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					                    OIE Reference Laboratory Reports
                                           Activities in 2011

 Name of disease for which you are                                   Salmonellosis
      a designated OIE Reference

                                 Address of laboratory:   OIE Reference Laboratory for Salmonellosis
                                                             Laboratory for Foodborne Zoonoses
                                                               Public Health Agency of Canada
                                                            110 Stone Road West, Guelph, Ontario
                                                                    N1G 3W4 CANADA

                                                  Tel.:                 519-822-3300

                                                  Fax:                  519-822-2280

                                        e-mail address:           cornelis.poppe@gmail.com

        Name of Head of Laboratory:                              Dr Roger Johnson

    Name of OIE Reference Expert:                                Dr Cornelis Poppe

Annual reports of OIE Reference Centres, 2011                                                          1

                  Part I: Summary of general activities related to the disease

1.   Test(s) in use/or available for the specified disease/topic at your laboratory

     The OIÉ Reference Laboratory for Salmonellosis is a national Salmonella surveillance centre, contributing
     laboratory support, Salmonella cultures and submissions data to the national enteric pathogen surveillance system
     of the Public Health Agency of Canada, the Canadian Integrated Program for Antimicrobial Resistance
     (http://www.phac-aspc.gc.ca/cipars-picra/index.html), and PulseNet Canada (the Canadian listserve for
     PFGE/molecular methods, bacterial outbreak investigations and surveillance). The Laboratory also supports
     provincial government Salmonella monitoring programs and research programs of government institutions and
     universities in Canada and internationally.

     Serotyping: 6323 Salmonella cultures isolated from animals and their environment, foods, feeds and other sources
     in Canada during the year 2011 were identified serologically using the antigenic formulae of Grimont and Weill
     (2007) and are shown in Table 1.

     Table 1. The commonest Salmonella serovars and number of isolates of each serovar

     Rank             Serovar      Number     Rank        Serovar      Number     Rank               Serovar         Number
            1         Kentucky         1616       5          Hadar         217         9            Mbandaka             123

            2        Enteritidis        883       6         Infantis       210        10                Derby            120

            3       Heidelberg          871       7    Senftenberg         126        11       Schwarzengrund            118

            4     Typhimuriuma          366       8      Orion var.        124        12        Other serovars          1549
                                                          15+, 34+
         Includes S. Typhimurium (154 isolates) and S. Typhimurium var. Copenhagen (212 isolates)

     Table 2. The commonest Salmonella serovars and number of isolates of each serovar from each species or source

     Specie               Commonest serovars and number of isolates of each serovar from each                           Total
     Chicken             Kentucky 1101     Heidelberg 496                            or source
                                                             Enteritidis 496species Hadar 96                          isolate
     source                                                                                                                 s
      Food -             Kentucky      345        Heidelberg     215    Enteritidis   178            Hadar    36         953
     Porcine         Typhimuriuma      120             Derby      83       Infantis    45      Worthington    27         528

         Avianb          Enteritidis    96        Heidelberg      64     Kentucky      35            Hadar    27         426

         Turkey         Orion var.      74    Schwarzengrund      45   Senftenberg     42            Hadar    36         382
         Bovine      Typhimuriumc       97       I:6,14,18:-:-    10    Enteritidis        9        Dublin       7       181

       Includes S. Typhimurium (40 isolates) and S. Typhimurium var. Copenhagen (80 isolates); b Avian species other
     than chicken and turkey; c Includes S. Typhimurium (27 isolates) and S. Typhimurium var. Copenhagen (70

     Phagetyping: The Salmonella Heidelberg, S. Enteritidis and S. Typhimurium strains were phagetyped with the
     typing schemes of Demczuk et al. (J Clin Microbiol 2003; 41:4279-4284), Ward et al. (Epidemiol Infect 1987;
     99:291-294), and Anderson et al. (J Hyg 1977; 78:297-300), respectively. A total of 2,193 strains were
     phagetyped. The results are shown in Table 3.

