Evidence for horizontal and vertical transmission in Campylobacter passage from hen to her progeny.

Campylobacter is an important human pathogen, and consumption of undercooked poultry has been linked to significant human illnesses. To reduce human illness, intervention strategies targeting Campylobacter reduction in poultry are in development. For more than a decade, there has been an ongoing national and international controversy about whether Campylobacter can pass from one generation of poultry to the next via the fertile egg. We recognize that there are numerous sources of Campylobacter entry into flocks of commercial poultry (including egg transmission), yet the environment is often cited as the only source. There has been an abundance of published research globally that refutes this contention, and this article lists and discusses many of them, along with other studies that support environment as the sole or primary source. One must remember that egg passage can mean more than vertical, transovarian transmission. Fecal bacteria, including Campylobacter, can contaminate the shell, shell membranes, and albumen of freshly laid fertile eggs. This contamination is drawn through the shell by temperature differential, aided by the presence of moisture (the "sweating" of the egg); then, when the chick emerges from the egg, it can ingest bacteria such as Campylobacter, become colonized, and spread this contamination to flock mates in the grow house. Improvements in cultural laboratory methods continue to advance our knowledge of the ecology of Campylobacter, and in the not-so-distant future, egg passage will not be a subject continuously debated but will be embraced, thus allowing the development and implementation of more effective intervention strategies.

Minimization of Salmonella contamination on raw poultry.

Many reviews have discussed Salmonella in poultry and suggested best practices to minimize this organism on raw poultry meat. Despite years of research and conscientious control efforts by industry and regulatory agencies, human salmonellosis rates have declined only modestly and Salmonella is still found on raw poultry. Expert committees have repeatedly emphasized the importance of controlling risk, but information about Salmonella in poultry is often limited to prevalence, with inadequate information about testing methods or strains of Salmonella that are detected by these methods and no information about any impact on the degree of risk. This review examines some assumptions behind the discussion of Salmonella in poultry: the relationships between sampling and cultural methodology, prevalence and numbers of cells, and the implications of serotype and subtype issues. Minimizing Salmonella contamination of poultry is not likely to reduce human salmonellosis acquired from exposure to contaminated chicken until these issues are confronted more systematically.

Isolation of Campylobacter from circulating blood of commercial broilers.

Campylobacter spp. are present in organs and tissues of broiler chickens but the dissemination route is unclear. The aim of the current study was to determine Campylobacter prevalence within circulating blood of commercial broilers. Broilers were acquired from 19 flocks originating from three commercial poultry processing companies. Using aseptic blood collection techniques, 5 ml of circulating blood was collected from each bird and the sample analyzed for Campylobacter. The Campylobacter colonization status of each bird was determined by aseptically sampling and analyzing the ceca. Campylobacter was recovered from 58% (11/19) of flocks sampled. From the 248 total birds sampled, 12% and 46% of the birds had Campylobacter in the blood and ceca, respectively. This study documents Campylobacter prevalence in the circulating blood of commercially raised broilers. Campylobacter presence in the circulatory system may indicate the path used by the organism for rapid dissemination to organs and tissues. From a processing viewpoint, Campylobacter presence in circulating blood of market-age broilers may increase the likelihood of cross-contamination between birds during slaughter.

Comparison of shell bacteria from unwashed and washed table eggs harvested from caged laying hens and cage-free floor-housed laying hens.

