Molecular detection and viability discrimination of zoonotic protozoan pathogens in oysters and seawater.

The presence of foodborne protozoan pathogens including Cryptosporidium parvum, Giardia duodenalis, Toxoplasma gondii, and Cyclospora cayetanensis in commercial shellfish has been reported across diverse geographical regions. In the present study, a novel multiplex nested polymerase chain reaction (PCR) assay was validated to simultaneously detect and discriminate these four targeted parasites in oyster tissues including whole tissue homogenate, digestive gland, gills, and hemolymph, as well as seawater where shellfish grow. To differentiate viable and non-viable protozoan (oo)cysts, we further evaluated reverse transcription quantitative PCR (RT-qPCR) assays through systematic laboratory spiking experiments by spiking not only dilutions of viable parasites but also mixtures of viable and non-viable parasites in the oyster tissues and seawater. Results demonstrate that multiplex PCR can detect as few as 5-10 (oo)cysts in at least one oyster matrix, as well as in 10 L of seawater. All parasites were detected at the lowest spiking dilution (5 (oo)cysts per extract) in hemolymph, however the probability of detection varied across the difference matrices tested for each parasite. RT-qPCR further discriminated viable from non-viable (heat-inactivated) C. parvum and T. gondii in seawater and hemolymph but did not perform well in other oyster matrices. This systematic spiking study demonstrates that a molecular approach combining multiplex PCR for sensitive and affordable screening of protozoan DNA and subsequent RT-qPCR assay for viability discrimination presents an important advance for accurately determining the risk of protozoal illness in humans due to consumption of contaminated shellfish.

Evidence of intrathecally-derived antibodies against Toxoplasma gondii in horses suspected of neurological disease consistent with equine protozoal myeloencephalitis.

Among the recognized neurologic diseases in horses, equine protozoal myeloencephalitis (EPM) has been reported around the world and still presents challenges in diagnosis and treatment. Horses can present with clinical neurologic signs consistent with EPM while testing negative for the two main causative agents, Sarcocystis neurona or Neospora hughesi, and may still be clinically responsive to anti-parasitic drug therapy. This context led to our hypothesis that another protozoal parasite, Toxoplasma gondii, which is known to cause toxoplasmosis in other mammalian species, is a potential pathogen to cause neurologic disease in horses. To evaluate this hypothesis, serum and cerebrospinal fluid (CSF) were collected from 210 horses presenting with clinical signs compatible with EPM, and the indirect immunofluorescent antibody test (IFAT) was used to detect antibody titers for T. gondii, S. neurona, and N. hughesi. Additionally, the serum to CSF titer ratio was calculated for T. gondii, S. neurona, and N. hughesi infections, suggesting intrathecally-derived antibodies for each of the three agents if the serum:CSF ratio was ≤ 64. There were 133 (63.3%) horses positive for serum T. gondii antibodies using a cutoff titer of 160, and 31 (14.8%) positive for CSF T. gondii antibodies using a cutoff titer of 5. Overall, 21 (10.0%) of EPM-suspect horses had a serum:CSF ratio ≤ 64 for antibodies for T. gondii, while 43 (20.5%) and 8 (3.8%) horses had a serum to CSF ratio ≤ 64 for antibodies for S. neurona and N. hughesi, respectively. A total of 6 (2.9%) animals presented evidence of concurrent intrathecally-derived antibodies for T. gondii and at least one other apicomplexan parasite in this study. Signalment and clinical signs were not different across the groups aforementioned. These data provide evidence of intrathecal production of anti-T. gondii antibodies, indicative of T. gondii infection in the brain and/or spinal cord of horses with EPM-like disease.

Quantification of viable protozoan parasites on leafy greens using molecular methods.

