An Improved PCR Protocol For Detection of Babesia duncanI In Wildlife and Vector Samples.

Human babesiosis is a tick-borne protozoal disease of increasing clinical significance in North America. Most cases in the eastern and Midwestern regions of the United States are reportedly due to Babesia microti infections. By contrast, most human infections reported in California and Washington have been attributed to a new species that was first identified in 1991 and subsequently named Babesia duncani. Although the tick vector and mammalian reservoir hosts for B. microti are well characterized, the vector and reservoir hosts for B. duncani are unknown. As a result, specific risk factors for human infections cannot be characterized. Identification of potential hosts and vector species has been hampered by the lack of specific and sensitive molecular diagnostic tools to amplify parasite DNA. To address this need, a nested PCR assay targeting the ß-tubulin gene, a well-conserved locus in piroplasm parasites with a highly variable intron region among species, was developed. The assay was evaluated by spiking tick and mammalian DNA extracts with DNA from a B. duncani isolate derived from a human patient (WA-1) as well as related Babesia spp. from Californian wildlife. This assay was highly specific, with a sensitivity of approximately 1 copy of template DNA in a background of tick DNA. At this level of detection B. duncani was detectable in larval tick samples, and the target locus allowed for visual differentiation between species by gel electrophoresis. This assay offers researchers a new tool for elucidating the natural transmission cycle of B. duncani.

Toxoplasma gondii seroprevalence and association with equine protozoal myeloencephalitis: A case-control study of Californian horses.

While toxoplasmosis is not commonly considered a clinical disease of equines, previous seroprevalence studies have reported differing background rates of Toxoplasma gondii infection in horses globally. The objective of this study was to evaluate possible associations between T. gondii seroprevalence and clinical signs of equine protozoal myeloencephalitis (EPM) in horses. Using a case-control study design, 720 Californian horses with neurologic signs compatible with EPM were compared to healthy, non-neurologic horses for the presence of T. gondii antibodies (using indirect fluorescent antibody tests [IFAT]). Toxoplasma gondii seroprevalence among cases and controls was determined at standard serum cut-offs: 40, 80, 160, 320, and 640. At a T. gondii titre cut-off of 320, horses with clinical signs compatible with EPM had 3.55 times the odds of a seropositive test compared to those without clinical signs (P<0.01) when adjusted for covariates. When restricted to the autumn season and at the same titre cut-off, an EPM suspect horse had 6.4 times the odds of testing seropositive to T. gondii, compared to non-neurologic horses. The association between high T. gondii titres and clinical signs compatible with EPM is potentially reflective of toxoplasmosis in equines. Serologic testing of cerebrospinal fluid and isolation of T. gondii in EPM suspect cases should be considered. Future studies investigating the relationship between T. gondii and EPM are warranted.

Comparison of human and southern sea otter (Enhydra lutris nereis) health risks for infection with protozoa in nearshore waters.

Cryptosporidium and Giardia spp. are waterborne, fecally-transmitted pathogens that cause economic loss due to gastroenteritis and beach closures. We applied quantitative microbial risk assessment (QMRA) to determine the health risks for humans and sea otters due to waterborne exposure of Cryptosporidium and Giardia spp. when swimming in three types of surface waters: river, stormwater and wastewater effluent during the wet and dry seasons in the central coast of California. This is the first application of QMRA to estimate both the probability of infection in Southern sea otters and the probability of illness in humans, using microbial source tracking (MST) as a variable. Children swimming close to stormwater discharges had an estimated Cryptosporidium-associated illness probability that exceeded the accepted U.S. EPA criteria (32 illnesses/1000 swimmers or 3.2%). Based on the assumption that sea otters are as susceptible as humans to Cryptosporidium infection, the infection probabilities were close to 2% and 16% when sea otters were swimming at the end of points of rivers and stormwater discharges, respectively. In the case of Giardia, infection probabilities of 11% and 23% were estimated for sea otters swimming at the end of point of wastewater discharges, assuming that sea otters are as susceptible as gerbils and humans, respectively. The results of this QMRA suggest that 1) humans and sea otters are at risk when swimming at outflow sites for rivers, stormwater and treated wastewater effluent; 2) reduced loads of viable protozoan cysts and oocysts in recreational water can lessen the probability of infection of humans and sea otters; and 3) the risk of infection of humans and sea otters can be reduced with the treatment of wastewater to decrease oocyst and cyst viability before effluent is released into the sea.

