Identification of a host cell target for the thiazolide class of broad-spectrum anti-parasitic drugs

The thiazolide nitazoxanide (NTZ) and some derivatives exhibit considerable in vitro activities against a broad range of parasites, including the apicomplexans Neospora caninum and Toxoplasma gondii tachyzoites. In order to identify potential molecular targets for this compound in both parasites, RM4847 was coupled to epoxy-agarose and affinity chromatography was performed. A protein of approximately 35 kDa was eluted upon RM4847-affinity-chromatography from extracts of N. caninum-infected human foreskin fibroblasts (HFF) and non-infected HFF, but no protein was eluted when affinity chromatography was performed with T. gondii or N. caninum tachyzoite extracts. Mass spectrometry analysis identified the 35 kDa protein as human quinone reductase NQO1 (P15559; QR). Within 8h after infection of HFF with N. caninum tachyzoites, QR transcript expression levels were notably increased, but no such increase was observed upon infection with T. gondii tachyzoites. Treatment of non-infected HFF with RM4847 did also lead to an increase of QR transcript levels. The enzymatic activity of 6-histidine-tagged recombinant QR (recQR) was assayed using menadione as a substrate. The thiazolides NTZ, tizoxanide and RM4847 inhibited recQR activity on menadione in a concentration-dependent manner. Moreover, a small residual reducing activity was observed when these thiazolides were offered as substrates.

Molecular characterization of Neospora caninum MAG1, a dense granule protein secreted into the parasitophorous vacuole, and associated with the cyst wall and the cyst matrix

SUMMARY: In Neospora caninum and Toxoplasma gondii, the parasitophorous vacuole (PV) is synthesized at the time of infection. During tachyzoite-to-bradyzoite stage conversion, the PV is later transformed into a tissue cyst that allows parasites to survive in their host for extended periods of time. We report on the characterization of NcMAG1, the N. caninum orthologue of T. gondii MAG1 (matrix antigen 1; TgMAG1). The 456 amino acid predicted NcMAG1 protein is 54% identical to TgMAG1. By immunoblotting, a rabbit antiserum raised against recombinant NcMAG1 detected a major product of approximately 67 kDa in extracts of N. caninum tachyzoite-infected Vero cells, which was stained more prominently in extracts of infected Vero cells treated to induce in vitro bradyzoite conversion. Immunofluorescence and TEM localized the protein mainly within the cyst wall and the cyst matrix. In both tachyzoites and bradyzoites, NcMAG1 was associated with the parasite dense granules. Comparison between NcMAG1 and TgMAG1 amino acid sequences revealed that the C-terminal conserved regions exhibit 66% identity, while the N-terminal variable regions exhibit only 32% identity. Antibodies against NcMAG1-conserved region cross-reacted with the orthologuous protein in T. gondii but those against the variable region did not. This indicates that the variable region possesses unique antigenic characteristics.

Neospora caninum immunoblotting improves serodiagnosis of bovine neosporosis

Neospora caninum ranges among the major causes of infectious abortion in cattle worldwide. The present study was designed to improve the serodiagnostic tools by complementing a conventional ELISA with a highly sensitive and species-specific N. caninum immunoblot. To evaluate this test combination, sera from several groups of cows were tested. The first group, consisting of experimentally infected calves, showed that immunoblot antibody reactivities were detectable 1 to 3 days earlier than those found in ELISA. The first immunodominant bands that appeared were a 29-kDa (NcSAG1) and a 36-kDa (NcSRS2) antigen. Other groups, based upon naturally infected cattle, were used to compare the diagnostic sensitivity of ELISA and immunoblotting. Overall, N. caninum immunoblotting exhibited a higher sensitivity (98%) than ELISA (87%). Conversely, immunoblotting also confirm in two other cases, true transient negativation in some animals. In general, banding patterns and band staining intensity correlated to the semiquantitative ELISA findings. On the other hand, the banding pattern could not be used to discriminate between sera from animals with a recent abortion and those of cows with latent N. caninum infection. We also addressed putative cross-reactions due to infection with Toxoplasma gondii. Sera from animals with a serologically proven T. gondii infection were either clearly negative by Neospora immunoblotting or they yielded a specific immunoblot antibody profile indicating a double infection with N. caninum. Sera from animals with positive findings in both Toxoplasma and Neospora ELISA thus provided dichotomic results in the immunoblot by allowing to confirm or to rule out the specificity of the antibody reaction in Neospora ELISA. Altogether, our findings demonstrate that N. caninum immunoblotting is a very sensitive and specific complementary tool to improve the serology for N. caninum infections in cattle.

