Research Note Comparison of Sampling Techniques for Detection of Arcobacter butzleri from Chickens J. D. Eifert,*,1 R. M. Castle,* F. W. Pierson,† C. T. Larsen,† and C. R. Hackney‡ *Department of Food Science and Technology, Virginia Tech, Blacksburg, Virginia 24061; †Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia 24061; and ‡College of Agriculture, Forestry and Consumer Sciences, West Virginia University, Morgantown, West Virginia 26506 ABSTRACT Arcobacter butzleri is a causative agent of human enteritis that has been recently differentiated from the genus Campylobacter. Previous work suggests that its transmission to humans is likely through a foodborne route with a substantial tendency to be located on poultry carcasses. For reducing the incidence of this pathogen on commercial poultry, improved protocols are needed to sample and identify A. butzleri from infected birds prior to slaughter. The purpose of this study was to compare sampling methods for this emerging pathogen from chickens that were artificially inoculated per os with A.
butzleri. We tested three sampling techniques commonly used to determine the microbiological quality of poultry: cloacal swabs, fecal samples, and environmental surface (drag) swabs collected when birds were 3, 5, or 7 wk old. These samples were cultured in Johnson-Murano enrichment broth and analyzed by PCR. Results indicate that environmental surface swabs yielded the highest recovery percentage. A detection rate of 75 to 100% was observed for each sampling period (age of chicken). Additionally, A. butzleri could not be isolated from the intestinal tract (jejunum, ileum, cecum, colorectum) of inoculated birds.
(Key words: Arcobacter, chicken, polymerase chain reaction, sampling) 2003 Poultry Science 82:1898–1902
INTRODUCTION Previous studies indicated that Arcobacter spp. are present on many retail poultry carcasses and other meat products (Festy et al., 1993; Collins et al., 1996; Lammerding, 1996; Marinescu et al., 1996; Schoeder-Tucker et al., 1996; Wesley, 1996; Atabay and Corry, 1997). The genus Arcobacter contains 4 different species: Arcobacter nitrofigilis isolated from plant roots, and A. cryaerophilus, A. skirrowii, and A. butzleri isolated from animals. A. butzleri, A. cryaerophilus, and A. skirrowii have been reported to cause human and animal illnesses, whereas A. butzleri has been isolated most frequently from cases of human enteritis (Kiehlbauch et al., 1991; Lerner et al., 1994). Most reports of Arcobacter in poultry meat have identified A. butzleri, but A. cryaerophilus and A. skirrowii have also been reported (Atabay et al., 1998). Although control of Arcobacter on the farm may reduce contamination at the processing and retail levels, the habitat of Arcobacter species in living birds is still unknown (Houf et al., 2002). Marinescu et al. (1996) indicated that
2003 Poultry Science Association, Inc. Received for publication March 31, 2003. Accepted for publication August 27, 2003. 1 To whom correspondence should be addressed:
[email protected].
the biotypes and serotypes of A. butzleri isolates from poultry products were similar to those isolated from humans with diarrheal illness. This was the first report that implied a link between human illness and contaminated food products. Limited information regarding the pathogenicity and epidemiology of A. butzleri is available. Arcobacter butzleri is a gram-negative, curved (vibriolike), nonsporeforming rod. It is approximately 0.2 to 0.9 µm wide and 1 to 3 µm long. It is motile due to a single polar unsheathed flagellum. It is capable of growth at a range of temperatures from 15 to 35°C, with its optimum range from 25 to 30°C. A. butzleri does not grow at temperatures higher than 35°C, unlike Campylobacter jejuni subspecies jejuni, which requires higher temperature of 37 to 42°C. Arcobacter and Campylobacter species have been found at similar locations on broiler carcasses and show a close genetic relationship (Vandamme et al., 1991). Additionally, Arcobacter has an ability to grow under aerobic conditions in which Campylobacter is inhibited from growing, but capable of survival. A. butzleri is microaerophilic upon initial isolation, but can be grown aerobically after subculturing. The organism does not ferment sugars, but instead uses pyruvate as an energy source. It has few biochemical properties that may aid in its identification;
Abbreviation Key: BPW = buffered peptone water; JMEB = JohnsonMurano enrichment broth.
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however, a multiplex PCR has proven useful for the detection of all Arcobacter species (Wesley and Baetz, 1999). The objectives of this study were 1) to determine an infective dose for colonization of A. butzleri in chickens, 2) to compare sampling methods and the optimum time for recovery of A. butzleri from inoculated, 1-d-old chickens, and 3) to determine the intestinal tract locations where A. butzleri may reside.