Molecular epidemiology of Cryptosporidium spp. and Giardia spp. in mussels (Mytilus californianus) and California sea lions (Zalophus californianus) from Central California.

Cryptosporidium and Giardia are of public health importance, with recognized transmission through recreational waters. Therefore, both can contaminate marine waters and shellfish, with potential to infect marine mammals in nearshore ecosystems. A 2-year study was conducted to evaluate the presence of Cryptosporidium and Giardia in mussels located at two distinct coastal areas in California, namely, (i) land runoff plume sites and (ii) locations near sea lion haul-out sites, as well as in feces of California sea lions (CSL) (Zalophus californianus) by the use of direct fluorescent antibody (DFA) detection methods and PCR with sequence analysis. In this study, 961 individual mussel hemolymph samples, 54 aliquots of pooled mussel tissue, and 303 CSL fecal samples were screened. Giardia duodenalis assemblages B and D were detected in hemolymph from mussels collected near two land runoff plume sites (Santa Rosa Creek and Carmel River), and assemblages C and D were detected in hemolymph from mussels collected near a sea lion haul-out site (White Rock). These results suggest that mussels are being contaminated by protozoa carried in terrestrial runoff and/or shed in the feces of CSL. Furthermore, low numbers of oocysts and cysts morphologically similar to Cryptosporidium and Giardia, respectively, were detected in CSL fecal samples, suggesting that CSL could be a source and a host of protozoan parasites in coastal environments. The results of this study showed that Cryptosporidium and Giardia spp. from the feces of terrestrial animals and CSL can contaminate mussels and coastal environments.

Individual subject meta-analysis of parameters for Giardia duodenalis shedding in animal experimental models.

Giardia duodenalis is a zoonotic protozoan parasite with public health importance worldwide. While articles about animal model infectivity have been published for G. duodenalis, the studies have used diverse protocols and parameters to evaluate the infectivity of this protozoan parasite. Hence, the objectives of this study were to (1) conduct a meta-analysis of published literature for cyst shedding and diarrhea outcomes in animal models and (2) develop recommendations to help standardize experimental dose response studies. Results showed that, for the outcome of cyst shedding in faeces, the covariates of infective stage (cyst versus trophozoite), Giardia dose, and the interactions between doses and infective stage, as well as dose and species of experimental host, were all significant (P value ≤ 0.05). This study suggests inoculation of the experimental host with cysts rather than trophozoites and administration of higher doses of Giardia will most likely result in cyst shedding. Based on the results of this meta-analysis, the infective stage (cyst versus trophozoite), parasite dose, and the interactions between dose and infective stage, as well as dose and species of experimental host, should be considered when designing experimental dose response studies that will assist in the study of zoonotic neglected tropical diseases globally.

Zoonotic fecal pathogens and antimicrobial resistance in county fair animals.

