Béatrice Grasland

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.

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.

Complete Genome Sequence of a Porcine Epidemic Diarrhea S Gene Indel Strain Isolated in France in December 2014

ABSTRACT We report the first and only case of a porcine epidemic diarrhea (PED) outbreak occurring in December 2014 in northern France, and we show using the full-length genome sequence of the French PED virus (PEDV) isolate that it was a PEDV indel strain close to German PEDV strains recently isolated.

Quantification of porcine circovirus type 2 (PCV-2) within-and between-pen transmission in pigs

PCV-2 within- and between-pen transmission was quantified by estimating the daily transmission rate beta and the basic reproduction ratio (R(0)) using a stochastic SEIR (Susceptible, Exposed, Infectious, Removed) model fitted on experimental data. Within-pen transmission was quantified by using four groups of eight SPF (specific pathogen-free) pigs (four infected and four susceptible pigs having direct contact). Between-pen transmission was studied in two groups of 16 SPF pigs (eight infected and eight susceptible pigs having indirect contact (10 cm distance)). Pigs were monitored twice a week (blood samples) and were tested for PCV-2 antibodies (ELISA test) and viral genome load in sera (real-time PCR). Transmission parameters beta(within) and beta(between) were estimated using a maximum likelihood method and the duration of infectiousness, to compute R(0), was estimated with a parametric survival model. Different assumptions were made to determine the end of infectiousness (seroconversion, seroconversion and decline in viral genome load, permanent infectiousness). R(0[within]) (8.9 (5.1-15.4)) was greater when the end of infectiousness was assumed to be related to both seroconversion and a decline of PCV-2 genome load in sera (average duration of infectiousness = 32 days) compared with only seroconversion as the indicator of recovery (R(0[within]) = 5.5 (3.3-9.0)). Whatever the assumption, between-pen R(0) (0.58 (0.23-1.47)) was always significantly lower than within-pen R(0). Only beta(within) was sensitive to the assumption on end of infectiousness and decreased with increasing duration of infectiousness. These results showed that PCV-2 transmission is influenced by contact structure that appears worth being taken into account in an epidemic model.

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].

Modification of PCV-2 virulence by substitution of the genogroup motif of the capsid protein.

Porcine circovirus type 2 (PCV-2) is the causal agent of the post-weaning multisystemic wasting syndrome (PMWS). PCV-2 are small single-stranded circular DNA viruses clustered into two main genogroups: PCV-2a and PCV-2b. Each genogroup present a specific highly-conserved motif of six amino acids (between amino acids 86 and 91) in the PCV-2 capsid protein. The aim of this study was to verify whether the motif located in the capsid protein and specific to each PCV-2 genogroup contributes to virulence. Two parental DNA clones, PCV-2a and PCV-2b, were constructed as well as two mutants DNA clones, PCV-2a/motif 2b and PCV-2b/motif 2a by exchanging the capsid motif of each genogroup. The four DNA clones were characterized in vitro as well as in vivo. Cells transfected by the four DNA clones produced infectious viruses. In specific-pathogen-free piglets transfected by the four infectious DNA clones, PCV-2b/motif 2a virulence was not attenuated while the PCV-2a/motif 2b virulence was drastically reduced compared to their parent virulence. These results suggest that the amino acids between positions 86 and 91 of the capsid protein are determinant for the virulence of isolates. However, the environment of this motif seems also involved.

Effect of granulocyte-macrophage colony-stimulating factor on post-weaning multisystemic wasting syndrome in porcine circovirus type-2-transfected piglets.

Post‐weaning multisystemic wasting syndrome (PMWS) is a complex disease syndrome in swine, affecting nursery and fattening pigs. Although ongoing evidence suggests that porcine circovirus type‐2 (PCV2) is the causal agent of PMWS, the host immune system appears to have a crucial role in the PMWS pathogenesis of PCV2‐affected pigs. Owing to difficulties in producing a biologically pure form of PCV2 devoid of the other viral agents commonly present in swine tissues, we decided to use a tandem‐cloned PCV2 DNA providing highly pure grade reagent in order to monitor the virulence of PCV2 alone or with an immunostimulating co‐factor, granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). A single intramuscular injection of tandem‐cloned PCV2 DNA into 5‐week‐old piglets produced plasmid to viral genome progeny and infectious particles as early as 8 days post‐injection in all the organs tested (the lung, the tonsil and the inguinal, mesenteric, bronchial and upper‐right axial lymph nodes). The initial plasmid load was not detected with the help of primers designed to specifically detect the acceptor plasmid, thus confirming the replication of the viral genome. Despite the presence of a high level of PCV2 genome copies in the lymphoid organs – the tonsil and the lung – and the presence of infectious particles, no detectable clinical manifestations or pathological lesions were observed in the transfected pigs over the period of observation, regardless of whether they had been co‐injected with plasmid containing GM‐CSF DNA or had received plasmid containing PCV2 DNA alone. GM‐CSF encoding DNA injection had no significant effect on viral replication or on the production of viral particles and appearance of the disease.

Factors associated with herd-level PRRSV infection and age-time to seroconversion in farrow-to-finish herds.