MATERIALS AND METHODS Culture Preparation Seventeen strains of A. butzleri, donated by the Texas A&M University Department of Animal Science, were used over the course of this study. These 17 different strains were isolated from retail poultry carcasses and positively identified by PCR and RFLP analysis. A. butzleri cultures were maintained in Brucella-FBP broth2 (campylobacter growth supplement: ferrous sulfate, sodium metabisulfite, sodium pyruvate). Each strain was grown separately in a shaker flask containing 50 mL of sterile Brucella-FBP broth and incubated aerobically at 30°C at 100 rpm for 24 h. Equivalent portions of each culture were combined into a single cocktail, with a concentration of approximately 2 × 109 cfu/mL. This cocktail was then diluted, as appropriate, for broiler inoculations.
Broiler Housing One-day-old, unsexed commercial broiler chickens were obtained from a local hatchery. They were placed in battery brooders for 1 wk and then transferred to 0.5 m wide × 0.9 m long × 0.6 m high (1.5 ft wide × 3.0 ft long × 2.0 ft high) cardboard boxes, 2 birds per box. These were converted into crude disposable isolators by covering the tops with plastic wrap and fitting side-cutouts with bacteriostatic filters that allowed passive air exchange. The bottoms of the isolators were covered with kiln-dried pine shavings; feed and water were provided ad libitum. Each isolator was equipped with a 40-W incandescent bulb to provide supplemental heat until birds were 3 wk of age. After this, they were maintained at ambient room temperature (25°C).
Administration of A. butzleri to Chickens To achieve artificial infection of A. butzleri, groups of 16 1-wk-old birds were administered 0.5 mL per os of A. butzleri cocktail. Each group received oral inoculations in one of three doses: 109, 106, or 103 cfu per bird. Another group of 16 birds were given the same volume of sterile Brucella-FBP broth to serve as a negative control.
Environmental Sample Collection Three types of environmental samples were collected at the ages of 3, 5, and 7 wk. These samples included 2
Oxoid, Inc. Ogdensburg, NY.
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fresh fecal material, cloacal swabs, and environmental surface (drag) swabs of the litter. For each sampling time, 5 out of 8 cages were randomly sampled from each of the 4 sample groups for a total of 15 cages and 5 negative control isolators. One fecal sample, one environmental surface sample, and two cloacal samples (one per bird) were collected per isolator. Two replicate experiments were conducted. Fecal Samples. Commercial plastic wrap was placed over the cage litter to ensure that fresh sample was collected. Fecal samples were collected no longer than 5 min after defecation. A sterile swab was used to collect fresh fecal matter from the top of the plastic. The inoculated swab was then placed into 10 mL of buffered peptone water (BPW). The sample was vortexed for 5 s, and 1 mL was withdrawn and added to 9 mL of Johnson-Murano enrichment broth (JMEB), which was then incubated at 30°C for 24 h (Johnson and Murano, 1999b). Cloacal Swabs. A sterile swab was used to sample the cloaca of each bird and then placed into 10 mL BPW. The sample was vortexed for 10 s, and 1 mL was dispensed into 9 mL JMEB, which was then incubated at 30°C for 24 h. Environmental Surface Samples. Sterile, 10.2 × 10.2cm (4 × 4 inch) gauze pads premoistened with 10 mL of BPW were dragged across the entire surface of the litter, one per isolator. The swab was placed in an 18-ounce Whirl-Pak bag and mixed with an additional 10 mL of BPW. One milliliter of this fluid was placed into 9 mL of JMEB, which was then incubated at 30°C for 24 h. In addition to those designated for the sample collection described above, other chickens were inoculated to demonstrate the ability of A. butzleri to colonize or persist in the litter. At 1, 4, and 6 wk of age, birds were orally inoculated with 109 cfu and placed into clean isolators (n = 5 for each bird age). The litter in these isolators was sampled daily for at least 1 wk.
Intestinal Tissue Sample Collection In separate experiments, additional birds were given an inoculum of 1 × 109 cfu/mL of the A. butzleri cocktail. A total of 60 birds (30 cages) were artificially inoculated per os. An additional 24 birds (12 cages) received sterile Brucella-FBP broth as negative controls. At 2, 4, and 6 wk of age, 10 inoculated birds (5 cages) and 4 uninoculated birds (2 cages) were killed by cervical dislocation. Jejunum, ileum, cecum, and colorectum were collected aseptically. They were transected lengthwise, placed into sterile stomacher bags with 100 mL of BPW and stomached for 2 min. One milliliter of this fluid was placed into 9 mL of JMEB which was incubated for 24 h at 30°C. Two replicate experiments were conducted.
