Food Safety Magazine

Inside Microbiology | October/November 2001

Campylobacter: Research Advances in Sourcing the Problem

By Nelson A. Cox, Ph.D.

Campylobacter: Research Advances in Sourcing the Problem

The Centers for Disease Control and Prevention (CDC) estimates that Campylobacter spp. is the leading cause of diarrheal illness in the U.S., causing 2.5 million cases per year. It is believed that 99% of these cases are caused by Campylobacter jejuni Last year, CDC’s FoodNet, which collects data about nine foodborne diseases in eight U.S. states, noted that in the five years since it began actively tracking infections caused by pathogens, Campylobacter was the most frequently diagnosed pathogen in all five years. As a result of the FoodNet findings, there are increased research efforts underway to pinpoint the source of Campylobacter in food and how to control it. Some industry professionals predict increased regulation by the Food Safety and Inspection Service (FSIS), testing and criteria for reducing levels of Campylobacter in the food supply, especially in ready-to-eat products that contain poultry.

According to the National Institute of Allergy and Infectious Diseases, most cases (more than 50% in one survey) of campylobacterosis result from people handling raw poultry or eating raw or undercooked poultry meat, and large outbreaks have been associated with drinking unpasteurized milk or contaminated water. Other foods that have been implicated include shellfish and fruits and vegetables. While fatalities are rare, Campylobacter-associated illnesses include pneumonia, meningitis and Guillain-Barre syndrome, a rare type of paralysis.

According to the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM), Campylobacter jejuni can survive two to four weeks under moist, reduced oxygen conditions at 4°C, surviving beyond the product’s shelf life, as well as two to five months at —20°C. When Campylobacter is damaged due to exposure to air, drying, low pH, heating, freezing or prolonged storage, its recovery and culturing is very difficult.

In this article, we are pleased to interview Nelson A. Cox, Ph.D., a microbiologist with the Poultry Microbiological Safety Research Unit of the U.S. Department of Agriculture—Agricultural Research Service (USDA-ARS) at the Russell Research Center in Athens, GA, about the state of research into this pathogen. Internationally recognized for his work involving the reduction of Salmonella on processed broiler carcasses, feed and in the growing animal’s intestinal tract over the past 32 years, Nelson has been involved for the past three years in the ARS project, “Campylobacter Epidemiology, Methods Development and Interventions in Poultry.” The objectives of the research are to collect data for a poultry risk assessment model describing the effects of production, transport and processing on Campylobacter contamination; identify sources (by DNA sequencing) in poultry operations; assess and characterize the potential for vertical spread and determine value in hatchery intervention; define antagonistic flora; and create a detection assay to selectively isolate and enumerate the pathogen.

A multi-award-winning microbiologist, Nelson has received the C.W. Upp Award, the Ralston Purina Research Award, and the National Broiler Research Award, among others, as well as receiving seven USDA Certificates of Merit. He is a Fellow of the American Academy of Microbiology.

Food Safety Magazine: Nelson, you are known for your work focusing on Salmonella in poultry. What impact has that research bad on your more recent endeavors in Campylobacter research?

Nelson Cox: First, there’s a lot to say about my Salmonella research as background to my Campylobacter research. I started researching Salmonella in 1966 in graduate school at Louisiana State University. My master’s degree was on the microbiology of crawfish, believe it or not. As a Ph.D. candidate, I joined a researcher who had a grant to research Salmonella in the laying hen, and I was inoculating it into the mouths of chickens trying to find the microorganism in their eggs. I was hired by USDA to work on Salmonella, and when I first arrived, we worked solely in the processing plants where pathogens like Salmonella wouldn’t die; i.e., you’ve got the scald temperature/scald water, but that isn’t high enough to kill it, and you’ve got to chill it (and you might even rinse it off), but you really didn’t have a cook or a kill step. So, during the first 10 years of my career I was dipping poultry carcasses coming out of the processing plant in just about everything I could think of to see what would kill Salmonella. The Catch-22 was that whenever a substance would indeed kill it, it wasn’t a recognized, acceptable compound that you could add without dealing with FDA hurdles for the next 20 years. We found that we could kill the Salmonella, but with many of the compounds researchers tried, no one could eat the poultry.

