Use of monoclonal antibodies in blocking ELISA detection of transmissible gastroenteritis virus in faeces of piglets.

Monoclonal antibodies (mAb) to the transmissible gastroenteritis virus (TGEV) nucleoprotein (N) and membrane protein (M) were prepared and used for the comparative assessment of three blocking ELISA variants to detect TGEV. The competitive blocking ELISA format showed the highest sensitivity, allowing detection of 10(3) TCID50 TGEV/ml in culture medium. Ninety-nine porcine field faecal samples obtained from 37 herds affected with diarrhoea were examined, and various TGEV levels were found in nine samples from six herds. However, only in three samples were significant TGEV concentrations demonstrated. The relationship between incidence of TGEV gastroenteritis and the spread of porcine respiratory coronavirus infection in pig farms is discussed.

An ELISA optimized for porcine epidemic diarrhoea virus detection in faeces.

Monoclonal antibodies to porcine epidemic diarrhoea virus (PEDV) membrane protein M were prepared and used for the comparative assessment of three blocking ELISA variants to detect PEDV. The competitive blocking ELISA (CB-ELISA) format showed the highest sensitivity, allowing detection of 10(2.5) plaque-forming units of PEDV/ml in culture medium. Its specificity was verified by inclusion of control samples containing transmissible gastroenteritis virus (TGEV) and rotavirus A in each analysis. Eighty porcine field samples of faeces obtained from 38 herds affected with diarrhoea were examined, and PEDV was found in 15 (19%) samples from 6 (16%) herds. The suitability of the CB-ELISA for the screening herds in epizootiologic situations is discussed.

Verification of sensitivity and specificity of group a rotavirus detection in piglets faeces with monoclonal blocking ELISA methods.

Monoclonal antibodies to group A rotavirus Vp6 protein were prepared and used for verification of three blocking enzyme-linked immunosorbent assay (ELISA) modifications to detect rotavirus A. Selected competitive blocking ELISA (CB-ELISA) and electron microscopy (EM) were used for examination of 194 field faecal samples of piglets affected with diarrhoea. Rotavirus was detected in 43 samples (22.2%) by CB-ELISA method, whereas in 26 (13.4%) samples by EM examination. However, of 26 samples positive by EM, rotavirus A was detected by CB-ELISA in 19 (73.1%) samples; indicating the share of group A rotavirus in all cases of gastroenteritis caused by rotavirus. The sensitivity and specificity of the CB-ELISA was verified both by inclusion of control samples containing transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhoea virus (PEDV) in each analysis and by comparative examination of samples with the commercial ELISA kit. The CB-ELISA sensitivity was positively affected by examination of samples in the presence of chelating agent.

Atypical myxomatosis--virus isolation, experimental infection of rabbits and restriction endonuclease analysis of the isolate.

Atypical form of myxomatosis, which caused non-lethal and clinically mild disease in domestic rabbits 1 month after immunization with a commercially available vaccine MXT, is described. The isolated myxoma virus designated as Litovel 2 (Li-2) did not induce systemic disease following subcutaneous and intradermal applications in susceptible experimental rabbits but led to the immune response demonstrated by ELISA. No severe disease was induced in those Li-2 inoculated rabbits by challenge with the virulent strains Lausanne (Lu) or Sanar (SA), while the control animals showed nodular form of myxomatosis with lethal course of the illness. Restriction fragment length polymorphism (RFLP) of genomic DNA with KpnI and BamHI endonucleases was used for genetic characterization of the Li-2 isolate, the vaccine strain MXT and both virulent strains Lu and SA, respectively. In general, RFLP analysis has shown to be informative for inferring genetic relatedness between myxoma viruses. Based on restriction endonuclease DNA fragment size distribution, it was evident that the pathogenic strain SA is genetically related to the reference strain Lu and the isolate Li-2 is more related, but not identical, to the vaccination strain MXT.

Rod-shaped virus-like particles in intestinal contents of pheasant chicks.

