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Journal of Food Protection, Vol. 75, No. 10, 2012, Pages 1896–1902 doi:10.4315/0362-028.JFP-11-322

General Interest Evidence for Horizontal and Vertical in Campylobacter Passage from Hen to Her Progeny

N. A. COX,1* L. J. RICHARDSON,1{ J. J. MAURER,2 M. E. BERRANG,1 P. J. FEDORKA-CRAY,1 R. J. BUHR,1 J. A. BYRD,3 M. D. LEE,2 C. L. HOFACRE,2 P. M. O’KANE,2 A. M. LAMMERDING,4 A. G. CLARK,5 S. G. THAYER,2 AND M. P. DOYLE6

1U.S. Department of Agriculture, Agricultural Research Service, Russell Research Center, 950 College Station Road, Athens, Georgia 30605; 2Department Downloaded from http://meridian.allenpress.com/jfp/article-pdf/75/10/1896/1686170/0362-028_jfp-11-322.pdf by guest on 02 October 2021 of Avian Medicine, University of Georgia, Athens, Georgia 30602; 3U.S. Department of Agriculture, Agricultural Research Service, Southern Plains Agricultural Research Center, College Station, Texas 77840; 4Public Health Agency of Canada, 160 Research Lane, Guelph, Ontario, Canada N1G B52; 5University of Toronto, Toronto, Ontario, Canada M5S 1A1; and 6Center for Food Safety, University of Georgia, 1109 Experiment Street, Melton Building, Griffin, Georgia 30223, USA

MS 11-322: Received 28 June 2011/Accepted 29 May 2012

ABSTRACT Campylobacter is an important human , and consumption of undercooked poultry has been linked to significant human illnesses. To reduce human illness, intervention strategies targeting Campylobacter reduction in poultry are in development. For more than a decade, there has been an ongoing national and international controversy about whether Campylobacter can pass from one generation of poultry to the next via the fertile egg. We recognize that there are numerous sources of Campylobacter entryintoflocksof commercial poultry (including egg transmission), yet the environment is often cited as the only source. There has been an abundance of published research globally that refutes this contention, and this article lists and discusses many of them, along with other studies that support environment as the sole or primary source. One must remember that egg passage can mean more than vertical, transovarian transmission. Fecal bacteria, including Campylobacter, can contaminate the shell, shell membranes, and albumen of freshly laid fertile eggs. This contamination is drawn through the shell by temperature differential, aided by the presence of moisture (the ‘‘sweating’’ of the egg); then, when the chick emerges from the egg, it can ingest bacteria such as Campylobacter, become colonized, and spread this contamination to flock mates in the grow house. Improvements in cultural laboratory methods continue to advance our knowledge of the ecology of Campylobacter, and in the not-so-distant future, egg passage will not be a subject continuously debated but will be embraced, thus allowing the development and implementation of more effective intervention strategies.

