Newly hatched broiler chicks may be exposed to Campylobacter from various sources in the hatchery and grow-out environments. Chicks may come in contact with Campylobacter in the air or in the droppings of other birds which chicks may eat or sit on. It is not clear how airborne, cloacal and oral exposure to Campylobacter may affect subsequent cecal colonization. In this study, a marker strain of Campylobacter coli, naturally gentamicin resistant (CcGR), was introduced into 585 day-of-hatch chicks through one of four body openings {mouth (with and without CaCO3 ), nasal passage, eye and cloaca} to simulate oral, cloacal and airborne exposure. Campylobacter coliGR was introduced by each route of exposure at three different inoculum levels (approximately 2 x 101 , 2 x 102 and 2 x 103 colony forming units (cfu)/bird). All chicks were humanely euthanized 7 d, ceca were removed and sampled for the presence and numbers of CcGR by serial dilution onto Campy-cefex agar plates with 200 ppm gentamicin. Three replications were conducted (n=225, n=225 and n=135 for experiments 1, 2 and 3 respectively). All routes of exposure that were tested resulted in cecal colonization of 7 d old broilers. The nasal passage produced the lowest level of cecal colonization requiring higher inoculum levels of CcGR for colonization. These data suggest that CcGR can readily colonize the ceca of day-of-hatch broiler chicks when exposed by mouth, cloaca, or breathing/blinking in airborne cells. Therefore, multiple intervention strategies may be required to interrupt exposure and colonization of young broilers by Campylobacter.

Cosby DE, Cox NA, Harrison MA, Berrang ME, Wilson JL (2017) Colonization of Day-Old Broiler Chicks with Campylobacter coli through Different Inoculation Routes. J Vet Med Res 4(7): 1096.

• Campylobacter coli
• Colonization
• Ceca
• Broiler chicks

mL: Milliliter; cfu: Colony Forming Unit; CcGR: Gentamicin Resisitant Campylobacter coli; h: Hours; d: Days; CCAG: Campylobacter Cefex Agar W/ Gentamicin; NB: Nutrient Broth; TB: Tecra® Broth; IU: Isolation Unit; w/v: Weight/Volume; C: Celsius

Campylobacter is a leading cause of bacterial induced diarrheal disease in the United States and worldwide [1,2]. Campylobacter jejuni is the leading organism isolated from humans and C. coli is the second leading causative agent for human diarrheal illness [3]. Poultry has long been implicated as a major source of transmission for Campylobacter to humans [4].

The routes of entry of this organism to broiler chickens have not been studied but Cox et al., [5] did examine the ability of Salmonella species to enter and colonize the ceca of broiler chicks by various body openings. They found that marker strains of nalidixic acid resistant Salmonella were able to enter and colonize ceca of day-of-hatch broiler chicks through the mouth, eyes, nares, and cloaca, consistently. Bailey et al. [6], found that after intracloacal and oral inoculation, Salmonella could be recovered from the thymus, spleen, liver/gall bladder, ceca and bursa in as little as 1 h post inoculation. Cox et al. [7], found naturally occurring Salmonella and Campylobacter in the internal organs of commercial broilers aged 6 and 8-week.

Research efforts to diminish the presence of this organism on processed poultry have had limited success partly because there is limited information regarding the entry of Campylobacter into commercial poultry flocks, particularly because it is difficult to detect naturally occurring Campylobacter until 2-3 weeks of age [8]. Husbandry, cleaning and disinfection of grow-out houses, ventilation, and colonization of parent flocks are factors often examined when trying to eliminate or delay the colonization of chicks in a broiler house [9, 10, 11, 12]. The goal would be to delay the event sufficiently to avoid any contaminated birds entering the processing plant.

The purpose of the experiment was to determine likely avenues of entry of Campylobacter into day of hatch broiler chicks.

Bacterial strains, growth and maintenance

Maintenance: Gentamicin resistant Campylobacter coliGR (CcGR) [13] was maintained on Campy-CEFEX agar with 200 µg/mL gentamicin (CCAG) added. CcGR were grown at 42o C for 48 h in a microaerobic atmosphere (85% N, 10% O2 , 5% CO2 ). CcGR were maintained frozen at -80o C in nutrient broth (NB, Becton-Dickinson, Franklin Lakes, NJ) with 15% glycerol (Sigma Chemical Co., St. Louis, MO).

