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Research Reports |
* Russell Research Center, USDA-ARS-PMSRU, 
Russell Research Center, USDA-ARS-PPRU, and Russell Research Center, USDA-ARS-BEAR, Athens, GA 30605; and Department of Poultry Science, University of Georgia, Athens 30602
Correspondence: 1 Corresponding author: ncox{at}saa.ars.usda.gov
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SUMMARY |
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 TOP SUMMARY DESCRIPTION OF PROBLEMMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONS AND APPLICATIONSREFERENCES AND NOTES |
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Key Words: Campylobacter • Salmonella • Enterobacteriaceae • unabsorbed yolk sac • broiler
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DESCRIPTION OF PROBLEM |
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TOPSUMMARYDESCRIPTION OF PROBLEMMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONS AND APPLICATIONSREFERENCES AND NOTES |
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Campylobacter and Salmonella contaminations are a major concern to the poultry industry due to the organisms being recognized as causes of acute bacterial gastroenteritis in humans [10, 11, 12]. Campylobacter and Salmonella are mainly associated with the alimentary tract of poultry. However, recent findings suggest that Campylobacter and Salmonella can be readily detected in numerous internal organs and tissues, as well as in the alimentary tract of broilers and broiler breeders [13, 14, 15, 16, 17, 18].
There is little, if any, published information on the natural bacteriology of unabsorbed yolk sacs, particularly concerning market-age broilers. However, there have been papers reporting the presence of Salmonella spp. in the remaining yolk of 1-d-old chicks and ostrich chicks [19, 20, 21]. Dzoma and Dorrestien [19] looked at the bacteriology of the unabsorbed yolk sacs of ostrich chicks that ranged from 1 to 21 d of age. Of the 80 yolk sacs they studied, 22% were infected with bacteria, and the same species were generally isolated from the liver (only rod-shaped bacteria were observed). No Campylobacter species, spore-forming bacteria, or fungal elements were observed. Escherichia coli was the most common isolate found in the unabsorbed yolks. However, they also found Pseudomonas mesophilia, Pseudomonas pneumonia, Serratia liquefaciens, Alcaligenes xylosopsid, Aeromonas hydrophila, and Enterobacter cloacae.
The objectives of this study were as follows: 1) to determine whether Campylobacter spp. and Salmonella serovars, along with other bacteria, are naturally present in the unabsorbed yolk sacs of market-age commercial broilers, and 2) to determine what bacterial species are common in these unabsorbed yolks.
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MATERIALS AND METHODS |
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TOPSUMMARYDESCRIPTION OF PROBLEMMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONS AND APPLICATIONSREFERENCES AND NOTES |
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For Experiment 1, three carcasses containing a free-floating yolk and yolk stalk and 3 carcasses containing an attached yolk were randomly selected from the 100 carcasses from 2 replications, and 5 carcasses each from the last replication were randomly selected from the 100 carcasses and analyzed for levels and types of total aerobic bacteria (APC), Enterobacteriaceae (ENT), Salmonella, and Campylobacter.
For Experiment 2, five carcasses containing a free-floating yolk and yolk stalk and 5 carcasses containing an attached yolk for all 4 replications were randomly selected from each of the 100 carcasses and analyzed for levels and types of APC, ENT, Salmonella, and Campylobacter spp. To reduce the possibility of cross-contamination among samples, the unabsorbed yolk sacs were aseptically removed before collection of the ceca. Individual samples (free-floating yolks, yolk stalks, attached yolk and yolk stalk, and ceca) were placed in sterile bags, packed on ice, and transported to the laboratory for evaluation. The samples within plastic bags were then macerated with a rubber mallet to ensure that the contents of the samples were exposed and standard laboratory procedures for Campylobacter, a modified laboratory procedure for recovery of Salmonella, and determination of ENT and APC were performed on the samples. Due to the size (<1 g to >15 g) of the sample tissues (unabsorbed yolks), Bolton’s broth was added to the samples to better evaluate incidence of Campylobacter, along with Salmonella, ENT, and APC. This procedure was validated in preliminary studies (data not shown) and was adequate for recovery of the organisms of interest. In addition, for samples that weighed >2 g, a ratio of 1:3 was used; for samples weighing <2 g, a standard of 6 mL was added to the sample bags to provide enough diluent for all enumerations.