Rod-shaped virus-like particles (RSV), 55-85 nm in length and 18 nm in diameter, with 5 to 10 segments or helical turns, were demonstrated in the intestinal contents of young diarrhoeic pheasants by examination of a fresh sample. The origin of RSV seems to be splitting tails of bacteriophages.

Colorimetric detection of lagomorphs' calicivirus genomic sequences by polymerase chain reaction incorporating digoxigenin dUTP.

A method of reverse transcription followed by polymerase chain reaction (RT-PCR) has been implemented for the demonstration of the rabbit haemorrhagic disease virus (RHDV) genome in organ suspensions, leukocytes and excretions of infected rabbits. RT-PCR has been tested with 10 RHDV strains isolated at various geographic sites and times using a pair of primers coming from the gene region coding for the capsid protein VP60. The same primers were effective in the amplification of 4 of 5 European brown hare syndrome (EBHS) virus isolates. Non-radioactive labelling of PCR products with digoxigenin during the amplification and a system of colorimetric assessment of hybridization reactions between a biotin-labelled RHDV capture probe and the chains of labelled amplicons (PCR ELISA) were used for specific analyses of nucleic acid synthesis. The sensitivity of the alternative procedure of analysis of the dig-labelled PCR products with PCR ELISA was two logs10 higher than that of conventional electrophoresis in agarose gel stained with ethidium bromide. The results of the hybridization reactions, carried out under various stringency conditions, have confirmed the presumption that the genomic similarity between the amplified and the probed areas of the capsid protein VP60 gene was not uniform within all the tested caliciviruses. A higher degree of heterogeneity was observed between the isolates of EBHSV and RHDV.

Isolation and identification of porcine reproductive and respiratory syndrome virus in cell cultures.

Three strains of porcine reproductive and respiratory syndrome virus (PRRSV) were isolated in porcine lung macrophage (PLM) cultures from three swine herds. This has been the first successful isolation of PRRSV in the Czech Republic and the strains received the designations CAPM V-501, CAPM V-502 and CAPM V-503, respectively. All the three isolates in PLM were identified by immunofluorescence and immunoperoxidase tests and the strain CAPM V-502 also by electron microscopy using the ultrathin section technique. The strain CAPM V-502 has been adapted to the cell line MARC-145. Viral RNA in PLM cultures infected with any of the isolated PRRSV strains was demonstrated by RT-PCR targeted to the more conserved ORF 7 genomic region encoding the nucleocapsid protein. The assessment of PCR products in agarose gel revealed a uniform size of 394 bp in all the three isolates and the European prototype strain Lelystad used as positive control.

[Detection of porcine epidemic diarrhea virus using electron microscopy in the Czech Republic]. / Elektronove mikroskopický prukaz viru epidemické diarrhoey prasat v Ceské republice.

Coronavirus-induced porcine epidemic diarrhoea (PED) was diagnosed in two swine herds. The causal agent was demonstrated in intestinal contents by electron microscopy and identified by immunoelectron microscopy using specific immune serum to the reference strain PED-CV77. Experimental transmission to hysterectomy-derived, colostrum-deprived piglets with an intestinal contents filtrate was successful. The virus was demonstrable by electron microscopy in the intestinal contents between 12th hour and 4th day, and in small intestinal epithelial cells 18 hours after infection. Scanning electron microscopy revealed shortening and fusion of villi of small intestinal mucosa.

Demonstration of parvovirus in diarrhoeic African cheetahs (Acinonyx jubatus jubatus Schreber, 1775).

Parvovirus was demonstrated in the intestinal content of diarrhoeic African cheetahs by electron microscopy. The virus was isolated in a feline kidney cell line inoculated with a filtrate of the intestinal content. Its growth characteristics, cytopathic effect, agglutination of porcine erythrocytes, structure, and results of immunoelectron microscopic examination were indistinguishable from those of feline panleukopenia virus.

Immunoelectron microscopy of rabbit haemorrhagic disease virus using monoclonal antibodies.