Then in 1981, researchers began to say that the problem with Salmonella had to do with the feed, specifically that the animals were eating it in their feed, and that if you could get the Salmonella out of the feed, you’d solve the problem. (This whole line of thinking actually began in 1955 when Dr. Erwin isolated living, viable Salmonella in commercial poultry feed, and having done that, everybody just assumed that was where the poultry was getting it from.) Then, we worked for five years trying to eliminate Salmonella from feed, which wasn’t a problem since all you have to do is to beef up all that conditioning and pelleting in the feed mill. However, the time and cost involved in doing all the things required to kill the Salmonella in the feed were prohibitive.

Of course, there is Salmonella in the feed, but was this really the source of infection in the poultry carcasses? We eat about nine billion chickens per year in this country, and of those nine billion chickens, we generate about one pound of guts, or offal, per chicken, which we don’t eat. If we didn’t have a place to put this offal, we’d fill the Grand Canyon with it in no time at all, so basically what we do with it is cook it down and dry it up and it becomes a protein source in the new chicken feed. In effect, then, the little chicks are eating the internal parts of the previously raised birds. The thinking was that the intestinal tracts of these animals harbor Salmonella and when the intestines were cooked and made into feed, the Salmonella was not completely killed and thus, the broiler chicks were eating it. So, it seemed simple to track the Salmonella to this source. It turns out that it is not that simple—we did a whole lot of studies in Puerto Rico (and they’ve done these studies all over the world) in which when they went and got Salmonella from the final carcass that was going to the supermarket and they fingerprinted it and serotyped it, it didn’t match anything coming from the feed. It was easier to match it to the breeder flocks and the hatcheries.

I am building this scenario about three decades of Salmonella research because I carried into my more recent Campylobacter research a bias that really led me to some solutions that I wouldn’t have come to unless that bias existed. All scientists are biased, whether they’ll ever admit it or not, and they carry these biases around with them. The problem is that they don’t know it or recognize it a great deal of the time and it creates problems in their research. But my bias, working with Salmonella for as many years as I had, was that the Salmonella is coming from the parents, the breeder birds.

Let me provide a little background on this bias. The bird that you purchase from the store is actually the fourth generation produced by the poultry industry. In other words, that bird has a parent, a grandparent and a great-grandparent generation before him. Now why does the poultry industry do that? So they can build into that animal whatever genetic material is desirable. Each of these levels of generations has their own hatcheries in which one rooster is placed with 10 hens to produce fertilized eggs. Once the egg is laid, it takes about four or five days for that egg to get transported and placed in an incubator. Twenty-one days later a little chick pops out of that egg, which is then taken to a chicken house with about 20,000 other little brothers and sisters. Chicks grow for about six weeks and then we take them to a processing plant. The bird that you buy in the store, then, took approximately 21 days to develop inside the egg and was hatched in a broiler hatchery.

When I was doing my research with Salmonella, I realized that the laying hen laid her egg through the same opening from which she defecates, and thus, as she laid the egg some feces would remain on the outside of the egg. Since a hen’s body is much warmer than mine and yours, about 107°F, if that hen laid her egg, say, anywhere but Kansas in July, chances were that the air temperature in the room was much cooler than her body temperature. This meant that the warm egg was laid with fecal material on its shell, and that the temperature differential sucked the bacteria into the egg. While the shell of an egg looks like a solid surface, in fact it is extremely porous and bacteria can work their way through it very easily, particularly in the presence of moisture, because the bacteria just get washed through it. This bacteria would get entrapped in the membrane just under the shell. There’s an outer membrane and an inner membrane. The outer membrane is kind of loose netting, if you will, so the bacteria work their way through that pretty easily, but then you hit this inner membrane, which is a really tight netting material, and bacteria get trapped in that membrane, as well. My Salmonella research showed that when that little chick gets ready to bite out of its egg after 21 days, it is eating the Salmonella from its mother before it ever gets into the world, because the developing chick is eating that membrane in the egg. At that time, scientists all over the world were looking for the source of Salmonella contamination in poultry, asking, ‘Is it the feed, is it the flies, is it the mice?’ It turns out that the great majority of Salmonella comes from the mother hen, and that basically, the critical source of Salmonella was coming from the breeder flocks and the hatcheries.

