Food Safety

The Problems with Agglutination Testing for Salmonella Serovars

Kelly Cassutt, PhD April 16, 2019

Most likely, the majority would say that they rely on traditional serology, specifically antisera agglutination. After all, it is the preferred method of the USDA.

While it may be the preferred method of governing bodies, is it really the best methodology for your food safety lab? Probably not.

In this blog post, we’ll look at the biggest problems with agglutination testing for Salmonella serovars, and we’ll explore alternative methods. But first, a refresher on how agglutination works.

Agglutination occurs when the appropriate concentration of Salmonella, expressing serovar-specific antibody binding sites (antigens), is mixed with an appropriate concentration of antibodies against these antigens.

Agglutinating antibodies are made by injecting animals with specific parts of the cell wall or flagella of a specific Salmonella serovar. Serum containing antibodies against multiple Salmonella binding sites is Polyclonal Serum; antibodies against one binding site, taken from one antibody-producing cell, are Monoclonal Antibodies.

The agglutination test starts by using a wax pencil to inscribe two ovals on a microscope slide. From an agar plate growing Salmonella, a loopful containing multiple Salmonella colonies is collected and emulsified in 2 mL of physiological saline.

To the top of each wax oval is added a drop of the Salmonella emulsion. To the bottom of one oval is added one drop of saline only; to the other oval is added a drop of Salmonella anti-serum only. For each oval the drops are mixed with a sterile needle and the slide is rocked back and forth for ten seconds.

By eye, the technician looks for agglutination and records results, one of the following possibilities:

Both negative: True negative
Both positive: Non-specific (Invalid test)
Serum positive, saline negative: True positive

The test is time sensitive and any observed agglutination is deemed positive.

Each serovar is described by the unique expression of cell wall and flagellar antigens. For example, Salmonella Enteritidis is 1,9,12:g,m:-, while Salmonella Infantis is 6,7:r:1,5.

To identify a specific serovar, a flowchart outlining serovars is executed, starting with a pool of anti-serum against multiple antigens and with positive results, individual antibody classes are retested. For the flagellar antigens, if one “phase” of Flagella antigen is present, a Salmonella colony is collected from the original agar plate and regrown on agar containing flagellar antibodies. The antibodies induce expression of the second class of flagellar antigens.

As you can see, the identification of a given serovar requires multiple rounds of agglutination assays along with colony incubation and re-incubation times. As a result, the agglutination assay is not conducive to:

1. Scale and automation – Agglutination is labor-intensive. Can you imagine a technician trying to do 300 tests at once? Furthermore, the process cannot be automated. It requires an experienced technician to oversee the entire workflow–from the identification of the Salmonella colonies to the interpretation of the results.

2. Fast turnaround times – Traditional serology cannot be performed directly from an enriched sample. It requires securing a pure isolate and that can take an additional five days after the initial screening test. That’s a long time to wait. As a result, some food manufacturers wait to perform serology so that they can batch their samples together.

3. Objectivity – Traditional serology is quite subjective. At the beginning of the process, the technicians must collect what looks like Salmonella colonies, and at the end of the process, they must eyeball the agglutination results.

Imagine that the initial screening indicates a positive result for Salmonella. Then, the sample goes through traditional serology, during which the technicians choose a collection of Citrobacter colonies instead of a collection of Salmonella colonies. Now, the technicians have a positive screening result for Salmonella and a non-specific serology result for Citrobacter. What do they do?

4. Mixed populations of serovars – Traditional serology can identify only one serovar at a time. However, oftentimes samples contain multiple serovars, meaning traditional serology gives food safety professionals either wrong serology results due to the mixed detection antigens or only a partial picture of their serovar ecology.

5. Maintaining the antibody stocks – It is challenging to maintain the antibody stocks which can lead to performance variation between different lots of the same serovar antibodies.

For close to a century, food safety professionals have recognized the importance of identifying Salmonella serovars, and the best test for serovar identification was the agglutination test.

In the 21st century, there’s a better way. It’s called next-generation sequencing (NGS). NGS platforms generate millions of sequences simultaneously, enabling greater resolution into the microbial ecology of food and environmental surfaces. In the last few years, advances in NGS technologies have revolutionized multiple food testing applications. While NGS used to be a costly endeavor, recent breakthroughs in read length and throughput have drastically reduced the costs related to identifying microbial diversity, including pathogens, in processed and unprocessed foods.

The use of NGS for microbial detection enables the generation of massive genetic information from multiple genes for more accurate pathogen detection. Having access to this amount of genetic information enables the user to interrogate the data from different angles and allows the customization of the test according to the need. When it comes to serotyping, NGS offers:

Fast turnaround times – Salmonella serotyping can be completed in about 24 hours for hundreds of samples in parallel. This includes multiple evidence from multiple genetic markers.
The highest accuracy – Instead of making subjective calls, NGS looks at multiple regions of the genome in order to correctly identify serotypes. NGS looks at the sequence variation within specific gene regions, not only presence/absence.
The identification of multiple serovars – Unlike traditional serology, NGS platforms detect multiple serovars at the same time, thus providing more complete information about your samples.
Flexible assays – With NGS, you can choose your level of specificity. Test for any combination of Salmonella species and available serotypes.
Automation – NGS protocols can be automated to test multiple food samples within the same day.
Ease of use and reproducibility – Automated NGS protocols eliminate the need for depending on the expertise of the technician to do the test and interpret the agglutination tests, which leads to more reliable, accurate results.