These studies evaluated the bacterial level of unwashed and washed shell eggs from caged and cage-free laying hens. Hy-Line W-36 White and Hy-Line Brown laying hens were housed on all wire slats or all shavings floor systems. On the sampling days for experiments 1, 2, and 3, 20 eggs were collected from each pen for bacterial analyses. Ten of the eggs collected from each pen were washed for 1 min with a commercial egg-washing solution, whereas the remaining 10 eggs were unwashed before sampling the eggshell and shell membranes for aerobic bacteria and coliforms (experiment 1 only). In experiment 1, the aerobic plate counts (APC) of unwashed eggs produced in the shavings, slats, and caged-housing systems were 4.0, 3.6, and 3.1 log(10) cfu/mL of rinsate, respectively. Washing eggs significantly (P < 0.05) reduced APC by 1.6 log(10) cfu/mL and reduced the prevalence of coliforms by 12%. In experiment 2, unwashed eggs produced by hens in triple-deck cages from 57 to 62 wk (previously housed on shavings, slats, and cages) did not differ, with APC ranging from 0.6 to 0.8 log(10) cfu/mL. Washing eggs continued to significantly reduce APC to below 0.2 log(10) cfu/mL. In experiment 3, the APC for unwashed eggs were within 0.4 log below the APC attained for unwashed eggs in experiment 1, although hen density was 28% of that used in experiment 1. Washing eggs further lowered the APC to 0.4 to 0.7 log(10) cfu/mL, a 2.7-log reduction. These results indicate that shell bacterial levels are similar after washing for eggs from hens housed in these caged and cage-free environments. However, housing hens in cages with manure removal belts resulted in lower APC for both unwashed and washed eggs (compared with eggs from hens housed in a room with shavings, slats, and cages).

Horizontal transmission of Salmonella and Campylobacter among caged and cage-free laying hens.

In each of five sequential trials, laying hens (56-72 wk of age) were challenged with Salmonella and Campylobacter, and 1 wk postinoculation, the challenged hens (n = 3) were commingled with nonchallenged hens (n = 12) in conventional wire cages, on all-wire slats, or on all-shavings floor housing systems. After 12 days, challenged and nonchallenged hens were euthanatized for sample collection. Ceca were aseptically collected from all hens, and the spleen, liver/gallbladder (LGB), lower (LRT) and upper (URT) reproductive tracts, and ovarian follicles (mature and immature) were collected from only the challenged hens after commingling. Samples were divided equally and cultured separately for Salmonella and Campylobacter. Differences in the horizontal transmission of the challenge Salmonella to nonchallenged hens housed in cages (12%), on slats (15%), and on shavings (14%) were not significantly different (P > 0.05) from the challenged pen-mate hens over the five trials. However, with the inclusion of residual environmental Salmonella, the recovery of Salmonella from nonchallenged hens housed in cages was lowest at 15%, intermediate for hens on slats at 20%, and highest for hens on shavings at 38%. Among challenged hens housed in cages, Salmonella was recovered from only 27% of the cecum and LRT samples. From challenged hens housed on slats, Salmonella was recovered from 38% of the cecum, 12% of the spleen, 19% of the LGB, 44% of the LRT, and 19% of the URT samples. From challenged hens housed on shavings, Salmonella was recovered from 31% of the cecum; 15% of the spleen, LGB, and URT; and 31% of the LRT samples. Horizontal transmission of Campylobacter among nonchallenged pen-mate hens was significantly lower for hens housed in cages at 28% than for hens on shavings at 47%, with hens on slats being intermediate at 36%. For challenged hens housed in cages, Campylobacter was recovered from 27% of the cecum, 13% of the LRT, 7% of the URT, and 17% of the follicle samples. Among the challenged hens housed on slats, Campylobacter was recovered from 44% of the cecum, 6% of the spleen, 19% of the LGB, 12% of the LRT, 6% of the URT, and 14% of the follicle samples. Among challenged hens housed on shavings, Campylobacter was recovered from 46% of the cecum, 8% of the LRT and URT, and 40% of the follicle samples. The overall results of this study indicate that the caged housing system provided the lowest horizontal transmission level of Salmonella and Campylobacter among egg-laying hens.

Colonization of a marker and field strain of Salmonella enteritidis and a marker strain of Salmonella typhimurium in vancomycin-pretreated and nonpretreated laying hens.

