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Consumption of shell eggs has been associated with Salmonella Enteritidis (SE) infections in humans in the United States. Because of this, the Pennsylvania Egg Quality Assurance Program (PEQAP) was developed and implemented in 1994. The PEQAP involves periodic flock testing and management practices to minimize SE contamination of shell eggs. Subsequently, the U.S. Food and Drug Administration (FDA) introduced a mandatory federal program in 2010 and 2012 for shell egg producers modeled closely after PEQAP to reduce the incidence and prevalence of SE during production, storage, and transport nationwide. In this study, a retrospective epidemiologic analysis was conducted by characterizing SE isolated from commercial layer environment samples and shell eggs submitted to the Animal Diagnostic Laboratory at The Pennsylvania State University using phage typing and pulsed-field gel electrophoresis (PFGE). The objective of this study was to determine the relatedness of SE isolates from hen house environments and shell eggs and to optimize the existing protocols of egg quality assurance programs by identifying the best layer-house environmental sampling time points in order to minimize SE contamination of shell eggs. A total of 94 SE isolates from 65 hen flocks on 35 premises in Pennsylvania recovered during 2007 to 2015 were used in this study. The SE phage type 8 and PFGE fingerprint type JEGX01.0004 most commonly associated with human SE infection was also the predominant type present in layer-house environments and shell eggs. This reconfirms hen house environmental monitoring is an effective method to identify SE-infected flocks. Further, the PEQAP program allowed SE detection of infected flocks earlier than the FDA program as it included an additional environmental test at 29-31 wk of age, enabling the earlier prevention of SE-contaminated shell eggs going to the market. Therefore, it is recommended to refine the sampling time points of the current FDA Egg Rule by adding hen house environmental testing at 29-31 wk of age.

Avian cholera is a significant disease of domestic and wild birds caused by the bacterium Pasteurella multocida (PM). In poultry, a major source of PM infection is chronic carriers, domestic birds that have become infected and recovered or had subclinical infections. Although outbreaks of avian cholera in ring-necked pheasants (Phasianus colchicus) have been reported, the potential for chronic carriers is unknown. To address this, we conducted surveillance for PM in a flock of captive ring-necked pheasants after an outbreak of avian cholera that responded positively to antibiotic treatment based on resolution of morbidity and mortality. At approximately 1 mo after antibiotic treatment, oropharyngeal swabs were collected from 300 pheasants (out of a total population of ~2300) in a single winter holding pen. All samples were tested for PM through routine aerobic bacterial culture, but none of the samples were positive. In addition, there were no additional outbreaks within this infected pen over the subsequent months. These data provide preliminary evidence to suggest that pheasants that respond to antibiotic therapy may be less likely to become chronic carriers of PM than other poultry species, such as chickens (Gallus domesticus). However, due to marked phenotypic and biologic differences between PM strains, additional studies are needed to further support or refute these findings and better understand avian cholera in this species.

