Routine fecal examination revealed novel coccidian oocysts in asymptomatic California sea lions ( Zalophus californianus ) in a rehabilitation facility. Coccidian oocysts were observed in fecal samples collected from 15 of 410 California sea lions admitted to The Marine Mammal Center between April 2007 and October 2009. Phylogenetic analysis using the full ITS-1 region, partial small subunit 18S rDNA sequence, and the Apicomplexa rpoB region identified 2 distinct sequence clades, referred to as Coccidia A and Coccidia B, and placed them in the Sarcocystidae, grouped with the tissue-cyst–forming coccidia. Both sequence clades resolved as individual taxa at ITS-1 and rpoB and were most closely related to Neospora caninum . Coccidia A was identified in 11 and Coccidia B in 4 of 12 sea lion oocyst samples successfully sequenced (3 of those sea lions were co-infected with both parasites). Shedding of Coccidia A oocysts was not associated with age class, sex, or stranding location, but yearlings represented the majority of shedders (8/15). This is the first study to use molecular phylogenetics to identify and describe coccidian parasites shed by a marine mammal.

abstract Enteric protozoal infection was identified in 5 stranded California sea lions ( Zalophus californianus ). Microscopically, the apical cytoplasm of distal jejunal enterocytes contained multiple stages of coccidian parasites, including schizonts with merozoites and spherical gametocytes, which were morphologically similar to coccidians. By histopathology, organisms appeared to be confined to the intestine and accompanied by only mild enteritis. Using electron microscopy, both sexual (microgametocytes, macrogamonts) and asexual (schizonts, merozoites) coccidian stages were identified in enterocytes within parasitophorous vacuoles, consistent with apicomplexan development in a definitive host. Serology was negative for tissue cyst-forming coccidians, and immunohistochemistry for Toxoplasma gondii was inconclusive and negative for Neospora caninum and Sarcocystis neurona . Analysis of ITS-1 gene sequences amplified from frozen or formalin-fixed paraffin-embedded intestinal sections identified DNA sequences with closest homology to Neospora sp. (80%); these novel sequences were referred to as belonging to coccidian parasites “A,” “B,” and “C.” Subsequent molecular analyses completed on a neonatal harbor seal ( Phoca vitulina ) with protozoal lymphadenitis, hepatitis, myocarditis, and encephalitis showed that it was infected with a coccidian parasite bearing the “C” sequence type. Our results indicate that sea lions likely serve as definitive hosts for 3 newly described coccidian parasites, at least 1 of which is pathogenic in a marine mammal intermediate host species.

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.

Three nematodes, Turgida turgida , Cruzia americana , and Didelphostrongylus hayesi , have been documented to cause morbidity and mortality in the Virginia opossum ( Didelphis virginiana ). The present study was designed to determine the frequency of infection of these nematodes in opossums at 2 study sites in California and to determine if there are risk factors associated with shedding of eggs or larvae in the feces. Turgida turgida and C. americana adults were found in 84.4% (stomach; n = 45) and 62.5% (intestinal wash and feces; n = 16) of sampled opossums. Eggs were present in opossum feces (n = 105) less frequently (40% T. turgida and 35.2% C. americana ). Didelphostrongylus hayesi larvae were found in 79.0% of opossum feces examined (n = 105). Adult age and wet season (December through April) were significant predictive factors for the presence of T. turgida eggs, whereas the dry season (May through November) was significantly associated with the presence of C. americana eggs in feces. Adult opossums were more likely to have eggs and larvae from all 3 nematodes in the feces.

Sera from 523 wild rodents were tested for Toxoplasma gondii antibodies using either an indirect fluorescent antibody test (IFAT) (rats and mice, with titer ≥80 considered positive) or a latex agglutination test (LAT) (voles, squirrels, and pocket mice, with titer ≥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 ≥200 m from residential housing and adult age.

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.

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.

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 co-infected 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.