This study was conducted to evaluate the influence of a vancomycin pretreatment on the ability of marker (nalidixic-acid resistant) Salmonella Enteritidis (SE(M)), field Salmonella Enteritidis (SE(E)), and marker Salmonella Typhimurium (ST(M)) strains to colonize within the intestinal and reproductive tracts and translocate to other organs of leghorn laying hens. In each of three trials, caged laying hens (76, 26, and 33 wk ofage) were divided into six groups designated to receive SE(M), SE(F), or ST(M), and half were pretreated with vancomycin (n = 11-12 hens). Vancomycin-treated hens received 10 mg vancomycin in saline/kilogram body weight orally for 5 days to inhibit Gram-positive bacteria within the intestines. On Day 6, all hens were concurrently challenged by oral, intravaginal, and intracolonal routes with Salmonella and placed into separate floor chambers by Salmonella strain. Two weeks postinoculation, all hens were euthanatized and the ceca, spleen, liver/gall bladder (LGB), upper (URT), and lower (LRT) reproductive tracts, and ovarian follicles were aseptically collected, and analyzed for Salmonella. Results did not differ for the three hen's ages and were therefore combined. The vancomycin pretreatment also had no significant effect on the colonization ability of SE(M), SE(F) or ST(M), and therefore results were combined within Salmonella strain. The marker strain of Salmonella Enteritidis was recovered from 21% of ceca, 4% of LGB, 9% of LRT, and 17% of the fecal samples. The field strain of Salmonella Enteritidis was recovered from 88% of ceca, 96% of spleen, 92% of LGB, 30% of LRT, 4% of URT, 13% of follicle, and 42% of the fecal samples. The marker strain of Salmonella Typhimurium was recovered from 100% of ceca, 74% of spleen, 91% of LGB, 30% of LRT, 9% of URT, 9% of follicle, and 100% of the fecal samples. Among ceca, spleen, LGB, and fecal samples, SE(F) and ST(M) colonization was significantly greater than SE(M) colonization. Overall prevalence of Salmonella in the reproductive tracts of challenged hens was relatively low, ranging from 4% to 30%.

Preliminary validation of a novel high-resolution melt-based typing method based on the multilocus sequence typing scheme of Streptococcus pyogenes.

The major limitation of current typing methods for Streptococcus pyogenes, such as emm sequence typing and T typing, is that these are based on regions subject to considerable selective pressure. Multilocus sequence typing (MLST) is a better indicator of the genetic backbone of a strain but is not widely used due to high costs. The objective of this study was to develop a robust and cost-effective alternative to S. pyogenes MLST. A 10-member single nucleotide polymorphism (SNP) set that provides a Simpson's Index of Diversity (D) of 0.99 with respect to the S. pyogenes MLST database was derived. A typing format involving high-resolution melting (HRM) analysis of small fragments nucleated by each of the resolution-optimized SNPs was developed. The fragments were 59-119 bp in size and, based on differences in G+C content, were predicted to generate three to six resolvable HRM curves. The combination of curves across each of the 10 fragments can be used to generate a melt type (MelT) for each sequence type (ST). The 525 STs currently in the S. pyogenes MLST database are predicted to resolve into 298 distinct MelTs and the method is calculated to provide a D of 0.996 against the MLST database. The MelTs are concordant with the S. pyogenes population structure. To validate the method we examined clinical isolates of S. pyogenes of 70 STs. Curves were generated as predicted by G+C content discriminating the 70 STs into 65 distinct MelTs.

Comparison of the statistics of Salmonella testing of chilled broiler chicken carcasses by whole-carcass rinse and neck skin excision.

Whether a required Salmonella test series is passed or failed depends not only on the presence of the bacteria but also on the methods for taking samples, the methods for culturing samples, and the statistics associated with the sampling plan. The pass-fail probabilities of the 2-class attribute sampling plans used for testing chilled chicken carcasses in the United States and Europe were compared by calculation and simulation. Testing in the United States uses whole-carcass rinses (WCR), with a maximum number of 12 positives out of 51 carcasses in a test set. Those numbers were chosen so that a plant operating with a Salmonella prevalence of 20%, the national baseline result for broiler chicken carcasses, has an approximately 80% probability of passing a test set. The European Union requires taking neck skin samples of approximately 8.3 g each from 150 carcasses, with the neck skins cultured in pools of 3 and with 7 positives as the maximum passing score for a test set of 50 composite samples. For each of these sampling plans, binomial probabilities were calculated and 100,000 complete sampling sets were simulated using a random number generator in a spreadsheet. Calculations indicated that a 20% positive rate in WCR samples was approximately equivalent to an 11.42% positive rate in composite neck skin samples or a 3.96% positive rate in individual neck skin samples within a pool of 3. With 20% as the prevalence rate, 79.3% of the simulated WCR sets passed with 12 or fewer positive carcasses per set, very near the expected 80% rate. Under simulated European conditions, a Salmonella prevalence of 3.96% in individual neck skin samples yielded a passing rate of 79.1%. The 2 sampling plans thus have roughly equivalent outcomes if WCR samples have a Salmonella-positive rate of 20% and individual neck skin samples have a positive rate of 3.96%. Sampling and culturing methods must also be considered in comparing the different standards for Salmonella.

