New Test Validation for Detection of S. Enteritidis in Poultry

Transcription

1 New Test Validation for Detection of S. Enteritidis in Poultry Kim Lam Chiok Salmonella and public health S. Enteritidis is associated with eggs due to vertical transmission; although around 2000 serotypes can cause human disease. About 2.2 billion eggs per year contain (CDC, 2013), 1 in 250 broiler chickens are also contaminated (USDA- FSIS, 2010). Ongoing outbreak: S. Heidelberg in chicken carcass 1.4 million cases/year? 400 fatal cases/year (CDC, 2013) 1

2 Salmonella and public health Salmonella incidence has not changed despite interventions What is the situa/on in poultry? Prevalence of MPPSTs in retail chicken meat from (NARMS, 2011) Non- serotypes are on the rise in poultry, How many of these can be transmitted through eggs? 2

3 Egg safety rule (July, 2010) Purpose: Reduce incidence of at farm level. What about non? How many tests are necessary under the rule? Environment tested at: pre-production (14-16 w), mid-production(40-45 w) and post-molt (4-6 w) 3

4 How do we identify Salmonella? Day 0 NPIP (2010) - equivalent to BAM for environmental samples - Sample Day 1 TT (1:10)/MSRV for 24±2h at 37 C Screening Day 2 XLT4 and BGN agar at 37 C for 24±2h Screening Day 3 Biochemistry and/or serogroup Confirmation Day 5-30 Serotype Disadvantages: time consuming, serotyping available only at a few laboratories, single targeted, not high throughput. Assays approved by FDA for detection of Romer RapidChek LECT Salmonella Applied Biosystems TaqMan Salmonella Enteritidis Neogen Reveal Salmonella Enteritidis BAX System PCR Assay: 10 4 CFU/ml in enriched samples 4

5 Assays approved by FDA for detection of Targets? One, or Salmonella only Similar timing:24 to 72 hours Enrichment required? YES Specificity and sensitivity: Similar Serotyping required? YES Can differentiate from vaccine antigens? NO Multiplex real time qpcr 5

6 Multiplex qpcr assay: Amplification profiles 500bp 400bp 300bp 200bp 250bp inva 215bp prot6e 146bp sdfi 100bp M ST PT4 How sensitive is the test? No. of Salmonella CFUs/PCR inva (Tx Red) prot6e (FAM) sdfi (HEX) ± ± ± ± ± ± NA NA NA Very sensitive: Detects at least 10 CFUs in pure culture 6

7 How specific is the test? Serotype inva + sdfi + prot6e + (n=36) Enteritidis Most prevalent Non- serotypes in (n=37) poultry Non C. jejuni Salmonella (n=12) E. coli APEC N 100% specific, able to discriminate from Non- and non-salmonella Does it work on environmental samples? Gauze pads soaked with milk Spiked with and non strains Enrichment DNA Extraction Sample prep/qpcr 96 well plate format Total time: 30 hours 7

8 Does it work on environmental samples? non-plasmid Non- CFU/ml 1 to Very sensitive: as little as 1 CFU in spiked environmental samples Does it work on clinical samples? Drag swabs from poultry house Un-spiked Spiked with Culture Enrichment DNA Extraction qpcr/ sample prep 96 well plate format Total time: hours 8

9 Does it work on clinical samples? Culture Status N inva + sdf + prot6e + inva + sdfi + prot6e + Sal sp. + + non spiked spiked Specificity: 100% Sensitivity: 100% Limit of detection: at least 4 CFU/sample Conclusions Developed assay is highly sensitive and specific for identification of. Saves time, effort and money: (+) samples are further investigated. Less environmental impact. Other serotypes can be detected, allowing for speedy serotyping = surveillance tool Whole process takes hours. Requires testing in different sample types and matrices for validation-> EGGS, carcass. Further research: likelihood of + isolation depending on numerical values. 9

10 What is Salmonella? 1.4 million cases/year? 400 fatal cases/year (CDC, 2013) 10

11 Challenges in Salmonella detection Foley et al., Salmonella challenges: Prevalence in swine and poultry and potential pathogenicity of such isolates. J Anim Sci, 86: E149-E162. Table 1: Salmonella serotypes isolated from poultry Group Serotypes D Enteritidis Gallinarum Pullorum B C E Typhimurium Heidelberg Agona Saint-paul Indiana Hadar Virchow Bovis morbificans Muenchen Newport Montevideo Kentucky Anatum Senftenberg Orion Binza Weltervreden Stanley Paratyphi B java Bredeney Chester Infantis Blockley Bareilly Livingstone Thompson Mbandaka Lille 11

