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.

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.

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.

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.

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.

A fresh look at the SarcoFluor antibody test for the detection of specific antibodies to Sarcocystis neurona for the diagnosis of equine protozoal myeloencephalitis.

Equine protozoal myeloencephalitis (EPM) is a challenging disease to diagnose in horses with neurological signs. To optimize contemporary diagnostic testing, including the use of serum:CSF antibody ratios, the SarcoFluor antibody test for Sarcocystis neurona requires revalidation. The SarcoFluor, a previously validated immunofluorescent antibody test (IFAT) for the detection of antibodies specific to S. neurona in serum and cerebrospinal fluid (CSF) of naturally infected horses was analyzed using recent data and considering a serum:CSF antibody ratio threshold. Utilization of serum and CSF phosphorylated neurofilament heavy protein (pNfH) concentrations in support of an EPM diagnosis was also evaluated. 172 horses were divided into three groups: EPM-positive horses (EPM+, n=42), neurological non-EPM horses (n=74) confirmed with non-EPM neurological diseases (cervical vertebral compressive myelopathy, equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy), and control horses (control, n=56) without neurological signs and neurological abnormalities on histology. Logistic regression was used to compare EPM diagnostic regimens. Specifically, EPM+ horses were compared with neurological non-EPM horses showing neurological signs. To consider diagnostic utility, post-test probabilities were calculated by titer. When differentiating between EPM and other neurological diseases, the combination of serum and CSF SarcoFluor testing added more information to the model accuracy than either test alone. Using serum and CSF for pNfH in support of an EPM diagnosis did not identify cutoffs with statistically significant odds ratios but increased the overall model accuracy when used with the IFAT. Utilization of IFAT titers against S. neurona in serum and CSF result in a high post-test probability of detecting EPM+ horses in a clinical setting.

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.

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.

Transplacental toxoplasmosis in a wild southern sea otter (Enhydra lutris nereis).

In September 2004, a neonatal sea otter pup was found alive on the beach in northern Monterey Bay, CA. Efforts to locate the mother were unsuccessful. Due to a poor prognosis for successful rehabilitation, the pup was euthanized. Postmortem examination revealed emaciation, systemic lymphadenopathy and a malformation of the left cerebral temporal lobe. On histopathology, free tachyzoites and tissue cysts compatible with Toxoplasma gondii were observed in the brain, heart, thymus, liver, lymph nodes and peri-umbilical adipose. The presence of T. gondii within host tissues was associated with lymphoplasmacytic inflammation and tissue necrosis. Immunofluorescent antibody tests using postmortem serum were positive for anti-T. gondii IgM and IgG (at 1:320 and 1:1280 serum dilution, respectively), but were negative for IgG directed against Sarcocystis neurona and Neospora caninum (<1:40 each). Brain immunohistochemistry revealed positive staining for tachyzoites and tissue cysts using antiserum raised to T. gondii, but not S. neurona or N. caninum. T. gondii parasite DNA was obtained from extracts of brain and muscle by PCR amplification using the diagnostic B1 locus. Restriction enzyme digestion followed by gel electrophoresis and DNA sequencing confirmed the presence of Type X T. gondii, the strain identified in the majority of southern sea otter infections.

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.

Effects of blood contamination of cerebrospinal fluid on results of indirect fluorescent antibody tests for detection of antibodies against Sarcocystis neurona and Neospora hughesi.

The purpose of this study was to determine the effect of blood contamination of cerebrospinal fluid (CSF) on the results of indirect fluorescent antibody tests (IFATs) for Sarcocystis neurona and Neospora hughesi. The in vitro study used antibody-negative CSF collected from non-neurologic horses immediately after euthanasia and blood samples from 40 healthy horses that had a range of IFAT antibody titers against S. neurona and N. hughesi. Serial dilutions of whole blood were made in seronegative CSF to generate blood-contaminated CSF with red blood cell (RBC) concentrations ranging from 10 to 100,000 RBCs/microl. The blood-contaminated CSF samples were then tested for antibodies against both pathogens using IFAT. Blood contamination of CSF had no detectable effect on IFAT results for S. neurona or N. hughesi at any serologic titer when the RBC concentration in CSF was <10,000 RBCs/microl. At concentrations of 10,000-100,000 RBCs/microl of CSF, positive CSF results (IFAT titer >or=5) for S. neurona and N. hughesi were detected only when the corresponding serum titers were >or=160 and >or=80, respectively. The IFAT performed on CSF is reliable for testing horses for equine protozoal myeloencephalitis caused by S. neurona or N. hughesi, even when blood contamination causes the RBC concentration in CSF to be up to 10,000 RBCs/microl.