Incidence of Neospora caninum and other intestinal protozoan parasites in populations of Swiss dogs

The protozoan parasite Neospora caninum is one of the most important abortifacient organisms in cattle worldwide. The dog is known to act as definitive host although its potential role as infection source for bovines still remains unelucidated. The aim of the present study was to compile initial epidemiological data on the prevalence and incidence of N. caninum in Swiss dogs acting as definitive hosts. Thus, 249 Swiss dogs were investigated coproscopically in monthly intervals over a period of 1 year. A total of 3289 fecal samples was tested by the flotation technique. Among these, 202 were shown to contain Sarcocystis sp. (6.1%), 149 Cystoisospora sp. (=Isospora sp.; 4.5%) and 25 Hammondia/Neospora-like oocysts (HNlO) (0.7%). All but one sample containing HNlO were from different dogs; one dog shed HNlO at two subsequent time points. Calculation of the yearly incidence for HNlO resulted in the surprisingly high value of 9.2%. Farm dogs exhibited a higher incidence for HNlO than urban family dogs. Thirteen out of the 25 HNlO-samples showed sporulation after 5 days incubation at room temperature. HNlO were further differentiated by species-specific PCR. However, all HNlO-samples were negative for N. caninum, Hammondia heydorni and Toxoplasma gondii. One reason may be the low oocyst density found in most fecal samples, which did not permit us to carry out PCR under optimal conditions. Three out of the 25 HNlO-cases contained enough oocysts to allow further enrichment and purification by the flotation technique. Subsequently, twenty to fifty sporulated HNlO-oocysts were orally administered to Meriones unguiculatus. All gerbils were seronegative for N. caninum at 5 weeks p.i. A N. caninum-seroprevalence of 7.8% was determined by ELISA upon 1132 serum samples collected from dogs randomly selected by veterinarians among their clinical patients.

The antibody response in experimental ocular toxoplasmosis

BACKGROUND: The dynamics of the humoral immune response in ocular toxoplasmosis (OT) are poorly understood. We therefore investigated this process in a rabbit model of the disease. MATERIALS AND METHODS: Of 24 infection-naïve adult rabbits, 12 were left untreated and 12 were systematically infected with 5,000 tachyzoites of the non-cyst-forming BK strain of Toxoplasma gondii. Three months later, all rabbits were inoculated transvitreally with 5,000 tachyzoites of Toxoplasma gondii. Paired samples of aqueous humor and serum were analyzed temporally for their total and specific IgG contents. RESULTS: In infection-naïve rabbits with primary OT, specific IgG reached detectable levels in the inoculated eyes between 5 and 15 days after inoculation. In infection-immunized rabbits with secondary OT, a significant increase in specific IgG was regularly detected after 5 days. The antibody ratio C was diagnostic (>/=3) from day 15 onward in primary OT and from day 21 onward in secondary OT. In the uninfected partner eyes, the antibody ratio C was found sporadically diagnostic from day 15 onward in primary OT, but at no time in secondary OT. Specific IgG persisted both locally and in the serum until the end of the monitoring period (100 days). CONCLUSION: Our findings relating to the rabbit model of OT reveal three features of clinical relevance: a diagnostic window precedes the establishment of a humoral immune response; specific antibodies persist long after the cessation of disease activity; and in primary OT, the antibody ratio C may also increase in the uninfected partner eye.

