Routes of transmission of Salmonella and Campylobacter in breeder turkeys

 C 2016 Poultry Science Association Inc.

Routes of transmission of Salmonella and Campylobacter in breeder turkeys M. D. Crespo,∗ S. Kathariou,† J. L. Grimes,∗ N. A. Cox,‡ R. J. Buhr,‡ J. G. Frye,‡ W. G. Miller,§ C. R. Jackson,‡ and D. P. Smith∗,1 ∗

Primary Audience: Researchers, Flock Supervisors, Quality Assurance and Laboratory Personnel, Veterinarians SUMMARY Salmonella and Campylobacter are frequent colonizers of the intestinal tracts of poultry and have often been associated with human foodborne illness. The entry, transmission, and prevalence of both pathogens have been extensively studied in chickens but little information is available for turkeys. This project monitored turkey breeder hens and toms from d of hatch to 65 wk of age with the objective of determining routes of transmission for Salmonella and Campylobacter throughout the turkey production cycle. Breeder poults were separated by sex and then into 2 groups (control and inoculated) for each sex. The inoculated group was orally gavaged with marker strains of both Salmonella and Campylobacter. The inoculated groups (toms and hens) were placed on the opposite side of a growout house from the uninoculated groups. Fecal samples, intestinal samples and organs, feed, drinkers, and potential vectors such as insects and mice, were analyzed at different times until 65 wk. Monitoring showed that Campylobacter spread rapidly and cross-contaminated turkeys throughout the growout house. For both Salmonella and Campylobacter, naturally occurring strains that were first isolated in control groups at wk 3 and 4, respectively, outcompeted marker strains several wk post inoculation and persisted in the flock. The most common naturally occurring strains were C. jejuni (tetracycline resistant), C. coli (kanamycin resistant), and S. Agona. Campylobacter and Salmonella also were isolated from flies and from a mouse, confirming the importance of proper pest control and biosecurity to reduce the spread of the bacteria. Key words: Salmonella, Campylobacter, breeder turkeys, routes of transmission 2016 J. Appl. Poult. Res. 0:1–19 http://dx.doi.org/10.3382/japr/pfw035

DESCRIPTION OF PROBLEM Salmonella and Campylobacter are foodborne zoonotic pathogens of high public health 1

Corresponding author: [email protected]

relevance worldwide, both ranking among the top 5 pathogens contributing to foodborne disease in the United States [1–3]. Frequent colonization of intestinal tracts of poultry with these bacteria makes poultry meat an important vehicle for infection [1, 2, 4]. Furthermore,

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Prestage Department of Poultry Science North Carolina State University 2711 Founders Drive, Raleigh 27695; † Department of Food, Bioprocessing, and Nutrition Sciences North Carolina State University 400 Dan Allen Drive, Raleigh 27695; ‡ USDA-ARS, Russell Research Center 950 College Station Road, Athens, GA 30605; and § USDA-ARS, Western Regional Research Center 800 Buchanan Street, Albany, CA 94710

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MATERIALS AND METHODS Inoculation and Monitoring This study was approved by the NCSU Institutional Animal Care and Use Committee (IACUC). A flock of 140 Nicholas turkey breeder poults [21] were placed in a growout house of the Turkey Research Unit at North Carolina State University, Raleigh [22]. Prior to beginning the project, the house was spraydisinfected [23], and environmental samples, including drinkers and feed, were analyzed for the presence of Salmonella and Campylobacter. At arrival, artificial straw from the transportation boxes that contained feces and fecal samples during the first wk of life were analyzed for both bacteria. Poults were placed into pens separated by sex and then separated further into 2 groups: Inoculated (82 hens and 22 toms) and control (28 hens and 8 toms) (Figure 1). Inoculated and control groups were separated by plastic curtains; a tray containing quaternary ammonium disinfecR tant (PI quat 20 ) for boot immersion was located at the end of the inoculated side for passing through when leaving this area. Control groups were serviced before inoculated groups to prevent cross-contamination. Moreover, boot covers and other personal protective equipment (gloves, coveralls) were required to enter the pens of inoculated turkeys and were removed when leaving the inoculated side of the house. Inoculated

Figure 1. Distribution of pens of control and inoculated turkeys in the growout house, wk 0 to 12. The number of turkeys per pen is indicated in parentheses. CT, control toms; CH, control hens; IT, inoculated toms; IH, inoculated hens.

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pre-harvest colonization of the flock by these pathogens has been found to be associated with contamination of poultry carcasses during processing [5–13]. Since 2011, USDA Food Safety and Inspection Service (FSIS) regulations require turkey processing companies not to exceed certain levels of Salmonella and Campylobacter in raw products, as monitored by testing programs [14]. A number of methods, including chemical antimicrobial interventions, have been used to control these pathogens at the processing plant. However, reducing or eliminating both pathogens from birds prior to processing is potentially more beneficial than excessive plant interventions. Further understanding about entry, transmission, and overall prevalence of both pathogens in the production chain may help to determine risk factors and lead to methods for prevention and/or reduction of pathogenic bacteria colonizing poultry. Many different routes of transmission for these pathogens into the flock such as vertical transmission, pests, wild animals, feed and water, farm workers, and environment have been previously considered and investigated, especially in chickens [15–20]. However, there is little information regarding turkey flocks. The objective of this project was to determine routes of transmission for Salmonella and Campylobacter throughout turkey production and processing. The focus of the current paper was horizontal transmission while vertical transmission will be evaluated in a different manuscript.

JAPR: Field Report

CRESPO ET AL.: TURKEY BREEDER PATHOGENS 1 T, tetracycline; S, streptomycin; K, kanamycin; Q, (fluoro) quinolones (nalidixic acid and ciprofloxacin); G, gentamicin; NAL, nalidixic acid. Acronyms indicate that the strain was resistant to these specific antibiotics but not to others used in the testing panel. Thus, GK indicates that the strain was resistant to gentamicin and kanamycin but susceptible to tetracycline, streptomycin, erythromycin, nalidixic acid and ciprofloxacin, while TSKQ indicates resistance to tetracycline, streptomycin, kanamycin, nalidixic acid and ciprofloxacin but susceptibility to erythromycin and gentamicin.

2 (5.6 × 108 ) 2 (3.6 × 108 ) 0.1 (9.4 × 106 ) 0.1 (1.2 × 107 ) S. Typhimurium NAL∗ S. Enteritidis NAL∗ 2 (1.3 × 108 ) 2 (4.8 × 108 ) 0.1 (9.2 × 106 ) 0.1 (1.4 × 107 ) C. jejuni 10882 TSKQ1 C. coli 12456 GK1 Toms Hens

Second inoculation (12 wk) First inoculation (10 d) Salmonella serotype (Nalidixic acid resistant) Second inoculation (12 wk) First inoculation (10 d) Campylobacter strain and antibiotic resistance profile Sex of inoculated turkeys

mL of inoculum gavaged per bird (cfu/mL) mL of inoculum gavaged per bird (cfu/mL)

Table 1. Campylobacter and Salmonella strains used for inoculation of turkeys.

