Necrotic Enteritis Research: It’s in the details

Necrotic enteritis (NE) is a devastating poultry disease that can strike within hours, leaving young birds with irreversible lesions causing death or poor growth. It’s a significant threat that Canadian broiler farmers manage with preventative antibiotics, but as the industry transitions away from this tool, new strategies are needed.

NE occurs when a pathogenic strain of the naturally occurring bacteria Clostridium perfringens teams up with specific farm conditions. Dr. Martine Boulianne, poultry research chair, Faculty of Veterinary Medicine of the University of Montreal, has completed work that has led to a more thorough understanding of the problematic pathogen, putting her team closer to developing vaccines to prevent NE.

“The more we study C. perfringens, the better we understand how this disease develops,” Boulianne says. “In the past, researchers believed the pathogenic strains were associated with the presence of specific toxins, but as we study the bacteria closely using bioinformatics tools, we have found that belief is not necessarily the case.”

Exploring vaccine delivery
One potential vaccine solution Boulianne’s team is investigating is a delivery mechanism from an unlikely source: “mini cells” that are produced by the pathogen itself. Under a microscope, she says the material, known as extracellular vesicles (EVs), appear under magnification as small bubbles on the surface of the bacteria.

“Many types of cells including bacteria will produce EVs, which are basically a bit of the membrane packaging a variety of lipids, nucleic acids and proteins from the bacteria. We see this as a communication pod, a way of sending information or messages to other bacteria in the environment,” Boulianne says.

It’s what’s outside that counts
Boulianne says it was the EV’s structure that got her team thinking about using EVs as potential vaccines. Her team has zeroed in on the value of using a byproduct of the bacteria to see if it could stimulate immunity.

“The membrane is the first point of contact for a host, and what a host encounters on the surface informs how it will mount its immune response,” Boulianne says. “When we saw that C. perfringens also produces EVs, we got the idea to use EVs as potential vaccines to protect against the pathogen. “

The team pursued the production and characterization of EVs from C. perfringens. In their lab, they were able to determine the EVs’ size, weight and various conditions where the bacteria was most productive. They sent their EVs to a partner for further analysis.

“We wanted to understand what sort of proteins the EVs contain, because if we know the proteins, we can better understand how the bacteria communicates,” Boulianne says.

Next, the team will administer a small amount of EVs to chicks orally, and monitor their blood response to see if antibodies develop. At three weeks of age, the chicks will be challenged to test if they have developed immunity against C. perfringens.

“This is the first time EVs are being used to protect chicks against C. perfringens, and it’s very exciting,” Boulianne says. “We are in the early stages, looking for proof of concept. If the work is successful, these EVs could be an interesting alternative to antibiotics.”

Reverse vaccinology approach
Boulianne says the EV project is complementary to her team’s other work, which involved developing three purified proteins that are ready for testing both in ovo and post-hatch vaccination, thanks to a “reverse and subtractive” vaccinology method.

“Researchers often work with a gene or protein to see if it is immunogenic,” Boulianne says. “But our work does the reverse, by looking at all the genes from pathogenic or harmful strains of the bacteria and comparing them with all the genes from the commensal or not harmful strains. It helps us understand what genes are important to the disease.”

The team categorized a total of 79 C. perfringensstrains from different geographical regions and tested each strain to determine whether it was pathogenic or commensal. They determined a list of criteria they wanted in a potential vaccine, and created a process of purifying the top three candidates.

Next, those proteins will be shared with a collaborator who will inject them into embryos as in ovo vaccination, while Boulianne’s team uses them to vaccinate day-old chicks. All birds will undergo a challenge study, and researchers will measure their immune response to NE.

“So much of our work is prediction based, and now it is time to test if our purified proteins are immunogenic and protective – because that’s what we want in a future vaccine,” Boulianne says. “We hope to see the embryos develop an immune response, and that in front of a challenge from the bacteria and the risk factors that would normally produce NE, our birds are protected.”

Both studies will be completed by March 2023.

What happens when
Boulianne’s team is working on another project she hopes will provide a more fundamental understanding of how NE develops in the body.

“We have a project that is looking not at DNA, but RNA – the message that the pathogenic bacteria sends at specific times,” Boulianne says. “We want to understand the arsenal that bacteria use at different times.”

Researchers have shown that within seven hours after exposure, toxins begin to produce, leading to intestinal lesions. Boulianne says the goal is to understand what happens at specific times within those seven hours.

“All of these projects complement each other and will help us to design better preventative and controlled means of controlling this disease in young birds,” Boulianne says. “If we plan to eliminate antibiotics, we need as much knowledge and as many tools as possible.”

Boulianne is working closely with Drs. Joshua Gong and Dion Lepp at Agriculture and Agri-Food Canada, Dr. Faizal Careem at the University of Calgary, her University of Montreal PhD student Nicolas Deslaurier and MSc student Laura Guerrero, in collaboration with Drs. Christopher Fernandez-Prada from University of Montreal and Dr Martin Olivier from McGill University.

This research was funded by the Canadian Poultry Research Council as part of the Poultry Science Cluster, which is supported by Agriculture and Agri-Food Canada (AAFC) as part of the Canadian Agricultural Partnership (CAP) program. Additional support was received Natural Sciences and Engineering Research Council of Canada (NSERC) and the Claire and Jean-Pierre Léger Foundation.