Source: UNIVERSITY OF ILLINOIS submitted to NRP
POLYMERASE CHAIN REACTION TECHNIQUES FOR DETECTION AND GENOTYPING OF SALMONELLA
Sponsoring Institution
Cooperating Schools of Veterinary Medicine
Project Status
COMPLETE
Funding Source
STATE
Reporting Frequency
Annual
Accession No.
0202132
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Jul 1, 2004
Project End Date
Jun 30, 2005
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA,IL 61801
Performing Department
VETERINARY PATHOBIOLOGY
Non Technical Summary
Using a sample of 509 Salmonella isolates from 17 swine production units in Illinois, PCR techniques for diagnosis (inv-A PCR) and genotyping (Rep-PCR) Salmonella will be evaluated with respect to their diagnostic accuracy and validity in detecting Salmonella and classifying its genetic variability. These studies will be compared with preliminary investigations to determine the generalizability of these diagnostic evaluation procedures.
Animal Health Component
75%
Research Effort Categories
Basic
25%
Applied
75%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
71235991100100%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
3599 - Swine, general/other;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
(1) A previous investigation conducted on 172 bacterial isolates from 3 Illinois swine farms [sample A] identified higher specificity (i.e., fewer false positives) in detecting Salmonella for the inv-A PCR than for the industry standard API-20E diagnostic test. The proposed study will determine the generalizability of these results by testing with the inv-A PCR 206 isolates collected from 11 additional farms [sample B], as well as 132 isolates collected from 3 other farms [sample C], identified by API-20E as Salmonella. The validity of inv-A as a Gold Standard will be evaluated by comparing the results of API-20E and inv-A PCR classification of isolates with serotyping. (2) A previous investigation conducted on 68 bacterial isolates from 9 Illinois swine farms [sub-sample from sample B] identified that repetitive sequence PCR (Rep-PCR) detected greater genetic diversity than pulsed field gel electrophoresis (PFGE), the Centers for Disease Control standard for Salmonella genotyping, in the classification of bacterial isolates identified by API-20E as Salmonella. The proposed study will determine the broader applicability of these results by conducting additional PFGE genotyping on 134 additional samples from these 10 farms [remainder of Sample B], 172 samples from the 3 farms [Sample A], and 131 samples from 3 farms [sample C], for which Rep-PCR has already been conducted. The validity of rep-PCR in differentiating Salmonella from other Enterobacteriacea will be assessed by testing these samples using inv-A PCR and serotyping.
Project Methods
There are 512 suspected Salmonella isolates (i.e., positive on API-20E) available for analysis that have been collected from 17 swine farms during 4 years in epidemiological investigations of Salmonella transmission. This study is designed to determine the generalizability of preliminary results on the accuracy of diagnostic tests and genotyping methods for Salmonella. A PCR targeted at the inv-A gene will be compared to the industry standard API-20E biochemical profiling system for Salmonella, in order to confirm preliminary analyses (172 samples from 3 farms) indicating a low 80% specificity (20% false positive rate) for API-20E. Serotyping will be used to reconcile differences in diagnosis between inv-A PCR and API-20E, with the hypothesis that inv-A negative samples are mostly non-typeable and thus confirm inv-A PCR as a preferable Gold Standard to API-20E for Salmonella diagnosis. Completed genotyping using repetitive sequence PCR (with BOX, ERIC, and REP primer sets) will be compared to the CDC standard PFGE (using the AvrII, Spe1, and Xba1 restriction enzymes) for genotyping, to confirm preliminary analysis (68 samples from 10 farms) indicating that Rep-PCR has high discriminative ability to detect genetic diversity. Genetic similarity between isolates will be determined by calculation of genetic distance based on similarity of DNA fragmentation patterns, which will then be used as input into a cluster analysis, using the complete linkage algorithm. Serotyping will be used to confirm the validity of Rep-PCR genetic classification by calculating the proportion of isolate clusters that are homogeneous for serotype, and comparing these results the tree structure produced for PFGE

Progress 07/01/04 to 06/30/05

Outputs
The commonly used Appareils et Procedes d'Identification 20 Enetrobacteriaceae (API 20E) test for identification of Salmonella enterica, uses biochemical traits for classification of bacterial isolates. Newer methods such as invA-PCR, targeting genetic traits not affected by variability in phenotypic expression, would be expected to provide more accurate diagnosis. In this study API 20E and invA-PCR were compared in their classification of Salmonella isolates, with validation of each method achieved by comparison to the gold standards of serotyping and 16S rRNA gene sequencing. Of 196 suspected Salmonella isolates from 3 swine farms, 174 were identified as S. enterica by API 20E at various likelihood levels and 125 by invA-PCR. All API 20E positive isolates were serotyped and sequenced. The highest agreement of API 20E with other tests was at the 99.9% likelihood level (API-99), with 99.2% of these isolates classified as Salmonella by invA-PCR, 98.4% by 16S rRNA sequencing, and 97.6% by serotyping. At lower likelihood levels, API 20E appeared to falsely classify samples as positive. The sensitivity and specificity of both API-99 and invA-PCR were 100% and 96%, respectively, when compared to 16S rRNA sequencing. Compared to serotyping, both API-99 and invA-PCR had 96% sensitivity; specificity was 86% for API-99 and 79% for invA-PCR. The high agreement of API-99 and invA-PCR with each other and with 16S rRNA sequencing, and the lower agreement of all these tests with serotyping indicate that both API 20E and invA-PCR are accurate methods for S. enterica identification, and suggest that serotyping may not be a reliable gold standard. API 20E may be a cost effective method for Salmonella identification in smaller laboratories with low sample submission rates. However, for larger well-equipped laboratories with high submission rates, invA-PCR is more efficient and economical.

Impacts
Validation of both the invA-PCR and API 20E (at the 99.9% likelihood level) as accurate diagnostic tests suggests that either can be used with similar results in a diagnostic laboratory. The selection of the invA-PCR versus API 20E depends in part upon cost considerations. The API 20E kit is relatively expensive, about $6 per sample at this time, compared to less than $2 current cost in laboratory materials per invA-PCR test. Thus, invA-PCR is a cost effective alternative to API 20E.

Publications

  • No publications reported this period