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.

Evaluation of parenteral vaccination methods with glycoproteins against Aujeszky's disease in pigs.

A comparative evaluation of vaccination methods with glycoproteins for the induction of immune responses and protection of the pig against Aujeszky's disease virus (ADV) was performed. Different vaccination routes (intradermal (i.d.) versus intramuscular (i.m.)), inoculation sites (the neck versus the back) and number of inoculation points (2 versus 6) per site were compared. Body weight (BW) changes and viral excretion after challenge were compared with virus-neutralizing titers, antigen-specific IgG and IgA responses in serum and virus-specific lymphoproliferative responses in peripheral blood during the immunisation period. According to BW changes better protection was obtained with six-point than two-point i.d. injections. i.d. vaccination in the back at six points gave similar results as i.m. vaccination in the neck but appeared inferior in the reduction of virus excretion. Regarding the immunological parameters, the virus-specific IgA response in serum gave the best indication for protection. It can be concluded that according to BW changes, six-point i.d. immunisation in the back and i.m. immunisation in the neck provided the best protection and that six-point i.d. injections resulted in a better vaccination than two-point i.d. injections.

Use of recombinant vaccinia-rabies virus for oral vaccination of fox cubs (Vulpes vulpes, L) against rabies.

Thirteen fox cubs were orally administered 10(7.2) plaque-forming units of live vaccinia-rabies glycoprotein recombinant virus. On Day 28 post-vaccination, all but 1 cub had produced rabies virus antibodies. Twelve animals were intramuscularly inoculated with 10(3.2) mouse intracerebral LD50 of rabies virus suspension on Days 33 (5 foxes), 180 (4 foxes) or 360 (3 foxes) after vaccination. Eleven of them resisted rabies challenge. Unvaccinated foxes, either put in contact with 1 vaccinated animal or used as controls, died after challenge applied on Day 33. The absence of horizontal transmission of this vaccine strain and its innocuity to cubs were also demonstrated.

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.

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.

[Biotechnology and animal health]. / Biotechnologie et santé animale.

The development of the first vaccines for use in animals, by Louis Pasteur at the end of the 19th Century, was an initial step in applying biotechnology to animal health. However, it is only much more recently that decisive progress has been made in finding applications for biotechnology, in both detecting and preventing infectious and parasitic diseases. This progress has shown the way to developing a range of procedures, the application of which will benefit the health of domestic and wild animals, enhance the well-being of companion animals, develop the performance of sporting animals and improve the productivity of farm animals, while also serving to protect human health. Such progress results from the increasingly rapid application of knowledge gained in the material and life sciences, all of which contribute to the multidisciplinary nature of biotechnology. Similarly, reagents and diagnostic techniques have been made more specific, sensitive, reproducible, rapid and robust by updating them through recent discoveries in immunology, biochemistry and molecular biology (monoclonal antibodies, nucleic probes, deoxyribonucleic acid amplification and many more). The development of new vaccines which combine efficacy, duration of protection, innocuity, stability, multivalence and ease of use (subunit vaccines, recombinant vaccines, synthetic vaccines and anti-idiotype vaccines) has resulted from recent progress in immunology, immunochemistry, molecular biology and biochemistry. Finally, the availability of new anti-infective, anti-parasitic agents and immunomodulatory therapeutic agents (capable of stimulating the specific and non-specific defence mechanisms of the body) demonstrates that biotechnology is continuing to find new applications in the field of animal health. New diagnostic techniques, vaccines and therapeutic substances are the most immediate applications of knowledge which may, in the future, extend to the development of transgenic animals of revised genetic potential, which will be more resistant to diseases and more productive. The ultimate aim of biotechnology applied to animal health and animal production is to protect human health, preserve the environment and ensure the health and well-being of animals.

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.

Vaccination against pseudorabies with glycoprotein gI+ or glycoprotein gI- vaccine.