Evaluation of two Toxoplasma gondii serologic tests used in a serosurvey of domestic cats in California.

We evaluated the sensitivity (Se) and specificity (Sp) of an IgG enzyme-linked immunosorbent assay (ELISA) and IgG indirect fluorescent antibody test (IFAT) for detection of Toxoplasma gondii-specific antibodies in sera from 2 cat populations using a Bayesian approach. Accounting for test covariance, the Se and Sp of the IgG ELISA were estimated to be 92.6% and 96.5%, and those of the IgG IFAT were 81.0% and 93.8%, respectively. Both tests performed poorly in cats experimentally coinfected with feline immunodeficiency virus and T. gondii. Excluding this group, Se and Sp of the ELISA were virtually unchanged (92.3% and 96.4%, respectively), whereas the IFAT Se improved to 94.2% and Sp remained stable at 93.7%. These tests and an IgM ELISA were applied to 123 cat sera from the Morro Bay area, California, where high morbidity and mortality attributable to toxoplasmosis have been detected in southern sea otters. Age-adjusted IgG seroprevalence in this population was estimated to be 29.6%, and it did not differ between owned and unowned cats. Accounting for Se, Sp, and test covariances, age-adjusted seroprevalence was 45.0%. The odds for T. gondii seropositivity were 12.3-fold higher for cats aged >12 mo compared with cats aged <6 mo.

Chemical inactivation of Toxoplasma gondii oocysts in water.

The protozoan parasite Toxoplasma gondii is increasingly recognized as a waterborne pathogen. Infection can be acquired by drinking contaminated water and conventional water treatments may not effectively inactivate tough, environmentally resistant oocysts. The present study was performed to assess the efficacy of 2 commonly used chemicals, sodium hypochlorite and ozone, to inactivate T. gondii oocysts in water. Oocysts were exposed to 100 mg/L of chlorine for 30 min, or for 2, 4, 8, 16, and 24 hr, or to 6 mg/L of ozone for 1, 2, 4, 8, or 12 min. Oocyst viability was determined by mouse bioassay. Serology, immunohistochemistry, and in vitro parasite isolation were used to evaluate mice for infection. Initially, mouse bioassay experiments were conducted to compare the analytical sensitivity of these 3 detection methods prior to completing the chemical inactivation experiments. Toxoplasma gondii infection was confirmed by at least 1 of the 3 detection methods in mice inoculated with all doses (10(5)-10(0)) of oocysts. Results of the chemical exposure experiments indicate that neither sodium hypochlorite nor ozone effectively inactivate T. gondii oocysts, even when used at high concentrations.

Experimental infection of Peromyscus californicus with Toxoplasma gondii.

Eight female Peromyscus californicus were infected with 10(2) or 10(4) Toxoplasma gondii culture-derived tachyzoites (Type II or X) isolated from southern sea otters. All but 2 mice survived infection and developed antibodies to T. gondii. The 2 fatally infected mice were inoculated with 10(4) tachyzoites of the Type X strain. Parasite detection by immunohistochemistry (IHC) and DNA amplification with 2 polymerase chain reaction (PCR) methods was compared for brain, heart, lung, liver, spleen, biceps muscle, and tongue, at a mean of 41 days postinfection. Parasites were detected most commonly by IHC in spleen (8/8) and brain (6/8). DNA amplification by PCR was most successful from brain, heart, and spleen.

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.

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.