Protozoan contamination in produce is of growing importance due to their capacity to cause illnesses in consumers of fresh leafy greens. Viability assays are essential to accurately estimate health risk caused by viable parasites that contaminate food. We evaluated the efficacy of reverse transcription quantitative PCR (RT-qPCR), propidium monoazide coupled with (q)PCR, and viability staining using propidium iodide through systematic laboratory spiking experiments for selective detection of viable Cryptosporidium parvum, Giardia enterica, and Toxoplasma gondii. In the presence of only viable protozoa, the RT-qPCR assays could accurately detect two to nine (oo)cysts/g spinach (in 10 g processed). When different proportions of viable and inactivated parasite were spiked, mRNA concentrations correlated with increasing proportions of viable (oo)cysts, although low levels of false-positive mRNA signals were detectable in the presence of high amounts of inactivated protozoa. Our study demonstrated that among the methods tested, RT-qPCR performed more effectively to discriminate viable from inactivated C. parvum, G. enterica and T. gondii on spinach. This application of viability methods on leafy greens can be adopted by the produce industry and regulatory agencies charged with protection of human public health to screen leafy greens for the presence of viable protozoan pathogen contamination.

Associations between faecal pathogen detection, E. coli concentrations and youth exhibitor biosecurity practices at California county fairs.

Interactions with livestock in public settings such as county and state fairs can expose people and other livestock to faecal material capable of spreading zoonotic enteric pathogens. The goal of this study was to understand these risks by screening livestock faeces (n = 245) and livestock bedding (n = 155) for common zoonotic pathogens (Giardia, Cryptosporidium, Salmonella and Campylobacter spp.) and by measuring faecal indicator, Escherichia coli, concentrations in drinking water (n = 153), feed containers (n = 124) and bedding material (n = 157) in four livestock species (cattle, sheep, goats and swine) from county fairs in California, USA. Results indicated that sheep were most likely to have pathogens detected in faeces and that Giardia was the most frequently detected pathogen in both faeces (11%) and bedding (21%) across all livestock species. Additionally, increasing the number of animals in a holding pen at fairs, increasing the stocking density of animals in transport trailers to fairs, and having access to water in transport trailers significantly increased the odds of detecting pathogens in livestock faeces of any animal species. Observing solid material in water, stale feed and soiled bedding was associated with detecting higher E. coli concentrations. These findings provide evidence of faecal pathogens present at county fairs and suggest that site observations can aid in assessing levels of faecal exposure. The findings also indicate that the use of biosecurity measures such as (a) routine changing of livestock drinking water, feed and bedding, (b) not overstocking animals in holding pens and trailers and (c) keeping species in separate holding areas may reduce the risk of humans and livestock being exposed to faecal pathogens.

Molecular detection of Sarcocystis neurona in cerebrospinal fluid from 210 horses with suspected neurologic disease.

An ante-mortem diagnosis of equine protozoal myeloencephalitis (EPM) is presently based on clinical presentation, immunodiagnostics performed on serum and cerebrospinal fluid (CSF), and ruling out other neurological disorders. Molecular techniques introduce a novel and promising approach for the detection of protozoal agents in CSF. Hypothesizing that real-time PCR (rtPCR) can be a useful complement to EPM diagnostics, 210 CSF samples from horses suspected of neurological disease with EPM included as a differential diagnosis were tested using rtPCR to detect Sarcocystis neurona DNA and immunodiagnostics targeting antibodies against the same pathogen, performed on serum and CSF samples. Molecular and immunological results were compared with respect to origin of the horse, time of the year, signalment, clinical signs and treatment history. Twenty-five horses tested positive in CSF for S. neurona by rtPCR only, while 30 horses had intrathecally-derived antibodies to S. neurona only (serum to CSF ratio ≤ 64 by indirect fluorescent antibody test - IFAT), and 13 horses tested rtPCR-positive in CSF with evidence of intrathecally-derived antibodies to S. neurona. Previous treatment for EPM was the only variable presenting statistical difference between the two testing modalities, highlighting that animals with history of anti-protozoal treatment were more likely to test positive solely in IFAT, while horses without treatment were more likely to test positive by rtPCR only. The results support the use of molecular diagnosis for EPM caused by S. neurona as a complement to immunodiagnostics. The use of rtPCR in CSF for the detection of S. neurona may improve the diagnostic work-up of neurologic disease suspected horses, especially in animals without previous anti-protozoal treatment.

