Eimeria ninakohlyakimovae casts NOX-independent NETosis and induces enhanced IL-12, TNF-α, IL-6, CCL2 and iNOS gene transcription in caprine PMN.

Eimeria ninakohlyakimovae represents a highly pathogenic coccidian parasite causing severe haemorrhagic typhlocolitis in goat kids worldwide. NETosis was recently described as an efficient defense mechanism of polymorphonuclear neutrophils (PMN) acting against different parasites in vitro and in vivo. In vitro interactions of caprine PMN with parasitic stages of E. ninakohlyakimovae (i. e. oocysts and sporozoites) as well as soluble oocyst antigens (SOA) were analyzed at different ratios, concentrations and time spans. Extracellular DNA staining was used to illustrate classical molecules induced during caprine NETosis [i. e. histones (H3) and neutrophil elastase (NE)] via antibody-based immunofluorescence analyses. Functional inhibitor treatments with DPI and DNase I were applied to unveil role of NADPH oxidase (NOX) and characterize DNA-backbone composition of E. ninakohlyakimovae-triggered caprine NETosis. Scanning electron microscopy (SEM)- and immunofluorescence-analyses demonstrated that caprine PMN underwent NETosis upon contact with sporozoites and oocysts of E. ninakohlyakimovae, ensnaring filaments which firmly entrapped parasites. Detailed co-localization studies of E. ninakohlyakimovae-induced caprine NETosis revealed presence of PMN-derived DNA being adorned with nuclear H3 and NE corroborating molecular characteristics of NETosis. E. ninakohlyakoimovae-induced caprine NETosis was found to be NOX-independent since DPI inhibition led to a slight decrease of NETosis. Exposure of caprine PMN to vital E. ninakohlyakimovae sporozoites as well as SOA resulted in up-regulation of IL-12, TNF-α, IL-6, CCL2 and iNOS gene transcription in stimulated PMN. Since vital E. ninakohlyakimovae-sporozoites induced caprine NETosis, this effective entrapment mechanism might reduce initial sporozoite epithelial host cell invasion during goat coccidiosis ultimately resulting in less macromeront formation and reduced merozoites I production.

Cryptosporidium Diagnostic Assays: Microscopy.

Stained microscopy of fecal smears was the cornerstone of Cryptosporidium diagnosis for many years, and still provides a low-cost method for detecting oocysts. The development and commercialization of improved enzyme immunosorbent assays (EIA) for coproantigen detection provided an automatable method for mass testing, and rapid diagnostics when incorporated onto a cartridge format. Similarly, immunochromatographic lateral flow assays (ICLF) enable rapid diagnostics. Nevertheless, it is important that positive reactions by EIA or ICLF are confirmed. Here we describe microscopical methods using tinctorial stains for the diagnosis of acute cryptosporidiosis, and using immunofluorescent reagents for diagnosis or for confirmation of EIA or ICLF positive reactions.

Oral inoculation of ultraviolet-irradiated Eimeria species oocysts protects chickens against coccidiosis.

Prevention of coccidiosis is one of the best ways of controlling disease. Therefore, the present study was carried out to evaluate the protective effect of ultraviolet (UV)-irradiated sporulated oocysts of Eimeria species against coccidiosis in layer chickens. One hundred forty-four one-day-old layer chicks were randomly divided into 4 groups (n = 36), including non-immunized/non-challenged negative control group (NC group), non-immunized/challenged control group (NIC group), non-irradiated sporulated oocyst/challenged group (CA group), and UV-irradiated sporulated oocyst/challenged (UV group). At the age of 4 days, chickens in groups UV and CA were both orally inoculated with 1.0 × 104 UV-irradiated and non-irradiated sporulated oocysts of Eimeria species, respectively. Chickens in groups NIC and NC were served as positive and negative controls, respectively. Chickens in all groups were orally challenged with 7.5 × 104 sporulated oocysts of Eimeria species except the NC group at the age of 21 days. The results revealed that chicks receiving UV-irradiated sporulated oocysts had no signs of illness with minimal or no changes in the cecal integrity and a significantly lower oocyst shedding (OPG) than in the NIC group. Additionally, the cytokine gene expression profiles were evaluated. Expression levels of IL-2, IL-12, and IFN-γ were significantly higher in the spleen of chicks in the UV and CA groups than in the NC group post-challenge. As expected, treatment with irradiated oocysts resulted in a significant reduction in oocyst shedding and maintenance of cecal mucosal integrity. Furthermore, the body weight was higher in chickens inoculated with UV-irradiated oocysts than their non-irradiated counterparts. In conclusion, our results demonstrate that inoculation with UV-irradiated sporulated oocysts of Eimeria species can produce a substantial reduction in infection symptoms.

