INSTITUTE FOR DIAGNOSIS AND ANIMAL HEALTH NATIONAL ... · used in official control – accredited SR EN ISO/CEI 17025:2005 . 1. Isolation and identification ofSalmonella SOP. 001/Bacteriol

INSTITUTE FOR DIAGNOSIS AND ANIMAL HEALTH NATIONAL REFERENCE LABORATORY

FOR ANIMAL SALMONELLOSIS ROMANIA

MAY 2017, ZAANDAM, NETHERLANDS National Reference Laboratory for Animal Salmonellosis, ROMANIA

• National Reference Laboratory (NRL) for animal salmonellosis

• Institute for Diagnosis and Animal Health (IDAH)

• National Sanitary Veterinary and Food

Safety Authority of Romania

National Reference Laboratory for Animal Salmonellosis


National Reference Laboratory for Animal Health Protocols used in official control – accredited SR EN

ISO/CEI 17025:2005

1. Isolation and identification of Salmonella SOP 001/Bacteriology

2. Serotyping of Salmonella SOP 008/Bacteriology 3. Identification and molecular subtyping of

main zoonotic Salmonella serovars SOP 029/Molecular Biology


NRL – animal salmonellosis ROMANIA

team

Maria Ionescu, DVM …………………… coordinator Larisa Tudor, Biologist …………………….deputy coordinator Viorica Vlaicu, DVM …………………… Bacteriology Department Lucia Iordache, DVM ………………….. Bacteriology Department Monica Vanghele, Biologist . . . . . . . . . . . Molecular Biology Department Oana Barbu, DVM . . . . . . . . . . . . . . . . Molecular Biology Department Adriana Răgălie, DVM . . . . . . . . . . . . . Imunology Department Gheorghe Gheorghe, DVM. . . . . . . . . Animal Health and Welfare Assessment Department Mihai Hurduc, DVM . . . . . . . . . . . . . . . Epidemiology Department


• elaboration and update Diagnostic Manual for animal salmonellosis; • elaboration of SOPs in accordance with European standards; • evaluation of performance characteristics of SOPs; • participation in the annual interlaboratory comparison studies

organized by EURL-Salmonella (Bilthoven, Netherlands); • the carrying out the tests for detection of Salmonella in National Programs for Control of zoonotic salmonellosis; • the carrying out the serotyping of Salmonella strains isolated by

official county labs; • organisation of training activities for county labs designated in official

control; • organisation of annual inter-laboratory comparative tests for checking

the diagnostic capability for salmonellosis in animals for all authorized county labs;

The major task of the NRL-animal salmonellosis ROMANIA


• evaluation and technical advising of county labs and proposal for designation of these by National Sanitary Veterinary and Food Safety Authority of Romania for testing of samples from National

Programs for Control of zoonotic salmonellosis; • coordination and monitoring the official county labs for the fulfillment

of the criteria for designation; • write down the periodical technical report regarding the incidence and

distribution of Salmonella serovars submitted to National Sanitary Veterinary and Food Safety Authority of Romania or EFSA;

• collaboration with EURL-Salmonella and other NRLs; • storage of Salmonella strains isolated in National Programs for Control

of zoonotic salmonellosis for 5 years; • participation (in field of competence) in revision of “Programme for

surveillance, prevention and control of animal diseases, those transmitted from animals to humans, animal and environmental protection” including the surveillance programs for animal salmonellosis;

The major task of the NRL-animal salmonellosis ROMANIA


Maria Ionescu - coordinator Monica Vanghele – Molecular Biology Department ● 5 - 9 March 2007: Detection of Salmonella in faeces according

with EN ISO 6579:2002/Amd.1:2007, Annex D. EURL- Salmonella, Bilthoven, Netherlands; ● 12 - 16 March 2007: Serotyping of Salmonella; EURL- Salmonella, Bilthoven, Netherlands; ● 20 Nov. – 1 Dec. 2006: Serotyping of Salmonella; Molecular subtyping of Salmonella by PFGE NRL-Salmonella, Clinical Science Institute, Galway, Irlanda.