Borrelia burgdorferi and Borrelia miyamotoi seroprevalence in California blood donors.

The western blacklegged tick, Ixodes pacificus, an important vector in the western United States of two zoonotic spirochetes: Borrelia burgdorferi (also called Borreliella burgdorferi), causing Lyme disease, and Borrelia miyamotoi, causing a relapsing fever-type illness. Human cases of Lyme disease are well-documented in California, with increased risk in the north coastal areas and western slopes of the Sierra Nevada range. Despite the established presence of B. miyamotoi in the human-biting I. pacificus tick in California, clinical cases with this spirochete have not been well studied. To assess exposure to B. burgdorferi and B. miyamotoi in California, and to address the hypothesis that B. miyamotoi exposure in humans is similar in geographic range to B. burgdorferi, 1,700 blood donor sera from California were tested for antibodies to both pathogens. Sampling was from high endemic and low endemic counties for Lyme disease in California. All sera were screened using the C6 ELISA. All C6 positive and equivocal samples and nine randomly chosen C6 negative samples were further analyzed for B. burgdorferi antibody using IgG western blot and a modified two ELISA test system and for B. miyamotoi antibody using the GlpQ ELISA and B. miyamotoi whole cell sonicate western blot. Of the 1,700 samples tested in series, eight tested positive for antibodies to B. burgdorferi (0.47%, Exact 95% CI: 0.20, 0.93) and two tested positive for antibodies to B. miyamotoi (0.12%, Exact 95% CI: 0.01, 0.42). There was no statistically significant difference in seroprevalence for either pathogen between high and low Lyme disease endemic counties. Our results confirm a low frequency of Lyme disease and an even lower frequency of B. miyamotoi exposure among adult blood donors in California; however, our findings reinforce public health messaging that there is risk of infection by these emerging diseases in the state.

Simultaneous detection of four protozoan parasites on leafy greens using a novel multiplex PCR assay.

Pathogen contamination of fresh produce presents a health risk for consumers; however, the produce industry still lacks adequate tools for simultaneous detection of protozoan parasites. Here, a simple multiplex PCR (mPCR) assay was developed for detection of protozoan (oo)cysts and compared with previously published real-time PCR assays and microscopy methods. The assay was evaluated for simultaneous detection of Cryptosporidium, Giardia, Cyclospora cayetanensis, and Toxoplasma gondii followed by parasite differentiation via either a nested specific PCR or a restriction fragment length polymorphism (RFLP) assay. Spiking experiments using spinach as a model leafy green were performed for assay validation. Leaf-washing yielded higher recoveries and more consistent detection of parasites as compared with stomacher processing. Lowest limits of detection using the nested mPCR assay were 1-10 (oo)cysts/g spinach (in 10 g samples processed), and this method proved more sensitive than qPCR for parasite detection. Microscopy methods were more reliable for visual detection of parasites in lower spiking concentrations, but are more costly and laborious, require additional expertise, and lack molecular confirmation essential for accurate risk assessment. Overall, the nested mPCR assay provides a rapid (<24 h), inexpensive ($10 USD/sample), and simple approach for simultaneous detection of protozoan pathogens on fresh produce.

Type X strains of Toxoplasma gondii are virulent for southern sea otters (Enhydra lutris nereis) and present in felids from nearby watersheds.

Why some Toxoplasma gondii-infected southern sea otters (Enhydra lutris nereis) develop fatal toxoplasmosis while others have incidental or mild chronic infections has long puzzled the scientific community. We assessed robust datasets on T. gondii molecular characterization in relation to detailed necropsy and histopathology results to evaluate whether parasite genotype influences pathological outcomes in sea otters that stranded along the central California coast. Genotypes isolated from sea otters were also compared with T. gondii strains circulating in felids from nearby coastal regions to assess land-to-sea parasite transmission. The predominant T. gondii genotypes isolated from 135 necropsied sea otters were atypical Type X and Type X variants (79%), with the remainder (21%) belonging to Type II or Type II/X recombinants. All sea otters that died due to T. gondii as a primary cause of death were infected with Type X or X-variant T. gondii strains. The same atypical T. gondii strains were detected in sea otters with fatal toxoplasmosis and terrestrial felids from watersheds bordering the sea otter range. Our results confirm a land-sea connection for virulent T. gondii genotypes and highlight how faecal contamination can deliver lethal pathogens to coastal waters, leading to detrimental impacts on marine wildlife.

