Study of specific immunodominant antigens in different stages of Neospora caninum, Toxoplasma gondii, Sarcocystis spp. and Hammondia spp.

The family Sarcocystidae includes several intracellular coccidial parasites such as Toxoplasma gondii, Neospora caninum, Sarcocystis spp. and Hammondia spp. with heteroxenous life cycles involving different parasitic stages (oocysts/sporocysts, tachyzoites and bradyzoites in tissue cysts). The aim of this work was to evaluate monoclonal antibodies (MAb) (anti NcSAG1, anti NcSAG4 and anti TgCC2) and/or polyclonal antibodies (PAb) (anti NcSAG4 and anti TgBAG1) to label specific immunodominant antigens in different parasitic stages of N. caninum (oocyst, bradyzoite and tachyzoite), T. gondii (oocyst, cyst and tachyzoite), H. heydorni (oocyst), S. cruzi (cyst and bradyzoite) and S. falcatula (sporocyst). It was observed that the MAb directed against NcSAG1 reacted exclusively with N. caninum tachyzoites. In contrast, the MAb directed against NcSAG4 did not react with any of the parasites tested at any stage. The MAb directed against NcSAG4 reacted with both N. caninum and T. gondii tachyzoites, T. gondii tissue cysts and S. cruzi tissue cysts and bradyzoites. As expected, the MAb directed against the T. gondii tissue cyst wall antigen TgCC2 reacted with T. gondii tissue cysts, N. caninum bradyzoites, but also with T. gondii and H. heydorni oocysts and S. falcatula sporocysts. Finally, the PAb directed against the T. gondii bradyzoite proteinTgBAG1 reacted with T. gondii tissue cysts, N. caninum bradyzoites, and also with S. cruzi tissue cysts and bradyzoites. These data reveal a wide range of cross-reactions between different species of protozoa and between different developmental stages, which should be taken into account in the design and evaluation of diagnostic tests, as well as in the assessment of vaccination and challenge studies.

Seroprevalence of Sarcocystis falcatula in Two Islands of Malaysia using Recombinant Surface Antigen 4.

Sarcocystosis was diagnosed worldwide by serodiagnostic tests utilising the whole parasite, for which the protozoa were maintained in vitro are more costly. In this study, antigenicity of Sarcocystis falcatula recombinant protein (rSfSAG4) was investigated towards the local communities of Pangkor and Tioman Islands and its seroprevalence was surveyed in these islands. A total of 348 human sera were tested using rSfSAG4 by Western blot and ELISA. High prevalence of sarcocystosis was observed in Tioman Island (80.6%) than in Pangkor Island (50.0%) by Western blot. In ELISA, the seroprevalence observed in Tioman Island was 45.9%, whereas in Pangkor Island 63.0%. In other parasitic infections, the prevalence was 34.0% by Western blot and 46.0% by ELISA. In healthy control group, 7% by Western blot and 8% by ELISA showed positivity to rSfSAG4. It is suggested SfSAG4 is a candidate antigen to measure seroprevalence of sarcocystosis.

Seroprevalence of sarcocystosis in the local communities of Pangkor and Tioman Islands using recombinant surface antigens 3 (rSAG3) of Sarcocystis falcatula.

OBJECTIVE: To investigate the seroprevalence of Sarcocystosis in the local communities of Pangkor and Tioman islands, Malaysia, by using antigenic recombinant surface antigens 2 and 3 from Sarcocystis falcatula (rSfSAG2 and rSfSAG3) as the target proteins via Western blot and ELISA assays. METHODS: SfSAG2 and SfSAG3 genes were isolated from S. falcatula and expressed in Escherichia coli expression system. A total of 348 serum samples [volunteers from both islands (n = 100), non-Sarcocystis parasitic infections patients (n = 50) and healthy donors (n = 100)] were collected and tested with purified SfSAGs in Western blot and ELISA assays to measure the seroprevalence of human sarcocystosis. RESULTS: None of the sera in this study reacted with rSfSAG2 by Western blot and ELISA. For rSfSAG3, relatively high prevalence of sarcocystosis was observed in Tioman Island (75.5%) than in Pangkor Island (34%) by Western blot. In ELISA, the different prevalence rate was observed between Tioman Island (43.8%) and Pangkor Island (37%). The prevalence rate in other parasitic infections (amoebiasis, cysticercosis, filariasis, malaria, toxocariasis and toxoplasmosis) was 30% by Western blot and 26% by ELISA. Only 8% (by Western blot) and 10% (by ELISA) of healthy donors showed reactivity towards rSfSAG3. CONCLUSION: This is the first study reporting a seroprevalence of sarcocystosis in Pangkor and Tioman Islands, Malaysia. The combination of Western blot and ELISA is suitable to be used for serodiagnosis of sarcocystosis. With further evaluations, SfSAG3 can potentially be used to confirm infection, asymptomatic screening, surveillance and epidemiological studies.

