A question that always seems to come up is: Where does campylobacter come from? Does it come from breeder chickens, the hatchery, the growout environment or the plant?
To answer this question, campylobacter are able to colonize the intestines of chickens, but before addressing how campylobacter is able to colonize chickens, it is important to discuss what campylobacter is and why it is of interest. Campylobacter is one of the most common causes of diarrheal illness in the United States. Most cases occur individually and it is not usually linked to large-scale outbreaks. Active surveillance through FoodNet indicates that about 13 cases are diagnosed each year for each 100,000 persons in the population1. Because cases go undiagnosed or unreported, campylobacteriosis is estimated to affect over 2.4 million people in the United States every year, or about 0.8% of the population. Campylobacteriosis occurs much more frequently in summer than in winter. Campylobacter infections in people do not usually result in death; however, it has been estimated that approximately 124 people die from campylobacter illness each year1. The following graph shows that campylobacter infections in the United States decreased from 1996 to 2002, but then remained stable until 200720.
Campylobacter bacteria are spiral-shaped. Most human illness is caused by one species, called Campylobacter jejuni, but human illness can also be caused by other species. Campylobacter jejuni grows best at the body temperature of a bird, and seems to be well adapted to birds, which carry it without becoming ill1. This is important for poultry producers because, although the birds are colonized with campylobacter, the birds are not sick and do not exhibit weight loss as a result. These bacteria are fragile. They cannot tolerate drying and can be killed by oxygen. They grow only in places with less oxygen than the amount in the atmosphere. Freezing reduces the number of campylobacter bacteria on raw meat1. A picture of campylobacter is shown below.
Is campylobacter transmitted vertically through the egg?
On the forefront of this area of research is the USDA-Agricultural Research Service (ARS).
For many years, scientists pointed to the possible sources of campylobacter as being:
- wild birds,
- well water,
- insects, and
By inoculating baby chicks with campylobacter, scientists have determined that the bacteria couldn't survive longer than an hour in dry conditions, eliminating bird feathers and hatchery transport paper pads from the list of possible sources2. Scientists then focused on poultry feces, which are moist and provide the environment needed for campylobacter to survive.
Dr. Kelli Hiett of USDA-ARS analyzed broiler production samples collected in the search for the source of campylobacter. Using a type of DNA fingerprinting, campylobacter from a broiler-breeder flock and its broiler offspring were found to be identical, even though the flocks were housed 20 miles apart2. The two flocks had no contact, other than that the eggs from the broiler-breeder operation were transported 20 miles to the broiler hatchery2. Evidence suggests that the only way the same campylobacter isolate could have traveled from one location to the other is for it to have been inside the moist confines of the egg2. A picture showing an egg “sweating” is shown below. This can become a serious problem because as the egg is moved into the cooler to synchronize all of the embryos, the contents of the egg will shrink, allowing bacteria to be sucked into the egg.
Sahin and Kobalka3 reported that vertical transmission of bacteria including campylobacter may take place by primary (contamination of the egg content in the hen's reproductive tract during the egg development) or secondary (contamination of the eggshell after the lay via fecal material containing the bacteria with the subsequent penetration of the agent inside the egg) infection of the egg. If vertical transmission of campylobacter through the secondary egg infection occurs, the organism must first penetrate through the eggshell and then maintain its viability once inside the egg until hatching. The authors stated that campylobacter has a limited ability to penetrate the eggshell3.
Studies regarding the survival of campylobacter in eggs have been somewhat conflicting. Research has shown that campylobacter would not survive more than six hours in any part of the eggs; however, in rare cases campylobacter managed to survive inside the eggs for long enough during incubation to contaminate a low percentage of chicks. Keep in mind that only a few chicks need to be colonized to result in widespread colonization within a growout facility. Viability of the bacterium on the shell surface is only 16 hours and the organism was not recovered from inside the eggs beyond two hours when the eggs were kept at room temperature3. Sahin and Kobalka3 reported that the high susceptibility of campylobacter to desiccation and atmospheric oxygen are thought to be the primary reason for its low survivability on the egg surface. The absence of campylobacter in the chicks hatched from surface-challenged eggs further supports the notion that campylobacter contamination of eggs by a secondary egg infection is rare, if it occurs at all, and is unlikely to result in chicks infected with campylobacter. These authors concluded that, “Therefore, control of campylobacter should focus on sources of infection that are not related to eggs.”
Researchers at USDA-ARS did not subscribe to the idea that, just because campylobacter could not be recovered from eggs, does not mean that they are not there. Thus, scientists at USDA began evaluating the reproductive tracts of breeder hens and they found that campylobacter was present. ARS microbiologist Dr. Nelson Cox stated, "Campylobacter was found all through the egg-making machinery of the breeding hens, though we still don't know the mode of action of the bacteria."
