Detection and identification of salmonellas from poultry-related samples by PCR

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Abstract

A polymerase chain reaction (PCR) assay was developed for the generic detection of Salmonella sp. and the identification of S. Enteritidis (SE), S. Gallinarum (SG), S. Pullorum (SP) and S. Typhimurium (ST) in material collected in the field from poultry. The specificity and sensitivity of the assay combined with Rappaport–Vassiliadis selective enrichment broth (PCR–RV) were determined, and field samples were analyzed to verify the validity of the method application. Specificity of the assay was tested using 29 SE, 11 SG, 10 ST and 10 SP strains, along with 75 strains of 28 other Salmonella serovars and 21 strains of other bacterial genera. The assay was 100% specific for Salmonella detection and ST identification. The primer pair for SE, SG and SP also detected S. Berta. PCR detection limits for Salmonella at the genus level were 2 ST, 8 SE, 1.1×103 SG and 1.8×105 SP cells. At the serovar level, detection limits were 7 ST, 1.2×103 SE, 4.4×107 SG and 1.8×106 SP cells. At the genus level, PCR–RV detected ≈128% more positive field samples than the standard microbiological techniques and results were ready in 48 h instead of 7 days. PCR–RV method is diagnostic of Salmonella at the genus level and ST at the serovar level, although other tests are needed to identify SE, SG and SP to serovar level.

Introduction

The primary motivation for controlling Salmonella infections in poultry has been to reduce disease losses, and this has lead to the implementation of extensive testing programs. Since poultry is one of the most important reservoirs of salmonellae that can be transmitted to humans through the food-chain, public health concerns have increasingly made the prevention of the foodborne transmission of disease to humans an urgent priority for poultry producers (Calnek, 1997).

Salmonellosis in chickens can be classified into three diseases: pullorum disease caused by Salmonella Pullorum, fowl typhoid caused by S. Gallinarum and paratyphoid infections due to a diverse group of serovars related to foodborne illness in humans. Salmonella Typhimurium and more recently S. Enteritidis have been the serovars most frequently isolated from cases of human food poisoning in which chicken products have been implicated.

Since the 1940s, there has been a rapid increase in the isolation of the non-host-specific Salmonella serovars from humans and animals (Poppe, 1999), and these serovars continue to cause significant disease losses in young poultry (Calnek, 1997). Worldwide, paratyphoid salmonellas are the most common agents of foodborne illness, and according to a WHO surveillance report, American, European and African health agencies have notified similar increases in such illnesses related to the consumption of eggs and poultry (Rodrigue et al., 1990).

The current standard laboratory procedure to culture and identify Salmonella serovars is laborious and can last up to 7 days. The polymerase chain reaction (PCR) represents a major advance in terms of the speed, sensitivity and specificity of diagnostic methods, and has been increasingly used to identify several bacterial species from food and clinical samples (Stone et al., 1994). Another advantage is that PCR is not dependent on utilization of a substrate or the expression of antigens, thereby circumventing the phenotypic variations in biochemical patterns and lack of detectable antigens (Hoorfar et al., 1999). During evolution, Salmonella serovars acquired many genes by horizontal transfer of plasmids or phages (Riley and Krawiec, 1987). Some of these genes now characterize serovars and are thus obvious candidates for the development of methods based on DNA analyses.

Non-selective and/or selective enrichment combined with PCR have been applied to the detection of many bacterial pathogens (Candrian, 1995, Schrank et al., 2001) to improve sensitivity and dilute PCR-inhibitory substances (Fluit et al., 1993).

The objective of the present work was to establish a specific, sensitive and rapid PCR protocol for the detection of Salmonella at the genus level and also for the identification of S. Typhimurium, S. Enteritidis, S. Gallinarum and S. Pullorum. The PCR protocol was tested in conjunction with selective enrichment in Rappaport–Vassiliadis broth (PCR–RV) and compared with standard microbiological techniques (SMTs) using field samples of poultry materials.

Section snippets

Bacterial strains and culture conditions

PCR specificity was determined using 156 strains comprising: 29 S. Enteritidis, 11 S. Gallinarum, 10 S. Pullorum, 10 S. Typhimurium, 75 strains belonging to 28 other Salmonella serovars and 21 strains of 16 non-Salmonella (Table 1). Salmonella (135 strains) and non-Salmonella (21 strains) were isolated at the Center of Diagnosis and Research in Avian Pathology (CDPA, UFRGS) from poultry derived materials or obtained from reference laboratories. Strains were kept at 4 °C in stock agar and were

Optimization of the assay

In a first trial, it was not possible to use the three primer pairs in one multiplex PCR; so identical conditions for the three primer pairs were tested to simplify the protocol. Magnesium chloride was used at 2.5 mM because this concentration gave the best sefA primers amplification efficiency and no significant changes in the sensitivity of the other two primer pairs.

Two methods of DNA preparation were compared: heat-induced bacterial lysis (which resulted in low sensitivity and some

Discussion

In the PCR assay for detection of Salmonella at the genus level, all 135 Salmonella strains gave positive results in the specificity test with the invA primer pair, while none of the strains of 13 other genera were positive. These results agree with the work of Rahn et al. (1992) except that these authors only detected nine out of 11 S. Senftenberg strains, while we detected all 13 tested strains of this serovar: a difference which could be due to the different amplification conditions or

Acknowledgements

We thank Cenbiot Enzimas from Centro de Biotecnologia da UFRGS for kindly providing the Taq DNA polymerase and D.P. Rodrigues from Fundação Oswaldo Cruz for providing Salmonella Dublin and S. Berta. S.D. Oliveira received a scholarship from Conselho Nacional de Pesquisa (CNPq). Financial support was provided by Fundação de Amparo a Pesquisa do Estado do Rio Grande do Sul (FAPERGS) and CNPq.

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