Multilocus characterization of Sarcocystis falcatula-related organisms isolated in Brazil supports genetic admixture of high diverse SAG alleles among the isolates.

In a previous study in Brazil, six isolates of Sarcocystis spp. recovered from budgerigars fed sporocysts excreted by opossums of the genus Didelphis were characterized by means of sequencing fragments of gene coding cytochrome B (CYTB), internal transcribed spacer 1 (ITS1), and surface antigen genes (SAG2, SAG3 and SAG4). The isolates shared identical ITS1 and CYTB sequences, but differed at SAG2, SAG3 and SAG4: three allele variants of SAG2, 3 allele variants of SAG3 and 2 allele variants of SAG4 were encountered in three multilocus genotypes (MLGs) (MLG1, MLG2, and MLG3). At ITS1 and CYTB, all the isolates from budgerigars were identical to the Sarcocystis falcatula-like isolate 59-2016-RS-BR that was detected in a barefaced ibis (Phimosus infuscatus) causing necrotizing meningoencephalitis in Brazil. At ITS1 locus, all the above isolates were clearly distinct from Sarcocystis neurona, Sarcocystis falcatula, Sarcocystis lindsayi, and Sarcocystis speeri, the four known species of Sarcocystis that use opossums of the genus Didelphis as definitive hosts. Here, we replicated the experiment above to identify additional MLGs or other species of Sarcocystis. Fifteen budgerigars were experimentally infected with sporocysts of Sarcocystis spp. from 12 opossums of the genus Didelphis. All the birds died 9-19 days after infection and tissue samples containing merozoites and schizonts of Sarcocystis spp. were recovered. Fractions of sequences coding for 18S ribosomal RNA gene (18S), CYTB, ITS1, SAG2, SAG3 and SAG4 were PCR amplified and sequenced from the infected lungs. In addition, fractions of 18S, SAG2, SAG3 and SAG4 were sequenced from the isolate 59-2016-RS-BR and fractions of 18S were sequenced from the six isolates from budgerigars described above. From the results, all the isolates shared identical 18S, ITS1 and CYTB sequences. Among the 15 new isolates from budgerigars, three allele variants of SAG2, 3 allele variants of SAG3 and 2 allele variants of SAG4 were encountered in five MLGs, of which four were novel (MLG1, MLG4, MLG5, MLG6 and MLG7). Isolate 59-2016-RS-BR was assigned to an eighth MLG (MLG8). Molecular data pointed that Sarcocystis assigned to MLGs 1 to 8 are variants of the same species, but the SAG-based trees of the isolates conflicted, which supports genetic admixture among them. The sarcocystinae studied have high diversity of SAG alleles per locus and the correlation of such an abundant variety of SAG alleles to host specificity and pathogenicity needs to be assessed. Remains to be elucidated if the parasites studied here and S. falcatula are variants of the same species that have diverged to the point of possessing differences at ITS1 level, but that are still capable of exchanging genes.

Immunoprotective responses against murine sarcocystosis by ß - Irradiated sporocysts.

This study aimed to induce protective immunity against infection with Sarcocystis muris in experimental mice using ß-irradiated sporocysts. Mice were vaccinated with 50 sporocysts of S. muris which were exposed to 1.84 µSv ß-irradiation for 2, 4 and 8 h. After challenge infection, different samples were taken for evaluation. Serum and intestinal wash were assayed for IFN-γ and IgA, respectively. Mesenteric lymph nodes (MLNs) and spleen were investigated for CD4+ and CD8+ T cells using immunohistochemistry. For liver, the morphological changes in parasitic stages and the count of infiltrated CD8+ T, NK1.1+ and FasL+ cells were also investigated. Real time (RT) - PCR was used for detection of liver MHC I, CD1d, IFN-γ, perforin and FasL as well as the parasite 18S ribosomal(r) RNA in liver and muscle tissues. Alterations of liver parasitic stages as well as a decrease in the infection with the parasite in both of liver and muscle tissues were dependent on radiation exposure time. An investigation for the mechanism of immunoprotection showed an increase in liver NK1.1+ & FasL+ cells, serum IFN-γ and intestinal IgA, while CD4+ and CD8+ T showed a remarkable increase in MLNs and spleen. FasL expression increased in the liver dependently on radiation exposure time, while perforin, MHC I and CD1d were not. ß-irradiated sporocysts with 1.84 µSv for 8 h s could induce the highest protection against infection with Sarcocystis. This could be largely relied on the increased infiltration of NK cells and associated higher expression of FasL in the liver.

