Jean Guard said Intergenic Sequence Ribotyping (ISR) is helping improve poultry production because it helps control Salmonella in the field and consumer poultry products.
ISR is being used to serotype strains within a particularly virulent group called Salmonella enterica, which is associated with foodborne illness.
There are about 175 sequences in the database, Guard told FoodQualityNews.
How ISR functions
ISR works by targeting one very small DNA region of the Salmonella genome, she said.
“The technician first isolates DNA from a culture suspected of being Salmonella, and then a polymerase chain reaction (PCR) is used to amplify the region using primers specific to the location. Then the amplified DNA is sequenced.
“The quality of sequencing must be high and we recommend getting results in both forward and reverse directions. Ambiguous base pairs, usually reported as “N” rather than as “A, T, C or G” must be removed from the ends.
“This should leave a sequence of about 600 letters. The trimmed sequence is then matched to the database for 100% similarity.”
Guard is with the USDA Agricultural Research Service (ARS) Egg Safety and Quality Research Unit at the Richard B. Russell Research Center in Athens, Georgia.
Traditional method comparison
The traditional method for serotyping Salmonella, called Kauffmann–White, (KW) is expensive and identifies a particular serotype in only 80% of cases, according to Guard.
“Most of the reasons have to do with the differences between the KW antisera-based test, which looks at the surface of the cell, and ISR, which targets DNA,” she said.
“It is important to note that ISR performed extremely similarly to DNA hybridization, which is also DNA based but which looks at multiple locations of the genome of Salmonella.
“Of the isolates tested, ISR was about 82.7% similar to KW but nearly 100% similar to DNA hybridization. A major problem with KW is that bacteria sometime stop producing surface antigens.
“DNA methods detect the genes regardless of their activity. ISR also detected mixtures of serotypes in culture, which was another reason it was more sensitive than KW.”
It takes about a week to process 48 isolates, but samples must be sent out for sequencing and it is possible to handle higher volumes, said Guard.
“DNA hybridization is faster, because no sequencing is required. However, DNA hybridization costs an average of $70 per isolate, whereas ISR costs $7. The two methods support each other,” she said.
“ISR is the screening method capable of handling lots of samples quickly while DNA hybridization is used for quality control and to double check sequences not yet in the database.
“There is no one perfect method, but two methods that work well together certainly improve accuracy. It has been our experience that other methods such as KW and other DNA approaches take at least a month.
“I have waited as long as three months for results and I have paid nearly $200 per isolate to get KW serotyping from private laboratories.”
Material Transfer Agreement
ISR is available to specialized laboratories, producers or other qualified users who sign a proprietary Material Transfer Agreement (MTA).
A producer’s lab technician can test a sample for Salmonella by culturing and if positive, submit it to a specialized lab—also an MTA holder—that uses the ISR tool for sequencing.
Producers and diagnostic consultants who hold an MTA can access private accounts to download sequences and compare to those in the ISR-based dataset.
The method is for use by anyone who wants to know if pathogenic Salmonella serotypes are present in their product, said Guard.
“ISR was not developed for use by regulators such as FSIS and FDA, because these groups already have whole-genome sequencing and other expensive, equipment-focused methods such as the gold standard Pulsed Field Gel Electrophoresis (PFGE) approved for use to meet their objectives,” she said.
“It was my goal to give the producers a tool so that they could do more testing on their own without waiting for regulators to come on farm.”
Guard said to control Salmonella requires effort from the farm, through the processing plant, to shipping product safely and then to safe preparation.
“I believe that most experts agree that it is important to keep pathogenic Salmonella out of the food supply,” she said.
“The method applies to any culture that is suspected of being Salmonella enterica subspecies I, which includes all the foodborne pathogens plus many others.
“Research to see if ISR could be applied to other pathogens is needed and is an area wide open for investigation.”