Identification of a new source of Campylobacter contamination in poultry: transmission from breeder hens to broiler chickens

Campylobacter jejuni, a foodborne pathogen closely associated with market poultry, is considered to be the most frequent agent of human gastroenteritis in the United States. The pathways involved in the contamination of poultry flocks, vertical transmission and/or horizontal transmission, are unclear. In this study, Campylobacter isolates from two independent commercial broiler breeder flocks, as well as from their respective progeny, were characterized and compared by PstI ribotype analysis and by DNA sequence analysis of the short variable region (SVR) of the flaA gene (flaA SVR). Campylobacter isolates originating from one set of breeder hens and the feces from their respective progeny demonstrated identical ribotype patterns as well as identical flaA SVR DNA sequences, thereby suggesting that these isolates were clonal in origin. Ribotype analysis of Campylobacter isolates from the second set of breeder hens and processed carcasses from their offspring resulted in two patterns. Sequence analysis placed these isolates into two closely related groups and one distant group, similar to the ribotype analysis. These results demonstrate that Campylobacter isolates from commercial broiler breeder flocks and from the respective broiler progeny may be of clonal origin and that breeder hens can serve as a source for Campylobacter contamination in poultry flocks.     

Presence of naturally occurring Campylobacter and Salmonella in the mature and immature ovarian follicles of late-life broiler breeder hens

Campylobacter and Salmonella are known to cause acute bacterial gastroenteritis in humans. Raw poultry products have been implicated as a significant source of these infections. Five trials were conducted to determine whether Campylobacter and Salmonella spp. exist naturally in the mature and immature ovarian follicles of late-life broiler breeder hens. Broiler breeder hens ranging from 60 to 66 wk of age were obtained from four different commercial breeder operations. For each trial, the hens were removed from the commercial operation and held overnight at the University of Georgia processing facility. The hens were euthanized, defeathered, and aseptically opened. To reduce the possibility of cross-contamination between samples, first the mature and immature ovarian follicles, then the ceca, were aseptically removed. Individual samples were placed in sterile bags, packed on ice, and transported to the laboratory for evaluation. Overall, Campylobacter was found in 7 of 55 immature follicles, 12 of 47 mature follicles, and 41 of 55 ceca. Campylobacter was found in at least one of each sample of mature follicles and in ceca in each of the five trials. Salmonella was found in 0 of 55 immature follicles, 1 of 47 mature follicles, and 8 of 55 ceca. In this study, the recovery rate of Salmonella from late-life broiler breeder hen ovarian follicles was relatively low. However, the recovery rate of Campylobacter from the hen ovarian follicles was reasonably high, suggesting that these breeder hens could be infecting fertile hatching eggs. Determining how Campylobacter contaminated these ovarian follicles and how many chicks could be colonized from this source are the next steps in helping to elucidate a better understanding of this ecology and the control of Campylobacter in poultry production.     

Presence of Campylobacter jejuni in various organs one hour, one day, and one week following oral or intracloacal inoculations of broiler chicks

Day-old broiler chicks (n=30) were obtained from a commercial hatchery and inoculated, either orally or intracloacally, with a characterized strain of Campylobacter jejuni. At 1 hr, 1 day, and 1 wk after inoculation, broilers (n = 5) from the orally and intracloacally inoculated groups along with control birds (n=4) were humanely killed by cervical dislocation. The broilers from the control and treatment groups were aseptically opened, and the thymus, spleen, liver/gallbladder, bursa of Fabricius, and ceca were aseptically removed and individually analyzed for C. jejuni. Overall, C. jejuni was isolated after oral inoculation from 13% (10/ 75), 17% (13/75), and 28% (14/50) of the 1-hr, 1-day, and 1-wk samples, respectively. Campylobacter jejuni was isolated from 10% (4/ 40), 8% (3/40), 10% (4/40), 25% (10/40), and 40% (16/40) of the thymus, spleen, liver/gallbladder, bursa of Fabricius, and ceca samples, respectively. Following the intracloacal route of inoculation, C. jejuni was recovered from 32% (24/75), 8% (6/75), and 16% (8/50) of the 1-hr, 1-day, and 1-wk samples, respectively. Campylobacter jejuni was isolated from 5% (2/40), 5% (2/40), 5% (2/40), 45% (18/40), and 40% (16/40) of the thymus, spleen, liver/gallbladder, bursa of Fabricius, and ceca samples, respectively, for all sampling periods. Campylobacter spp. were not recovered from sample sites examined from the control broilers from trial one, trial two, or trial three samples examined after 1 hr and 1 day. However, one control sample was positive from the 1-wk sampling from repetition three; therefore, those data were omitted. The rapid movement of Campylobacter to internal organs following both oral and intracloacal inoculation may be significant, particularly if it persists in these organs as reservoirs throughout the 65-wk life cycle of breeding birds.     