Livestock fairs present a unique opportunity for the public to experience close contact with animals, but may also expose people to zoonotic pathogens through contact with animal feces. The goal of this study was to screen cattle, sheep, goat, chicken, rabbit and horse feces from a livestock fair in California for the potentially zoonotic pathogens Escherichia coli O157:H7, Salmonella, Campylobacter, Vibrio, Cryptosporidium and Giardia spp., as well as determining the level of antimicrobial resistance in E. coli and Salmonella. Notably, E. coli O157:H7 was reported for the first time in a pig at a county fair in California. Campylobacter jejuni as well as Salmonella enterica serovars Derby and Thompson were also isolated from pigs, cattle, sheep, goats or chickens, whereas horses and rabbits were negative for all target pathogens. The prevalence of antimicrobial resistance as well as multi-drug resistance patterns were highest for E. coli and Salmonella spp. cultured from pigs and chickens, were generally widespread but at lower levels for other animal groups, and included resistance to ampicillin and streptomycin, two antimicrobial drugs of importance for human medicine. This study provides data that highlight the importance of practicing good hygiene in livestock fair settings to avoid transmission of zoonotic microbes, particularly pathogens with antimicrobial resistance, to fair visitors and among animal populations.

Individual subject meta-analysis of parameters for Cryptosporidium parvum shedding and diarrhoea in animal experimental models.

Cryptosporidium is a zoonotic protozoan parasite with public health importance worldwide. The objectives of this study were to (1) conduct a meta-analysis of published literature for oocyst shedding and diarrhoea outcomes, and (2) develop recommendations for standardization of experimental dose-response studies. Results showed that for the outcome of oocyst shedding in faeces, the covariates 'experimental species', 'immunosuppression', 'oocyst dose' and 'oocyst dose' × 'age' were all significant (P≤0.05). This study suggests that exposing mice, piglets, or ruminants, and using immunosuppressed experimental hosts, is more likely to result in oocyst shedding. For the outcome of diarrhoea in experimentally infected animal species, the key covariates 'experimental species', 'age' and 'immunosuppression' were significant (P≤0.2). Therefore, based on the results of this meta-analysis, these variables should be carefully reported and considered when designing experimental dose-response studies. Additionally, detection of possible publication bias highlights the need to publish additional studies that convey statistically non-significant as well as significant results in the future.

Methods to produce and safely work with large numbers of Toxoplasma gondii oocysts and bradyzoite cysts.

Two major obstacles to conducting studies with Toxoplasma gondii oocysts are the difficulty in reliably producing large numbers of this life stage and safety concerns because the oocyst is the most environmentally resistant stage of this zoonotic organism. Oocyst production requires oral infection of the definitive feline host with adequate numbers of T. gondii organisms to obtain unsporulated oocysts that are shed in the feces for 3-10 days after infection. Since the most successful and common mode of experimental infection of kittens with T. gondii is by ingestion of bradyzoite tissue cysts, the first step in successful oocyst production is to ensure a high bradyzoite tissue cyst burden in the brains of mice that can be used for the oral inoculum. We compared two methods for producing bradyzoite brain cysts in mice, by infecting them either orally or subcutaneously with oocysts. In both cases, oocysts derived from a low passage T. gondii Type II strain (M4) were used to infect eight-ten week-old Swiss Webster mice. First the number of bradyzoite cysts that were purified from infected mouse brains was compared. Then to evaluate the effect of the route of oocyst inoculation on tissue cyst distribution in mice, a second group of mice was infected with oocysts by one of each route and tissues were examined by histology. In separate experiments, brains from infected mice were used to infect kittens for oocyst production. Greater than 1.3 billion oocysts were isolated from the feces of two infected kittens in the first production and greater than 1.8 billion oocysts from three kittens in the second production. Our results demonstrate that oral delivery of oocysts to mice results in both higher cyst loads in the brain and greater cyst burdens in other tissues examined as compared to those of mice that received the same number of oocysts subcutaneously. The ultimate goal in producing large numbers of oocysts in kittens is to generate adequate amounts of starting material for oocyst studies. Given the potential risks of working with live oocysts in the laboratory, we also tested a method of oocyst inactivation by freeze-thaw treatment. This procedure proved to completely inactivate oocysts without evidence of significant alteration of the oocyst molecular integrity.

Endogenous transplacental transmission of Neospora hughesi in naturally infected horses.

