1. Salmonella Control in Poultry
Balázs Molnár – Key Account Manager Salmovac 440

2. Schedule of presentation
Future challanges of Salmonella on poultry flocks
The Salmonella vaccine
Differentiation of the active vaccine strain from wild strains

3. Salmonella in EU Future challanges
Where we see the development of Salmonella prevalances in poultry flocks and countries
The trend in regulations
Future challanges

4. Salmonella awareness Starting point for Salmonella awareness in EU:4
Starting point for Salmonella awareness in EU:
1988: UK Health minister Edwina Currie has provoked outrage
by saying most of Britain's egg production is infected with Salmonella
BBC: December 3rd, 1988
1990: National Egg Awareness Campaign (UK)
Rising awareness in media across Europe

5. Emergence of human cases of Salmonella Enteritidis
History of human Salmonella infections (EU level)
Emergence of human cases
of Salmonella Enteritidis
(Awareness)
After
Mandatory
Measures
*Monitoring of infection in food-production animals by S. Typhimurium and S. Enteritidis
*Plans to combat zoonoses (inc. slaughter).
*Measures for feed quality pertinent to breeding and laying flocks
*German Decree for Chicken Salmonella (Hühner-Salmonellen-Verordnung)
Source: The Community Summary Report on Food-borne Outbreaks in the European Union in 2010,The EFSA Journal (2011)

6. EFSA opinion on vaccination
EU summary report on zoonoses, zoonotic agents and food-borne outbreaks 2010 EFSA Journal 2012;10(3):2597
“The results above indicate that the reduction of S. Enteritidis in laying hen flocks and of Salmonella spp. in table eggs is likely to have contributed to the decline of S. Enteritidis cases in humans, since eggs are regarded to be the most important source of these infections. Increased voluntary and compulsory vaccination of laying hens, as well as other hygiene-based control measures, are likely to have contributed to this, driven by the economic consequences of egg restrictions and the requirement to heat treat eggs from positive flocks”.

7. Salmonella incidences in EU MSs, 2017
In 2017 the number of human salmonellosis cases was reported as
This means 19.7 cases / population in 2017
This was the highest since 2013 (due to report on case based data for the first time
Outbreak related data were higher than in 2015 but lower than in 2016

8. Overall prevalence of Salmonella target serovars relevant in various poultry populations, among all reporting MS, 2007–2017

9. % of 5 target serovars in breeder floks in EU MS, 2014-2016
In 2016, 13 MSs exceeded the prevalence target of 1% for all serovars.
A dual prevalence target for all serovars and for the selected high priority serovars could be proposed

10. Prevelance of the S. Enteritidis positive breeding hen flocks in 2017

11. Prevelance of the S. Enteritidis positive laying hen flocks in 2017

12. Prevalence of laying hen flocks in production period positive for S
Prevalence of laying hen flocks in production period positive for S. Enteritidis, S. Typhimurium
If a 1% target of Salmonella in laying hen flocks would be met, than the number of layer associated human salmonellosis is estimated to reduce by % (ie 254,400 cases less)
Recommendation to investigate:
the potential and reasons for under-detection of Salmonella
the effectiveness of administration of Salmonella vaccination programmes used in layering flocks and their protective effects

13. Prevalence of poultry flocks positive for target Salmonella serovars (SE, ST): EFSA Journal 2018

14. Prevalence of poultry flocks positive for target Salmonella serovars (SE, ST): EFSA Journal 2018

15. Prevalence of poultry flocks positive for target Salmonella serovars (SE, ST): EFSA Journal 2018

17. SE phagetypes across Europe
Image source:

18. New challenges:
different Serotypes, different Phagetypes

19. New challenges: Longer laying period: Ø 91 weeks
Progress in layer breeds
Low egg prices make longer production cycles more attractive
 Longer Salmonella protection is needed!
Alternative housing systems

20. What should a „better“ Salmonella vaccine offer?
Broad protection
to cover emerging Salmonella (like monophasic strain of S. Typhimurium)
Early and long protection
to cover the full laying period