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toms (IT) and inoculated hens (IH) were orally gavaged at 10 d of age with 0.1 mL of an inoculum containing the Salmonella marker strain (approx. 107 cfu/mL) and the Campylobacter strains (approx. 107 cfu/mL) (Table 1). S. enterica ser. Enteritidis resistant to nalidixic acid (S. Enteritidis NALR ) and C. coli resistant to gentamicin and kanamycin (GK) [24] were administered to IH, while nalidixic acid-resistant S. enterica ser. Typhimurium (S. Typhimurium NALR ) and a strain of C. jejuni resistant to tetracycline, streptomycin, kanamycin, and quinolones (nalidixic acid and ciprofloxacin) (TSKQ) were administered to IT. S. Enteritidis was chosen for inoculating hens due to the higher propensity of this serotype in colonizing the reproductive tract and transmission through shell eggs in layers [25, 26]. Control toms (CT) and hens (CH) were orally gavaged with the same volume of phosphate-buffered saline (PBS) [27] to simulate the same stressors, handling and gavage. Turkeys were fed diets formulated without antibiotics or growth promoters. At wk 12, birds in the same pen were distributed into 2 pens to reduce the number of turkeys per pen. The distribution of pens in the house and number of turkeys per pen is shown in Figure 2. Due to the reduction in number of positive fecal samples for the inoculated marker strains, IH and IT were inoculated again via gavage with 2 mL of an inoculum containing the same strains of bacteria used for the first inoculation but in higher concentration (approx. 108 cfu/mL) (Table 1). At wk 21 both IH and CH hens, were moved to a dark-out house where a step-down lighting program was applied. This house had solid side walls and light traps covering fans and air inlet systems for total light control. Hours of light were controlled by a time switch [28]. Hens received 8 h of light per d for the first five wk (wk 21 to 25), and then the h of light were reduced half an h per wkuntil wk 32, when they received 4.5 h of light per day. IH and CH were in 2 different rooms, but hens of each group coming from different pens in the breeder house (Figure 2) were placed together in a common pen. Thus, for this period of time fecal samples were reduced to 2 pooled samples for CH, and 3 to 4 pooled samples for IH.

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Inoculum Preparation S. enterica ser. Enteritidis and Typhimurium, both NALR , were grown overnight in brain heart infusion (BHI) broth [29] at 37◦ C in a water bath with agitation. Campylobacter strains were initially grown in Mueller-Hinton agar (MHA) plates [30] at 42◦ C/48 h in microaerobic conditions using zip-top bags filled with a gas mixture (5% O2 , 10% CO2 , 85% N) [31], and then transferred into Mueller-Hinton broth (MHB) [29] and incubated at 42◦ C /24 h in microaerobic conditions. To enumerate the number of bacteria in the inoculum, inoculum was serially diluted and plated (100 μL) onto brilliant green sulfa agar plates (BGS) [32] for Salmonella and on MHA for Campylobacter. For the first inoculation (d 10), after overnight growth, both marker inocula were diluted 1:10 in BHI (108 -109 cfu/mL). Then, one mL of each S. Enteritidis and C. coli inocula was added into 8 mL of PBS (dilution 1:10). The same dilution (1:10) was made for S. Typhimurium and C. jejuni. For inoculation at 12 wk, after overnight growth, both inocula were diluted 1:2 in BHI. Then, the same volumes of Salmonella and Campylobacter inocula were mixed. Fecal Analysis Pooled fecal droppings from each pen were analyzed weekly until wk 15 (from April 2012 to July 2012), biweekly in July (wk 17 and 19) and August (wk 21 and 23), and in September (wk 27), October (wk 32), December (wk 39)

2012, and May 2013 (wk 61). In wk 19 individual fecal droppings per pen were analyzed for Campylobacter, which increased the number of samples analyzed for Campylobacter (n = 200) in comparison with Salmonella (n = 184). For the last sample analyzed at wk 61, only the hens were available; toms were sacrificed and sampled at wk 50 (March 2013). Fecal droppings from the same pen were collected in a 50 mL centrifuge plastic tube, mixed with a sterile cotton swab, and directly streaked onto Campy Cefex agar (CCA) [32] containing gentamicin (200 μg/mL) or nalidixic acid (20 μg/mL), respectively, for the selective identification of marker strains of Campylobacter inoculated into the poults. CCA was no longer used after wk 21 due to excessive growth of background microflora, and modified cefoperazone charcoal deoxycholate agar (mCCDA) [30] was used instead. Enumeration of Campylobacter in fecal droppings was performed for occasional samples (Table 2). For enumeration, one g of feces was suspended in 9 mL of buffered peptone water (BPW) and serial dilutions were plated (0.1 mL) onto mCCDA; plates were incubated at 42◦ C for 48 h under microaerobic conditions. The detection limit was 100 cfu/g. One Campylobacter colony per plate was sub-cultured on MHA for purification and further characterization, including antibiotic susceptibility tests and species determination. For Salmonella identification, fecal samples were diluted in BPW in a ratio of 1:10 for a first pre-enrichment step [30]; the sample was stomached for 60 s and incubated at 37◦ C for

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Figure 2. Distribution of pens of control and inoculated turkeys in the growout house after second inoculation (wk 12). In parenthesis is indicated the number of turkeys per pen. CT, control toms; CH, control hens; IT, inoculated toms; IH, inoculated hens.

CRESPO ET AL.: TURKEY BREEDER PATHOGENS Table 2. Enumeration of Campylobacter from individual fecal samples performed occasionally. Group1

cfu/g levels (approx.)

4 4 4 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 47 47 47 47 47 47 61

IT IT IH IH IH IH IH IH IH IH IH IH IH IH IH IT IT IT CH CH CH IH IH IT CH

1.5 × 107 7.5 × 106 3 × 107 5.7 × 105 9.4 × 107 1.3 × 105 8.8 × 105 1.5 × 106 3 × 106 1.3 × 106 4.2 × 103 2.8 × 105 1.3 × 105 9.6 × 105 4 × 106 1.1 × 105 9 × 104 3 × 105 1.9 × 105 6 × 102 <1 × 102 2.6 × 105 1.3 × 105 1 × 102 1 × 104

1

IT, inoculated toms; IH, inoculated hens, CH, control hens.

24 hours. For selective enrichment, 0.5 mL of the pre-enrichment was added into tetrathionate (TT) broth base tubes [33] supplemented with 0.2 mL of iodine solution [33], and 0.1 mL was added into Rappaport-Vassiliadis (RV) broth [33]. Tubes were vortexed and incubated at 42◦ C for 24 hours. A loopful (10μL) from each of the tubes was then streaked onto BGS agar without antibiotics. For detection of the marker strains inoculated into the turkeys, selective enrichments (10μL) were streaked onto BGS containing NAL 200 μg/mL, and incubated at 37◦ C for 24 hours. Presumptive Salmonella colonies were stabbed and streaked to triple sugar iron (TSI) agar and lysine iron agar (LIA) slants, which were incubated for 24 h at 37◦ C. Isolates giving typical reactions for Salmonella were streaked onto nutrient agar (NA) for purification, and confirmed by serology with Salmonella Poly O [29] and Poly H [30] antiserum. Salmonella and Campylobacter isolates were preserved at -80◦ C in cryovials containing BHI broth with 20% glycerol.

