Determination of Antimicrobial Resistance Patterns in Salmonella from Commercial Poultry as Influenced by Microbiological Culture and Antimicrobial Susceptibility Testing Methods.

Monitoring antimicrobial resistance of foodborne pathogens in poultry is critical for food safety. We aimed to compare antimicrobial resistance phenotypes in Salmonella isolated from poultry samples as influenced by isolation and antimicrobial susceptibility testing methods. Salmonella isolates were cultured from a convenience sample of commercial broiler ceca with and without selective broth enrichment, and resistance phenotypes were determined for 14 antimicrobials using the Sensititre® platform and a qualitative broth breakpoint assay. The broth breakpoint method reported higher resistance to chloramphenicol, sulfisoxazole, and the combination of trimethoprim and sulfamethoxazole, and lower resistance to streptomycin as compared to the Sensititre® assay in trial one. Selective enrichment of samples containing Salmonella in Rappaport-Vassiliadis broth reported lowered detectable resistance to amoxicillin/clavulanic acid, ampicillin, azithromycin, cefoxitin, ceftriaxone, nalidixic acid, and meropenem, and increased resistance to streptomycin and tetracycline than direct-plating samples in trial one. Using matched isolates in trial two, the Sensititre® assay reported higher resistance to chloramphenicol and gentamicin, and lower resistance to nalidixic acid as compared to the broth breakpoint method. These results suggest methodology is a critical consideration in the detection and surveillance of antimicrobial resistance phenotypes in Salmonella isolates from poultry samples and could affect the accuracy of population or industry surveillance insights and intervention strategies.

Eimeria species and genetic background influence the serum protein profile of broilers with coccidiosis.

BACKGROUND: Coccidiosis is an intestinal disease caused by protozoal parasites of the genus Eimeria. Despite the advent of anti-coccidial drugs and vaccines, the disease continues to result in substantial annual economic losses to the poultry industry. There is still much unknown about the host response to infection and to date there are no reports of protein profiles in the blood of Eimeria-infected animals. The objective of this study was to evaluate the serum proteome of two genetic lines of broiler chickens after infection with one of three species of Eimeria. METHODOLOGY/PRINCIPAL FINDINGS: Birds from lines A and B were either not infected or inoculated with sporulated oocysts from one of the three Eimeria strains at 15 d post-hatch. At 21 d (6 d post-infection), whole blood was collected and lesion scoring was performed. Serum was harvested and used for 2-dimensional gel electrophoresis. A total of 1,266 spots were quantitatively assessed by densitometry. Protein spots showing a significant effect of coccidia strain and/or broiler genetic line on density at P<0.05-0.01 (250 spots), P<0.01-0.001 (248 spots), and P<0.001 (314 spots) were excised and analyzed by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry. Proteins were identified in 172 spots. A total of 46 different proteins were identified. Of the spots with a corresponding protein identification, 57 showed a main effect of coccidia infection and/or 2-way interaction of coccidia infection×broiler genetic line at P<0.001. CONCLUSIONS/SIGNIFICANCE: Several of the metabolic enzymes identified in this study are potential candidates for early diagnostic markers of E. acervulina infection including malate dehydrogenase 2, NADH dehydrogenase 1 alpha subcomplex 9, and an ATP synthase. These proteins were detected only in Line A birds that were inoculated with E. acervulina. Results from this study provide a basic framework for future research aimed at uncovering the complex biochemical mechanisms involved in host response to Eimeria infection and in identifying molecular targets for diagnostic screening and development of alternative preventative and therapeutic methods.

Multi-platform next-generation sequencing of the domestic turkey (Meleagris gallopavo): genome assembly and analysis.

A synergistic combination of two next-generation sequencing platforms with a detailed comparative BAC physical contig map provided a cost-effective assembly of the genome sequence of the domestic turkey (Meleagris gallopavo). Heterozygosity of the sequenced source genome allowed discovery of more than 600,000 high quality single nucleotide variants. Despite this heterozygosity, the current genome assembly (∼1.1 Gb) includes 917 Mb of sequence assigned to specific turkey chromosomes. Annotation identified nearly 16,000 genes, with 15,093 recognized as protein coding and 611 as non-coding RNA genes. Comparative analysis of the turkey, chicken, and zebra finch genomes, and comparing avian to mammalian species, supports the characteristic stability of avian genomes and identifies genes unique to the avian lineage. Clear differences are seen in number and variety of genes of the avian immune system where expansions and novel genes are less frequent than examples of gene loss. The turkey genome sequence provides resources to further understand the evolution of vertebrate genomes and genetic variation underlying economically important quantitative traits in poultry. This integrated approach may be a model for providing both gene and chromosome level assemblies of other species with agricultural, ecological, and evolutionary interest.