21. The Salmonella vaccine
Early, Long and broad protection against Salmonella in poultry
Preparation for the challanges

22. Salmovac 440 Early, long and broad protection against Salmonella in poultry

23. Salmovac 440 double-attenuated (adenine-histidine auxotrophic)
Salmovac 440
Active substances
double-attenuated (adenine-histidine auxotrophic)
Salmonella Enteritidis mutant, strain 441/014
1 - 8 x 108 CFU
Indication
&
target species
Active immunisation of healthy and susceptible chickens to reduce colonization, persistence and invasion of the intestinal tract and internal organs by Salmonella Enteritidis and Salmonella Typhimurium.

24. Salmovac 440: Broad protection
Salmovac 440: proven protection against:
SE Phagetypes: PT 1, PT 4, PT 8, PT 21
ST and monophasic strain of ST
Sven Springer, Thomas Lindner, Hans-Joachim Selbitz:
Testing of the immunogenicity of a Salmonella Enteritidis live vaccine against non-PT4 Salmonella Enteritidis strains and a multi-drug resistant monophasic Salmonella Typhimurium strain
International Symposium on Salmonella and Salmonellosis, St. Malo, France, 2013

25. Salmovac 440 double-attenuated (adenine-histidine auxotrophic)
Salmovac 440
Active substances
double-attenuated (adenine-histidine auxotrophic)
Salmonella Enteritidis mutant, strain 441/014
1 - 8 x 108 CFU
Indication
&
target species
Active immuniszation of healthy and susceptible chickens to reduce colonization, persistence and invasion of the intestinal tract and internal organs by Salmonella Enteritidis and Salmonella Typhimurium.
Vaccination scheme
One dose from first day of age, a second dose six weeks later and a third dose around 13 weeks of age 
 (At least 3 weeks before onset of lay)

26. Salmovac 440

27. Salmovac 440 Active substances
Salmovac 440
Active substances
double-attenuated (adenine-histidine auxotrophic)
Salmonella Enteritidis mutant, strain 441/014
1 - 8 x 108 CFU
Indication
&
target species
Active immuniszation of healthy and susceptible chickens to reduce colonization, persistence and invasion of the intestinal tract and internal organs by Salmonella Enteritidis and Salmonella Typhimurium.
Vaccination scheme
One dose from first day of age, a second dose six weeks later and a third dose around 13 weeks of age 
 (At least 3 weeks before onset of lay)
Onset of immunity
Within 6 days after first vaccination

28. Multiple response following oral vaccination
Salmovac 440:
Multiple response following oral vaccination
42%
13,7%
Unspecific Defence
(e.g. Colonization
Inhibition effect)
Humoral Immunity
(e.g. Secretory IgA)
Cell-mediated Immunity
(e.g. T-helper cells, IL-5, IFN-y)
21,9%
4,7%
5,9%
5,9%

29. Salmovac 440: Multiple response following oral vaccination
Early colonization
Inhibitory effect in the gut preventing colonization by other Salmonellae
No effect if already infected with Salmonella
First oral vaccination on 1st day of live!
Onset of immunity (OOI): 6d (AviPro: 14d)
Unspecific Defence
(e.g. Colonization
Inhibition effect)
42%
21,9%
4,7%
5,9%
13,7%

30. Salmovac 440: Multiple response following oral vaccination
5,9%
4,7%
21,9%
Unspecific Defence
(e.g. Colonization
Inhibition effect)
Cell-mediated Immunity
(e.g. T-helper cells, IL-5, IFN-y)
Humoral Immunity
(e.g. Secretory IgA)
Innate and adaptive immunity
Secretory IgA on mucous membrane  More important role than IgY (induced by inactivated vaccines)
Prevent Salmonella from entering macrophages
First line of defence
Protective immunity

