Diagnosis of canine parvovirus by rapid immunomigration on a membrane.

Rapid immunomigration on a membrane was applied to the diagnosis of canine parvovirus (CPV) in 128 samples of faeces containing four strains of parvovirus (two CPV-2a strains, including one vaccine strain, and two CPV-2b strains). The results were compared with the results of haemagglutination and ELISA sandwich techniques. The new test was quick and easy to use, and made it possible to identify both the CPV-2a and CPV-2b strains. Its detection thresholds per gram of faeces corresponded to specific haemagglutination titres of between 320 and 640 and a virus titre of between 10(4) and 10(5) CCID50 (dose required to infect 50 per cent of cell cultures).

Improvement of serological discrimination between herpesvirus-infected animals and animals vaccinated with marker vaccines.

Control/eradication plans of bovine herpesvirus 1 (BHV1) and suid herpesvirus 1 (SHV1) infections involve vaccination with inactivated or attenuated gE-deleted marker vaccines and associated companion serological tests to discriminate naturally infected from vaccinated animals. Blocking or competitive enzyme-linked immunosorbent assays (ELISAs) have been designed for the detection of specific antibodies against BHV1 or SHV1 gE glycoprotein. The antigen source usually consists of a crude viral preparation in which gE is associated with other envelope glycoproteins. Such assays suffer from a lack of specificity which is not due to serological cross-reactions with other pathogens. Interestingly, false-positive results occur with sera collected from multivaccinated cattle or pigs. After multivaccination with a marker vaccine, the binding of the conjugated monoclonal antibody used as a tracer, could be hampered by antibodies directed against the other viral glycoproteins. In order to validate the steric hindrance hypothesis, a simple preadsorption of such samples was carried out with a preparation of antigen devoid of gE, prior to the blocking ELISA itself. The decrease in antibody concentrations against the major glycoproteins, clearly leads to a better discrimination between positive and negative samples; that is between infected and multivaccinated animals, without significant loss of sensitivity. This experiment confirms the steric hindrance hypothesis, therefore serum preadsorption could be an easy way to improve the specificity of currently available diagnostic tests.

Circulating relaxin concentrations in pregnant and nonpregnant bitches: evaluation of a new enzymeimmunoassay for determination of pregnancy.

A new kit (ReproCHEK RELAXIN) intended for the diagnosis of pregnancy in bitches is now available for veterinary use. This assay measures relaxin concentrations in plasma and whole blood samples, and the presence of significant amounts of relaxin is indicative of pregnancy. A clinical trial was carried out to evaluate the performance of the test. Serial blood samples were collected on alternate days, and relaxin concentrations were determined from day 15 to day 35 after the LH surge (estimated by progesterone concentrations). Pregnancy was confirmed using ultrasonography. At the end of pregnancy, both the day of whelping and the size of the litter were recorded. Pregnancy was established in 61 bitches. The day that pregnancy was detected using the relaxin assay ranged from day 19 to day 28 after the LH surge and had a mean (+/- SD) of 25.4 +/- 2.5 days. The day of parturition was taken as a reference point, and pregnancy was detected from -46 to -38 days (mean -40.2 +/- 2.4 days) before parturition. False positives were not observed in pseudopregnant bitches (n = 16) or in the control group (30 anoestrous and ten unmated bitches). These results demonstrate that the new assay kit is an inexpensive, user-friendly and reliable technique for determining pregnancy.

A blocking ELISA using a monoclonal antibody for the serological detection of Mycoplasma hyopneumoniae.

