Evaluation of an immunochromatographic serologic test to detect the presence of anti-Toxoplasma gondii antibodies in cats.

BACKGROUND: Toxoplasmosis is a protozoan disease caused by Toxoplasma gondii. Different T. gondii confirmatory techniques, including serologic methods, are available to detect the presence of the parasite. Among serology techniques, immunochromatographic rapid testing could be a reliable alternative to serologic laboratory techniques. OBJECTIVE: This study evaluated a commercial immunochromatographic test (FASTest TOXOPLASMA g) in seronegative and seropositive cats. METHODS: Two indirect immunofluorescence antibody reference tests, an in-house technique, and a commercial test were used to classify 292 feline serum samples. The rapid test was evaluated in different groups of cats, including healthy seronegative cats (n = 121), seropositive cats with variable anti-Toxoplasma antibodies (n = 146), and cats with positive serologic results for other pathogens (n = 25). The sensitivity, specificity, accuracy, receiver operating characteristic curves, and kappa statistics were analyzed as performance measures. RESULTS: Of the 292 samples, 146 were classified as T. gondii seropositive and 146 as T. gondii seronegative. Concordant results were obtained for all samples using immunofluorescence antibody tests. The diagnostic measures of this rapid test showed 98.63% sensitivity and 100% specificity, and 99.32% accuracy. The kappa statistics value was 0.986, and the area under the receiver operating characteristic curve was 0.993. CONCLUSIONS: This rapid test showed diagnostic measurements similar to those of traditional quantitative serologic methods. In situations where laboratory techniques are not available, this test, under clinical conditions, could be a useful alternative to obtain accurate results rapidly.

Evaluation of total immunoglobulin G and subclass antibodies in an enzyme-linked immunosorbent assay for serodiagnosis of human amebic liver abscess.

Background: Amebic liver abscess (ALA) caused by Entamoeba histolytica is usually diagnosed based on its clinical symptoms, medical imaging abnormalities of the liver, and serological tests, the most common being the enzyme-linked immunosorbent assay (ELISA). For more than three decades, no investigation has evaluated the diagnostic performance of immunoglobulin G (IgG) subclasses in the serodiagnosis of ALA. Herein, we assessed the efficiencies of anti-amebic IgG and IgG subclasses for diagnosing ALA. Methods: A serological ELISA-based test was performed to assess its diagnostic performance using a total of 330 serum samples from ALA patients (n = 14), healthy individuals (n = 40), and patients with other diseases (n = 276). Results: ELISA targeting the total IgG antibody to E. histolytica antigen exhibited 100% sensitivity 95% CI [76.8-100.0] and 97.8% specificity 95% CI [95.5-99.1], whereas the assay targeting IgG1 showed the same sensitivity (100% 95% CI [76.8-100.0]) and a slightly higher specificity (99.1% 95% CI [97.3-99.8]). The other IgG subclasses (IgG2, IgG3, and IgG4) displayed a lower sensitivity and specificity. The sensitivity and specificity did not significantly differ between tests measuring total IgG and IgG1 (Exact McNemar's test; p > 0.05), with a concordance of 98.2%, represented by a Cohen's kappa of 0.83 (p < 0.001), indicating almost perfect agreement. Conclusion: ELISA targeting IgG1 can provide valuable information to clinicians in differentiating ALA from other parasitic diseases, cancers, cirrhosis, and viral hepatitis. However, enzyme-conjugated anti-human total IgG is cheaper than anti-human IgG subclasses. Therefore, we suggest that total IgG-based ELISA is sufficient for the routine serodiagnosis of human ALA and possibly other clinical manifestations of invasive amebiasis.

Unusual Toxoplasma infection of the eye and central nervous system in an HIV-positive patient.