Microbiological Analysis Confirming the presence of Arcobacter is difficult due to a lack of biochemical fermentation identification tests. Futhermore, recovery of this organism from a dry envi-
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EIFERT ET AL. TABLE 1. Proportions of environmental and live chicken samples positive for Arcobacter butzleri, all sampling times (3, 5 and 7 wk) combined Inocula (cfu/bird) Sample type Surface swab Fecal Cloacal Total
9
106
10
a
57/60 (95%) 48/60 (80%)a 26/120 (22%)a 131/240 (55%)a
103 a
28/30 (93%) 24/30 (80%)a 10/60 (17%)b 62/120 (52%)b
Total b
0/30 (0%) 0/30 (0%)b 0/60 (0%)c 0/120 (0%)c
85/120 (71%)x 72/120 (60%)y 36/240 (13%)z
Ratios within a row with the same letter are not significantly different; P ≤ 0.05. Ratios within a column with the same letter are not significantly different; P ≤ 0.05.
a–c
x–z
ronment can be difficult. Cox et al. (2001) suggested that a noncultural laboratory technique such as PCR should be used to verify the presence of dead, or viable but nonculturable, Campylobacter in dry poultry-associated samples. Johnson-Murano Enrichment Broth was used as an enrichment step before PCR analysis. JMEB and JohnsonMurano agar inhibit growth of Campylobacter and several other classes of bacteria by incorporating 5-fluorouracil (Johnson and Murano, 1999a,b). JMEB is a semisolid medium that allows a gradient of atmospheres. After sterilization and immediately before use, filter-sterilized solutions of cefoperazone (32 mg/L) and 5-fluorouracil (200 mg/L) were added. Brucella-FBP broth was used for general culture maintenance (Ransom and Rose, 1998). Inoculated JMEB tubes were then incubated at 30°C for 24 h. A 1-mL aliquot was withdrawn from a depth of approximately 0.5 cm below the top layer of inoculated JMEB tubes and transferred to a centrifuge tube. This aliquot was boiled for 15 min and centrifuged at 11,000 × g for 2 min then 5 µL of supernatant was removed for PCR analysis. A multiplex PCR assay3 was used to identify A. butzleri (Harmon and Wesley, 1997). ARCO I and ARCO II primers yield a 1,223-bp PCR product, which targets nucleotides at positions 244 to 1,446 of the 16s rRNA genes of Arcobacter species. This identifies all species of Arcobacter but does not differentiate between species. ARCO 2 and BUTZ primers yield a 686-bp PCR product. This targets nucleotides at positions 1,222 to 1,599 of the 23s rRNA genes, which are unique to A. butzleri. These primers differentiate A. butzleri from all other Arcobacter species. The PCR reaction conditions were similar to those described by Wesley and Baetz (1999). The reaction mixture contained 25 µL PCR Ready-Mix4 (1.5 U TAQ polymerase, 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, 0.001% gelatin, 0.2 mM dNTP), 5 µL of sample DNA, 4 µL of primer [1 µL (1.5 pmol) per each primer], and 16 µL of sterile water. This mixture was then overlaid with 50 µL sterile DNase- and RNase-free mineral oil and amplified in a DNA thermal cycler. The samples were subjected to an initial denaturation step (94°C for 4 min), followed by
3
Integrated DNA Technologies, Coralville, IA. ReadyMix Taq with MgCl2 Kit, Sigma Chemical Co., St. Louis, MO. SAS software, Version 6.12, SAS Institute, Inc., Cary, NC.
4 5
25 amplification cycles. Each cycle included a denaturation step (1 min at 94°C), an annealing step (1 min at 56°C), and an extension step (1 min at 72°C). A final extension step (7 min at 72°C) followed the last amplification cycle. A 20-µL aliquot of completed PCR reaction fluid was combined with 2 µL of loading dye. PCR amplification products were analyzed by electrophoresis in 1% agarose gels.
