Fecal Shedding of Zoonotic Food-Borne Pathogens by Wild Rodents in a Major Agricultural Region of the Central California Coast

Recent outbreaks of food-borne illness associated with the consumption of produce have increased concern over wildlife reservoirs of food-borne pathogens. Wild rodents are ubiquitous, and those living close to agricultural farms may pose a food safety risk should they shed zoonotic microorganisms in their feces near or on agricultural commodities. Fecal samples from wild rodents trapped on 13 agricultural farms (9 produce, 3 cow-calf operations, and 1 beef cattle feedlot) in Monterey and San Benito Counties, CA, were screened to determine the prevalence and risk factors for shedding of several food-borne pathogens. Deer mice (Peromyscus maniculatus) were the most abundant rodent species trapped (72.5%). Cryptosporidium species (26.0%) and Giardia species (24.2%) were the predominant isolates from rodent feces, followed by Salmonella enterica serovars (2.9%) and Escherichia coli O157:H7 (0.2%). Rodent trap success was significantly associated with detection of Salmonella in rodent feces, while farm type was associated with fecal shedding of Cryptosporidium and Giardia. Seasonal shedding patterns were evident, with rodents trapped during the spring and summer months being significantly less likely to be shedding Cryptosporidium oocysts than those trapped during autumn. Higher rodent species diversity tended to correlate with lower fecal microbial prevalence, and most spatiotemporal pathogen clusters involved deer mice. Rodents in the study area posed a minimal risk as environmental reservoirs of E. coli O157:H7, but they may play a role in environmental dissemination of Salmonella and protozoa. Rodent control efforts that potentially reduce biodiversity may increase pathogen shedding, possibly through promotion of intraspecific microbial transmission.     

Seasonal shedding of multiple Cryptosporidium genotypes in California ground squirrels (Spermophilus beecheyi)

Twelve percent of 853 California ground squirrels (Spermophilus beecheyi) from six different geographic locations in Kern County, Calif., were found to be shedding on average 44,482 oocysts g of feces(-1). The mean annual environmental loading rate of Cryptosporidium oocysts was 57,882 oocysts squirrel(-1) day(-1), with seasonal patterns of fecal shedding ranging from <10,000 oocysts squirrel(-1) day(-1) in fall, winter, and spring to levels of 2 x 10(5) oocysts squirrel(-1) day(-1) in summer. Juveniles were about twice as likely as adult squirrels to be infected and shed higher concentrations of oocysts than adults did, with particularly high levels of infection and shedding being found among juvenile male squirrels. Based on DNA sequencing of a portion of the 18S small-subunit rRNA gene, there existed three genotypes of Cryptosporidium species in these populations of squirrels (Sbey03a, Sbey03b, and Sbey03c; accession numbers AY462231 to AY462233, respectively). These unique DNA sequences were most closely related (96 to 97% homology) to porcine C. parvum (AF115377) and C. wrairi (AF115378). Inoculating BALB/c neonatal mice with up to 10,000 Sbey03b or Sbey03c fresh oocysts from different infected hosts did not produce detectable levels of infection, suggesting that this common genotype shed by California ground squirrels is not infectious for mice and may constitute a new species of Cryptosporidium.     

Lack of detectable shedding of Cryptosporidium parvum oocysts by periparturient dairy cattle

We examined whether periparturient dairy cattle shed Cryptosporidium parvum oocysts within 12 hr of calving on 3 commercial dairy farms endemic for calfhood cryptosporidiosis. Using a diagnostic method that can detect as few as 1 oocyst per gram of feces, we found no evidence of C. parvum oocysts in 86 fecal samples collected within 12 hr of calving from 43 dairy cows.     