Inoculum Preparation: Inocula were prepared according to laboratory standard operating procedures. Briefly, a culture was streaked from the stock culture maintained in the -80o C freezer onto CCAG, incubated at 42o C for 48 h under microaerobic conditions. Inocula were prepared from this plate by choosing several well isolated colonies and suspending these colonies in enough sterile 0.85% saline (NaCl, Sigma Chemical Co., St. Louis, MO) to create a suspension with an optical density of 0.20 at 540nm with a Spect-20 (Milton-Roy Spectrophotometer, Thermo Spectronics, Madison, WI). This provides approximately 2.0 x 108 colony forming units (cfu)/mL of solution as determined by use of a standard curve (data not shown). Serial dilutions were prepared to approximately one log cfu/mL above the desired inoculum levels of 101 , 102 and 103 cfu/bird; 0.1 mL of this solution was used to inoculate the chicks via various routes. Chicks (n=15 per isolation unit (IU)) were inoculated with 0.1 mL orally (with and without 5% CaCO3 buffer (Sigma Chemical Co.)), ocularly (through the eye, drop wise with the inoculum allowed to enter before another drop was administered), intranasally (through the nares, drop wise with the inoculum allowed to enter before another drop was administered) or intracloacally (by insertion of approximately 1 cm of a tuberculin syringe into the vent of the chick). The oral inoculation was administered with and without 5% CaCO3 . The addition of 5% CaCO3 was to mimic the effect of eggshell as a buffer when ingested with bacteria in a hatchery environment. The inocula were plated after appropriate serial dilutions onto CCAG for enumeration and incubated at 42o C for 48 h under microaerobic conditions.

Animals and animal husbandry

All day of hatch chicks were obtained from a local commercial broiler hatchery and transported on clean cardboard bedding pads in reusable chick transportation trays. Chick transport bedding pads (n=3) were sampled on day of placement by enrichment in 500 mL of Tecra Broth™ (TB, 3M Corporation, St. Paul, MN) and subsequent plating on Campy-CEFEX agar plates without gentamicin for detection of naturally occurring Campylobacter. Ceca from ten (n=10) chicks were aseptically removed and sampled as described below. Chicks were housed in separate IU on wire mesh flooring (15 chicks per IU) with ad libitum access to feed (non-medicated starter/grower crumbles and pellets, University of Georgia Poultry Science Feed Mill, Athens, GA) and water on a 24 h light regimen. Standard husbandry practices for growth were followed with birds being culled for disease and physical abnormalities which would lead to poor growth performance. At 7 d, all birds were humanely euthanized by cervical dislocation.

Isolation and enumeration

Seven (7) days post inoculation, all chicks per IU were humanely euthanized by cervical dislocation, ten chicks (n=10) were surface disinfected with 70% ethanol (Pharmco-Aaper®, Shelbyville, KY) and the ceca aseptically removed. Ceca were placed into Stomacher 80® sample bags (Seward Laboratory Systems, Inc., Port Saint Lucie, FL) and transported to the laboratory on ice. In the laboratory, ceca were weighed individually, macerated, diluted 1:10 (w/v) with TB, stomached for 60 s and serially diluted in 0.85% sterile saline and plated in duplicate on CCAG for enumeration. All plates were incubated in a microaerobic environment at 42o C for 48 h. Bags containing ceca and TB were likewise incubated in a microaerobic environment at 42o C for 48 h as an enrichment. Enriched cecal samples were streaked for isolation onto CCAG to detect low levels of Campylobacter spp. One of each colony type per plate was examined by wet mount microscopy and latex agglutination (Microgen Campylobacter Latex Agglutination Kit, Microbiology International, Frederick, MD) for confirmation as Campylobacter spp.