Over a 2-yr study period, we investigated possible endogenous transplacental transmission of Neospora hughesi in 74 mare and foal pairs following the diagnosis of neuronal neosporosis in a weanling foal. Presuckle and postsuckle serum of each foal, serum and colostrum of each periparturient mare, and serum of each mare and foal pair, collected at 3-mo intervals thereafter, were tested for N. hughesi using an indirect fluorescent antibody test (IFAT). Furthermore, whole blood and colostrum samples and placentae were tested for the presence of N. hughesi by real-time PCR. The mares' seroprevalence at foaling based on IFAT (titer ≥ 160) was 52 and 6% in 2006 and 2007, respectively. Colostral antibodies against N. hughesi were detected in 96 and 11% of the mares in the 2-yr study. With the exception of 3 foals, all remaining foals were born seronegative to N. hughesi. Passive transfer of colostral antibodies to N. hughesi was documented in 15 foals. Three foals born from 2 different mares had presuckle antibodies at a titer ranging from 2,560 to 20,480. All 3 foals were born healthy. Two foals were born to the same dam that also gave birth to the weanling diagnosed with neuronal neosporosis in 2005. The third foal was born to a second mare with no previous foaling history at the farm. Seroconversion was documented in 10 foals and 9 mares over the 2-yr study. All blood and colostrum samples tested PCR negative for N. hughesi. Only 1 placenta collected in 2007 from the mare with the 2 congenitally infected foals tested PCR positive for N. hughesi. In conclusion, N. hughesi persisted in this population via endogenous transplacental infection.

Limited genetic diversity among Sarcocystis neurona strains infecting southern sea otters precludes distinction between marine and terrestrial isolates.

Sarcocystis neurona is an apicomplexan parasite identified as a cause of fatal neurological disease in the threatened southern sea otter (Enhydra lutris nereis). In an effort to characterize virulent S. neurona strains circulating in the marine ecosystem, this study developed a range of markers relevant for molecular genotyping. Highly conserved sequences within the 18S ribosomal gene array, the plastid-encoded RNA polymerase (RPOb) and the cytochrome c oxidase subunit 1 mitochondrial gene (CO1) were assessed for their ability to distinguish isolates at the genus and species level. For within-species comparisons, five surface antigens (SnSAG1-SnSAG5) and one high resolution microsatellite marker (Sn9) were developed as genotyping markers to evaluate intra-strain diversity. Molecular analysis at multiple loci revealed insufficient genetic diversity to distinguish terrestrial isolates from strains infecting marine mammals. Furthermore, SnSAG specific primers applied against DNA from the closely related species, Sarcocystis falcatula, lead to the discovery of highly similar orthologs to SnSAG2, 3, and 4, calling into question the specificity of diagnostic tests based on these antigens. The results of this study suggest a population genetic structure for S. neurona similar to that reported for the related parasite, Toxoplasma gondii, dominated by a limited number of successful genotypes.

Radiofrequency-induced thermal inactivation of Toxoplasma gondii oocysts in water.

Toxoplasma gondii, a ubiquitous parasitic protozoan, is emerging as an aquatic biological pollutant. Infections can result from drinking water contaminated with environmentally resistant oocysts. However, recommendations regarding water treatment for oocyst inactivation have not been established. In this study, the physical method of radiofrequency (RF) power was evaluated for its ability to inactivate T. gondii oocysts in water. Oocysts were exposed to various RF energy levels to induce 50, 55, 60, 70 and 80 degrees C temperatures maintained for 1 min. Post-treatment oocyst viability was determined by mouse bioassay with serology, immunohistochemistry and in vitro parasite isolation to confirm T. gondii infections in mice. None of the mice inoculated with oocysts treated with RF-induced temperatures of > or =60 degrees C in an initial experiment became infected; however, there was incomplete oocyst activation in subsequent experiments conducted under similar conditions. These results indicate that T. gondii oocysts may not always be inactivated when exposed to a minimum of 60 degrees C for 1 min. The impact of factors such as water heating time, cooling time and the volume of water treated must be considered when evaluating the efficacy of RF power for oocyst inactivation.

