Salmonellosis
OIE Reference Laboratory Reports
Activities in 2011
Name of disease (or topic) for which you are a designated OIE Reference Laboratory: / SalmonellosisAddress of laboratory: / Viale dell’Università 12,
35020 Legnaro, Padova
ITALY
Tel.: / +39 0498084296
Fax: / +39 0498830268
e-mail address: /
website: / www.izsvenezie.it
Name (including Title and Position) of Head of Laboratory (Responsible Official): / Prof. Igino Andrghetto, Director General
Name(including Title and Position) of OIE Reference Expert: / Dr Antonia Ricci, Head of Department
Name (including Title and Position) of writer of this report
(if different from above):
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 Laboratory routinely performs isolation, quantification by mini-MPN and serotyping of Salmonella spp., phage typing of S. Enteritidis, S. Typhimurium and S. 4,[5],12:i:- , molecular detection of Salmonella spp. by Real-Time PCR, differentiation between S. Typhimurium and S. 4,[5],12:i:- by PCR, molecular typing for epidemiological investigations of Salmonella strains (specifically by using PFGE), phenotypic evaluation of the antimicrobial resistance level (by disk diffusion and MIC) and molecular characterization of antimicrobial resistance determinants.
Test / For / Specificity / TotalIsolation and identification / Salmonella detection / Genus / 3325
Quantification / MINI Most Probable Number for quantification of Salmonella spp. / Genus / 489
Slide agglutination / Serotyping of Salmonella strains / Serotype / 2037
Phage-typing / Phage-typing of S. Enteritidis, S. Typhimurium and S. 4,[5],12:i:- strains / Phage type / 1332
Disc diffusion test / Antimicrobial susceptibility of Salmonella strains / Antimicrobial resistance pattern / 130
MIC / Antimicrobial susceptibility of Salmonella strains / Antimicrobial resistance pattern / 397
PCR / Resolution of H1 antigenic complex / Serotype / 700
PCR / Differentiation between S. Typhimurium and monophasic variant (S. 4,[5],12:i:-) / Serotype / 830
PFGE / Sub-typing of Salmonella strains / Pulsotype / 300
Tests for live Salmonella Enteritidis vaccine / S. Enteritidis / Serotype – vaccinal strains / 30
Real Time PCR / Salmonella detection / Genus / 277
2. Production and distribution of diagnostic reagents
(including for own use) / Amount supplied to other countries
S. Typhimurium for the production of a commercial ELISA test / 2 ampoules of 10 ml / /
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
b) Organisation of inter-laboratory proficiency testing with laboratories other than OIE Reference Laboratories for the same pathogens and diseases to ensure equivalence of results
In 2011 the laboratory successfully participated in the following collaborative studies organized by the CRL Salmonella (Bilthoven, The Netherlands): a) Salmonella isolation from food b) Salmonella isolation from animal faeces c) Salmonella serotyping and d) Salmonella Enteritidis - Salmonella Typhimurium phage-typing.
In 2011 the Laboratory organized a ring trial on Salmonella spp. serotyping. The ring trial consisted in serovar identification of 20 Salmonella strains; 9 laboratories took part in it. Moreover, a ring trial on isolation of Salmonella spp. from poultry faeces was organized. The ring trial consisted in Salmonella detection from 12 samples and 8 controls; 25 laboratories took part in it.
Only national laboratories participated in the ring trials, but the participation could be extended to other laboratories outside the country in case some of them would be interested in.
4. Preparation and supply of international reference standards for diagnostic tests or vaccines
In 2011 the laboratory provided three laboratories, respectively from Belgium, Turkey and UK, with DNA templates obtained from 66 Salmonella strains belonging to from different Salmonella serovars (mainly S. Typhimurium and S. 4,[5],12:i).
Moreover, in 2011, the following strains were provided by the OIE laboratory:
- 10 strains of S. Typhimurium and 5 strains of S. Senftenberg isolated from poultry in 2010 (sent to an Italian laboratory)
- 19 strains of S. 4,[5],12:i, 1 strain of S. Glaucester, 1 strain of S. Agama, 1 strain of S. Lagos (sent to a laboratory in UK and a laboratory in Belgium)
- 1 strain of S. Typhimurium, 1 strain of S. 4,[5],12:i, 1 strain of S. Lagos, 1 strain of S. Agama (sent to an Italian Laboratory)
- 1 strain of S. Typhimurium, 2 strains of S. 4,[5],12:i (sent to the laboratory of an Italian hospital)
- 1 strain of S. 4,[5],12:i and 1 strain of S. Blockley (sent to an Italian laboratory)
5. Research and development of new procedures for diagnosis and control
In 2011 the laboratory completed the validation of a PCR protocol for the differentiation between S. Tyhimurium and its monophasic variant (S. 4,[5],12:i:-). Validation tests demonstrated that the method was repeatable (repeatability within and between days) and its specificity was equal to 100%.