2                                                                              Annual reports of OIE Reference Centres, 2011

     Table 3. The commonest phagetypes of S. Heidelberg, S. Enteritidis and S. Typhimurium
     Salmonella serovar                         PT       No.      PT       No.   PT    No.       PT    No.         Total
     Heidelberg                                  19      280       29      137    18    91    Other    349          857

     Enteritidis                                  8      310     13a       257    13    70    Other    217           854

     Typhimurium var. Copenhagen c              UT1       29     104        18   UT5    16    Other    184           247

     Typhimurium b                              104       30     108        26   132    17    Other    162           235

       PT denotes phagetype; b Includes S. Typhimurium var. Copenhagen-like strains with the antigenic formula:
     1,4,12:i:-. c Includes S. Typhimurium-like strains with the antigenic formula: 1,4,5,12:i:-;

     Four strains of Salmonella Enteritidis PT4 were identified.

2.   Production and distribution of diagnostic reagents

     Cultures are available from the Salmonella strain collection (which consists of more than 60,000 strains) for
     research and diagnostic purposes at laboratories nationally and in other OIE Member countries.

                    Part II: Activities specifically related to the mandate
                                of OIE Reference Laboratories

3.   International harmonisation and standardisation of methods for diagnostic testing or the
     production and testing of vaccines

     a)    Establishment and maintenance of a network with other OIE Reference Laboratories
           designated for the same pathogen or disease and organisation of regular inter-laboratory
           proficiency testing to ensure comparability of results

           The OIÉ Reference Laboratory has been a member of the WHO Global Foodborne Infections Network
           (who.int/gfn/en) since 2000 and is listed on its web page (who.int/gfn/members/en). The laboratory tested a
           blind panel of 100 Salmonella serovars from the Veterinary Laboratories Agency in the United Kindom using
           a Salmonella geno-serotyping array (SGSA) (see Section 12 below).

     b)    Organisation of inter-laboratory proficiency testing with laboratories other than OIE
           Reference Laboratories for the same pathogens and diseases to ensure equivalence of

           The Laboratory has successfully participated in a yearly External Quality Assurance System (EQAS) for
           Salmonella serotyping with other Global Foodborne Infections Network member laboratories since 2001.

4.   Preparation and supply of international reference standards for diagnostic tests or vaccines

     Salmonella antisera, Salmonella cultures and other diagnostic supplies are available to researchers and
     diagnosticians in Member Countries.

5.   Research and development of new procedures for diagnosis and control

     The following topics are being studied: The epidemiology, pathogenesis and control of salmonellosis in animals
     and humans; antimicrobial resistance and trends in resistance of Salmonella; the association between antimicrobial
     resistance and virulence in Salmonella; further characterization of Salmonella by serotyping, phagetyping, PFGE,
     plasmid profiles, and by PCR to identify antimicrobial resistance, virulence and other genes; sequencing of DNA
     and other molecular typing and research techniques; alternative molecular methods to traditional serotyping. The

Annual reports of OIE Reference Centres, 2011                                                                        3

     laboratory led an international scientific collaborative study to develop a Salmonella geno-serotyping array
     (SGSA) that rapidly generates an antigenic formula consistent with the White-Kauffmann-Le Minor scheme (see
     Section 12 and Reference 1). A genomic and proteomic characterization was carried out on the Salmonella
     bacteriophage PVP-SE1; the phage has a broad lytic spectrum against different Salmonella serovars (see
     Reference 4). Two Salmonella enterica serovar Typhimurium-based vaccines against necrotic enteritis were
     assessed to determine concomitant reduction in colonization of chickens by Salmonella serovars of different
     serogroups (see Reference 12).

6.   Collection, analysis and dissemination of epizootiological data relevant to international disease

     A recent study showed that Salmonella Enteritidis has emerged as the most prevalent cause of human
     salmonellosis in Canada. Recent trends of S. Enteriditis subtypes and their potential sources were described by
     integrating Salmonella data from several Canadian surveillance and monitoring programmes. A threefold increase
     in S. Enteritidis cases from 2003 and 2009 was primarily associated with phage types 13, 8 and 13a. Other
     common phage types (4, 1 and 6a) showed winter seasonality and were more likely associated with cases linked to
     international travel. Conversely, phage types 13, 8 and 13a had summer seasonal peaks and were associated with
     cases of domestically acquired infections. During agri-food surveillance, S. Enteriditis was detected in various
     commodities, most frequently in chicken (with phage types 13, 8 and 13a predominating). Antimicrobial
     resistance was low in human and non-human isolates. Continued integrated surveillance and collaborative
     prevention and control efforts are necessary to mitigate future illness (see Reference 17).

