Impact of ceftiofur injection on gut microbiota and Escherichia coli resistance in pigs.

Resistance to extended-spectrum cephalosporins (ESCs) is an important health concern. Here, we studied the impact of the administration of a long-acting form of ceftiofur on the pig gut microbiota and ESC resistance in Escherichia coli. Pigs were orally inoculated with an ESC-resistant E. coli M63 strain harboring a conjugative plasmid carrying a gene conferring resistance, bla CTX-M-1. On the same day, they were given or not a unique injection of ceftiofur. Fecal microbiota were studied using quantitative PCR analysis of the main bacterial groups and quantification of short-chain fatty acids. E. coli and ESC-resistant E. coli were determined by culture methods, and the ESC-resistant E. coli isolates were characterized. The copies of the bla CTX-M-1 gene were quantified. After ceftiofur injection, the main change in gut microbiota was the significant but transitory decrease in the E. coli population. Acetate and butyrate levels were significantly lower in the treated group. In all inoculated groups, E. coli M63 persisted in most pigs, and the bla CTX-M-1 gene was transferred to other E. coli. Culture and PCR results showed that the ceftiofur-treated group shed significantly more resistant strains 1 and 3 days after ESC injection. Thereafter, on most dates, there were no differences between the groups, but notably, one pig in the nontreated group regularly excreted very high numbers of ESC-resistant E. coli, probably leading to a higher contamination level in its pen. In conclusion, the use of ESCs, and also the presence of high-shedding animals, are important features in the spread of ESC resistance.

Methicillin-resistant Staphylococcus aureus ST398 contamination and transmission in pigs after a low dose inoculation.

AIMS: Methicillin-resistant Staphylococcus aureus (MRSA) ST398 has recently been described as a zoonotic agent. Its transmission between animals seems to be a pivotal factor in its emergence and dissemination. This experimental trial was performed to describe MRSA ST398 contamination and transmission in pigs after a low dose inoculation. METHODS AND RESULTS: Twelve specific pathogen-free (SPF) pigs were randomly divided between two separate pens. Three pigs in each pen received a nasal inoculation of 2 × 10(4) colony-forming units per animal, and three naïve pigs were left in contact with them. Every 2 days and at necropsy, different samples were screened for MRSA. It was detected in nasal swabs from five inoculated and three naïve contact pigs, as early as 1 day after inoculation. MRSA was also found in environmental wipes but never in faecal samples. At necropsy, MRSA was detected in the lymph nodes of two contact pigs and in the tonsils and lymph nodes of three inoculated pigs. Twelve other SPF pigs were included as negative control in a separate room. CONCLUSION: This experiment showed that inoculation of a low dose of MRSA ST398 could lead to the horizontal transmission of the bacterium between pigs, the contamination of mandibular lymph nodes and the contamination of the environment without faecal carriage. SIGNIFICANCE AND IMPACT OF THE STUDY: The minimal inoculated dose via nasal route to observe transmission of MRSA ST398 between pigs is equal or lower to 2 × 10(4) colony-forming units per animal, and faecal excretion seems not to be a necessary condition for horizontal transmission.

Modelling the time-dependent transmission rate for porcine circovirus type 2 (PCV2) in pigs using data from serial transmission experiments.

Six successive transmission trials were carried out from 4 to 39 days post inoculation (DPI) to determine the features of the infectious period for PCV2-infected pigs. The infectiousness of inoculated pigs, assessed from the frequency of occurrence of infected pigs in susceptible groups in each contact trial, increased from 4 to 18 DPI (0, 7 and 8 infected pigs at 4, 11 and 18 DPI, respectively) and then decreased slowly until 39 days post infection (4, 2 and 1 pigs infected at 25, 32 and 39 DPI, respectively). The estimated time-dependent infectiousness was fitted to three unimodal function shapes (gamma, Weibull and lognormal) for comparison. The absence of infected pigs at 4 DPI revealed a latency period between 4 and 10 DPI. A sensitivity analysis was performed to test whether the parametric shape of the transmission function influenced the estimations. The estimated time-dependent transmission rate was implemented in a deterministic SEIR model and validated by comparing the model prediction with external data. The lognormal-like function shape evidenced the best quality of fit, leading to a latency period of 8 days, an estimated basic reproduction ratio of 5.9 [1.8,10.1] and a mean disease generation time of 18.4 days [18.2, 18.5].