Horses seropositive for Toxoplasma gondii, Sarcocystis spp. and Neospora spp.: Possible risk factors for infection in Brazil.

Many parasitic diseases are considered asymptomatic, even though some studies have shown that they may cause pathological changes in the host. Therefore, the aim of this study was to evaluate the occurrence of antibodies against Toxoplasma gondii, Neospora spp. and Sarcocystis spp. in horses, and to identify the risk factors for disease. For this, 174 horses were studied, 90 males and 84 females aged between two and 20 years old. Blood samples were collected and stored in tubes without anticoagulant to obtain serum, which was subjected to serological tests for T. gondii, Sarcocystis spp., and Neospora spp. using indirect immunofluorescence assay (IFA). IFA results were as follows: Sarcocystis spp. 41.37% (72/174) (CI95%-34.05-49.09); T. gondii 32.18% (56/174) (CI95%-25.42-39.74) and Neospora spp. 48.27% (84/174) (CI95%-40.68.50-55.93). Out of 174 horses, 81 had simple infection, 61 had mixed infections with two or three of these pathogens, and therefore, only 32 horses showed no antibodies to any of these pathogens. No risk factors for Sarcocystis spp. and T. gondii infection were identified. However, there was a significant (1.22-CI95%-1.02-1.52) relationship between animal age and Neospora spp. infection, since older animals showed higher prevalence. Therefore, it was possible to conclude that T. gondii and Neospora spp. affect horses in Southern Brazil, however all the animals studied were asymptomatic without reproductive, neurological or locomotor problems.

Unusual Necrotizing Encephalitis in Raccoons and Skunks Concurrently Infected With Canine Distemper Virus and Sarcocystis sp.

Canine distemper virus commonly infects free-ranging, terrestrial mesopredators throughout the United States. Due to the immunosuppressive effects of the virus, concurrent opportunistic infections are also common. Among these, secondary systemic protozoal infections have been described in a number of species. We report an unusual presentation of necrotizing encephalitis associated withSarcocystissp in four raccoons and one skunk concurrently infected with canine distemper virus. Lesions were characterized by variably sized necrotizing cavitations composed of abundant mineral admixed with inflammatory cells and protozoa.Sarcocystissp was confirmed via immunohistochemistry using a monoclonal antibody toSarcocystis neurona The pathologic changes are similar to lesions in human AIDS patients infected withToxoplasma gondii.

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.

Serologic profiles for Sarcocystis sp. and Neospora caninum and productive performance in naturally infected beef calves.

Sarcocystis sp. and Neospora caninum infections affect cattle worldwide causing important economic losses. The objective of the present study was to trace serologic profiles for Sarcocystis sp. and N. caninum in naturally infected beef calves and analyze their relationship with transmission routes and productive performance. Samples were collected in two cow-calf operations located in Buenos Aires province, Argentina. In farm 1, 43 calves were bled and weighed three times. In farm 2, 69 calves were bled and weighed six times. Sarcocystis sp. and N. caninum immunofluorescence antibody test (IFAT) titers were averaged for each sampling point in order to trace serologic profiles for each infection. Categories were created to evaluate differences in daily weight gain. For S. cruzi antigen, animals were separated in a low-titer (< or = 200) and high-titer group (>200); for N. caninum, animals were grouped as infected and uninfected. Sarcocystis sp. antibody titer as well as the number of infected animals increased gradually over time in both farms. In farm 2 the low-titer group had significantly higher daily weight gain than the high-titer group. For N. caninum 44% (farm 1) and 65% (farm 2) of calves were considered infected, and the serological profile was horizontal or decreasing over time. However, seroprevalence increased in both farms and vertical and horizontal transmission frequency were estimated between 18.5%-29% and 22-25.5%, respectively. No differences were detected in daily weight gain between N. caninum groups from both farms. This is the first report of serological profiles for Sarcocystis sp. and N. caninum by IFAT in naturally infected beef calves and their relationship to different transmission routes and productive performance.