Chemical depletion of phagocytic immune cells in Anopheles gambiae reveals dual roles of mosquito hemocytes in anti-Plasmodium immunity.

Mosquito immunity is composed of both cellular and humoral factors that provide protection from invading pathogens. Immune cells known as hemocytes, have been intricately associated with phagocytosis and innate immune signaling. However, the lack of genetic tools has limited hemocyte study despite their importance in mosquito anti-Plasmodium immunity. To address these limitations, we employ the use of a chemical-based treatment to deplete phagocytic immune cells in Anopheles gambiae, demonstrating the role of phagocytes in complement recognition and prophenoloxidase production that limit the ookinete and oocyst stages of malaria parasite development, respectively. Through these experiments, we also define specific subtypes of phagocytic immune cells in An. gambiae, providing insights beyond the morphological characteristics that traditionally define mosquito hemocyte populations. Together, this study represents a significant advancement in our understanding of the roles of mosquito phagocytes in mosquito vector competence and demonstrates the utility of clodronate liposomes as an important tool in the study of invertebrate immunity.

Antibody and cytokine response to Cystoisospora suis infections in immune-competent young pigs.

BACKGROUND: To date, investigations on the immune response to Cystoisospora suis infections focused on suckling piglets, the age group clinically most affected. Actively immunizing piglets is unfeasible due to their immature immune system and the typically early infection in the first days after birth. Therefore, understanding and possibly enhancing the immune response of immune-competent animals is the prerequisite to develop a passive immunization strategy for piglets which currently rely on very limited treatment options. METHODS: To investigate antibody and cytokine responses of immune-competent animals and the impact of the oral immunization protocol on their immune response, growers with unknown previous exposure to C. suis (10-11 weeks-old) were infected one or three times with different doses (600 and 6000 or 200 and 2000, respectively) of C. suis oocysts, and compared to uninfected controls. Oocyst excretion was evaluated, and blood and intestinal mucus antibody titers were determined by IFAT. Systemic production of Th1, Th2, inflammatory and regulatory cytokines was determined in different immune compartments at mRNA and (after stimulation with a recombinant merozoite-protein) at protein level by PCR and multiplex fluorescent immunoassay, respectively. RESULTS: Infection generated significantly increased serum IgA and IgG levels against C. suis sporozoites and merozoites, irrespective of infection mode, with IgG against merozoites showing the strongest increase. No clinical signs and only occasional excretion were observed. The systemic cytokine response to C. suis was only weak. Nonetheless, in white blood cells, IL-4, IL-6 and IL-10 mRNA-levels significantly increased after infection, whereas IFN-É£, IL-2 and TGF-ß expression tended to decrease. In mesenteric lymph nodes (MLN), IL-10 and TNF-α levels were elevated while splenic cytokine expression was unaltered upon infection. Stimulated MLN-derived lymphocytes from infected pigs produced slightly more IL-12 and less IFN-α than controls. CONCLUSIONS: An infection and a subsequent systemic immune response can be induced in immune-competent animals by all evaluated infection models and growers can be used as models to mimic sow immunizations. The immune response to C. suis, although mild and with considerable variation in cytokine expression, was characterized by a Th2-associated and regulatory cytokine profile and antibody production. However, none of the parameters clearly stood out as a potential marker associated with protection. Antibody titers were significantly positively related with oocyst excretion and might thus serve as correlates for parasite replication or severity of infection.

Discovery of new Toxoplasma gondii antigenic proteins using a high throughput protein microarray approach screening sera of murine model infected orally with oocysts and tissue cysts.