HISTORICAL PERSPECTIVE place and affects the offspring, the acquirement of the disease is by transmission only’’ was disputed, contrary to known Transmission of Campylobacter spp. from the hen to its congenital infections like syphilis (70). This was the attitude progeny in commercial settings is presently debated. The in 1914 when Leo F. Rettger published his seminal paper implication is that the fertile egg can be the site of vertical demonstrating transovarian transmission of Bacterium pull- or transovarian transmission, or for the passage of these orum, the etiological agent of ‘‘white diarrhea,’’ or Pullorum organisms through the shell into the underlying membranes disease, in chickens. Six years earlier, he had published a of the freshly laid, fecally contaminated fertile egg. While article in which he proved the gram-negative bacilli B. is not the only route of Campylobacter isolated from necropsies of affected birds, was contamination for a broiler or broiler breeder flock, it is pullorum, responsible for the disease, fulfilling all of Koch’s postulates definitely one source. On the other hand, many accept that tying a suspect agent to this infection (71). It was noted in certain Salmonella enterica serovars are vertically transmit- earlier work that many of the affected birds failed to absorb ted from parents to offspring. There are several parallels in the yolk sac, a key observation to Rettger’s later 1914 article. our discussions of Campylobacter and vertical transmission with Salmonella and Escherichia coli (7). Leo Rettger was able to isolate B. pullorum, later renamed Salmonella Pullorum, from the yolk sac of chickens with Vertical transmission of an infectious agent, specifically white diarrhea (69). This lead Rettger to the hypothesis that Salmonella Pullorum, was a difficult concept to accept in the parent, particularly the hen, was transmitting the disease 1914. The scientific view then was that infections ‘‘are not to her offspring. Therefore, one would expect to be able inherited,’’ and even the idea that ‘‘germinal infection takes to isolate the pathogen from the ovaries and embryonated eggs in flocks with a history of Pullorum disease, which * Author for correspondence. Tel: 706-546-3484; Fax: 706-546-3772; E-mail: [email protected]. Leo Rettger demonstrated in his 1914 article. He also { Present address: The Coca-Cola Company, 1 Coca Cola Plaza N.W., experimentally infected young chicks with Salmonella Pull- Atlanta, GA 30313, USA. orum and recovered the organisms from the ovaries of those J. Food Prot., Vol. 75, No. 10 CAMPYLOBACTER TRANSMISSION IN EGGS 1897 birds that survived the infection. This approach became the looked. One reason is the difficulty in routinely culturing standard for determining transovarian transmission of an this organism from certain types of samples on a regular infectious agent: isolation of said pathogen from the basis. An ideal cultural procedure for the recovery and reproductive tissue, fertile eggs, and newly hatched offspring. isolation of Campylobacter is not available. Numerous Rettger might have made the fortuitous discovery of vertical plating media, enrichment broths, and procedures have been transmission because the agent Salmonella Pullorum is developed for recovery of Campylobacter. The majority of highly infectious, causes significant mortality, and is these methods were developed for Campylobacter recovery prevalent in tissues of the avian . Had the first Salmonella from fecal samples in which large populations exist; these proven to be vertically transmitted been Salmonella Galli- methods are not adequate for recovery of small numbers, narum, it might have been harder to prove because of the low sublethally injured or stressed cells, or viable nonculturable prevalence of the pathogen in eggs (56, 61). Salmonella cells (24). Better media and methodological approaches are Pullorum and white diarrhea set the precedent that Salmo- useful to evaluate more fully the ecology of Campylobacter nella could be vertically transmitted in poultry from parents within poultry flocks and routes of transmission. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/75/10/1896/1686170/0362-028_jfp-11-322.pdf by guest on 02 October 2021 to progeny. Another reason why the role of the fertile egg in Salmonella Enteritidis emerged in the 1980s to become Campylobacter transmission has not been fully accepted is the major Salmonella serovar causing human illnesses in the that in some cases, the opposite theory influences experi- United States and Europe (4, 5, 22). An epidemiological mental design and data analysis. Callicott et al. (16) stated link was made between human illnesses attributed to that they could not find any evidence for vertical transmission Salmonella Enteritidis and the consumption of grade A of Campylobacter spp. from grandparent flocks in Sweden table eggs (6, 22, 28, 79). However, screens of table and and their progeny in Iceland, despite Campylobacter from breeder eggs revealed Salmonella Enteritidis prevalence was grandparent birds and the parent birds showing identical low (0.04 to 6.5%) and intermittent, even for Salmonella genetic fingerprints. The authors concluded that this was Enteritidis–positive flocks (42, 43, 68). Even with the because of migrating birds rather than vertical transmission. pooling of eggs (10 eggs per pool), Salmonella Enteritidis However, broiler houses in Iceland are not accessible to the prevalence is low and variable (0 to 8.3%) in positive layer environment, so an influence by wild birds is unlikely. Acuff flocks (39). Similarly, Salmonella Enteritidis prevalence et al. (2) reported that no Campylobacter could be found in rates in the ovaries and oviducts of spent hens have also turkey eggs, but only 20 eggs were tested, and then with insensitive sampling methods. Fonseca et al. (33) sampled the been reported to be low, at 3 and 6%, respectively (68). meconium and a pooled sample of heart, spleen, and liver Experimentally, Salmonella Enteritidis egg transmission is from 117 chicks. When Campylobacter was not isolated, they very low (0.7 to 17%) and intermittent, influenced by many concluded that vertical transmission is a rare event. These factors including immunosuppression, strain type, etc. (34– types of studies leave the impression that the influence of the 37, 50, 62, 66, 77). While colonization of the avian fertile egg in the transfer of Campylobacter is highly reproductive tract with Salmonella Enteritidis is higher improbable. However, there are many reports that support in experimentally versus naturally infected birds (8 to the opposite conclusion. The poultry research community 100%), the infection is transient, diminishes with time, and must fairly consider all published works to better understand exhibits wide trial-to-trial, bird-to-bird variability. Other all possible sources of this microorganism and work toward egg-transmitted Salmonella serovars like Gallinarum and providing useful information for Campylobacter reduction in Pullorum also exhibit similar variability in colonization of commercial poultry. the reproductive tract (86). As discussed earlier, other As far back as 1985, it was shown that when 2-day-old factors appear to affect susceptibility to infection, including fertile chicken eggs were inoculated with Campylobacter by host genetics (11). pressure differential, 11% of the resulting chicks had the Other Salmonella serovars have also been shown to be inoculated microorganism in their intestinal tract on transmitted vertically from culture-based studies of naturally hatching (20). In 1986, Lindblom et al. (54) raised chickens (8, 67, 78, 85) or artificially (34, 37, 62) infected laying in isolation units without exposure to any farm environment hens or epidemiological investigations linking breeders as and they were still colonized by Campylobacter. Maruyama the source of Salmonella (22, 29, 51–53, 57, 75). Central to and Katsube in 1990 (55) demonstrated that Campylobacter these epidemiological studies has been identification of could be transmitted to the offspring via the fertile egg after phenotypic or genotypic traits that can further differentiate orally inoculating Japanese quail breeder hens. These Salmonella isolates to serovar or strain level (31, 38, 75, findings were supported further by Idris et al. (44). 80). Finally, the best proof that transmission is vertical is Studies using colony DNA hybridization indicated the when an intervention applied at the breeder level reduces carrier rate of Campylobacter in the cecal contents of newly transmission to eggs or progeny birds (12, 30, 81). hatched chicks to be as high as 35%, suggesting that the chicks were already contaminated with Campylobacter CAMPYLOBACTER ASSOCIATION WITH THE before they were delivered to the farm (19). In another FERTILE EGG study (26), Campylobacter isolates from two independent Relative to Salmonella, Campylobacter in poultry has commercial broiler breeder flocks, as well as their respective not been extensively studied. There are several reasons why progeny, were shown to be indistinguishable with both vertical transmission of Campylobacter has been over- ribotyping and DNA sequencing analysis. 1898 COX ET AL. J. Food Prot., Vol. 75, No. 10

