Comparison between acute oral/respiratory and chronic stomatitis/gingivitis isolates of feline calicivirus: pathogenicity, antigenic profile and cross-neutralisation studies.

Feline calicivirus (FCV) is a major oral and respiratory pathogen of cats, able to induce subclinical infection as well as acute disease. It is also characterized by a high degree of antigenic variation. This work sought to address the question of the existence of distinct biotypes of FCV. Eight French, 6 British and 9 American FCV isolates, responsible for acute oral/respiratory disease or chronic gingivitis/stomatitis, were compared for their pathogenicity, antigenic profiles and serological relationships. Antigenic profiles were assessed by an indirect immunofluorescence assay with a large panel of characterized monoclonal antibodies. Cross-neutralisation assays were performed with specific cat antisera collected at 30 days p.i., then analysed by calculation of antigenic bilateral relatedness and dominance. Whatever their pathogenic origin, all the isolates induced an acute upper-respiratory tract infection in oronasally infected SPF kittens. Their antigenic profiles were different and did not correlate with their geographical or pathological origin. Cross-neutralisation studies and calculation of the mean bilateral relatedness allowed us to distinguish chronic original isolates from acute original ones. This study did not confirm the existence of FCV biotypes but showed that the chronic carrier state is related to the emergence of antigenically distant viruses.

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.

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.

Neonatal immunity and immunisation in early age: lessons from veterinary medicine.

The objective of this paper is to review adaptive immunity of young animals using examples from my own experience and from the literature. Trials carried out by us with a modified live and inactivated canine parvovirus vaccine in newborn puppies provide evidence of the immune capacity of these puppies. With regard to transfer of immunity from mother to offspring, there is a role for transplacental and colostral immunity. Examples of passive protection of young animals against different infections include passive protection of kittens against the feline immunodeficiency virus. However, passive immunity, though very useful at an early age, varies in duration and makes implementation of standard vaccination schedules difficult. Other experiments demonstrate that, under certain conditions, it is possible to overcome residual maternally-derived antibodies and to induce post-vaccinal immunity.

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.

Spontaneous programmed cell death (PCD) process of lymphocytes of FIV-infected cats: pharmacological modulation in vitro.

We previously reported that unstimulated lymphocytes in culture 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, interleukins [interleukin (IL)1, Il2, IL6 and interferon-gamma (IFN gamma)] and after activation by phorbol ester [phorbol myristyl acetate (PMA)], superantigens [staphylococcal enterotoxin B (SEB), staphylococcal enterotoxin A (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). In this study, we studied the spontaneous programmed cell death (PCD)-inducing pathways, and the mechanisms of action of PMA, SEB and SEA. Spontaneous lymphocyte apoptosis of FIV-infected cats was inhibited by cycloheximide, ZnSO4 and N-acetyl-cystein. The preventive effect of SEB and SEA was inhibited by actinomycin, but not by inhibitors of kinases. Calyculin, an inhibitor of phosphatase, had no effect either on spontaneous apoptosis, or on the action of PMA, SEB and SEA. Ionomycin-induced apoptosis was found sensitive to PMA and cytokines. In FIV-infected cats, these data suggest that the mature lymphocytes appear programmed to die by apoptosis, unless rescued by specific agents, such as protein kinase C activators or growth factors, and that spontaneous PCD seems to be dependent of de nove protein synthesis (see effect of cycloheximide). The effects of PMA, SEB and SEA are probably mediated by de novo proteins which for PMA, undergo a phosphorylation involving serine-threonine and/or tyrosine groups. Our data suggest a clear difference between lymphocytes from FIV-infected cats and lymphocytes from HIV-infected humans, with regard to their metabolic regulations.

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.

Stability of recombinant vaccinia-rabies vaccine in veterinary use.