Training courses


Participation in interlaboratory comparison studies Detection of Salmonella in faeces – SR EN ISO

6579:2003/Amd.1:2007, Annex D

Organizer

Samples

RESULTS Bacteriology Real Time PCR

(optional) 2012 EURL-Salmonella,

Bilthoven, Netherlands

pig faeces Good perfomance

-

2013 EURL-Salmonella, Bilthoven,

Netherlands

samples from the primary

production (Boot socks)

Good perfomance

Good perfomance


Netherlands

chicken faeces

Good perfomance

Good perfomance


Organizer

Samples

Results Bacteriology Real Time PCR

(optional) 2015 EURL-Salmonella,


pig faeces Performance could not be

evaluated

-


Netherlands

samples from the primary production

(Boot socks)

Good perfomance

Good perfomance


Participation in interlaboratory comparison studies Detection of Salmonella in faeces – SR EN ISO

6579:2003/Amd.1:2007, Annex D

Interlaboratory comparison study. Detection of Salmonella in faeces –SR EN ISO 6579:2003/Amd.1:2007, Annex D


Interlaboratory comparison study. Detection of Salmonella in faeces –SR EN ISO 6579:2003/Amd.1:2007, Annex D


Participation in interlaboratory comparison studies. Serotyping of Salmonella

Organizer

Number of strains

Results

2012

EURL-Salmonella, Bilthoven,

Netherlands

21

Good

performance

2013


Netherlands

21

Good

performance

2014


Netherlands

21

Good

performance


Organizer

Number of strains

Results

2015

EURL-Salmonella,


21

Good

performance

2016

EURL-Salmonella,


21

Good

performance

Participation in interlaboratory comparison studies. Serotyping of Salmonella


Participation in interlaboratory comparison studies. Molecular subtyping of Salmonella –

PFGE

Organizer

Number of strains

2013

EURL-Salmonella,


10

PFGE – pilot study

2014

EURL-Salmonella,


10

PFGE – optionally

test


Training activities with specialists working in the authorized sanitary veterinary labs – Detection of Salmonella in faeces –SR EN ISO

6579:2003/Amd 1:2007, Annex D

● 2007-2016: 40 specialists. ● Number of activities :78 ● Period of training: 5 days Training activities with specialists working in 30 authorized sanitary veterinary labs designated in official control for zoonotic salmonellosis

in animals


County labs 2007-2013 2014 2015 2016 1. Alba 2007 X 2. Arad 2007, 2008, 2012

3. Bacău 2008, 2011

4. Bihor 2012 X X 5. Bistriţa-Năsăud 2008, 2012

6. Buzău 2009 X 7. Botoşani 2009 X 8. Brăila 2008, 2011 X 9. Călăraşi

2008, 2009,2012 X X X

10.Cluj 2008, 2009, 2011

11.Constanţa 2008, 2011 X X X


6579:2003/Amd 1:2007, Annex D


County labs 2007-2013 2014 2015 2016

12. Dâmboviţa 2011 X 13. Galaţi 2011, 2012 X 14. Giurgiu 2009, 2011 X 15. Gorj 2008 X 16. Hunedoara 2010, 2012

17. Ialomiţa 2009 X 18. Iaşi 2009, 2012 X 19. Maramureş

2008, 2012

20. Mureş 2007, 2013 X

21. Olt 2008, 2011


6579:2003/Amd 1:2007, Annex D


County labs 2007-2013 2014 2015 2016

22. Prahova 2009, 2011 X

23. Satu Mare 2007 X

24. Sălaj 2008, 2011, 2012

25. Suceava 2009

26. Teleorman 2008, 2011

27. Timiş 2007, 2011

28. Vaslui 2007 X

29. Vâlcea 2007, 2009 2011

X

30. Vrancea 2008 X


6579:2003/Amd 1:2007, Annex D


Training activities with specialists working in the authorized sanitary veterinary labs – Detection of Salmonella in faeces –SR

EN ISO 6579:2003/Amd 1:2007, Annex D


Training activities with specialists working in the authorized sanitary veterinary labs – Detection of Salmonella in faeces –SR