Blood, Organs, and Intestinal Samples Collection and Analysis Blood samples were taken at wk 11 after inoculation with marker strains from all groups, and wk 21 from both inoculated and control hens. Blood was drawn (8 to 10 mL) from the brachial wing vein. Skin was sprayed with 70% alcohol and allowed a 30 s contact time, followed by the application of povidone-iodine solution (10%) with at least one min contact time before vein puncture. Blood was directly plated (0.1 mL) onto CCA for Campylobacter detection, and 4 mL of blood were used for enrichment for each of the pathogens. For Campylobacter enrichment a 1:10 dilution using Bolton broth [30] was incubated in microaerobic conditions at 42◦ C for 24 to 48 h followed by plating of 0.1 mL onto CCA and incubation at 42◦ C for 48 hours. Salmonella detection was performed as described for fecal samples. Turkeys were periodically euthanized by electrocution between wk 11 and 65 (Table 3) for collection and analysis of internal organs and intestinal samples. The number of samples analyzed was limited due to the need of maintaining the flock for monitoring through the reproductive stages, including egg analysis and hatchability assessment (data to be presented in a separate manuscript). Absence of corneal reflex and vital signs was examined before spraying the carcass with 70% ethanol and opening the abdominal cavity. Spleen, liver and gallbladder, cecum and jejunum were aseptically collected, placed into sterile Whirl-pak bags, and packed on ice for transportation to the laboratory. Cecal and jejunum contents were directly streaked onto mCCDA agar plates as previous studies have reported that direct plating of fecal or cecal samples yielded better recovery of Campylobacter than enrichment [34–37]. Spleen was immersed in alcohol for 30 s, rinsed in saline solution, and then aseptically divided longitudinally into 2 pieces for the investigation of each pathogen. Liver was similarly aseptically divided into 2 pieces and the one containing the gallbladder was used for Campylobacter detection. Samples were individually weighed, macerated with a rubber mallet, and BPW was added in a ratio of 3 times the weight of the sample for the pre-enrichment of Salmonella. Subsequent

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Wk

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JAPR: Field Report

16 67 27 13 25 20 2 2 2

5 5 5 3 3 3 2 2 2 2

2 2 2

2 2 -

20 -

Total CH IH IT CT IT CT IT

IH

Jun (2013) wk 65 Mar (2013) wk 50 Dec (2012) wk 38 Nov (2012) wk 33

steps for selective enrichment and plating were as described above for the fecal samples. For Campylobacter enrichment, 3 times the weight of the sample of Bolton broth with selective supplements [30] and laked horse blood 5% [38] was added to the samples, stomached, and incubated in microaerobic conditions at 42◦ C for 48 hours. Samples were plated onto CCA or mCCDA agar plates. Vectors and Other Samples Feed (25g), swabs from drinkers (2 swabs per bell-drinker/pen), wood shavings (collected in a Whirl-pak bag), and other potential vectors of transmission such as insects and mice were periodically tested for Salmonella and Campylobacter (Table 4). Glue traps [39] were placed in several locations of both sides in the growout house, where inoculated and control turkeys were located. The number of insects analyzed was variable (Table 4). Whole insects and the 2 mice analyzed were initially macerated with a rubber mallet, and processed as previously described for organs and intestinal samples.

1 1 1 1 2 2 2 2 2 2 2 2 2 3 1 1 1 1 Blood Cecum Jejunum Spleen Liver-Gall-bladder

9 3 3 3 3

2 2 2 2 2

CT IT IH CH IT IH

Jun (2012) wk 11

Aug (2012) wk 20

Sept (2012) wk 25

Campylobacter and Salmonella Subtyping Species of Campylobacter isolates were determined by multiplex polymerase chain reaction (PCR) using hipO and ceuE primers for C. jejuni and C. coli, respectively [40–44]. Genomic DNA from C. coli D124 and C. jejuni NCTC 11168 were included as positive controls; negative controls (no DNA) were included each time. Salmonella isolates were serotyped by SMART, a multiplex PCR and capillary electrophoresis analysis [45]. Selected Campylobacter and Salmonella strains were subtyped by pulsed-field gel electrophoresis (PFGE) using SmaI and XbaI [46–49], respectively. Selected Campylobacter isolates also were subtyped by multi-locus sequence typing (MLST) [50, 51]. Campylobacter Antibiotic Resistance Determinations Campylobacter isolates were tested for resistance to a panel of antibiotics (tetracycline, streptomycin, erythromycin, kanamycin, nalidixic acid, ciprofloxacin, and gentamicin) based on

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Table 3. Number of blood, organs, and intestinal samples analyzed for Salmonella and Campylobacter at different time points during the project from the different groups of turkeys (IH, inoculated hens; IT, inoculated toms; CH, control hens; CT, control toms).

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CRESPO ET AL.: TURKEY BREEDER PATHOGENS

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Table 4. Vectors and other environmental samples analyzed during the project. Sampling month and year Mar 12 May 12 Sep 12 Oct 12 Oct 12 Oct 12

Campylobacter and/or Salmonella detected

Feed (25g), drinkers (2 swabs/drinker/pen), wood shavings Flies (10), feed (25g) Flies (5), cricket (1), roaches (3) Roaches (4), cricket (3), camel crickets (3) Flies (1), roaches (7), mouse (1), camel crickets (3) Flies (5), roaches (11), spiders (2), feed (25g), drinkers (2 swabs/drinker/pen) Feed (25g), drinkers (2 swabs/drinker/pen), wood shavings Mouse (1) Flies (5), roaches (5) Flies (6), roaches (4)

Negative Salmonella (flies) Negative Negative Negative Campylobacter (flies)

Table 5. Minimum inhibitory concentration of antibiotics tested for Campylobacter isolates. Isolates that yielded confluent growth at the indicated concentrations were considered resistant. Antibiotic Kanamycin Tetracycline2 Erythromycin2 Streptomycin2 Nalidixic Acid1 Ciprofloxacin1 Gentamicin1 2

banding patterns were calculated using BioNumerics [54].