DNA Sequence Similarity between California Isolates of Cryptosporidium parvum

We evaluated whether nucleic acid amplification with primers specific for Cryptosporidium parvum followed by automated DNA sequence analysis of the PCR amplicons could differentiate between California isolates of C. parvum obtained from livestock, humans, and feral pigs. Almost complete sequence identity existed among the livestock isolates and between the livestock and human isolates. DNA sequences from feral pig isolates differed from those from livestock and humans by 1.0 to 1.2%. The reference sequence obtained by Laxer et al. (M. A. Laxer, B. K. Timblin, and R. J. Patel, Am. J. Trop. Med. Hyg. 45:688–694, 1991.) differed from California isolates of C. parvum by 1.8 to 3.2%. These data suggest that DNA sequence analysis of the amplicon of Laxer et al. does not allow for differentiation between various strains of C. parvum or that our collection of isolates obtained from various hosts from across California was limited to one strain of C. parvum.     

Climate and on-farm risk factors associated with Giardia duodenalis cysts in storm runoff from California coastal dairies

Climatic factors and on-farm management practices were evaluated for their association with the concentrations (cyst/liter) and instantaneous loads (cysts/second) of Giardia duodenalis in storm-based runoff from dairy lots and other high-cattle-use areas on five coastal California farms over two storm seasons. Direct fluorescent antibody analysis was used to quantitate cysts in 350 storm runoff samples. G. duodenalis was detected on all five dairy farms, with fluxes of 1 to 14,000 cysts/liter observed in 16% of samples. Cysts were detected in 41% of runoff samples collected near cattle less than 2 months old, compared to 10% of runoff samples collected near cattle over 6 months old. Furthermore, the concentrations and instantaneous loads of cysts were > or =65 and > or =79 times greater, respectively, in runoff from sites housing young calves than in sites housing other age classes of animals. Factors associated with environmental loading of G. duodenalis included cattle age, cattle stocking number, and precipitation but not lot area, land slope, or cattle density. Vegetated buffer strips were found to significantly reduce waterborne cysts in storm runoff: each additional meter of vegetated buffer placed below high-cattle-use areas was associated with reductions in the concentration and instantaneous load of cysts by factors of 0.86 and 0.79 (-0.07 and -0.10 log(10)/m), respectively. Straw mulch, seed application, scraping of manure, and cattle exclusion did not significantly affect the concentration or load of G. duodenalis cysts. The study findings suggest that vegetated buffer strips, especially when placed near dairy calf areas, should help reduce the environmental loading of these fecal protozoa discharging from dairy farms.     

Salmonella spp., Vibrio spp., Clostridium perfringens, and Plesiomonas shigelloides in Marine and Freshwater Invertebrates from Coastal California Ecosystems

The coastal ecosystems of California are highly utilized by humans and animals, but the ecology of fecal bacteria at the land–sea interface is not well understood. This study evaluated the distribution of potentially pathogenic bacteria in invertebrates from linked marine, estuarine, and freshwater ecosystems in central California. A variety of filter-feeding clams, mussels, worms, and crab tissues were selectively cultured for Salmonella spp., Campylobacter spp., Escherichia coli-O157, Clostridium perfringens, Plesiomonas shigelloides, and Vibrio spp. A longitudinal study assessed environmental risk factors for detecting these bacterial species in sentinel mussel batches. Putative risk factors included mussel collection near higher risk areas for livestock or human sewage exposure, adjacent human population density, season, recent precipitation, water temperature, water type, bivalve type, and freshwater outflow exposure. Bacteria detected in invertebrates included Salmonella spp., C. perfringens, P. shigelloides, Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio alginolyticus. Overall, 80% of mussel batches were culture positive for at least one of the bacterial species, although the pathogens Campylobacter, E. coli-O157, and Salmonella were not detected. Many of the same bacterial species were also cultured from upstream estuarine and riverine invertebrates. Exposure to human sewage sources, recent precipitation, and water temperature were significant risk factors for bacterial detection in sentinel mussel batches. These findings are consistent with the hypothesis that filter-feeding invertebrates along the coast concentrate fecal bacteria flowing from land to sea and show that the relationships between anthropogenic effects on coastal ecosystems and the environmental niches of fecal bacteria are complex and dynamic.     

