Colonization, spread and persistence of Salmonella (Typhimurium, Infantis and Reading) in internal organs of broilers.

Transfer of Salmonella to internal organs of broilers over a 35 d grow-out period was evaluated. A total of 360 one-day old chicks were placed in 18 floor pens of 3 groups with 6 replicate pens each. On d 0, broilers were orally challenged with a cocktail of Salmonella (equal population of marked serovars; nalidixic acid-resistant S. Typhimurium, rifampicin-resistant S. Infantis, and kanamycin-resistant S. Reading) to have 3 groups: L (low; ∼2 log CFU/bird); M (medium; ∼5 log CFU/bird); and H (High; ∼8 log CFU/bird). On d 2, 7 and 35, 4 birds/pen were euthanized and ceca, liver, and spleen samples were collected aseptically. Gizzard samples (4/pen) were collected on d 35. The concentration of Salmonella in liver and spleen were transformed to binary outcomes (positive and negative) and fitted in glm function of R using cecal Salmonella concentrations (log CFU/g) and inoculation doses (L, M, and H) as inputs. On d 2, H group showed greater (P ≤ 0.05) cecal colonization of all 3 serovars compared to L and M groups. However, M group showed greater (P ≤ 0.05) colonization of all 3 serovars in the liver and spleen compared to L group. Salmonella colonization increased linearly in the ceca and quadratically in the liver and spleen with increasing challenge dose (P ≤ 0.05). On d 35, L group had greater (P ≤ 0.05) S. Infantis colonization in the ceca and liver compared to M and H groups (P ≤ 0.05). Moreover, within each group on d 35, the concentration of S. Reading was greater than those of S. Typhimurium and S. Infantis for all 3 doses in the ceca and high dose in the liver and gizzard (P ≤ 0.05). Salmonella colonization diminished in the ceca, liver, and spleen during grow-out from d 0 to d 35 (P ≤ 0.05). On d 35, birds challenged with different doses of Salmonella cocktail showed a similar total Salmonella spp. population in the ceca (ca. 3.14 log CFU/g), liver (ca. 0.54 log CFU/g), spleen (ca. 0.31 log CFU/g), and gizzard (ca. 0.42 log CFU/g). Estimates from the fitted logistic model showed that one log CFU/g increase in cecal Salmonella concentration will result in an increase in relative risk of liver and spleen being Salmonella-positive by 4.02 and 3.40 times (P ≤ 0.01), respectively. Broilers from H or M group had a lower risk (28 and 23%) of being Salmonella-positive in the liver compared to the L group when the cecal Salmonella concentration is the same (P ≤ 0.05). Oral challenge of broilers with Salmonella spp. with various doses resulted in linear or quadratic increases in Salmonella colonization in the internal organs during early age and these populations decreased during grow-out (d 35). This research can provide guidance on practices to effectively mitigate the risk of Salmonella from chicken parts and enhance public health.

Influence of peroxyacetic acid concentration, temperature, pH, and treatment time on antimicrobial efficacy against Salmonella on chicken wings.

Peroxyacetic acid (PAA) is commonly used during poultry processing to reduce the prevalence of Salmonella on carcasses and parts. Wash solutions containing PAA are used at varying concentrations during processing and processors use internally validated practices that best suit the needs of the individual establishment. This study was conducted to determine how temperature, pH, and contact time in combination with PAA concentration can affect the survival of Salmonella on poultry. The effectiveness of PAA in reducing the population of Salmonella on chicken wings was dependent on the concentration and temperature of the PAA solutions. The pH or contact time had no effects (P > 0.05) on total Salmonella or Salmonella Infantis reduction (log CFU/mL). Treatment with 0 ppm PAA at 27°C did not reduce (P > 0.05) total Salmonella or Salmonella Infantis compared to the inoculated, untreated control; in contrast, treatment at 4°C and 0 ppm PAA reduced (P < 0.05) total Salmonella and Salmonella Infantis. Treatments applied at 4°C significantly reduced (P < 0.05) total Salmonella at 50, 200, and 500 ppm PAA, compared to treatment at 27°C among the same PAA concentration. The population of Salmonella Infantis was significantly reduced (P < 0.05) at 4°C with 0, 50, 200, 500, and 1,000 ppm PAA among the same PAA concentration, compared to treatment at 27°C. Treatment conditions, such as temperature, can impact the effectiveness of PAA used as an antimicrobial treatment during poultry processing, and the results from this study can provide useful insights that could assist poultry processors to effectively incorporate PAA into antimicrobial intervention systems.

