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Résumés/Abstracts
Delegates/Participants
Présentation orale no. 21
Page 43
Proteomic analysis of outer membrane proteins of Actinobacillus pleuropneumoniae:
identification of immunogenic candidates for vaccine development.
Introduction and objectives
Gilt Jacqueline acclimatization W. Chung is one of (1), the Christopher most important Ng-Thow-Hing and effective (1), management Bernard F. Gibbs schemes (2), to John control H.E. PRRSV Nash infection (3), Mario (1).
It is also necessary as a preamble to eradication, and to prevent recirculation of the virus in the sow herd. Procedures
Jacques (4), and James W. Coulton (1)
that expose gilts to the homologous herd strains represent an approach that is being implemented in many countries.
Producers and veterinarians are using different methods to infect gilts during acclimatization (i.e. method to obtain
the virus, (1) Department inoculum of preparation, Microbiology and viral Immunology, dose, and McGill length University, of quarantine) 3775 University (2). The Street, objective Montreal, of QC, this Canada experiment H3A 2B4 was to
(2) Sheldon Biotechnology Center, McGill University, 3773 University Street, Montreal QC, Canada H3A 3B4
compare the immunological and virological response against PRRSV of pigs inoculated either with viremic serum or
(3) Institute for Biological Sciences, National Research Council of Canada, 100 Sussex Drive, Ottawa, ON, Canada K1A 0R6
an inoculm prepared from cell culture.
(4) Groupe de recherche sur les maladies infectieuses du porc, Département de pathologie et microbiologie, Faculté de
Materials and methods médecine vétérinaire, Université de Montréal, St-Hyacinthe, QC, Canada J2S 7C6
Forty five 30kg PRRSV negative barrows were randomly divided into four experimental (n=10) and one control group
(n=5). On day 0, groups 1 to 4 were intranasally inoculated with 102 TCID50, 104 TCID50 from viremic serum, 102 TCID50
and 104 The TCID50 Gram-negative from cell bacterial culture, respectively; pathogen Actinobacillus group 5 was pleuropneumoniae sham inoculated. (APP) On day causes 63 porcine animals received a
secondary homologous challenge with 10
pneumonia, a highly infectious respiratory disease that contributes to major economic losses in the swine industry.
With pressures to reduce the use of antibiotics in agricultural livestock, vaccination against bacterial pathogens has
emerged as a safer and more cost-effective approach for disease control. Current vaccines against APP provide
only partial protection, have little impact on morbidity, or are serotype-specific. Therefore an effective vaccine
offering cross-protection against different serotypes of APP is urgently needed. Outer membrane (OM) proteins,
localized at the bacterial cell surface, are attractive vaccine candidates because they are exposed as targets to
the immune system and play key roles in infection. Using the genome sequence of APP serotype 5b, we scanned in
silico for proteins predicted to be localized at the cell surface and constructed a consensus prediction list of 93 OM
proteins. We previously described proteomic analyses utilizing 1D gel electrophoresis, followed by identification with
LC-MS/MS. The outcome established the first OM proteome of APP grown under nutrient-rich conditions. We
identified 47 OM proteins representing 50% of the predicted OM proteome, most of which have not been
characterized. We now describe differences in OM protein profiles between APP grown under nutrient rich
conditions and under nutrient deprived conditions that resemble those in vivo: iron-restriction; growth-factor
nicotinamide adenine dinucleotide-restriction. Furthermore, we detected immunoreactive OM proteins by
immunoblot analyses of 1D and 2D gels probed with post-convalescent sera from infected animals. These studies
direct our selection of potential vaccine candidates for APP that are immunogenic and are expressed under
conditions that reflect infection in the porcine host.
