Identification and production of pestivirus proteins for diagnostic and vaccination purposes.

Using a panel of monoclonal antibodies (MAbs) previously characterized by seroneutralization, immunofluorescence and radioimmunoprecipitation, we have identified Pestivirus proteins useful for diagnostic purposes from the cytopathic Osloss isolate of bovine viral diarrhea virus (BVDV). Proteins that should be useful for vaccination have also been analysed. Cell-free translation of RNA from glycoprotein-coding cDNA fragments produced, when synthesized in the presence of canine pancreatic microsomes, two glycosylated proteins that were independently recognized and immunoprecipitated by two distinct classes of neutralizing MAbs. A similar in vitro procedure was carried out on nonstructural protein-coding sequences and allowed to identify a viral translation product that specifically reacted with MAbs directed against the 80 kDA protein of a number of Pestivirus strains. Its positioning within the polyprotein encoded by the viral genome was refined by epitope scanning using synthetic hexameric peptides. This viral antigen was further expressed in E. coli, produced as inclusion bodies and used successfully as an ELISA antigen in both competitive and indirect assays for the detection of BVD antibodies in cattle sera.

Evidence for a multiple innervation of cerebellar Purkinje cells by climbing fibers in adult ferrets infected at birth by a mink enteritis virus.

Newborn ferrets were inoculated with Mink Enteritis virus (parvovirus). They developed a cerebellar hypoplasia and presented severe ataxia. Electrophysiological study by intracellular recordings in the cerebellar cortex demonstrates that in these ferrets, like in other mammals, Purkinje cells deprived from granule cell input during development remain multiply innervated by climbing fibers in the adult.

Control of canine distemper.

Control of canine distemper can realistically only be achieved by the use of vaccination. The types of vaccine in current use are described, together with some of the problems encountered such as interference by maternal antibodies, and usage in species other than dogs. Modified live viral vaccines, as used for more than thirty years, have proved very effective. Nevertheless there is scope for some improvement in vaccine efficacy and recent developments in genetic recombinant methods are described.

Development of a simple, rapid and accurate in vitro whole blood technique for the detection and semi-quantification of FIV cellular viremia.

A new, simple, rapid and accurate culture technique is described for a semi-quantitative analysis of cellular viremia in FIV-infected cats. This assay can be carried out with small amounts of whole blood, and is based on the detection of FIV core gag antigen, which is released in culture supernatants. The amount of core antigen produced is measured with an enzyme-linked immunoassay using specific monoclonal antibodies. This whole blood technique (WB method) was compared with a culture method using isolated peripheral blood mononuclear cells (PBMC method). FIV could be detected in whole blood of all experimentally infected cats, but not from uninfected cats. This assay offers a number of advantages (small blood samples required, no leukocyte separation and lymphocyte purification procedures) and its reproducibility is very good. It provides a convenient in vitro cellular assay for viral semi-quantitation, well adapted for monitoring efficacy of prototype FIV vaccines or experimental antiviral drugs. Also, it could facilitate the study of the pathogenesis of FIV-related progressive immunodepression. Finally, it offers an alternative to serological techniques for diagnostic purposes in several circumstances: early viremia, maternal antibodies.

Comparative analysis of monoclonal antibodies against pestiviruses: report of an international workshop.

Thirty-three pestivirus strains were grown in cell culture and characterized by immunostaining with 19 monoclonal antibodies (MAbs) raised against hog cholera virus (HCV), with 42 MAbs against bovine viral diarrhoea virus (BVDV) and with 13 MAbs against border disease virus (BDV). Seven MAbs reacted with all pestivirus strains tested, eight MAbs detected only the seven HCV strains, three detected only the 16 BVDV strains. No MAb was found that was specific for BDV. BVDV and BDV strains were broadly cross-reactive with the MAbs, indicating a close relationship between these two species, whereas HCV strains were characterized as distinct from BVDV and BDV.

ELISA detection of bovine viral diarrhoea virus specific antibodies using recombinant antigen and monoclonal antibodies.