Subunit pseudorabies vaccines that contained only purified glycoproteins of either of 2 strains of pseudorabies virus (PRV) were prepared and subsequently tested for safety and efficacy. The strains of virus used for vaccine production differed in at least 2 properties. One strain (Kojnok) was virulent for pigs and was believed to code for the entire complement of viral glycoproteins. The other (Kaplan) was a deletion mutant that was unable to code for structural viral glycoproteins gI and gp63. Purified glycoproteins were dispersed in an oil-in-water emulsion and were administered IM to pigs. Both vaccines were found to be safe and effective immunogens. Neither caused any local or general reactions, as verified by examination of the injection site (local safety) and by vaccination of pregnant sows in PRV-infected and noninfected herds. Sows vaccinated with the gI+ or gI- vaccine protected their pigs at levels of 93 and 92%, respectively, against a severe challenge exposure that killed 98% of pigs born from nonvaccinated sows. Vaccinated pigs were tested for active immunity by intranasal challenge exposure with the NIA 3 strain. Protection was quantitated by measuring the relative daily weight difference, expressed in percent per day, between vaccinated and control pigs during the first week after challenge exposure (delta G7); the estimated differences were 2.25 and 2.13% for gI+ and gI- vaccines, respectively. The absence of gI and gp63 did not affect the efficacy of this type of subunit glycoprotein vaccines.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of recombinant vaccinia-rabies glycoprotein virus for oral vaccination of wildlife against rabies: innocuity to several non-target bait consuming species.

The pathogenicity of a vaccinia recombinant virus expressing the rabies glycoprotein (VVTGgRAB) was tested in several wild animal species which could compete with the natural rabies host, the red fox (Vulpes vulpes) in consuming vaccine baits in Europe. The following species were included in this study: wild boar (Sus scrofa), Eurasian badger (Meles meles), wood mouse (Apodemus sylvaticus), yellow-necked mouse (Apodemus flavicollis), bank vole (Clethrionomys glareolus), common vole (Microtus arvalis), field vole (Microtus agrestis), water vole (Arvicola terrestris), common buzzard (Buteo buteo), kestrel (Falco tinnunculus), carrion crow (Corvus corone), magpie (Pica pica) and jay (Garrulus glandarius). During the observation period, the 107 animals given the VVTGgRAB vaccine orally did not show any clinical signs. Daily monitoring for 28 days and postmortem examination did not result in the detection of pox lesions in the oral mucosa or the skin in mammals or the unfeathered portions of birds. VVTGgRAB seems to multiply in the mammalian species tested, since rabies seroconversion was observed in all of them. Birds failed to develop demonstrable rabies virus-neutralizing antibody. A seroconversion against vaccinia virus was observed in two of four wild boars. Serological results obtained in badgers and wild boars also demonstrates the absence of direct or indirect horizontal transmission of the recombinant virus. The potential of the recombinant virus for the immunization of badgers against rabies also was investigated. Only 50% of the badgers orally administered with 1 x 10(8.3) TCID50 of this vaccine were protected against rabies.

Efficacy of a baiting system for vaccinating foxes against rabies with vaccinia-rabies recombinant virus.

The efficacy of a vaccinia-rabies recombinant virus (10(8) TCID50) contained in a machine-made baiting system has been tested in 22 captive young foxes which were divided into three experimental groups of six and a control group of four foxes. Each fox in groups 1, 2 and 3 were fed one, two and three vaccine-baits, respectively, on successive days. The four unvaccinated foxes were housed separately. As shown by the incorporation of a tetracycline biomarker into their bones, all the baited foxes ingested at least one bait. Thirty days after baiting seroconversion to rabies was observed in 15 (83 per cent) of the foxes and seroconversion to vaccinia in 14 (78 per cent). Sixteen of the 18 (89 per cent) baited foxes resisted a rabies challenge 30 days after baiting. One cub was protected against rabies despite the absence of detectable anti-rabies antibody. The results demonstrate that the bait-sachet system permits a good release of the virus suspension into the mouth.

Efficacy of a canarypox virus-vectored vaccine against feline leukaemia.

Canarypox virus recombinant vaccines have a unique efficacy and safety profile for the vaccinated host because the canarypox virus is non-replicative in mammalian hosts. After the vaccination of a mammalian species, recombinant canarypox viruses express the inserted genes but cannot multiply in the host. They stimulate a strong immune response in the absence of any virus amplification in the host or any viral spread into the environment. A new canarypox-based recombinant vaccine is the canarypox-feline leukaemia virus (FeLV) vaccine (EURIFEL FeLV; Merial) that expresses the FeLV env and gag protective genes. This paper describes experiments which demonstrate that it is effective against any oronasal FeLV challenge. The protection was shown to be solid against an oronasal challenge one year after the initial vaccination, and was effective against a very severe 'in-contact' challenge. Furthermore, the canarypox virus-FeLV vaccine was effective without an adjuvant.

