Biomedical subjects

R Cariolet

Publications and source records attributed to R Cariolet.

At least 19 recordsLinked to original sources

PubMed2003

Protection of swine against post-weaning multisystemic wasting syndrome (PMWS) by porcine circovirus type 2 (PCV2) proteins.

Porcine circovirus type 2 (PCV2) is known to be associated with post-weaning multisystemic wasting syndrome (PMWS), a recently described disease of young pigs. Since no PCV2 vaccine was available so far, we have developed a specific PCV2 vaccine candidate. The Orf1-encoded replication protein and Orf2-encoded capsid protein of PCV2 were expressed and detected in either mammalian or insect expression systems. In a first trial, Orf2 protein was found to be a major immunogen, inducing protection in a prime-boost protocol; the piglets received a first injection with plasmids directing Orf2 protein and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression, followed by a second injection, a fortnight later, associated with baculovirus-expressed Orf2 protein. As evaluated by growth parameters, clinical signs (fever), seroconversion, the pigs were protected against a PCV2 challenge after vaccination. In a second trial, protection induced by a subunit vaccine was even better than the one induced by DNA vaccine, since PCV2 replication was completely inhibited.

Animals↗

PubMed2003

An ORF2 protein-based ELISA for porcine circovirus type 2 antibodies in post-weaning multisystemic wasting syndrome.

Porcine circovirus type 2 (PCV2) plays a crucial role in the pathogenesis of post-weaning multisystemic wasting syndrome (PMWS) in swine. As PCV2 displays significant homology with PCV1 (a non-pathogenic virus) at the nucleotide and amino-acid level, a discriminative antigen is needed for specific serological diagnosis. The ORF2-encoded capsid protein from PCV2 was used to develop an indirect enzyme-linked immunosorbent assay (ELISA). GST-fused capsid protein from PCV2 and GST alone (both expressed in recombinant baculovirus-infected cells) were used as antigens for serodiagnosis. The specificity of the ELISA for detection of PCV2 antibodies was demonstrated in sera from pigs experimentally infected with PCV1, PCV2 and other swine viruses. The semi-quantitative nature of the test was evaluated versus an immunoperoxidase monolayer assay (IPMA). The ELISA was performed on 322 sera from pigs in eight Brittany herds and compared with IPMA. The sensitivity (98.2%) and specificity (94.5%) of this test were considered suitable for individual serological detection. High PCV2 seroprevalence was found in sows and pigs at the end of the growth phase (18-19 weeks) in all eight herds. The seroprevalence in piglets (11-17 weeks) was statistically correlated with clinical symptoms of PMWS (93% in affected versus 54%, in non-affected farms). A cohort study performed in PMWS-free farms showed that 57% of piglets exhibited active seroconversion after 13 weeks, indicating that PCV2 infection occurred earlier in PMWS-affected piglets.

Animals↗

PubMed2001

Experimental airborne transmission of Streptococcus suis capsular type 2 in pigs.

Experimental airborne transmission of Streptococcus suis type 2 was studied in specific pathogen free piglets. Forty piglets were allotted to five groups of eight 7-week-old animals and housed in three separated units. Negative control pigs (group 1) were housed in unit A and infected batches were housed in units B (group 2) and C (groups 4). In units B and C, non-inoculated groups (groups 3 and 5, respectively), 40 cm distant from the respective inoculated group and without any physical contact between them, also took place. Six animals of groups 2 and 4 were inoculated intravenously with 2 x 10(8) colony forming units (cfu) of a mild and a high virulent S. suis strains, respectively. The remaining animals in these groups and pigs from groups 1, 3, 5 received broth medium in the same way. Differences among virulence of S. suis capsular type 2 were observed in inoculated pigs of groups 2 and 4. Pigs from group 2 became carriers, showing only mild symptoms. By contrast, animals from group 4 presented an acute form of the disease. All the indirect contact pigs in groups 3 and 5 had S. suis in palatine tonsils from day 6 after the infection and they presented clinical manifestations similar to those observed in experimentally infected pigs. Two direct contact animals were also contaminated in the upper respiratory tract but surprisingly they did not show any symptoms. Airborne transmission of S. suis in experimentally pigs was demonstrated in the present study. Indirect infections, as described in this study, are a more realistic way to infect pigs than other experimental procedures and may be used to further study the pathogenesis of the infection caused by this important pathogen.