How does campylobacter get on the egg in the first place?
Researchers at the USDA showed that campylobacter could be recovered from the ductus deferens of five of 101 broiler breeder roosters, and four of the five positive roosters had previously produced campylobacter-positive semen samples. Because of these findings, researchers set out to determine if inoculation route influenced the prevalence or level of campylobacter contamination of semen, the digestive tract, or reproductive organs4. Individually caged roosters, confirmed to be feces and semen negative for campylobacter, were challenged with a marker strain of Campylobacter jejuni either orally or by dropping a suspension of campylobacter on the everted phallus of roosters immediately after semen collection or by dip-coating a probe and then inserting the probe through the vent into the colon.
Six days after inoculation, individual feces and semen samples were collected and cultured for campylobacter. Seven days after inoculation, roosters were killed, the abdomen aseptically opened to expose the viscera, and one cecum, one testis, and both ductus deferens were collected. The samples were then tested for campylobacter.
Campylobacter was recovered six days after challenge from feces in 82% of samples, 85% of semen samples, and on the seventh day after inoculation from 88% of cecal samples. Campylobacter was not directly isolated from any testis sample but was detected following enrichment from 9% of ductus deferens samples. Roosters challenged with campylobacter orally, on the phallus, or by insertion of a campylobacter dip-coated ultrasound probe were all readily colonized in the ceca and produced campylobacter-positive semen and feces on day six after challenge. The low prevalence of recovery of campylobacter from the ductus deferens samples and failure to recover from any testis sample suggests that semen may become campylobacter-positive while traversing the cloaca upon the everted phallus. The production of campylobacter-positive semen could provide a route in addition to fecal-oral for the horizontal transmission of campylobacter from the rooster to the reproductive tract of the hen4.
The next logical step for researchers was to ask, “If campylobacter can be transmitted from the rooster’s semen to the reproductive system of the hen, where does it go from there?”To evaluate this, they obtained broiler breeder hens ranging from 60 to 66 weeks of age from four different commercial breeder operations. For each trial, the hens were removed from the commercial operation and held overnight at the University of Georgia processing facility5. The hens were euthanized, defeathered, and aseptically opened. To reduce the possibility of cross-contamination between samples, first the mature and immature ovarian follicles, then the ceca, were aseptically removed. Individual samples were placed in sterile bags, packed on ice, and transported to the laboratory for evaluation.
Campylobacter was found in seven of 55 immature follicles, 12 of 47 mature follicles, and 41 of 55 ceca4. The researchers reported that the recovery rate of campylobacter from the hen ovarian follicles was reasonably high, suggesting that these breeder hens could be infecting fertile hatching eggs.
What happens with the baby chick?
Researchers have found that when campylobacter comes into contact with intestinal cells in people they react differently than they do when they interact with chicken intestinal cells. Scientists conducted a study to compare the interaction of campylobacter with primary intestinal cells from humans and poultry to try to identify why they are different6. The researchers found that C. jejuni invaded primary human intestinal cells in a microtubule-, microfilament- and caveolin-dependent manner. Entry of C. jejuni into primary chicken intestinal cells also occurred. Chicken mucus, but not intestinal mucus of human origin, significantly reduced infection of primary human intestinal cells6.
The authors found that avian intestinal mucus appears to inhibit campylobacter from interacting with epithelial cell surfaces. This is a possible explanation for why chickens are not greatly impacted (get a debilitating infection) by colonization with campylobacter. Thus, campylobacter is considered to be a commensal organism in many avian species, such as chickens8. As a commensal organism, campylobacter colonizes the mucus layer on the intestinal lining in the crypts of the intestinal epithelium10.
In another study, USDA scientists inoculated day-old broiler chicks, either orally or intracloacally, with a characterized strain of Campylobacter jejuni 7. At one hour, one day, and one week after inoculation, broilers from the orally and intracloacally inoculated groups along with control birds were humanely killed, aseptically opened, and the thymus, spleen, liver/gallbladder, bursa of Fabricius, and ceca were aseptically removed and individually analyzed for campylobacter jejuni. Overall, C. jejuni was isolated from a high percentage of birds from the thymus, spleen, liver/gallbladder, bursa of Fabricius, and ceca samples after one-hour, one-day, or one week after inoculation orally or intracloacally7. The authors concluded that the rapid movement of campylobacter to internal organs following both oral and intracloacal inoculation may be significant, particularly if it persists in these organs as reservoirs throughout the 65-week life cycle of breeding birds7.