Cats and Toxoplasma: implications for public health.

Cats are popular as pets worldwide because they are easy to care for and provide companionship that enriches the lives of human beings. Little attention has been focused on their potential to contaminate the environment with zoonotic pathogens. One such pathogen, the protozoan parasite Toxoplasma gondii, rarely causes clinical manifestations in cats or immunocompetent humans; however, it can have serious adverse effects on human foetuses and immunocompromised patients. Many human infections are believed to be acquired from eating undercooked or raw meat, such as pork and lamb (Tenter et al. Int. J. Parasitol., 30, 2000, 1217; Dubey et al. J. Parasitol. 91, 2005, 1082). However, the prevalence of T. gondii infection in human populations that do not consume meat or eat it well-cooked suggests that the acquisition of infection from the environment, via oocysts in soil, water or on uncooked vegetables, is also important (Rawal. Trans. Royal Soc. Trop. Med. Hyg., 53, 1959, 61; Roghmann et al. Am. J. Trop. Med. Hyg., 60, 1999, 790; Chacin-Bonilla et al. Am. J. Trop. Med. Hyg., 65, 2001, 131). In the past 20 years, two changes occurred that significantly increased the size of the cat population in the USA. Pet cat ownership grew from 50 million to 90 million animals, and animal welfare activists created feeding stations for abandoned and free-roaming cats. As many cat owners allow their cats to deposit faeces outside and cats maintained in colonies always defecate outside, ample opportunity exists for T. gondii oocysts to enter the environment and be transmitted to humans. Prevention efforts should focus on educating cat owners about the importance of collecting cat faeces in litter boxes, spaying owned cats to reduce overpopulation, reducing the numbers of feral cats and promoting rigorous hand hygiene after gardening or soil contact.

Reducing microbial contamination in storm runoff from high use areas on California coastal dairies.

High use areas are a fundamental part of California coastal dairies and grazing livestock ranches as feeding areas, nurseries, and sick pens. High stocking densities and daily use in these areas lead to soil surfaces devoid of vegetation and covered in manure, with high potential for manure transport during winter rains to receiving waters regulated for shellfish harvesting and recreation. We characterized the association between California's Mediterranean climate and a series of existing and proposed management practices on fecal coliform bacteria (FCB) transport from high use areas on dairies and ranches. Results from 351 storm runoff samples collected below 35 high-use areas indicate that removal of cattle during winter, locating high use areas on level ground, application of straw and seeding, and vegetative buffer strip implementation were significantly associated with FCB concentration and load reductions. These results complement our findings for reductions of specific pathogens in runoff from these areas. These findings have practical significance because they document surface water quality benefits that the studied management practices provide in application on working farms and ranches. This direction is critical and timely for on-farm management efforts seeking to reduce microbial pollution in runoff and comply with indicator bacteria water quality criteria.

Ultrastructural and molecular confirmation of the development of Sarcocystis neurona tissue cysts in the central nervous system of southern sea otters (Enhydra lutris nereis).

In 2004, three wild sea otters were diagnosed with putative Sarcocystis neurona-associated meningoencephalitis by histopathology and immunohistochemistry. Schizonts, free merozoites and tissue cysts were observed in the brains of all three infected animals. Tissue cysts walls from sea otter 1 (SO1) stained positively using anti-S. neurona polyclonal antiserum. However, positive staining does not preclude infection by closely related or cross-reactive tissue cyst-forming coccidian parasites. Two immature tissue cysts in the brain of SO1 were examined using transmission electron microscopy. Ultrastructural features included cyst walls with thin villous projections up to 1 microm long with tapered ends and a distinctive, electron-dense outer lining layer composed of linearly-arranged, semi-circular structures with a "hobnailed" surface contour. Small numbers of microtubules extended down through the villi into the underlying granular layer. Metrocytes were short and plump with an anterior apical complex, 22 sub-pellicular microtubules, numerous free ribosomes and no rhoptries. Some metrocytes appeared to be dividing, with two adjacent nuclear profiles. Collectively these ultrastructural features were compatible with developing protozoal cysts and were similar to prior descriptions of S. neurona tissue cysts. Panspecific 18S rDNA primers were utilized to identify protozoa infecting the brains of these otters and DNA amplification and additional sequencing at the ITS1 locus confirmed that all three otters were infected with S. neurona. No other Sarcocystis spp. were detected in the brains or skeletal muscles of these animals by immunohistochemistry or PCR. We believe this is the first ultrastructural and molecular confirmation of the development of S. neurona tissue cysts in the CNS of any animal.

