Date of Award

5-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Microbiology

Committee Member

Xiuping Jiang, Committee Chair

Committee Member

Min Cao

Committee Member

Annel K. Greene

Committee Member

J. Michael Henson

Committee Member

Tzuen-Rong Jeremy Tzeng

Abstract

Poultry litter is usually recycled into the soil to improve the structure and fertility of agricultural land. As an important source of nutrients for crop production, poultry litter may possibly contain a variety of human pathogens that can threaten humans who consume the contaminated produce. The composting process can inactivate pathogens while creating a biological soil amendment beneficial for application to arable agricultural land. Some pathogens may have the potential to survive for long periods of time in raw poultry litter or its inadequately composted products after land application, and a small population of pathogenic cells may even regrow to high levels when the conditions are favorable for growth. Further thermal processing is a good choice for inactivating pathogens in poultry litter prior to land application. However, some microbial populations may become acclimatized to a hostile environment during composting or stockpiling and develop cross-protection against subsequent high temperature treatment. The objectives of this study were thus to 1) investigate the thermal inactivation of desiccation-adapted S. enterica in aged chicken litter, 2) study the effects of chicken litter storage time and ammonia content on thermal resistance of desiccation-adapted S. enterica, 3) develop a two-step heat treatment for inactivating desiccation-adapted S. enterica in aged chicken litter, 4) select the indigenous indicator microorganisms for validating desiccation-adapted S. enterica reduction in physically heat-treated poultry litter, and 5) study the thermal resistance and gene expression of both desiccation-adapted and rehydrated S. enterica Typhimurium in aged broiler litter. Thermal inactivation of desiccation-adapted Salmonella enterica in aged chicken litter was investigated in comparison with non-adapted cells to examine potential cross-tolerance of desiccation-adapted cells to heat treatment. A mixture of four Salmonella serovars was inoculated into the finished compost with 20, 30, 40, and 50% moisture contents for a 24-h desiccation adaptation. Afterwards, the compost with desiccation-adapted cells was inoculated into the aged chicken litter with the same moisture content for heat treatments at 70, 75, 80, 85 and 150°C. Recovery media were used to allow heat-injured cells to resuscitate. A 5-log reduction of the desiccation-adapted cells in aged chicken litter with 20% moisture content required >6, >6, 4~5, and 3~4 h exposure at 70, 75, 80, and 85°C, respectively. As a comparison, a 5-log reduction of non-adapted cells in the same chicken litter was achieved within 1.5~2, 1~1.5, 0.5~1, and <0.5 h at 70, 75, 80, and 85°C, respectively. Exposure time required to obtain a 5-log reduction in the desiccation-adapted cells gradually became shorter as temperature and moisture content were increased. At 150°C, desiccation-adapted Salmonella survived for 50 min in chicken litter with 20% moisture content, whereas control cells were detectable by enrichment until only 10 min. Our results demonstrated that the thermal resistance of Salmonella in aged chicken litter was increased significantly when the cells were adapted to desiccation. This study also validated the effectiveness of thermal processing being used for producing chicken litter free of Salmonella contamination. The effects of chicken litter storage time and ammonia content on thermal resistance of desiccation-adapted Salmonella were evaluated. Chicken litter was kept as a stacked heap on a poultry farm and samples were collected up to 9 months of storage. Chicken litter inoculated with desiccation-adapted Salmonella cells was heat-treated at 75, 80, 85, and 150°C. Salmonella populations decreased in all these samples during heat treatment, and the inactivation rates became slower in chicken litter when storage time was extended from 0 to 6 months. There was no significant difference (P>0.05) in thermal resistance of Salmonella in 6- and 9-month litter samples indicating that a threshold for thermal resistance was reached after 6 months. Overall, the thermal resistance of Salmonella in chicken litter was affected by the storage time of the litter. The changes in some chemical, physical, and microbiological properties during storage could possibly contribute to this difference. Moisture and ammonia could be two of the most significant factors influencing the thermal resistance of Salmonella cells in chicken litter. Our results emphasize the importance of adjusting time-temperature conditions for heat processing chicken litter when it is removed from the chicken house at different time intervals. The effectiveness of a two-step heat treatment for eliminating desiccation-adapted Salmonella spp. in aged chicken litter was evaluated. The aged chicken litter with 20, 30, 40, and 50% moisture contents was inoculated with a mixture of 4 Salmonella serotypes for a 24-h adaptation. Afterwards, the inoculated chicken litter was