Validation of a seminested PCR approach for rapid detection of Salmonella enterica subsp. enterica serovar Gallinarum

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Abstract

Salmonella enterica subsp. enterica serovar Gallinarum (S. Gallinarum) is the causative agent of fowl typhoid, one of the major causes of mortality and morbidity on poultry farms. Even though it has been substantially eradicated in many developed countries, the disease still remains endemic in Central and South America, in Africa and in the Mediterranean countries of Europe. This leads to the routine screening of flocks, mainly by cultivation and serological techniques, which are expensive, as well as time and labour-consuming. Here we describe a simple and specific PCR-based method for detecting S. Gallinarum. It relies on two seminested PCRs which use four pairs of primers designed on the basis of two genomic regions which appear to be exclusive to the pathogen. Furthermore, an internal positive control was devised in order to avoid any false negative results. We performed sensitivity and specificity tests, and our findings showed the cogency of the system and its potential effectiveness even for routine uses.

Introduction

Salmonella enterica subsp. enterica serovar Gallinarum (S. Gallinarum) is a non-motile and highly host-adapted salmonella which is the causative agent of fowl typhoid (Shivaprasad, 2003). This disease is one of the most important causes of poultry mortality, leading to considerable economic losses, so that many countries have adopted rigid control policies. To date, fowl typhoid has been substantially eradicated from North America and Australia. Nonetheless, the disease still remains endemic in many countries in Central and South America, Africa and Asia. Most European countries are considered fowl typhoid free, although the disease is still widespread in some regions, particularly those along Mediterranean coasts (Shah et al., 2005). Hence, to prevent the reintroduction of the disease, flocks are routinely screened for the presence of S. Gallinarum even in countries where fowl typhoid has been eradicated and this represents a major cost (Shivaprasad, 2003). Fowl typhoid is included by the World Organisation for Animal Health (OIE) among the notifiable avian diseases (OIE, 2010a). The Organisation also recommends specific measures for the prevention and the treatment of the disease, (OIE, 2010b) also requiring differential diagnostic procedures for motile and non-motile Salmonella. OIE also reports that molecular methods for detection of S. enterica ssp. enterica serovars Enteritidis (S. Enteritidis) and Typhimurium (S. Typhimurium) have been developed (OIE, 2010c).

By contrast, detection of S. Gallinarum mainly relies on cultural and serological tests, but they are both time- and labour-consuming since results are only available after 3–6 days (Jeon et al., 2007), even though the standard ISO 6579:2002 procedure takes at least 4–5 days, while the DIN EN 12824:1998 requires at least two additional days (Shönenbrücher et al., 2005).

Different molecular assays for detection of S. Gallinarum have been developed (Jeon et al., 2007, Kisiela et al., 2005, O'Reagan et al., 2008), but most of them either require expensive instruments or they sometimes lack high specificity.

In the light of this, the present study aimed to devise a molecular method for the detection of S. Gallinarum which could be suitable for routine field use. The sequencing of the S. Gallinarum chromosome (Thomson et al., 2008) allowed us to disclose potentially distinctive genomic regions and, consequently, to set up two seminested PCRs enabling the detection of S. Gallinarum in a few steps.

Section snippets

Bacterial strains

Thirty-five S. Gallinarum isolates, thirty-eight Salmonella non-Gallinarum isolates and twenty-five non-Salmonella strains were used in the specificity tests. Salmonella strains were mostly collected from hens in Southern Italy, identified by biochemical assays using API 20E strips (bioMérieux, Marcy l'Étoile, France) and serotyped according to the Kauffmann–Whyte Typing scheme (Popoff et al., 2005). Part of the non-Salmonella strains came from reference collections, while the others were

Genome comparison, primer design and PCR conditions

In order to discover distinctive regions of S. Gallinarum genome, we first compared the genomes of S. Gallinarum strain 287/91, S. Enteritidis strain P125109 and S. Typhimurium strain LT2. We selected S. Enteritidis and S. Typhimurium since these serovars are among those mostly recovered from chickens. Furthermore, their chromosomes were completely sequenced and they are available in GenBank. The comparison results led us to identify 66 large regions (exceeding 1000 bp) which overlaid S.

Conclusion

In summary, the approach described in this study may represent a suitable alternative to cultivation-based methods for detection of S. Gallinarum, since it has proved to be precise and accurate, other than rapid and affordable. Therefore it may be adopted for routine detection of S. Gallinarum directly from animal samples. The addition of an IPC to each reaction warrants the accuracy of negative results, avoiding false conclusions. Its feasibility for large-scale analysis should also make it

Acknowledgments

We thank Bayer Animal Health for financial support and Anna Caroli for excellent laboratory assistance. We would also like to thank Anthony Green for kindly reviewing the English in the manuscript.

References (23)

  • D. Kisiela et al.

    Differentiation of Salmonella Gallinarum biovar Gallinarum from Salmonella Gallinarum, biovar Pullorum by PCR-RFLP of the fimH gene

    J. Vet. Med. B Infect. Dis. Vet. Public Health

    (2005)
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