Campylobacter Lab Procedure
Standard laboratory methods for the recovery of Campylobacter spp. were performed utilizing Bolton’s enrichment broth (containing lysed horse blood) and Campy-Cefex agar [13]. All confirmed isolates were frozen on commercially available ceramic beads in cryopreservative fluid [22] and held at –80°C until species identification could be performed.
Campylobacter Speciation
Isolates were obtained from the –80°C freezer and placed onto Campy-Cefex agar and incubated for 48 h at 42°C in a microaerophilic condition (5% O2, 10% CO2, and 85% N). The isolates were then picked and placed onto blood agar plates and incubated at 42°C for 24 h. Template DNA was prepared by picking 3 to 4 colonies from a plate using a sterile disposable plastic loop and BAX PCR system [23] utilized for speciation of Campylobacter jejuni and Campylobacter coli.
Salmonella Lab Procedure
A validated laboratory procedure was utilized, in which a 1-mL aliquot of solution from the above Bolton’s enrichment broth after stomaching was added to 9 mL of buffered peptone water [24]. Standard laboratory procedures were then performed utilizing TT Broth (Hajna) and brilliant green S and modified Lys Fe agar for plating media [24]. Confirmed colonies were streaked onto Tryptic soy agar (TSA) [25] and incubated overnight at 37°C. Parafilm [26] was then wrapped around the top of each TSA tube and saved at room temperature.
Salmonella Speciation
All saved TSA slants containing Salmonella isolates were shipped to the USDA National Veterinary Services Laboratory in Ames, IA for serovar identification.
Aerobic Plate Count Lab Procedure
For Experiment 1 and 2, serial dilutions were made from the unabsorbed yolk sac samples after the addition of Bolton’s enrichment broth.
Aerobic populations were enumerated on plate count agar and incubated at 37°C for 24 h. Populations were counted and reported as the base-10 logarithm colony-forming units per gram of tissue sampled. From each plate, 3 to 5 colonies were picked from the plate count agar and streaked for isolation onto nutrient agar, and the VITEK 2 [27] was utilized for determination of colony types.
ENT Lab Procedure
For Experiments 1 and 2, serial dilutions were made from the unabsorbed yolk samples after the addition of Bolton’s enrichment broth.
Enterobacteriaceae was enumerated on violet red bile agar with 1% glucose added with an overlay. Plates were incubated at 37°C for 24 h. Presumptive colonies (purple-red colonies) were counted and reported as the base-10 logarithm colony-forming units per gram of tissue sampled. From each plate, 3 to 5 colonies were picked from the violet red bile agar and plated onto nutrient agar. Following incubation, Micro-ID [25] was utilized for determination of types of ENT present.
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RESULTS AND DISCUSSION |
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TOPSUMMARYDESCRIPTION OF PROBLEMMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONS AND APPLICATIONSREFERENCES AND NOTES |
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Dzoma and Dorrestein [19], when examining the bacteriology of the unabsorbed yolk sacs of ostrich chicks, found only rod-shaped bacteria, and they reported that E. coli was the most common isolate found in the unabsorbed yolks. However, they also found numerous other bacteria such as P. mesophilia, P. pneumonia, S. liquefaciens, A. xylosopsid, A. hydrophila, and E. cloacae. In a related study, APC and ENT were found in levels of up to log 3.5 cfu/g in inoculated, experimentally raised 6-wk-old broilers [18]. Therefore, these unabsorbed yolks, whether they are free-floating or attached, can harbor numerous types of bacteria.
For all experiments, Campylobacter spp. was found in 29% (10 out of 31) of the yolk stalks, 32% (10 out of 31) of the attached, and 13% (4 out of 31) of the free-floating yolk sacs (Table 1
). Salmonella serovars were found in 26% (8 out of 31) of the yolk stalks, 48% (16 out of 31) of the attached, and 23% (7 out of 31) of the free-floating yolk sacs. Campylobacter and Salmonella were simultaneously recovered from a yolk stalk and from 3 ceca from the 6-wk-old carcasses. In the 8-wk-old carcasses, Campylobacter and Salmonella were simultaneously recovered from 5 unabsorbed yolk sacs, 1 yolk stalk, and 2 ceca.