A panel of monoclonal antibodies was prepared by immunization of BALB/c mice with Moredun (BD) virus strains. These antibodies were characterized by immunofluorescence and seroneutralization against BD, BVD and hog cholera (HC) virus strains, and radioimmunoprecipitation of BVD-infected cells extracts. The MAbs reacting with the majority of the Pestivirus strains recognize the 80 kDa antigen of the BVD cytophathic strains. The 80 kDa antigen of the BVD/Osloss virus strain has been cloned and expressed in E. coli as a fusion protein with beta-galactosidase. The fusion protein has been purified from inclusion bodies and used successfully as an antigen for ELISA detection of BVDV specific antibodies in bovine sera. A competitive ELISA using MAbs is more specific than a direct assay. These results compare well with the ones obtained with antigen extracted from BVDV-infected cells.

Use of vaccinia rabies recombinant for oral vaccination of wildlife.

A vaccinia rabies recombinant virus was constructed and shown to induce the synthesis of rabies virus glycoprotein in infected cells and to induce rabies virus neutralizing antibodies and protection in susceptible animals. Active when orally administered, this recombinant is a good candidate for the development of vaccines for wild animal rabies vectors. This recombinant was found stable, safe for target and non-target animal species, and protective for most of the rabies vectors. After extensive experimental studies conducted under controlled conditions, it as used in limited field trials and in an extensive open field trial. The preliminary results confirmed its basic properties and potential for rabies eradication.

[Diagnostic problems posed by respiratory infections of dogs]. / Les problèmes de diagnostic posés par les infections respiratoires des chiens.

The following viruses as well as bacteria and mycoplasma have been isolated from dogs with contagious respiratory disease: canine distemper virus; Canine adenoviruses (type 1 and 2); Parainfluenza type 2 (SV5); Reovirus type 1; Canine Herpesvirus; Bordetella bronchiseptica, Streptococcus, Pasteurella, Staphylococcus and Mycoplasma. The occurrence of these agents can be in direct relationship with: the evolution of a systemic disease; respiratory disorders being a regular or inconsistant symptom of this disease; the evolution of a disease restricted to the respiratory tract; the tropism of the bacterial or viral agent is exclusively respiratory; secondary bacterial complications to a primary viral infection; saprophyte state or latency without pathologic significance. These various infectious agents are implicated alone or in mixed infections and the wide variety of clinical symptoms don't allow to precise a clinical diagnosis. We will try to bring some bases allowing, by the help of laboratory an etiologic diagnosis. This diagnosis is essential for providing an efficient prevention. We will approach some parameters which we have been confronted with as regards Canine Distemper and Canine Adenovirosis. Our purpose is, through these examples of the canine pathology, to confirm and complete some other similar situations which can appear in other animal species.

[Correlation between the NIH test and the seroneutralizing antibodies obtained after vaccination in the dog]. / Etude de la corrélation existant entre le test NIH et les anticorps séroneutralisants obtenus après vaccination chez le chien.

This work reports on two distinct studies on the analysis of correlation of results of potency tests of rabies vaccine intended for dogs. In a first study, the analysis compares the results of the conventional NIH test in mice with seroneutralizing antibodies obtained after 21 days of vaccination in dogs. Arithmetical analysis (109 NIH results, 466 antibody titres) did not reveal significant linear correlation between the results. In a second study, the analysis compares the results of the conventional NIH test and of the NIH test without booster in mice with seroneutralizing antibodies obtained after 21 days of vaccination in dogs. Arithmetical analysis (27 conventional NIH results (without booster), 118 antibody titres) did not reveal any significant linear correlation between the results.

[Haemagglutination inhibition test: improvements, limits and advantages of the method. Comparison with neutralization test]. / Test d'inhibition de l'hémagglutination de la rage amélioration, limites, intérêt de la methode. Comparaison avec les tests de neutralisation.

In this study, we report some technical improvements which enable us to envisage wider use of the haemagglutination test and haemagglutination inhibition of the rabies virus. Moderated trypsination of sensitive red blood cells and their counting are important parameters. An interesting application of the test was the haemagglutination competition enabling the amount of soluble glycoprotein (obtained during the fractionating of the virus components) to be evaluated. From antibody kinetics of vaccinated dogs, a study of the correlation which exists between the IHA titres, SN mice titres and SN-RFFIT titres was carried out. Satisfactory correlation was obtained between these three techniques.

Immunisation against panleukopenia: early development of immunity.

