Evaluation of early single dose vaccination on swine influenza A virus transmission in piglets: From experimental data to mechanistic modelling.

Swine influenza A virus (swIAV) is a major pathogen affecting pigs with a huge economic impact and potentially zoonotic. Epidemiological studies in endemically infected farms permitted to identify critical factors favoring on-farm persistence, among which maternally-derived antibodies (MDAs). Vaccination is commonly practiced in breeding herds and might be used for immunization of growing pigs at weaning. Althoughinterference between MDAs and vaccination was reported in young piglets, its impact on swIAV transmission was not yet quantified. To this aim, this study reports on a transmission experiment in piglets with or without MDAs, vaccinated with a single dose injection at four weeks of age, and challenged 17 days post-vaccination. To transpose small-scale experiments to real-life situation, estimated parameters were used in a simulation tool to assess their influence at the herd level. Based on a thorough follow-up of the infection chain during the experiment, the transmission of the swIAV challenge strain was highly dependent on the MDA status of the pigs when vaccinated. MDA-positive vaccinated animals showed a direct transmission rate 3.6-fold higher than the one obtained in vaccinated animals without MDAs, estimated to 1.2. Vaccination nevertheless reduced significantly the contribution of airborne transmission when compared with previous estimates obtained in unvaccinated animals. The integration of parameter estimates in a large-scale simulation model, representing a typical farrow-to-finish pig herd, evidenced an extended persistence of viral spread when vaccination of sows and single dose vaccination of piglets was hypothesized. When extinction was quasi-systematic at year 5 post-introduction in the absence of sow vaccination but with single dose early vaccination of piglets, the extinction probability fell down to 33% when batch-to-batch vaccination was implemented both in breeding herd and weaned piglets. These results shed light on a potential adverse effect of single dose vaccination in MDA-positive piglets, which might lead to longer persistence of the SwIAV at the herd level.

Different herd level factors associated with H1N1 or H1N2 influenza virus infections in fattening pigs.

Herd-level factors associated with European H1N1 or H1N2 swine influenza virus (SIV) infections were assessed by mean of a cross-sectional study carried out in 125 herds in France. Serum samples from 15 fattening pigs in each herd were tested by haemagglutination inhibition. Data related to herd characteristics, biosecurity, management and housing conditions were collected by questionnaire during the farm visit. Climatic conditions in the post-weaning and fattening rooms, where the sampled pigs were housed, were measured over 20 h. Factors associated with H1N1 or H1N2 sero-positive status of the herd were identified by logistic regressions for binary outcome. For both subtypes, the odds for a herd to be SIV sero-positive increased if there were more than two pig herds in the vicinity (OR=3.2, 95% confidence interval (95% CI): 1.4-7.6, p<0.01 and OR=3.5, 95% CI: 1.5-8.1 p<0.01 for H1N1 and H1N2 respectively). Different factors were specifically associated with either H1N1 or H1N2 SIV infections. The odds for a herd to be H1N1 sero-positive were significantly increased by having a large number of pigs per pen in the post-weaning room (OR=3.2, 95% CI: 1.2-8.6, p=0.02), temperature setpoints below 25 °C (OR=2.6, 95% CI: 1.1-6.4, p=0.03) and below 24 °C (OR=2.6, 95% CI: 1.1-6.1, p=0.03) for the heating device in the farrowing room and the ventilation controller, respectively, and moving the pigs to the fattening facility via a room housing older pigs (OR=3.3, 95% CI: 1.1-9.6, p=0.03). A H1N2 sero-positive status was associated with a brief down period in the farrowing room (OR=2.6, 95% CI: 1.1-6.3, p=0.03), small floor area per pig in the post-weaning pen (OR=2.9, 95% CI: 1.2-7.0, p=0.02), large-sized fattening room (OR=2.5, 95% CI: 1.1-5.9, p=0.03), lack of all-in all-out management in the fattening room (OR=2.4, 95% CI: 1.0-5.8, p=0.04) and a temperature range of less than 5 °C controlling ventilation in the fattening facilities (OR=3.2, 95% CI: 1.4-7.4, p<0.01). Factors related to external and internal biosecurity and to the control of inside climatic conditions should be considered together when implementing programmes to better control SIV infections.

Pre-infection of pigs with Mycoplasma hyopneumoniae induces oxidative stress that influences outcomes of a subsequent infection with a swine influenza virus of H1N1 subtype.

