Salmonella enterica serovar Enteritidis one of the main cause of food borne disease transmitted by eggs or eggcontaining

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2 Salmonella enterica serovar Enteritidis one of the main cause of food borne disease transmitted by eggs or eggcontaining products Egg contamination by both horizontal and vertical transmission Even though the proportion of positive eggs is low both in experimental and natural conditions

3 To investigate the incidence of Salmonella Enteritidis (SE) on egg shells and in egg contents laid by experimentally infected laying hens during the period of 40 days To detect the lenght of period between experimental SE inoculation and egg contamination To compare standard bacteriological method (ISO) and molecular method (Real-Time PCR)

4 TRIAL 1 40 Hyssex brown laying hens TRIAL 2 40 Lohmann brown laying hens 18 weeks old Not immunized against Salmonella Group A Group B Number of the Salmonella enterica subsp. enterica serovar animals per group Enteritidis 1.2 x 10 9 / p.o. 1 ml LENGHT OF BOTH TRIALS 40 DAYS

5 TRIALS 1 and 2 Animals kept in cages (0.5 x 0.5 m) in separated rooms Fed ad libitum with commercial feed for laying hens Housed under standard temperature/light conditions for commercial laying hens BEFORE INOCULATION: Aclimatization for 7 days in experimental environment Fecal samples and cloacal swabs taken to determine the health status and possible previous contamination with Salmonella Birds examined clinically on daily basis

6 EXPERIMENTAL INOCULATION o Single oral dose of 1.2 X 10 9 CFU Salmonella enterica subsp. enterica serovar Enteritidis (SE) o Farm strain, fagotipe 7, isolated from the liver of laying hen during an outbreak in Croatia o Having all genes encoding pathogenicity islands SPI-1 through SPI-5 (Federal Institute for Risk Assessment, Berlin). o Inoculated with the blunt metal probe, directly into the crop of each animal

7 TRIALS 1 AND 2 Experimental group every single egg during 40 days Egg shell and egg content of each egg were examined separately Control group pooled samples (one day - one sample) during 40 days Egg shells and egg contents were examined separately TRIAL 1 SE on egg shells and in egg contens was determined by: HR EN ISO 5679:2003 method Real-Time PCR method TRIAL 2 SE on egg shells and in egg contens were determined by: HR EN ISO 5679:2003 method

8 The samples of the egg shells were taken with the sterile swabs After the egg shells samples were taken, intact shells were cleaned with 70 % etanol and cracked with sterile instrument for sampling of the contents Both samples were then proceed as described by HR EN ISO 6579:2003

9 METHODS (Molecular Real time PCR) For Real-Time PCR detection, both samples in Buffered Peptone water were frozen after 24 hours of incubation (after first step of ISO method) After DNA isolation the following primers and probes were used: Primers 5-3 pmol/µl Forward primer CTC ACC AGG AGA TTA CAA CAT GG 10 Reverse primer AGC TCA GAC CAA AAG TGA CCA TC 10 Probes 5-3 pmol/µl Salmonella LNA (6FAM) CG+ACGGCG+AG+ACCG (BHQ1) 10 (Target probe) IAC probe (JOE) CAC ACG GCG ACG CGA ACG CTT (BHQ1) 10 THERMAL PROFILE: 1 cycle: 95 0 C for 3 minutes 40 cycles: 95 0 C for 30 s, 65 0 C for 60 s (annealing step), 72 0 C for 30 s Described by National Food Institute Technical University of Denmark, Malorney et al., 2004, and Kramer et al., 2011.

10 Days post-inoculation with at least one positive finding Trial 1 ISO method Trial 1 Real-Time PCR Trial 2 ISO method Egg shell Egg content Egg shell Egg content Egg shell Egg content Total* 8/262 0/262 7/262 0/262 0/258 1/258 (3.0%) (0,0%) (2.7%) (0.0%) (0.0%) (0.4%) *Number of positive/total number of eggs TRIAL 1: SE was found on: 6 egg shells on Day 10 One egg shell on Days 11 and 14 Altogether 8 egg shells of a total of 262 laid eggs TRIAL 2: SE was found in: One egg content on Day 7 of a total of 258 laid eggs All eggs from control groups were negative

11 (Molecular Real time PCR) Seven out of eight egg shells positive by ISO method were also positive by Real-Time PCR One sample collected on Day 14 positive by ISO method was negative by Real-Time PCR, probably due to the presence of inhibitors that may negatively affect the isolation of bacterial DNA All samples negative by ISO method were also negative by Real-Time PCR PROBE RESULT Ct VALUE positive 36 LNA negative > 40 IAC positive negative < 40

12 In two equal trials carried out with two different commercial lines of laying hens inoculated orally by highly pathogenic Salmonella Enteritidis (SE), SE was found both on egg shells and in egg content Positive egg shells in Trial 1 and positive content in Trial 2 suggest that eggs can be contaminated by both horizontal and vertical SE transmission Low proportion of positive eggs in both trials is in line with already published findings. The time period between SE inoculation and the first egg contamination seems to be the same in both trials (10 vs.7 days) Positive eggs were laid only in a short period of time, from day 7 to day 14 post infection Almost identical results were obtained by both ISO and molecular methods

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