Besnoitia besnoiti lytic cycle in vitro and differences in invasion and intracellular proliferation among isolates

Background Bovine besnoitiosis, caused by the protozoan Besnoitia besnoiti, reduces productivity and fertility of affected herds. Besnoitiosis continues to expand in Europe and no effective control tools are currently available. Experimental models are urgently needed. Herein, we describe for the first time the kinetics of standardised in vitro models for the B. besnoiti lytic cycle. This will aid to study the pathogenesis of the disease, in the screening for vaccine targets and drugs potentially useful for the treatment of besnoitiosis. Methods We compared invasion and proliferation of one B. tarandi (from Finland) and seven B. besnoiti isolates (Bb-Spain1, Bb-Spain2, Bb-Israel, Bb-Evora03, Bb-Ger1, Bb-France, Bb-Italy2) in MARC-145 cell culture. Host cell invasion was studied at 4, 6, 8 and 24 h post infection (hpi), and proliferation characteristics were compared at 24, 48, 72, 96, 120, and 144 hpi. Results In Besnoitia spp., the key parameters that determine the sequential adhesion-invasion, proliferation and egress steps are clearly distinct from those in the related apicomplexans Toxoplasma gondii and Neospora caninum. Besnoitia spp. host cell invasion is a rather slow process, since only 50 % of parasites were found intracellular after 3–6 h of exposure to host cells, and invasion still took place after 24 h. Invasion efficacy was significantly higher for Bb-France, Bb-Evora03 and Bb-Israel. In addition, the time span for endodyogeny to take place was as long as 18–35 h. Bb-Israel and B. tarandi isolates were most prolific, as determined by the tachyzoite yield at 72 hpi. The total tachyzoite yield could not be predicted neither by invasion-related parameters (velocity and half time invasion) nor by proliferation parameters (lag phase and doubling time (dT)). The lytic cycle of Besnoitia was asynchronous as evidenced by the presence of three different plaque-forming tachyzoite categories (lysis plaques, large and small parasitophorous vacuoles). Conclusions This study provides first insights into the lytic cycle of B. besnoiti isolates and a standardised in vitro model that allows screening of drug candidates for the treatment of besnoitiosis.

Inhibition of apoptosis by intracellular protozoan parasites

Protozoan parasites which reside inside a host cell avoid direct destruction by the immune system of the host. The infected cell, however, still has the capacity to counteract the invasive pathogen by initiating its own death, a process which is called programmed cell death or apoptosis. Apoptotic cells are recognised and phagocytosed by macrophages and the parasite is potentially eliminated together with the infected cell. This potent defence mechanism of the host cell puts strong selective pressure on the parasites which have, in turn, evolved strategies to modulate the apoptotic program of the host cell to their favour. Within the last decade, the existence of cellular signalling pathways which inhibit the apoptotic machinery has been demonstrated. It is not surprising that intracellular pathogens subvert these pathways to ensure their own survival in the infected cell. Molecular mechanisms which interfere with apoptotic pathways have been studied extensively for viruses and parasitic bacteria, but protozoan parasites have come into focus only recently. Intracellular protozoan parasites which have been reported to inhibit the apoptotic program of the host cell, are Toxoplasma gondii, Trypanosoma cruzi, Leishmania sp., Theileria sp., Cryptosporidium parvum, and the microsporidian Nosema algerae. Although these parasites differ in their mechanism of host cell entry and in their final intracellular localisation, they might activate similar pathways in their host cells to inhibit apoptosis. In this respect, two families of molecules, which are known for their capacity to interrupt the apoptotic program, are currently discussed in the literature. First, the expression of heat shock proteins is often induced upon parasite infection and can directly interfere with molecules of the cellular death machinery. Secondly, a more indirect effect is attributed to the parasite-dependent activation of NF-kappaB, a transcription factor that regulates the transcription of anti-apoptotic molecules.