Wildlife vaccination depends on vaccines which can be orally administered by a baiting system. Therefore only two possibilities exist: either the use of attenuated strains of viruses, or recombinant vector viruses. As far as rabies is concerned, the choice of the recombinant vaccinia-rabies virus was made because it was safer and more stable. An in vitro stability study of the recombinant product compared to wild rabies virus at different temperatures (4 degrees C, 20 degrees C, 37 degrees C, 45 degrees C) showed that the recombinant virus was more stable. The stability of the recombinant virus was also tested under field conditions; besides natural freezing and thawing cycles, the virus titre remained unchanged in the bait for a month. Taking into account the fact that all baits are eaten by wild animals within this period, one can assume that the vaccine is efficacious for all baiting animals in field conditions. The stability of the recombinant vaccinia-rabies vaccine is of considerable interest in such uncontrolled conditions.

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.

Localization of antigenic sites of the S glycoprotein of feline infectious peritonitis virus involved in neutralization and antibody-dependent enhancement.

The S glycoprotein of feline infectious peritonitis virus (FIPV) has been shown to contain the antigenic sites responsible for eliciting both neutralization and antibody-dependent enhancement. To determine the region of S responsible, overlapping DNA fragments spanning the entire S gene were cloned and expressed as fusion proteins by in vitro transcription and translation. Fusion proteins containing relevant epitopes were identified by radioimmunoprecipitation with neutralizing and enhancing FIPV-specific monoclonal antibodies (MAbs). A region spanning residues 509 to 673 reacted with most MAbs tested. Translation in the presence of microsomal membranes did not enhance reactivity, suggesting that glycosylation is not essential for recognition by the MAbs. To localize the antigenic sites further, several MAb-resistant (mar) mutants of FIPV were cloned and sequenced. Amino acid residues that contribute to the neutralizing and enhancing epitopes were localized to two regions, designated A1 and A2, which show partial overlap with the homologous antigenic site A of transmissible gastroenteritis virus. Site A1 contains residues 568 and 591 and is homologous with part of subsite Aa of transmissible gastroenteritis virus. Site A2 contains residues 643, 649, and 656. Double mutations in sites A1 and A2 were found in mar mutants derived from neutralizing and enhancing MAbs 23F4.5 and 18A7.4, while a single mutation in site A2 was found in a mar mutant derived from MAb 24H5.4, which is neutralizing but not enhancing. The data suggest that site A2, which includes residues 643 to 656, is a dominant neutralizing site of FIPV and that sites A1 and A2 may act in concert to induce antibody-dependent enhancement.

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.

The safety and use of canarypox vectored vaccines.

ALVAC recombinants have been administered to humans and animals by parenteral and oral routes without giving signs of replication, systemic dissemination or severe reaction. In principle, it should be impossible for canarypox recombinants to disseminate in the environment as they would not be synthesised in mammalian cells as complete virus. Canarypox vectors have been safe for humans, in whom there has been no evidence of replication, but more work needs to be done to prove absence of replication. Recombinants are immunogenic by the intramuscular and subcutaneous routes. They are also immunogenic when given orally, but the dose required is still under study. Canarypox recombinants effectively prime the immune system for induction of antibodies and CD8 cell-mediated cytotoxicity by protein antigens. Antibody responses are not influenced by prior inoculation of canarypox, of subunit vaccine corresponding to the gene insert, or of vaccinia. Canarypox virus is attenuated for canaries, in which species it is already widely used. In principle, it is non-infectious for humans or other mammals. It may be infectious for other birds.

Applications of canarypox (ALVAC) vectors in human and veterinary vaccination.

This paper presents data derived from safety and efficacy studies of ALVAC-based rabies and feline leukemia virus (FeLV) vaccine candidates in target species. Inoculation of the ALVAC-RG recombinant was well tolerated in all species including humans and very young dogs. Protection induced in dogs against rabies challenge was long-lasting and could be elicited in the face of high levels of maternally-derived neutralizing antibody. Parenteral inoculation of cats with an ALVAC-FeLV recombinant was safe and induced protection against persistent infection following oro-nasal FeLV challenge.