Comparison of neck skin excision and whole carcass rinse sampling methods for microbiological evaluation of broiler carcasses before and after immersion chilling.

Sampling protocols for detecting Salmonella on poultry differ among various countries. In the United States, the U.S. Department of Agriculture Food Safety and Inspection Service dictates that whole broiler carcasses should be rinsed with 400 ml of 1% buffered peptone water, whereas in the European Union 25-g samples composed of neck skin from three carcasses are evaluated. The purpose of this study was to evaluate a whole carcass rinse (WCR) and a neck skin excision (NS) procedure for Salmonella and Escherichia coli isolation from the same broiler carcass. Carcasses were obtained from three broiler processing plants. The skin around the neck area was aseptically removed and bagged separately from the carcass, and microbiological analysis was performed. The corresponding carcass was bagged and a WCR sample was evaluated. No significant difference (alpha

Campylobacter species occurrence within internal organs and tissues of commercial caged Leghorn laying hens.

Campylobacter spp. are frequently present in the intestinal tract and internal tissues of broiler breeder and broiler chickens. Campylobacter spp. ecology in commercial Leghorn laying hens has not been extensively studied. The objectives of the current study were to determine 1) Campylobacter spp. presence in the reproductive tract, lymphoid organs, liver-gallbladder, and ceca of commercial Leghorn laying hens; 2) species of Campylobacter present; and 3) antimicrobial resistance pattern of Campylobacter isolates. In study 1, three flocks ranging from 94 to 105 wk of age were sampled from a commercial laying complex. In study 2, two flocks, 82 and 84 wk of age, were sampled from a separate complex. Hens were killed, defeathered, aseptically necropsied, and the spleen, liver-gallbladder, ovarian follicles, and upper (infundibulum, magnum, and isthmus) and lower (shell gland and vagina) reproductive tracts were aseptically removed before the ceca. Samples were packed on ice and transported to the laboratory for evaluation. For speciation, a standard BAX real-time PCR method was used while susceptibility testing was performed using US National Antimicrobial Resistance Monitoring System (NARMS) standards and recommended quality control organisms. Isolates were examined for susceptibility using a semi-automated testing system (Sensititer) to the following 9 antimicrobials: azithromycin, clindamycin, ciprofloxacin, erythromycin, florfenicol, gentamicin, nalidixic acid, telithromycin, and tetracycline. In study 1, the isolation rate was 13, 67, 53, 3, 13, and 57% from the ovarian follicles, lower reproductive tract, upper reproductive tract, spleen, liver-gallbladder, and ceca, respectively. In study 2, the isolation rate was 17, 43, 33, 20, 17, and 73% from the ovarian follicles, lower reproductive tract, upper reproductive tract, spleen, liver-gallbladder, and ceca, respectively. Overall, 50% of isolates were Campylobacter jejuni, 49% Campylobacter coli, and 1% Campylobacter lari. In study 1, all of the isolates were pan-susceptible. In study 2, thirty-seven percent of the isolates were resistant to tetracycline. Commercial table egg laying hens housed in colony cages on wire floors had diverse Campylobacter spp. recovered from different tissues and these isolates were not resistant to a broad range of antimicrobials.

Campylobacter coli naturally resistant to elevated levels of gentamicin as a marker strain in poultry research.