OBJECTIVE: To report on a unique combination of cerebral toxoplasmosis and ocular toxoplasmosis in an HIV-positive patient in Slovakia. METHODS: A 35-year-old heterosexual patient who presented with headache and major seizures underwent computed tomography (CT) and magnetic resonance imaging (MRI). Based on clinical findings, serological tests for toxoplasmosis were performed on serum and ocular fluid specimens. PCR was also used to detect Toxoplasma gondii and cytomegalovirus DNA. Goldmann and Witmer coefficient calculation was applied to demonstrate the synthesis of intraocular IgG antibodies. RESULTS: CT and MRI revealed cystic lesions suspected of metastasis in the occipital and temporal regions, and we searched for the primary tumor. After vision loss in the left eye, which rapidly progressed to complete blindness, an eye examination detected macular edema. Anti-edema treatment was initiated. HIV positivity with a very low CD4 T-cell count (20/μL) was found, and the viral load was 100 400 HIV-RNA copies/ml. The serum was positive for anti-Toxoplasma IgG antibodies (> 200 IU/mL), IgM negative, and IgA borderline. As toxoplasmic encephalitis and retinitis were suspected, antitoxoplasmic therapy with pyrimethamine, spiramycin, and folinic acid was started. The ophthalmologist considered cytomegalovirus retinitis, which was not confirmed by serology or PCR. In contrast, the presence of IgG antibodies in ocular fluid and serum with the calculation of the Goldmann-Witmer coefficient (GW = 32) as well as PCR DNA positivity pointed to Toxoplasma gondii as the etiological agent. Follow-up MRI scan confirmed regression of the pathological lesions, neurological deficit also improved, CD4 T-lymphocytes increased above 200/μL, but blindness of the left eye persisted. CONCLUSION: CT and MRI scans offered no clue as to Toxoplasma etiology of the brain and eye involvement in an HIV-positive patient, which was only confirmed by laboratory tests. Due to the delay in the diagnosis of toxoplasmosis, time from the epileptic seizure to treatment initiation was 16 days, which assumedly caused irreversible blindness in the patient.

Antibodies against Toxoplasma gondii positive in serum and aqueous humor to diagnose clinically suspected ocular toxoplasmosis: A case report.

INTRODUCTION: Previously, diagnosis of ocular toxoplasmosis is based on clinical symptoms and Toxoplasma serology. Checking serological indicators often cannot reflect the real intraocular situation, and may even mislead clinicians to make wrong judgments. PATIENT CONCERNS: A 38-year-old male patient visited our ophthalmology clinic with a chief complaint of decreased vision for about 5 days in his right eye. DIAGNOSIS: Aqueous humor sample analysis found Toxoplasma DNA detectable, and Toxoplasma immunoglobulin G (IgG) and immunoglobulin M (IgM) to be positive. His serum Toxoplasma IgG was also positive (10.04 IU/mL; reference range: 0 to 7.2 IU/mL). Therefore, the final diagnose was ocular toxoplasmosis involving his right eye. INTERVENTIONS: Oral prednisone 60 mg/day and azithromycin 0.25 g/day were started. Oral antibiotic treatment for toxoplasma was continued for 4 weeks, and prednisone followed by weekly stepwise tapering in steps of 10 mg/day. OUTCOMES: The BCVA and fundus of right eye remained stable after treatment at follow-up. CONCLUSIONS: This article reported a case of ocular Toxoplasma gondii infection diagnosis by serum and aqueous humor antibody tests. We provide some additional information on the T gondii infection diagnosis.

YKL-40 as a novel diagnostic biomarker in Toxoplasmosis.

Toxoplasmosis is one of the most globally prevalent zoonotic infection caused by an obligate, intracellular parasite called Toxoplasma gondii. Toxoplasmosis actively triggers an acute immune response and inflammatory reactions, which causes serious pathological changes in various tissues in the human body, and more evidently localizes in different nervous tissues of various body organs. The YKL-40 is a glycoprotein secreted by numerous cell types in different patterns associated with various pathological processes such as inflammatory reactions, tissue remodeling, and fibrosis, and is a disease-specific biomarker of neuroinflammation. Therefore, this study aimed to determine whether the YKL-40 is markedly increased in toxoplasmosis or not and whether its level is different between the acute and chronic phases of the infection to determine if it can be used as a clinically useful biomarker in the diagnosis, and determination of disease severity and follow-up of toxoplasmosis. Accordingly, a total of 80 serum samples were collected from previously diagnosed female patients of different ages with toxoplasmosis. In addition, serum samples of 10 healthy females were used as the control. Patients were first divided into two groups (30 patients with acute infection, and 50 patients with chronic infection) depending on the results of detection of specific anti-Toxoplasma IgM and IgG by enzyme-linked immunosorbent assay (ELISA). The level of YKL-40 was then measured in the patients' serum by ELISA. The statistical analysis of data clearly disclosed very highly significant differences (P < 0.001) between the level of YKL-40 in the acute infection group and healthy controls, chronic infection group and healthy controls, and between the groups with acute and chronic infections. These findings led to conclude that YKL-40 classify as a unique and sophisticated biomarker in the diagnosis of toxoplasmosis where it can vitally be used to detect the stage of the disease, whether acute or chronic, besides its ability to detect the infection.