31. Salmovac 440 Active substances
Salmovac 440
Active substances
double-attenuated (adenine-histidine auxotrophic)
Salmonella Enteritidis mutant, strain 441/014
1 - 8 x 108 CFU
Indication
&
target species
Active immuniszation of healthy and susceptible chickens to reduce colonization, persistence and invasion of the intestinal tract and internal organs by Salmonella Enteritidis and Salmonella Typhimurium.
Vaccination scheme
One dose from first day of age, a second dose six weeks later and a third dose around 13 weeks of age 
 (At least 3 weeks before onset of lay)
Onset of immunity
Within 6 days after first vaccination
Duration of Immunity
Duration of immunity for Salmonella Enteritidis is 63 weeks after third vaccination when used according to the recommended vaccination schedule. Duration of immunity for Salmonella Typhimurium is 60 weeks after third vaccination when used according to the recommended vaccination schedule.

32. Salmovac 440: Efficacy testing – SE and STm challenge, DOI
Up to 11 weeks longer protection against Salmonella Enteritidis
and 14 weeks longer protection against Salmonella Typhimurium.
Salmovac 440

33. Salmovac 440 Active substances
Salmovac 440
Active substances
double-attenuated (adenine-histidine auxotrophic)
Salmonella Enteritidis mutant, strain 441/014
1 - 8 x 108 CFU
Indication
&
target species
Active immuniszation of healthy and susceptible chickens to reduce colonization, persistence and invasion of the intestinal tract and internal organs by Salmonella Enteritidis and Salmonella Typhimurium.
Vaccination scheme
One dose from first day of age, a second dose six weeks later and a third dose around 13 weeks of age 
 (At least 3 weeks before onset of lay)
Onset of immunity
Within 6 days after first vaccination
Duration of Immunity
Duration of immunity for Salmonella Enteritidis is 63 weeks after third vaccination when used according to the recommended vaccination schedule. Duration of immunity for Salmonella Typhimurium is 60 weeks after third vaccination when used according to the recommended vaccination schedule.
Shedding = active vaccine strain
Vaccinated chickens excrete the vaccine strain up to six weeks post vaccination. In studies, the vaccine strain can be found in the environment for up 5 weeks after the third vaccination.

34. Detection of S. Enteritidis at sock swabs by ISO and PCR methods
The active vaccine strain is found in samples many weeks after vaccination
It assures the equal distribution of protection to those animals which did not drink in time of vaccination
It invasively competes with wild strains in the environment
The veterinarian can measure the success of vaccination by sampling the litter, feaces or environment
Results on comparison between molecular and cultural based detection of Salmonella enteritidis live vaccine strain (Slamovac 440) during rearing period in pullets; September 2016-February 2017

35. Differentiation methods
Laboratory
Differentiation of Salmovac 440 vaccine strain from wild strains
Differentiation methods

36. Salmovac 440 - Diagnostics
Bacteriological verification for Salmonella has to follow the ISO 6579:2002
Our Vaccine strain (Salmovac 440) grows in the used selective media  „false positive“

37. Salmovac 440 - Diagnostics
Live Salmonella vaccines for which the manufacturer does not provide an appropriate method to distinguish bacteriologically wild-type strains of salmonella from vaccine strains shall not be used in the framework of national control programmes adopted pursuant to Article 6 of Regulation (EC) No 2160/2003.

38. Salmovac 440 - Diagnostics
Field strain or Vaccine strain?
Differentiation based on Adenin and Histidin Auxotrophy

39. IDT Salmonella Diagnostic kit
The IDT Salmonella Diagnostic Kit RTU two ready-to-use media – Medium A and Medium B – for differentiation of Salmonella Dublin, Salmonella Typhimurium and Salmonella Enteritidis IDT vaccine strains from wild type strains of the same serotype.
Medium A : Various salts, Glucose, and Nicotinamide
Medium B : Various salts, Glucose, Nicotinamide and nutrients dependent on vaccine strain.
Wild type strains of S. Dublin, S. Typhimurium and S. Enteritidis from Salmonella enterica subsp. Enterica show a definitive growth (turbidity) in both, Medium A and B of the IDT Salmonella Diagnostic Kit RTU.
The IDT vaccine strains, however, only grow in Medium B but not in Medium A, due to their special requirements for nutrients as auxotrophic strains.
In very rare cases, the appearance of auxotrophic wild strains has been observed. These wild strains will not grow - neither in Medium A nor in Medium B of this kit.