12 Challenges in Salmonella detection Foley et al., Salmonella challenges: Prevalence in swine and poultry and potential pathogenicity of such isolates. J Anim Sci, 86: E149-E162. Egg safety rule (July, 2010) 1. Purpose: Reduce incidence of at farm level. 2. Actions: Birds only from suppliers who monitor for Salmonella Pest control and biosecurity measures Conduct testing in the poultry house and eggs for Clean and disinfect poultry houses that have tested positive Refrigerate eggs at 45 F during storage and transportation no later than 36 hours after eggs are laid. 12

13 Nucleic acid based assays for detection of Salmonella Source: Bacteriological Analytical Manual, Chapter 5, Salmonella. December 2007 Edition. Past 10 years: high number of new nucleic acid based assays Nucleic acid based assays for detection of Salmonella Gene Target Function Specificity Format Matrix Reference hila invasion protein regulator of SPI-I Salmonella spp end-point, singleplex Culture Miras et al., 1995 inva needle complex type III secretion system Salmonella spp end-point, singleplex food samples Cheng et al., 2008 sdfi chromosomally encoded real time, singleplex mice tissue Deng et al, 2008 sdfi chromosomally encoded end-point, singleplex Culture Deng et al, 2009 sefa Fimbrial antigen F14 serogroup D end-point, singleplex pooled shell eggs Day et al., 2009 sefa Fimbrial antigen F14 serogroup D real time, singleplex pooled shell eggs Seo et al., 2004 orgc located in spii typhimurium, end-point, singleplex pooled shell eggs Day et al., 2010 inva needle complex type III secretion system Salmonella spp hila invasion protein regulator of SPI-I Salmonella spp sefa Fimbrial antigen F14 serogroup D sdfi chromosomally encoded in inva needle complex type III secretion system Salmonella spp prot6e 60kb virulence plasmid acek flux of isocitrate through ATC Salmonella spp sefa Fimbrial antigen F14 serogroup D flic phase 1 flagellin (H1antigen) typhimurium, kentucky sdfi chromosomally encoded inva needle complex type III secretion system Salmonella spp flic phase 1 flagellin (H1antigen) typhimurium, kentucky prot6e 60kb virulence plasmid end-point, multiplex real time, multiplex real time, multiplex real time, multiplex human stool samples pooled shell eggs, carcass rinse chicken skin and meat Culture Trafny et al., 2006 Malorny et al., 2007 O' Regan et al., 2008 Hadjimicolaou et al.,

14 Table 1: Oligonucleotide primers for the serotype-specific detection of Target sdfi prot6e inva Conditions: Development and Standardization of Taqman qpcr Forward Primer (5-3 ) CATCGTAAGTACCAGA AGA GGTCAAACTGAACGTT TG GTGTCCTTTGGTATTA ATCC Reverse Primer (5-3 ) CGTCAGTCACTATTTG AAC CCCAAATTATTATTACC ATAAGC GTCTGAGCACTTCTTT AAG 50 ng of template DNA 200 nm inva 400 nm sdf 500 nm of prot6e 5 mm MgCl 2 bp Probe (5-3 ) HEX*CCTCTCAACACTGATGC GGC*BHQ 6- FAM*CCAGCACGAGACGACA TTCTTG*BHQ Texas Red*ATCCATCAGCAAGGTAG CAGTCA*BHQ 2. Cycling conditions: 50 C/2 min 95 C/3 min 95 C/10s, 35 cycles 54 C/10s 72 C/30s Taqman qpcr Sensitivity levels 3 strains TTB 37 C for 24h Total time aprox.: 30 hours Dneasy tissue kit Boiling for 10 min Bio Rad CFX manager qpcr set up 10ng/µl 14

15 Taqman qpcr: Diagnostic ability in field samples + + TTB 25ml Overnight shipment 12ml uninoculated 12ml: 4-8 CFU Total time aprox.: hours 130ml TTB - 37 C x 24h Bio Rad CFX manager qpcr set up 10ng/µl DNA extraction However Latest outbreak: Salmonella Heidelberg from chicken carcass is multidrug-resistant (CDC, 2013). Number of cases: 362 (CDC, Oct 30) 15