Seroprevalences of anti-Sarcocystis neurona and anti-Neospora hughesi antibodies among healthy equids in the United States.

OBJECTIVE To describe the general seroprevalence of anti-Sarcocystis neurona and anti-Neospora hughesi antibodies among healthy equids by use of indirect fluorescent antibody tests and determine potential risk factors for seropositivity. DESIGN Cross-sectional study. SAMPLE Whole blood samples collected from 5,250 equids (1 sample/animal) across 18 states in the United States during October 2013. PROCEDURES Information regarding potential risk factors (geographic region, breed, primary use, sex, and age) was collected along with the blood samples. For each equid, an indirect fluorescent antibody test was used to determine serum titers of antibody against each of the 2 protozoal parasites. Mixed-effects logistic regression models were created to determine ORs for seropositivity. RESULTS The overall seroprevalence of anti-S neurona and anti-N hughesi antibodies in the tested equids was 78% and 34%, respectively. Of the equids, 31% were seropositive and 18% were seronegative for antibodies against both parasites. Factors associated with equids being seropositive for anti-S neurona antibodies were residence in the South, warmblood breed, and age > 5 years. Seroprevalence of anti-N hughesi antibodies did not differ among equids in different states across the country, but warmblood breed and age > 5 years were associated with seropositivity. CONCLUSIONS AND CLINICAL RELEVANCE With regard to risk factors for S neurona and N hughesi exposure and antibody response among tested equids, older age was not unexpected; however, the influences of warmblood breed and geographic location on seropositivity for anti-S neurona antibody but not for anti-N hughesi antibody deserve further investigation.

Detection and characterization of diverse coccidian protozoa shed by California sea lions.

Tissue-cyst forming coccidia in the family Sarcocystidae are etiologic agents of protozoal encephalitis in marine mammals including the federally listed Southern sea otter (Enhydra lutris). California sea lions (Zalophus californianus), whose coastal habitat overlaps with sea otters, are definitive hosts for coccidian protozoa provisionally named Coccidia A, B and C. While Coccidia A and B have unknown clinical effects on aquatic wildlife hosts, Coccidia C is associated with severe protozoal disease in harbor seals (Phoca vitulina). In this study, we conducted surveillance for protozoal infection and fecal shedding in hospitalized and free-ranging California sea lions on the Pacific Coast and examined oocyst morphology and phenotypic characteristics of isolates via mouse bioassay and cell culture. Coccidia A and B were shed in similar frequency, particularly by yearlings. Oocysts shed by one free-ranging sea lion sampled at Año Nuevo State Park in California were previously unidentified in sea lions and were most similar to coccidia infecting Guadalupe fur seals (Arctocephalus townsendi) diagnosed with protozoal disease in Oregon (USA). Sporulated Coccidia A and B oocysts did not replicate in three strains of mice or in African green monkey kidney cells. However, cultivation experiments revealed that the inoculum of fecally-derived Coccidia A and B oocysts additionally contained organisms with genetic and antigenic similarity to Sarcocystis neurona; despite the absence of detectable free sporocysts in fecal samples by microscopic examination. In addition to the further characterization of Coccidia A and B in free-ranging and hospitalized sea lions, these results provide evidence of a new role for sea lions as putative mechanical vectors of S. neurona, or S. neurona-like species. Future work is needed to clarify the distribution, taxonomical status, and pathogenesis of these parasites in sea lions and other marine mammals that share their the near-shore marine environment.

Dual congenital transmission of Toxoplasma gondii and Sarcocystis neurona in a late-term aborted pup from a chronically infected southern sea otter (Enhydra lutris nereis).