Another line of epidemiological evidence for vertical interior egg contents and 3.8% of the egg surfaces were transmission of Campylobacter from breeder flocks to the contaminated with this microorganism. Furthermore, Cam- progeny comes from a few observations that the isolates pylobacter was culturally isolated from one of the from both the breeders and broilers had the same serotypes commercial incubators in that study. or genotypes. Pearson et al. (64, 65) performed multiyear Cultural isolation of Campylobacter from commercial studies with over 1.4 million broilers and published hatchery tray liners (15) strongly suggests the possibility of evidence for the involvement of both vertical and horizontal egg transmission. Two thousand tray liners were sampled, transmission (64). Once the conditions for horizontal which represented a screening of approximately 200,000 transmission were under control, a pattern of intermittent broiler chicks. By using a modified methodology procedure shedding within the same broiler flock and the lack of that incorporated a preenrichment step, these researchers diversity of types isolated during the entire study period were able to culturally detect Campylobacter from three of became apparent, strongly suggesting a common source of the eight different commercial hatcheries. Ordinarily, a Campylobacter, most likely egg transmission. Furthermore, preenrichment step is not a part of the normal protocol. Two the isolation rate of Campylobacter in market-age broilers of the three positive hatcheries were sampled, and breeder Downloaded from http://meridian.allenpress.com/jfp/article-pdf/75/10/1896/1686170/0362-028_jfp-11-322.pdf by guest on 02 October 2021 supplied by hatchery B (42.9%) was significantly higher lots were positive for Campylobacter in both. Overall, live than that in broilers supplied by hatchery A (17.6%) in the Campylobacter organisms were cultured from 0.75% of same study. In two instances, when both hatcheries were the tray liners. Taking into account only samples in which used to hatch chicks to stock the same farm flock, the breeder lots were positive, 1.6% of the tray liners were Campylobacter was found only in those sheds with chicks positive. From this study, the incidence rate of egg from hatchery B. Together, the results suggested that there transmission could be as high as 1 per 133 eggs or as was a common source of infection for the broiler farm. low as 1 per 13,333 eggs. Hatcheries receiving eggs laid by Through molecular testing, Campylobacter has been positive broiler breeder lots could have a much higher found in hatchery fluff, intestinal tracts of developing incidence rate (15). When evaluating just positive breeder embryos, and newly hatched chicks (18, 19, 40, 41). In one lots, the incidence rate could range from 1 per 61 to 1 per of these studies (41), PCR with a set of primers specific for 6,133 eggs. Overall, the incidence rate is more likely on the the Campylobacter flaA short variable region was used to low end, explaining why, in some studies, the recovery of detect the presence of Campylobacter in 40 of 40 fluff Campylobacter from the hatchery or from the fertile egg has samples, and 28 of 40 eggshell samples. These 40 samples not been successful. were taken from four separate commercial broiler-hatching Approximately 9 billion fertile eggs are hatched each cabinets. In a follow-up study by the same group (40), the year in the United States; this could hypothetically mean presence of Campylobacter DNA was detected in the that 675,000 broilers could have acquired Campylobacter majority of stomached intestinal contents of unhatched through the fertile egg, by a conservative evaluation of the chicken embryos at 7, 15, and 19 days of incubation. results obtained in the Byrd et al. study (15). It has been In another very convincing study, researchers (44) shown that even if a single bird in a flock is colonized, then wanted to determine whether the presence of Campylobacter the spread of Campylobacter to adjacent rearing mates DNA in young chicks was because of ingestion of the could reach 100% within a few weeks (58, 84). Therefore, a bacteria in food or water. They obtained commercial broiler transmission rate of even 1 in 13,333 should be considered hatching eggs and incubated them in a research facility until significant, as more than 20,000 broilers represent a flock the chicks hatched. Analysis of the ileal, cecal, and yolk within a single modern broiler house. contents of the day-of-hatch chicks revealed that Campylo- It has been stated that the 2- to 3-week log phase in bacter DNA was present before the chicks consumed any observable infection is evidence for refuting vertical food or water. In the same study, in order to determine transmission (60). We believe this is incorrect. The fact whether the same strains of bacteria were present in multiple that a low transmission rate exists, along with an insensitive sites associated with the integrated broiler company, method of sampling a flock (drag swab), more than likely Campylobacter was cultured from a flock of broiler breeders and