Concentration (μg/mL) 1

1

Negative Salmonella Negative Negative

25 16 10 15 20 4 200

[28]. [78].

their growth in the presence of specific amounts of the indicated antibiotic (Table 5), as described [52]. For gentamicin, the level of resistance of the marker strain C. coli was used (200 μg/mL). Plates were spotted in duplicate and examined after 48 h of microaerobic growth on MHA at 42◦ C; isolates yielding confluent growth in both spots were considered resistant. All isolates were simultaneously also spotted on MHA to ensure viability. C. jejuni ATCC 33560 (purchased from the American Type Culture Collection; sensitive to all tested antibiotics) was included each time as quality control strain. Statistical Analysis Frequencies of detection observed for both pathogens were reported. Frequencies of detection in ceca and jejuna were compared using 2-sided Fisher’s exact test. Fisher’s test was performed using JMP 11 software [53]. Significance was defined at P ≤ 0.05. Clonal relationships of Campylobacter isolates based on PFGE

RESULTS AND DISCUSSION Monitoring of Salmonella in Fecal Samples Prior to the placement of the poults in the pens, environmental samples, drinkers, and feed samples analyzed were negative for Salmonella, as were samples of artificial straw containing feces from the boxes where the birds were shipped, and fecal samples during the first 2 weeks. However, nalidixic acid-susceptible strains of Salmonella (S. Agona) were isolated from CH at wk 3 (wk 1 after the first inoculation), and at wk 9 from CT (wk 7 after inoculation). The same serotype (S. Agona) was first detected in IH at wk 7 (wk 6 after inoculation). In the IT, nalidixic acid-susceptible Salmonella was detected at wk 10 (wk 8 after inoculation); however, this isolate was not subtyped. Table 6 shows the number of positive fecal samples for Salmonella per group. A total of 184 fecal samples was analyzed for Salmonella from wk 3 to 61. Of those, 102 (55.4%) were positive. Marker strains (nalidixic acid-resistant) were isolated from 45 (44.1%) of the positive samples while the remaining 57 (55.9%) were susceptible to nalidixic acid (NALS ) and presumed to be naturally occurring strains (Table 6). A subset of 29 representative NALS isolates, including isolates from each group and from different dates was selected for subtyping at the Russell Research Center (USDA-ARS, Athens, GA). Serotyping of these 29 isolates revealed

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Feb 13 Mar 13 Jun 13 Jun 13

Vectors-environmental sample

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Table 6. Marker and naturally occurring Salmonella recovered from fecal samples analyzed from the different groups of turkeys (hens and toms, control and inoculated) from wk 3 (April 2012) to wk 61 (May 2013). Group1 CT CH IH IT Total 1 2

Fecal samples 20 36 96 32 184

POS (%) 14 (70) 21 (58) 50 (52) 17 (53) 102 (55)

Marker (% of isolates from positive samples)

Naturally occurring (susceptible to NAL2 ) (% of isolates from positive samples)

0 0 (0) 31 (62) 14 (82) 45 (44)

14 (100) 21 (100) 19 (38) 3 (18) 57 (56)

CT, control toms; CH, control hens; IH, inoculated hens; IT, inoculated toms. NAL, nalidixic acid 200 μg/mL.

Figure 4. PFGE results showing the band pattern of S. Typhimurium marker strains (Marker), the two nalidixic acid sensitive S. Typhimurium isolates (NC18 and NC8), both isolated on wk 13 from fecal samples collected in 2 different pens of inoculated toms, and 2 other isolates (S. Agona-NC14 and S. Liverpool-NC1), isolated from fecal samples collected in 2 different pens of inoculated hens on wk 10. PFGE performed at the Russell Research Center (USDA-ARS), Athens, GA.

that the majority (26/29, 90%) were serotype Agona while 2 (7%) and one (3%) were serotypes Typhimurium and Liverpool, respectively (Figure 3). Further characterization by PFGE revealed that the 2 NALS S. Typhimurium isolates from IT had the same PFGE profile as the NALR

marker strain inoculated into the toms (Figure 4), suggesting that they may have been derived from the inoculated marker strain upon loss of nalidixic acid resistance. Although nalidixic acid resistance of Salmonella is frequently associated with point mutations in the quinolone resistance

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Figure 3. Naturally occurring Salmonella serotypes isolated from fecal samples of each group of breeder turkeys. Control toms (CT), control hens (CH), inoculated hens (IH), and inoculated toms (IT). Relative frequencies were calculated from the total number of naturally occurring isolates per group (CT = 7, CH = 8, IH = 11, IT = 3).

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Table 7. C. jejuni and C. coli strains recovered from fecal samples of the different groups. GROUP1 CT

CH

IH

IT

Total

27 (13.5)

38 (19.0)

99 (49.5)

36 (18.0)

200

19 (70.4)

27 (71.1)

75 (75.8)

23 (63.9)

144 (72)

11 (69)

9 (39)

30 (40)

7 (30)

57 (44)

5 (31)

14 (61)

38 (51)

16 (70)

73 (56)

3 (16)

4 (15)

7 (9)

0 (0)

14 (10)

4 (36)

1 (11)

14 (47)

2 (29)

21 (37)

3 (60)

3 (21)

20 (53)

12 (75)

38 (52)

7 (44)

4 (17)

34 (50)

14 (61)

59 (45)

1

CT, control toms; CH, control hens; IH, inoculated hens; IT, inoculated toms. G, gentamicin; K, kanamycin; T, tetracycline; S, streptomycin; Q, (fluoro)quinolones (nalidixic acid and ciprofloxacin). Acronyms indicate that the strain was resistant to these specific antibiotics but not to others used in the testing panel. Thus, GK indicates that the strain was resistant to gentamicin and kanamycin but susceptible to tetracycline, streptomycin, erythromycin, nalidixic acid, and ciprofloxacin, while TSKQ indicates resistance to tetracycline, streptomycin, kanamycin, nalidixic acid, and ciprofloxacin, but susceptibility to erythromycin and gentamicin.

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determining regions (QRDR) [55, 56], resistance to quinolones also can be mediated by plasmids [57, 58]. However, the origin of the resistance in the marker strain used in this study is unknown. After the first inoculation, Salmonella marker strains (NALR ) could be recovered from fecal samples for up to wk 5 (hens) and wk 10 (toms), and were not isolated from control birds at any time. However, naturally occurring strains were isolated from control hens at wk 3 (wk 1 after the first inoculation) and from control toms at wk 9 (wk 7 after inoculation). After the second inoculation marker strains were recovered from fecal samples of the inoculated turkeys up to wk 19 (7 wk after the second inoculation), while the control birds yielded only NALS Salmonella isolates. Serotype Agona found in the majority of these NALS isolates (Figure 3) has been previously reported to be commonly colonizing swine, broilers, and turkeys [59, 60]. Monitoring of Campylobacter in Fecal Samples Prior to the placement of poults in the pens, environmental samples, drinkers, and feed

samples were analyzed for Campylobacter, as were samples of artificial straw containing feces from the shipping boxes; all tested samples were negative (data not shown). Campylobacter strains other than the marker strains used in the study were detected in both groups of control birds at wk 4 (wk 2 after inoculation), and at wk 7 (wk 6 after inoculation) in IH. A total of 200 fecal samples were analyzed for Campylobacter from wk 3 to wk 61. Of these, 144 (72%) were Campylobacter-positive. Multiplex PCR-based speciation of 130 isolates indicated that 73 (56%) and 57 (44%) were C. jejuni and C. coli, respectively. Moreover, 59 of the 144 positive samples (41%) yielded marker strains. A detailed analysis of the isolates from each of the 4 groups of turkeys, i.e., CT, CH, IH, and IT, is shown in Table 7. C. jejuni was detected at higher rates than C. coli for all groups, except for CT in which C. coli was more frequently found (Table 7). Enumeration analysis for selected fecal samples indicated that in wk 4 and 19 Campylobacter was found in levels of 103 to 107 cfu/g of feces, while numbers were generally lower (between 102 to 105 cfu/g) in older birds at wk 47 and 61 (Table 2).

