Aim:  To determine the prevalence of Listeria monocytogenes and associated risk factors among beef operations (cow‐calf and feedlot) in central and southern California. Methods and Results:  A repeated cross‐sectional study where faecal and environmental samples were collected from 50 operations three times a year at different seasons was carried out. Samples were tested for presence of L. monocytogenes using a combination of enrichment and polymerase chain reaction tests. Data on putative risk factors were also collected. Listeria monocytogenes was detected in faecal samples from cows, calves and other animals on calf‐cow operations at proportions of 3·1%, 3·75% and 2·5%, respectively. The organism was detected in 5·3% of cut‐grass, 5·3% of soil, 14·3% of irrigation ditches, 3·1% of the ponds and 6·5% of water troughs samples. Listeria monocytogenes was less common in faecal (0·3%) and soil (0·75%) samples collected from feedlots. Conclusions:  Listeria monocytogenes was present at a higher proportion among cow‐calf operations than feedlots. There was no significant seasonal variation in the occurrence of this pathogen within the two types of operations. Significance and Impact of the Study:  If risk mitigation strategies were implemented to reduce the public health risk these should focus in cow‐calf operations.

We reexamined the finding of Neumann et al. (10) that intact Cryptosporidium parvum oocysts obtained after in vitro excystation were infectious for neonatal CD-1 mice. We used both established excystation protocols and our own protocol that maximized excystation (2). Although intact oocysts isolated after any of three protocols were infectious for neonatal CD-1 mice, the infectivity of intact oocysts isolated with our optimized excystation protocol was significantly lower than the infectivity of intact oocysts isolated after established protocols or from fresh oocysts. Excystation should not be considered a valid measure of C. parvum viability, given that it is biologically implausible for oocysts to be nonviable and yet infectious.The use of excystation as an indicator of Cryptosporidium parvum oocyst viability has come under criticism, given the observation that intact oocysts isolated after in vitro excystation were infectious for CD-1 neonatal mice (10). The presumption is that if an oocyst cannot excyst, it will be incapable of the physical and biochemical steps involved in initiating asexual multiplication (6, 7). Using established in vitro procedures, previous studies have found a wide discrepancy in the proportion of oocysts that excyst, ranging from 50 to 95% depending on which technique was used to stimulate excystation, such as pretreatment of oocysts with acid (2, 4, 5, 10, 12). Such a wide discrepancy in excystation rates for the same batch of oocysts indicates that some excystation protocols fail to fully stimulate oocysts to excyst, resulting in excessive amounts of intact oocysts following the procedure. This result not only leads to underestimating the proportion of oocysts that are presumably viable but also biases experiments that use intact oocysts isolated after in vitro excystation.Our concern with the conclusion that intact oocysts isolated after in vitro excystation are infectious for CD-1 neonatal mice (10) is that the process of excystation appeared not to be optimized, resulting in substantial numbers of excystable (i.e., viable) oocysts failing to excyst, thereby explaining the infectious potential of such oocysts for neonatal mice. Our goal for this project was to reexamine the finding that intact C. parvum oocysts obtained after in vitro excystation were infectious for neonatal CD-1 mice, using a protocol that maximized the proportion of oocysts that excysted (2), resulting in a more valid population of intact, presumably nonviable oocysts for in vivo experimentation.C. parvum oocysts. Feces were collected from naturally infected calves at 9 to 21 days of age from three local commercial dairies in Tulare, Calif., which served as the source of wild-type C. parvum oocysts for these experiments. These oocysts were previously classified as bovine genotype A, using the genotyping scheme described by Xiao et al. (13). After an acid faststaining procedure was used to detect oocysts (9), samples having more than 25 oocysts per microscopic field (400ϫ) of fecal smears were washed through a s...

This study measured the inactivation rate of bovine genotype A Cryptosporidium parvum oocysts attributable to diurnal oscillations of ambient temperature and solar radiation typical of California rangelands and dairies from spring through autumn. We first measured the relationship between air temperature and the internal temperature of bovine feces exposed to sunlight on commercial operations throughout California. Once maximum air temperature exceeded the mid 20 degrees C, diurnal thermal regimes of bovine fecal material exhibited peaks of over 40, 50, 60, and 70 degrees C. These diurnal thermal regimes were emulated using a thermocycler, with oocysts suspended in distilled water or fecal-water mix. Using oral inoculations of 10(5) C. parvum oocysts per neonatal Balb/c mouse (>1000-fold the ID50), no infections were observed using 1 to 5-day cycles of these thermal regimes. Loss of infectivity induced bythese thermal regimes was primarily due to partial or complete in vitro excystation during the first 24-h diurnal cycle and secondarily to thermal inactivation of the remaining intact or partial oocysts. These results suggest that as ambient conditions generate internal fecal temperatures > or = 40 degrees C via conduction, radiation, and convection, rapid environmental inactivation occurs at a rate of > or = 3.27 log reduction d(-1) for C. parvum oocysts deposited in the feces of cattle.

The present work calculated the rate of inactivation of Cryptosporidium parvum oocysts attributable to daily oscillations of low ambient temperatures. The relationship between air temperature and the internal temperature of bovine feces on commercial operations was measured, and three representative 24-h thermal regimens in the ϳ15°C, ϳ25°C, and ϳ35°C ranges were chosen and emulated using a thermocycler. C. parvum oocysts suspended in deionized water were exposed to the temperature cycles, and their infectivity in mice was tested. Oral inoculation of 10 3 treated oocysts per neonatal BALB/c mouse (ϳ14 times the 50% infective dose) resulted in time-and temperature-dependent reductions in the proportion of infected mice. Oocysts were completely noninfectious after 14 24-h cycles with the 30°C regimen and after 70 24-h cycles with the 20°C regimen. In contrast, oocysts remained infectious after 90 24-h cycles with the 10°C regimens. The estimated numbers of days needed for a 1-log 10 reduction in C. parvum oocyst infectivity were 4.9, 28.7, and 71.5 days for the 30, 20, and 10°C thermal regimens, respectively. The loss of infectivity of oocysts induced by these thermal regimens was due in part to partial or complete in vitro excystation.It is well recognized that the protozoan parasite Cryptosporidium parvum causes waterborne enteric disease and poses a significant threat to public health. Fecal contamination from infected hosts, such as humans and some species of livestock and wildlife (17), can lead to elevated concentrations of C. parvum oocysts in drinking, recreational, and irrigation water supplies (6,8). Once excreted, C. parvum oocysts can be eluted from fresh fecal matrices during precipitation events that generate surface flow or runoff conditions (4,5,12,21,32). During cool moist conditions oocysts can persist for months in the environment (10,11,25,30), but factors such as extremes of temperature, exposure to UV radiation, and desiccation can substantially reduce the number of infective oocysts prior to waterborne transport (2,7,9,11,19,24,25,29,30).To examine thermal stress, most studies have used constant thermal regimens to investigate the effect of temperature on the viability or infectivity of Cryptosporidium oocysts (11,14,20,28,30). To complement this work, we previously investigated the impact of large daily changes in the ambient temperature on C. parvum oocyst infectivity, using spring through autumn thermal regimens and temperatures measured inside bovine fecal pats that were exposed to solar radiation at cowcalf and dairy production facilities (23). Under California's summer climatic conditions, internal fecal pat temperatures range from 45°C to 75°C during the day and decrease 10 to 60°C during the night. Exposing oocysts to these large thermal fluctuations results in Ͼ3.3-log 10 reductions in oocyst infectivity in each 24-h cycle (23). The present study was conducted in order to measure the effect of exposure to oocysts to coolseason daily temperatures (with peaks at temperatures greater t...