BACKGROUND: Toxoplasma gondii is an obligate intracellular protozoan parasite that causes congenital toxoplasmosis, as well as other serious clinical presentations in immune compromised humans. The parasite has also been recently linked to behavioral diseases in humans and other mammalian hosts. New antigens are being evaluated to develop a diagnostic kit for the diagnosis of acute infection or a protective vaccine. METHODS: In this study, we have focused on the discovery of new antigenic proteins from T. gondii genomic data using a high throughput protein microarray screening. To date, microarrays containing > 2870 candidate exon products of T. gondii have been probed with sera collected from patients with toxoplasmosis. Here, the protein microarrays are probed with well-characterized serum samples from animal models administered orally with oocysts or tissue cysts. The aim was to discover the antigens that overlap in the mouse profile with human antibody profiles published previously. For this, a reactive antigen list of 240 antigens recognized by murine IgG and IgM was identified using pooled sera from orally infected mice. RESULTS: Analyses of screening data have identified plenty of antigens and showed strong immunogenicity in both mouse and human antibody profiles. Among them, ROP1, GRA2, GRA3, GRA4, GRA5, GRA6, GRA7, GRA8, GRA14, MIC1, MIC2 and MAG1 have shown strong immunogenicity and used as antigen in development of vaccines or serological diagnostic assays in previous studies. CONCLUSION: In addition to the above findings, ROP6, MIC12, SRS29A and SRS13 have shown strong immunogenicity but have not been tested in development of a diagnostic assay or a vaccine model yet.

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.

Natural killer cell activity irreversibly decreases after Cryptosporidium gastroenteritis in neonatal mice.

Cryptosporidiosis causes persistent diarrhoea in infants, immunocompromised patients and elderly persons. Long-term consequences of the disease include increased risk of malignancy, cardiomyopathy and gastrointestinal inflammation. This study aimed to investigate prolonged effects of cryptosporidiosis on innate immunity and growth in neonatal C3HA mice. The disease was challenged by Cryptosporidium parvum oocyst inoculation into 7-day-old animals. The mice whose intestine smears contained 3-5 or 6 and more oocysts per microscopic field at the day 5 after infection were considered as mildly or severely infected, correspondingly. To determine natural killer cell (NK) activity, we applied 3 H-uridine cytotoxic assay to the animals at 5-68 days after infection using K562 cells as targets. At severe infection, there was a statistically significant 1.5-2.0 fold decline of body mass, spleen mass and spleen cellularity that persisted in animals of all ages. Accordingly, NK cytotoxicity showed even more drastic drop reaching 2.7-3.0 folds that was statistically significant in all animals. At mild infection, the discovered effects were less pronounced and reached significance only in some age groups. Thus, our study provides evidence that NK cells show long-term cytotoxic activity decrease following Cryptosporidium infection in neonatal mice, particularly in severe disease.

Avirulence and lysozyme secretion in Paneth cells after infection of BALB/c mice with oocysts of Toxoplasma gondii strains TgCatCHn2 (ToxoDB#17) and TgCatCHn4 (ToxoDB#9).

Toxoplasma gondii has a complex life cycle and pathogenic mechanisms. Acute T. gondii infections in mice often result in death, whereas in chronic infections, the parasites may persist in the host tissues as intraneuronal or intramuscular cysts. However, the virulence of T. gondii strains in mice varies with its genetic background. The present study investigated the pathogenicity and pathological lesions of two T. gondii isolates from China: namely, TgCatCHn2 (ToxoDB#17) and TgCatCHn4 (ToxoDB#9). The virulent (ToxoDB#216) and avirulent (VEG) strains were employed as controls. Toxoplasmosis was induced by inoculating BALB/c mice with oocysts of the strains of T. gondii VEG, ToxoDB#216, TgCatCHn2 (ToxoDB#17), and TgCatCHn4 (ToxoDB#9), respectively. As a result, one oocyst of ToxoDB#216 could kill a mouse within 10 days post inoculation (DPI). The survival time of the mice for T. gondii TgCatCHn2 and TgCatCHn4 was >60 DPI for 106/mL oocysts, but this concentration (106/mL oocysts) of VEG strain could kill mice within 11 DPI. Compared with the strains of T. gondii ToxoDB#216 and VEG, the lesions in the small intestines of the strains of TgCatCHn2- and TgCatCHn4-infected mice were significantly smaller (P < 0.01). The positive area of T. gondii antigen in the ileum of mice infected with the strains of T. gondii VEG, TgCatCHn2 and TgCatCHn4 were significantly lower than that T. gondii ToxoDB#216 at 8 DPI (P < 0.01). Paneth cells (PCs) in the small intestines was eliminated by ToxoDB#216 (5-6 DPI) (P < 0.05). The strains of T. gondii TgCatCHn2-, TgCatCHn4- and VEG-infected mice, the number of PCs and granules decreased in the intestines, compared to T. gondii free mice, but the difference was not significant (8 DPI, P > 0.05). However, the granules in the PCs showed negative lysozyme expression in the intestines of mice infected with T. gondii TgCatCHn2 and TgCatCHn4. Thus, T. gondii strains of TgCatCHn2 (ToxoDB#17) and TgCatCHn4 (ToxoDB#9) were avirulent strains, they triggered an inhibition of lysozyme expression in the granules of PCs of the mouse intestine. These effects may in turn lead to intestinal dysbiosis, which may be related to further parasitic invasion of the intestines. The findings of the present study further expand the spectrum of the pathogenic features of various Chinese isolates of T. gondii.

