Methods for serological and PCR detection of Salmonella enteritidis in chickens.
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Date
2003
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Abstract
Salmonella enteritidis (S. enteritidis) is a bacterial pathogen of chickens, and is currently
one of the leading causes of human food poisoning in the world. It is believed that
contaminated poultry products, especially eggs and egg products, have been responsible
for the dramatic increase in the incidence of this Salmonella serotype. Detection of
S. entertidis has conventionally involved bacteriological examination of samples, yet these
procedures are time-consuming which could lead to the rapid spread of S. enteritidis
through commercial flocks and potentially cause a human health risk. A number of
alternative detection techniques, mostly based on serological methods, have been reported
as effective diagnostic assays. However, some of these reports have not been supported by
representations of SDS-PAGE gels or Western blots. The objective of this study was the
evaluation of these serological techniques as well as a PCR amplification technique, which
has been reported to show promising results as a diagnostic method. The techniques
discussed in these reports were evaluated with regards to how rapid they were, their
specificity and their potential for use in local diagnostic laboratories.
Antigens from the outer surface of S. enteritidis were purified by several methods and their
antigenicity was tested by separating the antigens by means of SDS-PAGE, followed by
Western blotting using sera of chickens infected with S. enteritidis. A high degree of cross
reactivity was observed with many of the antigens tested, especially the
lipopolysaccharides (LPSs) and outer membrane proteins (OMPs) which had previously
been reported as containing antigens which could be used for specific detection of
S. enteritidis. This cross-reactivity could be explained by the conserved nature of many of
the LPS and OMP antigens among the Salmonella serotypes tested. A fimbrial antigen,
SEF14, which has been reported as a novel antigen, was seen as a prominent band at 14.3
kDa and was found to react with antibodies against S. enteritidis, yet not to the specificity
levels described in previous reports.
PCR amplification of the sefA gene sequence, which encodes for the SEF14 fimbrial
antigen, was found to give a predicted product of 310 bp when using a previously
described oligonucleotide primer pair. This amplified product was found to be specific for
S. enteritidis and other serogroup D Salmonella serotypes that are not poultry pathogens The cross-reactivity observed with many of the serological techniques used in this study,
meant that detection of S. enteritidis infection in chickens was considerably hindered.
However, the identification of further novel antigens by serological means, could result in the development of new vaccines. The specificity and speed afforded by PCR amplification indicated that this technique showed excellent potential for use in local diagnostic laboratories.
Description
Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.
Keywords
Salmonella enteritidis., Chicken--Diseases., Poultry--Infections., Polymerase chain reaction--Methodology., Pathogenic bacteria., Theses--Biochemistry.