Utility of 2 immunological tests for antemortem diagnosis of equine protozoal myeloencephalitis (Sarcocystis neurona Infection) in naturally occurring cases.

BACKGROUND: Antemortem diagnosis of equine protozoal myeloencephalitis (EPM) is challenging. Limited information is available regarding a commercial test (surface antigen 1 [SAG-1] ELISA). Performance of another commercial test (indirect fluorescent antibody test [IFAT]) using samples from an independent group has not been well described. HYPOTHESIS/OBJECTIVES: The primary goal was to evaluate the SAG-1 ELISA and IFAT using naturally occurring EPM cases. A secondary goal was to obtain more information regarding clinical presentation. ANIMALS: Hospital cases were admitted over 20 months and classified into 4 groups. Confirmed positive cases (n = 9) had asymmetric or multifocal neurologic deficits or both and postmortem lesions consistent with EPM. Confirmed negative cases (n = 17) had variable clinical signs and postmortem lesions consistent with another neurologic disease (or no lesions). Suspected positive cases (n = 10) had asymmetric or multifocal deficits or both, marked improvement after treatment for EPM, and other likely diseases excluded. Suspected negative cases (n = 29) had orthopedic disease and no neurologic deficits. METHODS: Results of immunological testing (SAG-1 ELISA and IFAT on serum or cerebrospinal fluid [CSF] or both), neurologic examinations, CSF analyses, and postmortem examinations were analyzed retrospectively. RESULTS: SAG-1 ELISA sensitivity was 12.5% (95% CI, 1.6-38.4) and specificity was 97.1% (95% CI, 84.7-99.9) using serum. IFAT sensitivity was 94.4% (95% CI, 72.7-99.9) and specificity was 85.2% (95% CI, 66.3-95.8) using serum; sensitivity was 92.3% (95% CI, 64.0-99.8) and specificity was 89.7% (95% CI, 72.7-97.8) using CSF. CONCLUSIONS AND CLINICAL IMPORTANCE: Low sensitivity of the SAG-1 ELISA limited its usefulness for antemortem diagnosis of EPM in this patient population.

Survey of Toxoplasma gondii, Neospora caninum and Sarcocystis neurona antibodies in wild red-tailed Amazon parrots (Amazona brasiliensis).

Toxoplasma gondii, Neospora caninum and Sarcocystis neurona are obligate intracellular parasites within the phylum Apicomplexa. The red-tailed Amazon parrot (Amazona brasiliensis) is a near-threatened species of psittacine that is endemic to the Atlantic Forest of Brazil and has been designated as a bioindicator because of its sensitivity to environmental qualitative status and changes. The aim of this study was to evaluate the presence of antibodies against T. gondii, N. caninum and S. neurona in wild red-tailed Amazon parrot nestlings on Rasa Island, Brazil. Blood samples were collected from 51 parrots and plasma samples were stored at - 20 °C until immunofluorescence antibody tests (IFAT) were performed. Antigen slides were prepared using tachyzoites of T. gondii (RH strain) and, N. caninum (NC-1 strain) and using merozoites of S. neurona (SNR37 strain). Plasma samples were tested at initial dilutions of 1:16 for T. gondii, 1:50 for N. caninum and 1:5 for S. neurona. An anti-chicken antibody conjugated with FITC was used as a secondary antibody at 1:50 dilution. No antibodies for any of these three protozoa were found, thus suggesting that these wild red-tailed Amazon parrot nestlings had not been exposed to these parasites.

Serologic reactivity of canine sera to Sarcocystis neurona and Sarcocystis cruzi antigens.