The study objective was to assess the risk of transplacental transmission of Sarcocystis neurona and Neospora hughesi in foals from 4 California farms during 3 foaling seasons. Serum of presuckle foals and serum and colostrum of periparturient mares were tested using indirect fluorescent antibody tests for S. neurona and N. hughesi . Serum antibody titers were ≤10 in 366 presuckle foals tested. There was no serologic or histologic evidence of either parasite in aborted fetuses or placentas examined. Positivity for S. neurona and N. hughesi in mares increased with age. Mares ≤9 yr that originated from Kentucky were 3.8 and 1.4 times more likely to be positive for S. neurona and N. hughesi , respectively, than mares from California. The strength of association between positivity to either parasite and state of birth decreased as age increased. Mares positive for S. neurona and N. hughesi were 2.2 and 1.7 times more likely, respectively, to have a previous abortion than negative mares, adjusted for age and state of birth. The annual mortality rate for mares was 4%. The annual incidence rate of equine protozoal myeloencephalitis was 0.2%. In conclusion, there was no detectable risk of transplacental transmission of S. neurona and N. hughesi . Prevalence of antibodies against both parasites in mares increased with age.

The objectives of this study were to evaluate the accuracy of the indirect fluorescent antibody test (IFAT) using serum and cerebrospinal fluid (CSF) of horses naturally and experimentally infected with Sarcocystis neurona , to assess the correlation between serum and CSF titers, and to determine the effect of S. neurona vaccination on the diagnosis of infection. Using receiver-operating characteristic analysis, the areas under the curve for the IFAT were 0.97 (serum) and 0.99 (CSF). Sensitivity and specificity were 83.3 and 96.9% (serum, cutoff 80) and 100 and 99% (CSF, cutoff 5), respectively. Titer-specific likelihood ratios (LRs) ranged from 0.03 to 187.8 for titers between <10 and 640. Median time to conversion was 22–26 days postinfection (DPI) (serum) and 30 DPI (CSF). The correlation between serum and CSF titers was moderately strong ( r = 0.6) at 30 DPI. Percentage of vaccinated antibody-positive horses ranged from 0 to 95% between 0 and 112 days after the second vaccination. Thus, the IFAT was reliable and accurate using serum and CSF. Use of LRs potentially improves clinical decision making. Correlation between serum and CSF titers affects the joint accuracy of the IFAT; therefore, the ratio of serum to CSF titers has potential diagnostic value. The S. neurona vaccine could possibly interfere with equine protozoal myeloencephalitis diagnosis.

NcSUB1 (formerly known as NC-p65) is the first molecularly described proteolytic enzyme of the intracellular protozoan parasite Neospora caninum . This report describes the characterization of a rabbit anti-N54, which is an antiserum generated against an internal fragment of NcSUB1 (amino acids 649–783). In immunofluorescence studies rabbit anti-N54 labeled the apical end of the fixed parasite. By immuno-gold electron microscopy, the antibody bound primarily to the microneme organelles of the parasite. Analysis of secreted parasitic proteins indicated that a protein of molecular weight 65 kDa (reduced) or 55 kDa (nonreduced) was recognized by the antibody. The same secreted proteins were affinity purified with rabbit anti-N54–coupled resins and were shown to contain major proteolytic activity by zymography. Thus, rabbit anti-N54 is the first antibody developed for N. caninum that binds to the microneme proteins and recognizes a major secreted enzyme.

Neospora hughesi is a newly recognized protozoan pathogen in horses that causes a myeloencephalitis similar to Sarcocystis neurona . There are no validated serologic tests using the gold standard sera that are currently available to detect specific N. hughesi antibodies and, thus, no tests available to detect antemortem exposure or estimate seroprevalence in the horse. The objectives of the present study were to establish a bank of gold standard equine sera through experimental infections with N. hughesi and to assess several serologic tests for the detection of related protozoan antibodies. Seven horses were inoculated with N. hughesi tachyzoites, and 7 horses received uninfected cell culture material. The horses were monitored, and blood and cerebrospinal fluid were collected repeatedly over a 4-mo period. With the sera, 4 different serologic techniques were evaluated, including a whole-parasite lysate enzyme-linked immunosorbent assay (ELISA), a recombinant protein ELISA, a modified direct agglutination test, and an indirect fluorescent antibody test. Qualitative and quantitative evaluation of the results showed that the N. hughesi indirect fluorescent antibody test (IFAT) consistently discriminated between experimentally infected and noninfected horses, using a cutoff of 1:640. Sera from 3 naturally infected horses had titers >1:640. Cerebrospinal fluid in all but 1 infected horse had very low N. hughesi IFAT titers (<1:160), starting at postinoculation day 30.

