Roland Cariolet

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.

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.

IMMUNE RESPONSES IN PIGS INFECTED WITH PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS (PRRSV)

Abstract 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.

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

Abstract 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.

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.

Protection of European domestic pigs from virulent African isolates of African swine fever virus by experimental immunisation

African swine fever (ASF) is an acute haemorrhagic disease of domestic pigs for which there is currently no vaccine. We showed that experimental immunisation of pigs with the non-virulent OURT88/3 genotype I isolate from Portugal followed by the closely related virulent OURT88/1 genotype I isolate could confer protection against challenge with virulent isolates from Africa including the genotype I Benin 97/1 isolate and genotype X Uganda 1965 isolate. This immunisation strategy protected most pigs challenged with either Benin or Uganda from both disease and viraemia. Cross-protection was correlated with the ability of different ASFV isolates to stimulate immune lymphocytes from the OURT88/3 and OURT88/1 immunised pigs.

Effect of low dose of fumonisins on pig health: immune status, intestinal microbiota and sensitivity to Salmonella.

The objective of this study was to measure the effects of chronic exposure to fumonisins via the ingestion of feed containing naturally contaminated corn in growing pigs infected or not with Salmonella spp. This exposure to a moderate dietary concentration of fumonisins (11.8 ppm) was sufficient to induce a biological effect in pigs (Sa/So ratio), but no mortality or pathology was observed over 63 days of exposure. No mortality or related clinical signs, even in cases of inoculation with Salmonella (5 × 104 CFU), were observed either. Fumonisins, at these concentrations, did not affect the ability of lymphocytes to proliferate in the presence of mitogens, but after seven days post-inoculation they led to inhibition of the ability of specific Salmonella lymphocytes to proliferate following exposure to a specific Salmonella antigen. However, the ingestion of fumonisins had no impact on Salmonella translocation or seroconversion in inoculated pigs. The inoculation of Salmonella did not affect faecal microbiota profiles, but exposure to moderate concentrations of fumonisins transiently affected the digestive microbiota balance. In cases of co-infection with fumonisins and Salmonella, the microbiota profiles were rapidly and clearly modified as early as 48 h post-Salmonella inoculation. Therefore under these experimental conditions, exposure to an average concentration of fumonisins in naturally contaminated feed had no effect on pig health but did affect the digestive microbiota balance, with Salmonella exposure amplifying this phenomenon.

Transmission of pathogenic respiratory bacteria to specific pathogen free pigs at slaughter.

The purpose of this study was to evaluate the transmission of pathogenic respiratory bacteria to thirteen 5-month-old specific pathogen free (SPF) pigs, during the slaughtering process in a commercial slaughterhouse. Before transportation, the SPF pigs and the lorry were checked to confirm the absence of pathogenic respiratory bacteria. Nine SPF pigs (group 1) were in contact in a conventional slaughterhouse with finishing pigs, during 4h before slaughtering. Four SPF pigs (group 2) were slaughtered immediately at arrival in the slaughterhouse. Five bacterial pathogens (Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Pasteurella multocida, Haemophilus parasuis and Streptococcus suis) were detected by PCR, after slaughtering, from nasal cavities, tonsils and trachea in the two groups of pigs. Lung samples were PCR negative. Three and four bacterial species were isolated from the pigs of group 2 and group 1, respectively. Cultures were negative from the lungs. All the bacterial species present in the SPF pigs were detected by PCR. P. multocida was isolated, from three samples of scalding water before the onset of slaughtering. Our results suggest that the SPF pigs became contaminated mainly by the slaughterhouse environment and the scalding water. Histological examinations revealed that during scalding, contaminated water could reach the trachea and the lungs of pigs. Checks conducted at slaughter for respiratory disorders have to be carried on, but nasal cavities and tonsils are not appropriate for bacteriological investigations. Moreover, bacteriological results obtained from the lungs of slaughtered pigs have to be used with carefulness.

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.

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.