Animals↗

PubMed2001

An experimental model for post-weaning multisystemic wasting syndrome (PMWS) in growing piglets.

Post-weaning multisystemic wasting syndrome (PMWS) is a comparatively new disease of swine, and known to occur in France since 1996. A porcine circovirus type 2 (PCV2) is found in the lesions of affected piglets. Six piglets aged 10-13 weeks were obtained from a French PMWS-affected farm. Two showing characteristic signs of PMWS (palor, weakness and emaciation) remained in poor condition and were finally killed 6 and 9 days after their arrival in the experimental unit. Tissue homogenates from these two piglets were used to reproduce mild PMWS in specific pathogen-free (SPF) piglets. This mild PMWS consisted of pyrexia (up to 41.7 degrees C) and growth retardation (up to 30% of weight reduction compared with controls) commencing 1 week after infection and lasting 3 weeks. In seven additional trials, pyrexia, growth retardation and lesions characteristic of PMWS were consistently produced in SPF and conventional piglets. However, only four of 55 inoculated SPF piglets (7.2%) showed severe wasting disease. One died and the others had to be killed 3 to 4 weeks after inoculation. None of the inoculated animals developed antibodies to any common swine viruses or bacteria, but clear evidence of PCV2 seroconversion was obtained. Our results therefore strongly suggest that PCV2 is the primary aetiological agent of PMWS.

Animals↗

PubMed2001

Experimental infection of specific pathogen free piglets with French strains of Streptococcus suis capsular type 2.

A standardized model of Streptococcus suis type 2 infection in specific-pathogen-free piglets, housed in high-security barns, was used to compare the virulence of 3 French field strains of S. suis serotype 2 isolated from tonsils of a healthy pig (strain 65) or from diseased pigs (meningitis, strain 166', or septicemia, strain 24). In one of the 2 trials, 7-week-old pigs, in 3 groups of 8, were inoculated intravenously with 2 x 10(8) colony-forming units of S. suis type 2. In each group, 1 uninfected animal was a sentinel. Eight animals were also used as negative control group. The experiment was repeated under similar conditions with strains 65 and 166'. Virulence differed markedly among these S. suis strains when clinical signs, zootechnical performances, lesions, and bacteriological data were analyzed. Strain 65 did not induce clinical signs in inoculated pigs. In contrast, pigs infected with the other 2 strains exhibited clinical signs and typical lesions of S. suis type 2 infections. Differences in virulence were also observed between the 2 virulent strains. Sentinel animals exhibited the same manifestations as those recorded in inoculated piglets. Results were similar in the second trial, indicating that under the present experimental conditions, results were reproducible. The standardized conditions described in this study could be a useful tool to further study about the S. suis infection.

Animals↗

PubMed2000

A PCR assay used to study aerosol transmission of Actinobacillus pleuropneumoniae from samples of live pigs under experimental conditions.

The study describes a polymerase chain reaction (PCR) assay for the detection of Actinobacillus pleuropneumoniae. The test is based on the amplification of the omlA gene coding for an outer membrane protein of A. pleuropneumoniae. To test the specificity of the reaction, 19 other bacterial species related to A. pleuropneumoniae or isolated from pigs were assayed. They were all found negative in the PCR assay. The detection threshold of the test was 10(2) A. pleuropneumoniae CFU/assay. The test was then applied to the detection of A. pleuropneumoniae from tonsillar biopsies and tracheobronchial lavage fluids of pigs without a culture step. The detection of A. pleuropneumoniae in these samples was performed by PCR, by conventional culture and by bacteriology with immunomagnetic beads. The number of samples that were found positive by PCR was almost three times higher than the number of samples from which A. pleuropneumoniae was isolated by both bacteriological techniques. The detection of A. pleuropneumoniae in these samples allowed us to demonstrate its aerosol transmission to pigs under experimental conditions. The trial involved 18 specific pathogen free pigs. Six pigs, infected with A. pleuropneumoniae, were located in a unit A, together with four non-infected animals (contact pigs). Eight non-infected pigs (reporter pigs) were located in a unit B, adjacent to A. We detected A. pleuropneumoniae in samples from infected animals but also from 'contact' (unit A) and 'reporter' (unit B) pigs. The results of this study show that the simple preparation of the samples followed by the PCR assay may be a useful tool for epidemiological studies.