Seroprevalence of toxoplasmosis and current thinking regarding screening in pregnancy: A review of the literature and case study discussion

The approach to the diagnosis and treatment of congenital toxoplasmosis has been one of flux and debate, fueled by lack of knowledge, lack of consensus, different methods of screening and different national policies for screening in different parts of the world. Countries with higher prevalence of disease such as in Europe and South America have a heightened awareness of the need to screen and treat for this parasitic infection during pregnancy. In contrast, in the United States, it is a condition scarcely discussed and has been largely ignored except in some large centers and by a few researchers. Policies and research strategies for any condition should start with obtaining good data. The aims of this thesis included a review of prevalence studies conducted in the United States, focused on the past 20 years, combined with a description of original research conducted by the author several years ago. The latter was a cross-sectional study performed in Houston, one of the largest American cities with a great ethnic mix. The study analyzed prevalence rates of Toxoplasma gondii IgG antibody in sera of women of reproductive age. Overall seroprevalence was 12.3%. In keeping with other studies, higher prevalence correlated with lower socioeconomic status, Black and Hispanic and Asian ethnicities, and increasing age. A literature search revealed only three prevalence studies performed in the United States over the past 20 years, with another four studies only referred to as personal communications or within a textbook, without further study detail available. The literature review also revealed a lack of consensus on whether or not to screen for toxoplasmosis in pregnancy, and even whether or not treatment in utero is worthwhile.^ Proponents of screening and treatment in pregnancy site studies both in the United States and France, emphasize that treatment reduces disease manifestations in infants. Opponents cite other studies that show only marginal benefits, together with potential side effects of medication regimens and generation of anxiety in parents. What is agreed on so far is the value of educating pregnant women on how to avoid contracting toxoplasmosis, and educating physicians on making the best use of reference laboratories before major treatment decisions are made. Further research to reevaluate the literature critically, review new treatment regimens and examine costs and benefits of screening and treatment of toxoplasmosis in pregnancy, bringing together European and American researchers, is needed.^

Altered lymphocyte responses and cytokine production in mice deficient in the X-linked lymphoproliferative disease gene SH2D1A/DSHP/SAP

We have introduced a targeted mutation in SH2D1A/DSHP/SAP, the gene responsible for the human genetic disorder X-linked lymphoproliferative disease (XLP). SLAM-associated protein (SAP)-deficient mice had normal lymphocyte development, but on challenge with infectious agents, recapitulated features of XLP. Infection of SAP− mice with lymphocyte choriomeningitis virus (LCMV) or Toxoplasma gondii was associated with increased T cell activation and IFN-γ production, as well as a reduction of Ig-secreting cells. Anti-CD3-stimulated splenocytes from uninfected SAP− mice produced increased IFN-γ and decreased IL-4, findings supported by decreased serum IgE levels in vivo. The Th1 skewing of these animals suggests that cytokine misregulation may contribute to phenotypes associated with mutation of SH2D1A/SAP.

Toxoplasma invasion: the parasitophorous vacuole is formed from host cell plasma membrane and pinches off via a fission pore.

Most intracellular pathogens avoid lysing their host cells during invasion by wrapping themselves in a vacuolar membrane. This parasitophorous vacuole membrane (PVM) is often retained, serving as a critical transport interface between the parasite and the host cell cytoplasm. To test whether the PVM formed by the parasite Toxoplasma gondii is derived from host cell membrane or from lipids secreted by the parasite, we used time-resolved capacitance measurements and video microscopy to assay host cell surface area during invasion. We observed no significant change in host cell surface area during PVM formation, demonstrating that the PVM consists primarily of invaginated host cell membrane. Pinching off of the PVM from the host cell membrane occurred after an unexpected delay (34-305 sec) and was seen as a 0.219 +/- 0.006 pF drop in capacitance, which corresponds well to the predicted surface area of the entire PVM (30-33 microns2). The formation and closure of a fission pore connecting the extracellular medium and the vacuolar space was detected as the PVM pinched off. This final stage of parasite entry was accomplished without any breach in cell membrane integrity.