Genotype analyses of Campylobacter isolated from the gastrointestinal tracts and the reproductive tracts of broiler breeder roosters

Campylobacter is considered to be the leading bacterial etiologic agent of acute gastroenteritis in humans. Evidence implicates poultry as a major source of the organism for human illness; however, the pathways involved in Campylobacter contamination of poultry flocks, horizontal transmission and/or vertical transmission, remain unclear. Recent evidence implicates breeders as a potential source for Campylobacter contamination of the subsequent broiler offspring. In this investigation, Campylobacter isolated from feces, cloacal swabs, ceca, semen, and vas deferens of 12 breeder broiler roosters were genotyped by both flagellin A short variable region (flaA SVR) DNA sequence analysis and repetitive element (rep)-polymerase chain reaction (PCR). In 9 of 12 roosters, Campylobacter was isolated from multiple sites sampled. Comparison of multiple isolates obtained from individual roosters revealed variable results. In five of the nine roosters, all Campylobacter isolated demonstrated closely related flaA SVR DNA sequences as well as rep-PCR patterns; isolates from these roosters were collected from both the gastrointestinal and the reproductive tracts or from the gastrointestinal tract alone. The remaining four roosters had Campylobacter that were distinct by both typing methods. Isolates from two of these four roosters originated from both the gastrointestinal and the reproductive tracts. Isolates from the remaining two roosters originated from only the reproductive tract. Comparisons of all Campylobacter isolates recovered from a distinct sample type within either the reproductive tract or the gastrointestinal tract (feces, semen, cloaca, vas deferens, or ceca) were quite diverse. No relationship between the genotypes and the sample type could be ascertained. Further investigation is needed to determine the route of contamination and if the presence of Campylobacter within the rooster leads to contamination of the broiler offspring via the fertilized egg.     

Direct polymerase chain reaction detection of Campylobacter spp. in poultry hatchery samples

A rapid, sensitive, and specific polymerase chain reaction (PCR) assay was developed for the direct detection of Campylobacter in environmental samples from hatcheries. PCR, with a set of primers specific for the Campylobacter flaA short variable region (SVR), detected the presence of Campylobacter in both fluff and eggshell samples; however, a determination of whether the organism was living or dead could not be made. Conventional cultural methods detected no Campylobacter from the same samples. An additional benefit of the direct PCR assay is it allows for the production of a product that can be sequenced to provide further epidemiologic information.     

Natural presence of Campylobacter spp. in various internal organs of commercial broiler breeder hens

Campylobacter are known to cause acute bacterial gastroenteritis in humans. Poultry products have been implicated as a significant source of these infections. Six experiments were performed to determine whether Campylobacter could be isolated naturally from the primary and secondary lymphoid organs, liver/gallbladder, and ceca of commercial broiler breeder hens. Broiler breeder hens were acquired from different commercial sources during the early, middle, and late lay cycles. The birds were euthanatized, defeathered, and aseptically opened. To reduce the possibility of cross-contamination between samples, the thymus, spleen, and liver/gallbladder were aseptically removed prior to removal of the ceca. Individual samples were placed in sterile bags, packed on ice, and transported to the laboratory for evaluation. In this study Campylobacter were found in 11 of 43 thymii, eight of 43 spleens, four of 43 liver/gallbladders, and 30 of 43 ceca. Overall, 28 of 53 isolates from the above samples were Campylobacter coli and 25 of 53 isolates were found to be Campylobacter jejuni.     

