Talking Turkey Health with an NC State Avian Immunology Expert

In the United States this month, the poultry industry will sell 50% of a year’s worth of turkeys for Thanksgiving, and with North Carolina being the nation’s second-largest turkey producer, keeping the birds healthy is critical.

The NC State College of Veterinary Medicine plays a vital role in safeguarding and improving poultry health for the state’s leading agricultural sector. The poultry industry generates more than $40 billion in economic impact a year, supporting about 150,000 jobs and more than 5,700 farms.

Dr. Ravi Kulkarni, associate professor in poultry immunology and microbiology in the Department of Population Health and Pathobiology, has focused his considerable research on developing vaccines to common poultry diseases to cut down on antibiotic use and on creating oral vaccines using probiotics, which also would improve gut health and protect birds against bacterial infections. 

His research on Clostridial dermatitis, a bacterial disease that produces severe skin lesions and frequently causes death within days of infection, has led to a patent and a vaccine that has been shown to prevent the disease in turkeys in a research setting. 

Kulkarni says he has replicated the process for chickens and anticipates that his paper outlining the research results will be published this year. He already has contributed to four other research papers during the past year related to his dermatitis research

Kulkarni has spent several years working in both human and veterinary immunology and vaccine research, including stints at a pharmaceutical company in his native India and at the Emory University Vaccine Center in Atlanta, Georgia. He received his DVM-equivalent degree from Karnataka Veterinary, Animal and Fisheries Sciences University, India, and a master’s in immunology and parasitology from the Indian Veterinary Research Institute. 

He pursued a Ph.D. in immunology and microbiology at the University of Guelph Veterinary College, Canada, and completed post-doc training in avian immunology at Guelph on a fellowship from the Canadian Ministry of Research and Innovation. After working at Emory, he returned to Guelph as an adjunct professor and associate graduate faculty in avian health and disease control. 

NC State recruited him in 2019. He also teaches immunology to first-year veterinary students and poultry infectious diseases to third-years.

We talked with Dr. Kulkarni, the recipient of 2024 Bayer-Snoeyenbos New Investigator Award by the American Association of Avian Pathologists and also an NC State Goodnight Early Career Innovator, about his important work.

Q: How did you come to be developing vaccines for poultry diseases? 

Dr. Ravi Kulkarni: The important aspect of vaccine discovery research is that, until you understand how the disease is produced and how the immune system reacts to a particular infectious agent, it is hard to make an efficacious vaccine. These Clostridium are very clever bugs and have been a big-time problem to the poultry industry, both in chickens and turkeys. My approach has always been to understand the immunology of disease and then develop the vaccine and go from there.

Traditionally, poultry were raised with feed supplemented with a very small amount of antibiotics to keep these intestinal bad bugs under control. After people started getting aware of and talking about antimicrobial resistance, the poultry consumer perception started to change toward a demand for poultry raised without antibiotics. There has already been a regulatory ban on using medically important antibiotics in poultry. So, poultry producers are moving more and more toward organic and no-antibiotic-ever farming.

Now, think about when you had those minute amounts of antibiotics in the feed on a daily basis, all these bugs were keeping quiet. Now you take antibiotics away, and they become opportunists, and their opportunism has brought me back into the research game. What I have been trying to do for the past five years, very actively, is telling the industry as well as the federal funding agencies such as US Poultry and USDA that I want to take a very rational, logical approach to develop alternatives to antibiotics. 

There are several antibiotic alternatives you can think about, but my lab works in two streams. One is vaccines; another is probiotics. For the diseases I’m working on, we do not have a vaccine which can give 100% protection and neither do we have probiotics that can provide 100% protection. Through collaborations, we are combining these two to make newer vaccines that can yield successful outcomes and patentable novel discovery research.

Q: What do you consider your biggest research breakthrough?

Kulkarni: My big breakthrough was the injectable dermatitis vaccine in turkeys published in PLOS One. I then constructed food-grade probiotic-based vaccines that were given orally to turkeys, and just like a movie sequel, this was our research sequel publication in the journal Vaccine as a proof of concept study. We have patented this work, which has opened a novel oral vaccine development research strategy to use poultry-specific probiotic bacteria as vectors to deliver vaccine antigens. An oral vaccine could be put into the water or in the feed, which is critical for poultry due to its mass application capacity. 

Q: How are vaccines made and how do they work?

Kulkarni: Our vaccine work is aimed at preventing diseases caused by bacteria, so understanding how those diseases occur is critical. We found that the Clostridial dermatitis bacteria produce a toxin and that, if you make this toxin functionless, these bugs can’t produce disease. We dug deeper into the gene sequence of this toxin and identified two gene segments that were devoid of toxicity and made a vaccine called a “recombinant protein-based vaccine” containing the two gene products.

To test whether these vaccines work, we developed a turkey model to reproduce the disease that closely mimics how it breaks out in the field. We vaccinated the model birds, and our vaccine candidates showed a great potential to prevent the disease. 

We believe the vaccine works by causing the body to produce antibodies and immunity that can kill the bacteria as well as neutralize the toxin’s activity, which protects our birds from death and severe skin pathology. 

So far we have studied the immunology of this disease, developed a model and made two research-based vaccines, one injectable and one oral, and both were found to be effective, as reported in our publications. The injectable vaccine is protein-based while the oral vaccine is probiotic-based. Both vaccines deliver the same antigens, but by different routes, and work toward the ultimate goal of protection against the disease. 

We measured this protection by comparing birds that had been vaccinated with those that had not and seeing whether the vaccinated birds had fewer deaths, less nausea and less weight loss and, importantly, less or no skin pathology, including gangrene. 