After working with Salmonella from 1966 until about 1998, I went into my boss’s office and said, ‘We know where Salmonella is coming from, we virtually know what we have to do to stop it, and the regulators, the poultry industry and the lawyers now can decide what’s going to be done.’ I said I’d like to move into Campylobacter research because, in my opinion, no one knows how Campylobacter is getting into these poultry flocks. He agreed. I told him, ‘The first thing I’m going to do is go look in the hatchery,’ to which he replied that others had already tried this approach and that Campylobacter is not in the hatchery. But, remember, I’m hardheaded and biased, and because of my 32 years of Salmonella research I was convinced that these breeder flocks and hatcheries were going to be a main playing field for Campylobacter.

FSM: How have you gone about proving (or disproving) the bias?

Cox: First, I sent technicians to the nearest hatchery to collect various kinds of samples, including the small egg shell fragments from when the chicks bite out of the eggs, the paper pads that had been placed underneath the chicks to collect their droppings, and the chicks’ fluff from the hatching cabinets. Now, if you go in and take these samples and test for Salmonella, you would find it in a great number of samples. But when we tested for Campylobacter, these 300 or 400 samples were all negative. We either needed to pull a larger number of samples, I thought, or we needed to collect larger sized samples, so I sent the technicians back to the hatchery. By the third or fourth time of sampling without any positives, everybody in my unit was laughing in their hands when I walked into the room. Now, remember, I said I have a bias from my Salmonella research, believing that this organism was also in the breeder flocks and the hatcheries, but even so, I was not going to keep sending my technicians to do the same thing and expect different results.

Now two questions arose: Are we unable to find Campylobacter in the hatchery because it really isn’t there, or is Campylobacter in the hatchery, but these materials that we are sampling are so dry and the water activity so low that this bacteria, which is a fragile organism when it comes to dryness, is either viable but non-culturable, or the dryness is killing them? So, I sent my technicians to the hatchery one more time to get samples—the fluff, the egg shell fragments and the paper pads of droppings—and we inoculated 100,000 or a million Campylobacter on all of these samples. After a few hours, we found that we could not isolate the Campylobacter we had just put in the samples, even though we inoculated the samples with a large number of bacteria. This demonstrated that we really didn’t know if Campylobacter was in those hatcheries, but that our weak culture methods were not good enough to recover them. There really is not a nonselective recovery media that allows a Campylobacter cell that has been stressed by either dryness or heat or injured in some way to be recovered.

Once I saw that we were inoculating these dry samples and Campylobacter couldn’t survive long enough to be cultured at the lab bench, we needed to find another way to demonstrate that it is passing through this fertile egg. I decided to go to the parent breeder flocks of these fourth-generation broilers and collect wet droppings in which Campylobacter can live.

In the lab, I isolated the Campylobacter and characterized those isolates with ribotyping and polymerase chain reaction (PCR) gene sequencing using some high technology methodology to fingerprint the isolates. Then I went to the house where their offspring were being raised and I collected their wet droppings and isolated the Campylobacter. When I compared the fingerprints of the isolates of the parent birds with the offspring, they matched incredibly well, showing that these were the same Campylobacter. This indicated that the Campylobacter was transmitted through the fertile egg.

When I reported these findings at an international Campylobacter meeting, the naysayers noted that since the the breeder house where the parent birds were and the house where the little broilers were only about 20 miles apart and thus, the bacteria could have been transmitted via mice or birds, for example. However, in the next experiment I did, the parent and broiler houses were about 1,700 miles apart and we still got the same results. Unless a transportation company was involved in a conspiracy, I don’t believe that a mouse had anything to do with that transmission.