Experimental design

Three replications were conducted. In replications 1 and 2 three IU (n=15 birds) were used for each of three inoculum levels (101 , 102 and 103 cfu/bird) and each of four body openings (n=225/replication). For each replication four body openings (mouth, eye, nares and cloaca) were used as the routes of of entry for the inoculum. Ten chicks (n=10) per treatment/IU were sampled for the presence of CcGR by serial dilution and enrichment to determine presence or absence of CcGR in the ceca. Average cfu/ mL of cecal material was calculated by counting the appropriate dilution. Samples with no growth on enumeration plates were streaked for isolation from the enriched samples onto fresh CCAG plates and incubated at 42o C for 48 h, microaerobically. Any sample with no visible growth after incubation of the enriched sample was determined to be negative and assigned an average colonization value of 0. Based on the data showing 100% colonization with the intracloacal route of inoculation in Replications 1 and 2, Replication 3 used only two IU (n=15 birds) and two inoculum levels, 101 and 103 cfu/bird for the oral, ocular and nasal routes of entry and only one IU (n=15 birds) for the 103 cfu/bird for the intracloacal route. This reduction of animals used is in line with the USDA, ARS guidelines for the humane use of animals.

Cox et al. [13], identified a marker strain of CcGR which was found to have a stable resistance to gentamycin and to be a reliable colonizer of day of hatch broiler chicks and this strain was used in this experiment. Results of inoculation by oral gavage with and without CaCO3 are shown in Table 1. When day old broiler chicks were orally gavaged with 101 cfu/bird, only birds in one of the three groups were colonized. Birds in the subsequent groups which received 102 or 103 cfu/bird were colonized and with an average of log 7.2 cfu/mL of CcGR marker in the ceca. When CaCO3 was included in the inoculation broth, all birds challenged with CcGR were colonized by the marker strain with an average of log10 7.7 cfu/mL in the cecal material. Calcium carbonate appears to serve as a buffering agent against the hostile pH of the gizzard which Cox et al. [14], determined to be approximately pH 1.9 by direct measurements. These results are somewhat different than those observed with a Salmonella marker when CaCO3 was added to the inoculum [5]. Calcium carbonate did not increase the chances of a chick becoming colonized with Salmonella but it appears to increase the chance a chick may be colonized with Campylobacter. This is understandable since Campylobacter is a more fragile microorganism and may be more sensitive to the acidic conditions in the provetnriculus and/or gizzard.

Intraocular inoculation of CcGR ranged from 15 to 2000 cfu/bird and resulted in the colonization of 70 of the 80 chicks challenged through this route (Table 2). Additionally, this suggests it is possible to produce potential seeder birds by entry of low numbers of bacteria such as Campylobacter and Salmonella [5] into the eyes of young chicks. This route of entry is significant in that there may be many airborne bacteria present in the various environments that young chicks are exposed to, such as hatching cabinets, hatcheries and grow-out houses. This provides an ideal environment for early colonization of birds without an oral challenge.

Table 3 shows the cecal colonization of day of hatch chicks when CcGR were introduced into the trachea via the nares. This route of inoculation produced fewer colonized chicks with none colonized at the lowest inoculation level of approximately 101 cfu/bird (statistical difference was not determined). Similar to intraocular inoculation this route of entry may be of biological significance because of airborne microorganisms in the chick’s environment and the nares provide a moist body opening for bacteria to enter the chick’s body. Cox et al. [5], used two different methods for inoculating the respiratory tract of chicks, using a small tube through the cleft palette and by fogging with a fine mist, and the results with Salmonella obtained were similar to the data in this experiment where the lower inoculum levels did not consistently result in colonization.

When CcGR was introduced intracloacally, all of the chicks were colonized even when the inocula contained only 15 cfu/ bird (Table 4). This route of entry is significant because of the anti-peristalsis present in the cloaca of the young chicks [15]. Microorganisms on any surface could be introduced by this antiperistaltic action into the cloaca and not be subject to the low pH, high acidity, of the proventriculus and/or gizzard of the chick’s intestinal tract.

Regardless of the route or source, preventing the exposure of newly hatched chicks to Campylobacter is critical because the young bird lacks a mature gut microflora and as such is highly susceptible to colonization. This study clearly demonstrates that a marker strain of Campylobacter (CcGR) can colonize the intestinal tract of young broiler chicks through an assortment of body openings. There is potential for seeder birds to be produced by bacterial contamination which may occur through any of the four body openings utilized in this study and may occur early in the chick’s life. This contamination can then be spread throughout the broiler house to both the outside and/or inside of the flock mates present. Further studies are needed to develop intervention strategies to prevent colonization by any and/or all of these openings of the body.