We found that our vaccines prevented deaths, reduced disease pathology and helped birds resist the worst of the disease.

Our vaccine work involved a series of animal studies and a lot of molecular biology and immunology. In the past few years, my laboratory has been working in collaboration with people at the NC State College of Agriculture and Life Sciences, Dr. Rodolphe Barrangou, who is an internationally acclaimed scientist leading the CRISPR lab, and Dr. Sarah O’Flaherty, a senior scientist in Barrangou lab, who are experts in molecular biology and bacterial genetics and immensely helping my projects. 

We began working on a “two birds, one stone” approach, so you have poultry-specific probiotics, which are good bacteria, and I knew from our work that we have excellent vaccine candidates that can actually protect against the disease. So now the idea is to take the genes that make these vaccine proteins and put them into probiotics and develop a probiotic-based vaccine for poultry.

I have talked to the turkey producers about our vaccines. Turkeys up to 6 weeks old are in one house, which is called the brooder, then they go to the grower, so they said that if your vaccine works well with one injection given at 6 weeks, when we are transferring the birds, we can give the vaccine, if you can produce in large scale. We’re not sure if one shot will work, but those things are in the pipeline as well.

Q: What exactly does your patent cover?

Kulkarni: Our vaccine patent intellectually protects the discovery of the two gene segments, called the ntATX (non-toxic Alpha Toxin) domain 1 and domain 2, which we have shown to be excellent vaccine candidates, especially the domain 2. We have also included all our vaccine testing work in the patent that was published recently.

Q: You clearly love birds. Why?

Kulkarni: If you are an immunologist who studies immune systems, the bird immune system is very, very interesting, because birds do not have all the immune defense machinery that we have, but having less of them, they are so robustly effective in defending against varieties of pathogens. 

Think about birds. I mean, if I were a bird, oh, my gosh, there are so many environments I would get exposed to. And if you think about migratory birds, they fly miles and miles and visit several habitats, and they need to adapt; Darwin’s principles and struggle for existence and survival of the fittest, right? And you will survive if you have a robust immune system. At times, my wife jokingly tells me that since I chose poultry for my master’s research, we have been migratory birds, too.

You have heard about lymph nodes, right? We have tonsils, and a lot of other lymph nodes in the body. Chickens, or birds for that matter, do not have lymph nodes, but they have something else, which is lymphoid aggregates. The physiological and immunological anatomy of birds has distinctly different features. For example, they have air sacs for flight, but when you have air sacs, you’re much more vulnerable to inhaled microbes, and some can be pretty harmful.

I teach DVM first-years avian immunology, and I do get feedback from my students, some who say, “Dr. Kulkarni, we can see that excitement in you when you’re talking about avian immunology!” I start that lecture by saying, “Less is more, right?” We often say that. And I tell my students that I’ll prove it, that less is more when it comes to avian immunology. I also tell them, “You know, birds don’t have teeth, but they can gulp nuts like anything, right? Then think about if I take a bunch of chickpeas and just gulp them, I don’t think my tummy will like it, but Mother Nature, for birds, has given two types of stomachs. One that softens the food, and the other grinds it. They have something called a muscular stomach, which works like a mill.” I can go on and on. 

Q: What’s your ultimate goal?

Kulkarni: When I left India for a Ph.D., the original plan was not to stay in North America forever. I told my father that I’ll do my Ph.D. and go back to India, but man proposes and God disposes. Every time I talk to him, at least half an hour on Sundays, I feel like I learn something from him. He often tells me that you think you are leading your life, but actually life leads you. 

You think many, many times, “This is what I’m going to do.” There are lots of ups and downs in our lives. I talk to him when I’m down, and what he says is — I’m trying to translate a little bit here — if you get what you wish is good. If you don’t get what you wish is even better, because something much better is waiting for you. 

Like, for example, we live in this world of grant writing, and every time you write, you want to get it. With my first grant proposal, I didn’t get anything, but I always remember my dad and say to myself, “Well, I wished I’d get it, but I didn’t. That means something even better.”

So I changed my research strategy and took a step back and started diversifying my research directions but with an ultimate goal of developing novel and long-lasting disease control strategies for our birds. Poultry are an important backbone of animal-ag, and they feed the world and, at the least, we need to take care of their health. Through productive collabs and constant interactions with the local and national poultry industry, we want to build a novel vaccine design and development platform that can be used to counter many disease problems faced by our birds. It is ambitious, but ambitions are good. There’s nothing wrong in dreaming big, right?  

Q: Do you have any other projects in the works?

Kulkarni: What’s next is a project I’ve submitted to the USDA on enteritis in chickens. It causes necrosis (tissue death) of intestines, leading to some deaths. It’s a huge deal for the broiler industry. Once that is done, I want to jump to another disease and so on. I’m not going to be here forever. My ambitious plan is to develop one or two successful vaccine platforms that can prevent multiple diseases. And then pray, pray that it works.

But there is one thing to remember. We try to counter pathogens. Pathogens are disease-causing agents, but these infectious agents also evolve strategies to beat what we do. So this is a never, ever ending process. 

The world’s population is expected to reach around 9 or 10 billion by 2050, and population is not going to go down and human longevity will increase. Who will feed the world? The food security will fall back onto animal proteins, and if you think about it, chicken and turkey meat and table eggs and various other poultry products are huge. And the United States is one of the world’s top poultry producers and exporters. As a vet school, I think it is our responsibility for some of us to engage ourselves in food animal or poultry research.

This post was originally published in Veterinary Medicine News.