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

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

Cystoisospora suis merozoite development assay for screening of drug efficacy in vitro.

Cystoisospora suis is a common diarrheal pathogen of piglets and typically controlled by metaphylactic toltrazuril application. Recently, toltrazuril resistance has been reported in the field; however, both evaluation of toltrazuril efficacy against field isolates and the anticoccidial drug development for pigs is hampered by costs and labor of animal experimentation. Therefore an in vitro merozoite development assay was developed to evaluate the efficacy of compounds against C. suis in vitro. Monolayers of IPEC-1 cells were infected with sporozoites derived from oocysts of defined C. suis laboratory strains and the optimal infection dose as well as concentration, time point and duration of treatment were evaluated by quantitative real-time PCR. Cell cultures were treated with bumped kinase inhibitor (BKI) 1369 at different time points to evaluate the possibility to delineate effects on different developmental stages in vitro during invasion and early infection, and to determine different inhibitory concentrations (IC50, IC95). BKI 1369 had an IC50 of 35 nM and an IC95 of 350 nM. Dose- and duration-dependent efficacy was seen when developing stages were treated with BKI 1369 after infection (days 0-1, 2-3 and 2-5) but not when sporozoites were pre-incubated with BKI 1369 before infection. Efficacies of further BKIs were also evaluated at 200 nM. BKI 1318, 1708, 1748 and 1862 had an efficacy comparable to that of BKI 1369 (which is also effective in vivo). BKI 1862 showed a more pronounced loss of efficacy in lower concentrations than BKI 1369, signifying pharmacokinetic differences of similar compounds detectable in vitro. In addition, the effects of toltrazuril and its metabolites, toltrazuril sulfoxide and toltrazuril sulfone, on a toltrazuril sensitive and a resistant strain of C. suis were evaluated. Inhibition of merozoite growth in vitro by toltrazuril and its metabolites was dose-dependent only for toltrazuril. Clear differences were noted for the effect on a toltrazuril-sensitive vs. a resistant strain, indicating that this in vitro assay has the capacity to delineate susceptible from resistant strains in vitro. It could also be used to evaluate and compare the efficacy of novel compounds against C. suis and support the determination of the optimal time point of treatment in vivo.

Characterization of Cystoisospora suis sexual stages in vitro.

BACKGROUND: The porcine coccidium Cystoisospora suis is characterized by a complex life-cycle during which asexual multiplication is followed by sexual development with two morphologically distinct cell types, the micro- and macrogametes. Genes related to the sexual stages and cell cycle progression were previously identified in related Apicomplexa. Dynein light chain type 1 and male gamete fusion factor HAP2 are restricted to microgametes. Tyrosine-rich proteins and oocyst wall proteins are a part of the oocyst wall. The Rad51/Dmc1-like protein and Nima-related protein kinases are associated with the cell cycle and fertilization process. Here, the sexual stages of C. suis were characterized in vitro morphologically and for temporal expression changes of the mentioned genes to gain insight into this poorly known phase of coccidian development. METHODS: Sexual stages of C. suis developing in vitro in porcine intestinal epithelial cells were examined by light and electron microscopy. The transcriptional levels of genes related to merozoite multiplication and sexual development were evaluated by quantitative real-time PCR at different time points of cultivation. Transcription levels were compared for parasites in culture supernatants at 6-9 days of cultivation (doc) and intracellular parasites at 6-15 doc. RESULTS: Sexual stage of C. suis was detected during 8-11 doc in vitro. Microgamonts (16.8 ± 0.9 µm) and macrogamonts (16.6 ± 1.1 µm) are very similar in shape and size. Microgametes had a round body (3.5 ± 0.5 µm) and two flagella (11.2 ± 0.5 µm). Macrogametes were spherical with a diameter of 12.1 ± 0.5 µm. Merozoite gene transcription peaked on 10 doc and then declined. Genes related to the sexual stages and cell cycle showed an upregulation with a peak on 13 doc, after which they declined. CONCLUSIONS: The present study linked gene expression changes to the detailed morphological description of C. suis sexual development in vitro, including fertilization, meiosis and oocyst formation in this unique model for coccidian parasites. Following this process at the cellular and molecular level will elucidate details on potential bottlenecks of C. suis development (applicable for coccidian parasites in general) which could be exploited as a novel target for control.