Two species of protozoans were isolated from a harbor seal with fatal meninogoencephalitis. Serologic reactivity was detected to both Sarcocystis neurona and Toxoplasma gondii . Parasites associated with brain inflammation and necrosis reacted only with immunohistochemical stains utilizing polyclonal antisera raised against Sarcocystis neurona . However, 2 distinct parasites were observed in cell cultures derived from the seal's brain tissue. These parasites were separated by mouse passage and limiting dilution. Purified zoites from 1 isolate (HS1) reacted strongly with polyclonal antiserum to S. neurona and with the harbor seal's own serum (1:2,560 for each) on indirect immunofluorescent antibody tests (IFAT), but weakly to antisera to T. gondii and Neospora caninum (1:40). Zoites from the second isolate (HS2) reacted positively with T. gondii polyclonal antiserum (1:81,920) and with the harbor seal's own serum (1:640), but weakly to S. neurona and N. caninum antisera (1:80 or less). Amplification and sequence analysis of protozoal DNA encoding portions of the 18s ribosomal RNA (18s rDNA) and the adjacent first internal transcribed spacer (ITS1) were performed for both isolates, and resulting sequences were compared to those from similar protozoans. Based on molecular characterization, parasite morphology, serologic reactivity, histology, and immunohistochemistry, HS1 was indistinguishable from S. neurona , and HS2 was indistinguishable from T. gondii .

Babesia leo, a small piroplasm isolated from lions in South Africa is described as a distinct species based on a phylogenetic analysis of the 18S rRNA gene. Intraerythrocytic trophozoite and merozoite stages of B. leo are morphologically indistinguishable from other small piroplasms of felids. Previous studies showed that B. leo was biologically and antigenically distinct from B. felis, which is known to infect wild and domestic felids in South Africa. Molecular characterization showed strong support for the phylogenetic seperation of B. leo as a distinct species from B. felis and other felid piroplasms. Phylogenetic analysis also showed that Babesia microti and all of the felid piroplasms from Africa with known 18S rRNA gene sequences available, including B. leo, formed a single, separate clade, sister to the other babesial and theilerial piroplasm parasites.

An epidemiologic study designed to identify the small mammal reservoir for the zoonotic WA1-type babesial parasite resulted in the discovery of a small, intraerythrocytic piroplasm in smeared blood from dusky-footed woodrats ( Neotoma fuscipes ) in northern California. The woodrat parasites were isolated and compared to other piroplasm parasites based on their morphology, antigenicity, and genetic characteristics. These studies indicated that the woodrat parasites were not the WA1-type babesial agent but were of the genus Theileria. We accordingly named it Theileria youngi. The prevalence in the woodrat population was high (61%). Infection was unrelated to gender or age of the woodrats. Potential vectors for this tick-transmitted parasite included 3 species of ticks recovered from the woodrats, Dermacentor occidentalis, Ixodes woodi, and Ixodes pacificus. Mostly larval or nymphal stages were recovered, suggesting transstadial transmission is possible. This is the first piroplasm fully characterized from a dusky-footed woodrat.

We characterized the cytokine response and T-cell requirements of mice infected with the intraerythrocytic parasites Babesia microti and WA1. WA1 infections were fatal, whereas B. microti infections were resolved. We measured production of tumor necrosis factor (TNF)-α, interferon-γ, interleukin (IL)-10, and IL-4 by splenic CD4 + , CD8 + , and γδ + T cells using flow cytometry. WA1 inoculation stimulated TNF-α production, whereas resolving B. microti infections were characterized by increased IL-10 and IL-4. The role of TNF-α in WA1 infections was further investigated by inoculating TNFRp55 −/− mice with a lethal dose of WA1. A survival rate of 90% in the TNFRp55 −/− mice indicated that a disruption in the TNF-α pathway abrogated the pathologic mechanism of WA1. Inoculation of WA1 into CD4 −/− and CD8 −/− mice resulted in survival rates of 60% and 78%, respectively, whereas WA1 infection in γδ −/− and control mice was fatal. These results suggest that CD8 + T cells may contribute to the WA1-associated disease. Babesia -infected CD4 −/− mice experienced a longer duration of parasitemia, indicating that CD4 + T cells participate in parasite elimination. These studies demonstrate differences in immune responses during fatal or resolving Babesia infections, and they identify TNF-α as an important mediator of the WA1-associated pathogenesis.