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Number of samples analyzed per group (% of isolates from the total 200 samples) Campylobacter-positive samples (% of isolates from the 144 total samples analyzed/group) C. coli (% of isolates from the 57 total speciated isolates) C. jejuni (% of isolates from the 73 total speciated isolates) Isolates not further characterized (% of isolates from the positive samples) C. coli GK2 (marker inoculated into IH) (% of isolates from the total C. coli) C. jejuni TSKQ2 (marker inoculated into IT) (% of isolates from the total C. jejuni) Total marker strains (% of isolates from the total speciated isolates)

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jejuni [61], (W. G. Miller and S. Kathariou, unpublished). In contrast, the C. coli GK strain used for inoculating the hens was originally recovered from retail chicken meat (USDA-ARSRRC, Athens, GA) and has ST-1068 (Table 9). Even though ST-1068 was recently (2014) identified in C. coli from turkeys in the course of an unrelated study (L. Rynde, S. Kathariou and W. G. Miller, unpublished data), it was previously encountered only among cattle and swine isolates [51]. Host-associated as well as generalist genotypes of Campylobacter spp. have indeed been reported [62, 63], and putative genomic attributes associated with host specificity have been identified (M. D. Crespo, E. Altermann, J. Olson, R. M. Siletzky and S. Kathariou, unpublished data). In addition to the marker strains, which are discussed above, the naturally occurring strains C. jejuni T (tetracycline resistant) and C. coli K (kanamycin resistant), also were retrieved with various frequencies from all 4 groups (Figure 5). Field strains were first detected at wk 4 in control groups. C. coli K was recovered at higher frequencies than C. jejuni T for all groups except for CH (Figure 5). Selected naturally occurring isolates from different dates and groups were analyzed by PFGE (data not shown) and MLST (Table 8). The ST of the fly-derived isolate (ST-1017) also was detected among several feces-derived isolates from all 4 groups and also had been detected earlier in feces from turkeys grown in the same facility (R. M. Siletzky, M. D. Crespo and S. Kathariou, unpublished). All ST-1017 isolates were C. coli that were resistant only to kanamycin (antimicrobial resistance profile K), while all C. coli isolates that were resistant only to gentamicin and kanamycin (antimicrobial resistance profile GK) exhibited the same ST (ST-1068) (Table 8). ST1017 also was detected in cecal C. coli isolates with antimicrobial resistance profile K from the different groups of turkeys (Table 8). In contrast to C. coli isolates with antimicrobial resistance profile K, all of which had the same ST, 3 different STs were identified among C. jejuni isolates with antimicrobial resistance profile T (resistant only to tetracycline): ST-51, ST-862 and ST- 7629. Most frequently encountered was ST-51, which also was detected previously in isolates from fecal samples of poults

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Marker strains of Campylobacter were isolated from fecal samples until wk 6 and 7 in IT and IH, respectively (5 and 6 wk after the first inoculation in IT and IH, respectively). However, at wk 4 (wk 3 after the first inoculation) the strain inoculated into the toms (C. jejuni) was isolated from hens’ fecal droppings, while the marker strain inoculated into the hens (C. coli) was recovered from the toms’ fecal droppings. Both marker strains also were recovered from control hens and toms at wk 4 (wk 3 after inoculation). Regardless of their source, the isolates exhibited the same PFGE profiles (data not shown) and MLST-based sequence type (ST) as the corresponding marker strain (Table 9). Campylobacter species and antibiotic susceptibility profile determinations were made for 16 and 23 isolates from CT and CH, respectively. Of the 16 CT isolates, seven (44%) were marker strains: 3 were C. jejuni TSKQ, the marker strain inoculated into IT, while 4 were C. coli GK, the marker strain inoculated into IH (Table 7). The marker strains also were identified among the isolates recovered from the CH group (Table 7). After the second inoculation, C. coli GK (marker strain inoculated into IH) was no longer recovered from fecal samples of IH or any of the other groups; however, C. jejuni TSKQ (the marker strain for IT) was isolated from fecal samples of IH up to wk 21 (9 wk after the second inoculation). In IT, C. jejuni TSKQ was recovered in 12 of the 23 speciated isolates (75% of the total C. jejuni), and 2 isolates were C. coli GK (29% of the C. coli recovered from IT) (Table 7). In IH, 14 from the 68 speciated isolates were the marker strain C. coli GK (representing a 47% of the total C. coli from IH), while the C. jejuni TSKQ marker strain was detected among 53% of the C. jejuni isolates recovered from IH (Table 7). Thus, even though it was inoculated twice into IH, the marker strain C. coli GK constituted a smaller portion of the Campylobacter isolates than the C. jejuni TSKQ marker strain, which was not deliberately introduced into IH (Table 7). Host association could be one explanation for the apparently higher colonization potential of the C. jejuni TSKQ strain. This strain was originally recovered from turkeys and its sequence type (ST), ST-1839 (Table 9) has been frequently encountered among turkey-derived C.

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grown in the same facility and was detected as late as in wk 39 in the current study (Table 8). Several distinct STs also were detected among pansensitive C. coli strains (C. coli P), specifically ST-21 (most common), ST-52, and ST-353, which was encountered only once (Table 8). As mentioned earlier, isolates with the same species and antimicrobial susceptibility profile as the marker strains also shared the marker strain ST. All tested C. jejuni TSKQ had ST-1839 as did the marker strain, and a C. coli GK isolate from feces of the control toms group had ST-1068, as did the C. coli GK marker strain (Table 8). The detection of the same strains of Campylobacter colonizing turkeys at different times and locations in the facility may indicate the persistence of certain strains in the environment [64, 65]. These findings are in accordance with previous studies in broilers showing that environmental reservoirs may play a key role in transmission of Campylobacter into the flocks [18, 64]. A high prevalence of Campylobacter resistant to tetracycline and kanamycin in both conventional and organic poultry production have been previously reported [66]. Our findings indicate that Campylobacter spread rapidly throughout the growout house and

the study groups became colonized both by the marker strains and by several naturally occurring strains of Campylobacter, which seem to successfully compete with the marker strains after a few wk and to persist in the flock. It is not known how these naturally occurring strains reached the pens, but transport via vectors such as flies or via human traffic may have taken place. It seems evident that all noted antimicrobial resistance profiles, including tetracycline and kanamycin resistance in natural C. jejuni and C. coli, respectively, can persist in the facility’s environment without current antibiotic selection pressure, as previously suggested by some authors [66]. In fact, these antibiotics had not ever been employed at the facility in the current study. Further investigation is needed to understand the ecology and mechanisms of survival of these Campylobacter strains in the environment. Frequencies of Detection of Campylobacter and Salmonella Over Time To assess potential temporal trends in prevalence of Salmonella and Campylobacter in the course of the study, the number of positive samples per mo and group was determined relative

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Figure 5. Campylobacter species and antibiotic susceptibility profile of isolates from fecal samples. Control toms (CT), control hens (CH), inoculated hens (IH), and inoculated toms (IT). Frequencies were calculated based on the total positive isolates characterized (species and antibiotic susceptibility profile) per group (CT = 16, CH = 23, IH = 68, IT = 23). T, tetracycline; S, streptomycin; K, kanamycin; Q, quinolones (nalidixic acid and ciprofloxacin); P, pansensitive; G, gentamicin. Acronyms indicate that the strain was resistant to these specific antibiotics but not to others used in the testing panel. Thus, GK indicates that the strain was resistant to gentamicin and kanamycin but susceptible to tetracycline, streptomycin, erythromycin, nalidixic acid, and ciprofloxacin, while TSKQ indicates resistance to tetracycline, streptomycin, kanamycin, nalidixic acid, and ciprofloxacin, but susceptibility to erythromycin and gentamicin.