4 TCID50 from viremic serum. On day 0,1,2,3,7,14,21,28,49,63,64,65,70 and 77
post inoculation (pi) blood was collected from the jugular vein. Briefly, viremic serum was obtained from blood
samples from viremic animals during a PRRS outbreak in a commercial farm. Serum was harvested by centrifugation,
filtered, and gentamycin was added to prevent growth of bacterial contaminants, finally the serum was diluted with
modified Eagle medium (MEM) to obtain a concentration of 102 and 104 COMPARISON OF SEROLOGICAL AND VIROLOGICAL RESPONSE OF PIGS AGAINST PORCINE REPRODUCTIVE AND
RESPIRATORY SYNDROME VIRUS (PRRSV) WITH VIREMIC SERUM OR AN INOCULM PREPARED ON CELL CULTURE
L. Batista
TCID50 per mL. Inoculum from cell culture
was obtained by inoculating sera in MARC-145 continuous cell lines and porcine alveolar macrophages. After
propagation PRRSV will was tittered and diluted as explained above. Viremic serum and the final inoculum from cell
culture were sent to the diagnostic laboratory of the University of Montreal to confirm freedom of bacterial
contamination by culture and available PCR’s and adventitious viruses by cell culture and available PCR’s were also
performed. Clinical signs (i.e. depression, anorexia, fever and respiratory distress) were observed once a day for the
first 7 days after the primary and secondary challenge; mortality was also recorded throughout the duration of the
experiment. Sera was assessed for viremia by quantitative real timePCR (Tetracore Inc., Rockville, MD), and the
presence of PRRSV antibodies by IDEXX ELISA (IDEXX Laboratories Westbrook, ME).
Results: Percentage of PCR and ELISA positive samples from day 0 to 77 pi are presented in table 1 & 2 respectively.
Table 1. Percentage of PCR positive samples from day 0 to 77 pi
VS= Viremic serum
CC= Cell culture
NC= Negative control
Table 2. Percentage of ELISA positive samples from day 1 to 77 pi.
There was no difference in clinical signs and/or mortality between the different experimental groups.
Discussion and conclusions
As shown in Table 1, group 1 and 2 inoculated with viremic serum did not present 100% viremia until day 3 and 2
respectively. However animals inoculated with cell culture, independently of the viral dose, presented viremia in
100% of the animals after day 1 pi. Nevertheless, all animals were positive to ELISA by day 14. After secondary
homologous challenge on day 63 pi, viremia could no be detected in any of the 4 experimental groups,
independently of the type of inoculum and/or viral dose. This finding corroborated that even if initial viremia pi was
slower in the pigs inoculated with viremic serum, sterilizing immunity against homologous challenge on 63 days pi was
equal amongst the 4 experimental groups. The results of this experiment show that controlled exposure to PRRSV is an
effective and safe tool to generate adequate immunity against homologous challenge. However, caution should be
exercised in order to adequately prepare and assure the safety of the inoculum, and the authors recommend that
this procedure should be supervised and monitored by a consulting veterinarian. Also, it is important to consider the
sanitary status of the country, region, and/or farm were this technique is planned to be put into operation;
particularly attention should focus on the presence of other important viruses such as foot and mouth disease virus,
classical swine fever virus, amongst others that could jeopardize the health of the exposed animals. Further studies
are performed by this group to demonstrate the effectiveness of this technique both to a homologous and/or
heterologous challenge in pregnant animals. Finally, regulatory and ethical considerations should analyzed and
discussed before implementing this technique on a large scale basis.
References: (1) Dee SA. An overview of production system to prepare naïve replacement gilts for impeding PRRSV
challenge: A global perspective. Swine Health Prod 1997; 5: 231-239.. (2) Batista L, Torremorell M, Pijoan C.
Experimental exposure to porcine reproductive and respiratory syndrome virus (PRRSV) in gilts during acclimatization.
1
COMPARISON OF SEROLOGICAL AND VIROLOGICAL RESPONSE OF PIGS AGAINST PORCINE REPRODUCTIVE AND
RESPIRATORY SYNDROME VIRUS (PRRSV) WITH VIREMIC SERUM OR AN INOCULM PREPARED ON CELL CULTURE
L. Batista1, S. D'Allaire1, J. Harel1, C. Gagnon 1, M. Gottschalk1 1
Université de Montréal, St. Hyacinthe, Canada