A blocking ELISA was developed by using a monoclonal antibody (4082-05-344-18) which specifically detected an epitope on the Mycoplasma hyopneumoniae 40 kDa membrane protein without cross-reacting with M flocculare or M hyorhinis. The results obtained with sera from specific pathogen-free pigs inoculated with M flocculare or M hyorhinis confirmed the specificity of the assay. An immunoblotting procedure was used to characterise the antibody response of pigs experimentally infected with M hyopneumoniae. Antibodies to the 40 kDa antigen were detected two weeks after infection and remained as major markers for at least 20 weeks. Cross-reacting antibodies to this antigen were not detected in convalescent sera from piglets infected with M flocculare or M hyorhinis. Sera from experimentally infected pigs were compared by means of the blocking ELISA and an indirect ELISA. The kinetics of ELISA antibodies after experimental inoculation were also studied. The detection of antibody was rather more stable for a longer time with the blocking ELISA than with the indirect ELISA. In an evaluation of more than 1000 sera from the field there was excellent agreement between the two methods.

Control of bovine virus diarrhoea-mucosal disease in cattle: examples of the combined use of serological screening, viral antigen detection and vaccination.

As part of the preparatory phase of a disease control programme in three herds infected with bovine virus diarrhoea-mucosal disease (BVD-MD) virus (demonstrated by virus isolation), initial serological screening was performed on all livestock older than six months (351 animals) by blocking enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody recognising a common epitope to the different strains of BVD-MD virus. The presence of immunotolerant, persistently-infected animals was strongly suspected, as a high percentage (334 = 95.2%) of cattle showed positive serological reactions, while the other members of the herd (17 = 4.8%) continued to give negative results, even after vaccination with a live vaccine. Whole blood samples from all cattle were then tested individually for viral antigen by an ELISA technique which had previously been tested successfully. As a result, a total of nine viraemic animals were identified in the three herds. A confirmatory test was performed by the reference amplification method on cell culture with virus identification using specific fluorescein isothiocyanate-labelled monoclonal antibodies. The identification and elimination of the persistently-infected animals led to the recovery of a negative serological status for the herds. It was therefore recommended that protective measures should be taken to avoid the reappearance of viraemic animals, involving vaccination and systematic viral testing before introducing any new animal into the herd. It was advisable that these measures should be maintained until all the potential reservoirs and vectors of BVD-MD virus are better known.

Five hours to identify immunotolerant cattle, persistently infected with bovine virus diarrhoea virus.

Detection of animals which are persistently-infected with bovine virus diarrhoea virus (BVDV) is of prime importance in the control of pestivirus infections in cattle, as these animals constitute the main reservoir of the virus. Identification of such animals can be readily performed using crude whole blood samples with a sandwich enzyme-linked immunosorbent assay (ELISA) requiring only approximately five hours. This ELISA uses a combination of monoclonal antibodies as the capture agent and an immunological amplification step of the specific signal for detecting the non-structural 80/120 kDa protein of BVDV. The degree of correlation between this ELISA and virus isolation as the reference method is 100% for animals older than six months.

Monoclonal antibodies against an immunodominant and neutralizing epitope on hepatitis A virus antigen.

Two monoclonal antibodies (813 and 10.09) were raised against hepatitis A virus (HAV). They recognize an immunodominant epitope and a neutralizing site on HAV.

Immunopathologic aspects of woodchuck hepatitis.

The natural history of infection with woodchuck hepatitis virus (WHV) has been studied in a colony of 38 Marmota monax. Besides serologic assessment for WHV markers, light-microscopic findings of 61 liver biopsies were correlated with the results of immunofluorescence analysis for nucleocapsid (WHcAg) and surface (WHsAg) antigens. Twenty-four chronic WHsAg carriers all featured signs of continuous viral replication. Two major immunomorphologic patterns were observed in their livers: 1) portal hepatitis in which WHcAg accumulated in the cytoplasm and WHsAg was associated with the hepatocyte membrane and 2) periportal hepatitis in which WHcAg shifted toward nuclear localization and WHsAg became mostly intracytoplasmic. Progression from portal to periportal hepatitis, observed in 7 woodchucks, appeared to be induced by a partial recovery of specific immune reactivity to WHV, insufficient, however, to interrupt WHV replication. Deposits of WHsAg and immunoglobulins were present in the kidney and spleen of animals with severe hepatitis.