Hammondia heydorni: Oocyst shedding by dogs fed in vitro generated tissue cysts, and evaluation of cross-immunity between H. heydorni and Neospora caninum in mice.

Hammondia heydorni is a coccidian parasite believed to be nonpathogenic for naturally-infected animals, but it is biologically and genetically related to Neospora caninum, a worldwide cause of abortion in cattle. The major aim of the present work was to determine whether dogs shed H. heydorni oocysts after consuming in vitro generated tissue cysts of the parasite. In addition, we investigated cross-immunity between H. heydorni and N. caninum in mice. Two dogs were fed cultured cells containing tissue cysts of H. heydorni mixed with canned dog food, and a third dog (negative control) received only non-infected cells mixed with canned food. The two dogs that consumed in vitro produced tissue cysts shed high numbers of oocysts, which were induced to sporulate and tested positive for H. heydorni by a species-specific PCR. The third uninfected dog did not shed H. heydorni oocysts in the feces. Oocysts shed by the dogs induced the formation of encysted bradyzoites of H. heydorni on KH-R cells. Nineteen BALB/c mice were employed in the cross-immunity study. Nine mice were orally inoculated with 1×105 sporulated oocysts of H. heydorni and challenged with N. caninum tachyzoites 30days after infection with H. heydorni. Other ten mice, which did not receive H. heydorni oocysts, were infected with 2×105N. caninum tachyzoites. Thirty days after challenging with N. caninum, all mice were euthanized and N. caninum DNA in their tissues was quantified by real time PCR. No statistically significant difference in N. caninum DNA concentrations were observed between the two groups. We concluded that in vitro generated cysts of H. heydorni are biologically active, because they induced oocyst shedding in dogs. As no cross-protection occurred in mice inoculated with H. heydorni and challenged with N. caninum, it is suspected that these parasites do not express significant numbers of homologous proteins during infection, or the immune response of BALB/c mice after H. heydorni infection was not sufficient.

Evaluation of novel oocyst wall protein candidates of Toxoplasma gondii.

Oocyst stage of Toxoplasma gondii is characterized by a durable wall that confers a strong protection to this protozoan parasite in face of harsh environmental conditions. Thus, it is considered the key for transmission of T. gondii. Analysis of oocyst wall composition is mandatory therefore; the aim of this study was to identify novel T. gondii oocyst wall proteins and test their use in detection of these oocysts in environmental samples. Five candidates of novel T. gondii oocyst wall proteins (TgOWPs) were identified and named TgOWP8 through TgOWP12. Recombinant protein of TgOWP8 was expressed in E. coli using glutathione S-transferase as fusion protein. Polyclonal antibody was produced and validated by indirect immunofluorescence antibody assay (IFA). For detection by IFA, we used different methods for fixation and permeabilization of oocysts to improve the antigen-antibody detection. Specificity to wall of T. gondii oocyst was confirmed and revealed absence of cross reactivity with bradyzoite cyst wall and tachyzoites. Although some TgOWPs were identified previously, our study represents a continuation of molecular investigations of oocyst wall proteins as an essential structure for the longevity and infectivity of this stage and also provided new trial to improve T. gondii oocysts detection.

rROP2 from Toxoplasma gondii as a potential vaccine against oocyst shedding in domestic cats.

