Preharvest Food Safety: Determining The Potential Of Salmonella Serotype Heidelberg To Systemically Contaminate Meat After Oral Challenge Of Broiler Chickens

<p>S. Heidelberg inoculum preparation:The ATCC and broiler meat-associated outbreak strains will be induced for resistance to nalidixic acid (NA - 50µg/ml), separately. Each strain will be grown in 1L of tryptic soy broth (TSB) containing NA for 24 hours at 37oC with shaking. The culture will be sedimented by centrifugation, and the resultant pellet reconstituted in phosphate buffered saline (PBS; pH 7.2) and diluted appropriately for use as inoculum (Kollanoor Johny et al., 2008, 2010a, 2012a, b, c).b) Ethics Statement:The project is approved by the IACUC (1403-31368A) for experiment with the ATCC strain. A modification involving experiments with the outbreak strain is awaiting approval.c) Experimental groups and S. Heidelberg challenge:Experimental groups explainedGroups-Birds-Challenge-ReasonGroup 1- 20 birds- No (100 CFU/bird) - Negative controlGroup 2- 20 birds - Yes (101 CFU/bird) - Treatment Dose 1Group 3- 20 birds - Yes (102 CFU/bird) - Treatment Dose 2Group 4- 20 birds - Yes (103 CFU/bird) - Treatment Dose 3Group 5- 20 birds - Yes (104 CFU/bird) - Treatment Dose 4Group 6- 20 birds - Yes (105 CFU/bird) - Treatment Dose 5Group 7- 20 birds - Yes (106 CFU/bird) - Treatment Dose 6Group 8- 20 birds - Yes (107 CFU/bird) - Treatment Dose 7Group 9- 20 birds - Yes (108 CFU/bird) - Treatment Dose 8Group 10- 20 birds - Yes (109 CFU/bird) - Treatment Dose 9200 birds/experiment X 3 replicates X 2 age groups (3- and 6-weeks) X 2 bacterial strains (ATCC and outbreak) = 2400 birdsCFU - colony forming units, a measure of bacterial numberFor the 3-week trials, 200 day-old broiler chicks (Ross X Ross) will be purchased and housed at the University of Minnesota BL2 research facility. All birds will be provided access ad libitum to Salmonella-free water and feed. Ten birds from the incoming flock will be randomly selected and screened for Salmonella using Sero-Quick Group kit. Birds will be weighed, wing banded, and grouped randomly into 10 groups of 20 birds per inoculum level as given in Table 1.On day 7, all birds in the inoculated groups will receive S. Heidelberg ranging from 101 to 109 CFU/bird by crop gavage (directly to the crop) (Kollanoor-Johny et al., 2009, 2012a, b, c). The control birds (10o CFU) will receive sterile PBS without bacteria (Kollanoor Johny et al., 2009). The birds will be maintained for 21 days when they will be euthanized for sample collection. For the 6-week trials, similar protocol will be followed except that the birds will be challenged with S. Heidelberg on day 25, and maintained until the end of experiment (day 42). Separate experiments will be carried out for the ATCC and outbreak strains.d) Sample analysis and measurements:d.</p><p><ol><li> Microbiological analysis: After 24 hours of challenge, 6 birds from each group will be euthanized to ensure colonization of the pathogen in the cecum and internal organs, liver and spleen. For this, 2-5 g of samples will be collected in PBS at 4oC during necropsy and homogenized. The homogenized mixture will be diluted ten-fold in PBS, and 0.1 ml portions plated on XLD-NA plates. The plates will be incubated at 37oC for 48 hours until characteristic colonies appear. Representative colonies from XLD-NA plates will be confirmed using the Salmonella rapid detection kit. Moreover, colonies will be confirmed as S. Heidelberg using the proprietary Taqman® PCR procedure after enrichment (Life Technologies). In addition, plasma Salmonella titer (from wing vein peripheral blood) will be determined (Nandre et al., 2011) using the Chicken IgG ELISA quantitation set (Bethyl Laboratories, TX). Similarly, on day 21 (last day of the 3-week trial) the remaining birds will be slaughtered and samples of cecum, liver, spleen, blood and meat (~5 g total from breasts, thighs and drumstick) will be collected aseptically for bacteriological analysis and PCR analysis as described above. Each muscle sample will be cut into small pieces using sterile scalpel blades to resemble a finished ground product before dilution or enrichment. Furthermore, if no colonies were detected by plating, samples will be tested for surviving cells by enrichment for 48 hours at 37oC in 100 ml selenite cysteine broth (SCB) (Fernandez et al., 2002; Filho et al., 2000) containing NA, followed by streaking on XLD-NA plates. Confirmation of colonies will be done using PCR as described above. For the 6-week experiments, similar protocol will be followed except that after 5 days of challenge, i.e. on day 30, 6 birds from each group will be euthanized to ensure colonization by the pathogen in the cecum, liver and spleen. Later, on day 42, remaining birds will be euthanized for end point analysis.d. </li><li> Histopathology and immunohistochemistry: Liver and muscle tissues (breast, thighs and drumsticks) from 6 birds in each group from both 3- and 6-week trials will be fixed in 10% neutral-buffered formalin for 24 hours. All tissues will be externally wiped with 100% ethanol to remove any live or attached bacteria on the surface. Tissues will be processed by standard methods to obtain 4µm sections, and stained with hematoxylin and eosin (HE). Further, the sections will be used for immunohistochemistry analysis using anti-Salmonella LPS antibody by avidin-biotin complex method in Dr. Porter's laboratory as described by Gonzalez-Escobedo et al. (2013). Salmonella LPS staining could determine if infection has proceeded into these tissues, especially in the muscles.d. </li><li> Growth measurements: The average feed consumption and body weights of birds will be determined for all groups in all experiments. Birds will be weighed individually at the beginning and at weekly intervals. The average feed consumption per bird will be calculated as done previously (Kollanoor Johny et al., 2012a, b, c).e. Power, Model and Data Analysis:Based on the power analysis, a total of 2400 broilers are required to complete the experiments. The broiler experiments will follow a completely randomized design factorial treatment structure. Pen will be the experimental unit and the experiments will be repeated three times. The data will be analyzed using the PROC-MIXED procedure of the statistical analysis software (version 9.3, SAS Institute Inc., Cary, NC). Differences among the least square means will be detected using Fisher's least significant different test. A P value of 0.05 will be considered statistically significant. The data on body weight and feed consumption will be analyzed similarly. For histopathology, inflammation will be scored from 0-4, with 0 being normal, 1 being focal infiltrates with no necrosis, 2 being multifocal infiltrates with necrosis, 3 being widespread inflammation with extensive necrosis, and 4 being widespread infiltrates with coalescing areas of necrosis (Gonzalez-Escobedo et al., 2013). A presence or absence of S. Heidelberg by visual analysis after H&E staining will be also determined. A Student's t test will be used to detect the differences of mean histopathology scores at P<0.05.f. Efforts:The results from this study will be published in Journal of Poultry Science that has wide range of readers, including the target population. Moreover, a graduate student will be also directly benefited from the project. The presentation made at the professional meetings will deliver scientific information to the peers, industry, government officials and others who are interested. The results will be used for procuring other extensive research/integrated funds from the USDA.g.Evaluation/Milestones:A graduate student will successfully finish and defend a research thesis under the PD's guidance. A peer-reviewed publication will be produced. A presentation based on the results will be made in a professional meeting. The results will be used for preparing an integrated USDA grant proposal determining the potential of viable strategies to control systemic transfer ofSalmonellato meat.</li></ol></p>