Although I am not going to argue that Campylobacter doesn’t also transmit through nonchlorinated water or via a mouse, I now had evidence that it is transmitting through the egg. In this country, we take birds and put them right back on the same litter as the previous flock, and if Campylobacter can survive in that litter in a non-culturable way and get eaten by the new chicks, that’s probably contributing a little, as well. But I am convinced that the breeder flocks and the fertile eggs are contributing a great deal to the Campylobacter in broilers. This was the hypothesis that most scientists had written off. Why? Because their microbiological methods were not able to culture this organism in the hatchery.

FSM: Where did your research take you?

Cox: I said, now that I’ve proved it, let’s prove it even further. So I went and collected several commercial breeder hens that were close to the ends of their lives. I pulled a few dozen female breeders from different flocks that had everted vaginas, meaning these birds were actively laying fertile eggs which produce chicks for you and me to eat. I took these back to the laboratory and cut out their reproductive tracts to see whether Campylobacter could be found there.

At the top of the female reproductive tract is a little “basket” if you will, where the yolks reside. As a fertilized yolk slides down the tract, the albumin is added followed by the membrane and shell gland. It takes about 22 to 23 hours for the shell to be made, after which it moves down past the vagina and is presented by the hen outside of the body as a laid egg. The hen’s reproductive tract is made by Mother Nature in such a way that anything that gets past the vagina gets sucked all the way up to that basket. Why? Because Mother Nature wants the sperm to get all the way up there to fertilize that yolk.

When we began cutting into these breeder hens’ reproductive tracts, we found Campylobacter throughout. Now, we have to determine whether it is coming from the hen’s feces, which is getting sucked up that reproductive tract, or whether it is coming from inside the hen; for example, because much of the yolk material comes from the liver and so forth, and we know that Campylobacter is attracted to the liver-gall bladder. Since we did not know which end it was coming from, we started taking isolates from the reproductive tract and the feces of the hen. Our research has shown that quite a few of these isolates do not match up, and so we’re looking for another source besides the fecal material of the hen. Dr. Jeff Buhr, Dr. Kelli Hiett and Dr. Norman Stem are assisting in this area of my research.

FSM: Did you look at the male reproductive system to see whether roosters are contributing to the problem?

Cox: Yes. I’m working with Dr. Jeanna Wilson, an expert in the male reproductive systems of poultry, sampling semen to see if it has Campylobacter in it. We fast the birds so they are not defecating, since the penis is so close to the cloaca and could contaminate the sample, and then sample their droppings. Also, Dr. Wilson swabs all around the outside of the penis to clean it, puts a tube right next to the penis and bleeds the semen into it, just like milking the venom from a snake’s fang. These are very clean samples.

We’ve found about 10% to 15% Campylobacter-positive semen samples in these commercial breeder males. Unlike the female reproductive tract, which draws everything up, the male reproductive tract pushes everything out. If you look at the literature, any scientist who has ever gone into the rooster’s vas deferens has found this area to be sterile; in other words, they found no microorganisms in there at all. However, none of these scientists ever went looking for Campylobacter. Why? Because they were convinced that it wasn’t involved with the egg or the hatchery. Once again, they were biased in the other direction from my own bias, and as such, they had written this off as a potential source.

I started cutting out vas deferens and, of course, we now are finding Campylobacter in appreciable numbers. We are starting to track these roosters, mostly from 49 to 65 weeks of age, sampling the same roosters in individual cages over a period of time. Currently, we are finding that every time we sample the semen in some birds, it is positive, but that in others, we find samples that are sometimes positive and sometimes negative.