Molecular cloning and functional characterization of avian interleukin-19.

The present study describes the cloning and functional characterization of avian interleukin (IL)-19, a cytokine that, in mammals, alters the balance of Th1 and Th2 cells in favor of the Th2 phenotype. The full-length avian IL-19 gene, located on chromosome 26, was amplified from LPS-stimulated chicken monocytes, and cloned into both prokaryotic (pET28a) and eukaryotic (pcDNA3.1) expression vectors. The confirmed avian IL-19 amino acid sequence has 66.5% homology with human and murine IL-19, with a predicted protein sequence of 176 amino acids. Analysis of avian IL-19 amino acid sequence showed six conserved, structurally relevant, cysteine residues as found in mammals, but only one N-glycosylation residue. The recombinant IL-19 (rChIL-19) expressed in the prokaryotic system was purified by Ni(+)-resin column followed by endotoxin removal. Using purified avian rChIL-19, expression of Th2 cytokines was measured in splenocytes using quantitative real-time PCR (qRT-PCR). In the presence of rChIL-19, expression levels of IL-4 and IL-13, as well as IL-10, were significantly increased after 6- and 12 h treatments. This was confirmed by treating splenocytes with supernatants from IL-19 transfected cells. Also, avian monocytes incubated with rChIL-19 displayed increased expression of IL-1beta, IL-6, and IL-19. This study represents the first report for the cloning, expression, and functional characterization of avian IL-19. Taken together, avian IL-19 function seems to be conserved and similar to that of mammals and may play an important role in responses to intracellular poultry pathogens like bacteria and protozoa.

Evaluation of the Polymerase Chain Reaction for the Detection of Salmonella enteritidis in Experimentally Inoculated Eggs and Eggs from Experimentally Challenged Hens.

Current recommendations for identification of Salmonella enteritidis (SE)-contaminated eggs, as outlined by the USDA SE Task Force, require the combination of yolk and albumen from several eggs for room-temperature enrichment for 3 days prior to culture on solid medium. We have previously reported the development of a technique involving enzymatic digestion and chemical reduction of pools of egg albumen allowing for the concentration of low numbers of SE by centrifugation. This technique allowed for detection of Salmonella with sensitivity comparable to conventional culture. Importantly, this technique allowed presumptive identification of SE at least 48 h sooner than conventional culture. We presently describe the use of this technique for the concentration of low numbers of SE in albumen pools for detection by the polymerase chain reaction (PCR). Three experiments performed with experimentally inoculated eggs indicated the PCR to be similar in sensitivity to the room temperature culture procedure for the detection of SE. Additionally, 5 experiments were performed in which hens were experimentally challenged with SE, and eggs were collected at selected times postchallenge. While few positive egg pools were detected by either method, data suggested that the PCR did not falsely identify positive eggs. These experiments provide preliminary evidence that the PCR is comparably sensitive to the room-temperature culture technique, while providing presumptive identification of SE 72 h sooner than conventional culture.

Evaluation of a Centrifugation Method for the Detection of Salmonella enteritidis in Experimentally Contaminated Chicken Eggs.

Current recommendations for identification of Salmonella enteritidis ( SE ) contaminated eggs, as outlined by the USDA SE Task Force, require the combination of yolk and albumen from several eggs for room-temperature enrichment for 3 days prior to culture on solid medium. We describe the use of enzymatic digestion and chemical reduction to reduce viscosity and allow for concentration of Salmonella cells in egg albumen by centrifugation prior to enrichment and plating. In these studies, sanitized, intact, fresh shell eggs were inoculated with low numbers of viable SE . Albumen and yolk from six eggs in each group were separated; the albumen was pooled, treated with the proteolytic enzyme papain and the reducing agent N-acetyl-l-cysteine, and centrifuged, and the resulting pellet was incubated in tetrathionate broth (5 ml) at 37°C for 24 h prior to plating on brilliant green agar (BOA). For comparison, yolk and albumen from six eggs in each group were pooled, thoroughly mixed, and incubated at room temperature for 3 days prior to direct plating on BOA. Results from three trials indicated that the two culture techniques were equally sensitive for detection of Salmonella (range: 72 to 0.6 colony-forming units (CFU) of SE per pool). While as sensitive as the technique recommended by the SE Task Force, the centrifugation technique allowed more rapid (48 h) presumptive identification of Salmonella .