Sarcocystis neurona, a coccidian parasite shed by opossums (Didelphis spp.) in the Americas, is the major cause of equine protozoal myeloencephalitis (EPM) and induces disease in other domestic and wild animal species, including domestic dogs. Sarcocystis cruzi, despite its low pathogenicity for cattle (intermediate hosts), is worldwide distributed and uses mostly dogs as definitive hosts. The aims of this study were to test serological reactivities of dog sera to S. neurona and S. cruzi antigens, and to investigate potential serological cross-reactivity to these parasites. Sera from 353 Brazilian dogs were obtained from rural areas in the municipality of Ilhéus, Bahia, and examined by immunofluorescent antibody tests (IFAT). Antigens used in serological reactions consisted of S. neurona merozoites from a North American strain (SN138), and bradyzoites of S. cruzi obtained from Brazilian bovine hearts, with parasite species identity confirmed by PCR and sequencing of the 18S gene of the rDNA. Seropositivity to S. neurona and to S. cruzi were detected in 3.39% (12/353) and 4.81% (17/353) of the dogs, respectively. Ten canine sera reacted solely to S. neurona and 15 serum samples reacted only to S. cruzi. Two serum samples were simultaneously positive for both parasites. Sera from 14 dogs that tested positive by IFAT (9 for S. neurona and 3 for S. cruzi) and from two dogs that were negative by IFAT for the two parasites, were examined by Western blot using S. neurona as antigen; these sera reacted to a great number of protein bands, including antigens on the 16 and 30 KDa positions, which encompass immunodominant antigens for S. neurona in horses. Western blot did not show any specific pattern for S. neurona infection/exposure using canine sera. Dogs act as definitive hosts for several Sarcocystis spp. that infect farm animals, including horses, sheep, goats, water buffaloes and pigs, and for this reason, should contain antibodies to a broad repertoire of Sarcocystis spp. antigens. In conclusion, low percentages of dogs from rural areas of Ilhéus, Bahia, were reactive to both S. neurona and S. cruzi antigens. It is possible that other Sarcocystis species, besides S. neurona and S. cruzi, might have contributed for the seropositivity observed in this study. IFAT was more specific than Western blot to differentiate canine serological reactions to S. neurona and S. cruzi antigens.

Anti-Sarcocystis Antibodies in Lambs Deprived of Colostrum.

INTRODUCTION: The objective of this study was to evaluate the presence of anti-Sarcocystis spp. specific IgG antibodies in serum samples from precolostral lambs to determine the occurrence of transplacental transmission of Sarcocystis spp. in sheep. METHODS: Blood samples were collected from 80 ewes and their respective lambs, immediately after lambing and before colostrum ingestion, respectively. The presence of anti-Sarcocystis spp. IgG was evaluated in serum samples using the indirect fluorescent antibody test (IFAT). Positive samples of the lambs were submitted to titration and IFAT to detect anti-T. gondii and anti-N. caninum specific IgG. RESULTS: Anti-Sarcocystis spp. IgG was detected in 62.5% of the ewes (50/80) and in 4% of the lambs of the seropositive ewes (2/50). None of the lambs from seronegative ewes were positive. The final titers of the positive lambs were 80. No cross reaction was detected among the positive samples to anti-Sarcocystis spp., anti-N. caninum, and anti-T. gondii IgG. The detection of anti-Sarcocystis spp. antibodies in serum samples of lambs deprived of colostrum suggests transplacental transmission of infection. Thus, the vertical transmission may be an alternative route of infection of Sarcocystis spp. also in sheep. Further studies are warranted to confirm transplacental transmission in sheep and to explain the importance of this infection pathway.

In silico identification of immunotherapeutic and diagnostic targets in the glycosylphosphatidylinositol metabolism of the coccidian Sarcocystis aucheniae.

Meat of the South American camelids (SACs) llama and alpaca is an important source of animal protein and income for rural families in the Andes, and a product with significant growth potential for local and international markets. However, infestation with macroscopic cysts of the coccidian protozoon Sarcocystis aucheniae, a parasitosis known as SAC sarcocystosis, significantly hampers its commercialization. There are no validated methods to diagnose the presence of S. aucheniae cysts other than carcass examination. Moreover, there are no available drugs or vaccines to cure or prevent SAC sarcocystosis. Identification of relevant molecules that act at the host-pathogen interface can significantly contribute to the control of this disease. It has been shown for other pathogenic protozoa that glycosylphosphatidylinositol (GPI) is a critical molecule implicated in parasite survival and pathogenicity. This study focused on the identification of the enzymes that participate in the S. aucheniae GPI biosynthetic pathway and the repertoire of the parasite GPI-anchored proteins (GPI-APs). To this aim, RNA was extracted from parasite cysts and the transcriptome was sequenced and translated into amino acid sequences. The generated database was mined using sequences of well-characterized GPI biosynthetic enzymes of Saccharomyces cerevisiae and Toxoplasma gondii. Eleven enzymes predicted to participate in the S. aucheniae GPI biosynthetic pathway were identified. On the other hand, the database was searched for proteins carrying an N-terminal signal peptide and a single C-terminal transmembrane region containing a GPI anchor signal. Twenty-four GPI-anchored peptides were identified, of which nine are likely S. aucheniae-specific, and 15 are homologous to membrane proteins of other coccidians. Among the latter, 13 belong to the SRS domain superfamily, an extensive group of coccidian GPI-anchored proteins that mediate parasite interaction with their host. Phylogenetic analysis showed a great degree of intra- and inter-specific divergence among SRS family proteins. In vitro and in vivo experiments are needed to validate S. aucheniae GPI biosynthetic enzymes and GPI-APs as drug targets and/or as vaccine or diagnostic antigens.