40. Salmovac 440 - Diagnostics
Vaccine strain:
Medium A is completely clear, text is fully legible = no bacterial growth
Medium B is turbid = bacterial growth due to the special nutrients it contains
IDT Diagnostikum:
 48 h
Wild strain:
Medium A is turbid = bacterial growth
Medium B is turbid = bacterial growth
Auxhotrophic wild strain:
Medium A is completely clear, text is fully legible = no bacterial growth
Medium B is completely clear, text is fully legible = no bacterial growth

41. Salmovac 440 - Diagnostics
KYLT® DIVA 1: Real Time PCR
Differentiation field strain / Salmovac (Gallivac) vaccine strain within 2-3 hours after Salmonella-isolation
Salmovac 440

42. System for Salmonella Monitoring and Control in Poland
Identification spp.
Sample from farm
Accredited Laboratory
No Salmonella spp.
+Salmonella spp.
Testing flow
Identification strain
Not SE or ST
Suspicious SE or ST
Differentiation test
- IDT-Diagnostikum or PCR (DIVA)
Vaccine strain
Field strain
Veterinarian Authorithy
communication with farm veterinarian
Reference Laboratory
final control of field strain
Information flow

43. Accredited Laboratories
System for Salmonella Monitoring and Control in Lithuania
Identification spp.
Sample from farm
Accredited Laboratories
National Reference Laboratory
Identification strain
Differentiation test
- IDT-Diagnostikum or PCR (DIVA)
No Salmonella spp.
Not SE or ST
+Salmonella spp.
Vaccine strain
Suspicious SE or ST
Field strain
This process shall be as short as possible < 1 day
The egg producing farm shall be not banned from sales of eggs untill the vaccine strain differentiation is not concluded
Veterinarian Authorithy
communication with farm veterinarian
Vaccine starin cannot be considered as a contamination (EU regulation)
Testing flow
Information flow

44. Take home messages
It is not possible to eradicate Salmonella because the wide range of contamination sources: Natural reservoirs (e,g, poultry, pigs, cattle, pets ), Vectors (e.g. rodents, flies, darkling beetles) and other sources (e.g. feed, raw materials, fomites)
Salmonella is still and will be a problem in the future
Vaccination in general is the major intervention tool to prevent Salmonella infections  significant decrease of Salmonella prevalence in EU-27

45. Salmovac Early, long and broad protection against Salmonella in poultry  OOI: 6 days after 1st vaccination
AviPro Duo: 14d

46. Salmovac Early, long and broad protection against Salmonella in poultry  S.E.: 63 weeks  S.T.: 60 weeks
AviPro Duo: 52 weeks
AviPro Duo: 46 weeks

47. Salmovac Early, long and broad protection against Salmonella in poultry  different phage types of S.E.  S.T., also monophasic S.T.

48. Salmovac The active vaccine strain can be found by sampling It can be differentiated by ISO and PCR methods in laboratories

51. Thank you very much for your attention!

52. FAQ: Are Antibodies important for day old chicks?

53. FAQ: Are Antibodies important for day old chicks?
What do we know about IgY:
Induced by inactivated vaccines
Easy to measure in the blood
Transmitted to their offspring via the egg
But: High level of IgY does not correlate with good protection against salmonella
Much more important: active protection with a live vaccine that has early onset of protection
unspecific defense, cell-mediated immunity, IgA antibodies

54. FAQ: Do maternal antibodies interfere with an early live vaccination?
Springer et al. (2006)
Vaccine strain in the liver on day 14 after hatching
in log c.f.u. / g
Group
Liver
1 (parents vaccinated with SALENVAC®)
1.99 ± 0.50
2 (parents unvaccinated)
1.74 ± 0.35
No significant differences
Maternally-induced antibodies had no influence on the colonisation of the liver by the vaccine strain
Protective effect against virulent field strain?