But here’s the take-home message: We’ve not only demonstrated that the Campylobacter can transmit from one generation to another through the fertile egg when it comes to the broiler industry in this country, but we’ve demonstrated the natural presence of this organism in the reproductive tract of both the female and the male breeder birds. Campylobacter is in those hatcheries, it’s in those cabinets, it’s in the breeder birds, it’s in that fertile egg, and it is coming into the flock through that source. Again, I’m not about to say that this is the only source, because as we all know, mice and beetles in the litter, flies and other things have been associated with these organisms. It is up to us not only to show conclusively how this is contributing and how we can stop it, but also to pay attention to other possible or proven sources, because until we can close them all down, they still pose an infection problem.

FSM: In what areas are you now focusing your Campylobacter experiments?

Cox: At present, I’ve got 200 experiments designed, but we can only do about 25 or 30 at a time. Basically we’re trying to determine now is where Campylobacter comes from; can one of the sexes transmit it to the other and how can we stop it? Let’s say these breeder birds are getting it from their parents through the egg, we still don’t know how they’re getting in their reproductive tracts. That means sampling the chicks beginning on the first day of their lives. They won’t start breeding until they are about 25 weeks, and if we find Campylobacter in their droppings, we’re going to sample their parents to see if the chicks got it from there. What we think might be happening is that as the breeder hen comes into egg production at 20—25 weeks, a very stressful situation on the animal’s body, there may be so much stress on the hen that if Campylobacter is residing in an internal organ in the animal’s body like the liver-gall bladder then it might become expressed in her reproductive tract through the lymph system or the blood. Right now, we don’t know, but we’re looking at all of these aspects. Remember, at first we thought we’re were going to find it in the feces and it was getting pulled into the reproductive tract, but these isolates are not matching up, so the story is now getting more interesting. Since we have banks of negative roosters with no Campylobacter in their droppings or semen, we’re now going to match them up with a bank of individually caged positive hens and characterize all those Campylobacter to see whether the female can transmit the bacteria to the male. We’ve also got the reverse experiment set up in which we’ve got the positive males copulating with the negative females.

At the same time, we’re trying to determine the source: Where does the first Campylobacter get into either one of these breeder birds—is it from their parents, is it from their environment, is it from their parents’ environment and stored in their body and expressed later? All of these birds are actively breeding and laying eggs that become the chicks we will eventually eat, and a significant percent of these birds are carrying significant numbers of Campylobacter in their reproductive systems. You can still argue all you want about egg transmission, but why are those organisms in those bodies, in the egg manufacturing machinery of the female bird, and why is it found throughout the male reproductive system? I think that we very well may show that some of these organisms might originate from the feces, and some might originate from other sources such as the parents, but until we know where it is coming from we can’t stop it, and so that’s what I’m working really hard to do with all these experiments.

FSM: How do you think your findings will ultimately affect the poultry industry?

Cox: I am probably more excited about my research now than in the last 30 years. It is pretty clear to us that a significant source of Campylobacter is through the fertile egg. Eventually, when the the regulators start doing mega-regs on Campylobacter, as they’ve done with Salmonella, the poultry industry is going to need help when processing operations start failing these tests and wondering how they can reduce and/or eliminate Campylobacter. Well, we’re not going to be able to help them if we don’t fully understand what is going on in these breeder birds and in these hatcheries. Ultimately, my job as a researcher is to intervene, to figure out how to stop it. Of course, assessment of the problem is the first step, and thus far, it has been extremely exciting with this organism, partly because it is so fragile and so difficult to culture that it lends itself to researchers arriving at erroneous conclusions; they get negative results and assume that the organism is not there when, in fact, it is there.

The fact is, the integrated poultry industry in the U.S. is huge. We are a big poultry-consuming nation, and we are not likely to quit eating poultry. Americans eat 300 to 400 million turkeys per year, about 70 to 80 billion eggs, and nine billion chickens. It is up to researchers to discover how we’re going to assure safe products and production. If I can help find ways to reduce Campylobacter and Salmonella counts so that fewer people are getting sick, then I’ve earned my paycheck.

 

Categories: Food Types: Meat/Poultry; Testing and Analysis: Methods, Microbiological