Gentamycin resistant Campylobacter coli can readily colonize the ceca of day-of-hatch chicks by different routes of inoculation and in particular, routes other than the fecal – oral route.

As few as 101 cfu/ bird are able to colonize the ceca of dayof-hatch chicks through three of the four body openings tested.

Calcium carbonate appears to act as a buffer to the low pH of the gastric system allowing CcGR to more easily colonize the ceca.

Preventing Campylobacter species from colonizing chicks and chickens is going to take more research in laboratories, in the hatcheries, on farms and in the processing plant. Having this gentamycin resistant marker will make this research easier.

1. Havelaar AH, Ivarsson S, Löfdahl M, Nauta MJ. Estimating the true incidence of campylobacteriosis and salmonellosis in the European Union, 2009. Epidemiol Infect. 2013; 141: 293-302.

2. Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson MA, Roy SL, et al. Foodborne illness acquired in the United States--major pathogens. Emerg Infect Dis. 2011; 17: 7-15.

3. Gillespie IA, O’Brien SJ, Frost JA, Adak GK, Horby P, Swan AV, et al. A case-case comparison of Campylobacter coli and Campylobacter jejuni infection: a tool for generating hypotheses. Emerg Infect Dis. 2002; 8: 937-942.

4. Engberg J. Contributions to the epidemiology of Campylobacter infections. A review of clinical and microbiological studies. Dan Med Bull. 2006; 53: 361-389.

5. Cox NA, Bailey JS, Berrang ME. Alternative routes for Salmonella intestinal tract colonization of chicks. J Appl Poult Res. 1996; 5: 282- 288.

6. Bailey JS, Cox NA, Cosby DE, Richardson LJ. Movement and persistence of Salmonella in broiler chickens following oral or intracloacal inoculation. J Food Prot. 2005; 68: 2698-2701.

7. Cox NA, Richardson LJ, Buhr RJ, Northcutt JK, Bailey JS, Cray PF, et al. Recovery of Campylobacter and Salmonella serovars from the spleen, liver and gall bladder, and ceca of six and eight week old commercial broilers. J Appl Poult Res. 2007; 16: 477-480.

8. Stern NJ, Fedorka-Cray P, Bailey JS, Cox NA, Craven SE, Hiett KL, et al. Distribution of Campylobacter spp. in selected U.S. poultry production and processing operations. J Food Prot. 2001; 64: 1705-1710.

9. Humphrey TJ, Henley A, Lanning DG. The colonization of broiler chickens with Campylobacter jejuni: some epidemiological investigations. Epidemiol Infect. 1993; 110: 601-607.

10. Jacobs-Reitsma WF, Bolder NM, Mulder RW. Cecal carriage of Campylobacter and Salmonella in Dutch broiler flocks at slaughter: a one-year study. Poult Sci. 1994; 73: 1260-1266.

11. RE Smitherman, CA Genigeorgis and TB Farver. Preliminary observations on the occurrence of Campylobacter jejuni at four California chicken ranches. J Food Prot. 1984; 47: 293-298.

12. Van De Giessen AW, Tilburg JJ, Ritmeester WS, van der Plas J. Reduction of campylobacter infections in broiler flocks by application of hygiene measures. Epidemiol Infect. 1998; 121: 57-66.

13. Cox NA, Richardson LJ, Berrang ME, Fedorka-Cray RJ, Buhr RJ. Campylobacter coli naturally resistant to elevated levels of gentamicin as a marker strain in poultry research. J Food Prot. 2009; 72: 1288- 1292.

14. Cox NA, Davis BH, Watts AB, Colmer AR. Salmonella in the laying hen. 2. The effect of simulated digestive tract pH levels on the survival of the three species of Salmonella. Poult Sci. 1972; 51: 1268-1270.

15. Schaffner T, Mueller J, Hess MW, Cottier H, Sordat B, Ropke C. The bursa of Fabricius: A central organ providing for contact between the lymphoid system and intestinal contents. Cell Immunol. 1974; 13: 304-312.