55. Salmovac 440 in (broiler) breeders
Reduced colonization, persistence and invasion of internal organs
 Reduced shedding  Clean parent flock
No vertical transmission of Salmonella via the egg
Best protection against a pathogen is the absence of the pathogen

56. Top down approach for Salmonella control Vertical Transmission
Elite/Great Grand parent farm
Grand parent farm
Layer/Broiler breeder farm
Hatchery
Layer or Broiler farm
Processing plant / Wholesalers

57. FAQ: Are there advantages in using 3x Salmovac 440 instead of 2x live vaccine + 1x inactivated?

58. Comparative study: 3x live vaccine – 2x live, 1x inactivated

59. Comparative study: 3x live vaccine – 2x live, 1x inactivated
 Higher level of protection against Salmonella Enteritidis

60. Comparative study: 3x live vaccine – 2x live, 1x inactivated
 At least same level of protection against Salmonella Typhimurium

61. FAQ: Are there advantages in using 3x Salmovac 440 instead of 2x live vaccine + 1x inactivated?
Cross protection against
different phage types of Salmonella Enteritidis (PT 1, 4, 8, 21)
Salmonella Typhimurium, also the monophasic strain
Salmonella Infantis
Salmonella Gallinarum: Reduced Mortality (week 20, week 32)
Long protection: S.E. 63 weeks, S.T. 60 weeks
Less stress: birds don’t have to be caught
Lower costs: employees, inactivated vaccine

62. Salmovac 440: Cross protection S. Gallinarum
S. Enteritidis and S. Gallinarum belong to same Salmonella group D
Better protection if 3x Salmovac 440
No official claim in the SPC

63. FAQ: Can the vaccine be sprayed on day old chicks?

64. FAQ: Can the vaccine be sprayed on day old chicks?
Yes!
Coarse spray application
Chicks are covered with vaccine, preen their feathers  “drinking” the vaccine
But: Disclaimer
Off-label-use (no registration)
Collected data is not yet published in a peer reviewed journal
No official promotion

65. Field Spra-Vac 10 l tank 40.000 chicks / hour
Intermittent or continuous spray
Rechargeable battery: 4 hours (continuous)

66. Field Spra-Vac 4 different nozzles
Coarse spray: droplet size > 100 µm
Do not use fine spray or aerosol
Pressure: 1,5 bar (20 psi)
Distance: about 50 cm

67. Field Spra-Vac
Dilution: 300 ml  1000 ID (+ 5% surplus) 1,5 l  5000 ID (+ 5% surplus)
1 spray dose = 15 ml 2 spray doses = 30 ml  100 chicks or 1 box

68. Field Spra-Vac 1.
Box with 100 day old chicks 2.

69. Field Spra-Vac Content of vaccinal strain in liver and caeca Liver
Comparative study:
24 chicks
Vaccination on 1st day
Investigation 8 days after vaccination
No significant differences

70. Field Spra-Vac
Easy and efficient administration of live vaccines to day old chicks (small hatcheries or on the farm)
Early vaccination (1st day) possible, although day old chicks do not drink much  Early onset of protection
Disclaimer remark

71. Impfpflicht und Subvention
Country
Compulsory
Subsidy
Breeder
Layer
Baltic countries No
Belgium
Yes (60% of total cost)
Bulgaria
Cyprus
SE; ST SE
Yes CZ
France
Germany
Some Federal States
Greece
Hungary
(2 doses)
Italy
Yes (if previous flock is +) NL
Poland
Portugal
Skandinavia
Prohibited
Spain
Yes (> 9%)
United Kingdom
(SE; ST)
Brazil
4,1%
10,6%
43,7%
12,3%
14,4%
5,3%

73. Thank you very much for your attention!