Then, the laboratory worked on the development of an in house Real Time PCR protocol to identify Salmonella spp. in foodstuffs and samples collected at primary production level. The protocol set up was based on the detection of Salmonella-specific invA gene.
Moreover, an in house Real Time PCR protocol to identify and differentiate S. Typhimurium-its monophasic variant S. 4,[5],12:i:- and S. Enteritidis was set up. The targets of the protocol were a region of the gene STM4497 specific for S. Typhimurium- S. 4,[5],12:i:- and a region of the sefA gene specific for S. Enteritidis. For both these last protocols the laboratory is working on the validation.
6. Collection, analysis and dissemination of epizootiological data relevant to international disease control
The laboratory in 2011 carried on the coordination of the Enter-vet net, which collects information concerning Salmonella typing results at national level. The network involves ten Italian institutes that collect Salmonella strains and their epidemiological information. In 2011 the laboratory elaborated data collected during the previous year. In 2010, in the framework of the network, 4639 Salmonella strains were notified. The great majority of strains was isolated from animal samples (2140) and foodstuffs (1604). Strains collected from poultry, pigs and turkeys accounted for 60.9% of the Salmonella isolates. The most common serovar was the monophasic variant of S. Typhimurium (frequency equal to 10.26%), followed by S. Typhimurium and S. Enteritidis (frequency equal to 10.13% and 5.35% respectively ). As far as phage-typing is concerned for S. Typhimurium the most common phage type was DT104, from the monophasic variant it was DT193 and for S. Enteritidis it was PT4. The laboratory is currently finalising the publication of the report about data collected during 2010.
As far as scientific projects is concerned ,a study aimed at investigating epidemiology of S. Typhimurium and its monophasic variant and comparing strains of these two serovars isolated from animals (pigs, poultry and cows) and humans started in 2011. The project, lasting 3 years, will allow to identify possible sources of human infections due to these relevant serovars. The OIE laboratory will take part at the project as coordinator, then, the reference Italian Institute in charge of collecting and analysing human Salmonella strains, and a Danish University will participate as partners.
Moreover, in 2011 the laboratory collaborated with the poultry industry in a project aimed at evaluating the effect of different domestic storage conditions on the presence and quantity of Salmonella spp. in poultry meat products.
7. Maintenance of a system of quality assurance, biosafety and biosecurity relevant to the pathogen and the disease concerned
The laboratory is accreditated according to the ISO 17025 by the Italian Accreditation Body. In 2011 the laboratory maintained the accreditation for the following procedures (identification of Salmonella from samples collected at the primary production level Annex D ISO 6579), serotyping of Salmonella and phage typing of S. Enteritindis and S. Typhimurium (internal methods).
8. Provision of consultant expertise to OIE or to OIE Member Countries
Following a request from OIE, the laboratory has prepared, in collaboration with the OIE Reference Laboratory for Salmonellosis in UK, a review: Belluco S., Cibin V., Davies R., Ricci A, Wales A. “A review of the scientific literature on the control of Salmonella spp. in food producing animals other than poultry”, which has been provided to the OIE Animal Production Food Safety Working Group, in order to provide a basis for a decision on future priorities for standard development in this field.
Dr A. Ricci participated as invited speaker in the OIE regional seminar for Animal Production Food Safety Focal Points which was held in Brescia, Italy, on 8-10 November 2011.
Dr A. Ricci., in 2011 participated in the following working groups:
· Panel Biohaz, EFSA;
· “QMRA of new target for Salmonella in Laying Hens” EFSA
· “Analysis of the Salmonella Baseline Survey in Breeding Pigs” EFSA
· “QMRA of new target for Salmonella in broilers” EFSA
· “Monitoring and assessment of the public health risk of “Salmonella Typhimurium-like” strains” EFSA
· “Estimation of the the public health hazards to be covered by inspection of meat from poultry” EFSA
· “Estimation of the public health impact of setting a new target for the reduction of Salmonella in turkeys”
Moreover other experts from the laboratory took part in Task Force meetings on “Zoonoses data collection”, EFSA
9. Provision of scientific and technical training to personnel from other OIE Member Countries
In November 2011 the laboratory hosted two veterinarians (Dr. Elvira Beli and. Dr. Renis Maci) from Albania and provided them with a training on Salmonella isolation, serotyping, determination of the antimicrobial resistance profile and different molecular procedures for Salmonella detection and subtyping.