Campylobacter inoculation studies are limited without a suitable marker strain. The lurpose of this study was to screen Campylobacter strains (n=2073) obtained from poultry carcass rinses through the Centers for Disease Control and Prevention's National Antimicrobial Resistant Monitoring System for resistance to gentamicin and evaluate one strain's efficacy as a marker. A C. coli strain was found resistant to gentamicin at >32 microg/ml. Gentamicin was incorporated into media (Campy-Cefex agar, Brucella agar, and blood agar) from 0 to 1000 microg/ml, and the upper level of gentamicin resistance was determined. C. coli strain's upper level of growth on Campy-Cefex plates, blood agar plates, and Brucella agar plates was 400, 300, and 200 pg/ml, respectively. Ceca and postpick carcass rinses were obtained and streaked onto Campy-Cefex agar at the above gentamicin levels to evaluate background microflora exclusion. Campy-Cefex agar containing gentamicin at 100 ag/ml prevented from the ceca, and reduced from the rinse, background microflora. The C. coli strain was orally or intracloacally inoculated into chicks. At 1, 3, and 6 weeks of age, inoculated broilers were removed and several tissue types sampled for the presence of the marker strain. At 6 weeks of age, 10 additional noninoculated penmates were sampled. The C. coli strain colonized chicks, disseminated to body tissues, colonized penmates, and persisted throughout the 6-week grow-out. The C. coli strain's unique characteristic, being resistant to high levels of gentamicin, allows for a marker that can be used in a wide range of Campylobacter research projects.

Evaluation of TECRA broth, Bolton broth, and direct plating for recovery of Campylobacter spp, from broiler carcass rinsates from commercial processing plants.

The purpose of this study was to compare a conventional culture broth method (Bolton enrichment), a newly developed proprietary broth method (TECRA Campylobacter enrichment), and direct plating for recovery of Campylobacter spp. from chicken carcass rinsates. Whole carcass rinses were taken from 140 carcasses at rehang (immediately after defeathering but before evisceration) and from 140 carcasses at postchill from eight different processing plants in the United States. The rinsate samples were packed in ice and shipped overnight to the laboratory. Aliquots of the rinsate were transferred into Bolton and TECRA enrichment broths and were direct plated. Standard laboratory procedures with Campy-cefex plates were followed for recovery of Campylobacter spp. For rehang carcasses, 94% were positive for Campylobacter spp. with the TECRA enrichment broth and 74% were positive with the Bolton enrichment broth. For postchill carcasses, 74% were positive for Campylobacter spp. with the TECRA enrichment broth and 71% were positive with the Bolton enrichment broth. Compared with the Bolton enrichment broth, TECRA enrichment broth significantly suppressed non-Campylobacter microflora (P < 0.05). Overall, TECRA enrichment broth yielded an 11% higher total number of Campylobacter-positive samples compared with the Bolton enrichment broth. Campylobacter spp. detection in postchill samples was significantly greater (P < 0.05) by enrichment (84%) than by direct plating (19%). The high number of Campylobacter-positive samples obtained with all procedures indicated that 99% of the carcass rinsates obtained at rehang and 84% obtained at postchill contained Campylobacter spp.

Transmission of Salmonella to broilers by contaminated larval and adult lesser mealworms, Alphitobius diaperinus (Coleoptera: Tenebrionidae).

The ability of the lesser mealworm, Alphitobius diaperinus (Panzer), commonly known as the darkling beetle, to transmit marker Salmonella Typhimurium to day-of-hatch broiler chicks was evaluated, as well as the spread to nonchallenged pen mates. In trial 1, day-of-hatch chicks were orally gavaged with 4 larval or 4 adult beetles that had been exposed to marker Salmonella-inoculated feed for 72 h. In addition, chicks were gavaged with the marker Salmonella in saline solution. These chicks were then placed into pens to serve as challenged broilers. In trial 2, all pens received 2 challenged chicks that were gavaged with larvae or beetles that had been exposed to marker Salmonella-inoculated feed for 24 h and then removed from the inoculated feed for a period of 7 d. At 3 wk of age, cecal samples from the marker Salmonella-challenged broilers and from 5 pen mates in trial 1, or 10 pen mates in trial 2, were evaluated for the presence of the marker Salmonella in their ceca, and at 6 wk of age, all remaining pen mates were sampled. To monitor the presence of the marker Salmonella within pens, stepped-on drag swab litter samples were taken weekly. For the Salmonella-saline pens, 29 to 33% of the broilers that had been challenged and 10 to 55% of the pen mates were positive at 3 wk of age, and only 2 to 6% had positive ceca at 6 wk. For the pens challenged with adult beetles, 0 to 57% of the challenged broilers and 20 to 40% of the pen mates had positive ceca at 3 wk, and 4 to 7% were positive at 6 wk. The pens challenged with larvae had the greatest percentage of marker Salmonella-positive broilers; 25 to 33% of the challenged broilers and 45 to 58% of pen mates were positive at 3 wk, and 11 to 27% were positive at 6 wk. These results demonstrated that ingestion of larval or adult beetles contaminated with a marker Salmonella could be a significant vector for transmission to broilers.