The time necessary to obtain the immunity of cats against Panleukopenia has been studied by means of a modified live vaccine. This vaccine makes it possible to obtain a very early post-vaccinal immunity: the full immunity is reached 72 hr after the inoculation of the vaccine by the subcutaneous route. Furthermore, we have demonstrated that a sensitive kitten can be admitted in a contaminated environment immediately after vaccination without showing any clinical evidence of the disease.

[Modalities of production and immunity conferred by an inactivated rabies vaccine originating from cell culture]. / Modalités de production et immunité conferée par un vaccin antirabique inactivé provenant de culture cellulaire.

Further to guidelines advised by the World Health Organization, an inactivated Rabies vaccine was prepared from virus propagated on cell culture. This vaccine is presented either in the freeze-dried form or in the liquid form together with an immunity adjuvant. The specific and nonspecific immunity of the vaccine is excellent. The potency, tested in laboratory animals and in species for which the vaccine is intended, satisfies recommendations published by the W.H.O. The immunity persistence, evaluated by the titration of serum antibodies and by challenge with a pathogenic virus, proves to be excellent 3 years following primovaccination. Finally, the stability of this vaccine is an interesting factor for its application, especially in the form of a combined vaccine.

Rabies virus pathogenicity and challenge. Influence of the method of preparation, the route of inoculation, and the species. Comparison of the characteristics of the modified, fixed and wild strains.

The challenge we carried out, which regularly brought about the death of the control animals, led us to study the different factors influencing the pathogenicity of the rabies virus. So, the method of preparation is important. The strain NYC, prepared from the salivary glands is extremely pathogenic for dogs; however, when it is prepared from mouse brains after nine passages have been made, it proves to be already partially modified. One then notices a death-rate which is less severe, even when larger quantities of virus are employed, and also the presence of some aberrant phenomena. (The survival of some of the infected animals and the nature of their inapparent forms of infection were confirmed by serology.) The significance of the route of inoculation in the different species of animals was studied. The injection in the crotaphytes was reserved for dogs, the cervical muscles for cats and the masseters for sheep. Paradoxically, cats prove to be most resistant to the challenge under our conditions. Finally the numerical data, allowing us to compare the different strains, modified, fixed or wild, was established. The data was based on the incubation period and on the differences between the titres obtained via intramuscular routes and intracerebral routes in the mice.

Spontaneous programmed cell death (PCD) process of lymphocytes of FIV-infected cats: cellular targets and modulation.

Unstimulated lymphocytes from FIV-infected cats undergo spontaneous apoptosis in vitro as indicated by internucleosomal DNA fragmentation and hypodiploid DNA content of nuclei. Unlike what is reported in HIV-infected individuals, we observed that cell death of cat lymphocytes was inhibited by activation. Spontaneous apoptosis was reduced by the addition of cat serum and after activation by phorbol ester (PMA), superantigens (SEB, SEA), and to a lesser extent by mitogens such as concanavalin A and pokeweed mitogen. In contrast, apoptosis of lymphocytes from FIV-infected, but not from control cats was increased in the presence of calcium ionophore (ionomycin). Analysis of the phenotype of cells undergoing apoptosis revealed that cell death is not restricted to a cell subpopulation but involved all lymphocyte subsets. These data suggest that the mature lymphocytes of FIV-infected cats appear programmed to die by apoptosis unless rescued by specific agents, such as protein kinase C activators or mitogens.

Propagation of bovine rotavirus by young dogs.

Ten young dogs were experimentally infected twice with different isolates of bovine rotavirus and 2 uninfected dogs were kept in contact with them. None of the animals developed diarrhoea, but all of them excreted rotavirus in their faeces over a period of up to 10 days after each inoculation, as shown by counterimmunoelectro-osmophoresis and virus isolation. Dogs may thus play a role in the epizootiology of rotavirus diarrhoea in calves. Seroconversion occurred in 6 of the 10 infected dogs but in neither of the 2 contact controls.

Classical swine fever: the European experience and a guide for infected areas.