[Demonstration of cell death process by apoptosis in cat lymphocytes infected by FIV (feline immunodeficiency virus)]. / Mise en évidence d'un phénomène de mort cellulaire par apoptose dans les lymphocytes de chats infectés par le FIV (feline immunodeficiency virus).

Feline immunodeficiency virus (FIV), the causative agent of feline AIDS, induces a disease syndrome in cats characterized by a decreased lymphocyte-proliferative response to mitogens at all stages of infection and selective depletion of CD4 lymphocyte subsets. In this work, we report that peripheral blood lymphocytes isolated from FIV-infected cats undergo a spontaneous death, in vitro, according to a programmed cell death (PCD) or apoptosis. This phenomenon has also been seen in peripheral blood lymphocytes from HIV-infected humans and SIV-infected macaques. Four different techniques were used to document PCD in FIV-infected cats. DNA gel electrophoresis has shown a DNA fragmentation pattern with DNA fragments displaying sizes corresponding to multiples of oligonucleosomes DNA length unit (180 bp). Transmission electron microscopy revealed condensation of both nuclear chromatin and cytoplasm. An increase in the percentage of fragmented DNA was demonstrated by Burton's technique. In addition, flow cytometric analysis detected a cell population with condensed chromatin. The spontaneous PCD in FIV-infected cats could not be inhibited by RNA synthesis inhibitors or protein synthesis inhibitors. Our results could have implications for understanding the pathogenesis of FIV-infection and establishing specific strategies against apoptosis in cats and humans.

Expression of the bovine viral diarrhoea virus Osloss p80 protein: its use as ELISA antigen for cattle serum antibody detection.

The putative gene encoding the cytopathic bovine viral diarrhoea virus (BVDV) Osloss strain p80 protein was amplified by PCR and inserted into a T7 promoter-based vector for expression in Escherichia coli. Bacterial expression led to cytoplasmic insoluble inclusion bodies which were denatured by urea treatment and renatured by dialysis. Rabbit antisera were raised against this p80 recombinant antigen and assayed for the immunoprecipitation of either p120 or p80 protein from cytopathic or non-cytopathic BVDV biotype-infected bovine cells. The p80 gene sequence was also integrated into a baculovirus genome for its expression in Spodoptera frugiperda insect cells. The recombinant proteins isolated from bacteria or insect cells showed distinct antigenic properties when analysed by ELISA. Their ability to detect anti-BVDV specific antibodies was examined in a monoclonal antibody-based competitive ELISA performed on a series of field cattle sera. This comparative assay revealed the superiority of the insect cell-mediated expression to mimic the natural BVDV antigen produced by cell culture. The baculovirus/insect cell recombinant antigen gave the highest correlation between the ELISA-detected antibodies and the corresponding virus neutralization data.

Nucleotide sequence of the bovine viral diarrhoea virus Osloss strain: comparison with related viruses and identification of specific DNA probes in the 5' untranslated region.

The nucleotide sequence of the cytopathic Osloss isolate of bovine viral diarrhoea virus (BVDV) was deduced from overlapping cDNA clones and from PCR products. The Osloss genome is an RNA molecule of positive polarity containing 12,480 nucleotides and having the capacity to code for a polyprotein of 3975 amino acids. The presence of the previously described internal stop codon in this viral sequence was disproved after direct sequencing of the appropriate PCR-amplified fragment. Except for the previously reported insertion of a sequence coding for a ubiquitin-like protein, the viral genome shares great similarity with those of three other strains of the pestivirus genus. Computer-assisted sequence analyses and comparisons of known pestiviral genomic sequences led us to identify selected PCR primers in the 5' untranslated region. These primers were used successfully to amplify 18 distinct pestivirus isolates and potential DNA probes were noted from the deduced sequences. The possible use of a well conserved 26 base fragment as a diagnostic probe was confirmed in hybridization experiments. The 5' untranslated region was further studied and compared with those of other members of the Flaviviridae family, which includes the flaviviruses and the hepatitis C virus group. These sequence analyses support the possibility of discrimination amongst the closely related ruminant pestiviruses, border disease virus and BVDV.

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.