Comparison of four sampling methods for the detection of Salmonella in broiler litter.

Experiments were conducted to compare litter sampling methods for the detection of Salmonella. In experiment 1, chicks were challenged orally with a suspension of naladixic acid-resistant Salmonella and wing banded, and additional nonchallenged chicks were placed into each of 2 challenge pens. Nonchallenged chicks were placed into each nonchallenge pen located adjacent to the challenge pens. At 7, 8, 10, and 11 wk of age the litter was sampled using 4 methods: fecal droppings, litter grab, drag swab, and sock. For the challenge pens, Salmonella-positive samples were detected in 3 of 16 fecal samples, 6 of 16 litter grab samples, 7 of 16 drag swabs samples, and 7 of 16 sock samples. Samples from the nonchallenge pens were Salmonella positive in 2 of 16 litter grab samples, 9 of 16 drag swab samples, and 9 of 16 sock samples. In experiment 2, chicks were challenged with Salmonella, and the litter in the challenge and adjacent nonchallenge pens were sampled at 4, 6, and 8 wk of age with broilers remaining in all pens. For the challenge pens, Salmonella was detected in 10 of 36 fecal samples, 20 of 36 litter grab samples, 14 of 36 drag swab samples, and 26 of 36 sock samples. Samples from the adjacent nonchallenge pens were positive for Salmonella in 6 of 36 fecal droppings samples, 4 of 36 litter grab samples, 7 of 36 drag swab samples, and 19 of 36 sock samples. Sock samples had the highest rates of Salmonella detection. In experiment 3, the litter from a Salmonella-challenged flock was sampled at 7, 8, and 9 wk by socks and drag swabs. In addition, comparisons with drag swabs that were stepped on during sampling were made. Both socks (24 of 36, 67%) and drag swabs that were stepped on (25 of 36, 69%) showed significantly more Salmonella-positive samples than the traditional drag swab method (16 of 36, 44%). Drag swabs that were stepped on had comparable Salmonella detection level to that for socks. Litter sampling methods that incorporate stepping on the sample material while in contact with the litter appear to detect Salmonella in greater incidence than traditional sampling methods of dragging swabs over the litter surface.

Presence of inoculated Campylobacter and Salmonella in unabsorbed yolks of male breeders raised as broilers.

Day-old male broiler breeder chicks were obtained from a commercial hatchery and raised as broilers. For Experiment 1, at 5 wk of age, the broilers were orally inoculated with a 10(6) cfu/ml of a characterized strain of Campylobacter jejuni and a cocktail (three naladixic acid-resistant strains) of Salmonella serovars. One week after inoculation, the birds were euthanatized and defeathered. The abdominal cavity was examined and any unabsorbed yolk material (and remaining yolk stalk) and ceca were aseptically removed for microbiological analyses. For each pooled sample (two birds per pool), an aerobic plate count (APC), an Enterobacteriaceae (ENT) count, and a test for the presence of Campylobacter and Salmonella was performed. For Experiment 2, at 5 wk of age, the broilers were orally inoculated with 10(5) cfu/ml of a characterized strain of Campylobacter jejuni. One week after inoculation, the birds (n = 20) were killed, defeathered, and the yolk stalk, attached yolk, or free-floating yolk and ceca were individually analyzed for presence of Campylobacter. For Experiment 1, the Salmonella-inoculated birds had 2/12 ceca and 0/12 unabsorbed yolk samples positive for Salmonella. The average yolk APC was log10 3.4 cfu/g and the average ENT was log10 1.9 cfu/g. For the Campylobacter-inoculated birds, 12/12 ceca and 9/12 unabsorbed yolk samples were positive for Campylobacter. The average yolk APC was log10 3.5 cfu/g and the average ENT was log10 3.1 cfu/g. For Experiment 2, the inoculated Campylobacter birds had 19/20 ceca, 5/20 free floating yolks, and 19/20 yolk stalks positive. In Experiment 1, the inoculated Campylobacter colonized the ceca in every instance and were present in 75% of the unabsorbed yolks. Alternatively, the inoculated Salmonella were not found in any of the unabsorbed yolks and only rarely in the ceca. In Experiment 2, the inoculated Campylobacter was found in very high numbers in the yolk and internal body samples. Determining to what extent these internal bodies and unabsorbed yolks play in bacterial colonization and contamination of the birds at processing has not been determined. The next step will be to determine the incidence of unabsorbed yolks and presence of Campylobacter and Salmonella in these bodies of commercial broilers at processing.