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12 Table 8. MLST-based designations of Campylobacter isolates. Date isolated

Origin

C. jejuni (marker strain) CT IH 24 IT 23 IH C74 C. coli (marker strain) CT CH C4 IH C4 CH P6 IH P7 IH C6 IH IH C8 IH 20 Pen 15 IH CH IT CT Flies CH C1 CH P10 IH P4 IT IH 22 CH IH 24 Pen 16 CT IT IT IH 24 IH 22

9/11/2005 wk 6 wk 19 wk 19 wk 13 NA wk 4 wk 13 wk 13 wk 13 wk 13 wk 13 wk 23 wk 13 wk 19 12/16/20115 wk 23 wk 23 wk 33 wk 33 wk 32 wk 13 wk 13 wk 13 wk 19 wk 19 wk 23 wk 39 12/16/20115 wk 33 wk 27 wk 27 wk 19 wk 10

Feces Feces Feces Feces Cecum Chicken meat, retail Feces Cecum Cecum Cecum Cecum Cecum Jejunum Cecum Feces Feces Cecum Cecum Cecum Cecum Fly Cecum Cecum Cecum Feces Feces Jejunum Feces Feces Jejunum Jejunum Cecum Feces Feces

Species2 Cj Cj Cj Cj Cj Cc Cc Cj Cj Cj Cj Cj Cj Cj Cj Cc Cc Cc Cc Cc Cc Cc Cc Cc Cj Cj Cj Cj Cj Cj Cj Cj Cj Cj

Antibiotic resistance profile3

ST

TSKQ TSKQ TSKQ TSKQ TSKQ GK GK P P P P P P P P K K K K K K K K K T T T T T T T T T T

1839 1839 1839 1839 1839 1068 1068 353 21 21 21 21 52 52 52 1017 1017 1017 1017 1017 1017 1017 1017 1017 862 862 862 51 51 51 51 51 51 7629

1

CT, control toms; CH, control hens; IH, inoculated hens; IT, inoculated toms; number and letters P and C indicate pen location. Cj, C. jejuni; Cc, C. coli. 3 T, tetracycline; S, streptomycin; K, kanamycin; Q, quinolones (nalidixic acid and ciprofloxacin); G, gentamicin; P, pansensitive. Acronyms indicate that the strain was resistant to these specific antibiotics but not to others used in the testing panel. Thus, GK indicates that the strain was resistant to gentamicin and kanamycin but susceptible to tetracycline, streptomycin, erythromycin, nalidixic acid, and ciprofloxacin, while TSKQ indicates resistance to tetracycline, streptomycin, kanamycin, nalidixic acid, and ciprofloxacin, but susceptibility to erythromycin and gentamicin. 4 C. jejuni IH C7 exhibited a different PFGE profile than the marker strain but the same ST and antibiotic resistance profile. 5 These Campylobacter strains were isolated prior to the beginning of this study from poults raised in the same house for unrelated investigations. 2

to the total number of fecal samples analyzed. A complicating factor was that the number of pens per group was different, leading to a larger number of samples of IH (6 pens), in contrast to only one pen of CT. There was a tendency for Salmonella and Campylobacter to peak at different times (Figures 6 and 7, respectively). Campylobacter had a maximum number of pos-

itive samples detected in April 2012 (wk 3 to 6), right after inoculation with the marker strains, but this was also true for the control groups, which were colonized with naturally occurring strains. In June (wk 11 to 14) the number of positive samples was lower or Campylobacter was absent/undetectable in some groups (Figure 7). There was an increase in background microflora

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Campy isolate ID1

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Figure 7. Relative frequencies of detection of Campylobacter in fecal samples on the different groups of turkeys. Fecal samples were analyzed from April 2012 to May 2013. CT, control toms; CH, control hens; IT, inoculated toms; IH, inoculated hens.

on the plates that could have had a negative effect on Campylobacter detection; nevertheless, in the control groups 2 different media were used (mCCDA and Campy Cefex) and Campylobacter could not be detected on either. In contrast, Salmonella detection seemed to increase in April and May (except for the CH group) and then reached maximum detection in June for all groups, coinciding with decreases in Campylobacter (Figure 7). Frequency of detection of Campylobacter was higher in July (wk 15-1719) with a second peak in August (wk 21 to 23), and prevalence of Salmonella in turn decreased in July, reaching low levels or decreasing below detection levels from August 2012 to February 2013 (wk 47) (Figures 6 and 7). The only exception was the CT group in which Salmonella detection remained high for a longer time, until December 2012 (wk 39), and then became un-

detectable in February 2013 (Figure 6). The lack of detection of Salmonella in hens, especially in IH, was intriguing since artificial insemination using semen containing marker strains of Salmonella (S. Enteritidis NALR ) and Campylobacter (C. coli GK) was performed between December 2012 and January 2013 (wk 39 to 43), which should have increased the possibilities of infection (the investigation of vertical transmission after artificial insemination using semen harboring marker strains of both bacteria will be reported in a different manuscript). Also, C. coli GK was not detected in feces during that period. Prevalence of Campylobacter in both groups of toms, CT and IT, was similarly high between August and October, but not detected in December, and again high in February (Figure 7). In hens, both CH and IH, frequencies of detection were high from August to February 2013 and

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Figure 6. Relative frequencies of detection of Salmonella in fecal samples on the different groups of turkeys.

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Table 9. Prevalence of Campylobacter and Salmonella in ceca and jejuna from turkeys of the 4 groups. GROUP1 Pathogen and source Campylobacter Ceca2 Campylobacter Jejuna Salmonella Ceca Salmonella Jejuna 1 2

CT Positive/ analyzed (%)

CH analyzed (%)

IH analyzed (%)

IT analyzed (%)

Total Positive/ Total analyzed (%)

6/6 (100) 1/6 (17) 2/6 (33) 1/6 (17)

16/22 (73) 1/2 (50) 0/22 (0) 0/2 (0)

21/30 (70) 2/10 (20) 4/30 (13) 0/10 (0)

8/9 (89) 3/9 (33) 1/9 (11) 0/9 (0)

51/67 (76) 7/27 (26) 7/67 (10) 1/27 (4)

CT, control toms; CH, control hens; IH, inoculated hens; IT, inoculated toms. There was a significant difference (P < 0.05) between the carriage of Campylobacter in ceca and jejuna.