Factors associated with porcine reproductive and respiratory syndrome virus (PRRSV) infection were investigated in 109 herds. Serums from four batches of pigs (4, 10, 16 and 22 weeks, 15 pigs/batch) were tested by ELISA for PRRSV antibodies. Infection by Mycoplasma hyopneumoniae (Mhp), Actinobacillus pleuropneumoniae, H1N1 and H1N2 swine influenza A viruses (swIAV) and PCV2 were detected by specific serological or PCR tests. Data related to herd characteristics, biosecurity, management housing and climatic conditions were collected during a herd visit. Factors associated with the herds PRRSV seropositive status were identified by logistic regression. Large herd size, the lack of disinsectisation in the gestation facilities, on-farm semen collection, a short time-period for gilt quarantine and a low temperature setpoint for the ventilation controller in the fattening room significantly increased the odds of a herd being seropositive for PRRSV. Infection by Mhp and H1N2 swIAV were associated with a PRRSV seropositive status. A Cox proportional hazards model was used to identify the factors associated with the age-time to seroconversion in infected herds. Joint housing for the gilts and sows when lactating, a large nursery pen, a small number of pens per fattening room and lack of all-in all-out management in the fattening section significantly reduced the age-time to seroconversion. A small range of temperatures controlling ventilation rate in the nursery room was also associated with time to PRRSV seroconversion. Infection by Mhp and a high PCV2 infection pressure were associated with a shorter time to seroconversion. Biosecurity measures minimising the risk of introducing PRRSV into the herd, management practices reducing contacts between animals from different batches and within batches and favourable climatic conditions should be implemented to better control PRRSV infection.

A commercial PCV2a-based vaccine significantly reduces PCV2b transmission in experimental conditions.

Transmission characteristics of PCV2 have been compared between vaccinated and non-vaccinated pigs in experimental conditions. Twenty-four Specific Pathogen Free (SPF) piglets, vaccinated against PCV2 at 3weeks of age (PCV2a recombinant CAP protein-based vaccine), were inoculated at 15days post-vaccination with a PCV2b inoculum (6⋅10(5) TCID50), and put in contact with 24 vaccinated SPF piglets during 42days post-inoculation. Those piglets were shared in six replicates of a contact trial involving 4 inoculated piglets mingled with 4 susceptible SPF piglets. Two replicates of a similar contact trial were made with non-vaccinated pigs. Non vaccinated animals received a placebo at vaccination time and were inoculated the same way and at the same time as the vaccinated group. All the animals were monitored twice weekly using quantitative real-time PCR and ELISA for serology until 42days post-inoculation. The frequency of infection and the PCV2 genome load in sera of the vaccinated pigs were significantly reduced compared to the non-vaccinated animals. The duration of infectiousness was significantly different between vaccinated and non-vaccinated groups (16.6days [14.7;18.4] and 26.6days [22.9;30.4] respectively). The transmission rate was also considerably decreased in vaccinated pigs (β=0.09 [0.05-0.14] compared to β=0.19 [0.11-0.32] in non-vaccinated pigs). This led to an estimated reproduction ratio of 1.5 [95% CI 0.8 - 2.2] in vaccinated animals versus 5.1 [95% CI 2.5 - 8.2] in non-vaccinated pigs when merging data of this experiment with previous trials carried out in same conditions.

The expression level of gC1qR is down regulated at the early time of infection with porcine circovirus of type 2 (PCV-2) and gC1qR interacts differently with the Cap proteins of porcine circoviruses.

Porcine circoviruses (PCV) are small, non-enveloped single-stranded DNA-viruses. Porcine circovirus type 2 (PCV-2) is the causal agent of post-weaning multisystemic wasting syndrome (PMWS) whereas porcine circovirus of type 1 (PCV-1) is non- pathogenic. gC1qR is a membrane-located receptor of the complement protein subunit C1q and interacts with PCV capsid proteins. The mechanisms associated with the triggering of PMWS are not well known and gC1qR may have a role in the life cycle and eventually in the pathogenicity of PCV. The objectives of this study were to determine the level of expression of gC1qR during early PCV-2 infection, to determine the region of PCV-2 capsid protein (Cap) required for the interaction with gC1qR and to evaluate the interaction of gC1qR with Cap proteins of different PCV strains. The results indicate that gC1qR transcripts are downregulated in the tonsils and the tracheo-bronchial lymph nodes of piglets infected by PCV-2 at the early time of the infection. The N-terminal amino acids (a.a. 1-59) of PCV-2b Cap, an arginine rich region, are involved in the interaction with gC1qR. Porcine gC1qR interacts with Cap proteins of two pathogenic viral strains, PCV-2a and PCV-2b, while interaction has been observed with only one Cap protein of two investigated strains of PCV-1. The amino acids 30 and 49 of PCV-1Cap, solely, were not responsible of the difference of interaction observed. We have also shown that gC1qR interacts strongly with PCV-2Caps and PCV-1 GER Cap. This result suggests that the different interaction of gC1qR with PCV Cap proteins may have an impact on the pathogenicity of the PCV.