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.

Detection of Campylobacter jejuni in various lymphoid organs of broiler breeder hens after oral or intravaginal inoculation.

Two studies were conducted to determine whether Campylobacter jejuni could rapidly spread and reside in the internal organs of adult broiler breeder hens. In Study 1, university-housed broiler breeders at 22 wk of age were obtained and placed in individual cages. Each hen was intravaginally inoculated weekly from 23 to 32 wk of age with a characterized strain of C. jejuni. At wk 23, 27, and 32, 4 d postinoculation, the hens were euthanized, defeathered, and aseptically opened. In Study 2, university-housed broiler breeder hens were obtained at 42, 53, and 56 wk of age, placed in individual cages, and inoculated either orally or intravaginally with a characterized strain of C. jejuni. To reduce the possibility of cross-contamination among samples, the thymus, spleen, liver, and gallbladder were aseptically removed, prior to the ceca. In both studies, all samples were individually analyzed. In Study 1, at 23 wk of age, C. jejuni was recovered from 4/7 thymii, 2/7 spleens, 5/7 livers and gallbladders, and 6/7 ceca. At 27 wk of age, C. jejuni was recovered from 1/7 thymii and 1/7 ceca. At 32 wk of age, C. jejuni was recovered from 4/11 thymii, 1/11 livers and gallbladders, and 2/11 ceca. In Study 2, C. jejuni was recovered from 2/6 thymii and 5/6 ceca after oral inoculation and 1/6 spleens, 1/6 livers and gallbladders, and 4/6 ceca after vaginal inoculation of 43-wk-old hens. Campylobacter jejuni was recovered from 2/5 thymii, 3/5 spleens, 3/5 livers and gallbladders, and 2/5 ceca after oral inoculation of 53-wk-old hens and 1/5 thymii and 1/5 livers and gallbladders after vaginal inoculation. Campylobacter jejuni was recovered from 1/4 thymii, 2/4 livers and gallbladders, and 1/4 ceca and was not detected in any vaginally inoculated birds of 57-wk-old hens. This study provides evidence that C. jejuni can reside in the internal organs of broiler breeder hens following oral or intravaginal inoculation.

Incidence of unabsorbed yolk sacs in broilers, broiler breeder roosters, white Leghorn hens, and Athens-Canadian randombred control broilers.

