White-Nose Syndrome-Affected Litttle Brown Myotis (Myotis Lucifugus) Increase Grooming and Other Active Behaviors During Arousals from Hibernation

White-nose syndrome (WNS) is an emerging infectious disease of hibernating bats linked to the death of an estimated 5.7 million or more bats in the northeastern United States and Canada. White-nose syndrome is caused by the cold-loving fungus Pseudogymnoascus destructans (Pd), which invades the skin of the muzzles, ears, and wings of hibernating bats. Previous work has shown that WNS-affected bats arouse to euthermic or near euthermic temperatures during hibernation significantly more frequently than normal and that these too-frequent arousals are tied to severity of infection and death date. We quantified the behavior of bats during these arousal bouts to understand better the causes and consequences of these arousals. We hypothesized that WNS-affected bats would display increased levels of activity (especially grooming) during their arousal bouts from hibernation compared to WNS-unaffected bats. Behavior of both affected and unaffected hibernating bats in captivity was monitored from December 2010 to March 2011 using temperature-sensitive dataloggers attached to the backs of bats and infrared motion-sensitive cameras. The WNS-affected bats exhibited significantly higher rates of grooming, relative to unaffected bats, at the expense of time that would otherwise be spent inactive. Increased self-grooming may be related to the presence of the fungus. Elevated activity levels in affected bats likely increase energetic stress, whereas the loss of rest (inactive periods when aroused from torpor) may jeopardize the ability of a bat to reestablish homeostasis in a number of physiologic systems.

Sudden Increase and Regrowth of Fecal Coliforms and Escherichia Coli in Wastewater Biosolids After High Solids Centrifugation

Treatment plants that operate either thermophilic or mesophilic anaerobic digesters with centrifugal dewatering processes have consistently observed densities of fecal coliform and Escherichia coli, both indicator bacteria, that decrease during digestion but then increase after dewatering and storage. The increases have been characterized as two separate phenomena to explain this observation: 1) “Sudden Increase,” or SI, which is defined as the increase that occurs immediately after dewatering and 2) “regrowth,” which is defined as an increase during storage of cake samples over a period of hours or days. The SI observation appears to be more prevalent with biosolids that are generated with thermophilic processes and dewatered by centrifugation. Both thermophilic and mesophilic digesters with centrifuge dewatering processes have observed the regrowth phenomena. This research hypothesizes that the SI phenomenon is due to the presence of viable nonculturable (VNC) bacteria that are reactivated during dewatering. In other words, the bacteria were always present but were not enumerated by standard culturing methods (SCM). Analysis of the E. coli density in thermally treated solids by SCMs and quantitative real-time polymerase chain reaction (qPCR) indicated that E. coli densities are often underestimated by SCM. When analyzed with qPCR, the E. coli density after digestion can be 4-5 orders of magnitude greater than the non-detect levels identified by SCMs, which supports the non-culturable hypothesis. The VNC state describes a condition where bacteria are alive but unable to sustain the metabolic process needed for cellular division. Supplements added to culturing media were investigated to determine if the resuscitation of VNC bacteria could be enhanced. The autoinducer molecules Nhexanoyl- L-Homoserine lactone (C6-HSL), 3-oxo-N-octanoyl-L-Homoserine lactone (3-oxo- C8-HSL), and norepinephrine were unable to induce the resuscitation of VNC E. coli. Additional sampling was performed to determine if autoinducer molecules, peroxides, or other as of yet unknown inhibitory agents and toxins could be removed from biosolids during SCM. Culture media supplemented with the peroxide degrading compounds catalase, α-ketoglutaric acid, and sodium pyruvate was unable to resuscitate non-culturable E. coli. The additions of bentonite and exponential growth phase E. coli cell-free supernatant to culturing media were also unable to increase the culturability of E. coli. To remove inhibitory agents and toxins, a cell washing technique was employed prior to performing SCM; however, this cell washing technique may have increased cellular stresses that inhibited resuscitation since cell densities decreased. A novel laboratory-scale dewatering process was also investigated to determine if the SI and regrowth phenomena observed in full-scale centrifugal dewatering could be mimicked in the laboratory using a lab shearing device. Fecal coliform and E. coli densities in laboratory prepared cake samples were observed to be an order of magnitude higher than full-scale dewatered cakes. Additionally, the laboratory-scale dewatering process was able to resuscitate fecal coliforms and E. coli in stored sludge such that the density increased by 4-5 orders of magnitude from nondetect values. Lastly, the addition of aluminum sulfate during centrifuge dewatering at a full-scale utility produced an increased regrowth of fecal coliforms and E. coli that was sustained for 5 days.