A Twinning Project with the Central Veterinary Institute in Vietnam CVVI (lasting 36 months from July 2011) has been approved by OIE. The main activity planned in the framework of the project is to provide training to CVVI on laboratory techniques for Salmonella detection by using traditional microbiology and molecular methods, serotyping and determination of antimicrobial resistance. During November 2011, the OIE Laboratory visited the CVVI in order to perform a baseline assessment of the CVVI Laboratory. Briefly,: needs for CVVI capacity building (media, equipment, personnel training) were identified; sampling plans for the assessment of Salmonella prevalence in poultry and pig farms in Vietnam, as well as the procedures to analyze samples collected were defined; and the timetable for the initial training of CVVI bacteriologists at the OIE Laboratory was shared. A detailed description of the activities carried out can be found in the interim report sent to OIE in January 2012.
10. Provision of diagnostic testing facilities to other OIE Member Countries
The laboratory collaborated with Dr. Michael Agbaje, from the Department of Veterinary Microbiology and Parasitology, College of Veterinary Medicine, University of Agriculture, Abeokuta, Nigeria and with Dr. Nancy Sati from the National Veterinary Research Institute, Vom, Nigeria, who sent 146 Salmonella strains previously isolated in their laboratories, to be confirmed as Salmonella spp., serotyped and analyzed for the antimicrobial resistance profile.
11. Organisation of international scientific meetings on behalf of OIE or other international bodies
None
12. Participation in international scientific collaborative studies
The laboratory is participating in a project entitled “Restrictive use of antibiotics in organic animal farming – a potential for safer, high quality products with less antibiotic resistant bacteria” – SafeOrganic. The project is coordinated by the DTU –Denmark, and includes 5 partners from 5 different countries. In the framework of the project it will be documented whether the organic pigs in different European countries does show lower levels of antibiotic resistant bacteria compared to the conventional pigs. An important part of the project will be aimed at investigating to which degree antibiotic resistant bacteria from conventional raised animals is transferred to organic meat during processing along the slaughter line. Then, it will be investigated if bacterial antibiotic resistance patterns and genotypes can be used as markers for the consumption of antibiotics in organic animal production.
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
Ricci A., presentation entitled “Monitoring and intervention strategies of Salmonella in poultry: the European experience” Seminar for the OIE National Focal Points for Animal Production Food Safety, organizzed by OIE at the Istituto Zooprofilattico Sperimentale della Lombardia e Emilia Romagna, Brescia, Italy, 8-10/11/2011
Mancin M., presentation entitiled «Proficiency testing in Food Microbiology AQUA according to ISO/IEC 17043:2010 and ISO/TS 22117:2010» Workshop “Eurachem 2011 - 7th Workshop - Proficiency Testing in anlytical chemistry, microbiology and laboratory medicine - Current Practice and Future Directions” organized by the working group EURACHEM Proficiency testing con CITAC, Istanbul, Turkey, 03-06/10/2011
Ricci A., presentation entitled “Antibiotic resistance in the food chain in the EU” Workshop “Antimicrobial resistance in a food safety perspective (World health day celebration)” Organized by Technical Officer/Food Safety - WHO EURO, Country Office, Tirana, Albania, 6-8/04/2011
Lettini A.A., presentation entitled «Comparison of DNA extraction methods to detect Salmonella spp. from pig faeces and pork» SafePork Conference 2011, Maastricht, The Netherlands 19-22/06/2011; p. 40
Ricci A., presentation entitled “Management of pre-harvest and control measures:importance and alternatives” International Seminar on Avian Salmonellosis” organized by Ubabef e Ala, Rio De Janeiro, Brazil, 27/06-01/07/2011
Ricci A., presentation entitled «Using molecular biology in the diagnosis of Salmonella:future prospects», International Seminar on Avian Salmonellosis” organized by Ubabef e Ala, Rio De Janeiro, Brazil 27/06-01/07/2011
Lettini A.A., presentation entitled “Trends and limitations of diagnostic tools” “22° Congresso Brasileiro de Avicoltura” Sao Paulo, Brazil, 25-27/10/2011
¡ Scientific publications in peer-reviewed journals
Barco L, Lettini AA, Ramon E, Longo A, Saccardin C, Pozza MC, Ricci A. A rapid and sensitive method to identify and differentiate Salmonella enterica serotype Typhimurium and Salmonella enterica serotype 4,[5],12:i:- by combining traditional serotyping and multiplex polymerase chain reaction. Foodborne Pathog Dis. 2011 Jun;8(6):741-3