Detection and identification of salmonellas from poultry-related samples by PCR
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.
References (23)
Polymerase chain reaction in food microbiology
J. Microbiol. Meth.
(1995)- et al.
Diagnostic potential of sefA DNA probes to Salmonella enteritidis and certain other O-serogroup D1 Salmonella serovars
Mol. Cell. Probes
(1996) - et al.
Amplification of an invA sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella
Mol. Cell. Probes
(1992) - et al.
Influence of enrichment media and application of a PCR based method to detect Salmonella in poultry industry products and clinical samples
Vet. Microbiol.
(2001) - et al.
Characterization of monoclonal antibodies against a fimbrial structure of Salmonella enteritidis and certain other serogroup D salmonellae and their application as serotyping reagents
Res. Vet. Sci.
(1992) Rapid and alternative methods for the detection of salmonellas in foods: a review
J. Appl. Bacteriol.
(1993)- Calnek, B.W., 1997. Diseases of Poultry. Iowa State University Press, Ames, IA, pp....
- et al.
A rapid, sensitive and automated method for detection of Salmonella species in foods using AG-9600 AmpliSensor analyzer
J. Appl. Microbiol.
(1997) - et al.
Rapid detection of Salmonellae in poultry with the magnetic immuno-polymerase chain reaction
Appl. Environ. Microbiol.
(1993) - et al.
Molecular and functional characterization of the Salmonella invasion gene invA: homology of invA to members of a new protein family
J. Bacteriol.
(1992)
Prevalência de sorovares de Salmonella isolados de aves no Brasil
Pesq. Vet. Bras.
Cited by (153)
Antimicrobial tolerance, biofilm formation, and molecular characterization of Salmonella isolates from poultry processing equipment
2021, Journal of Applied Poultry ResearchThe effect of vegetation barriers at reducing the transmission of Salmonella and Escherichia coli from animal operations to fresh produce
2021, International Journal of Food MicrobiologyPet birds as potential reservoirs of virulent and antibiotic resistant zoonotic bacteria
2021, Comparative Immunology, Microbiology and Infectious DiseasesCitation Excerpt :Meanwhile, S. aureus isolates were identified by the amplification of the nuc gene [20], while methicillin-resistant S. aureus (MRSA) isolates were confirmed by the amplification of the mecA gene [21]. Salmonella spp. were identified via the invA primers specific for Salmonella spp., fliC gene for S. Typhimurium and sefA gene for S. Enteritidis [22]. Furthermore, Chlamydophila psittaci [23] and Pasteurella multocida [24] were identified by direct PCR of the collected samples.
How to rapidly and sensitively detect for Escherichia coli O157:H7 and Salmonella Typhimurium in cabbage using filtration, DNA concentration, and real-time PCR after short-term enrichment
2020, LWTCitation Excerpt :Various rapid detection methods have been developed, such as using agar to compensate for weaknesses of a long detection time in common microbial analysis methods. Numerous studies have been conducted on immunological methods (enzyme-linked immunosorbent assay: ELISA), biosensor-based methods (loop-mediated isothermal amplification: LAMP), and nucleic acid-based molecular biological methods (polymerase chain reaction: PCR) (Chen, Wang, Beaulieu, Stein, & Ge, 2011; Li et al., 2009; Oliveira et al., 2002). One of the most commonly used immunological methods is ELISA, which has the advantage of high selectivity and a labor-saving procedure due to its automation (Rennard, Berg, Martin, Foidart, & Robey, 1980).