Farm factors associated with reducing Cryptosporidium loading in storm runoff from dairies.

A systems approach was used to evaluate environmental loading of Cryptosporidium oocysts on five coastal dairies in California. One aspect of the study was to determine Cryptosporidium oocyst concentrations and loads for 350 storm runoff samples from dairy high use areas collected over two storm seasons. Selected farm factors and beneficial management practices (BMPs) associated with reducing the Cryptosporidium load in storm runoff were assessed. Using immunomagnetic separation (IMS) with direct fluorescent antibody (DFA) analysis, Cryptosporidium oocysts were detected on four of the five farms and in 21% of storm runoff samples overall. Oocysts were detected in 59% of runoff samples collected near cattle less than 2 mo old, while 10% of runoff samples collected near cattle over 6 mo old were positive. Factors associated with environmental loading of Cryptosporidium oocysts included cattle age class, 24 h precipitation, and cumulative seasonal precipitation, but not percent slope, lot acreage, cattle stocking number, or cattle density. Vegetated buffer strips and straw mulch application significantly reduced the protozoal concentrations and loads in storm runoff, while cattle exclusion and removal of manure did not. The study findings suggest that BMPs such as vegetated buffer strips and straw mulch application, especially when placed near calf areas, will reduce environmental loading of fecal protozoa and improve stormwater quality. These findings are assisting working dairies in their efforts to improve farm and ecosystem health along the California coast.

Novel and canine genotypes of Giardia duodenalis in harbor seals ( Phoca vitulina richardsi).

Feces of harbor seals (Phoca vitulina richardsi) and hybrid glaucous-winged/western gulls (Larus glaucescens / occidentalis) from Washington State's inland marine waters were examined for Giardia and Cryptosporidium spp. to determine if genotypes carried by these wildlife species were the same genotypes that commonly infect humans and domestic animals. Using immunomagnetic separation followed by direct fluorescent antibody detection, Giardia spp. cysts were detected in 42% of seal fecal samples (41/97). Giardia-positive samples came from 90% of the sites (9/10) and the prevalence of positive seal fecal samples differed significantly among study sites. Fecal samples collected from seal haulout sites with over 400 animals were 4.7 times more likely to have Giardia spp. cysts than samples collected at smaller haulout sites. In gulls, a single Giardia sp. cyst was detected in 4% of fecal samples (3/78). Cryptosporidium spp. oocysts were not detected in any of the seals or gulls tested. Sequence analysis of a 398 bp segment of G. duodenalis DNA at the glutamate dehydrogenase locus suggested that 11 isolates originating from seals throughout the region were a novel genotype and 3 isolates obtained from a single site in south Puget Sound were the G. duodenalis canine genotype D. Real-time TaqMan PCR amplification and subsequent sequencing of a 52 bp small subunit ribosomal DNA region from novel harbor seal genotype isolates showed sequence homology to canine genotypes C and D. Sequence analysis of the 52 bp small subunit ribosomal DNA products from the 3 canine genotype isolates from seals produced mixed sequences at could not be evaluated.

Type X Toxoplasma gondii in a wild mussel and terrestrial carnivores from coastal California: new linkages between terrestrial mammals, runoff and toxoplasmosis of sea otters.