Spread among hatch mates is rapid if infected birds are introduced into the population8. In a laboratory setting, only thee days are required for the majority of the brood to become colonized because very young chicks (up to two weeks) are highly susceptible to colonization. Chickens are coprophagic (eat feces) and this facilitates the spread via fecal-oral route. However, Montrose and others9 reported that the rapid shift from uncolonized to 100% colonized birds is likely due to their communal drinking water source. Other implicated mechanisms of rapid transfer of campylobacter from bird to bird during growout include:
- wild birds,
- fecal contact, and
- farm personnel on their boots11.
Another factor is that the birds are in close proximity to one another during growout as is shown in the following picture.
Chickens in close proximity during growout.
What about environmental sources?
A study was conducted to determine the prevalence of campylobacter in wild birds around broiler chicken houses12. Intestinal samples and cloacal swabs were obtained from European starlings and house sparrows. Most of the samples collected consisted of wild bird droppings found on or near the houses. Samples were collected from each of four farms of a broiler integrator during a grow-out cycle: a cycle in the summer for farm A, fall for farm B, and spring, summer, fall, and winter for farms C and D.
Of the 25 wild bird intestinal and fecal samples collected from a broiler house on farm A during a grow-out cycle in July-August 1997, 4% were positive for Campylobacter jejuni. Of the nine fecal samples collected from broiler house B in a grow-out cycle in September-November 1997, 11% were positive. For farms C and D, of the 23 samples collected in March-April 1998, 11% were positive for C. jejuni. Of 24 samples collected in August-October 1998, 5% were positive for C. jejuni and of 14 samples collected December 1998-January 1999, 50% of the samples had C. jejuni. The prevalence of campylobacter in wild birds near the broiler chicken houses suggests that wild birds that gain entry to poultry grow-out houses have the potential to transmit these pathogens to poultry12.
Once campylobacter positive birds have been introduced into the growout facility, the organisms are spread very quickly to other birds, often reaching a prevalence level of 100% by the end of growout. Additionally, the number of these bacteria in the gut, fecal material, and in the litter can be extremely high with counts as high as 9.0 log10 cfu/g8. Because the levels may be very high on birds coming into the plant, it is very difficult to eliminate the pathogen completely from carcasses.
Controlling campylobacter during growout
Competitive exclusion - Scientists have attempted to use competitive exclusion cultures (defined bacterial cultures) to reduce camplylobacter in chickens. In this study, the reduction of C. jejuni colonization in chicks by oral administration of defined competitive exclusion (CE) cultures, 2.5% dietary carbohydrates, or CE cultures and carbohydrates was examined14. Prevention, elimination, or direct challenge of campylobacter infection was simulated by administering treatments before, after, or at the same time as that of the campylobacter inoculation. Additionally, the effect of maintaining high levels of protective bacteria was evaluated by administering CE cultures on days one and four (booster treatment).
All treatments evaluated were able to reduce C. jejuni colonization. Protection by aerobically grown CE cultures was not statistically different from that by anaerobically grown CE cultures. A combination of Citrobacter diversus 22, Klebsiella pneumoniae 23, and Escherichia coli 25 (CE 3) was the most effective CE treatment14. Interestingly, boosting the CE cultures by giving the chickens an additional exposure to the CE organisms at day four did not affect the reduction in campylobacter. C. jejuni was not detected in the ceca of birds receiving the prevention treatment (CE culture with mannose) representing a 62% reduction in the colonization rate. The authors found that fructo-oligosaccharide supplementation alone strongly prevented campylobacter colonization. Only 8% of the chicks in this group were colonized.
When lactose was fed in combination with the CE culture, reduced colonization from 80% of chicks (controls) to only 5%14. The CE approach appears to be a very successful means for controlling this organism; however, its cost and difficulty in achieving FDA approval may prevent its widespread use.
Vaccines for prevention of colonization
Researchers have developed a formalin inactivated, Campylobacter jejuni whole cell vaccine (with or without Escherichia coli heat labile toxin as a mucosal adjuvant), that can be administered orally to broiler chickens15. In three different trials where the number of exposures to the vaccine, the time of administration, and the inclusion and dose of E.coli heat labile toxin were conducted, the overall reductions of C. jejuni colonization in the vaccinated chickens ranged from 16 to 93%, when compared with non-vaccinated controls. Enhanced levels of anti-C. jejuni secretory IgA antibodies were demonstrated in vaccinated chickens.
Interestingly, the inclusion of the E. coli heat labile toxin in the vaccine regimen did not appear to boost the immunogenicity of the vaccine. The authors concluded that future development of successful oral vaccines for the control of enteropathogenic campylobacter in poultry is feasible15. The next picture depicts a spray vaccination system that could be used to deliver a campylobacter vaccine to baby chicks at the hatchery.
Spray vaccinator for chicks23.