EN ISO 6579:2003/Amd 1:2007, Annex D


Results of interlaboratory comparative tests obtained by county labs designated in official control for zoonotic salmonellosis in animals

Detection of Salmonella in faeces – SR EN ISO 6579:2003/Amd.1:2007

County labs

2012 2013 2014 2015 2016

1. Alba Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory

2. Arad Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory

3. Bacău Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory

4. Bihor Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory

5. Bistriţa-Năsăud

Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory

6. Buzău Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory

7. Botoşani Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory

8. Brăila Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory

9. Călăraşi Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory

10. Cluj Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory

11. Constanţa Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory


County labs

2012 2013 2014 2015 2016

12. Dâmboviţa Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory

13. Galaţi Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory

14. Giurgiu Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory

15. Gorj Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory

16. Hunedoara Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory

17. Ialomiţa Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory

18. Iaşi Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory

19. Maramureş Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory

20. Mureş Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory

21. Olt Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory




County labs

2012 2013 2014 2015 2016

22. Prahova Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory

23. Satu Mare Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory

24. Sălaj Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory

25. Suceava Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory

26. Teleorman Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory

27. Timiş Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory

28. Vaslui Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory

29. Vâlcea Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory

30. Vrancea Satisfactory Satisfactory Satisfactory Satisfactory Satisfactory




Protocol for detection of Salmonella in faeces – SR EN ISO 6579:2003/Amd.1:2007, Annex D

● PRE-ENRICHMENT: - 25 g faeces in 225 ml BPW -

incubation at 37°C± 1°C, for 18 ± 2 h


• ENRICHMENT - MSRV: 0.1 ml culture in BPW, incubation at 41.5ºC±1ºC, for 24 – 48 h



• Isolation on selective media (XLD, MC, BGA)



• Biochemical identification: TSI, MIU, Lizină, VP, ONPG, API 20E



• Serotyping – slide agglutination test



Workflow according SR EN ISO 6579:2003/Amd.1:2007

PRE-ENRICHMENT In BPW

Real Time-PCR for detection of Salmonella

ENRICHMENT on MSRV

SELECTIVE ISOLATION

on selective media (XLD, MC, BGA)

BIOCHEMICAL IDENTIFICATION: TSI, MIU, API 20E

SEROTYPING: poli “O“, poli “H“ şi “Vi“

SEROTYPING serovar identification

MOLECULAR SUBTYPING

PFGE


Serotyping of Salmonella strains


548 samples examinated by NRL in National Programs for Control of zoonotic serovars of Salmonella – 2012

BROILERS 146 samples

Own check 107

Positive 0

Official 39

Positive 1

S. Infantis 1

LAYING HENS 226 samples

Own check 170

Positive 3

S. Enteritidis 1 S. Mbandaka 2

Official 56

Positive 10

S. Enteritidis 10

BREEDING HENS 176 samples

Own check 51

Positive 1

S. Infantis 1

Official 125

Positive 0



BROILERS 58 samples

Own check 33

Positive 0

Official 25

Positive 0


Own check 89

Positive 9

S. Enteritidis 9

Official 99

Positive 0


Own check 36

Positive 0

Official 132

Positive 0



BROILERS 84 samples

Own check 53

Positive 6

S. Infantis 4 S. Orion 2

Official 31

Positive 1

S. Infantis 1


Own check 76

Positive 0

Official 72

Positive 3

S. Enteritidis 3


Own check 60

Positive 0

Official 162

Positive 0



BROILERS 126 samples

Own check 95

Positive 44

S. Enteritidis 11 S. Infantis 4 S. Tennessee 1 S. Brandenburg 1

Official 31

Positive 2

S. Infantis 2


Own check 201

Positive 25

S. Enteritidis 22 S. Infantis 1 S. Kentucky 1 S. Liverpool 1

Official 81

Positive 1

S. Kottbus 1


Own check 72

Positive 0

Official 36

Positive 0


Serotyping of Salmonella strains isolated in National Control Programs - 2012 (956 strains)