Actinobacillus Infections↗

PubMed2000

Experimental models of porcine post-weaning colibacillosis and their relationship to post-weaning diarrhoea and digestive disorders as encountered in the field.

The aim of this study was to develop a reliable model system of porcine post-weaning colibacillosis, and in doing so to assess the primary relationship of enterotoxigenic Escherichia coli to post-weaning diarrhoea and digestive disorders as encountered in the field. Six sequential experiments were carried out using 168 SPF piglets weaned into an optimal controlled environment at 28 days of age. The piglets were allocated to 23 treatment groups, 17 of which were inoculated either orally or intragastrically with enterotoxigenic strains of E. coli (LT+, STI+, STII+) possessing adhesive factors including K88 (F4). The piglets were challenged either once (Day 4 post-weaning) or on several days post-weaning, with the challenge load for each inoculation varying from 10(8) to 10(12) CFU. Overall 14.5% of inoculated pigs developed severe illness and died: these had lesions in their digestive tracts typical of colibacillosis. Diarrhoea occurred on at least 1 day in 50% of inoculated pigs, but was transient (1.7 days on average), appeared very soon after challenge (sometimes within half a day), and was accompanied by signs of depression and low weight gain. Generally a prompt recovery then occurred. In the second 2 weeks post-inoculation daily weight gain reached the same level in most inoculated groups of pigs as in the uninoculated controls. Only a small number of pigs developed a chronic enteritis lasting several days, as is typically observed in field cases. Diarrhoea was more common in the piglets that were tested adhesive positive to the K88 fimbriae receptor, but the disorders were no more severe in these animals. The response of all pigs depended primarily on the inoculum used, and especially on the challenge load. Although enterotoxigenic E. coli are clearly important in the aetiology of post-weaning diarrhoea, other factors are also required for the production of the chronic post-weaning digestive disorders and ill-thrift that are commonly encountered in commercial piggeries.

Animals↗

PubMed2000

Single inoculation of replication-defective adenovirus-vectored vaccines at birth in piglets with maternal antibodies induces high level of antibodies and protection against pseudorabies.

In neonates, one limitation of vaccination is its inhibition by maternal antibodies. We show that piglets vaccinated intramuscularly once at birth with recombinant replication-defective adenoviruses developed comparable neutralizing antibody response against pseudorabies virus, independently of the presence or absence of maternal antibodies, and were partially protected against challenge 16 weeks later.

Adenoviridae↗

PubMed2000

Induction of porcine cytokine mRNA expression after DNA immunization and pseudorabies virus infection.

Injection of plasmid DNA encoding pseudorabies virus (PRV) glycoprotein into pig muscle has been shown to result in protective immunity against lethal infection. Here, pigs were vaccinated by a single coinjection of three plasmids encoding PRV glycoproteins gB, gC, and gD, with plasmid expressing porcine granulocytemacrophage colony-stimulating factor (GM-CSF) or porcine interferon-alpha (IFN-alpha). DNA immunization induced a primary T cell-mediated response characterized by low rates of IFN-gamma, interleukin-2 (IL-2), and IL4 mRNA in peripheral blood mononuclear cells (PBMC). Very low rates of PRV-specific IgG1 and the absence of IgG2 were obtained. Codelivery of plasmid expressing GM-CSF or IFN-alpha had no effect on cytokine mRNA expression or on B cell response. After a high virulent challenge, high levels of cytokine mRNA, mainly IFN-gamma, and high secondary antibody (Ab) response were induced in all DNA-vaccinated pigs. Codelivery of GMCSF gene significantly increased both Th immune response (i.e., IFN-gamma and IL-4 mRNA expression) and clinical protection but had no effect on secondary B immune response. Codelivery of IFN-alpha gene had no beneficial effect on secondary T and B cell immune responses.

Animals↗

PubMed2000

Differential recognition of ORF2 protein from type 1 and type 2 porcine circoviruses and identification of immunorelevant epitopes.