Bacteriophages to reduce colonization
A new approach includes the use of naturally occurring viruses that specifically attack and kill campylobacter as a means of eliminating the pathogen from the intestines of chickens. Experimental models of campylobacter colonization of broiler chickens were established by using low-passage C. jejuni isolates from United Kingdom broiler flocks. Fifty-three different lytic bacteriophage isolates were tested against a panel of 50 different campylobacter isolates from broiler chickens and 80 different campylobacter strains isolated after human infection. The scientists identified two phage candidates with an ability to kill campylobacter16.
These phages were orally administered in a suspension, at different dosages, to 25-day-old broiler chickens that were experimentally colonized with the C. jejuni broiler isolates. Phage treatment of C. jejuni-colonized birds resulted in campylobacter counts falling between 0.5 and 5 log10 CFU/g of cecal contents compared to untreated controls over a five-day period after administration of the phages. These reductions were dependent on the phage-campylobacter combination used, the dose of phage applied, and the time elapsed after administration. Campylobacters resistant to bacteriophage infection were recovered from phage-treated chickens at a frequency of <4%16. These resistant types were compromised in their ability to colonize experimental chickens and rapidly reverted to a phage-sensitive phenotype in vivo. The selection of appropriate phage and their dose optimization are key elements for the success of phage therapy to reduce campylobacters in broiler chickens16. This line of research is very interesting and has great potential in eliminating campylobacter colonization of chickens. However, the cost, and consumer concern over feeding viruses to chickens may limit its acceptance as a viable option. The following picture shows a bacteriophage.
Bacteriophage (Virus that attacks a bacterium)24
Bacteriophage attacking and killing a bacterium25.
Would eradication reduce production costs?
In a new study conducted in Holland13, the Dutch government decided to evaluate whether it is cost effective to control campylobacter during production. In order to reduce campylobacter infections in the Dutch population, CARMA (Campylobacter Risk Management and Assessment), a multidisciplinary project, was developed to provide a scientific basis for risk management decisions. Within CARMA, information and methods from risk assessment, epidemiology, and economics were integrated when evaluating the effectiveness and efficiency of potential interventions in the chicken meat chain, which is considered one of the major routes of human campylobacter infections identified.
They evaluated the costs associated with bacteriophage therapy (feeding viruses to chickens that will kill campylobacter in their intestines) and carcass decontamination by chemical substances such as, for example, lactic acid, were estimated to cost up to €100,000 each day. All other interventions would cost more than €100,000 each day, either because the intervention would have little effect or because the intervention was expensive. Interventions with little effect were the decontamination of the scald tank and consumer education with respect to improved kitchen hygiene and home freezing. Expensive interventions were further improvement of farm hygiene, crust freezing, and irradiation. The CARMA group concluded that the eradication of campylobacter in animal production would not result in reduced production costs13.
Transmitted during transport in coops?
Microbiologists from the USDA-ARS have evaluated the role of transport coops as a critical point at which campylobacter contamination of broilers may occur17. The research team found that feces from campylobacter-positive birds can contaminate the feathers and skin of campylobacter-negative birds later placed in the same soiled transport coop. The authors reported that, because campylobacter cells are susceptible to drying, allowing the coops to dry for at least 48 hours before reuse dramatically lowered campylobacter numbers17. The following picture shows a transport coop with dried feces, which may transmit campylobacter from bird to bird.
U.S. trend of contamination on carcasses
In 1992, Most retail chicken was found to be contaminated with Campylobacter jejuni. The study by Stern and Line18 found that campylobacter could be isolated from 98% for retail chicken meat18. Additionally, Campylobacter jejuni counts on this meat were very high and often exceeded 103 per 100 g18.
Since that time, the poultry industry in the United States has done an incredible job of lowering the prevalence of this organism on broiler carcasses. Scientists recently conducted a large study to determine campylobacter prevalence on broiler carcasses in the United States19. In this study, 10 of the largest U.S. poultry integrators cooperatively determined the incidence and counts of campylobacter on processed broiler carcasses. Among each of the 13 participating poultry complexes, rinses from 25 randomly selected, fully-processed carcasses were sampled monthly from individual flocks. Among 4,200 samples, approximately 74% of the carcasses yielded no countable campylobacter cells. This means that only 26% of carcasses were positive for campylobacter. Campylobacter spp. were isolated from approximately 3.6% of all commercially processed broiler carcasses at more than 105 CFU per carcass. Nevertheless, this survey indicates industry recognition of its responsibility to assess and reduce public exposure to campylobacter through broiler chickens19.
The industry faces a daunting task in attempting to control campylobacter colonization of poultry. However, as more studies are conducted that identify how campylobacter is able to colonize poultry and novel methods are developed to control campylobacter, the industry has good news on the horizon. Moreover, the poultry industry has accomplished a Herculean task in lowering total prevalence of campylobacter on poultry from 98% in 1972 to 26% today. The industry is to be commended for such efforts to keep food safe for the American public.
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