Five monoclonal antibodies (MoAbs) to rabbit haemorrhagic disease virus (RHDV), prepared and tested in ELISA, immunoperoxidase (IP) and immunofluorescence (IF) test previously, reacted specifically in immunoelectron microscopy (IEM), too. No differences in binding of individual MoAbs with full or empty RHDV particles were found by IEM.

Application of control measures against viral haemorrhagic disease of rabbits in the Czech and Slovak Federal Republic.

The first outbreaks of viral haemorrhagic disease (VHD) of rabbits were reported from eastern Slovakia in 1987. In 1988, the infection spread throughout the Czech and Slovak Federal Republic. Electron microscopy was used by the Veterinary Research Institute in Brno to diagnose the disease during the early stage of infection. At present, the regional laboratories of the veterinary investigation services use the haemagglutination and the direct immunofluorescence tests as the principal methods to demonstrate the causal agent. Indirect immunofluorescence and immunoperoxidase techniques have been developed to demonstrate VHD virus, while the enzyme-linked immunosorbent assay (ELISA) has been used to detect antibodies. Diagnostic kits, allowing a wide use of these methods, are now available commercially. Two types of inactivate vaccines were developed and produced in 1988 and 1989. VHD is controlled by vaccination of exposed rabbit colonies. This is accompanied by other preventive and protective measures, directed by district veterinary officers following instructions from federal authorities.

Rabbit haemorrhagic disease: an investigation of some properties of the virus and evaluation of an inactivated vaccine.

An inactivated vaccine against rabbit haemorrhagic disease (RHD), developed and tested in our laboratory, is produced commercially by Bioveta, Ivanovice, Czechoslovakia. Rabbits developed full protection against infection 3 weeks after the administration of a single dose. Antibodies were detectable from day 5 after vaccination. Naturally acquired antibodies were demonstrated in some rabbits kept on commercial farms. The virus survived at least 225 days in an organ suspension kept at 4 degrees C, at least 105 days in the dried state on cloth at room temperature (around 20 degrees C), and at least 2 days at 60 degrees C, both in organ suspension and in the dry state. Experimental infection of rabbits younger than 2 months was successful in some animals. Hares, guinea pigs, white mice, golden and Chinese hamsters, chinchillas and hysterectomy-derived, colostrum-deprived piglets were resistant to infection.

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.

Enzyme-linked immunosorbent assay of antibodies to rabbit haemorrhagic disease virus and determination of its major structural proteins.

An ELISA was developed for the determination of antibodies to rabbit haemorrhagic disease virus (RHDV) in whole blood and blood serum of rabbits. Naturally acquired antibodies were detected in 19.4% of blood samples collected from 1461 rabbits in 43 farms apparently free of the disease, 19.7% samples were doubtful and 60.9% of the rabbits were free of antibodies to RHDV. Their presence has a considerable effect on the resistance of rabbits to infection with RHDV. Antibodies were also found in rabbit blood serum samples collected up to 12 years before the first outbreaks of RHD were reported. Up to 14 viral protein antigens were determined by PAGE and Western blot analysis, of which three with Mr values of 61K, 38K and 52K were major proteins, the 61K being dominant. Our hyperimmune sera, a Chinese reference serum and sera with positive antibody titres, including those collected several years before the first outbreaks of RHD, reacted identically with these antigens in the Western blot analysis. The data obtained suggest that naturally acquired antibodies are a product of a specific response to prior infection with an avirulent strain of the virus.

Electron and immunoelectron microscopy of rabbit haemorrhagic disease virus (RHDV).

Rabbit haemorrhagic disease virus (RHDV) had a calicivirus-like structure and a diameter of 31.5-33.0 nm. Antigenic relationship between the investigated RHDV strain and the causal agent of RHD in China was demonstrated by immunoelectron microscopy.

Experimental transmission and electron microscopic demonstration of the virus of haemorrhagic disease of rabbits in Czechoslovakia.