Two types of porcine circovirus (PCV) have been isolated and are referred to as PCV1 and PCV2. PCV1 represents an apathogenic virus, whereas PCV2 is associated with post-weaning multisystemic wasting syndrome. The two PCVs are related, since they display about 70% identity based on nucleotide sequences. In order to discriminate between common and type-specific antigens, an immunocytological approach was used following transfections with cloned circovirus DNAs, as well as recombinant proteins expressed by either baculovirus or plasmid vectors. The ORF1-encoded proteins in the two viruses were shown to be antigenically related, whereas the ORF2 proteins were recognized differentially by polyclonal anti-PCV2 antibodies. Furthermore, PEPSCAN analysis performed on overlapping fragments of the genes encoding part of ORF1 and the entire ORF2 and ORF3 led to the identification of five dominant immunoreactive areas, one located on ORF1 and four on ORF2. However, only some ORF2 peptides proved to be immunorelevant epitopes for virus type discrimination. The potential use of ORF2-derived antigens as diagnostic tools is demonstrated.

Amino Acid Sequence↗

PubMed2000

Experimental aerosol transmission of Actinobacillus pleuropneumoniae to pigs.

In order to demonstrate the possible role of aerosol in the transmission of Actinobacillus pleuropneumoniae, an experiment including 18 specific pathogen-free (SPF), 10-week-old piglets, randomly distributed into 2 adjacent units, was carried out. In these facilities, air was forced through absolute filters to prevent any contact with infectious agents. During the first 6 d post inoculation, the 2 units were connected by a rectangular opening and the air circulation was forced by the ventilation system from unit A (inoculated pigs) to unit B (non-inoculated pigs). The A. pleuropneumoniae strain (biovar 1 serovar 9) was isolated in France from an outbreak of porcine pleuropneumonia. Two different infecting doses, 10(7) cfu/animal and 10(8) cfu/animal, were inoculated by intranasal route in 6 pigs of unit A. The infection spread quickly from the inoculated pigs to the non-inoculated pigs. Clinical signs were acute during the 4 d post inoculation: hyperthermia, respiratory distress and, sometimes, death (6 pigs of the unit A and 2 pigs of the unit B). All pigs seroconverted against A. pleuropneumoniae serovar 9 within 2 weeks. Lung lesions were severe: fibrinous pleurisy and lung hemorrhages in the acute stage, pleural adherences and focal pulmonary necrosis in the chronic stage. Actinobacillus pleuropneumoniae was isolated from the tonsils and/or lungs in 16 animals. It could be also isolated from the air of the experimental unit. This study showed that A. pleuropneumoniae was readily transmitted through aerosol over a distance of at least 2.5 m.

Actinobacillus Infections↗

PubMed1998

Immune responses in pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV).

In three successive experiments, the immune functions of pigs persistently infected with the porcine reproductive and respiratory syndrome virus (PRRSV) have been evaluated. Non-specific immune responses were analyzed over a period of 12 weeks post-infection (PI). In addition, the capacity of PRRSV-infected pigs to develop an efficient immune response against pseudorabies virus (PRV) glycoproteins and to resist to a subsequent virulent challenge was investigated. Our results demonstrate that PRRSV produced minor effects on the immune system of pigs. The skin delayed type hypersensitivity (DTH) in response to phytohemagglutinine injection was slightly diminished one week after challenge, but was restored thereafter. However, three weeks after the infection, the total white blood cell count, and the number of CD2+, CD8+ and IgM+ cells were enhanced. The increase in numbers of CD8+ cells persisted for three consecutive weeks. Serum immunoglobulins in infected pigs also increased by week 3 PI and up to 8 weeks PI. These results show that PRRSV may have stimulating effects on the pig immune system during the phase of long-lasting infection. After immunization with PRV glycoproteins, the production of anti-PRV antibodies and skin DTH response against PRV glycoproteins were not affected. On the contrary, following a virulent PRV challenge, PRRSV-infected pigs developed a better secondary antibody response and their resistance to the infection was as effective as in control pigs. Taken together, our data do not support a systemic immunosuppressive effect of PRRSV, during the persistent phase of infection. Other mechanisms may therefore apply to explain the emergence of secondary infections in endemically infected herds.