7.   Maintenance of a system of quality assurance, biosafety and biosecurity relevant to the
     pathogen and the disease concerned

     The OIÉ Reference Laboratory for Salmonellosis has been accredited by the Standards Council of Canada since
     1997 and its diagnostic procedures comply with the requirements of the ISO/IEC Guide 17025. Besides
     participating in the yearly External Quality Assurance System (EQAS) for Salmonella serotyping with other
     Global Foodborne Infections Network member laboratories (see Section 3b), the laboratory continued its
     participation in a joint inter-laboratory quality assurance (QA) program with the Ontario Agency for Health
     Protection and Promotion, Toronto, Ontario, Canada and the Laboratoire d’épidémiosurveillance animale du
     Québec, Saint-Hyacinthe, Québec, Canada. It also carried out an internal QA program. A total of 270 isolates
     belonging to 78 serovars unique to the QA program and to other more commonly occurring serovars were
     serotyped, and 16 strains were phagetyped as part of the QA program.

8.   Provision of consultant expertise to OIE or to OIE Member Countries

     The OIE Expert on Salmonellosis at our Laboratory completed an OIE questionnaire on salmonellosis in
     connection with an attempt to prioritize animal diseases in East Africa. He answered inquiries from OIE member
     countries about procedures and protocols regarding the isolation, identification, virulence, antimicrobial resistance
     and host-specificity of Salmonella isolates.

9.   Provision of scientific and technical training to personnel from other OIE Member Countries


10. Provision of diagnostic testing facilities to other OIE Member Countries

     The OIE Salmonellosis Reference Laboratory has had a long-standing relationship with researchers and
     epidemiologists in Ethiopia and serotyped and determined antimicrobial resistance of bovine, human and
     environmental Salmonella strains isolated in Ethiopia (see Reference 22).

11. Organisation of international scientific meetings on behalf of OIE or other international bodies


4                                                                              Annual reports of OIE Reference Centres, 2011

12. Participation in international scientific collaborative studies

     The laboratory led an international scientific collaborative study to develop a Salmonella geno-serotyping array
     (SGSA) that rapidly generates an antigenic formula consistent with the White-Kauffmann-Le Minor scheme,
     currently the gold standard for Salmonella serotyping. A set of 287 strains representative of 133 Salmonella
     serovars was assembled to validate the array and to test the array probes for accuracy, specificity, and
     reproducibility. Initially, 76 known serovars were utilized to validate the specificity and repeatability of the array
     probes and their expected probe patterns. The SGSA generated the correct serovar designations for 100% of the
     known subspecies I serovars tested in the validation panel and an antigenic formula consistent with that of the
     White-Kauffmann-Le Minor scheme for 97% of all known serovars tested. Once validated, the SGSA was
     assessed against a blind panel of 100 Salmonella enterica subsp. I samples serotyped using traditional methods
     correctly identifying all samples as Salmonella, and successfully identifying 92% of the antigens found within the
     unknown samples. Antigen- and serovar-specific probes, in combination with a pepT PCR for confirmation of S.
     enterica subsp. enterica determinations, generated an antigenic formula and/or a serovar designation consistent
     with the White-Kauffmann-Le Minor scheme for 87% of unknown samples tested with the SGSA. Although the
     current array has been validated for detection of the 76 target serovars it is important to note that the combination
     of probes on the current layout has the potential to type 1026 subspecies I. A tri-site collaborative validation of
     the SGSA is currently underway involving testing of >770 strains representing >150 serovars to determine the
     specificity, sensitivity, repeatability and robustness of the assay. This will be followed by parallel testing of
     approximately 700 incoming Salmonella isolates at each of the three locations in an effort to demonstrate the
     versatility and utility of this array as a public health tool in the identification of Salmonella (see Reference 1). To
     increase the detection capabilities of future array layouts the group has sequenced more than 20
     Salmonella isolates which are undergoing assembly, gap closure and annotation. An additional 40 strains are
     scheduled for sequencing in 2012 which will include representatives from all previously unsequenced serogroups,
     and allow for differentiation of highly homologous serotypes.

13. Publication and dissemination of information relevant to the work of OIE (including list of
    scientific publications, internet publishing activities, presentations at international conferences)

         Presentations at international conferences and meetings
     1.   Yoshida C, Franklin K, Lingohr E, Kostic T, Anjum M, Clark C, Kropinski A. Blind assessment of human,
          animal and environmental samples using the Salmonella Geno-Serotyping Array (SGSA). FEMS, Geneva,
          Switzerland. June 26-30, 2011. Poster Presentation.