Detection of Acanthamoeba spp. using carboxylesterase antibody and its usage for diagnosing Acanthamoeba-keratitis.

Contact lens usage has contributed to increased incidence rates of Acanthamoeba keratitis (AK), a serious corneal infection that can lead to blindness. Since symptoms associated with AK closely resemble those incurred by bacterial or fungal keratitis, developing a diagnostic method enabling rapid detection with a high degree of Acanthamoeba-specificity would be beneficial. Here, we produced a polyclonal antibody targeting the carboxylesterase (CE) superfamily protein secreted by the pathogenic Acanthamoeba and evaluated its diagnostic potential. Western blot analysis revealed that the CE antibody specifically interacts with the cell lysates and conditioned media of pathogenic Acanthamoeba, which were not observed from the cell lysates and conditioned media of human corneal epithelial (HCE) cells, Fusarium solani, Staphylococcus aureus, and Pseudomonas aeruginosa. High titers of A. castellanii-specific antibody production were confirmed sera of immunized mice via ELISA, and these antibodies were capable of detecting A. castellanii from the cell lysates and their conditioned media. The specificity of the CE antibody was further confirmed on A. castellanii trophozoites and cysts co-cultured with HCE cells, F. solani, S. aureus, and P. aeruginosa using immunocytochemistry. Additionally, the CE antibody produced in this study successfully interacted with 7 different Acanthamoeba species. Our findings demonstrate that the polyclonal CE antibody specifically detects multiple species belong to the genus Acanthamoeba, thus highlighting its potential as AK diagnostic tool.

Detection of Sarcocystis spp. and Toxoplasma gondii in swine and detection of DNA of these protozoa in tissues and sausages / Soroprevalência de Sarcocystis spp. e Toxoplasma gondii em suínos e detecção do DNA desses protozoários em tecidos e embutidos

Abstract The seroprevalence of Sarcocystis spp. and Toxoplasma gondii was researched in swine raised in Santa Maria, RS, Brazil. Serum samples from 84 pigs from 31 farms were tested using indirect immunofluorescence assay (IFA) for both agents. Additionally, 53 samples of pork sausages and tissues destined for human consumption, including: salami, sausage, black pudding, heart, tongue, brain, and rib muscle, were submitted to PCR to detect DNA for each agent. The frequency of anti-Sarcocystis spp. antibodies was 36.9% (31/84), with titers ranging from 32 to 1024, and 25% (21/84) for anti-T. gondii antibodies, with titers ranging from 64 to 2048. Sarcocystis spp. and T. gondii DNA were detected in 67.9% (36/53) and 13.2% (7/53) of samples, respectively. The presence of antibodies and the detection of DNA from Sarcocystis spp., and T. gondii suggests that the pigs were infected and may serve as an important reservoir for both parasites. The infection by these protozoa in the swine population is relevant to public health due to their zoonotic potential.

