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.

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.

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.

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.

[Ecotoxicologic relations on a large pig-fattening farm located in a lignite mining area and near and solid-fuel electical power plant]. / Ekotoxikologické vztahy ve velkovýkrmne prasat lokalizované v oblasti tezby lignitu a tepelné elektrárny.

Major contaminants identified in 1983-1984 on a pig fattening farm with an output of 60,000 pigs per annum, located in a lignite mining area and near a solid fuel power plant, were mercury, cadmium, lead, chromium and aflatoxin B1 (Tab. I, II, III, IV). Feed samples were collected from througs to assess the contamination load at feed uptake. Permissible concentrations of mercury, chromium, cadmium, aflatoxin B1, lead and atrazin in the feed were exceeded in 56, 50, 31, 19, 6 and 6% samples, respectively (Tab. I). Stable dust deposits, in which the contaminants concentrate, (Tab. I) proved to be a suitable material for assessing the type and level of environmental contamination. Permissible concentrations of mercury, cadmium and lead in porcine muscles were exceeded in 65, 51 and 24% samples, respectively (Tab. III). Corresponding values of mercury, cadmium, lead and aflatoxin B1 in the liver were 27, 27, 16 and 3%, respectively (Tab. III) and those of mercury, lead and chromium in kidneys 24, 22 and 5%, respectively (Tab. III). Rather surprisingly, elevated pancreatic concentrations of aflatoxin B1 were found (Tab. IV). Pigs fattened in the contaminated environment (i.e. fed contaminated feed mixtures, inspiring contaminated dust and absorbing percutaneously contaminants form dust deposits on the body surface) showed: 1) Impairment of the genetic apparatus (percentage of aberrant peripheral leucocytes elevated to 6.2%); 2) a certain degree of immunosuppression (concentrations of IgG, IgM and IgA reduced by 16.9, 45.1 and 45.0%, respectively); 3) higher feed consumption per 1 kg weight gain (4 kg) and lower average daily weight gain (0.57 kg); 4) increased incidence of health disorders (dermatitis in 25%, pancreatopathy in 13%, liver dystrophy in 8% and femoral fracture in 6% of the pigs). Unfortunately, the authors were not allowed to analyse ash and solid emissions from the power plant. Therefore the share of the emissions in the overall environmental contamination on the fattening farm could not be quantified. The personnel, working in the contaminated environment for a prolonged period, is endangered most of all by stable dust, being exposed to its mechanical, chemical, allergic and infectious effects (Tab. I). In addition to the chemical contaminants, 21 mould genera and species, six mite species and numerous saprophytic and some pathogenic bacteria were demonstrated in stable dust samples in our earlier experiments. Consumption of meat and organs from pigs fattened in a contaminated environment is associated with the risk of an increased uptake of various contaminants.(ABSTRACT TRUNCATED AT 400 WORDS)

[Experimental chronic phenylmercuric chloride poisoning in pigs]. / Experimentální chronická intoxikace prasat fenylmerkurichloridem.

Four gilts, sisters from one litter, aged 70 days and weighing 20-24 kg, were used for a trial. Two experimental gilts (P) were administered an experimental feed mixture containing phenylmercury chloride (40 mg/kg). Two control gilts (K) were fed the same mixture but without phenylmercury chloride. P gilts began to lag behind in their growth from day 60 of the experiment, they manifested nonphysiological postures (dog's sitting posture), paresis of hind limbs and uncoordinated movements. P gilts had cloudy, orange-brown urine from day 70 and from day 75 they began to suffer from diarrhoea. Mercury (Hg) contents in urine and blood serum of P gilts were irregularly variable: urine 0.58-2.15 mg/l, blood serum 0.02-0.37 mg/l. Hg content in excrements of P gilts fluctuated from 23 to 26 mg/kg. Vitamin A concentrations in blood serum and liver decreased in P gilts. Phenylmercury chloride feeding caused mutagenic changes in peripheral lymphocytes of P gilts (an increase in the number of aberrant cells from 2-3% to 8-9%) and reduced IgA, IgM and IgG immunoglobulin levels in blood serum. Pathological lesions were observed in the colon, kidneys and liver. None of the above-mentioned changes were observed in K gilts. Increased resistance to the negative effects of Hg was found in one experimental gilt. In comparison with K gilts, Hg concentrations in P gilts after 130 days of the experiment increased as follows: 427 times in kidneys, 333 times in liver, 106 times in guts, 71 times in pancreas, 53 times in ovaries, 50 times in muscles, 47 times in bristles and 16 times in the brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Genomic 3' terminal sequence comparison of three isolates of rabbit haemorrhagic disease virus.

Comparison of sequence data is necessary in older to investigate virus origins, identify features common to virulent strains, and characterize genomic organization within virus families. A virulent caliciviral disease of rabbits recently emerged in China. We have sequenced 1100 bases from the 3' ends of two independent European isolates of this virus, and compared these with previously determined calicivirus sequences. Rabbit caliciviruses were closely related, despite the different countries in which isolation was made. This supports the rapid spread of a new virus across Europe. The capsid protein sequences of these rabbit viruses differ markedly from those determined for feline calicivirus, but a hypothetical 3' open reading frame is relatively well conserved between the caliciviruses of these two different hosts and argues for a functional role.

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.