Sea otters in California are commonly infected with Toxoplasma gondii. A unique Type X strain is responsible for 72% of otter infections, but its prevalence in terrestrial animals and marine invertebrates inhabiting the same area was unknown. Between 2000 and 2005, 45 terrestrial carnivores (lions, bobcats, domestic cats and foxes) and 1396 invertebrates (mussels, clams and worms) were screened for T. gondii using PCR and DNA sequencing to determine the phylogeographic distribution of T. gondii archetypal I, II, III and Type X genotypes. Marine bivalves have been shown to concentrate T. gondii oocysts in the laboratory, but a comprehensive survey of wild invertebrates has not been reported. A California mussel from an estuary draining into Monterey Bay was confirmed positive for Type X T. gondii by multilocus PCR and DNA sequencing at the B1 and SAG1 loci. This mussel was collected from nearshore marine waters just after the first significant rainfall event in the fall of 2002. Of 45 carnivores tested at the B1, SAG1, and GRA6 typing loci, 15 had PCR-confirmed T. gondii infection; 11 possessed alleles consistent with infection by archetypal Type I, II or III strains and 4 possessed alleles consistent with Type X T. gondii infection. No non-canonical alleles were identified. The four T. gondii strains with Type X alleles were identified from two mountain lions, a bobcat and a fox residing in coastal watersheds adjacent to sea otter habitat near Monterey Bay and Estero Bay. Confirmation of Type X T. gondii in coastal-dwelling felids, canids, a marine bivalve and nearshore-dwelling sea otters supports the hypotheses that feline faecal contamination is flowing from land to sea through surface runoff, and that otters can be infected with T. gondii via consumption of filter-feeding marine invertebrates.

Novel symptomatology and changing epidemiology of domoic acid toxicosis in California sea lions (Zalophus californianus): an increasing risk to marine mammal health.

Harmful algal blooms are increasing worldwide, including those of Pseudo-nitzschia spp. producing domoic acid off the California coast. This neurotoxin was first shown to cause mortality of marine mammals in 1998. A decade of monitoring California sea lion (Zalophus californianus) health since then has indicated that changes in the symptomatology and epidemiology of domoic acid toxicosis in this species are associated with the increase in toxigenic blooms. Two separate clinical syndromes now exist: acute domoic acid toxicosis as has been previously documented, and a second novel neurological syndrome characterized by epilepsy described here associated with chronic consequences of previous sub-lethal exposure to the toxin. This study indicates that domoic acid causes chronic damage to California sea lions and that these health effects are increasing.

Cryptosporidium and Giardia in marine-foraging river otters (Lontra canadensis) from the Puget Sound Georgia Basin ecosystem.

Species of Cryptosporidium and Giardia can infect humans and wildlife and have the potential to be transmitted between these 2 groups; yet, very little is known about these protozoans in marine wildlife. Feces of river otters (Lontra canadensis), a common marine wildlife species in the Puget Sound Georgia Basin, were examined for species of Cryptosporidium and Giardia to determine their role in the epidemiology of these pathogens. Using ZnSO4 flotation and immunomagnetic separation, followed by direct immunofluorescent antibody detection (IMS/DFA), we identified Cryptosporidium sp. oocysts in 9 fecal samples from 6 locations and Giardia sp. cysts in 11 fecal samples from 7 locations. The putative risk factors of proximate human population and degree of anthropogenic shoreline modification were not associated with the detection of Cryptosporidium or Giardia spp. in river otter feces. Amplification of DNA from the IMS/DFA slide scrapings was successful for 1 sample containing > 500 Cryptosporidium sp. oocysts. Sequences from the Cryptosporidium 18S rRNA and the COWP loci were most similar to the ferret Cryptosporidium sp. genotype. River otters could serve as reservoirs for Cryptosporidium and Giardia species in marine ecosystems. More work is needed to better understand the zoonotic potential of the genotypes they carry as well as their implications for river otter health.