Broilers: 784 strains– 22 serovars LAYING HENS : 140 strains – 16 serovars

BREEDING HENS: 32 strains – 11 serovars

S. Agona 24 S. Agona 4 S. Agona 2

S. Amsterdam 4 S. Albany 5 S. Amsterdam 1

S. Bredeney 3 S. Blockley 3 S. Infantis 4

S. Ghester 1 S. Bredeney 3 S. Kentucky 2

S. Cubana 1 S. Corvalis 8 S. Liverpool 2

S. Derby 2 S. Enteritidis 66 S. Montevideo 2

S. Enteritidis 10 S. Glostrup 3 S. Senftenberg 5

S. Glostrup 2 S. Hadar 9 S. Taksony 2

S. Hadar 17 S. Infantis 15 S. Tennessee 4

S. Infantis 270 S. Kentucky 4 S. Typhimurium 1

S. Kentucky 68 S. Mbandaka 4 S. Uganda 7

S. Liverpool 68 S. Montevideo 1

S. Livingstone 47 S. Senftenberg 1

S. Mbandaka 14 S. Tennessee 5

S. Montevideo 1 S. Thompson 5

S. Newport 2 S. Typhimurium 4

S. Rissen 4

S. Senftenberg 44

S. Taksony 14

S. Tennessee 184

S. Typhimurium 1

Incidence of zoonotic serovars for the three production categories- 2012

Broilers: 784 strains

Laying hens: 140 strains Breeding hens: 32 strains

S. Enteritidis 1.27% S. Enteritidis 47.14% S. Enteritidis 0.0%

S. Typhimurium 0.12% S. Typhimurium 2.85% S. Typhimurium 3.12%

S. Infantis 34.43% S. Infantis 10.71% S. Infantis 12.5%

S. Hadar 2.16% S. Hadar 6.42% S. Hadar 0.0%

S. Virchow 0.0% S. Virchow 0.0% S. Virchow 0.0%


Serotyping of Salmonella strains isolated in National Control Programs 2013 – 1237 strains

