Survival of a Pathogenic Leptospira Serovar in Response to Combined in vitro pH and Temperature Stresses and An Evaluation of Methods for the Detection of Leptospira from Water Samples

Abstract

Abstract:Leptospira interrogans is a zoonotic pathogen hosted commensally by a variety of mammalian species and is endemic to most tropical regions. Leptospirosis in humans is a febrile hemorrhagic illness and in severe cases can result in debilitating illness with possible long-term health consequences and even death. Human infection most commonly occurs when an individual with an abrasion of the skin comes in contact with water or moist soil contaminated by the urine of an infected host. Very little is known about the survival and transport of Leptospira spirochetes (leptospires) in the environment. Both aspects are key parts of developing strategies to reduce the potential for infection in streams that may be affected by drainage contaminated by infected hosts. This research focused on two aspects of assessing the risk of contamination: occurrence of leptospires in water and survival of leptospires after exposure to environmental stresses. The distribution and transport of Leptospira within the environment has not been well characterized. This is largely due to the difficulty associated with isolating and detecting leptospires from water samples, especially with the interference of sediment. We adapted molecular diagnostic tools involving PCR to develop and evaluate a method for the rapid detection of pathogenic leptospires from environmental water samples. L. icterohaemorrhagiae and L. Pomona are both known to act as human pathogens. We enumerated suspensions of both serovars in distilled deionized water during laboratory trials and successfully detected leptospires at concentrations as low as 100 cells/ml by filtration through 0.45 micron Millipore filters using the lipL32 primer set and PCR as our analytical endpoint. We applied these methods to environmental water samples during field trials on the islands of Oahu and Kauai in Hawaii. We were not able to detect wildtype Leptospira during our field trials however we successfully identified the presence of L. icterohaemorrhagiae from seeded environmental water samples as positive controls during our field trials.We also evaluated the probability of survival of pathogenic leptospires in response to the independent and combined stresses of pH and temperatures commonly observed in composted piggery waste. We treated the results of culturing as a binary variable (successful or not) and used logistic regression to model results. The results indicate that high temperatures (> 45 degrees C) are nearly 100% effective in killing leptospires, that pH slightly enhances the effect of temperature and that duration of exposure has an insignificant effect on survival. The results indicate that a correctly constructed and maintained composting facility could be very effective as a treatment to minimize the potential for releasing viable leptospires in drainage from piggeries.