Intramuscular administration of the filtrate of organ suspensions, prepared from a dead rabbit, killed 62.9% of inoculated rabbits within 1 to 5 days, while 93.3% died after intranasal administration of the same inoculum. The virus survived freeze-drying and was resistant to treatment with 0.4% formaldehyde when incubated at 37 degrees C for 1 hour and 4 degrees C for the subsequent 12 hours, but lost its infectivity when the treatment was prolonged to 3 hours at 37 degrees C and 3 days at room temperature. Its infectivity was also inhibited by reconvalescent serum. The virus could not be detected after 3 passages in primary rabbit kidney cell cultures. Electron microscopy of negatively stained preparations demonstrated icosahedral virus particles with a diameter of 29 to 33 nm without an envelope. Accurate morphological classification has not yet been completed. Incubation with a reconvalescent serum, diluted 1:20 or 1:40, resulted in the formation of immune complexes, detectable by electron microscopy.

[The effect of colostral immunity on the active formation of antibodies in pigs after the administration of inactivated vaccine against Aujeszky's disease]. / Vliv kolostrální imunity na aktivní tvorbu protilátek u prasat po aplikaci inaktivované vakcíny proti Aujeszkyho chorobe (ACH).

In a large herd of pigs where a trial was performed to cure the animals from Aujeszky's disease (AD) by applying to all animals an inactivated vaccine, a post-vaccination antibody response was studied in piglets coming from the sows that were vaccinated several times. When the piglets were vaccinated at the age of eight weeks (the average virus-neutralizing titer (VNT) of colostral antibodies was 1:11.4) and revaccinated at the age of 11 weeks, 73% of the forty-five animals (examined at the age of 17 weeks) did not have any virus-neutralizing (VN) antibodies in the blood serum. After the third vaccination dose (at the age of 17 weeks), 11% of piglets did not have any VN antibodies if they were examined at the age of 22 weeks (the average antibody VNT was 1:15.3). Applying the ELISA procedure, the antibodies were demonstrated in the sera of all piglets after three vaccination doses. Shifting the time intervals of vaccination (at the age of 8, 13 and 19 weeks), the VN antibodies were found out after three vaccination doses in the sera of all piglets examined at the age of 23 weeks (the average VNT was 1:56.4). After three vaccination doses at the age of 12, 17 and 23 weeks, the VN antibodies were also demonstrated in all piglets at the age of 27 weeks (the average VNT was 1:208).

Four-layer enzyme immunoassay (EIA) detection of differences in IgG, IgM and IgA antibody response to Aujeszky's disease virus in infected and vaccinated pigs.

The use of the four-layer enzyme immunoassay (EIA) for the detection of IgG, IgM and IgA antibodies against Aujeszky's disease virus in blood and oropharyngeal swabs of infected and vaccinated pigs is described. Mean antibody titres obtained using the four-layer EIA were 6.1 and 3829 times higher compared with the indirect enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VN) test, respectively. The VN test detected mainly IgG antibodies, while the IgM antibodies did not react. Using the EIA, the first antiviral antibodies in sera were demonstrated on Days 5-7 after infection or vaccination. Up to the 7th day, demonstrable antibodies were almost exclusively of the IgM class. In infected pigs high titres of IgM antibodies were still detected on Day 18, while in vaccinated animals they were absent by this time. Antibodies of the IgG class appeared in infected pigs sooner (Day 7) than in vaccinated pigs (Day 10) and reached higher mean titres. Antibodies of the IgA class were demonstrable from Day 10 only in samples from infected pigs. Similar antibody dynamics and distribution were detected in oropharyngeal swabs, except that the IgG and IgM titres were roughly 100 times lower than in sera. However, titres of IgA antibodies in oropharyngeal swabs were two times higher than in sera. The greatest differences between both groups of animals were recorded on Day 18; in the infected pigs, IgG, IgM and IgA antibodies were present in sera and oropharyngeal swabs at that time, while in vaccinated pigs only IgG antibodies were demonstrable. The effect of infection and vaccination on the pattern of the immune response as well as the importance of the detection of individual immunoglobulin classes for the specificity of the enzyme immunoassay are discussed.