André Jestin

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.

Characterisation of PCV-2 isolates from Spain, Germany and France

The new isolated circovirus variant PCV-2 is discussed to be the etiological agent of a new emerging swine disease with a variable morbidity and high lethality, postweaning multisystemic wasting syndrome (PMWS). PMWS has been diagnosed in North America and West Europe. Clinical signs include dyspnea, loss of weight, lymph node enlargement and lymphocyte depletion in lymphoid tissues. This report describes the characterisation of PCV-2 isolates from animals affected with PMWS from Germany, Spain and France. We could demonstrate the presence of circovirus by electron microscope, in situ hybridisation and PCR. PCR revealed incidence of PCV-2 in many tissues of one infected animal with the exception of heart and nervous system. The phylogenetic analysis of all PCV-2 isolates yet published in the database, showed relationship of isolates from Spain, Germany and France, with three sequences from Canada determined recently and two isolates from Taiwan, while other North American sequences display a separate cluster. PCR screening of randomly collected organ samples from German pigs not affected with PMWS, revealed a rate of infection with PCV-1 of 5% and with PCV-2 of 26.8%, while blood samples showed a lower incidence.

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.

Measurement of digestive disorders in the piglet at weaning and related risk factors

A cohort study was carried out in France about postweaning digestive disorders in the piglet. One hundred and six farrow-to-finish farms were involved. In each of them, a batch of contemporary piglets was considered. A total of 12,034 piglets were ear-notched, evaluated during the suckling phase and weighed at weaning, at 14 and 28 days postweaning. Postweaning diarrhoea and mortality were recorded daily. Data were collected about diet composition and feed intake, housing and husbandry throughout the period. Weaning weight was 8.1 kg and weaning age was 27.2 days on average. Diarrhoea occurred in the pens after a 3-4-day latency period. Prevalence was maximum around 7 to 9 days after weaning and remained high until 21 days after weaning. Mortality was moderate (1.9%). Average daily gains were 283 and 489 g for the two subsequent 14-day periods postweaning. Descriptive multivariate methods indicated a strong pattern between diarrhoea, mortality and growth. The main risk factors associated with the digestive disorders were determined. The hygiene level at the reception of the piglets (cleanliness, level of temperature), management and husbandry level (air quality, group size and stocking procedure) were found to be important factors leading to risky or secure profiles. In addition, the feed intake of the piglet during the first week postweaning was strongly associated with the severity of the digestive disorders over the whole 28-day postweaning period of observation. It is concluded that prevention of postweaning digestive disorders could be based on the control of zootechnical conditions.

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.

Post-weaning multisystemic wasting syndrome (PMWS) in pigs in France: clinical observations from follow-up studies on affected farms

Abstract A disease affecting weaned pigs and known as Postweaning Multisystemic Wasting Syndrome (PMWS) is described on French farms. Follow-up studies were designed on cohorts of pigs on a group of 12 severely affected farrow-to-finish operations. Three of them were free of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection. Three thousand and seventy eight pigs were included in the study. Mortality, from weaning to slaughter, was 11% and weeks 11–13 were the most critical. The first clinical sign reported was unthriftiness, then pallor and often fever with associated respiratory or digestive disorders. Wasting could follow rapidly and when clearly established in an individual the prognosis was grave especially when the sick pigs were kept with their penmates. Antibiotics were administered without real efficacy. The lesions affected several viscera including lungs, spleen, kidneys and lymph nodes with a severe lymphoid depletion. The disease did not show a collective impact. From preliminary epidemiological investigations, a strong litter effect on disease susceptibility was suspected. PRRS virus was excluded as a major agent. On the other hand, a porcine circovirus (PCV2) was found associated to the lesions. The environment was suspected as an important determining factor for the effect of PMWS in the herds.

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.

Identification of an immunorelevant ORF2 epitope from porcine circovirus type 2 as a serological marker for experimental and natural infection

Summary. Post-weaning multisystemic wasting syndrome (PMWS) is a recently identified disease of pigs linked to the emergence of a new porcine circovirus (PCV2). We report here the characterization of immunorelevant linear B-cell epitopes of the Open Reading Frame 2-encoded protein (Orf2) from PCV2 by an enzyme-linked immunosorbent assay (ELISA) using experimental antisera collected from pigs inoculated with a PCV2 isolate. Two epitopes spanning residues 69 to 83 and 117 to 131 were specific to PCV2. Antibodies to the 117 to 131 epitope (B-133) were detected in 22% and 100% of specific pathogen-free (SPF) pig sera 6 and 11 weeks post inoculation, respectively. Cross-sectional studies performed with field sera collected from PMWS-affected herds showed B-133 antibodies in 5% of 8 to 10 week-old pigs, 38% of 13–14 week-old pigs, 62% of 16 to 19 week-old pigs, 56% of 20 to 25 week-old pigs and 45% of 26 to 31 week-old pigs. All these data suggest that epitope B-133 is a serological marker of PCV2 infection that could be used for the detection of PCV2 antibody response.

Detection of Newcastle disease virus RNA in infected allantoic fluids by in vitro enzymatic amplification (PCR).

SummaryThe Polymerase Chain Reaction (PCR) procedure was applied in order to identify the Newcastle disease virus (NDV), an avian paramyxovirus (A-PMV 1). The sequence selected for amplification consists of 238 bp lying in the gene encoding the fusion protein F. A pair of 19-mer and 18-mer oligonucleotides, flanking this sequence, were used as primers. Following RNA extraction by the proteinase K method, a cDNA was prepared using the previous 19-mer oligonucleotide as the primer. The amplification reaction product was analyzed by electrophoresis and ethidium bromide staining, using the restriction enzymes Hae III, Mbo II, and Nar I. The PCR was performed on cDNA prepared from 30 A-PMV 1 and 3 other strains (A-PMV 2, A-PMV 3, A-PMV 4). It was thereby demonstrated that the selected sequence was highly specific and constant. However, two of the PMV 1 strains isolated from feral ducks, are thought to present a deletion of about 25 bp inside this fragment as shown by the smaller length of the corresponding amplified product and the disappearance of the Nar I restriction site.The advantages of this technique, as a first step in evaluating virulence by means of molecular biology, is discussed.

Epidemiology and transmission of porcine circovirus type 2 (PCV2).

PCV2 has been highly prevalent in the pig population for decades, prior to the emergence of associated clinical disease manifestations that severely affected the pig production worldwide in the late 90s. PCV2 can be further subdivided into several genotypes. From descriptive epidemiologic data, there is evidence of a global shift of the main PCV2 genotypes in different countries from PCV2a to PCV2b, which is generally associated with more severe disease. In addition, from analytic epidemiologic studies, the modified within-herd PCV2 dynamics of infection is strongly related to the increased incidence of clinical disorders associated with PCV2 infection. Because PCV2 is shed for a long time by an extremely large variety of routes, it easily spreads within the population both through horizontal and vertical transmission. Even if airborne transmission cannot be formally excluded, direct contact is certainly the most efficient infectious route due to the simultaneous exposure of susceptible pigs to contaminated respiratory, digestive, and urinary secretions since the probability of transmission is strongly limited by the distance between infectious and susceptible animals. Consequently, farm to farm transmission is restricted to the introduction of infected animals or infected animal products such as semen. More information would be required to assess the risk of other vehicles such as vaccines or feed ingredients since the probability of these products to be contaminated by PCV2 is unknown. However, owing to its transmission characteristics, PCV2 is able to be maintained within pig farms for years without any further need for re-introduction due to the population dynamics of modern pig operations, which continually renew the pool of the susceptible population through replacements and pig movements between compartments.