Parasitos de importância para a caprinocultura e a ovinocultura potiguar

The goat and sheep industry shows up as an agricultural activity of great importance for the semiarid Northeast. However, the sheep and goats production is made with various difficulties. Among them, parasitic infections, particularly helminth infections of the gastrointestinal tract, the eimeriosis and toxoplasmosis; this one related to problems in reproduction. For this reason, the aim of this study is to to make a survey of the occurrence and some determinants of parasitic diseases that affect small ruminant flocks of the microregions Natal, Macaíba, Litoral Sul, Angicos, Vale do Açu and Borborema Potiguar. Thereunto, epidemiological tools were applied with producers, keepers or guardians of herds and also held collections of blood and feces of animals in eight properties located in seven municipalities of these microregions. The parasite load of the animals was determined through eggs and oocysts counting per gram of feces EPG and OPG, respectively. In addition, the recovery of infective larvae was made. Blood samples were used to measure the globular cell volume and the search for anti-Toxoplasma gondii IgG in sheep serum, by Enzyme Linked Immune Sorbent Assay (ELISA). For categorical variables, the statistical analysis was performed using Poisson regression, with significance level of 0.05. The analysis of the instruments showed that ivermectin is the anthelmintic used in 85,71% of properties. From the total of feces samples of the sheep (n = 179), 53,07% were positive for helminth eggs and 48,04% were positive for oocysts of Eimeria. From the samples of faeces of goats (n = 133), 72,18% were positive for helminth eggs and 96,99% for oocysts of Eimeria. The lowest EPG and OPG count was observed in the micro region of Angicos. Most of the EPG count was found in the micro region Litoral Sul and the OPG count in the micro-region Borborema Potiguar. Both cases the difference was statistically significant(p- value0,000)The most prevalent helminth genus found was Haemonchus, present in 49,87% of the sheep and 80,42% of goats. The average of hematocrit ranged from 22,91 to 33,25 in sheep and from 22,62 to 28,25 in goats. The prevalence of anti-Toxoplasma gondii IgG ranged from 63,33% to 100,00%. The goats showed to be more susceptible to infections by parasites of the gastrointestinal tract than the sheep. In all the properties was observed high prevalence of infection by T. gondii, with the lowest percentages recorded in the micro regions Angicos and Borborema Potiguar.

Manipulação gênica do protozoário Neospora caninum como ferramenta para o estudo das interações parasito-hospedeiro

Neospora caninum is an obligate intracellular parasite classified in the phylum Apicomplexa, characterized by the presence of the apical complex composed by micronemes proteins, rhoptries and dense granules, used by parasite during the adhesion and invasion process of the host cell. This is the mean event in infection pathogenesis generated by N. caninum and other parasites from the phylum Apicomplexa, promoting influence in the parasite biology and the interface between the parasite and its host. Therefore, molecular tools have been developed in order to identify and characterize these possible virulence factors. Thus, the present study sought to establish a specific system of genetic manipulation of N. caninum, searching for the improvement of the genetics manipulation of this parasite. So, we developed genetically depleted N. caninum to Rop9 rhoptry using the pU6-Universal CRISPR-Cas9 plasmid of T. gondii modified by the insertion of Ku80. The Rop9 depleted parasite showed important during initial phase of invasion and replication of the parasite, however it was not characterized as a potential virulence fator for N. caninum. Furthermore, T. gondii proteins were expressed in N. caninum by the use of specific vectors for this parasite, showing an heterologous system for the study of Toxoplasma proteins, due to the fact that Gra15 or Gra24 of type II T. gondii and Rop16 of type I T. gondii were expressed in N. caninum tachyzoites in a stable way and keept its biological phenotype, as already presented the former parasite, that naturaly expresses these proteins. In addition, it was observed that N. caninum induced an inflammasome activation through NLRP3, ASC and Caspase-1. IL-1R/MyD88 demonstrated an indirect pathway in the control of parasite replication. Furthermore, it was observed that this activation is dependent of the potassium efflux and that different strains of N. caninum keep this activation profile. However, T. gondii strains block this activation, making necessary a prior signal in order to active the inflamosome pathway. Type I T. gondii Rop16 was identified as responsible for blocking this activation, in a dependent way to the STAT3 activation. Therefore, the development of molecular tools and their application in N. caninum may prove to be useful to identify and characterize virulent factors involved in the pathogenesis by these two protozoans.

