​AMI members develop rapid test for bacterium that costs poultry industry billions globally

Scientists have developed a rapid, sensitive and specific test for a bacterial pathogen that is responsible for necrotic enteritis in poultry, a disease that causes billions in global economic losses annually.

The research, which resulted in the development of a loop-mediated isothermal amplification (LAMP) assay, was led by Deepa Chaudhary, a graduate student from the Department of Pathobiology at the School of Veterinary Medicine, Auburn University. She carried out the work as a graduate student at Mississippi State University.

The research, published in the Journal of Applied Poultry Research, was carried out under the supervision of advisor Dr. Li Zhang from the Department of Poultry Science, Mississippi State University and other colleagues from multiple institutions. Both scientists are members of Applied Microbiology International.

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Necrotic enteritis (NE) in poultry, caused by Clostridium perfringens, results in huge economic losses of approximately $6 billion per annum globally. 

Rapid detection method

“To address this issue, we developed a rapid detection method targeting the cpa gene, which is present in all C. perfringens strains and serves as a major virulence factor. The study addressed overcoming limitations of traditional culture-based and PCR methods, which are time-consuming and require specialized equipment,” said Ms Chaudhary.

“Additionally, the study addresses a technique suitable for on-site testing in poultry farms; and creating a cost-effective and user-friendly diagnostic tool and clear, easily interpretable results. By optimizing a LAMP assay for C. perfringens, we aimed to provide a rapid, sensitive, and field-deployable method to detect C. perfringens in poultry production.”

Two primer sets targeting the cpa gene were designed and optimized for temperature, time, and primer concentration. The assay was optimized to run at 63°C for 30 minutes, with primer concentrations of 8 μM for F3/B3 and 48 μM for FIP/BIP. The assay performed at 63°C for 30 minutes.

Performance of assay

The researchers evaluated the assay’s performance through inclusivity tests on 55 C. perfringens strains and exclusivity tests on other Clostridium and non-Clostridium bacteria, utilizing both standardized ATCC reference strains and DNA extracted from field samples. They also compared the sensitivity of the LAMP assay to PCR using serially diluted genomic DNA from C. perfringens ATCC 13124.  

The optimized CP-LAMP assay demonstrated high specificity and sensitivity comparable to PCR. It achieved 100% inclusivity for C. perfringens strains and high exclusivity for other bacteria. The assay’s optimal conditions were 63°C for 30 minutes, with specific primer concentrations.

These results suggest that the developed LAMP assay offers a rapid, sensitive, and specific method for C. perfringens detection, potentially enhancing surveillance and management of C. perfringens in poultry farms.

Surprising finding

One surprising finding was an unexpected cross-reactivity with Paeniclostridium sordellii (formerly Clostridium sordellii) in the LAMP assay designed for Clostridium perfringens detection. While the assay demonstrated 100% specificity against non-Clostridium strains, P. sordellii tested positive, resulting in a specificity of approximately 83.33% for other Clostridium species.

This surprising result occurred due to the close genetic relationship between P. sordellii and C. perfringens, illustrating the challenges in developing highly specific molecular diagnostic tools for closely related bacterial species.

The study addresses key challenges in detecting C. perfringens in poultry. It enables rapid, on-site detection without specialized equipment, offers early disease identification for timely intervention, provides high sensitivity and specificity to reduce false results, and presents a cost-effective screening method for routine use.

This matters in the real world because it can reduce economic losses from necrotic enteritis in poultry farms. It can also improve food safety in poultry production and boost overall poultry health and welfare by allowing faster diagnosis. Overall, this assay can lead to better poultry health management, food safety, and economic sustainability in the poultry industry.

Next steps

Ms Chaudhary said future research will focus on improving the specificity of the assay, including whole genome sequencing of P. sordelli to better understand its cross reactivity.

Another focus will be optimization of the CP-LAMP assay targeting the Net-B gene, which is responsible for causing necrotic enteritis in poultry.

“Field validation will be done to evaluate the performance of the CP-LAMP assay in an actual poultry farm. This should include using different types of samples (e.g., fecal matter, tissue samples, environmental swabs) and assessing the assay’s effectiveness across various farm environments and seasons,” Ms Chaudhary said.

“We can use the portable real time LAMP assay for the quantification of C. perfringens in field samples and focus on the simple and convenient methods of DNA extraction in the field that is suitable for LAMP assay applications.”

The study was led by Deepa Chaudhary as a graduate student at Mississippi State University, supervised by Dr. Li Zhang from the Department of Poultry Science at Mississippi State University, and involved colleagues from multiple institutions. Funding was provided by the National Institute of Food and Agriculture, U.S. Department of Agriculture, through Hatch project MIS-322430/NE2442, as well as the U.S. Department of Agriculture’s Agricultural Research Service, project numbers USDA-ARS NACA 58-6064-3-017 and 59-6064-4-004.