用PCR技术从家禽孵化样本中检测空肠弯曲杆菌

空肠弯曲杆菌 ,一种革兰氏阴性、微需氧细菌 ,现在被认为是人类急性胃肠炎的一种主要病源。在美国每年大约有 2 4 0万例由空肠弯曲杆菌引起的胃肠炎 ,占总人口的 1 %～ 2 %。接触或食用禽类产品被认为是主要感染途径 ,据调查 ,市场上出售的 75 %活禽和 80 %的禽肉中含有弯曲杆菌 ,如此高的病源检出率和人类大量的临床感染 ,已经促使针对弯曲杆菌的快速检测进行了大量的工作。弯曲杆菌的传染源和传染途径主要有鸡舍环境 ,垃圾 ,水源 ,进出人员以及畜牧场内的各种小动物如苍蝇和各种啮齿动物。但由于我们用传统的方法不能从孵化样本或新孵出仔…     

Isolation of Campylobacter spp. from semen samples of commercial broiler breeder roosters

Pooled semen samples from 12 groups of mature commercial broiler breeder roosters were analyzed for the presence of Campylobacter. Each of the 12 groups was comprised of eight individuals and was sampled weekly for five consecutive weeks. Once a day, roosters were allowed to have a restricted amount of feed after the semen samples were collected by abdominal massage. This feeding schedule reduced the amount of fecal contamination in and around the vent as well as in the semen sample. For replications 1, 2, and 3, the numbers of Campylobacter-positive groups were 8, 5, and 5, respectively, out of 12. For replications 4 and 5, 6 of 8 and 6 of 11 groups were positive, respectively. Only two groups were positive for Campylobacter at all sampling times, two groups were negative each time, and eight groups produced variable results. Also, fecal droppings, external swabs of the genitalia, and semen samples were taken from individual roosters between 49 to 65 wk of age. Of the total 275 semen samples collected, 9.47% contained naturally occurring Campylobacter, whereas 9.6% of 114 fecal droppings and 7.9% of the 114 genital swabs were positive. Levels of the organism present in the fecal samples ranged from 1.0 to 4.2 log colony-forming units (CFU)/g with an average of 2.9 log CFU/g feces. For semen, the levels ranged from as low as enrichment recovery only to as high as 3.1 log CFU/ml of semen with an average of 1.2 log CFU/ml. For swabs of genitalia, the levels of Campylobacter were so low that recovery was achieved only through enrichment. These data suggest that rooster semen may serve as a vehicle for transmission of Campylobacter to the reproductive tract of the hen and subsequently to the fertile egg.     

Recovery of Campylobacter from segments of the reproductive tract of broiler breeder hens

Three groups of >60-wk-old broiler breeder hens were assessed for the presence of Campylobacter within segments of the reproductive tract. In the first group, after stunned, the hens were bled, scalded, and defeathered, the reproductive tracts were aseptically excised from 18 hens, six from each of three adjacent floor pens that were feces positivefor Campylobacter. The reproductive tract segments (infundibulum, magnum-isthmus, shell gland, vagina, and cloaca) were pooled by pen. In the second group, 10 individual hens were sampled from the pens; the reproductive tract was divided into the following segments: magnum, isthmus, shell gland, vagina, and cloaca. For the third group, hens were obtained from two commercial farms that had been determined to be feces positive for Campylobacter, and the reproductive tract was divided into five segments, as described for the second group. Segments of the reproductive tract were placed into sterile plastic bags and suspended 1:3 (w/v) in Bolton enrichment broth, and serial dilutions were plated (0.1 ml) onto Campy-Cefex agar. The agar places were incubated at 42 C for 24 hr in a microaerobic atmosphere. In group 1, the pooled reproductive tract segments for hens from pen A were Campylobacter positive for the shell gland, vagina, and cloaca; hens from pen B were positive for the cloaca only; and hens from pen C were positive for the magnum-isthmus and doaca. In the second group, 9 of 10 cloaca samples were Campylobacter positive. Commercial hens in group 3 had campylobacter-positive cloaca samples (12/12), vagina (10/12), shell gland (7/12), isthmus (2/12), and magnum (4/12). Campylobacter colonization of the reproductive tract of the hen could enable vertical transmission of Campylobacter from the hen to the chick.