Statistical Analysis The qualitative data are reported as proportions or ratios of positive samples to negative samples. Ratio data are not normally distributed; therefore an arcsin transformation of the ratio data means was performed to achieve constancy of variance (Hinkelmann and Kempthorne, 1994). The transformed means were further analyzed by least squares difference tests (α = 0.05) to determine if the differences in recovery ratios were statistically significant for any sample types or sampling times.5
RESULTS AND DISCUSSION A. butzleri was recovered from cloacal, environmental, and fecal samples only when birds were inoculated with at least 106 cfu. A similar proportion of positive samples was found when birds were inoculated with either 106 or 109 cfu. Table 1 displays the proportion of Arcobacter positive samples from each of these sample types for all sampling times (3, 5, and 7 wk) combined. No sample type yielded 100% recovery, but A. butzleri was identified from at least 80% of the environmental surface and fecal swabs collected from birds inoculated with 106 or 109 cfu. Recovery of A. butzleri from cloacal swabs was only 22% from birds inoculated at the highest level. Analysis of these data showed that the percentage of positive identifications for environmental surface swabs was significantly higher than that for fecal swabs. Recovery of A. butzleri was similar across sampling times for cloacal, environmental surface swabs, and fecal samples. The proportion of positive samples increased slightly, from 90 to 100%, for environmental swabs collected at 3 to 7 wk of age (Table 2). The proportion of positive samples increased slightly, from 70 to 90%, for fecal swabs collected at 3 wk to 7 wk of age (Table 2). The percentage of positives identified from samples collected at 7 wk was significantly higher than the percentage
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RESEARCH NOTE TABLE 2. Proportions of environmental and live bird samples positive for Arcobacter butzleri (inoculum is 109 cfu/bird) Bird age Sample type Surface swab Fecal Cloacal Total
3 wk 18/20 14/20 8/40 40/80
(90%) (70%) (20%) (50%)a
5 wk 19/20 16/20 10/40 45/80
7 wk
(95%) (80%) (25%) (56%)ab
20/20 18/20 8/40 46/80
(100%) (90%) (20%) (58%)b
Total 57/60 (95%)x 48/60 (80%)y 26/120 (24%)z
Ratios within a row with the same letter are not significantly different; P ≤ 0.05. Ratios within a column with the same letter are not significantly different; P ≤ 0.05.
a–c
x–z
collected at 3 wk but not at 5 wk. Environmental surface swabs collected when birds were 3, 5, or 7 wk old and fecal swabs collected when birds were 7 wk old yielded the highest percentages (>90%) of A. butzleri positive samples. Environmental and fecal samples were the most effective sources for successful detection of A. butzleri. At least 90% of the environmental swab samples were positive when birds were age 3, 5 or 7 wk. At least 70% of the fecal samples were positive when birds were age 3, 5 or 7 wk. Due to the high recovery rate of A. butzleri observed with this study and the possibility that A. butzleri could be an environmental contaminant, surface (drag) swabs may be well suited for A. butzleri detection. Our results suggest that A. butzleri does not colonize chickens, but that it may persist in the environment, e.g., litter. As shown in Table 3, A. butzleri could not be isolated from any of the intestinal tract samples (n = 360). Analyses of the jejunal, ileal, cecal, and colorectal samples were performed only for birds inoculated with 109 cfu. Although Arcobacter spp. have been found on poultry carcasses, it appears that they may not colonize the poultry intestinal tract (Corry and Atabay, 2001). For example, Atabay and Corry (1997) did not recover Arcobacter strains form the gizzard, small intestine, or cecum from 15 abattoir broilers. These researchers did isolate Campylobacter from the cecum of each of these broilers. One explanation for our observations was that A. butzleri did not colonize the birds after inoculation but passed through the digestive system and contaminated the cage. Because A. butzleri thrives at 25 to 30°C, it is feasible that the high body temperature of the birds (41°C) inhibited or suppressed growth such that it was incapable of colonizing the intestinal tract. The ambient temperature of the animal housing was 25°C and provided a more suitable growth temperature for A. butzleri; therefore, survival
TABLE 3. Proportion of intestinal samples positive for Arcobacter butzleri (inoculum is 109 cfu/bird) Bird age Sample type
2 wk
4 wk
6 wk
Jejunum Ileum Cecum Colorectum
0/30 0/30 0/30 0/30
0/30 0/30 0/30 0/30
0/30 0/30 0/30 0/30
within the litter was a distinct possibility. In addition, because birds were confined to a restricted area, a constant supply of growth medium was provided by the continual addition of feces by the birds. If the litter was a source of A. butzleri, it is likely that the feathers and skin of the bird would be contaminated as well. In this study, the feathers from 20 of the inoculated birds were collected after they were killed for intestinal sample dissection. All 20 feather samples (100%) were positive for A. butzleri. This result is consistent with the high environmental contamination rate (>90% positive) among the samples and could explain why some cloacal samples were positive, especially if swabs contacted the external cloaca in the sampling process. Additional chickens were inoculated to demonstrate the ability of A. butzleri to contaminate and persist in the litter. For 1-wk-old birds orally inoculated with A. butzleri at 1 wk and introduced into clean isolators, each isolator was positive for A. butzleri after 3 d. For birds inoculated with 109 cfu at 4 and 6 wk of age and placed into clean isolators, each isolator became positive for A. butzleri within 3 to 4 d. Furthermore, these isolators remained positive until birds were euthanized at 7 wk of age, and A. buztleri could be detected for up to 4 wk after birds were removed. These observations suggest that A. butzleri is capable of surviving in litter with or without birds, and could therefore be problematic in operations where builtup litter is used. If A. butzleri is an environmental pathogen, then the implementation of on-farm best management practices might play a substantial role in reducing its prevalence in commercial poultry.
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