The aim of the present study was to evaluate oocyst shedding in cats immunized by nasal route with T. gondii proteins ROP2. Twelve short hair cats (Felis catus) were divided in three groups G1, G2 and G3 (n=4). Animals from G1 received 100 µg of rROP2 proteins plus 20 µg of Quil-A, G2 received 100 µg of BSA plus 20 µg of Quil-A, and the G3 only saline solution (control group). All treatments were done by intranasal route at days 0, 21, 42, and 63. The challenge was performed in all groups on day 70 with ≅ 800 tissue cysts of ME-49 strain by oral route. Animals from G1 shed less oocysts (86.7%) than control groups. ELISA was used to detect anti-rROP2 IgG and IgA, however, there were no correlation between number of oocyst shedding by either IgG or IgA antibody levels. In the present work, in spite of lesser oocysts production in immunized group than control groups, it was not possible to associate the use of rROP2 via nostrils with protection against oocyst shedding. For the future, the use of either other recombinant proteins or DNA vaccine, in combination with rROP2 could be tested to try improving the efficacy of this kind of vaccine.

rROP2 from Toxoplasma gondii as a potential vaccine against oocyst shedding in domestic cats / rROP2 de Toxoplasma gondii como potencial vacina contra a eliminação de oocistos em gatos domésticos

Abstract The aim of the present study was to evaluate oocyst shedding in cats immunized by nasal route with T. gondii proteins ROP2. Twelve short hair cats (Felis catus) were divided in three groups G1, G2 and G3 (n=4). Animals from G1 received 100 μg of rROP2 proteins plus 20 μg of Quil-A, G2 received 100 μg of BSA plus 20 μg of Quil-A, and the G3 only saline solution (control group). All treatments were done by intranasal route at days 0, 21, 42, and 63. The challenge was performed in all groups on day 70 with ≅ 800 tissue cysts of ME-49 strain by oral route. Animals from G1 shed less oocysts (86.7%) than control groups. ELISA was used to detect anti-rROP2 IgG and IgA, however, there were no correlation between number of oocyst shedding by either IgG or IgA antibody levels. In the present work, in spite of lesser oocysts production in immunized group than control groups, it was not possible to associate the use of rROP2 via nostrils with protection against oocyst shedding. For the future, the use of either other recombinant proteins or DNA vaccine, in combination with rROP2 could be tested to try improving the efficacy of this kind of vaccine.

Resumo O objetivo do presente estudo foi avaliar a eliminação de oocistos de Toxoplasma gondii em gatos imunizados pela via nasal com proteínas ROP2 de T. gondii. Doze gatos sem raça definida (Felis catus) foram divididos em três grupos experimentais G1, G2 e G3 (n = 4). Os animais do G1 receberam 100 μg de proteínas de rROP2 mais 20 μg de Quil-A, G2 recebeu 100 μg de albumina de soro bovino (BSA) junto com 20 μg de Quil-A, e o G3 recebeu apenas solução salina (grupo de controle). Todos os tratamentos foram realizados pela via intranasal nos dias 0, 21, 42 e 63. O desafio foi realizado em todos os grupos no dia 70 com aproximadamente 800 cistos de tecido da cepa ME-49 por via oral. Os animais de todos os grupos tiveram as suas fezes examinadas e o número de oocistos foi determinado durante 20 dias após o desafio. Os animais de G1 eliminaram menos oocistos (86,7%) do que os grupos controles. O ELISA foi utilizado para detectar IgG e IgA anti-rROP2, no entanto, não houve correlação entre o número de eliminhação de oocistos com os níveis de anticorpos IgG ou IgA. No presente trabalho, apesar da menor produção de oocistos no grupo imunizado (G1) em relação aos grupos controles (G2 e G3), não foi possível associar o uso de rROP2 pela via nasal com proteção contra eliminação de oocistos de T. gondii. Para o futuro, a utilização de outras proteínas recombinantes, ou mesmo vacina de DNA, em combinação com rROP2 poderia ser utilizada para tentar melhorar a eficácia deste tipo de vacina.