Campylobacter and Salmonella in Blood, Organs, and Intestinal Samples Neither Campylobacter nor Salmonella were detected in blood or spleens analyzed. Liver samples also failed to yield Salmonella but C. jejuni was recovered from a single CH sample of liver-gallbladder. Although organs were aseptically collected before ceca to prevent crosscontamination, samples were further manipulated in the laboratory, which could have increased the risk of external cross-contamination. Salmonella and Campylobacter were successfully recovered from organs of broilers in previous studies [68–72]. Differences in systemic colonization kinetics can be attributed to different strains of bacteria, host breed, age, and the length of time after inoculation [73–75]. In the current study, internal organ samples were analyzed when poults were 11 wk and older; the extent to which age of the turkeys may impact the systemic dissemination and survival of the bacteria remains uncharacterized. In the case of intestinal samples, 51 of the 67 ceca analyzed (76%) were positive for Campylobacter, in contrast with 7 (10%) positive for Salmonella (Table 9). Similar rates of cecal colonization by both pathogens were previ-

ously described in broilers [76]. The carriage of Campylobacter and Salmonella in jejuna was lower than in ceca. From the 27 jejuna samples analyzed, 7 (26%) were positive for Campylobacter, while only one (4%), from a CT bird, was positive for Salmonella (Table 9). S. Agona was isolated from ceca of IH, ceca of CT and IT, and a jejunum sample of CT. A significant difference (P < 0.05) between the carriage rates in ceca and jejuna was evident for Campylobacter, but not for Salmonella. However, difference in colonization between hens and toms was not significant (Table 9). These findings confirm that cecal analysis was a good indicator of Campylobacter colonization as shown in previous studies [5, 77]. Analysis of the antibiotic resistance profiles of intestinal isolates suggested absence of the C. coli GK marker strain, while C. jejuni TSKQ was recovered only from one cecal sample, of IH (sample ID: IHC7). Interestingly, the PFGE profile of this isolate was different from that of the marker strain (data not shown), but the MLST-based ST was the same (ST-1839) (Table 8). It is possible that the strain may have acquired genomic differences accounting for the altered PFGE profile, possibly via infection with temperate bacteriophage as reported in other studies [78, 79]. Alternatively, isolate IHC7 may represent a natural isolate with ST1839, acquired from an unidentified source. The intestinal samples yielded primarily the naturally occurring C. coli K; such isolates were recovered from 6 of 7 (86%) cecal samples characterized from IT, 3 of 4 (75%) from CT, 8 of 16 (50%) from CH, and 8 of 21 (38%) from IH (Figure 8). C. coli K was also recovered from one of 3 (25%) jejunum samples from IT, but was not detected in any other jejunum samples (Figure 9). On the other hand, C. jejuni

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then there was a decrease in May 2013 (wk 61) (Figure 7). In IH, an opposite tendency was observed again at this time point (May 2013), with an increase in Salmonella detection (Figure 6). This pattern has been observed for Salmonella in previous studies in which Salmonella colonizing ceca was reduced as the juvenile animals developed into adults [16, 67]. In general, these observations point to the high complexity of the population dynamics in intestinal tract colonization and subsequent detection.

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Figure 9. Species and antibiotic resistance profile of Campylobacter isolated from jejunum. Relative frequencies were calculated from the total positive samples characterized per group (CT = 1, CH = 2, IH = 2, IT = 3). T, tetracycline; K, kanamycin; P, pansensitive. Acronyms indicate that the strain was resistant to these specific antibiotics but not to others used in the testing panel. Thus, GK indicates that the strain was resistant to gentamicin and kanamycin but susceptible to tetracycline, streptomycin, erythromycin, nalidixic acid, and ciprofloxacin, while TSKQ indicates resistance to tetracycline, streptomycin, kanamycin, nalidixic acid, and ciprofloxacin, but susceptibility to erythromycin and gentamicin.

T constituted 100% of the jejunum isolates from CT and CH, 67% from IT and 50% from IH, in contrast with a low frequency of detection in cecal samples (Figure 9). Pan-sensitive C. jejuni were encountered in 50 and 43% of cecal isolates from CH and IH, respectively, as well as in a single isolate from jejunum from a hen, but were

not recovered from any of the cecal or jejunum samples from toms (Figures 8 and 9). This may reflect sample biases, as the majority of these C. jejuni pan-sensitive isolates were detected at the end of the study after the toms had been removed from the study. It was intriguing to note the low frequency of detection of pan-sensitive strains

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Figure 8. Species and antibiotic resistance profile of Campylobacter isolates from ceca. Relative frequencies were calculated from the total positive characterized cecal samples per group (CT = 4, CH = 16, IH = 21, IT = 7). AB ND, antibiotic susceptibility not determined. T, tetracycline; S, streptomycin; K, kanamycin; Q, quinolones (nalidixic acid and ciprofloxacin); G, gentamicin; P, pan-sensitive. Acronyms indicate that the strain was resistant to these specific antibiotics but not to others used in the testing panel. Thus, GK indicates that the strain was resistant to gentamicin and kanamycin but susceptible to tetracycline, streptomycin, erythromycin, nalidixic acid, and ciprofloxacin, while TSKQ indicates resistance to tetracycline, streptomycin, kanamycin, nalidixic acid, and ciprofloxacin, but susceptibility to erythromycin and gentamicin.

16 of C. jejuni in fecal samples (Figure 5), and the higher frequency of detection in ceca later in the study (at 65 wk) (Figure 8). Pan-sensitive isolates exhibited 3 different STs; while ST-21 was the most common, ST-352 was noted only in wk 19 and 23 (Table 8). The findings may reflect a low level of colonization by pan-sensitive strains of ST-21 at the beginning of the study, with ST-352 being possibly introduced later via new contamination events.

Both C. jejuni and C. coli were isolated from flies in one sample analyzed in October 2012, but not in any other insects analyzed (Table 4). Antibiotic resistance testing showed that the C. coli isolate was resistant to kanamycin (C. coli K). Unfortunately, fly-derived C. jejuni was not further characterized because isolates were inadvertently not preserved. Campylobacter and Salmonella were not detected in any of the tested samples from feed or the drinkers. Salmonella (S. Agona) was isolated from flies and from a mouse caught in the proximity of the control hens’ pens; the latter isolate was S. Enteritidis NALR , as was one of the Salmonella marker strains (Table 4). One difficulty found during the detection of Campylobacter from vector and other environmental samples was the use of enrichment media, which led to the overgrowth of background microflora in the plates. This difficulty can be addressed in the future by complementing the enrichments with direct plating of the sample on selective media such as mCDA. Challenges Associated with Co-inoculation and Other Components of Study Design In this study, poults were inoculated with both organisms at the same time. The final objective was to establish and maintain a Salmonella and Campylobacter-positive flock through the reproductive stages, allowing assessment of vertical transmission of both bacteria from the breeders to the offspring. The study also intended to monitor the occurrence of extraintestinal infection, or dissemination and colonization of other organs and tissues such as spleen, liver, gallbladder, and the reproductive tract. One pitfall