Animals↗

PubMed1997

Development of a RT-PCR test coupled with a microplate colorimetric assay for the detection of a swine Arterivirus (PRRSV) in boar semen.

Transmission of porcine reproductive and respiratory syndrome virus (PRRSV) through boar semen has been demonstrated, stressing the need for a reliable semen PRRSV detection test. A diagnostic assay was developed based on amplification of the PRRSV RNA by reverse transcription and polymerase chain reaction (RT-PCR) followed by detection of the amplification products by hybridization and colorimetric assay in microwell plates. A highly reproducible and efficient method of viral RNA isolation from semen samples was set up. A combined RT-PCR procedure was performed, incorporating the use of uracil-N-glycosylase (UNG) in combination with dUTP instead of dTTP to prevent false positive results due to carry-over contamination. An RNA internal control was added during the RNA isolation procedure to detect false negative results. The colorimetric detection in microwell plates of amplification products from either PRRSV or IC RNA gave specific and objective results and was automated. A cut-off value of 1000 RNA copies or 10 TCID50 of PRRSV per ml of semen samples could be detected with this assay. Semen samples collected from experimentally-infected boars were tested with this assay and showed PRRSV excretion early after infection and for an extended period.

Animals↗

PubMed1997

Effective priming of neonates born to immune dams against the immunogenic pseudorabies virus glycoprotein gD by replication-incompetent adenovirus-mediated gene transfer at birth.

One of the main limitations of the vaccination of neonates from vaccinated or infected mothers is the interference by inherited maternal antibodies, which are known to inhibit the immune response against both live and inactivated vaccines. The efficiency of bypassing this inhibition by the transfer of an immunogenic glycoprotein gene, the gD gene of pseudorabies virus (PRV), into neonates was explored. The experiments were conducted in 1-day-old piglets, which are immunocompetent at birth. The same transcription unit (gD of PRV under the control of the adenovirus major late promoter) was delivered intramuscularly at birth either in the form of naked DNA or cloned in the genome of a replication-defective adenovirus. A booster injection of a conventional live PRV vaccine strain was given at 10 weeks of age, the replication of which was greatly restricted by the residual amounts of colostral antibodies in control animals. Piglets were challenged at the age of 16 weeks with a virulent PRV strain. The replication-defective adenovirus was able to efficiently prime piglets born to immune dams against gD in such a way that inoculation with the Bartha strain protected them against a subsequent challenge with the same level of efficacy in piglets born to naive or immune dams. In contrast, piglets born to immune dams into which the gD gene was not transferred, or transferred as naked DNA at birth, were not protected. These results open the way for early immunization of neonates born to vaccinated or infected mothers.

Adenoviridae↗

PubMed1997

Minimisation of microbial contamination for potential islet xenografts using specific pathogen-free pigs and a protected environment during tissue preparation.

One major risk of islet xenotransplantation is transmission of infections. We thus compared microbial contamination during preparation of islets from 4 pigs conventionally breeded and slaughtered or 8 specific pathogen free (SPF) pigs, and different environmental conditions during pancreas excision. Pancreas harvested in a slaughterhouse (for conventional pigs) or in a protected autopsy room (for SPF pigs) were soaked in betadine solution and submitted to enzymatic digestion with collagenase. Islets were purified on histopaque gradient with a COBE 2991 processor. For each step of the process, a 10 ml aliquot was harvested and microbial contamination was analysed. For all animals, contamination of livers, which were not soaked in betadine solution, was also examined. Analysis of livers from the 4 conventional pigs showed polymicrobial contaminations (1,122 +/- 841 CFU/mg) with several species of Staphylococcus, Streptococcus, Bacillus and Enterobacteriaceae. For these conventional pigs, soaking of pancreas in betadine solution and presence of antibiotics in all media decreased the pancreatic contamination compared to hepatic contamination, but were unable to suppress it, as transport solution and crude suspension obtained after the digestion step with collagenase showed persistent contamination (9.7 +/- 2.4 and 10.5 +/- 4 CFU/ml, respectively). After islet purification by histopaque gradient, no medium remained contaminated. During analysis of the 8 SPF pigs, no liver exhibited contamination. Analysis of medium from each preparation step showed complete absence of contamination for 7 pancreases. Only one contamination with Staphylococcus simulans was observed for one pancreas in transport solution (6 CFU/ml), and persisted in digestion medium (16 CFU/ml). Finally, all purified suspensions were completely sterile. In conclusion, breeding conditions of pig islet donors, and controlled environment for pancreas excision, considerably influence the risk of microbial contamination. In order to limit the risk, SPF pigs are a suitable and compulsory source of islets.