added into the chicken litter with the adjusted moisture content for a 1-h moist-heat treatment at 65°C and 100% relative humidity inside a water bath, followed by a dry-heat treatment in a convectional oven at 85°C for 1 h to the desired moisture level (<10~12%). After moist-heat treatment, the populations of Salmonella in aged chicken litter at 20 and 30% moisture contents declined from ca. 6.70 log cfu/g to 3.31 and 3.00 log cfu/g, respectively. And after subsequent 1-h dry-heat treatment, the populations further decreased to 2.97 and 2.57 log cfu/g, respectively. Salmonella cells in chicken litter with 40 and 50% moisture contents were only detectable by enrichment after 40 and 20 min of moist-heat treatment, respectively. Moisture contents in all samples were reduced to <10% after 1-h dry-heat process. Our results demonstrated that the two-step heat treatment was effective in reducing >5.5 logs of desiccation-adapted Salmonella in aged chicken litter with moisture content at or above 40%. Clearly, the findings from this study may provide chicken litter processing industry with an effective heat treatment method for producing Salmonella-free chicken litter. The indigenous indicator microorganisms for validating desiccation-adapted Salmonella reduction in physically heat-treated poultry litter was selected. The thermal resistance of desiccation-adapted S. Senftenberg 775/W was compared with those of indigenous enterococci and total aerobic bacteria in poultry litter. Aged broiler litter and composted turkey litter with 20, 30, 40, and 50% moisture contents were inoculated with desiccation-adapted S. Senftenberg 775/W, and then heat-treated at 75 and 85°C. Compared to total aerobic bacteria, there were better correlations between mean log reductions of desiccation-adapted S. Senftenberg 775/W and indigenous enterococci in broiler litter samples with 20, 30, 40, and 50% moisture contents at 75°C (R2>0.91), and 20, 30, and 40% moisture contents at 85°C (R2>0.87). The mean log reductions of S. Senftenberg 775/W were better-correlated with those of indigenous enterococci in turkey litter samples with 20, 30, 40, and 50% moisture contents at 75°C (R2>0.88), and 20 and 30% moisture contents at 85°C (R2=0.83) than those of total aerobic bacteria, which had a better correlation in turkey litter sample with 40% (R2=0.98) moisture content at 85°C. Indigenous enterococci may be used to validate the thermal processing of poultry litter as it mimics the survival behavior of Salmonella under some treatment conditions. This study provides some scientific data for poultry litter processors when validating the effectiveness of thermal processing. The thermal resistance and gene expression of both desiccation-adapted and rehydrated S. Typhimurium in aged broiler litter was investigated. S. Typhimurium cells were desiccation-adapted in aged broiler litter with 20% moisture content (aw: 0.81) for 3, 12, or 24 h at room temperature and then rehydrated for 3 h. Four genes (rpoS, proV, dnaK, and grpE) were up-regulated under desiccation stress (P<0.05) and they could be induced within an even shorter period of time (after 1 h but less than 2 h). Following rehydration, fold changes of these four genes became significantly lower (P<0.05). Desiccation-adapted ΔrpoS Salmonella mutant was less heat-resistant at 75°C than desiccation-adapted wild type (P<0.05), whereas there were no differences in heat resistance between desiccation-adapted Salmonella mutants in two non-regulated genes (otsA and PagfD) and desiccation-adapted wild type (P>0.05). Survival characteristics of desiccation-adapted ΔPagfD (rdar morphotype) and ΔagfD (saw morphotype) were similar (P>0.05). The trehalose synthesis in 3-, 12-, or 24-h desiccation-adapted wild type was not significantly induced as compared to non-adapted cells (P>0.05). Our results demonstrated the importance of rpoS, proV, dnaK, and grpE genes in the desiccation survival of S. Typhimurium. Moreover, rpoS gene was identified to be involved in the cross-protection of desiccation-adapted S. Typhimurium against high temperature, while trehalose synthesis or rdar morphology did not play a significant role in this phenomenon. S. Typhimurium could respond rapidly to the low-aw condition in aged broiler litter while developing the cross-protection against high temperature. Our results suggested the thermal resistance of desiccation-adapted Salmonella in poultry litter could be affected by litter storage time and ammonia content. Desiccation-adapted Salmonella in poultry litter could produce cross-protection against subsequent thermal stress. And rpoS has been found to play a significant role in this cross-protection. A two-step thermal processing technique was developed to rapidly inactivate desiccation-adapted Salmonella in poultry litter. Indigenous enterococci can be used as an indicator microorganism to validate the thermal processing of poultry litter, as it mimics the survival behavior of desiccation-adapted Salmonella during heat treatment. This study provides some valuable information for poultry litter processors to control human pathogens in poultry litter as biological soil amendments.

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