detection of proviral DNA by semi-nested polymerase chain reaction and phylogenetic analysis of Czech Maedi-Visna isolates based on gag gene sequences.

A semi-nested polymerase chain reaction (snPCR) for detecting proviral DNA of ovine lentivirus (OvLV) in peripheral blood mononuclear cells was developed. Primers for snPCR were situated within the gag gene of the Maedi-Visna virus (MVV) genome. A comparison between the snPCR and serological tests (agar gel immunodiffusion test, immunoblot) were performed using 98 ovine blood samples. Thirty (30.6%) of the 98 sheep examined had antibodies specific for the MVV. PCR showed 21 of them to be positive and nine seropositive animals to be PCR negative. Six of the 68 serologically negative sheep were found to be PCR positive, probably due to delayed seroconversion. The PCR amplification products of these six sheep were sequenced and subjected to phylogenetic analysis. The resulting phylogenetic tree of partial gag gene sequences confirmed that the ovine lentivirus genotype in the Czech Republic is more closely related to the prototype MVV isolates than to the caprine arthritis encephalitis viruses.

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.

Serologic diagnosis of ovine lentiviruses by whole virus ELISA and AGID test.

Enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) are the most widely used serological tests for Maedi-Visna diagnostics. The purpose of the present study was to develop an indirect whole virus ELISA and an immunodiffusion test and compare their sensitivity. A total of 747 ovine serum specimens were analysed for antibodies against this ovine lentivirus. The number of positive results in the ELISA was 430 (57.56%). In the AGID test, a positive result was found in 380 samples (50.87%). In the group of discordant results 78 (10.4%) samples tested positive by the ELISA and negative by the AGID test and 28 sera (3.7%) were found to be positive by the AGID test and negative by the ELISA. The data in this report show the ELISA to be more sensitive than the AGID test, but accurate serological diagnostics should be based on a combination of the two tests.

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.

[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.