The severity of swine influenza is highly variable and can be exacerbated by many factors, such as a pre-infection of pigs with Mycoplasma hyopneumoniae (Mhp). The aim of this study was to investigate the oxidative stress induced by Mhp and the impact of this stress on the evolution of an infection with the European avian-like swine H1N1 influenza virus. Two experimental trials (E1 and E2), which differed only by the feed delivered to the animals, were conducted on SPF pigs. In each trial, one group of nine 6-week-old pigs was inoculated intra-tracheally with Mhp and H1N1 at 21 days intervals and a mock-infected group (8 pigs) was included. Clinical signs were observed, blood samples were collected throughout the study and pathogens were detected in nasal swabs and lung tissues. Results indicated that Mhp infection induced an oxidative stress in E1 and E2, but its level was more important in E2 than in E1 three weeks post-Mhp inoculation, before H1N1 infection. In both trials, a strong inflammatory response and a response to the oxidative stress previously induced by Mhp appeared after H1N1 infection. However, the severity of influenza disease was significantly more marked in E2 as compared to E1, as revealed by prolonged hyperthermia, stronger reduction in mean daily weight gain and earlier viral shedding. These results suggested that severity of flu syndrome and reduction in animal performance may vary depending on the level of oxidative stress at the moment of the influenza infection, and that host responses could be influenced by the feed.

Pre-infection of pigs with Mycoplasma hyopneumoniae modifies outcomes of infection with European swine influenza virus of H1N1, but not H1N2, subtype.

Swine influenza virus (SIV) and Mycoplasma hyopneumoniae (Mhp) are widespread in farms and are major pathogens involved in the porcine respiratory disease complex (PRDC). The aim of this experiment was to compare the pathogenicity of European avian-like swine H1N1 and European human-like reassortant swine H1N2 viruses in naïve pigs and in pigs previously infected with Mhp. Six groups of SPF pigs were inoculated intra-tracheally with either Mhp, or H1N1, or H1N2 or Mhp+H1N1 or Mhp+H1N2, both pathogens being inoculated at 21 days intervals in these two last groups. A mock-infected group was included. Although both SIV strains induced clinical signs when singly inoculated, results indicated that the H1N2 SIV was more pathogenic than the H1N1 virus, with an earlier shedding and a greater spread in lungs. Initial infection with Mhp before SIV inoculation increased flu clinical signs and pathogenesis (hyperthermia, loss of appetite, pneumonia lesions) due to the H1N1 virus but did not modify significantly outcomes of H1N2 infection. Thus, Mhp and SIV H1N1 appeared to act synergistically, whereas Mhp and SIV H1N2 would compete, as H1N2 infection led to the elimination of Mhp in lung diaphragmatic lobes. In conclusion, SIV would be a risk factor for the severity of respiratory disorders when associated with Mhp, depending on the viral subtype involved. This experimental model of coinfection with Mhp and avian-like swine H1N1 is a relevant tool for studying the pathogenesis of SIV-associated PRDC and testing intervention strategies for the control of the disease.

Longitudinal study of respiratory infection patterns of breeding sows in five farrow-to-finish herds.

A longitudinal study was carried out in five French farrow-to-finish herds differently affected by respiratory diseases to describe the carrying and infection patterns of batches of sows to various respiratory pathogens during gestation and lactation. An entire batch of sows was followed during two successive reproduction cycles. Nasal, tonsillar and oro-pharyngeal swabs and blood samples were taken from each sow 9 and 4 weeks before farrowing and 1 and 4 weeks after farrowing. Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Pasteurella multocida, Haemophilus parasuis and Streptococcus suis were detected from swab samples using PCR assays. Blood samples were tested for antibodies against M. hyopneumoniae, A. pleuropneumoniae serotypes 1-9-11 and 2, Porcine Circovirus type-2 (PCV-2) and Porcine Reproductive and Respiratory Syndrome virus (PRRSV) by ELISA tests. Antibodies against H(1)N(1), H(1)N(2) and H(3)N(2) Swine Influenza Viruses (SIV) of European lineages were tested by hemagglutination inhibition assay. The results indicated that S. suis is widespread among sows (67.1% of PCR-positive sows). A. pleuropneumoniae, P. multocida, and H. parasuis were detected by PCR in 30.9%, 24.6% and 23.4% of the sows, respectively. Antibodies against M. hyopneumoniae were recovered from more than 55% of the sows in all herds whereas the micro-organism was detected in 2.4% of the sows. Although PCV-2 and SIV infections were highly prevalent, the PRRSV infection patterns ranged from no infection in farms mildly affected by respiratory diseases to active circulation in more severely affected herds. The sow population thus constitutes a reservoir for a continuous circulation of respiratory pathogens and needs to be properly considered in control strategies.

Longitudinal serological responses to Salmonella enterica of growing pigs in a subclinically infected herd.