Vaccination of porcine circovirus type 2 (PCV2)-infected sows against porcine Parvovirus (PPV) and Erysipelas: effect on post-weaning multisystemic wasting syndrome (PMWS) and on PCV2 genome load in the offspring.

The effect of different Parvovirus+Erysipelas vaccination schemes in PCV2-infected sows on PMWS outcome in the offspring was investigated under experimental conditions. Six PCV2-free sows were first infected oro-nasally with PCV2 two months before insemination (D0; "Day 0") and then by the intra-uterine route at insemination (D62). On D21 and D42, vaccinated sows received either the two commercial monovalent vaccines, A1(PPV) and A2(Erysipelas), or the bivalent vaccine B (PPV+Erysipelas). In addition, three SPF sows (foster-sows) were synchronized for farrowing dates to enable them to foster piglets born to infected sows and removed at birth before colostrum intake. A significantly higher proportion of mummified fetuses was obtained from PCV2-infected non-vaccinated sows than from vaccinated sows. Acute myocarditis lesions were found in their piglets, together with a high PCV2 genome load. The latter was significantly higher than in those born to PCV2-infected vaccinated sows. Sentinel PCV2-negative piglets, born to SPF foster-sows, seroconverted at almost the same time as piglets without PCV2 passive immunity and born to infected sows. Sixteen of the 84 liveborn piglets born to infected sows and foster-sows were affected by a syndrome possibly related to PMWS, as judged by clinical signs and histological lesions. Most were born to PCV2-infected non-vaccinated sows and 12/16 did not receive PCV2 passive immunity. The probability of PCV2 infection and the number of PCV2 genome copies per gram of tissue were significantly increased in piglets that did not receive PCV2 passive immunity.

Immune and protective abilities of ubiquitinated and non-ubiquitinated pseudorabies virus glycoproteins.

Plasmids encoding ubiquitinated (ubi-) or non-ubiquitinated (non-ubi-) glycoproteins of Pseudorabies virus (PRV) were used for vaccination of pigs. We found that the fusion of ubiquitin to viral glycoproteins increased their degradation in proteasomes in vitro, in which ubiquitin plays a key role. In the animals immunized with the plasmids encoding PRV ubi-glycoproteins and then challenged with PRV, we detected a slightly decreased cellular immune response on days 13 and 19 after immunization and a reduced nasal excretion of infectious virus on day 2 after the challenge. Afterwards, no effect of the ubiquitination of the glycoproteins on humoral or cellular immunity and on excretion of infectious virus was observed. Similarly, no effect of the ubiquitination on protective abilities of PRV glycoproteins was found.

Impact of two different colistin dosing strategies on healthy piglet fecal microbiota.

Colistin is often used in piglets but underdosing and overdosing are frequent. The impact of such administrations on fecal microbiota was studied. Piglets were given either underdoses of colistin by oral gavage for five days or overdoses by in-feed medication for 14days. The composition of fecal microbiota was studied by quantitative PCR, 16S rRNA sequencing, culture of Enterobacteriaceae, and quantification of short-chain fatty acids (SCFAs). The mean colistin concentrations during the treatment for underdosed and overdosed groups were 14.4µg/g and 64.9µg/g of feces respectively. Whatever the piglet and the sampling day, the two main phyla were Firmicutes and Bacteroidetes, The main families were Lactobacillaceae, Clostridiales, Lachnospiraceae and Ruminococcaceae. The main perturbation was the significant but transitory decrease in the Escherichia coli population during treatment, yet all the E. coli isolates were susceptible to colistin. Moreover, colistin did not affect the production of SCFAs. These results show that under- or overdoses of colistin do not result in any major disturbance of piglet fecal microbiota and rarely select for chromosomal resistance in the dominant E. coli population.