Occurrence of Neospora caninum antibodies in capybaras (Hydrochaeris hydrochaeris) from Sao Paulo State, Brazil

Capybara (Hydrochaeris hydrochaeris) is a large rodent distributed throughout tropical America. Antibodies to Neospora caninum in 213 feral capybaras from 11 counties of the State of Sao Paulo, Brazil. were assessed using the indirect immunofluorescent antibody test (titer >= 1:25) and found in 20 (9.4%), with titers of 1:25 in 4, 1:50 in 7, and 1:100 in 9 animals. This is the first report of occurrence of N. caninum antibodies in capybaras.

VIABILITY OF SPORULATED OOCYSTS OF NEOSPORA CANINUM AFTER EXPOSURE TO DIFFERENT PHYSICAL AND CHEMICAL TREATMENTS

The aim of the present study was to evaluate the viability of Neospora caninum sporulated oocysts after various chemical and physical treatments. Bioassays in gerbils and molecular techniques (PCR-RFLP) were used for identification of the oocysts shed by experimentally infected dogs. Sporulated oocysts were purified and divided into 11 treatment groups as follows: absolute ethanol for 1 hr; 20 C for 6 hr; 4 C for 6 hr; 60 C for 1 min; 100 C for 1 min; 10% formaldehyde for 1 hr; 10% ammonia for 1 hr; 2% iodine for 1 hr; 10% sodium hypochlorite for I hr; 70% ethanol for I hr; and one group was left untreated and kept as a positive control. All chemical treatments were performed at room temperature (37 C). A total of 33 gerbils, or 3 gerbils per treatment, were used for bioassays. After treatment, the oocysts were divided into aliquots of 1,000 oocysts and orally administered to gerbils. After 63 days, the gerbils were anesthetized and killed with 0.2 ml of T61; blood and tissue samples were collected for serological (IFAT and western blotting), molecular (real-time PCR), histopathology, and immunohistochemical tests. Treatments were considered effective only if all 5 detection techniques tested negative. High temperatures at 100 C for 1 min and 10% sodium hypochlorite for 1 hr were the only treatments that met this condition, effectively inactivating all oocysts.

Placentation in Sigmodontinae: a rodent taxon native to South America

Background: Sigmodontinae, known as ""New World rats and mice,"" is a large subfamily of Cricetidae for which we herein provide the first comprehensive investigation of the placenta. Methods: Placentas of various gestational ages ranging from early pregnancy to near term were obtained for five genera, i.e. Necromys, Euryoryzomys, Cerradomys, Hylaeamys, and Oligoryzomys. They were investigated by means of histology, immunohistochemistry, a proliferation marker, DBA-lectin staining and transmission electron microscopy. Results: The chorioallantoic placenta was organized in a labyrinthine zone, spongy zone and decidua and an inverted yolk sac persisted until term. The chorioallantoic placenta was hemotrichorial. The interhemal barrier comprised fetal capillary endothelium and three layers of trophoblast, an outermost, cellular layer and two syncytial ones, with interspersed trophoblast giant cells (TGC). In addition, accumulations of TGC occurred below Reichert's membrane. The junctional zone contained syncytial trophoblast, proliferative cellular trophoblast, glycogen cells and TGC that were situated near to the maternal blood channels. In three of the genera, TGC were also accumulated in distinct areas at the placental periphery. PAS-positive glycogen cells derived from the junctional zone invaded the decidua. Abundant maternal uNK cells with positive response to PAS, vimentin and DBA-lectin were found in the decidua. The visceral yolk sac was completely inverted and villous. Conclusion: The general aspect of the fetal membranes in Sigmodontinae resembled that found in other cricetid rodents. Compared to murid rodents there were larger numbers of giant cells and in some genera these were seen to congregate at the periphery of the placental disk. Glycogen cells were found to invade the decidua but we did not identify trophoblast in the walls of the deeper decidual arteries. In contrast these vessels were surrounded by large numbers of uNK cells. This survey of wild-trapped specimens from five genera is a useful starting point for the study of placentation in an important subfamily of South American rodents. We note, however, that some of these rodents can be captive bred and recommend that future studies focus on the study of time dated pregnancies.