Resumo A soroprevalência de Sarcocystis spp. e Toxoplasma gondii foi pesquisada em suínos criados em Santa Maria, RS, Brasil. Amostras de soro de 84 suínos de 31 fazendas foram testadas pela reação deimunofluorescência indireta (IFA) para ambos os agentes. Adicionalmente, 53 amostras de embutidos suínos e tecidos cárneos destinados ao consumo humano, incluindo: salame, linguiça, morcela, coração, língua, cérebro e músculo da costela foram submetidas à PCR para detecção de DNA para cada agente. A frequência de anticorpos anti-Sarcocystis spp. foi de 36,9% (31/84), com títulos variando de 32 a 1.024; e 25% (21/84) para anticorpos anti-T. gondii, com títulos variando de 64 a 2048. A presença de DNA de Sarcocystis spp. e T. gondii foi detectada em 67,9% (36/53) e 13,2% (7/53) das amostras avaliadas, respectivamente. A detecção de anticorpos e DNA de Sarcocystis spp. e T. gondii sugere que os suínos foram infectados e podem servir como um importante reservatório de ambos os parasitas. A circulação desses agentes na população suína é relevante para a saúde pública devido ao seu potencial zoonótico.

Antibody signatures of asymptomatic Plasmodium falciparum malaria infections measured from dried blood spots.

BACKGROUND: Screening malaria-specific antibody responses on protein microarrays can help identify immune factors that mediate protection against malaria infection, disease, and transmission, as well as markers of past exposure to both malaria parasites and mosquito vectors. Most malaria protein microarray work has used serum as the sample matrix, requiring prompt laboratory processing and a continuous cold chain, thus limiting applications in remote locations. Dried blood spots (DBS) pose minimal biohazard, do not require immediate laboratory processing, and are stable at room temperature for transport, making them potentially superior alternatives to serum. The goals of this study were to assess the viability of DBS as a source for antibody profiling and to use DBS to identify serological signatures of low-density Plasmodium falciparum infections in malaria-endemic regions of Myanmar. METHODS: Matched DBS and serum samples from a cross-sectional study in Ingapu Township, Myanmar were probed on protein microarrays populated with P. falciparum antigen fragments. Signal and trends in both sample matrices were compared. A case-control study was then performed using banked DBS samples from malaria-endemic regions of Myanmar, and a regularized logistic regression model was used to identify antibody signatures of ultrasensitive PCR-positive P. falciparum infections. RESULTS: Approximately 30% of serum IgG activity was recovered from DBS. Despite this loss of antibody activity, antigen and population trends were well-matched between the two sample matrices. Responses to 18 protein fragments were associated with the odds of asymptomatic P. falciparum infection, albeit with modest diagnostic characteristics (sensitivity 58%, specificity 85%, negative predictive value 88%, and positive predictive value 52%). CONCLUSIONS: Malaria-specific antibody responses can be reliably detected, quantified, and analysed from DBS, opening the door to serological studies in populations where serum collection, transport, and storage would otherwise be impossible. While test characteristics of antibody signatures were insufficient for individual diagnosis, serological testing may be useful for identifying exposure to asymptomatic, low-density malaria infections, particularly if sero-surveillance strategies target individuals with low previous exposure as sentinels for population exposure.

Ten-year follow-up of the largest oral Chagas disease outbreak. Laboratory biomarkers of infection as indicators of therapeutic failure.

Trypanosoma cruzi uses various mechanisms of infection to access humans. Since 1967, food contaminated with metacyclic trypomastigotes has triggered several outbreaks of acute infection of Chagas disease by oral transmission. Follow-up studies to assess the effectiveness of anti-parasitic treatment of oral outbreaks are rather scarce. Here, we report a 10-year laboratory follow-up using parasitological, serological, and molecular tests of 106 individuals infected in 2007 of the largest known outbreak of orally transmitted Chagas disease, which occurred in Caracas city, Venezuela. Before treatment (2007), specific IgA, IgM and IgG, were found in 71% (75/106), 90% (95/106) and 100% (106/106), respectively, in addition to 21% (9/43) parasitemia, Complement Mediated Lysis (CML) in 98% (104/106) and 79% (34/43) parasitic DNA for PCR. Blood culture detected parasitemia up to 18 months post-treatment in 6% (6/106) of the patients. In 2017, the original number of cases in the follow-up decreased by 46% and due to the country's economic situation, not all the trials could be carried out in the entire population. During follow-up, IgA and IgM disappeared promptly, with IgM persisting in 19% (20/104) of the patients three years after treatment. The anti-T. cruzi IgG remained positive 10 years later in 41% (20/49) of the individuals evaluated. CML remained positive seven years later in 79% (65/82) of the cases. PCR positive cases decreased after treatment but progressively recovered, being positive in 69% (32/46) of the individuals evaluated in 2017. The group of children (under 18 years of age) showed the highest PCR positivity with 76% (26/34) of the cases, but their parasitic load tended to diminish, while in adults the parasitic load regained their initial values. The simultaneous evaluation of serological tests and PCR of the patients allowed us to separate patients among responders and non-responders to the anti-parasitic treatment, and this information prompted us to apply a second anti-parasitic treatment in the group of non-responders. In this population not subjected to the like lihood of re-infection, adult patients were more likely to be non-responders when compared to children. These results suggest that rigorous laboratory follow-up with T. cruzi infectious biomarkers is essential to detect cases of parasite persistence.