Capture and Retention of Cryptosporidium parvum Oocysts by Pseudomonas aeruginosa Biofilms

The association of Cryptosporidium oocysts with biofilm communities can influence the propagation of this pathogen through both environmental systems and water treatment systems. We observed the capture and retention of C. parvum oocysts in Pseudomonas aeruginosa biofilms using laboratory flow cells. Biofilms were developed in two different growth media using two different strains of P. aeruginosa, a wild-type strain (PAO1) and a strain that overproduces the exopolysaccharide alginate (PDO300). Confocal laser-scanning microscopy was used in conjunction with image analysis to assess the structure of the biofilms prior to introducing oocysts into the flow cells. More oocysts were captured by the biofilm-coated surfaces than the abiotic glass surface in both media. There was no significant difference in capture across the two strains of P. aeruginosa biofilm, but the fraction of oocysts captured was positively related to biofilm roughness and surface-area-to-volume ratio. Once captured, oocysts were retained in the biofilm for more than 24 h and were not released after a 40-fold increase in the system flow rate. We believe the capture and retention of oocysts by biofilm communities can impact the environmental transmission of C. parvum, and this interaction should be taken into consideration when predicting the migration of pathogens in the environment.     

Comparison of Sensitivity of Immunofluorescent Microscopy to That of a Combination of Immunofluorescent Microscopy and Immunomagnetic Separation for Detection of Cryptosporidium parvum Oocysts in Adult Bovine Feces

A direct immunofluorescence assay (DFA) (Merifluor; Meridian Diagnostics, Inc., Cincinnati, Ohio) was compared to an immunomagnetic separation (IMS) assay (Dynabeads; Dynal, Inc., Lake Success, N.Y.) coupled with immunofluorescent microscopy (Waterborne, Inc., New Orleans, La.) for their ability to detect low concentrations of Cryptosporidium parvum oocysts in adult bovine fecal material. IMS-DFA resulted in a 2-log-unit increase in sensitivity (10 oocysts/g) compared to DFA alone (1,000 oocysts/g). The higher sensitivity obtained with IMS-DFA resulted from testing 2 g of fecal material instead of the 13 to 19 mg of fecal material tested in the DFA; the increased sensitivity was not attributable to a higher percent recovery.     

Polymorphisms in the beta-tubulin gene of Cryptosporidium parvum differentiate between isolates based on animal host but not geographic origin

Polymerase chain reaction primers were designed to target a region of the Cryptosporidium parvum beta-tubulin gene spanning an intron. Amplification products contained 11 polymorphic positions, representing a sequence divergence of 1.8%, which discriminated between isolates of C. parvum found solely in humans (genotype 1) and those found in humans and animals (genotype 2). Seven of the polymorphic sites were located outside of the intron and the polymorphism between isolates was readily demonstrated by HaeIII restriction digestion. However, all of the sequences from genotype 1 human-derived oocysts isolated in the United States and Australia were conserved. Also, there were no sequence differences between bovine isolates obtained from both continents. Therefore, isolates could not be differentiated based on geographic source of origin.     

Clams (Corbicula fluminea) as bioindicators of fecal contamination with Cryptosporidium and Giardia spp. in freshwater ecosystems in California

This study evaluated clams as bioindicators of fecal protozoan contamination using three approaches: (i) clam tissue spiking experiments to compare several detection techniques; (ii) clam tank exposure experiments to evaluate clams that had filtered Cryptosporidium oocysts from inoculated water under a range of simulated environmental conditions; (iii) sentinel clam outplanting to assess the distribution and magnitude of fecal contamination in three riverine systems in California. Our spiking and tank experiments showed that direct fluorescent antibody (DFA), immunomagnetic separation (IMS) in combination with DFA, and PCR techniques could be used to detect Cryptosporidium in clam tissues. The most analytically sensitive technique was IMS concentration with DFA detection of oocysts in clam digestive gland tissues, which detected 10 oocysts spiked into a clam digestive gland 83% of the time. In the tank experiment, oocyst dose and clam collection time were significant predictors for detecting Cryptosporidium parvum oocysts in clams. In the wild clam study, Cryptosporidium and Giardia were detected in clams from all three study regions by IMS-DFA analysis of clam digestive glands, with significant variation by sampling year and season. The presence of C. parvum DNA in clams from riverine ecosystems was confirmed with PCR and DNA sequence analysis.