Salmonella contamination of laying hen flocks and shell eggs is associated with various management and environmental factors. Foodborne outbreaks of human salmonellosis have been traced back to consumption of Salmonella-contaminated shell eggs. In the present study, a systematic literature review was conducted to identify and provide an evidence-based overview of potential risk factors of Salmonella contamination of laying hens, layer premises, and shell eggs. This systematic literature search was conducted using AGRICOLA, CAB Abstracts, and PubMed databases. Observational studies that identified risk factors for Salmonella contamination of layer flocks and shell eggs were selected, and best evidence was synthesized to summarize the results. Altogether, 13 cross-sectional studies and four longitudinal studies published in English were included in the review. Evidence scores were assigned based on the study design and quality of the study to grade the evidence level. The strength of association of a risk factor was determined according to the odds ratios. In this systematic review, the presence of previous Salmonella infection, absence of cleaning and disinfection, presence of rodents, induced molting, larger flock size (>30,000 hens), multiage management, cage housing systems, in-line egg processing, rearing pullets on the floor, pests with access to feed prior to movement to the feed trough, visitors allowed in the layer houses, and trucks near farms and air inlets were identified as the risk factors associated with Salmonella contamination of laying hen premises, whereas high level of manure contamination, middle and late phase of production, high degree of egg-handling equipment contamination, flock size of >30,000, and egg production rate of >96% were identified as the risk factors associated with Salmonella contamination of shell eggs. These risk factors demonstrated strong to moderate evidence of association with Salmonella contamination of laying hens and shell eggs. Eggshells testing positive for Salmonella were 59 times higher when fecal samples were positive and nine times higher when floor dust samples were positive. Risk factors associated with Salmonella Enteritidis infection in laying hens were flock size, housing system, and farms with hens of different ages. As a summary, this systematic review demonstrated that Salmonella contamination of laying hen flocks and shell eggs in layer production systems is multifactorial. This study provides a knowledge base for the implementation of targeted intervention strategies to control Salmonella contamination of laying hen flocks and shell eggs.

Streptococcus gallolyticus, previously known as Streptococcus bovis biotypes I and II/2, is a well-known cause of sepsis and meningitis in humans and birds. The present case report describes an outbreak of fatal septicemia associated with S. gallolyticus subsp. pasteurianus (S. bovis biotype II/2) in 11 turkey flocks in Pennsylvania between 2010 and 2013. Affected poults were 2-3 wk of age. Major clinical observation was sudden increase in mortality among turkey poults without any premonitory clinical signs. Postmortem examination findings revealed acute septicemia with lesions such as fibrinous pericarditis, meningitis, splenic multifocal fibrinoid necrosis, hepatitis, osteochondritis, myositis, and airsacculitis. Gram-positive cocci were isolated from several organs by routine bacterial culture. Biotyping identified bacteria as streptococci, whereas 16S ribosomal RNA gene sequencing identified them as S. gallolyticus subsp. pasteurianus. Antibiotic susceptibility profiles revealed that all the strains isolated were sensitive to penicillin and erythromycin with different sensitivity profiles for other antibacterial agents tested. The present study reports the first confirmed case of acute septicemia in turkey poults caused by S. gallolyticus subsp. pasteurianus.

The present study describes an experimental infection model for avian pathogenic Escherichia coli (APEC)-induced egg peritonitis in layer chickens. First, a pilot study which consisted of two separate experiments was carried out to compare two routes of inoculations of APEC to induce peritonitis and to examine if the presence of egg yolk in the peritoneum would facilitate APEC-induced peritonitis. This study showed that the presence of egg yolk in the peritoneum facilitated the development of egg peritonitis when the APEC was inoculated via the intra-uterine (IU) route. Based on the results of the pilot study, 56-wk-old white leghorn hens were divided into two groups of five chickens, Group G (inoculated with E. coli APECO78 strain) and Group H (control). Both groups were inoculated with 2-3 ml of egg yolk via the intraperitoneal route (IP). Subsequently, hens in Group H were inoculated with only egg yolk whereas the hens in Group G were inoculated with 1 x 10(9) colony-forming units of APECO78 bacteria via the IU route. Parameters such as mortality, clinical signs (anorexia, depression, and egg production efficiency), gross lesion scores, bacterial loads in internal organs, and histopathology of ovary and oviduct were assessed to evaluate the success of the infection model. Group G showed 40% acute mortality, severe depression, and anorexia with markedly reduced egg production and developed peritonitis-associated lesions such as accumulation of yellowish caseous fluid in the peritoneum, salpingitis, and oophoritis. Histopathologically, ovarian and oviduct tissues from group G exhibited severe inflammatory changes such as infiltration of mononuclear cells and edema. Group G also showed significant bacterial loads in the peritoneum, ovary, and oviduct. Interestingly, deceased birds from group G had also developed mild perihepatitis and pericarditis with heavy bacterial loads in the internal organs. On the other hand, group H birds did not exhibit any of the clinical signs and remained healthy until the end of the experiment. To summarize, our results demonstrate that IP administration of egg yolk followed by IU inoculation of APECO78 induced peritonitis in laying hens. Experimental infection models are often required to understand the mechanisms of disease pathogenesis. Therefore, the present infection model will aid in the studies of pathogenesis of layer peritonitis caused by APEC and in evaluating vaccine candidates to control the disease.