Diagnosis and prevention of equine infectious diseases: present status, potential, and challenges for the future.

The frequent transfers of horses, whether on a permanent or temporary basis, make strict control of infectious diseases essential. Such control needs a reliable and rapid means to accurately diagnose the relevant diseases. Indirect diagnosis based on antibody detection remains certainly the best method to secure the epidemiologic surveillance of the diseases at regional, national, or even world level, while direct diagnosis is the only way to diagnose a new outbreak. New diagnostic methods resulting from advances in biochemistry, molecular biology, and immunology are now available. As far as antibody detection is concerned, the new methods are mainly based on immunoassays, especially ELISAs. Regarding the identification of the pathogens, while isolation is still of importance, much progress has been made with immunocapture tests including capture ELISA based on monoclonal antibodies. DNA probes and amplification tests such as PCR or RT-PCR are representing a real breakthrough. Factors common to all of these tests are specificity, sensitivity, rapid implementation, and quick results. Such tests are, however, often still at the development stage. They absolutely need to be validated under multicentric evaluations prior to being used on a larger scale. At the same time there is an obvious need for the standardization of the reagents used. The technical and economic impact of a false (either positive or negative) diagnosis justifies such an harmonization which could effectively be achieved worldwide under the aegis of the Office International des Epizooties (OIE), which is itself the primary source of disease information. Vaccines are also essential for the control of equine infectious diseases. Most vaccines used in the prevention of viral or bacterial diseases are inactivated adjuvanted vaccines, which may cause unacceptable side effects. Also, their efficacy can sometimes be questioned. Subunit vaccines, when available, represent significant advances especially with regards to safety. Greater progress is expected from the use of new technologies taking advantage of recent developments in molecular biology (recombinant DNA technology) and in immunology (immunomodulators). Significant results have been obtained with subunit vaccines or with live vectored vaccines using recombinant DNA technology. Good results are on the way to be achieved with genetic (or naked-DNA) vaccines. It is therefore possible to expect the availability of a new generation of vaccines in the rather short term. Such vaccines will not only be safer and more efficacious, but they will also make it possible to differentiate vaccinated from infected animals, which will contribute to better control of the infection. Whatever the quality of the vaccines of the future may be, vaccination alone will never be sufficient to control infectious diseases. It is therefore essential to keep on making the animal owners and their veterinarians aware of the importance of the management and the hygiene in the diseases control and to organize them under "Common Codes of Practice."

[Determination of antigen structures of Pasteurella multocida: the preparation of vaccines]. / Determination des structures antigeniques de Pasteurella multocida: intérêt pour la préparation des vaccins.

The discovery of the role played by somatic antigens of the Pasteurella multocida A and D capsular types in the immunization of susceptible species makes it necessary to type the somatic antigens of the isolated strains prior to any action to induce active or passive immunity. Only immunoprecipitation in gel was chosen out of the various typing techniques proposed, and a correlation was established between the capsular types (according to Carter), somatic types (according to Namioka) and types determined by immunoprecipitation in gel (according to Heddleston). Parallel to this, the same technique was developed for strains of Pasteurella multocida B and E capsular types, and the immunogenicity of these capsular antigens was proven.

[Cloning and expression of the genetic determinants encoding for the serological variants of the adhesive factor K 88 carried by enterotoxinogenic Escherichia coli strains of porcine origin]. / Clonage et expression des déterminants génétiques codant pour les sous-types du facteur d'attachement K 88 porté par les souches d'Escherichia coli entérotoxinogènes d'origine porcine.

The existence of homologous sequences on plasmids carried by 3 enterotoxinogenic strains of Escherichia coli of porcine origin, and encoding for adhesive factors of different antigenic specificities: K 88 ab, K 88 ac, K 88 ad; has been demonstrated by DNA-DNA hybridization. The use as probe of a DNA fragment carrying a piece of structural genes of the adhesive factor K 88 ab taken out of a recombinant plasmid expressing this factor, has allowed us to localize the genetic determinants which encode for the adhesive factors K 88 ac and K 88 ad. Cloned in the plasmid pBR 322, these determinants have allowed us to obtain the expression, by the host bacteria of the corresponding adhesive factors.

[Comparative study of mycobactin-dependent strains of mycobacteria isolated from the wood-pigeon with Mycobacterium avium and M. paratuberculosis: study of biological and antigenic characteristics]. / Etude comparative de souches de mycobactéries mycobactine-dépendantes, isolées de pigeon ramier, avec Mycobacterium avium et M. paratuberculosis: étude des caractères biologiques et antigéniques.