Oocyst-Derived Extract of Toxoplasma Gondii Serves as Potent Immunomodulator in a Mouse Model of Birch Pollen Allergy.

INTRODUCTION: Previously, we have shown that oral infection with Toxoplasma gondii oocysts prevented type I allergy in mice. Here we investigated whether the application of a T. gondii oocyst lysate antigen (OLA) could also reduce allergy development. BALB/c mice were immunised twice with OLA followed by sensitisation with the major birch pollen (BP) allergen Bet v 1 and an aerosol challenge with BP extract. METHODS: First, we tested OLA in vitro. Stimulation of splenocytes and bone marrow-derived dendritic cells (BMDC) with OLA led to the production of pro-inflammatory and regulatory cytokines such as IL-6, IFN-γ and IL-10. Moreover, BMDC exposed to OLA upregulated the maturation markers CD40, CD80, CD86, and MHCII. Furthermore, OLA was recognised by TLR2-transfected human embryonic kidney cells. RESULTS: Immunisation of mice with OLA induced high levels of Toxoplasma-specific IgG antibodies in sera along with increased production of IFN-γ and IL-10 in Toxoplasma-antigen restimulated splenocytes. OLA reduced allergic airway inflammation as manifested by significant reduction of eosinophils in bronchoalveolar fluids, decreased cellular infiltrates and mucus production in the lungs. Accordingly, Bet v 1-specific IgE was decreased in OLA-pretreated mice. The reduced allergic immune responses were accompanied by increased numbers of CD4+CD25highFoxp3+ regulatory T cells in spleens as well as by increased numbers of granulocytic myeloid-derived suppressor cells in lungs when compared to sensitised controls suggesting that these two cell populations might be involved in the suppression of the allergic immune responses. CONCLUSION: Our data demonstrate that pretreatment with the oocyst extract can exert anti-allergic effects comparable to T. gondii infection. Thus, the immunomodulatory properties of the parasite extract indicate that this extract and in the future defined molecules thereof might serve as immunomodulatory adjuvants in allergy treatment and prophylaxis.

Characterization of an IgG monoclonal antibody targeted to both tissue cyst and sporocyst walls of Toxoplasma gondii.

Toxoplasma gondii infects animals habiting terrestrial and aquatic environments. Its oocysts and tissue cysts are important for the horizontal transmission of this parasite. The oocyst and tissue cyst walls are crucial for the ability of the parasite to persist in the environment or in animal tissues, respectively. However, the composition of these walls is not well understood. We report the generation of monoclonal antibodies directed against wall components using mice immunized with oocyst antigens of T. gondii. One monoclonal antibody (mAb) G1/19 reacted solely with T. gondii sporozoites. The respective antigen had a relative molecular weight (Mr) of 30 kDa. MAb G1/19 failed to react with sporozoites of any other coccidian parasite species tested (Hammondia hammondi, Hammondia heydorni, Cystoisospora felis, Eimeria bovis, Sarcocystis sp.). Another mAb, designated K8/15-15, recognized antigens in sporocyst walls of the parasite and in the walls of in vivo or in vitro produced tissue cysts, as demonstrated by immunofluorescence and immunoblot assays. Antigens of 80 to a high molecular weight protein of about 350 kDa Mr were recognized by this antibody using antigen extracts from sporocysts, and from in vitro or in vivo generated tissue cysts of the parasite. Tissue cyst and sporocyst walls of H. hammondi and H. heydorni, and tissue cysts of Neospora caninum were also recognized by mAb K8/15-15. Sporocyst walls of C. felis also reacted to this mAb. The cyst walls of Sarcocystis sp. and Besnoitia besnoiti were not recognized by mAb K8/15-15. Reactivity by a single mAb against T. gondii antigens in tissue cysts and sporocysts had not been reported previously. MAb K8/15-15 may be a practical tool for the identification of both cysts and sporocysts of the parasite, and may also be potentially employed in proteomic studies on the identification of new components of the cyst and sporocyst walls of T. gondii.