Effect of a carrageenan/chitosan coating with allyl isothiocyanate on microbial spoilage and quality of chicken breast.

Approximately 3.7% of poultry meat is lost due to spoilage each year in the United States. The objective of this study was to determine the efficacy of a layered carrageenan/chitosan coating in combination with an application of two concentrations of allyl isothiocyanate (AITC) against lactic acid bacteria, aerobic bacteria, and yeast and mold during storage of chicken breast for 21 d. Additionally, the rancidity, color, and pH of the chicken breast as indicators of non-microbial quality were evaluated. The combination of carrageenan/chitosan coating with 20 and 200 ppm AITC reduced (P ≤ 0.05) yeast and mold populations by 3 log10 CFU/g at d 21 compared to the untreated control. The carrageenan/chitosan coating with 20 and 200 ppm AITC delayed aerobic spoilage by 3 and 12 d, respectively, compared to the untreated control; aerobic bacteria populations on the samples treated with 200 ppm AITC remained below the threshold for spoilage (∼6 log10 CFU/g) for the duration of storage. The pH of the 20 ppm and 200 ppm AITC-treated chicken breast was unaltered (P > 0.05) at the end of storage and was lower than the pH of the untreated and coating-only-treated control chicken breast at d 18 through the end of storage (P ≤ 0.05). The application of the coating alone did not (P > 0.05) affect L*, a*, and b* values of the chicken breast at the end of storage compared to the uncoated control. The carrageenan/chitosan coating with 20 and 200 ppm AITC prevented decreases in the lightness (L* values) of the chicken breast at the end of storage (P ≤ 0.05) compared to the control and coating-only-treated samples. The coating alone or with AITC did not (P > 0.05) impact the rancidity of the chicken breast over the 21-d storage period, thus showing potential to be used as antimicrobial packaging to increase shelf life of fresh poultry.

Evaluating the efficacy of peracetic acid on Salmonella and Campylobacter on chicken wings at various pH levels.

Peracetic acid (PAA) is commonly used as an antimicrobial aid during poultry processing to reduce the pathogen load on poultry and poultry products. However, limited research is available on the effects of pH on the efficacy of PAA against Salmonella and Campylobacter. Therefore, the objective of this study was to determine the efficacy of PAA in reducing Salmonella and Campylobacter populations on chicken wings adjusted to various pH levels. Chicken wings (0.454 kg each) were inoculated with nalidixic acid-resistant (200 ppm) Salmonella Typhimurium (∼7 log10 cfu/mL) and gentamicin-resistant (200 ppm) Campylobacter coli (∼6-7 log10 cfu/mL). Inoculated wings were treated with PAA by immersion for 10 s or 60 min at 4°C to 6°C. The treatments included 50 ppm (0.005%) and 500 ppm (0.05%) PAA at 3 pH levels (8.2, 10, and 11) or sodium hydroxide (NaOH, pH 11). Surviving populations of Salmonella and Campylobacter were determined by sampling the chicken wings after treatments. Irrespective of concentration and pH of PAA, higher (P ≤ 0.05) reductions of Salmonella were observed subsequent to 60 min exposure as compared with 10 s of immersion. Immersion time and the higher pH of antimicrobial solutions did not affect (P > 0.05) the antimicrobial efficacy of PAA (50 or 500 ppm) against Campylobacter. The antimicrobial efficacy of PAA was not affected by pH of the antimicrobial solutions, and longer exposure time and higher PAA concentrations improve the antimicrobial efficacy.

Genomic features shaping the landscape of meiotic double-strand-break hotspots in maize.