Avian pathogenic Escherichia coli (APEC) causes morbidity in chickens and exhibits zoonotic potential. Understanding host transcriptional responses to infection aids the understanding of protective mechanisms and serves to inform future colibacillosis control strategies. Transcriptomes of spleen and peripheral blood leukocytes (PBLs) of the same individual birds in response to APEC infection were compared to identify common response patterns and connecting pathways. More than 100 genes in three contrasts examining pathology and infection status were significantly differentially expressed in both tissues and similarly regulated. Tissue-specific differences in catalytic activity, however, appear between birds with mild and severe pathology responses. Early expression differences, between birds with severe pathology and uninfected controls, in the mitogen-activated protein kinase pathway in PBLs precede spleen responses in the p53 and cytokine-cytokine receptor pathways. Tissue bianalysis is useful in identifying genes and pathways important to the response to APEC, whose role might otherwise be underestimated in importance.

Avian pathogenic Escherichia coli (APEC) cause colibacillosis, a disease which is responsible for significant losses in poultry. Control of colibacillosis is problematic due to the restricted availability of relevant antimicrobial agents and to the frequent failure of vaccines to protect against the diverse range of APEC serogroups causing disease in birds. Previously, we reported that the increased serum survival gene (iss) is strongly associated with APEC strains, but not with fecal commensal E. coli in birds, making iss and the outer membrane protein it encodes (Iss) candidate targets for colibacillosis control procedures. Preliminary studies in birds showed that their immunization with Iss fusion proteins protected against challenge with two of the more-commonly occurring APEC serogroups (O2 and O78). Here, the potential of an Iss-based vaccine was further examined by assessing its effectiveness against an additional and widely occurring APEC serogroup (O1) and its ability to evoke both a serum and mucosal antibody response in immunized birds. In addition, tissues of selected birds were subjected to histopathologic examination in an effort to better characterize the protective response afforded by immunization with this vaccine. Iss fusion proteins were administered intramuscularly to four groups of 2-wk-old broiler chickens. At 2 wk postimmunization, chickens were challenged with APEC strains of the O1, O2, or O78 serogroups. One week after challenge, chickens were euthanatized, necropsied, any lesions consistent with colibacillosis were scored, and tissues from these birds were taken aseptically. Sera were collected pre-immunization, postimmunization, and post-challenge, and antibody titers to Iss were determined by enzyme-linked immunosorbent assay (ELISA). Also, air sac washings were collected to determine the mucosal antibody response to Iss by ELISA. During the observation period following challenge, 3/12 nonimmunized chickens, 1/12 chickens immunized with 10 microg of GST-Iss, and 1/12 chickens immunized with 50 microg of GST-Iss died when challenged with the O78 strain. No other deaths occurred. Immunized chickens produced a serum and mucosal antibody response to Iss and had significantly lower lesion scores than nonimmunized chickens following challenge, regardless of the challenge strain. This study expands on our previous report of the value of Iss as an immunoprotective antigen and demonstrates that immunization with Iss can provide significant protection of chickens against challenge with three different E. coli strains.