Serological prevalence and risk factors of Toxoplasma gondii in Zoo Mammals in Chile.

Toxoplasma gondii is a zoonotic cosmopolitan protozoan that causes a high mortality rate among zoo mammals such as New World primates, meerkats, marsupials and Pallas' cat. It has been recently reported in Chile, mainly among wild populations, but also as the cause of death of a kangaroo and a mara. However, there has not been a T. gondii report at a Zoo population level in Chile in the last 35 years. The aim of the study was to estimate the seroprevalence and risk factors associated with T. gondii infection in mammals housed in a zoo located in the Metropolitan Region of Chile between 2011 and 2018. In this study, we analyzed 350 samples, from 324 animals, belonging to 57 species of carnivores, non-human primates, macropodids, ungulates and rodents to detect the presence of anti-T. gondii antibodies. Additionally, 20 animals were longitudinally sampled to evaluate intra-zoo infection. Using a commercial indirect Enzyme-Linked Immuno Sorbent Assay (ELISA) test, we detected T. gondii antibodies in 72 (22.2 %) samples. The overall seroprevalence estimates were 48.4 % in felines, 22.9 % in non-feline carnivores, 21.1 % in ungulates and 15.0 % in non-human primates. There were no positive samples from rodents or marsupials. Of animals sampled longitudinally, only a culpeo fox (Lycalopex cualpaeus) became seropositive along the study indicating exposition inside the facility. T. gondii seroprevalence differed significantly in taxonomic groups (p = 0.003), felines are statistically different from non-feline carnivores (NFC) (p = 0.040), ungulate (p = 0.027) and non-human primates (NHP) (p = 0.009). Annual prevalence comparison was performed showing no statistical difference (p = 0.941). A multivariable logistic regression was performed to ascertain the effect of taxonomic groups, proximity to water sources, diet, sex and type of housing on seropositivity. Only taxonomic group was statistically significant, indicating that NFC (OR = 0.35; 95 % CI = 0.15 - 0.83; p = 0.017), ungulates (OR = 0.30; 95 % CI = 0.13 - 0.69; p = 0.005), and NHP (OR = 0.25; 95 % CI = 0.09 - 0.72; p = 0.010) have lower risk of positivity to T. gondii compared to felines. Additionally, a black-faced spider monkey (Ateles chamek) and a siamang (Symphalangus syndactylus) were seropositive, being the first description of T. gondii infection in these species worldwide. As seen in previous studies, the widespread presence and exposure of T. gondii in zoo mammals was confirmed, and there may be contact with the agent and transmission within the zoo, which was confirmed by one animal became seropositive over the time. This fact could be a health problem for animals susceptible to fatal toxoplasmosis.

Classification Criteria for Toxoplasmic Retinitis.