Classical swine fever (CSF) (hog cholera) virus infection is still of world-wide concern, either because of the direct effects of the disease on swine breeding in areas where the virus is epizootic or enzootic, or as a threat in areas where the virus has been eradicated. The authors provide an overview of the characteristics of the disease. Special emphasis is placed on the chronic form of disease, particularly in the late stages of eradication programmes. In the early 1980s, the European Union (EU) was composed of countries which were officially free of the disease (absence of infection and no vaccination) and countries in which vaccination was either permitted or was compulsory. To ensure free trade between the Member States, an eradication plan was agreed upon and implemented. Initially, the plan consisted of a combination of vaccination with the Chinese strain of the virus and slaughter and removal of infected herds. Consequently, when the number of infected herds was low, vaccination was abandoned and the control of CSF was conducted exclusively by eradication (removal and slaughter). The United Kingdom, Austria, Denmark, Ireland, Luxembourg, Finland and Sweden ceased vaccination before 1980. In the other countries, vaccination was useful in controlling the last epidemics and was finally ceased as follows: France in 1983, the Netherlands in 1986, Belgium, Spain and Greece in 1988, Germany in 1989 and Italy in 1990. From 1990 onwards, no vaccination against CSF has been performed in the EU. New techniques for the diagnosis of CSF (for example, the enzyme-linked immunosorbent assay based on the detection of the p125 antigen of the virus) have been shown to be of value in the early detection of infected animals. In enzootic areas, the use of vaccines based on the Chinese strain has been successful. Vaccines with at least 100 PD50 of virus per dose are able to significantly limit the replication of virulent virus in the tonsils. Consequently, shedding of virus after infection can be reduced considerably. In heavily infected areas, vaccination plays a crucial role. The European experience shows that eradication may be achieved when vaccination with highly effective vaccines is combined with effective identification of swine, movement control, early diagnosis and the rapid elimination of infected herds.

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.

Serological survey of selected canine viral pathogens and zoonoses in grizzly bears (Ursus arctos horribilis) and black bears (Ursus americanus) from Alaska.

Between 1988 and 1991, 644 serum samples were collected from 480 grizzly bears (Ursus arctos horribilis) and 40 black bears (Ursus americanus) from Alaska, United States of America, and were tested for selected canine viral infections and zoonoses. Antibody prevalence in grizzly bears was 0% for parvovirus, 8.3% (40/480) for distemper, 14% (68/480) for infectious hepatitis, 16.5% (79/480) for brucellosis, 19% (93/480) for tularaemia and 47% (225/478) for trichinellosis. In black bears, prevalence ranged from 0% for distemper and parvovirus to 27.5% for trichinellosis and 32% for tularaemia. Antibody prevalence for brucellosis (2.5%) and tularaemia (32%) were identical for grizzly bears and black bears from the geographical area of interior Alaska. Links between differences in prevalence and the origin of the grizzly bears were observed. Antibodies to canine distemper virus and infectious hepatitis virus were mainly detected in grizzly bears from Kodiak Island and the Alaskan Peninsula. Brucellosis antibodies were prevalent in grizzly bears from western and northern Alaska, whereas tularaemia antibodies were detected in grizzly bears from interior Alaska and the Arctic. There was a strong gradient for antibodies to Trichinella spp. from southern to northern Alaska. For most diseases, antibody prevalence increased with age. However, for several infections, no antibodies were detected in grizzly bears aged from 0 to 2 years, in contrast to the presence of those infections in black bears. Grizzly bears served as excellent sentinels for surveillance of zoonotic infections in wildlife in Alaska.

Field use of a vaccinia-rabies recombinant vaccine for the control of sylvatic rabies in Europe and North America.

During recent years, most research on the control of sylvatic rabies has concentrated on developing methods of oral vaccination of wild rabies vectors. To improve both the safety and the stability of the vaccine used, a recombinant vaccinia virus, which expresses the immunising glycoprotein of rabies virus (VRG), has been developed and tested extensively in the laboratory as well as in the field. From 1989 to 1995, approximately 8.5 million VRG vaccine doses were dispersed in Western Europe to vaccinate red foxes (Vulpes vulpes), and in the United States of America (USA) to vaccinate raccoons (Procyon lotor) and coyotes (Canis latrans). In Europe, the use of VRG has led to the elimination of sylvatic rabies from large areas of land, which have consequently been freed from the need for vaccination. Nevertheless, despite very good examples of cross-border cooperation, reinfections have occurred in some regions, due to the difficulty of co-ordinating vaccination plans among neighbouring countries. In the USA, preliminary data from field trails indicate a significant reduction in the incidence of rabies in vaccinated areas.