P fimbrial adhesins may be associated with the virulence of avian pathogenic Escherichia coli (APEC). However, most APECs are unable to express P fimbriae even when they are grown under conditions that favor P fimbrial expression. This failure can be explained by the complete absence of the pap operon or the presence of an incomplete pap operon in Pap-negative APEC strains. In the present study, we analyzed the pap operon, specifically the papA gene that encodes the major fimbrial shaft, to better understand the pap gene cluster at the genetic level. First, by PCR, we examined a collection of 500 APEC strains for the presence of 11 genes comprising the pap operon. Except for papA, all the other genes of the operon were present in 38% to 41.2% of APEC, whereas the papA was present only in 10.4% of the APEC tested. Using multiplex PCR to probe for allelic variants of papA, we sought to determine if the low prevalence of papA among APEC was related to genetic heterogeneity of the gene itself. It was determined that the papA of APEC always belongs to the F11 allelic variant. Finally, we sequenced the 'papA region' from two papA-negative strains, both of which contain all the other genes of the pap operon. Interestingly, both strains had an 11,104-bp contig interruptingpapA at the 281-bp position. This contig harbored a streptomycin resistance gene and a classic Tn10 transposon containing the genes that confer tetracycline resistance. However, we noted that the papA gene of every papA-negative APEC strain was not interrupted by an 11,104-bp contig. It is likely that transposons bearing antibiotic resistance genes have inserted within pap gene cluster of some APEC strains, and such genetic events may have been selected for by antibiotic use.

P or Pap fimbriae of avian pathogenic Escherichia coli (APEC) have previously been shown to be important in the pathogenesis of colibacillosis. However, no study has been done to ascertain the effects of deletion of the genes responsible for P fimbrial biogenesis on APEC's in vitro and/or in vivo characteristics. In the present study, all 11 genes of the pap gene cluster were deleted from APEC O1, the recently sequenced APEC strain, and the wild-type strain was compared to the mutant (ECPAP1) for changes in certain phenotypic characteristics and virulence for chickens. Both APEC O1 and ECPAP1 demonstrated mannose-sensitive agglutination of guinea pig and chicken erythrocytes, but only APEC O1 demonstrated mannose-resistant hemagglutination and P-receptor binding properties. The in vivo experiments revealed that ECPAP1 was markedly attenuated as compared to its wild-type strain APEC O1. These findings suggest that P fimbriae are involved in the virulence of APEC O1.

Colibacillosis caused by avian pathogenic Escherichia coli (APEC) is a leading cause of economic loss to the poultry industry worldwide. The ability of APEC to cause disease is determined by certain virulence markers, some of which are located on pathogenicity islands (PAls). We recently described one such PAI in an APEC O1:K1 strain (APEC-O1). This PAI, termed PAI I(APEC-O1), carries the genes of the pap operon, a region similar to the tia invasion determinant of enterotoxigenic E coli; ireA, a gene that encodes an iron-responsive element; and a novel 1.5-kb region, ORF 54. Here, the occurrence of six selected loci of PAI I(APEC-O1) (papA, papC, papG, ireA, tia, and ORF 54) among APEC and fecal E. coli strains from apparently healthy chickens (avian commensal E. coli) was determined using polymerase chain reaction (PCR) techniques. None of the commensal E. coli was positive for all six traits, whereas 7.2% of the APEC isolates were positive for all the traits. Although there was no significant difference in the occurrence of ORF 54 among APEC and commensal E. coli, tia, ireA, papC, and papG genes were predominantly present in APEC rather than in avian commensal E. coli. papA was detected in only 6.3% of APEC, perhaps because of the presence of allelic variants of the gene. Additionally, the presence of all six traits was tested with PCR in APEC isolates collected in the 1980s, and these results were compared with those obtained with the APEC isolated in the 1990s. There was no significant difference in the occurrence of tia, ireA, papC, papG, and ORF 54 between APEC isolates collected during the different decades. However, papA was more frequently present in APEC from the 1980s than it was in APEC from the 1990s. Phylogenetic group of an isolate did not correlate with pathogenicity or the presence of PAI traits, except that more APEC of the low-pathogenicity group belonged to the phylogenetic group B1. However, PAI traits occurred more frequently in isolates belonging to the intermediate- and high-pathogenicity groups than in isolates of low pathogenicity.