Toxoplasma gondii and Sarcocystis neurona are protozoan parasites with terrestrial definitive hosts, and both pathogens can cause fatal disease in a wide range of marine animals. Close monitoring of threatened southern sea otters (Enhydra lutris nereis) in California allowed for the diagnosis of dual transplacental transmission of T. gondii and S. neurona in a wild female otter that was chronically infected with both parasites. Congenital infection resulted in late-term abortion due to disseminated toxoplasmosis. Toxoplasma gondii and S. neurona DNA was amplified from placental tissue culture, as well as from fetal lung tissue. Molecular characterization of T. gondii revealed a Type X genotype in isolates derived from placenta and fetal brain, as well as in all tested fetal organs (brain, lung, spleen, liver and thymus). This report provides the first evidence for transplacental transmission of T. gondii in a chronically infected wild sea otter, and the first molecular and immunohistochemical confirmation of concurrent transplacental transmission of T. gondii and S. neurona in any species. Repeated fetal and/or neonatal losses in the sea otter dam also suggested that T. gondii has the potential to reduce fecundity in chronically infected marine mammals through parasite recrudescence and repeated fetal infection.

Daily feeding of diclazuril top dress pellets in foals reduces seroconversion to Sarcocystis neurona.

Thirty-three foals from a farm with a high exposure rate to Sarcocystis neurona were assigned to either an untreated or a diclazuril-treated group. Treated foals received daily 0.5 mg/kg of diclazuril pellets from 1 to 12 months of age. Monthly blood was tested for IgG against S. neurona using the indirect fluorescent antibody test. Following ingestion of colostral antibodies to S. neurona, there was a steady and continuous decline in seroprevalence to S. neurona until foals from both groups reached weaning age. Thereafter, the untreated foal group showed a significant increase in monthly seroprevalence compared to the diclazuril-treated foal group. The difference in temporal seroprevalence could be explained by the successful reduction of S. neurona infection in foals receiving a daily low-dose diclazuril.

Serological investigation of transplacental infection with Neospora hughesi and Sarcocystis neurona in broodmares.

The aim of the present study was to investigate the likelihood of transplacental transmission of Neospora hughesi and Sarcocystis neurona in foals, born from seropositive mares. Three broodmares with persistent N. hughesi infection gave birth to eight healthy foals over a period of 7 years. These foals were seropositive to N. hughesi prior to colostrum ingestion, with titers ranging between 640 and 20,480, measured by indirect fluorescence antibody test (IFAT). Of 174 foals born at another farm to mares with a high seroprevalence to S. neurona, only one (with a pre-colostrum antibody titer of 80) tested seropositive. Transplacental transmission of N. hughesi seems to occur from latently infected mares to their foals, while this route of transmission does not seem to occur commonly for S. neurona.

Surveillance for zoonotic and selected pathogens in harbor seals Phoca vitulina from central California.

The infection status of harbor seals Phoca vitulina in central California, USA, was evaluated through broad surveillance for pathogens in stranded and wild-caught animals from 2001 to 2008, with most samples collected in 2007 and 2008. Stranded animals from Mendocino County to San Luis Obispo County were sampled at a rehabilitation facility: The Marine Mammal Center (TMMC, n = 175); wild-caught animals were sampled at 2 locations: San Francisco Bay (SF, n = 78) and Tomales Bay (TB, n = 97), that differed in degree of urbanization. Low prevalences of Salmonella, Campylobacter, Giardia, and Cryptosporidium were detected in the feces of stranded and wild-caught seals. Clostridium perfringens and Escherichia coli were more prevalent in the feces of stranded (58% [78 out of 135] and 76% [102 out of 135]) than wild-caught (42% [45 out of 106] and 66% [68 out of 106]) seals, whereas Vibrio spp. were 16 times more likely to be cultured from the feces of seals from SF than TB or TMMC (p < 0.005). Brucella DNA was detected in 3.4% of dead stranded harbor seals (2 out of 58). Type A influenza was isolated from feces of 1 out of 96 wild-caught seals. Exposure to Toxoplasma gondii, Sarcocystis neurona, and type A influenza was only detected in the wild-caught harbor seals (post-weaning age classes), whereas antibody titers to Leptospira spp. were detected in stranded and wild-caught seals. No stranded (n = 109) or wild-caught (n = 217) harbor seals had antibodies to phocine distemper virus, although a single low titer to canine distemper virus was detected. These results highlight the role of harbor seals as sentinel species for zoonotic and terrestrial pathogens in the marine environment.