their 6-week-old progeny residing on a commercial explains why Campylobacter is not detected within a broiler broiler farm. The broiler breeders had been given fluoro- flock until chicks are almost 4 weeks of age. quinolone antibiotics, and researchers wanted to determine HORIZONTAL TRANSMISSION OF whether the same fluoroquinolone-resistant strain was present CAMPYLOBACTER in their progeny. Isolates were typed by pulse-field gel electrophoresis, which confirmed that the parental and Clearly, horizontal transmission is also a source. Some progeny flocks contained an indistinguishable strain of of the potential environmental sources are other farm fluoroquinolone-resistant Campylobacter coli. animals, domestic pets, wildlife, insects, houseflies, farm Campylobacter has been detected by traditional culture workers, vehicles and equipment on the farm, old litter, methods in 4 of 12 samples of hatchery debris (fluff) in feed, and water. With reference to other farm animals such the United States (10) and in New Zealand (9), where 2 of as cattle, identical genotypes were found from the cattle and 8 samples were culture positive for Campylobacter. In the broilers from the same farm; therefore, the cattle were addition, in 2005, Acevedo (1) sampled 960 commercial believed to be a source of infection for the broilers (82). breeder eggs for Campylobacter and found that 1.6% of the However, the authors realized that the mode of spread was J. Food Prot., Vol. 75, No. 10 CAMPYLOBACTER TRANSMISSION IN EGGS 1899 not known, and it could have been from the broilers to the rRNA-PCR, it did not appear that litter was responsible cattle. In another study, Campylobacter jejuni isolated from for the transfer of contamination to successive flocks in the cattle was found to differ from the isolates recovered from same poultry house (63). the broilers on the same farm, thereby disputing the role of cattle as a source of poultry infection (45, 59). In still other ASSOCIATION OF CAMPYLOBACTER WITH studies (46, 82), no significant association was found THE REPRODUCTIVE TRACT between colonization of broilers by C. jejuni and presence AND INTERAL ORGANS of pigs on the same farm. In addition, pigs are generally Traditionally, Campylobacter was thought to colonize infected with C. coli instead of C. jejuni. Other farm animals primarily the chicken’s intestinal tract (i.e., ceca and ileum). such as cats, dogs, horses, etc., can also be infected with In the past decade, evidence has emerged that clearly Campylobacter, but their role as a source of broiler demonstrates that Campylobacter colonization is not limited contamination has not been established. to the gastrointestinal tract, but also appears in many tissues Insects such as flies, beetles, etc., can act as mechanical

and organs throughout a bird’s body. Campylobacter spp. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/75/10/1896/1686170/0362-028_jfp-11-322.pdf by guest on 02 October 2021 vectors and can transmit Campylobacter from various were cultured from primary (thymus) and secondary animals to chickens (45, 76). Even though identical (spleen) lymphoid organs, as well as from the livers and serotypes and genotypes of Campylobacter have been gallbladders of commercial broiler breeder hens (25). From isolated from insects and broilers within broiler houses, 43 samples taken, Campylobacter was found to occur the direction of spread was not determined (3, 13, 74). In naturally in 11, 8, and 4 of the thymus, spleen, and liver and fact, the insects in a chicken house were usually not positive gallbladder samples, respectively. C. coli was isolated more for Campylobacter until after the broilers were determined frequently than C. jejuni. Twenty-eight of the 53 isolates to be positive (59). Therefore, insects might not be were C. coli, while only 25 were C. jejuni. In an earlier important as an original source of Campylobacter for a study, Campylobacter was cultured from all the various broiler house (73, 76). sections of the reproductive tract of actively laying Rodents can carry Campylobacter in their intestines, commercial breeder hens (14). In this study, the number and they have been considered sources of contamination in of positive samples decreased as sampling ascended the broiler houses (49, 59); however, Jones et al. (48) reported reproductive tract, and this suggests fecal contamination that Campylobacter was not isolated from rodents found could be playing an important role in this trend. In another near broiler houses. When one considers both the effective study (23), naturally occurring Campylobacter was isolated vermin-control programs in most commercial poultry from 26% of the mature and 12% of the immature ovarian production facilities and the limited access of rodents into follicles of