High prevalence of anti-toxoplasma antibodies and absence of Toxoplasma gondii infection risk factors among pregnant women attending routine antenatal care in two Hospitals of Addis Ababa, Ethiopia.

OBJECTIVE: Toxoplasmosis is an infection caused by the protozoan parasite Toxoplasma gondii. It is acquired mainly by eating raw or undercooked meat containing Toxoplasma gondii tissue cyst, eating food or water contaminated with oocyst, and acquiring congenital infection through the placenta. This study was conducted to determine the prevalence of toxoplasmosis and assess possible risk factors associated with the infection among pregnant women in Addis Ababa, Ethiopia. METHOD: Cross sectional study was designed, and 288 serum samples were collected from November 1(st) 2010 to January 2011. The serum samples were tested for anti- Toxoplasma gondii antibodies using latex agglutination test. The risk factors were tested for significance using Bivariate and multivariate analysis. P-value <0.05 was considered statistically significant. RESULT: 85.4% were positive for anti Toxoplasma gondii antibody. No significant association was observed between seroprevalence and age, gestational age, socio demographic characters, history of abortion, consumption of raw or undercooked meat, consumption of raw vegetable, owning of cat, and blood transfusion. CONCLUSION: Prevalence of toxoplasmosis among pregnant women in Addis Ababa, Ethiopia is higher than that reported from other countries. Efforts to describe risk factors for toxoplasma infection among Ethiopians should focus in children.

Identification and molecular characterization of microneme 5 of Eimeria acervulina.

In the present study, the microneme 5 gene of Eimeria acervulina (E. acervulina) (EaMIC5) was cloned and characterized. Specific primers for the rapid amplification of cDNA ends (RACE) were designed based on the expressed sequence tag (EST, GenBank Accession No. EH386430.1) to amplify the 3'- and 5'-ends of EaMIC5. The full length cDNA of this gene was obtained by overlapping the sequences of 3'- and 5'-extremities and amplification by reverse transcription PCR. Sequence analysis revealed that the open reading frame (ORF) of EaMIC5 was 336 bp and encoded a protein of 111 amino acids with 12.18 kDa. The ORF was inserted into pET-32a (+) to produce recombinant EaMIC5. Using western blotting assay, the recombinant protein was successfully recognized by the sera of chicks experimentally infected with E. acervulina, while the native protein in the somatic extract of sporozoites was as well detected by sera from rats immunized with the recombinant protein of EaMIC5. Immunofluorescence analysis using antibody against recombinant protein EaMIC5 indicated that this protein was expressed in the sporozoites and merozoites stages of E. acervulina. Animal challenge experiments demonstrated that the recombinant protein of EaMIC5 could significantly increase the average body weight gains, decrease the mean lesion scores and the oocyst outputs of the immunized chickens, and presented anti-coccidial index (ACI) more than 160. All the above results suggested that the EaMIC5 was a novel E. acervulina antigen and could be an effective candidate for the development of a new vaccine against this parasite.

Evaluation of an experimental irradiated oocyst vaccine to protect broiler chicks against avian coccidiosis.

The current study investigates the use of irradiated oocysts to protect broiler chicks, raised on litter, from infection with multiple species of Eimeria. In order to determine the optimum radiation dose for each Eimeria species, 1-day-old chicks were immunized with oocysts of Eimeria maxima, Eimeria acervulina, or Eimeria tenella exposed to gamma radiation ranging from 0-500 Gy. The litter oocyst counts at 7 days postimmunization, and the effect on weight gain following a challenge infection, decreased with an optimum dose between 150-200 Gy. Based on this finding, broiler chicks were immunized with a mixture of E. maxima, E. acervulina, and E tenella that had been exposed to 150 or 200 Gy. This resulted in more than a 100-fold reduction in litter oocyst counts and significant protection from a challenge infection, as measured by improved weight gain and feed conversion ratio (FCR). Immunization of birds with oocysts receiving 200 Gy was less effective in providing protection from a challenge infection. An additional formulation of vaccines containing two different oocyst doses of the three species that had been irradiated with 150 Gy were evaluated in their ability to attenuate oocyst output and convey protection to challenge. Results were similar with both high and low numbers of irradiated oocysts. Immunized chicks shed less oocysts at 7 days postimmunization and were protected from negative effects of challenge infection as measured by FCR, changes in weight gain, lesion scores, and measurement of body composition. However, the level of protection was somewhat less than that achieved by immunization with nonirradiated oocysts. The overall conclusion is that an irradiated oocyst vaccine developed in this study can effectively protect chicks that are raised on litter from challenge infection with multiple species of Eimeria, comparable to vaccines with virulent or precocious strains.

