François Madec

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.

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.

High prevalence of Hepatitis E virus in French domestic pigs

The importance of the domestic pig reservoir for Hepatitis E virus (HEV) was assessed by estimating the seroprevalence and prevalence of HEV contaminated livers in French slaughter-aged pigs. 6565 sera and 3715 livers were randomly sampled from 186 pig farms throughout the country. Taking the sampling design into account, the farm-level seroprevalence was 65% (95% CI 57-74) and 31% (95% CI 24-38) of the slaughter-aged pigs had antibodies against HEV. The individual prevalence of HEV RNA positive livers was 4% (95% CI 2-6) and 24% (95% CI 17-31) of the farms had at least 1 positive liver. Most isolates were of genotype 3f (76.7%) with smaller amounts of 3c (18.6%) and 3e (4.6%). The high prevalence of HEV in pigs and the similarities between HEV subtypes from pigs and humans corroborates the possible zoonotic origin of some HEV autochthonous infections.

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.

Infectious agents associated with respiratory diseases in 125 farrow-to-finish pig herds: A cross-sectional study

A study was carried out in 125 farrow-to-finish pig herds to assess the relationships between pathogens involved in respiratory disorders and to relate these findings to clinical signs of respiratory diseases and pneumonia and pleuritis at slaughter. Clinical examination and sampling were carried out on four different batches in each herd (pigs aged 4, 10, 16 and 22 weeks). Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, swine influenza viruses (SIV), porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) were detected by serological or PCR tests. Pneumonia-like gross lesions and pleuritis were scored at the slaughterhouse. The results indicate that the percentage of pigs PCR-positive for PCV2 at 4, 10 and 16 weeks old was associated with the percentage of pigs PCR-positive for M. hyopneumoniae at these ages. On the other hand, the percentage of pigs with antibodies against PRRSV at 10, 16 and 22 weeks was positively correlated with the percentage of pigs seropositive for M. hyopneumoniae at 22 weeks, with the percentage of pigs with antibodies against SIV H1N1 and SIV H1N2 and the percentage of pigs sero-positive for A. pleuropneumoniae serotype 2. The findings also indicate that, within the five studied pathogens, M. hyopneumoniae, PRRSV and SIV H1N1 are the major pathogens involved in pneumonia-like gross lesions even though PCV2 may play a role. A. pleuropneumoniae serotype 2, in association with PRRSV, is significantly associated with extensive pleuritis. Respiratory diseases could be significantly reduced by implementing measures including appropriate management practices to control these pathogens.

Virological Surveillance and Preliminary Antigenic Characterization of Influenza Viruses in Pigs in Five European Countries from 2006 to 2008

This study presents the results of the virological surveillance for swine influenza viruses (SIVs) in Belgium, UK, Italy, France and Spain from 2006 to 2008. Our major aims were to clarify the occurrence of the three SIV subtypes – H1N1, H3N2 and H1N2 – at regional levels, to identify novel reassortant viruses and to antigenically compare SIVs with human H1N1 and H3N2 influenza viruses. Lung tissue and/or nasal swabs from outbreaks of acute respiratory disease in pigs were investigated by virus isolation. The hemagglutinin (HA) and neuraminidase (NA) subtypes were determined using standard methods. Of the total 169 viruses, 81 were classified as ‘avian‐like’ H1N1, 36 as human‐like H3N2 and 47 as human‐like H1N2. Only five novel reassortant viruses were identified: two H1N1 viruses had a human‐like HA and three H1N2 viruses an avian‐like HA. All three SIV subtypes were detected in Belgium, Italy and Spain, while only H1N1 and H1N2 viruses were found in UK and Northwestern France. Cross‐hemagglutination inhibition (HI) tests with hyperimmune sera against selected older and recent human influenza viruses showed a strong antigenic relationship between human H1N1 and H3N2 viruses from the 1980s and H1N2 and H3N2 human‐like SIVs, confirming their common origin. However, antisera against human viruses isolated during the last decade did not react with currently circulating H1 or H3 SIVs, suggesting that especially young people may be, to some degree, susceptible to SIV infections.

A survey of group-level antibiotic prescriptions in pig production in France.

There is world-wide concern that antimicrobial use in food-producing animals might contribute to antimicrobial resistance both in animals and in humans. The relationship between antimicrobial use and resistance is likely to be related to frequency of prescription of the compound, dose and duration of treatment. Routine collection of that information is not possible today in France. A postal survey of French pig veterinarians therefore was carried out in October 2000. The questionnaire focused on the last antibiotic group-level prescription made; data were collected on the type of animals, presumptive clinical diagnosis and drug prescription. The list frame was defined using a veterinary yearbook. All practitioners with mention of pig in the treated species or with employment in intensive animal production were sent the questionnaire. Out of the 431 selected practitioners, 303 responded to the self-administered questionnaire (overall return proportion 70%). 159 prescriptions were received and analysed (response proportion 37%). Their repartitions according to indications and active compounds were summarised. Mean prescribed daily doses and mean treatment length were calculated for four antibiotics: amoxicillin, colistin, oxytetracycline, tylosin. Prescribed daily dose were in the range of dosages used and recommended in Europe. High variations were encountered in treatment length: from 3 to 21 days.