Comparison of prevalence factors in horses with and without seropositivity to Neospora hughesi and/or Sarcocystis neurona.

Equine protozoal myeloencephalitis is a commonly diagnosed neurological disease of horses in North America and is caused by infection with Sarcocystis neurona or Neospora hughesi. The aim of this study was to compare prevalence factors among horses seropositive or seronegative to N. hughesi and/or S. neurona. A total of 3123 submissions were included in the study, with horses originating from 49 States. Thirty-eight animals from 21 States tested seropositive for N. hughesi only, 840 horses from 40 States were seropositive for S. neurona only, 25 horses from 14 States were seropositive for both protozoa, and 2220 horses from 49 States tested seronegative for both parasites. Significant associations were found between geographical location (State), month of submission, breed and serological status.

Serum antibodies from a subset of horses positive for Babesia caballi by competitive enzyme-linked immunosorbent assay demonstrate a protein recognition pattern that is not consistent with infection.

Tick-borne pathogens that cause persistent infection are of major concern to the livestock industry because of transmission risk from persistently infected animals and the potential economic losses they pose. The recent reemergence of Theileria equi in the United States prompted a widespread national survey resulting in identification of limited distribution of equine piroplasmosis (EP) in the U.S. horse population. This program identified Babesia caballi-seropositive horses using rhoptry-associated protein 1 (RAP-1)-competitive enzyme-linked immunosorbent assay (cELISA), despite B. caballi being considered nonendemic on the U.S. mainland. The purpose of the present study was to evaluate the suitability of RAP-1-cELISA as a single serological test to determine the infection status of B. caballi in U.S. horses. Immunoblotting indicated that sera from U.S. horses reacted with B. caballi lysate and purified B. caballi RAP-1 protein. Antibody reactivity to B. caballi lysate was exclusively directed against a single ∼50-kDa band corresponding to a native B. caballi RAP-1 protein. In contrast, sera from experimentally and naturally infected horses from regions where B. caballi is endemic bound multiple proteins ranging from 30 to 50 kDa. Dilutions of sera from U.S. horses positive by cELISA revealed low levels of antibodies, while sera from horses experimentally infected with B. caballi and from areas where B. caballi is endemic had comparatively high antibody levels. Finally, blood transfer from seropositive U.S. horses into naive horses demonstrated no evidence of B. caballi transmission, confirming that antibody reactivity in cELISA-positive U.S. horses was not consistent with infection. Therefore, we conclude that a combination of cELISA and immunoblotting is required for the accurate serodiagnosis of B. caballi.

A protozoal-associated epizootic impacting marine wildlife: mass-mortality of southern sea otters (Enhydra lutris nereis) due to Sarcocystis neurona infection.

During April 2004, 40 sick and dead southern sea otters (Enhydra lutris nereis) were recovered over 18km of coastline near Morro Bay, California. This event represented the single largest monthly spike in mortality ever recorded during 30 years of southern sea otter stranding data collection. Because of the point-source nature of the event and clinical signs consistent with severe, acute neurological disease, exposure to a chemical or marine toxin was initially considered. However, detailed postmortem examinations revealed lesions consistent with an infectious etiology, and further investigation confirmed the protozoan parasite Sarcocystis neurona as the underlying cause. Tissues from 94% of examined otters were PCR-positive for S. neurona, based on DNA amplification and sequencing at the ITS-1 locus, and 100% of tested animals (n=14) had elevated IgM and IgG titers to S. neurona. Evidence to support the point-source character of this event include the striking spatial and temporal clustering of cases and detection of high concentrations of anti-S. neurona IgM in serum of stranded animals. Concurrent exposure to the marine biotoxin domoic acid may have enhanced susceptibility of affected otters to S. neurona and exacerbated the neurological signs exhibited by stranded animals. Other factors that may have contributed to the severity of this epizootic include a large rainstorm that preceded the event and an abundance of razor clams near local beaches, attracting numerous otters close to shore within the affected area. This is the first report of a localized epizootic in marine wildlife caused by apicomplexan protozoa.