Meiotic recombination is the most important source of genetic variation in higher eukaryotes. It is initiated by formation of double-strand breaks (DSBs) in chromosomal DNA in early meiotic prophase. The DSBs are subsequently repaired, resulting in crossovers (COs) and noncrossovers (NCOs). Recombination events are not distributed evenly along chromosomes but cluster at recombination hotspots. How specific sites become hotspots is poorly understood. Studies in yeast and mammals linked initiation of meiotic recombination to active chromatin features present upstream from genes, such as absence of nucleosomes and presence of trimethylation of lysine 4 in histone H3 (H3K4me3). Core recombination components are conserved among eukaryotes, but it is unclear whether this conservation results in universal characteristics of recombination landscapes shared by a wide range of species. To address this question, we mapped meiotic DSBs in maize, a higher eukaryote with a large genome that is rich in repetitive DNA. We found DSBs in maize to be frequent in all chromosome regions, including sites lacking COs, such as centromeres and pericentromeric regions. Furthermore, most DSBs are formed in repetitive DNA, predominantly Gypsy retrotransposons, and only one-quarter of DSB hotspots are near genes. Genic and nongenic hotspots differ in several characteristics, and only genic DSBs contribute to crossover formation. Maize hotspots overlap regions of low nucleosome occupancy but show only limited association with H3K4me3 sites. Overall, maize DSB hotspots exhibit distribution patterns and characteristics not reported previously in other species. Understanding recombination patterns in maize will shed light on mechanisms affecting dynamics of the plant genome.

Evolution of meiotic recombination genes in maize and teosinte.

BACKGROUND: Meiotic recombination is a major source of genetic variation in eukaryotes. The role of recombination in evolution is recognized but little is known about how evolutionary forces affect the recombination pathway itself. Although the recombination pathway is fundamentally conserved across different species, genetic variation in recombination components and outcomes has been observed. Theoretical predictions and empirical studies suggest that changes in the recombination pathway are likely to provide adaptive abilities to populations experiencing directional or strong selection pressures, such as those occurring during species domestication. We hypothesized that adaptive changes in recombination may be associated with adaptive evolution patterns of genes involved in meiotic recombination. RESULTS: To examine how maize evolution and domestication affected meiotic recombination genes, we studied patterns of sequence polymorphism and divergence in eleven genes controlling key steps in the meiotic recombination pathway in a diverse set of maize inbred lines and several accessions of teosinte, the wild ancestor of maize. We discovered that, even though the recombination genes generally exhibited high sequence conservation expected in a pathway controlling a key cellular process, they showed substantial levels and diverse patterns of sequence polymorphism. Among others, we found differences in sequence polymorphism patterns between tropical and temperate maize germplasms. Several recombination genes displayed patterns of polymorphism indicative of adaptive evolution. CONCLUSIONS: Despite their ancient origin and overall sequence conservation, meiotic recombination genes can exhibit extensive and complex patterns of molecular evolution. Changes in these genes could affect the functioning of the recombination pathway, and may have contributed to the successful domestication of maize and its expansion to new cultivation areas.

Recombination patterns in maize reveal limits to crossover homeostasis.

During meiotic recombination, double-strand breaks (DSBs) are formed in chromosomal DNA and then repaired as either crossovers (COs) or non-crossovers (NCOs). In most taxa, the number of DSBs vastly exceeds the number of COs. COs are required for generating genetic diversity in the progeny, as well as proper chromosome segregation. Their formation is tightly controlled so that there is at least one CO per pair of homologous chromosomes whereas the maximum number of COs per chromosome pair is fairly limited. One of the main mechanisms controlling the number of recombination events per meiosis is CO homeostasis, which maintains a stable CO number even when the DSB number is dramatically altered. The existence of CO homeostasis has been reported in several species, including mouse, yeast, and Caenorhabditis elegans. However, it is not known whether homeostasis exists in the same form in all species. In addition, the studies of homeostasis have been conducted using mutants and/or transgenic lines exhibiting fairly severe meiotic phenotypes, and it is unclear how important homeostasis is under normal physiological conditions. We found that, in maize, CO control is robust only to ensure one CO per chromosome pair. However, once this limit is reached, the CO number is linearly related to the DSB number. We propose that CO control is a multifaceted process whose different aspects have a varying degree of importance in different species.