Sarcocystosis: a clinical outbreak in dairy calves.

Death and illness in a pen of eight yearling dairy heifers was caused by the protozoan parasite Sarcocystis. All animals had weight loss, weakness, marginal anemia, and elevated serum enzymes. Affected animals had high hemagglutinating antibody titers to Sarcocystis antigen. Affected tissues of the two animals that died demonstrated schizonits and young cysts during pathologic examination. The resident farm dog was shedding Sarcocystis sporocysts and was incriminated as the source of infection.

Lambs infected with UV-attenuated sporocysts of Sarcocystis ovicanis produced abnormal sarcocysts and induced protective immunity against a challenge infection.

The present study surveyed the prevalence of natural infection of the sheep esophagus muscle with sarcocysts of Sarcocystis ovicanis and examined induction of protective immunity using UV-attenuated sporocysts. The overall prevalence of natural infection of the sheep was 95%. Infectivity of the collected sarcocysts was confirmed by shedding of sporulated oocysts after feeding infected esophageal tissues to dogs. To induce protective immunity, lambs were immunized 3 times (once a week) with 1.5 x 10(4) sporocysts exposed to UV-light for 30 min (UV-30 group) or 60 (UV-60 group) min and then challenged with 1.5 x 10(4) normal sporocysts at the 3rd week post the 1st vaccination. These lambs showed high survival and less clinical signs of sarcocystosis than normal infected lambs. The attenuated sporocysts produced abnormal cysts; small in size and detached from the muscle fiber. These abnormalities were more obvious in UV-60 group than UV-30 group. Also, the IFN-gamma level and lymphocyte percentage were increased while the total leukocyte count was decreased in the UV-60 group compared with other groups. The high level of IFN-gamma may be an evidence for the induction of Th1 responses which may have protective effect against a challenge infection.

Serosurvey of Toxoplasma gondii, Sarcocystis sp. and Neospora caninum in geese (Anser sp.) from urban parks and captivity.

Geese, ducks, mallards, and swans are birds of the order Anseriformes, which are found in the wild, in zoos and parks, and raised for meat consumption. Toxoplasma gondii, Sarcocystis sp., and Neospora caninum are protozoans of several species of animals. Wild and domestic birds can serve as intermediate hosts, disseminators and potential sources of infection of these protozoa to humans through contaminated meat. The aims of this study were: (i) to perform a serological survey of T. gondii, Sarcocystis sp. and N. caninum in geese (Anser sp.) from public parks and from captivity and (ii) to compare seroprevalence between these two locations. Antibodies were detected by Immunofluorescence antibody test using the serum of 149 geese. Antibodies to Sarcocystis sp., T. gondii, and N. caninum were detected in 28.18%, 18% and 0.67% of geese, respectively; 57% of geese from urban parks and 26.53% of geese from captivity were seropositive for at least one protozoa. The results indicate environmental contamination, particularly for the occurrence of antibodies against T. gondii - a zoonosis that causes toxoplasmosis and is transmitted through oocyte ingestion. This is the first serological survey of T. gondii, Sarcocystis sp. and N. caninum in geese from urban parks in Curitiba, Brazil.

Low prevalence of infection by Sarcocystis neurona in horses from the State of Alagoas, Brazil.

The aim of this study was to determine the prevalence of infection by Sarcocystis neurona in horses and identify potential risk factors. Were analyzed 427 samples from 36 farms in 21 municipalities in the Alagoas State, Brazil. Presence of anti-S. neurona antibodies was diagnosed by indirect immunofluorescence antibody test (IFAT) and was confirmed using the immunoblot test. Risk factors were assessed through investigative questionnaires on animal management on the farms. The prevalence of anti-S.neurona antibodies was 2.8% (confidence interval, CI: 1.5-4.9%) from IFAT and 1.6% (CI:0.8-3.34%) from immunoblot, and there were positive horses on 16.6% of the studied farms. None of the variables studied presented associations with serological status for S. neurona. This is the first report on infection by S. neurona in horses reared in Alagoas, Brazil showing a low exposure to S. neurona in this region, but with significant numbers of foci.