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In Experiment 1, Salmonella serovars were isolated from 3 out of 11 attached yolk sacs and from 1 out of 11 ceca of the attached yolk sac sampled carcasses. Salmonella serovars were isolated from 3 out of 11 free-floating yolk sacs, 5 out of 11 yolk stalks, and 1 out of 11 ceca of the free-floating and yolk stalk sampled carcasses. In replication 1, all samples tested were negative for the presence of Salmonella serovars. In replication 2, three of the attached yolk samples were positive, but the corresponding ceca were negative. This was also seen in replication 3, in which an unabsorbed yolk was positive and the corresponding ceca were negative, and, as with Campylobacter, Salmonella may have been in the ceca, but it was not culturally detected. All of the Salmonella isolates were Salmonella Typhimurium (9).
In Experiment 2 (8-wk-old broilers), Campylobacter spp. was isolated from 5 out of 20 attached yolk sacs and 4 out of 20 ceca from the attached yolk sampled carcasses. Campylobacter spp. was isolated from 1 out of 20 free-floating yolk sacs, 4 out of 20 yolk stalks, and 5 out of 20 ceca from the free-floating yolk and stalk sampled carcasses. In replication 2 and 3, all samples tested were negative for the presence of Campylobacter. Campylobacter again was found in some of the unabsorbed attached yolk samples but not in the ceca. All isolates were determined to be C. jejuni except a single attached yolk isolate from replication 4, which was found to be C. coli.
In Experiment 2, Salmonella serovars were isolated from 12 out of 20 attached yolk sacs and 15 out of 20 ceca from the attached yolk sampled carcasses. Salmonella serovars were isolated from 7 out of 20 free-floating yolk sacs, 7 out of 20 yolk stalks, and 12 out of 20 ceca from the free-floating yolk and stalk sampled carcasses. In replication 1, four of the unabsorbed attached yolk samples were positive for Salmonella, whereas the corresponding ceca were negative. The majority of Salmonella isolates were S. Typhimurium (n = 24). However, a few isolates were Salmonella Montevideo (9), Salmonella Manchester (7), Salmonella Kentucky (6), Salmonella Thompson (3), Salmonella Berta (2), Salmonella London (1), and Salmonella Schwarzengrund (1).
Campylobacter and Salmonella are mainly associated with the alimentary tract of poultry [28, 29, 30]. However, these findings and previous findings suggest that Campylobacter and Salmonella may be readily detected in numerous internal tissues of broilers in addition to the alimentary tract. The mechanism by which these bacteria reach and colonize these unabsorbed free-floating yolks and internal organs has not been determined. However, in studies by Cox et al. [17] and Bailey et al. [30], it was found that inoculated Campylobacter and Salmonella could disseminate within 1 h after inoculation to the lymphoid-like organs in 1-d-old chicks that were either inoculated by oral or cloacal routes. This could suggest that the dissemination or presence of Campylobacter and Salmonella in these internal bodies is due to immune, systemic, or macrophage functions. In addition, Campylobacter have been found in the mature and immature follicles of adult broiler breeder hens [15]. However, it was not determined in that study if the liver played a role in follicle contamination.
In the present study, the significance of these unabsorbed yolks as possible reservoirs for these human foodborne enteropathogens and contamination points for broilers being processed is yet to be determined. The yolk stalk of chickens retains its lumen opening into the small intestine by a small papilla. It has been shown that the yolk stalk plays a role in immune function, due to lymphoid cells accumulating and partially occluding the stalk [31, 32]. The lymphoid tissue appears when yolk absorption is nearly complete and regression of the yolk sac quickens. However, yolk stalk closure occurs at different ages, and, in some species, lymphoid invasion does not occur; therefore, it is unknown if or how the yolk stalks close [20, 32]. The yolk stalk may enable the internal cavity of birds to become contaminated with different types of bacteria. If a yolk sac becomes free-floating within the abdominal cavity and the yolk stalk does not completely close, then bacteria originating from the intestines could be excreted into the abdominal cavity of the bird and become peritoneal. Regardless of the cause of contamination of internal bodies, developing an understanding of the viability and presence of Campylobacter and Salmonella in these internal organs and tissues of broilers could prove to be very beneficial in developing intervention strategies at the farm and processing levels.
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CONCLUSIONS AND APPLICATIONS |
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TOPSUMMARYDESCRIPTION OF PROBLEMMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONS AND APPLICATIONSREFERENCES AND NOTES |
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ACKNOWLEDGMENTS |
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REFERENCES AND NOTES |
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TOPSUMMARYDESCRIPTION OF PROBLEMMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONS AND APPLICATIONSREFERENCES AND NOTES |
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