Congenital transmission of Toxoplasma gondii in deer mice (Peromyscus maniculatus) after oral oocyst infection.

To investigate how different routes of Toxoplasma gondii transmission influence the antibody response and infection status of deer mice (Peromyscus maniculatus), 80 mice were orally infected with 1, 5, 10, or 100 T. gondii oocysts. Ten weeks postinfection, 15 T. gondii -seropositive female mice were bred and allowed to produce 2 litters. Evidence of persistent T .gondii infection in orally infected mice was detected by serology and DNA amplification in mice from all 4 oocyst treatment groups, including those that received only a single T. gondii oocyst. Congenital transmission of T. gondii was detected by polymerase chain reaction (PCR) in 7/8 first and 4/7 second litters. Toxoplasma gondii was also detected by PCR in 9/30 congenitally infected offspring 16 wk after birth, despite the fact that detectable serological titers had waned. These findings raise questions about the applicability of serological testing to assess the prevalence of T. gondii infection in deer mice and other rodents in the wild. Additionally, the detection of frequent congenital transmission suggests that deer mice could help maintain T. gondii in the environment even in the absence of definitive feline hosts.

Prevalence and risk factors associated with Sarcocystis neurona infections in opossums (Didelphis virginiana) from central California.

Sarcocystis neurona, a protozoal parasite shed by opossums (Didelphis virginiana), has been shown to cause significant morbidity and mortality in horses, sea otters, and other marine mammals. Over the course of 3 years (fall 2005-summer 2008), opossums from central California were tested for infection with S. neurona. Of 288 opossums sampled, 17 (5.9%) were infected with S. neurona based on the molecular characterization of sporocysts from intestinal scrapings or feces. Risk factors evaluated for association with S. neurona infection in opossums included: age, sex, location, season, presence of pouch young in females, concomitant infection, and sampling method (live-trapped or traffic-killed). Multivariate logistic regression analysis identified that opossums in the Central Valley were 9 times more likely to be infected than those near the coast (p=0.009). Similarly, opossum infection was 5 times more likely to be detected during the reproductive season (March-July; p=0.013). This first investigation of S. neurona infection prevalence and associated risk factors in opossums in the western United States can be used to develop management strategies aimed at reducing the incidence of S. neurona infections in susceptible hosts, including horses and threatened California sea otters (Enhydra lutris neries).

Risk factors for Toxoplasma gondii infection in wild rodents from central coastal California and a review of T. gondii prevalence in rodents.

Sera from 523 wild rodents were tested for Toxoplasma gondii antibodies using either an indirect fluorescent antibody test (IFAT) (rats and mice, with titer >or=80 considered positive) or a latex agglutination test (LAT) (voles, squirrels, and pocket mice, with titer >or=32 considered positive). Seventeen percent (88/523) of the rodents, including 26% (85/328) of the Peromyscus sp. and 8% (3/37) of Spermophilus beecheyi, were seropositive. Fourteen percent (23/161) of rodents captured in trap sites next to Morro Bay (California) and 15% (16/109) of rodents from sites adjacent to riparian habitats had antibodies to T. gondii, compared to 19% (49/253) of rodents captured in habitats not associated with water; this difference was not statistically significant (P = 0.32). Significantly fewer rodents were captured <200 m from residential housing compared to locations further away (11% vs. 30%, respectively). Factors associated with an increased risk for T. gondii seropositivity in rodents were capture location >or=200 m from residential housing and adult age.