BROILERS:1087 strains – 25 serovars

Laying hens: 121 strains – 18 serovars

Breeding hens: 25 strains – 7 serovars

Turkeys: 4 strains - 2 serovars

S. Agona 103 S. Agona 6 S. Agona 12 S. Infantis 2

S. Amsterdam 3 S. Albany 7 S. Kentucky 2 S. Newport 2

S. Blockley 1 S. Blockley 1 S. Kottbus 1

S. Bovismorbificans 1 S. Bredeney 5 S. Liverpool 2

S. Bredeney 17 S. Corvalis 1 S. Newport 1

S. Enteritidis 35 S. Derby 1 S. Senftenberg 5

S. Galiema 6 S. Enteritidis 46 S. Tennessee 2

S. Glostrup 3 S. Glostrup 2

S. Hadar 7 S. Hadar 1

S. Infantis 545 S. Infantis 29

S. Kentucky 90 S. Kentucky 4

S. Kottbus 7 S. Kottbus 4

S. Liverpool 72 S. Liverpool 1

S. Livingstone 10 S. Mbandaka 2

S. Mbandaka 2 S. Newport 2

S. Montevideo 3 S. Senftenberg 6

S. Newport 1 S. Tennessee 1

S. Orion 8 S. Rissen 3 S. Uganda 2

S. Senftenberg 39 S. Taksony 11

S. Tennessee 84 S. Thompson 11

S Typhimurium 13 S Uganda 12

Incidence of zoonotic serovars for the four production categories - 2013

Broilers 1087 strains

Laying hens 121 strains

Breeding hens 25 strains

Turkeys 4 strains

S. Enteritidis 3.21% S. Enteritidis 38.01% S. Enteritidis 0.0% S. Enteritidis 0.0%

S. Typhimurium 1.19% S. Typhimurium 0.0% S. Typhimurium 0.0% S. Typhimurium 0.0%

S. Infantis 50.13% S. Infantis 23.96% S. Infantis 0.0% S. Infantis 50.0%

S. Hadar 0.64% S. Hadar 0.82% S. Hadar 0.0% S. Hadar 0.0%

S. Virchow 0.0% S. Virchow 0.0% S. Virchow 0.0% S. Virchow 0.0%



Broilers: 993 strains – 23 serovars Laying hens: 69 strains – 12 serovars


S. Abony 4 S. Albany 2 S. Amsterdam 2

S. Agona 9 S. Enteritidis 23 S. Infantis 8

S. Anatum 15 S. Fillmore 3 S. Livingstone 2

S. Bredeney 1 S. Glostrup 1 S. Meleagridis 2

S. Chester 10 S. Gloucester 2 S. Senftenberg 6

S. Dabou 1 S. Hadar 3 S. Taksony 8

S. Enteritidis 23 S. Infantis 9

S. Fillmore 2 S. Kentucky 4

S. Hadar 2 S. Kottbus 10

S. Infantis 626 S. Livingstone 3

S. Kentucky 79 S. Newport 3

S. Kottbus 20 S. Typhimurium 6

S. Liverpool 9

S. Livingstone 5

S. Mbandaka 25

S. Montevideo 13

S. Newport 2

S. Orion 6

S. Senftenberg 88

S. Taksony 2 S. Tennessee 43

Incidence of zoonotic serovars for the three production categories - 2014

Broilers: 993 strains Laying hens: 69 strains Breeding hens: 28 strains

S. Enteritidis 2.31% S. Enteritidis 33.33% S. Enteritidis 0.0%

S. Typhimurium 0.50% S. Typhimurium 8.69% S. Typhimurium 0.0%

S. Infantis 63.04% S. Infantis 13.04% S. Infantis 28.57%

S. Hadar 0.20% S. Hadar 4.34% S. Hadar 0.0%




Broiler: 1086 strains – 21 serovars Laying hens: 165 strains – 16 serovars


S. Abony 1 S. Agona 29 S. Amsterdam 3

S. Agona 3 S. Albany 3 S. Enteritidis 3

S. Brandenburg 2 S. Cubana 1 S. Infantis 3

S. Bredeney 8 S. Enteritidis 49 S. Kottbus 1

S. Chester 1 S. Fyris 1 S. Liverpool 6

S. Enteritidis 70 S. Glostrup 2 S. Mbandaka 1

S. Glostrup 3 S. Hadar 7 S. Orion 1

S. Havana 5 S. Infantis 11 S. Senftenberg 2

S. Infantis 672 S. Kentucky 5 S. Typhimurium 4

S. Kentucky 50 S. Kottbus 4 S. Uganda 2

S. Kottbus 20 S. Liverpool 1

S. Liverpool 32 S. Mbandaka 16

S. Livingstone 15 S. Montevideo 6

S. Mbandaka 1 S. Newport 17

S. Montevideo 8 S. Senftenberg 4

S. Ohio 1 S. Typhimurium 9

S. Senteftenberg 60

S. Taksony 11

S. Tennessee 64

S. Thompson 43 S. Typhimurium 16

Incidence of zoonotic serovars for the three production categories – 2015

Broilers: 1086 strains

Laying hens: 165

strains

Breeding hens: 26

strains

S. Enteritidis 6.44%

S. Enteritidis 29.69% S. Enteritidis 11.53%

S.Typhimurium 1.47%

S. Typhimurium 5.45% S. Typhimurium 15.38%

S. Infantis 61.87%

S. Infantis 6.66% S. Infantis 11.53%

S. Hadar 0.0%

S. Hadar 4.24% S. Hadar 0.0%



Other activities

• Detection of Salmonella by Real Time - PCR on the pre-

enrichment samples in BPW; - optional, in the annual interlaboratory comparison studies

organized by EURL-Salmonella (Bilthoven, Netherlands); • Molecular subtyping of main zoonotic serovars of Salmonella by

PFGE; - discrimination between wild strains of Salmonella Enteritidis

and vaccinal strains • Identification of main zoonotic serovars of Salmonella (S. Enteritidis şi S. Typhimurium);


National Reference Laboratory for animal salmonellosis

ROMANIA

Thank you!


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Documents

INSTITUTE FOR DIAGNOSIS AND ANIMAL HEALTH NATIONAL ... · used in official control – accredited SR EN ISO/CEI 17025:2005 . 1. Isolation and identification ofSalmonella SOP. 001/Bacteriol