     2.   Yoshida C, Lingohr EJ, Franklin K, Bodrossy L, Anjum M, Clark CG, Kropinski AM. A Salmonella Geno-
          Serotyping Array (SGGSA) for the rapid classification of serovars. National Molecular Microbiology
          Diagnostic Users Group Annual Fall Meeting (NMG), Toronto, Canada. October 17-18, 2011. Oral

         Scientific publications in peer-reviewed journals
     1.   Franklin K, Lingohr EJ, Yoshida C, Anjum M, Bodrossy L, Clark CG, Kropinski AM, Karmali MA. Rapid
          genoserotyping tool for classification of Salmonella serovars. J Clin Microbiol 2011; 49:2954-2965.

     2.   Laing C, Villegas A, Taboada EN, Kropinski, A, Thomas JE, Gannon VP. Identification of Salmonella
          enterica species- and subgroup-specific genomic regions using Panseq 2.0. Infect Genet Evol 2011; 11:2151-

     3.   Anany H, Lingohr EJ, Villegas A, Ackermann H-W, She Y-M, Griffiths MW, Kropinski AM. A Shigella
          boydii bacteriophage which resembles Salmonella phage ViI. Virology Journal 2011; 8:242.

     4.   Santos SB, Kropinski AM, Ceyssens PJ, Ackermann H-W, Villegas A, Lavigne R, Krylov VN, Carvalho CM,
          Ferreira EC, Azeredo J. Genomic and proteomic characterization of the broad-host-range Salmonella phage
          PVP-SE1: creation of a new phage genus. J Virol 2011; 85:11265-11273.

     5.   Kropinski AM, Arutyunov D, Foss M, Cunningham A, Ding W, Singh A, Pavlov AR, Henry M, Evoy S,
          Kelly J, Szymanski CM. Genome and proteome of Campylobacter jejuni bacteriophage NCTC 12673. Appl
          Environ Microbiol 2011; 77:8265-8271.

Annual reports of OIE Reference Centres, 2011                                                                             5

    6.   Kutter EM, Skutt-Kakaria K, Blasdel B, El-Shibiny A, Castano A, Bryan D, Kropinski AM, Villegas A,
         Ackermann H-W, Toribio AL, Pickard D, Anany H, Callaway T, Brabban AD. Characterization of a ViI
         phage specific to Escherichia coli O157:H7. Virology Journal 8:430.

    7.   Martin LC, Weir EK, Poppe C, Reid-Smith RJ, Boerlin P. Characterization of blaCMY-2 plasmids in
         Salmonella and E. coli from food animals in Canada. Appl Environ Microbiol 2011; Dec 9 [Epub ahead of

    8.   Jardine C, Reid-Smith RJ, Janecko N, Allan M, McEwen SA. Salmonella in raccoons (Procyon lotor) in
         southern Ontario, Canada. J Wildl Dis 2011; 47:344-351.

    9.   Allen SE, Boerlin P, Janecko N, Lumsden JS, Barker IK, Pearl DL, Reid-Smith RJ, Jardine C. Antimicrobial
         resistance in generic Escherichia coli isolates from wild small mammals living in swine farm, residential,
         landfill, and natural environments in southern Ontario, Canada. Appl Environ Microbiol 2011; 77:882-888.

    10. Cook A, Reid-Smith RJ, Irwin RJ, McEwen SA, Young V, Ribble C. Antimicrobial resistance in
        Campylobacter, Salmonella, and Escherichia coli isolated from retail grain-fed veal meat from Southern
        Ontario, Canada. J Food Prot 2011; 74:1245-1251.

    11. Mather AE, Matthews L, Mellor DJ, Reeve R, Denwood MJ, Boerlin P, Reid-Smith RJ, Brown DJ, Coia JE,
        Browning LM, Haydon DT, Reid SW. An ecological approach to assessing the epidemiology of antimicrobial
        resistance in animal and human populations. Proc Biol Sci 2011; Nov 16 [Epub ahead of print].

    12. Jiang Y, Kulkarni RR, Parreira VR, Poppe C, Roland KL, Prescott JF. Assessment of 2 Salmonella enterica
        serovar Typhimurium-based vaccines against necrotic enteritis in reducing colonization of chickens by
        Salmonella serovars of different serogroups. Can J Vet Res 2010; 74:264-270.