Silencing of a metaphase I-specific gene results in a phenotype similar to that of the Pairing homeologous 1 (Ph1) gene mutations.

Although studied extensively since 1958, the molecular mode of action of the Pairing homeologous 1 (Ph1) gene is still unknown. In polyploid wheat, the diploid-like chromosome pairing is principally controlled by the Ph1 gene via preventing homeologous chromosome pairing (HECP). Here, we report a candidate Ph1 gene (C-Ph1) present in the Ph1 locus, transient as well as stable silencing of which resulted in a phenotype characteristic of the Ph1 gene mutants, including HECP, multivalent formation, and disrupted chromosome alignment on the metaphase I (MI) plate. Despite a highly conserved DNA sequence, the C-Ph1 gene homeologues showed a dramatically different structure and expression pattern, with only the 5B copy showing MI-specific expression, further supporting our claim for the Ph1 gene. In agreement with the previous reports about the Ph1 gene, the predicted protein of the 5A copy of the C-Ph1 gene is truncated, and thus perhaps less effective. The 5D copy is expressed around the onset of meiosis; thus, it may function during the earlier stages of chromosome pairing. Along with alternate splicing, the predicted protein of the 5B copy is different from the protein of the other two copies because of an insertion. These structural and expression differences among the homeologues concurred with the previous observations about Ph1 gene function. Stable RNAi silencing of the wheat gene in Arabidopsis showed multivalents and centromere clustering during meiosis I.

Chromatin immunoprecipitation for studying chromosomal localization of meiotic proteins in maize.

Chromatin immunoprecipitation (ChIP) is a method that allows identification of chromosomal sites occupied by specific proteins. In this technique, chromatin is extracted from cells, sheared, and, using a specific antibody, enriched in fragments that contain a protein of interest. Genomic location of the protein can then be identified by hybridization of the resulting DNA to tiling microarrays or by sequencing. Thanks to advances in high-throughput sequencing methods, studying protein localization using ChIP has become possible even in species with relatively large genomes. Here, we describe a ChIP protocol that we developed to examine localization of meiotic proteins in maize.

Fine structure mapping of a gene-rich region of wheat carrying Ph1, a suppressor of crossing over between homoeologous chromosomes.

The wheat gene-rich region (GRR) 5L0.5 contains many important genes, including Ph1, the principal regulator of chromosome pairing. Comparative marker analysis identified 32 genes for the GRR controlling important agronomic traits. Detailed characterization of this region was accomplished by first physically localizing 213 wheat group 5L-specific markers, using group 5 nulli-tetrasomics, three Ph1 gene deletion/insertion mutants, and nine terminal deletion lines with their breakpoints around the 5L0.5 region. The Ph1 gene was localized to a much smaller region within the GRR (Ph1 gene region). Of the 61 markers that mapped in the four subregions of the GRR, 9 mapped in the Ph1 gene region. High stringency sequence comparison (e < 1 x10(-25)) of 157 group 5L-specific wheat ESTs identified orthologs for 80% sequences in rice and 71% in Arabidopsis. Rice orthologs were present on all rice chromosomes, although most (34%) were on rice chromosome 9 (R9). No single collinear region was identified in Arabidopsis even for a smaller region, such as the Ph1 gene region. Seven of the nine Ph1 gene region markers mapped within a 450-kb region on R9 with the same gene order. Detailed domain/motif analysis of the 91 putative genes present in the 450-kb region identified 26 candidates for the Ph1 gene, including genes involved in chromatin reorganization, microtubule attachment, acetyltransferases, methyltransferases, DNA binding, and meiosis/anther specific proteins. Five of these genes shared common domains/motifs with the meiosis specific genes Zip1, Scp1, Cor1, RAD50, RAD51, and RAD57. Wheat and Arabidopsis homologs for these rice genes were identified.