PURPOSE: To determine classification criteria for toxoplasmic retinitis. DESIGN: Machine learning of cases with toxoplasmic retinitis and 4 other infectious posterior uveitides / panuveitides. METHODS: Cases of infectious posterior uveitides / panuveitides were collected in an informatics-designed preliminary database, and a final database was constructed of cases achieving supermajority agreement on diagnosis, using formal consensus techniques. Cases were split into a training set and a validation set. Machine learning using multinomial logistic regression was used on the training set to determine a parsimonious set of criteria that minimized the misclassification rate among the infectious posterior uveitides / panuveitides. The resulting criteria were evaluated on the validation set. RESULTS: Eight hundred three cases of infectious posterior uveitides / panuveitides, including 174 cases of toxoplasmic retinitis, were evaluated by machine learning. Key criteria for toxoplasmic retinitis included focal or paucifocal necrotizing retinitis and either positive polymerase chain reaction assay for Toxoplasma gondii from an intraocular specimen or the characteristic clinical picture of a round or oval retinitis lesion proximal to a hyperpigmented and/or atrophic chorioretinal scar. Overall accuracy for infectious posterior uveitides / panuveitides was 92.1% in the training set and 93.3% (95% confidence interval 88.2, 96.3) in the validation set. The misclassification rates for toxoplasmic retinitis were 8.2% in the training set and 10% in the validation set. CONCLUSIONS: The criteria for toxoplasmic retinitis had a low misclassification rate and seemed to perform sufficiently well for use in clinical and translational research.

Diagnostic usefulness of immunohistochemical evaluation of CD1a antigen and polyclonal anti-leishmania antibodies in cutaneous leishmaniasis.

BACKGROUND: Different immunohistochemical markers to detect amastigotes in cutaneous leishmaniasis have been proposed with variable diagnostic usefulness. OBJECTIVES: To evaluate the diagnostic usefulness of immunohistochemical amastigotes identification by specific polyclonal anti-Leishmania antibodies and CD1a expression (clone EP3622) in a series of PCR confirmed cutaneous leishmaniasis. MATERIALS AND METHODS: Thirty-three skin samples corresponding to PCR confirmed cutaneous leishmaniasis were included in the study. All samples were stained with Hematoxylin-eosin and Giemsa. Moreover, immunohistochemical studies with anti-CD1a and anti-Leishmania antibodies were performed. The patients clinical features and the observed histopathological features were also recorded. RESULTS: From the selected 33 biopsies, Leishmania spp. amastigotes were detected in 48.4% of cases with conventional Hematoxylin-eosin stain and in 57.5% of cases by Giemsa staining. In 31/33 cases, anti-CD1a allowed us to identify parasitic structures, and in 33/33 cases amastigotes were detected with anti-Leishmania antibodies. Concordance between both techniques, anti-CD1a and anti-Leishmania, was 94% [CI 95%: (79,8%-99,3%)] ; p value <0.05. The sensitivity of anti-CD1a in comparison with the PCR was 94%, with a positive predictive value of 100%. Two cases of low parasitic index were negative for CD1a immunostaining. In cases with high parasitic index, anti-CD1a stained amastigotes in superficial and deep dermis. Only a few cases were originally diagnosed with the available histological techniques, needing PCR for Leishmania spp. CONCLUSIONS: Anti-CD1a antibody seems to be a useful technique to identify amastigotes when PCR and anti-Leishmania antibodies are not available. The sensitivity to detect amastigotes is increased when the CD1a immunostaining is added to the classical Haematoxylin - eosin and Giemsa staining.

Experimental infection of tachyzoites of the NC1 strain of Neosporacaninum in female swine.

Neospora caninum is a protozoan that can cause reproductive problems in several animal species. Although N. caninum infection has been reported in swine, the pathogenesis and clinical signs are not fully known in this species. The objective of this work was to evaluate the effect of experimental infection with tachyzoites of the N. caninum strain Nc1 in swine matrices at different stages of gestation. For that purpose, 12 gilts, seronegative for N. caninum and T. gondii, were selected and allocated into four groups of three animals each. Animals in group A were not inoculated (control) and animals in groups B, C, and D were inoculated intravenously with of 2.9 × 107 tachyzoites, 30 days before conception, and at 45 and 90 days of gestation, respectively. Temperature, heart rate, blood, saliva, and vaginal mucus samples from the animals were collected periodically until the time of delivery for the investigation of IgG and IgM antibodies against N. caninum using IFAT and PCR to detect the parasite DNA. All gilts sero-converted from 5 and 7 DPI (days postinoculation) to IgM and IgG, respectively. Two gilts showed hypothermia on the 5th and 7th DPI, and five inoculated animals had leukocytosis on the 7th DPI. It was possible to detect DNA of N. caninum in samples of saliva (33/84), vaginal mucus (17/84), and blood (2/84). Based on serology (IgM) and PCR, three animals in group B showed evidence of reappearance of the infection during pregnancy. It is concluded that N. caninum can cause clinical signs in infected swine females, in addition to indicating saliva as a suitable diagnostic biological material for the detection of N. caninum DNA in this animal species.