    13. Farzan A, Friendship RM, Dewey CE, Poppe C, Funk J. Evaluation of the risk factors for shedding
        Salmonella with or without antimicrobial resistance in swine using multinomial regression method. Zoonoses
        Public Health 2010; 57: Suppl 1:85-93.

    14. Garcia-Migura L, Sunde M, Karismose S, Veldman K, Schroeter A, Guerra B, Granier SA, Perrin-Guyomard
        A, Gicquel-Bruneau M, Franco A, Englund S, Teale C, Heiska H, Clemente L, Boerlin P, Moreno MA,
        Daignault D, Mevius D, Hendriksen RS, Aarestrup FM. Establishing streptomycin epidemiological cut-off
        values for Salmonella and Escherichia coli. Microb Drug Resist 2011; Jul 12 [Epub ahead of print].

    15. Jokinen C, Edge TA, Ho S, Koning W,Laing C, Mauro W, Medeiros D, Miller J, Robertson W, Taboada E,
        Thomas JE, Topp E, Ziebell K, Gannon VP. Molecular subtypes of Campylobacter spp., Salmonella enterica,
        and Escherichia coli O157:H7 isolated from faecal and surface water samples in the Oldman river watershed,
        Alberta, Canada. Water Res 2011; 45:1247-1257.

    16. Wilkes G, Edge TA, Gannon VP, Jokinen C, Lyautey E, Neumann NF, Ruecker N, Scott A, Sunohara M,
        Topp E, Lapen DR. Associations among pathogenic bacteria, parasites, and environmental and land use
        factors in multiple mixed-use watersheds. Water Res 2011; 45:5807-5825.

    17. Nesbitt A, Ravel A, Murray R, McCormick R, Savelli C, Finley R, Parmley J, Agunos A, Majowicz SE,
        Gilmour M and the Canadian Integrated Program for Antimicrobial Resistance Surveillance Public Health
        Partnership and the Canadian Public Health Laboratory Network. Integrated surveillance and potential sources
        of Salmonella Enteritidis in human cases in Canada from 2003 to 2009. Epidemiol Infect 2011; Dec 14:1-16
        [Epub ahead of print].

    18. Davidson VJ, Ravel A, Nguyen TN, Fazil A, Ruzante JM. Food-specific attribution of selected
        gastrointestinal illnesses: estimates from a Canadian expert elicitation survey. Foodborne Pathog Dis 2011;

    19. Ruzante JM, Majowicz SE, Fazil A, Davidson VJ. Hospitalization and deaths for select enteric illnesses and
        associated sequelae in Canada, 2001-2004. Epidemiol Infect 2011; 139:937-945.

    20. Bucher O, Fazil A, Rajić A, Farrar A, Wills R, McEwen SA. Evaluating interventions against Salmonella in
        broiler chickens: applying synthesis research in support of quantitative exposure assessment. Epidemiol Infect
        2011; Jul 25:1-21 [Epub ahead of print].

6                                                                           Annual reports of OIE Reference Centres, 2011

     21. Bucher O, Farrar AM, Totton SC, Wilkins W, Waddell LA, Wilhelm BJ, McEwen SA, Fazil A, Rajić A. A
         systematic review-meta-analysis of chilling interventions and a meta-regression of various processing
         interventions for Salmonella contamination of chicken. Prev Vet Med 2012; 103:1-15 [Epub 2011 Oct 10].

     22. Sibhat B, Molla Zewde B, Zerihun A, Muckle A, Cole L, Boerlin P, Wilkie E, Perets A, Mistry K, Gebreyes
         WA. Salmonella serovars and antimicrobial resistance profiles in beef cattle, slaughterhouse personnel and
         slaughterhouse environment in Ethiopia. Zoonoses Public Health 2011; 58:102-109.

     23. Leonard EK, Pearl DL, Finley RL, Janecko N, Reid-Smith RJ, Peregrine AS, Weese JS. Comparison of
         antimicrobial resistance patterns of Salmonella spp. and Escherichia coli recovered from pet dogs from
         volunteer households in Ontario (2005-06). J Antimicrob Chemother 2012; 67:174-181 [Epub 2011 Oct 20].

         Other communications



Annual reports of OIE Reference Centres, 2011                                                                    7

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