commercial broiler breeder hens. broiler houses, the role of rodents as a common source of From a total of 275 semen samples collected from infection for multiple broiler flocks becomes questionable commercial broiler breeder roosters, 9.47% contained (32). The same can be said for wild birds. Campylobacter is substantial numbers of Campylobacter organisms. Levels prevalent in wild birds (17, 87); however, birds are not were found to be as high as 3.1 log CFU/ml of semen, with normally found in well-maintained modern broiler houses. an average of 1.2 log CFU/ml (27). By using electron In addition, wild birds near broiler houses are often microscopy, C. jejuni was shown to attach to chicken contaminated with a Campylobacter that is different from spermatozoa (83). Fecal contamination of the semen and not those of chicken origin (59, 74). the male reproductive organs was thought to be the primary Farm workers have been implicated as an environmen- source of the Campylobacter, as 96.6% of pooled semen tal source of Campylobacter for the broiler flock. This samples were positive for Campylobacter; however, Cam- would include both the farm worker that manages several pylobacter was culturally isolated from the testes (11.8 ) flocks on a daily basis as well as farm workers that load % and vas deferens (47.1 ) of turkey toms (21). birds for transport prior to slaughter. The organism has been % isolated from footbath water, farmers’ boots, and transport In a recent study (72), 223 market-age commercial crates (45, 82); therefore, it is reasonable to assume that broilers were sampled with aseptic techniques, and from 25 Campylobacter can spread between flocks via personnel if of these broilers, Campylobacter was cultured from the the staff does not change clothes and boots when going from bloodstream, providing insight to a possible means by one flock to another (13). In contrast, a recent study did which this organism is able to disseminate rapidly to an show that two adjacent broiler houses that lacked biosecur- assortment of tissues and organs within a bird. Clearly, this ity procedures were colonized with different genotypes of C. demonstrates that Campylobacter is not strictly limited to jejuni, even though the houses shared equipment and the the digestive and reproductive tracts. farmer worked both houses wearing the same boots (59). SUMMARY Feed, litter, and water have also been mentioned as possible sources of environmental contamination of broilers Although transovarian transmission occurs, it is not with Campylobacter. Because of their low moisture content, the only means of egg contamination. Feces can easily feed and fresh litter are unlikely sources for the introduction contaminate the surface of an egg, because the egg and feces of Campylobacter into broiler houses (47, 82). In addition, both pass through the cloaca. Bacterial contamination, in a study in which Campylobacter isolates were typed by including Campylobacter, can be drawn through the shell by randomly amplified polymorphic DNA-PCR and 235 temperature differential, aided by the presence of moisture 1900 COX ET AL. J. Food Prot., Vol. 75, No. 10

(the sweating of an egg). Campylobacter could have a 16. Callicott, K. A., V. Frioriksdottir, J. Reierson, R. Lowman, J. R. difficult time surviving on the dry shell or in the hostile Bisaillon, E. Gunnarsson, E. Berndtson, K. L. Hiett, D. S. Needle- man, and N. J. Stern. 2006. Lack of evidence for vertical transmission albumen, but certainly can survive in the moist eggshell of Campylobacter spp. in chickens. Appl. Environ. Microbiol. 72: membranes. Then, when the chick emerges from the egg, it 5794–5798. can ingest the Campylobacter entrapped in the shell 17. Chuma, T., S. Hashimoto, and K. Okamoto. 2000. Detection of membranes and become colonized; it can then spread this thermophilic Campylobacter from sparrows by multiplex PCR: the contamination to flock mates, possibly through ingestion of role of sparrows as a source of contamination of broilers with cecal droppings. Thus, egg passage can occur without Campylobacter. J. Vet. Med. Sci. 62:1291–1295. 18. Chuma, T., K. Makino, K. Okamoto, and H. Yugi. 1997. Analysis of involving the reproductive tract or the follicles of the hen. distribution of Campylobacter jejuni and Campylobacter coli in This is expected to occur in a relatively small number of broilers by using restriction fragment length polymorphism of birds placed in a grow house, making it unlikely that an flagellin gene. J. Vet. Med. Sci. 59:1011–1015. insensitive sampling method such as a drag swab would 19. Chuma, T., T. Yamada, K. Yano, K. Okamoto, and H. Yugi. 1994. A reveal Campylobacter immediately. survey of Campylobacter jejuni in broilers from assignment to

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