Serologic cross-reactivity between Sarcocystis neurona and Sarcocystis falcatula-like in experimentally infected Mongolian gerbils.

Sarcocystis neurona is the major cause of the equine protozoal myeloencephalitis (EPM) in the Americas and has opossums of the genus Didelphis as definitive hosts. Most isolates of Sarcocystis sp. shed by opossums in Brazil differ genetically from the known species of Sarcocystis. These Brazilian isolates behave similarly as Sarcocystis falcatula, which causes sarcocystosis in birds, and for this reason, have been classified as Sarcocystis falcatula-like. Genes coding for the immunodominant surface antigens SAG2, SAG3 and SAG4 of S. falcatula-like are similar to those from S. neurona. It is unknown the Sarcocystis species that causes EPM in Brazil, as S. neurona has never been genetically confirmed in Brazilian horses. All cases associated with EPM in Brazil were diagnosed by immunological tests, which are not specific for S. neurona infection. It is possible that S. falcatula-like may infect horses in Brazil. The aims of the current study were to test the susceptibility of gerbils (Meriones unguiculatus) to experimental infections with S. neurona and S. falcatula-like, and to investigate potential serologic cross-reactivity to these parasites by immunofluorescent antibody test (IFAT) and Western blot (WB). A total of 27 gerbils, distributed in five experimental groups (G1-G5), were employed in this work (G1: 4 negative controls; G2: 6 infected with S. neurona merozoites, G3: 6 infected with S. falcatula-like merozoites; G4 and G5 (5 and 6, respectively, infected with different doses of sporocysts). None of the 17 animals that seroconverted for the parasites in IFAT presented any visualized organism or Sarcocystis DNA in the examined tissues. No serologic cross-reactivity was observed using IFAT. However, sera from animals infected with S. falcatula-like and S. neurona presented the same pattern of antigenic recognition when S. neurona merozoites were used as antigen in WB, including reactivity to proteins of 30 and 16 kDa, regarded as specific markers for S. neurona-infected animals. Gerbils did not sustain infection by these parasites, although produced antibodies after inoculation. These results are suggestive that other animal species that are exposed to S. falcatula-like, including horses, may present serologic cross-reactivity to S. neurona in WB. IFAT was demonstrated to be more specific that WB for the detection of antibodies to S. falcatula-like and S. neurona in the experimental conditions of this study.

Parasite survey on a captive wolf population using classical techniques and ELISA coproantigen detection, USA.

As laws change around the United States, wildlife that were once kept as companion animals are now often confiscated by local authorities. They are then euthanized unless a home is found for them at a sanctuary. Wolf sanctuaries are, therefore, becoming increasingly important for their conservation and management. However, little data is available on best practices for the health management of captive wolves, including data on parasitic diseases. Our objective was to assess the prevalence of parasites of captive wolves combining classical coprological techniques and immunoassays based on the detection of coproantigen of selected canid parasites. Fecal samples of 39 animals were collected upon observation of individual animals defecating. All samples were processed using the Fecal Dx® tests, a suite of coproantigen ELISAs for detection of ascarid, hookworm, whipworm, and Giardia (IDEXX Laboratories Inc.). Out of the 39 samples, 38 were processed using the double-centrifugation sugar flotation (DCSF) and 34 using a modification of the Baermann technique. Twenty-eight samples (71.8%) were positive for hookworm, and none positive for the other parasites tested using coproantigen ELISA. Ancylostoma sp. (26, 68.4%), Eucoleus boehmi (13, 34.2%), and Trichuris sp. (2; 5.3%), and Sarcocystis sp. (13, 34.2%) were detected using DCSF. No metastrongyloid lungworm larvae were found. The Cohen's kappa index (0.97) showed excellent agreement between the hookworm coproantigen ELISA and the DCSF using feces preserved in ethanol for a short period of time. This study provides a baseline on the parasites of captive wolves, and shows that recent innovative diagnostics in veterinary parasitology, developed and optimized for dogs, may be used for assessing the health of wolves.