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.

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.

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.

Vaccination of pigs against Aujeszky's disease by the intradermal route using live attenuated and inactivated virus vaccines.

Inactivated and live Aujeszky's disease virus vaccines were administered intradermally using a special device without a needle. The 88 pigs were vaccinated at the beginning of the fattening period, both under experimental conditions and in commercial herds. All the pigs were challenged at the end of the fattening period in isolation units. The same vaccines were also injected intramuscularly. Vaccination by the intradermal route induced good protection, similar to that conferred with live virus vaccine injected intramuscularly. The inactivated virus vaccine was not as effective when it was injected by the intradermal route. In animals vaccinated intradermally, there were no local lesions in the meat, but very small nodules were found in the dermis; these do not affect carcass quality. The effects of challenge exposure depended on the initial health of the animals, and a synthetic value (delta G) was used to interpret the data. In fattening pigs, intradermal vaccination required less animal constraint than intramuscular injection; administration could be verified by the presence of a papule at the site of inoculation, and pigs could be vaccinated while they were feeding. Injection without a needle also helps avoid bacterial contamination under farm conditions.

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.

Experimental infection of pigs with the porcine respiratory coronavirus (PRCV): measure of viral excretion.

Twelve pigs were experimentally infected with a porcine respiratory coronavirus (PRCV) by the oronasal route. Viral excretion was measured daily by two means-deep nasal swabs and air samples obtained in a cyclone sampler. Clinical signs were very slight on infected pigs. Airborne virus could be recovered from day 1 to day 6 post-infection in the cyclone sampler as well as in petri dishes placed in the same loose-box. Viral titres obtained from nasal swabs were significantly correlated with those obtained from air samples. Different collection media were compared. The most efficient media for the collection of infectious viral particles contained a protective agent such as foetal calf serum.

Level of virulent virus excreted by infected pigs previously vaccinated with different glycoprotein deleted Aujeszky's disease vaccines.

Seven deleted Aujeszky's disease vaccines were compared for their ability to induce an immunity which suppresses virus excretion. For each vaccine, the levels of clinical protection and viral excretion were compared. Groups of eight pigs were vaccinated twice with attenuated deleted Aujeszky's disease vaccines (which do not express certain glycoproteins: gI, gX or gp63). Pigs were vaccinated at the beginning of the fattening period and challenge took place at the end of it when the pigs were 18-19 weeks old. Live virus vaccines were suspended in water or in an oil-in-water emulsion. The experiment was performed in three successive assays of two groups of eight pigs (except three groups for the first assay). At each assay, a control unvaccinated group of eight pigs was added to compare the effects of challenge between vaccinated and unvaccinated animals. In total, 80 pigs were involved in this experiment. All the vaccinated pigs excreted virus from 3 to 9 d after challenge. However the level of viral excretion and the duration of the period of excretion were reduced after vaccination and especially, when oil-in-water emulsion was used. There were obvious differences between vaccines. With some vaccines, when the level of viral excretion was low, the level of clinical protection was high. However, in other cases, the level of clinical protection could be good despite a higher level of viral excretion. The seroneutralizing titres were significantly and inversely related to a low level of viral excretion but not to the level of clinical protection.

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.

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.

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.

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.

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.

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.

Air sampling procedure for evaluation of viral excretion level by vaccinated pigs infected with Aujeszky's disease (pseudorabies) virus.

Five groups of eight fattening pigs were vaccinated and then infected with Aujeszky's disease virus. Viral excretion was evaluated by two means: deep nasal swabbing and air sampling. It appeared that infectious airborne virus could be recovered from day 1 to day 6 after infection in the isolated units where control animals were raised. In vaccinated animals, airborne particles were also detected but the amount and duration varied in relation to their immune status at the day of virulent challenge: viral excretion was significantly lower in pigs presenting a high antibody level (1/16 to 1/64) just before infection. Results obtained with nasal swabs and with air samples were closely related. Despite its low sensitivity, the air sampling procedure could be considered as an efficient tool for reflecting infectious viral pressure in a confined atmosphere.