was the unbalanced design of the project, with different numbers of birds and pens per group (IH = 82, IT = 22, CH = 28, CT = 8), which also constituted a handicap for statistical analysis purposes. This was also an impediment for a more extended analysis of organs at the beginning of the project since it was a requirement to keep the flock until reproduction for assessment of vertical transmission. These aspects may be considered in future studies, in which a balanced design would allow a better analysis and comparison among groups and a group size big enough to allow periodic culling for internal organ assessments. Potential drawbacks of inoculating both pathogens include the fact that one cannot evaluate the effects that colonization by one pathogen may have in the subsequent colonization by the other. Future studies also must take into account the possibility of colonization with field strains of Campylobacter and Salmonella, especially upon prolonged maintenance of the birds as would be needed for longitudinal studies such as the current one, and the effect of competition between these field strains and those that were deliberately inoculated into the birds. In our study, some of the field Campylobacter strains also were isolated from turkeys in previous studies at the same facility, which may indicate a certain endemic nature of the isolates. The repetitive isolation of the same strains from successive flocks has been described [80]. Field strains can be better adapted to a particular environment, or be better at colonizing turkeys than the marker strains used, due to a number of still poorly understood determinants, including host association [62, 81].

CONCLUSIONS AND APPLICATIONS 1. Monitoring done in this study indicated that Campylobacter spread more rapidly than Salmonella and cross-contaminated turkeys throughout the growout house. 2. Naturally occurring strains (field isolates) of both pathogens seemed to outcompete marker strains after a few wk and to persist longer in the flock. Such field isolates of Salmonella and Campylobacter were detected in the inoculated groups by wk 7

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Vectors and Other Samples

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M. Picherot, J. Santolini, S. Bougeard, G. Salvat, and M. Chemaly. 2011. Campylobacter contamination of broiler caeca and carcasses at the slaughterhouse and correlation with Salmonella contamination. Food Microbiol. 28:862– 868. 10. Corry, J. E. L., V. M. Allen, W. R. Hudson, M. F. Breslin, and R. H. Davies. 2002. Sources of Salmonella on broiler carcasses during transportation and processing: Modes of contamination and methods of control. J. Appl. Microbiol. 92:424–432. 11. Berrang, M. E., D. P. Smith, W. R. Windham, and P. W. Feldner. 2004. Effect of intestinal content contamination on broiler carcass Campylobacter counts. J. Food Prot. 67:235– 238. 12. Berrang, M. E., R. J. Buhr, J. A. Cason, and J. A. Dickens. 2001. Broiler carcass contamination with Campylobacter from feces during defeathering. J. Food Prot. 64:2063– 2066. 13. Heyndrickx, M., D. Vandekerchove, L. Herman, I. Rollier, K. Grijspeerdt, and L. De Zutter. 2002. Routes for Salmonella contamination of poultry meat: Epidemiological study from hatchery to slaughterhouse. Epidemiol. Infect. 129:253–265. 14. Federal Register Notice, Vol. 75, No. 93, 27288, May 14, 2010 (Docket No. FSIS-2009-0034), “New Performance Standards for Salmonella and Campylobacter in Young Chicken and Turkey Slaughter Establishments.” 15. Pearson, A. D., M. H. Greenwood, R. K. Feltham, T. D. Healing, J. Donaldson, D. M. Jones, and R. R. Colwell. 1996. Microbial ecology of Campylobacter jejuni in a United Kingdom chicken supply chain: Intermittent common source, vertical transmission, and amplification by flock propagation. Appl. Environ. Microbiol. 62:4614–4620. 16. Jacobs-Reitsma, W. F., N. M. Bolder, and R. W. A. W. Mulder. 1994. Cecal carriage of Campylobacter and Salmonella in Dutch broiler flocks at slaughter: A one-year study. Poult. Sci. 73:1260–1266. 17. Pearson, A. D., M. Greenwood, T. D. Healing, D. Rollins, M. Shahamat, J. Donaldson, and R. R. Colwell. 1993. Colonization of broiler chickens by waterborne Campylobacter jejuni. Appl. Environ. Microbiol. 59:987–996. 18. Hiett, K. L., N. J. Stern, N. A. Cox, M. T. Musgrove, and S. Ladely. 2002. Molecular subtype analyses of Campylobacter spp. from Arkansas and California poultry operations. Appl. Environ. Microbiol. 68:6220–6236. 19. Cox, N. A., L. J. Richardson, J. J. Maurer, M. E. Berrang, P. J. Fedorka-Cray, R. J. Buhr, J. A. Byrd, M. D. Lee, C. L. Hofacre, P. M. O’Kane, A. M. Lammerding, A. G. Clark, S. G. Thayer, and M. P. Doyle. 2012. Evidence for horizontal and vertical transmission in Campylobacter passage from hen to her progeny. J. Food Prot. 75:1896– 1902. 20. Jacobs-Reitsma, W. F., A. W. van de Giessen, N. M. Bolder, and R. W. Mulder. 1995. Epidemiology of Campylobacter spp. at two Dutch broiler farms. Epidemiol. Infect. 114:413–421. 21. Aviagen Turkeys, Inc. Lewisburg, West Virginia, USA. 22. Turkey Research Unit (TEU). 4601 Mid Pines Road, Raleigh, NC (NCSU). R 23. VirkonS. Dupont. Distributed by Neogen, Lexington, KY. 24. Cox, N. A., L. J. Richardson, M. E. Berrang, R. J. Fedorka-Cray, and R. J. Buhr. 2009. Campylobacter coli

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(6 wk after gavage with marker strains); they were detected noticeably earlier (by wk 3 and 4 for Salmonella and Campylobacter, respectively) in the control groups. Salmonella showed a reduction in prevalence over time while a high prevalence of Campylobacter was observed throughout. Naturally occurring strains of both pathogens were isolated from ceca and jejuna of older breeder hens and toms, suggesting a persistent reservoir. Moreover, cecal carriage of Campylobacter was higher than observed for Salmonella. Both pathogens were isolated from several vectors present in the facility confirming that pests and poor biosecurity may contribute to dissemination of the bacteria. A better understanding of the ecology and relation between these 2 pathogens is needed and may contribute to the implementation of effective intervention strategies to prevent the colonization of the flocks on farm, and, consequently, a reduction in foodborne disease risks related to poultry products.

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Acknowledgments The present study was supported in part by the North Carolina Agricultural Foundation and the US Poultry and Egg Association (USPEA). We thank Robin Siletsky, Mike Mann, and Christina Shenton for technical support and assistance, John Barnes (Veterinary Medicine School, North Carolina State University) for assistance during necropsy, Jodie Plumblee and Lari Hiott, (USDA-ARS-RRC, Athens, GA) for helping with species determination of Campylobacter isolates, and serovar determination and PFGE of isolates. The assistance of the farm crew is sincerely appreciated.

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