Glycan moieties in Entamoeba histolytica ubiquitin are immunodominant.

The ubiquitin-proteasome system plays a central role performing several functions to maintain parasite homeostasis. We have reported the partial characterization of N-linked glycosylation profile in E. histolytica ubiquitin (EhUb). Here we examined the immunogenicity and antigenicity of carbohydrates in EhUbiquitin. Rabbits were immunized with purified EhUbiquitin or purified recombinant rUb expressed by E. coli. Using Western Blot, we explored the immunogenicity and antigenicity of protein portion and carbohydrates moiety. Interestingly, immunized rabbits produced antibodies to both Ub glycoprotein and rUb; but antibodies against carbohydrates were immunodominant, rather than antibodies to the protein moiety of EhUbiquitin. In addition, we observed that antibodies to protein moiety are not conserved in serum unless antigen is continually administrated. Conversely, anti-Ub glycoprotein antibodies are well maintained in circulation. In humans, infection with Entamoeba histolytica induces strong IgG anti-Ub response. The human antibodies recognize both, the protein moieties and the glycosylated structure. Entamoeba histolytica ubiquitin is immunogenic and antigenic. The glycan moieties are immunodominant and induces IgG. These data open the door to use carbohydrates as potential targets for diagnose tests, drugs and vaccine to prevent this parasitic disease.

Characterization of a Plasmodium falciparum PHISTc protein, PF3D7_0801000, in blood- stage malaria parasites.

During intraerythrocytic development Plasmodium falciparum deploys numerous proteins to support erythrocyte invasion, intracellular growth and development, as well as host immune evasion. Since these proteins are key for parasite intraerythrocytic survival and propagation, they represent attractive targets for antimalarial vaccines. In this study we sought to characterize a member of the PHISTc family of proteins, PF3D7_0801000, as a potential vaccine target. Using the wheat germ cell-free system we expressed the N-terminal region of PF3D7_0801000 (G93-L494, PF3D7_0801000N) and generated specific immune sera. We observed that PF3D7_0801000 localizes in merozoites, and antibodies against PF3D7_0801000N modestly inhibit P. falciparum parasite growth in in vitro culture. Sliding window analysis of the coding sequence revealed that pf3d7_0801000n is relatively conserved among African parasite isolates. Antibody profiles in a malaria-exposed Ugandan population revealed that PF3D7_0801000N is strongly immunoreactive with antibody acquisition increasing with age. Taken together, these findings suggest the need for further evaluation of PF3D7_0801000 for its role in merozoite invasion and utility as an asexual blood-stage vaccine candidate antigen.

Immune blotting as a method for diagnosis of toxoplasmosis.

Test kit for detection of IgG-antibodies to individual antigens of Toxoplasma gondii by immune blotting («Western blot¼ format) has been developed. Laboratory testing with first international WHO standard «Anti-toxoplasma serum (IgG), human, Lyophilized, 20 IU / ampoule¼ (NIBSC, Great Britain) demonstrated the analytical sensitivity of the new kit equal to 10 IU / ml. Study of the diagnostic efficiency of the new kit showed its high sensitivity, equal to 98.51 - 100%, and high specificity, equal to 99.5 - 100%. New test kit is intended for confirmatory testing in laboratory diagnostics of toxoplasmosis.

Prevalence of Toxoplasma gondii Antibodies in Stray Dogs from Various Locations in West and East Malaysia.