Unabsorbed yolk sacs are being investigated as a possible reservoir for internal Campylobacter and salmonellae contamination of processed poultry carcasses. However, it is unknown at what frequency that unabsorbed yolk sacs persist at the time of processing of broilers and spent breeders. Seven sets of 100 broiler carcasses (at 6 or 8 wk of age) were obtained from commercial processing plants. In addition, 100 52-wk-old broiler breeder males, 100 102-wk-old Leghorn hens, and 300 8-wk-old Athens-Canadian randombred control (ACRBC) broilers were euthanized, and their abdominal cavities were opened for determination of the presence of unabsorbed yolk sacs. Carcasses with obliterated yolk stalks or stalks with no detectable yolk material were categorized as normal. Those with unabsorbed yolk sacs were further separated into 2 groups: 1) attached by the yolk stalk to the small intestine or 2) unattached within the abdominal cavity. Yolk sacs were further classified by size: 1) small was <2 mm in diameter, 2) medium was 2 to 10 mm, and 3) large was >10 mm. From the 300 commercial broiler carcasses that were 6 wk old, 54% were categorized as normal with no detectable yolk sac, 35% had an unabsorbed yolk sac attached to the yolk stalk, and 12% had unattached yolk sacs. From the 400 commercial broiler carcasses that were 8 wk old, 49% of the carcasses were normal, 31% had attached unabsorbed yolk sacs, and 20% had unattached yolk sacs. From the 100 rooster carcasses sampled, 73% were normal, 8% had attached unabsorbed yolk sacs, and 19% had unattached yolk sacs. From the 100 White Leghorn hen carcasses sampled, 88% were normal, 8% had attached unabsorbed yolk sacs, and 4% were unattached yolk sacs. From the 300 ACRBC carcasses sampled, 76% were normal, 4% had attached unabsorbed yolk sacs, and 20% were unattached yolk sacs. The incidence of unabsorbed yolk sacs in present day commercial broilers appears twice as high as for mature roosters, hens, or ACRBC broilers.

Apparent attachment of Campylobacter and Salmonella to broiler breeder rooster spermatozoa.

It has been demonstrated that horizontal and vertical transmission of Salmonella and Campylobacter can occur in broiler breeder flocks. The mechanism of this transmission is still unclear. Previously negative broiler breeder flocks have been reported to become positive with Salmonella, Campylobacter, or both after the introduction of "spike" roosters at 45 wk of age. To determine whether the rooster semen is a possible source of transmission to hens for colonization, we evaluated the association of both Salmonella and Campylobacter spp. to segments (head, midpiece, and tail) of individual spermatozoa after artificial inoculation. Salmonella typhimurium, Salmonella heidelberg, and Salmonella montevideo, or Campylobacter jejuni (in 0.85% saline) was added to a freshly collected (by abdominal massage) aliquot of pooled semen from roosters housed in individual cages. The semen and bacteria solutions were incubated 1 h at room temperature. Samples were fixed using Karnosvsky and Zamboni fixatives for 24 h prior to centrifuging and rinsing in 0.1 M cacodylate x HCl buffer. Individual aliquot samples were then subjected to both scanning (JSM-5800) and transmission (JEM-1210) electron microscopy. The scanning electron microscopy showed that Salmonella was associated with all 3 segments (head, midpiece, and tail) of the spermatozoa and apparently equally distributed. Campylobacter was mainly associated with the midpiece and tail segments; few isolates were located on the head segment. The transmission electron microscopy showed apparent attachment of Salmonella and Campylobacter to the spermatozoa.

Movement and persistence of Salmonella in broiler chickens following oral or intracloacal inoculation.

The dissemination of Salmonella into various lymphoid-like organs in young broiler chicks after oral and intracloacal inoculation was studied. A three-strain cocktail of Salmonella Typhimurium, Salmonella Montevideo, and Salmonella Enteritidis was administered either orally or intracloacally to day-old chicks. After 1 h, 1 day, or 1 week, the ceca, thymus, liver and gallbladder, spleen, and bursa were sampled for the presence of Salmonella. There was a marked difference in the recovery of Salmonella 1 h postinoculation. Only 6 of 50 samples from orally inoculated chicks were positive compared with 33 of 50 samples from cloacally inoculated samples. In comparison, 24 h and 1 week after inoculation, there was no difference in the number of positive samples between oral or cloacal inoculation. The rapidity of the translocation of the Salmonella from the cloacal inoculum compared to the oral inoculum is likely due to the transient time required for Salmonella to move through the alimentary tract. The method of inoculation did not affect the distribution of serogroups. Of the three serotypes in the composite inoculum, the Salmonella Enteritidis (group D) was recovered only twice in replication 1 and not at all in replication 2. Both the Salmonella Typhimurium (serogroup B) and the Salmonella Montevideo (serogroup C1) were recovered extensively throughout the study.