Animals↗

PubMed1996

Genetic immunization of seronegative one-day-old piglets against pseudorabies induces neutralizing antibodies but not protection and is ineffective in piglets from immune dams.

Immune response inhibition by maternal antibodies is a major impediment to the vaccination of the young born to immune dams. This study explored the efficiency of genetic immunization of the neonates at bypassing this inhibition, by testing the muscular inoculation of the gD glycoprotein gene of pseudorabies virus (PRV) in piglets. Plasmid DNA (400 micrograms) was inoculated in four groups of one-day-old piglets, from sows vaccinated or not against PRV. Half of the groups received a booster injection on day 42. All piglets were challenged on day 115. Only piglets from non-immune sows and which received a booster injection developed a medium level of neutralizing antibodies, but they were not significantly protected against the challenge. Piglets from immune sows neither developed an antibody response nor were primed against PRV, as demonstrated by the antibodies kinetics after challenge. It can therefore be concluded that genetic immunization was inefficient at efficiently preventing the immune response inhibition by colostral antibodies.

Animals↗

PubMed1995

Influence of passive immunity on pig immunization with deleted Aujeszky's disease vaccines measured by the amount of wild virus excretion after challenge.

Four attenuated glycoprotein I deleted Aujeszky's disease virus (ADV) vaccines were compared on the basis of their ability to induce immunity in the presence of passive antibodies. The relative severity of clinical disease and amount of viral excretion following experimental challenge with virulent ADV were determined among groups of eight pigs that were unvaccinated or vaccinated with one of the four ADV vaccines. Vaccinated pigs received two vaccine doses, the first administered when passively acquired serum antibodies were still detectable at 10 weeks of age, and the second four weeks later. The experiment was divided into two trials, with vaccinated and unvaccinated control groups in each trial. Challenge with virulent ADV took place at 18 weeks of age for the first lot and 19 weeks of age for the second. Differences among the vaccines were observed with regard to clinical protection and viral excretion. Virulent virus was excreted by most of the vaccinated pigs from two to seven days after challenge. In the case of two of the vaccines, no virus excretion was detected in several of the pigs. It was confirmed that mean serum neutralizing titers at challenge are inversely associated with amount of viral excretion post-challenge. Difficulties in the standardization of vaccine trials with passive antibodies were discussed.

Animals↗

PubMed1994

Immune response and persistence of the porcine reproductive and respiratory syndrome virus in infected pigs and farm units.

The kinetics of the serum antibody response to porcine reproductive and respiratory syndrome (PRRS) virus and the persistence of the virus after infection were determined in experimentally and naturally infected pigs. In an experimental study, four specific pathogen free (SPF) sows were infected with a French strain of PRRS virus at 90 days of gestation, and their piglets (the test piglets) were monitored for 29 weeks from birth. In one litter, antibodies against PRRS virus were absent before the piglets had ingested colostrum. Four days after birth, passive antibodies were present in the serum of these piglets, but they had disappeared by three weeks (just before weaning) when clinical signs were observed in a minority of the pigs. In a second litter, most of the piglets had no detectable antibodies until they were four weeks old, and clinical signs were observed during their second week of life. By eight weeks, antibodies were detected in all the pigs, and they persisted until observations ceased at 29 weeks. Two groups of three SPF pigs were placed in direct contact with the test piglets when they were four weeks old and a group of five SPF pigs was placed in indirect contact when they were 13 weeks old. The first two groups showed clinical signs and seroconverted but the third group did not. At 22 weeks old, two of the test piglets were subjected to movement stress and were given exogenous corticosteroids, after which the in-contact SPF pigs developed clinical signs and seroconverted.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