Diagnostic value of a Rec-ELISA using Toxoplasma gondii recombinant SporoSAG, BAG1, and GRA1 proteins in murine models infected orally with tissue cysts and oocysts.

Toxoplasma gondii causes congenital toxoplasmosis in newborns resulting with fetal anomalies. Determining the initiation time of infection is very important for pregnant women and current serological assays have drawbacks in distinguishing the recently acute toxoplasmosis. Diagnosis of recently acute infection may be improved by using stage specific antigens in serological assays. In the present study, the diagnostic value of sporozoite specific SporoSAG, bradyzoite specific BAG1 proteins and GRA1 protein expressed by all forms of the parasite have been evaluated ELISA using sera systematically collected from mice administered orally with tissue cyst and oocysts. The anti-SporoSAG IgM antibodies in sera obtained from mice infected with oocysts peaked significantly at days 1, 10, and 15 (P<0.01). The anti-BAG1 IgM antibodies in sera obtained from mice infected with tissue cysts peaked significantly at days 15, 40, and 120 (P<0.05). The anti-GRA1 IgM antibodies in sera obtained from mice infected with oocysts peaked significantly at days 2, 10, and 40 (P<0.01). The anti-GRA1 IgM antibodies in sera obtained from mice infected with tissue cysts peaked significantly only at day 40 (P<0.05). The anti-SporoSAG, anti-BAG1, and anti-GRA1 IgG titers of mice showed significant increases at day 40 (P<0.05) and decrement started for only anti-GRA1 IgG at day 120. The presence of anti-SporoSAG IgM and IgG antibodies can be interpreted as recently acute infection between days 10-40 because IgM decreases at day 40. Similarly, presence of anti-BAG1 IgM and absence of IgG can be evaluated as a recently acute infection that occurred 40 days before because IgG peaks at day 40. A peak in anti-GRA1 antibody level at first testing and reduction in consecutive sample can be considered as an infection approximately around day 40 or prior. Overall, recombinant SporoSAG, BAG1 and GRA1 proteins can be accepted as valuable diagnostic markers of recently acute toxoplasmosis.

An experimental Toxoplasma gondii dose response challenge model to study therapeutic or vaccine efficacy in cats.

High numbers of Toxoplasma gondii oocysts in the environment are a risk factor to humans. The environmental contamination might be reduced by vaccinating the definitive host, cats. An experimental challenge model is necessary to quantitatively assess the efficacy of a vaccine or drug treatment. Previous studies have indicated that bradyzoites are highly infectious for cats. To infect cats, tissue cysts were isolated from the brains of mice infected with oocysts of T. gondii M4 strain, and bradyzoites were released by pepsin digestion. Free bradyzoites were counted and graded doses (1000, 100, 50, 10), and 250 intact tissue cysts were inoculated orally into three cats each. Oocysts shed by these five groups of cats were collected from faeces by flotation techniques, counted microscopically and estimated by real time PCR. Additionally, the number of T. gondii in heart, tongue and brains were estimated, and serology for anti T. gondii antibodies was performed. A Beta-Poisson dose-response model was used to estimate the infectivity of single bradyzoites and linear regression was used to determine the relation between inoculated dose and numbers of oocyst shed. We found that real time PCR was more sensitive than microscopic detection of oocysts, and oocysts were detected by PCR in faeces of cats fed 10 bradyzoites but by microscopic examination. Real time PCR may only detect fragments of T. gondii DNA without the presence of oocysts in low doses. Prevalence of tissue cysts of T. gondii in tongue, heart and brains, and anti T. gondii antibody concentrations were all found to depend on the inoculated bradyzoite dose. The combination of the experimental challenge model and the dose response analysis provides a suitable reference for quantifying the potential reduction in human health risk due to a treatment of domestic cats by vaccination or by therapeutic drug application.