A longitudinal survey was conducted in France in a subclinically Salmonella-infected farrow-to-finish pig farm to describe the time-course of the serological response to Salmonella enterica in growing pigs. We used three batches of sows and their corresponding litters (n = 31 litters). Among these, 256 pigs randomly selected and individually identified were followed from the first week of age until slaughter. Serial individual blood samples were submitted to indirect Salmonella-ELISA testing. Salmonella shedding was investigated by bacteriological testing of faecal material regularly collected from the sows and pigs and by environment swabs taken from the pens. Caecal contamination was checked at the slaughterhouse. Information about litter composition (filiation), location of the pigs in post-weaning and fattening pens, sanitary events, sex and body weights was recorded. 11.6% of the pigs shed S. enterica; 52% of pigs seroconverted before slaughter. The age-related variation of the natural logarithm of calibrated optical densities (COD) of pigs was described with two linear mixed models. From 8 to 61 days of age, the decrease in COD with age was fitted with a model including random effects of the animal and the dam on the intercept and slope, a batch random effect on the intercept and an individual birth-weight fixed effect on the intercept. The dam random effect was explained by the parity of the sow, Salmonella shedding by the sow during the farrowing phase and the value of the optical density of colostrum collected at parturition. A second model fitting the increase in COD from 61 days of age until slaughter included the random effect on intercept of the batch and the random effects on slope and intercept of the animal, the dam and the pen in which the followed animals were located during the fattening phase and the environmental contamination as fixed effect. In this second model, no relation was found between individual slaughter-bacteriological results and increasing COD values. Considering seroconversion time between 61 days of age and slaughter, survival function were constructed using the Cox proportional-hazards model. Both methods suggested that seroconversions generally occurred during the last third of the fattening phase (from 140 days of age to slaughter), while shedding was observed during the first half of the fattening period. The fitted models suggest the existence of clusters (such as pen and litter of origin).

Feasibility of fluorescent detection of pathogens on pork carcasses.

The direct immunofluorescent detection of pathogens on pork skin is evaluated. Calibrate contamination of pork skin with Salmonella Typhimurium (ST) and Listeria monocytogenes (Lm) is developed in 2 h at 4 degrees C. Then a specific indirect immunofluorescent staining protocol is optimized in order to obtain specific and intensive signals able to be detected by electronic cameras (deported microscopy). Despite the individual staining of ST and Lm is possible on pork skin and is specific and bright, the deported microscopy failed to detect these particles. After respectively 3 and 6 h, we obtain micro-colonies of ST and Lm. Due to the limited power of the video camera used, only the microscope permits the detection on the skin. However, our work gives standard conditions to mime the pathogens contamination and staining directly on a biological matrix such as pork skin. This work is a first step in the development of direct and rapid detection of pathogens on biological matrix.

Tracing of Salmonella spp. in two pork slaughter and cutting plants using serotyping and macrorestriction genotyping.

AIMS: The origin of Salmonella contamination of pork products is not well established. In order to further this knowledge, the transmission of Salmonella spp. from live pigs to pork cuts was investigated in two pork slaughter and cutting plants. METHODS AND RESULTS: Salmonella spp. were isolated from both pork (pigs, carcasses, cuts) and the environment before and during slaughterhouse activities. Eight serotypes were identified. XbaI and SpeI macrorestriction distinguished 20 genotypes of Salmonella Typhimurium and 16 genotypes of Salmonella Derby. A major cluster of Salmonella Typhimurium genotypes was common to both plants and all pig-related genotypes, while a predominant pig-related Salmonella Derby genotype was common to both plants. CONCLUSION: None of the Salmonella strains persisted for long periods in the pork-processing environments. SIGNIFICANCE AND IMPACT OF THE STUDY: This work shows that contaminated live pigs, because of bacterial spread due to the process and ineffective cleaning procedures, are involved in Salmonella contamination.

Listeria monocytogenes in pork slaughtering and cutting plants. Use of RAPD, PFGE and PCR-REA for tracing and molecular epidemiology.

In order to determine the origin of pork cuts contamination by Listeria monocytogenes, 287 isolates, collected from five French pork slaughtering and cutting plants, from live pigs to pork cuts, were characterised using three molecular typing methods: random amplification of polymorphic DNA (RAPD) carried out with five different primers, genomic macrorestriction using ApaI with pulsed-field gel electrophoresis (PFGE) and a PCR-restriction enzyme analysis (PCR-REA) based on the polymorphism existing within the inlA and inlB genes. Results obtained from RAPD and PFGE were closely related and distinguished respectively 17 RAPD types (r1-r17) and 17 PFGE types (a1-a17) among the 287 isolates, whereas the PCR-REA analysis only yielded two profiles (p1 and p2). Considering the combined results obtained with the three molecular typing methods, 19 Listeria monocytogenes genotypes (1-19) were distinguished. Serotyping led at least four serotypes being distinguished: 1/2a, 3a, 1/2c and 3c. The application of genotyping identified the predominance of a Listeria monocytogenes strain of type (1) and other very closely related ones (5, 9, 10, 12, 13, 14, 16 and 19) which were present on pork as well as in the environment within the five investigated plants. This study also pointed out the presence of these closely related Listeria monocytogenes strains over a 1-year period in the environments of two plants, even after cleaning and disinfection procedures. This highlights the possibility for some Listeria monocytogenes strains to persist in pork processing environments and raises the problem of the efficiency of cleaning and disinfection procedures used in pork slaughterhouses, chilling and cutting rooms.