Toxoplasmosis is caused by an obligate intracellular protozoan parasite; Toxoplasma gondii, which is one of the most important zoonotic parasite worldwide. In dogs, the sexual reproductive cycle of T. gondii is lacking, and the animals are not widely consumed as food, but they are vital in the mechanical transmission of the parasite. However, there is no present data on the exposure of stray dogs to T. gondii in Malaysia. The objective of this serological survey was to determine the prevalence of T. gondii antibodies (IgG) and associated factors in stray dogs in East and West Malaysia. Antibodies to T. gondii were determined in serum samples from 222 stray dogs from 6 different states in East and West Malaysia (Peninsular Malaysia) using an Indirect ELISA. The seroprevalence for T. gondii was 23.4% (Confidence interval: CI 17.8-29.2%). Stray dogs from Selangor and Kuala Lumpur had the highest seroprevalence (32.4%; CI 13.2-45.5%) and lowest in those from Penang and Kedah (12.5%; CI 1.3-23.5%). Gender and breed were not associated with T. gondii seropositivity. However, adult dogs were more likely to be seropositive for T. gondii (OR=2.89; CI 1.1-7.7) compared with younger dogs. These results revealed that T. gondii is prevalent in stray dogs in the studied areas in Malaysia, and indicative of the level of environmental contamination of this parasite especially in urban areas.

Pathological, immunohistochemical, and molecular findings of equine protozoal myeloencephalitis due to Sarcocystis neurona infection in Brazilian horses.

Equine protozoal myeloencephalitis (EPM) is an important neurologic disease of horses in the American continent caused by Sarcocystis neurona and Neospora hughesi infection. This study describes the pathological, immunohistochemical, and molecular findings of fatal cases of EPM in southern Brazil. A review was performed on a total of 13 cases compatible with EPM, which were diagnosed by postmortem examination in the period of 2010-2017. Epidemiological information was obtained from necropsy reports. Gross and histological lesions were characterized, and cases were subjected to immunohistochemistry anti-Sarcocystis neurona, Toxoplasma gondii, and Neospora spp. Molecular search was performed using ITS-1 gene PCRs. Microscopic lesions were multifocal in all cases, and more frequently observed in the spinal cord segments and in the rhombencephalon. Intralesional protozoans were histologically detected in five horses, while a positive immunostaining for S. neurona was observed in eleven cases (11/13). Through molecular techniques, six positive cases for the ITS-1 gene were detected, and obtained sequences presented highest similarity with S. neurona. EPM due to S. neurona infection represents an important neurologic disease of horses in Brazil and this disease should be considered as a main differential diagnosis in horses presenting neurologic signs.

Epidemiological surveillance of Toxoplasma gondii in small ruminants in southern Spain.

Toxoplasmosis, caused by the protozoan Toxoplasma gondii, is a worldwide zoonotic disease, which affects most warm-blooded species. Besides the zoonotic relevance, toxoplasmosis is one of the major causes of reproductive disorders in small ruminants. A cross-sectional study was carried out to determine the seroprevalence, spatial distribution and risk factors associated with T. gondii infection in sheep and goats in southern Spain. During 2015-2017, a total of 1,943 small ruminants (998 sheep and 945 goats) from 127 flocks were tested for antibodies against T. gondii using the modified agglutination test (MAT, cut-off 1:25). Antibodies against T. gondii were detected in 464 of the 998 sheep (46.5 %; CI95 %: 43.4-49.6%) and 362 of the 945 goats (38.3 %; CI95 %: 35.2-41.4%) tested. The farm prevalence was 98.4 % (CI95 %: 95.4-100%) for sheep and 93.7 % (CI95 %: 87.6-99.7%) for goats. The generalized estimating equation analysis showed that presence of cats and existence of previous reproductive disorders were risk factors potentially associated with T. gondii seropositivity in small ruminants. Two statistically significant spatial clusters (P < 0.001) were identified. The seroprevalence observed in the present study indicates a widespread exposure to T. gondii in sheep and goats in southern Spain, which might have important implications for animal and public health. Management measures should be implemented in small ruminant farms in this region in order to reduce the risk of T. gondii infections, particularly in those areas identified in the spatial analysis.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay.

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.