[Presence of 2 different epitopes on the human beta 2-microglobulin defined by monoclonal antibodies]. / Présence de deux épitopes différents sur la beta 2-microglobuline humaine définis par des anticorps monoclonaux.

Two monoclonal antibodies recognizing monomorphic determinants on class I antigens of MHC were characterized. They were shown to recognize beta 2m by cytotoxicity inhibition studies. The epitopes recognized were different, as assayed by cross-inhibition and phylogenetical studies. One antibody recognized free as well as HLA-associated beta 2m. The other reacted with a conformational epitope which was well conserved during evolution.

[Release of hepatitis A virus into the culture medium, during its replication in PLC/PRF/5 cells]. / Libération du virus de l'hépatite A dans le milieu de culture, lors de sa réplication dans des cellules PLC/PRF/5.

Hepatitis A virus (HAV), obtained from human feces collected 3 days after the onset of jaundice, was propagated in human hepatocarcinoma cells (PLC/PRF/5). In the course of 5 passages, it was released into the supernatant without cytopathic effect. Released HAV was propagated after inoculation into PLC/PRF/5 cells. Its biophysical properties (buoyant density in CsCl: 1, 33 g/cm3; sedimentation coefficient: 155 S) were similar to those of the infectious particle present in the cells or in the stools of patients.

[Analysis of various isoferritins with monoclonal antibodies]. / Analyse de différentes isoferritines par des anticorps monoclonaux.

Balb/c Mice were immunized with splenic isoferritin. Spleen cells from immunized Mice were fuzed with SP2O myeloma cells. Four monoclonal antibodies designated respectively M29, M211, M386 and B8 were selected. Various isoferritins were analysed by immunodiffusion (Ouchterlony technic). With these monoclonal antibodies and with Rabbit polyclonal sera: human basic and acidic isoferritins and Horse spleen ferritin. Ferritin could be precipitated by these monoclonal antibodies. These results confirm that the ferritin molecule consists of repeating antigenic sites. No immunoreactivity difference could be detected between acidic and basic human ferritine. Species specificity was recognized. The high affinity constant of the M29 monoclonal antibody allowed development of a standardized radioimmunossay of ferritin.

Monoclonal antibodies as a tool for phylogenetic studies of major histocompatibility antigens and beta 2-microglobulin.

The cross-reactivity of several monoclonal antibodies recognizing monomorphic determinants of human HLA-A, B, C, and DR antigens and human beta 2-microglobulin (beta 2m) has been studied on peripheral blood leukocytes in 24 different species. An monoclonal HLA-A-, B-, and C-specific antibody and four monoclonal HLA-DR-specific antibodies cross-reacted with cells from all the primate species tested. Furthermore, antibodies HLA-DR-specific were positive with peripheral blood leukocytes (PBL) from cows, goats, sheep, horses, and dogs. Two monoclonal beta 2m-specific antibodies, which were positive with PBL from certain primates, also reacted with cells from cows, goats, sheep, horses, and dogs. Two other beta 2-m-specific antibodies reacted only with PBL from chimpanzees. No reaction could be detected with all our reagents in other classes tested (birds, reptiles, amphibians, and Teleostei).

[Production of monoclonal antibodies against HBs (author's transl)]. / Production d'anticorps monoclonaux anti-HBs.

Two BALB/c mice were immunized 4 times with a mixture of adw2 and ayw4 subtypes of HBs antigens. Their spleens were then hybridized with mouse myeloma cell line NS1. Using three different radioimmunoassays (RIA), 264 independent hybridomas were screened for anti-HBs activity. By at last one of these techniques, 95% of the colonies were positive. Selected colonies were cloned and supernatants studied by RIA and immunodiffusion techniques for specificity characterisation. Some clones recognised the common "a" subtype, and other were directed to more restricted specificities. Ascites fluid was active on RIA up to 10(7) dilution (up to 29.000 UI/ml). These monoclonal antibodies may be powerful reagent for the diagnosis and understanding of viral hepatitis B.