In this investigation three mycobacterial strains isolated in our laboratory from wood-pigeons were compared with one strain isolated by Matthews and another by Jorgensen from, respectively, a wood-pigeon and a roe-deer. The strains were also compared with various strains of Mycobacterium avium and M. paratuberculosis. The strains isolated from the wood-pigeons formed a relatively homogeneous group, which could be distinguished from M. avium and M. paratuberculosis. It was interesting to verify that most of the cultural and biochemical properties of the wood-pigeon mycobacteria were similar to those of M. paratuberculosis. The strains formed rough colonies and grew slowly in special mediums containing M. phlei extracts or mycobactin. However, one must recall that mycobactin dependence was also reported by Matthews for some strains of M. avium. The tween hydrolysis test (10 days), negative for M. avium, was positive for both the wood-pigeon mycobacteria and M. paratuberculosis. The trehalase test, which appears to be regularly positive for M. avium, was also positive for the wood-pigeon mycobacteria and M. paratuberculosis. In respect to drug susceptibility, no significant differences were observed. The organisms were resistant to most drugs studied, but were also resistant to cycloserine, in contrast to most M. avium strains. On the other hand, the organisms were not distinguished using sensitins, prepared from wood-pigeon mycobacteria and M. paratuberculosis using specifically sensitized guinea-pigs. However, the wood-pigeon mycobacteria could be clearly differentiated from M. paratuberculosis and M. avium using serology methods. Indeed, the wood-pigeon mycobacteria did not agglutinate in the presence of any of the sera defining the M. avium-intracellulare serovars (serovars 1 to 28); and by immunodiffusion in agar the wood-pigeon mycobacteria antigens formed two precipitation lines which were absent from M. paratuberculosis. Judging from our findings, the wood-pigeon mycobacteria are more closely related to M. paratuberculosis than to M. avium. It appears that biochemical and antigenic properties are not sufficient to completely differentiate these bacteria. Further studies are needed, and we plan to investigate in the near future their pathogenicity for rabbits, chicken and calves.

Efficacy studies on a canarypox-rabies recombinant virus.

Recombinant avipox viruses have been developed expressing the rabies glycoprotein gene. A fowlpox-rabies recombinant has previously been shown to be protective against live rabies virus challenge in a number of non-avian species. This report describes the development of a canarypox-rabies recombinant. A comparison is made of the protective efficacy of this recombinant with other pox-rabies recombinants.

Protection of cats against feline leukemia virus by vaccination with a canarypox virus recombinant, ALVAC-FL.

Two ALVAC (canarypox virus)-based recombinant viruses expressing the feline leukemia virus (FeLV) subgroup A env and gag genes were assessed for their protective efficacy in cats. Both recombinant viruses contained the entire gag gene. ALVAC-FL also expressed the entire envelope glycoprotein, while ALVAC-FL(dl IS) expressed an env-specific gene product deleted of the putative immunosuppressive region. Although only 50% of the cats vaccinated with ALVAC-FL(dl IS) were protected against persistent viremia after oronasal exposure to a homologous FeLV isolate, all cats administered ALVAC-FL resisted the challenge exposure. Significantly, protection was afforded in the absence of detectable FeLV-neutralizing antibodies. These results represent the first effective vaccination of cats against FeLV with a poxvirus-based recombinant vector and have implications that are relevant not only to FeLV vaccine development but also to developing vaccines against other retroviruses, including human immunodeficiency virus.

Recombinant fowlpox virus inducing protective immunity in non-avian species.

The natural host of fowlpox virus is limited to avian species. When inoculated into non-avian tissue culture cells, however, fowlpox virus can initiate an abortive infection. A fowlpox virus was engineered to express rabies virus glycoprotein. On inoculation of the recombinant virus into either avian (permissive) or non-avian (non-permissive) cells, the rabies glycoprotein was expressed as a membrane-associated antigen. Inoculation of the fowlpox virus recombinant into six different species of mammal resulted in specific immune responses to both fowlpox antigens and to rabies glycoprotein. In mice, cats and dogs the immune response was sufficient to protect against a live rabies virus challenge. The results demonstrate the utility of a fowlpox virus vector in immunizing non-avian species against rabies in the absence of productive viral replication of the fowlpox vector.