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Loop-mediated isothermal amplification: rapid molecular detection of virulence genes associated with avian pathogenic Escherichia coli in poultry.

ABSTRACT: Infections with pathogenic Escherichia coli can lead to different animal- and human-associated diseases. E. coli infections are common in intensive poultry farming, and important economic losses can be expected during infections with avian pathogenic E. coli (APEC) strains followed by colibacillosis. Loop-mediated isothermal amplification (LAMP) assays were developed for rapid detection of 3 APEC-associated virulence genes: sitA, traT, and ompT. All 3 LAMP assays are shown to be specific, repeatable, and reproducible. High sensitivities of the assays are shown, where as few as 1,000 bacterial cells/mL can be detected in different matrices. On-site applicability of this LAMP method is demonstrated through testing of different sample types, from animal swabs and tissues, and from environmental samples collected from 6 commercial poultry farms. All 3 virulence genes were detected at high rates (above 85%) in samples from layer and broiler chickens with clinical signs and, interestingly, high prevalence of those genes was detected also in samples collected from clinically healthy broiler flock (above 75%) while lower prevalence was observed in remaining 3 clinically healthy chicken flocks (less than 75%). Importantly, these virulence genes were detected in almost all of the air samples from 11 randomly selected poultry houses, indicating air as an important route of E. coli spread. Three LAMP assays that target APEC-associated virulence genes are shown to be sensitive and robust and are therefore applicable for rapid on-site testing of various sample types, from animal swabs to air. This on-site LAMP testing protocol offers rapid diagnostics, with results obtained in <35 min, and it can be applied to other important microorganisms to allow the required prompt measures to be taken.

SUBMITTER: Kogovsek P

PROVIDER: S-EPMC6377436 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Loop-mediated isothermal amplification: rapid molecular detection of virulence genes associated with avian pathogenic Escherichia coli in poultry.

Kogovšek Polona P Ambrožič-Avguštin Jerneja J Dovč Alenka A Dreo Tanja T Hristov Hristo H Krapež Uroš U Ravnikar Maja M Slavec Brigita B Lotrič Marjetka M Žel Jana J Zorman Rojs Olga O

Poultry science 20190301 3

Infections with pathogenic Escherichia coli can lead to different animal- and human-associated diseases. E. coli infections are common in intensive poultry farming, and important economic losses can be expected during infections with avian pathogenic E. coli (APEC) strains followed by colibacillosis. Loop-mediated isothermal amplification (LAMP) assays were developed for rapid detection of 3 APEC-associated virulence genes: sitA, traT, and ompT. All 3 LAMP assays are shown to be specific, repeat ...[more]

PMID: 30476321

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Project description:Salmonella enterica, a leading cause of food-borne gastroenteritis worldwide, may be found in any raw food of animal, vegetable, or fruit origin. Salmonella serovars differ in distribution, virulence, and host specificity. Salmonella enterica serovar Kentucky, though often found in the food supply, is less commonly isolated from ill humans. The multidrug-resistant isolate S. Kentucky CVM29188, isolated from a chicken breast sample in 2003, contains three plasmids (146,811 bp, 101,461 bp, and 46,121 bp), two of which carry resistance determinants (pCVM29188_146 [strAB and tetRA] and pCVM29188_101 [bla(CMY-2) and sugE]). Both resistance plasmids were transferable by conjugation, alone or in combination, to S. Kentucky, Salmonella enterica serovar Newport, and Escherichia coli recipients. pCVM29188_146 shares a highly conserved plasmid backbone of 106 kb (>90% nucleotide identity) with two virulence plasmids from avian pathogenic Escherichia coli strains (pAPEC-O1-ColBM and pAPEC-O2-ColV). Shared avian pathogenic E. coli (APEC) virulence factors include iutA iucABCD, sitABCD, etsABC, iss, and iroBCDEN. PCR analyses of recent (1997 to 2005) S. Kentucky isolates from food animal, retail meat, and human sources revealed that 172 (60%) contained similar APEC-like plasmid backbones. Notably, though rare in human- and cattle-derived isolates, this plasmid backbone was found at a high frequency (50 to 100%) among S. Kentucky isolates from chickens within the same time span. Ninety-four percent of the APEC-positive isolates showed resistance to tetracycline and streptomycin. Together, our findings of a resistance-conferring APEC virulence plasmid in a poultry-derived S. Kentucky isolate and of similar resistance/virulence plasmids in most recent S. Kentucky isolates from chickens and, to lesser degree, from humans and cattle highlight the need for additional research in order to examine the prevalence and spread of combined virulence and resistance plasmids in bacteria in agricultural, environmental, and clinical settings.

Dataset Information

Loop-mediated isothermal amplification: rapid molecular detection of virulence genes associated with avian pathogenic Escherichia coli in poultry.

Publications

Loop-mediated isothermal amplification: rapid molecular detection of virulence genes associated with avian pathogenic Escherichia coli in poultry.

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