Cystoisospora felis infection in a captive jaguar cub (Panthera onca) in Michoacán, México.

Cystoisospora felis (Wenyon 1923) was identified in a 3-month-old, captive jaguar (Panthera onca) presenting with signs of gastrointestinal distress. The cub was fed beef, chicken, and commercial diet. Examination of fresh feces detected large (47.8 µm × 35 µm) unsporulated oocysts. Sporulated oocysts contained 2 sporocysts, each with 4 sporozoites. Oocyst morphometrics agreed with published features of C. felis described from domestic felines. Partial mitochondrial cytochrome c oxidase subunit I (mtCOI) gene was PCR-amplified and sequenced; the resulting sequence showed 100% identity to a C. felis isolate from a domestic cat. This is the first molecularly confirmed report of C. felis infecting and producing clinically evident, enteric disease in a jaguar cub.

A model for chronic bovine besnoitiosis: Parasite stage and inoculation route are key factors.

In this work, an experimental model for chronic besnoitiosis in bovine was developed and characterized. Using a previously established calf model, two new variables (parasite stage and inoculation route) were combined and used. Twelve Holstein Friesian 3-month-old male calves were randomly divided into four groups of three animals each. Bradyzoites were obtained from a chronically infected bull and used for inoculation via three different inoculation routes. Three groups were inoculated with 106 bradyzoites by intravenous (G1), subcutaneous (G2) and intradermal (G3) routes, and a non-infected control group (G4) was inoculated with PBS. The trial lasted for 90 days and included daily clinical monitoring as well as weekly skin biopsies and blood sampling. Sera were obtained to analyse both cellular and humoral responses. Once the calves were euthanized, tissues from the skin, eyes, respiratory and reproductive tracts, among others, were collected to study presence of the parasite. Clinically, the infection was classified as mild to moderate for the acute stage since all infected calves showed lymphadenopathy from four days post-infection (pi) and fever from one week pi until 24 days pi. However, the most relevant results were achieved during the chronic stage that was classified as moderate to severe. In fact, pathognomonic conjunctival cysts were observed in all infected calves from 40 days pi onwards and were more abundant in G3. Moreover, one calf from this group developed skin lesions (49 days pi). The microscopic tissue cysts and Besnoitia DNA were detected primarily in skin, reproductive tract and respiratory tissue samples, and parasite load was higher in G3. In conclusion, the parasite stage (bradyzoite) and the inoculation route are key factors that influence the outcome of an infection. In particular, the intradermal route led to more severe clinical signs of the chronic phase in the inoculated calves.

Lytic cycle of Besnoitia besnoiti tachyzoites displays similar features in primary bovine endothelial cells and fibroblasts.

BACKGROUND: Bovine besnoitiosis, caused by the cyst-forming apicomplexan parasite Besnoitia besnoiti, is a chronic and debilitating cattle disease that continues to spread in Europe in the absence of control tools. In this scenario, in vitro culture systems are valuable tools to carry out drug screenings and to unravel host-parasite interactions. However, studies performed in bovine target cells are scarce. METHODS: The objective of the present study was to obtain primary bovine aortic endothelial cells (BAECs) and fibroblast cell cultures, target cells during the acute and the chronic stage of the disease, respectively, from healthy bovine donors. Afterwards, expression of surface (CD31, CD34 and CD44) and intracellular markers (vimentin and cytokeratin) was studied to characterize cell populations by flow cytometry. Next, the lytic cycle of B. besnoiti tachyzoites was studied in both target cells. Invasion rates (IRs) were determined by immunofluorescence at several time points post-infection, and proliferation kinetics were studied by quantitative PCR (qPCR). Finally, the influence of bovine viral diarrhea virus (BVDV) co-infection on the host cell machinery, and consequently on B. besnoiti invasion and proliferation, was investigated in BAECs. RESULTS: Morphology and cytometry results confirmed the endothelial and fibroblast origins. CD31 was the surface marker that best discriminated between BAECs and fibroblasts, since fibroblasts lacked CD31 labelling. Expression of CD34 was weak in low-passage BAECs and absent in high-passage BAECs and fibroblasts. Positive labelling for CD44, vimentin and cytokeratin was observed in both BAECs and fibroblasts. Regarding the lytic cycle of the parasite, although low invasion rates (approximately 3-4%) were found in both cell culture systems, more invasion was observed in BAECs at 24 and 72 hpi. The proliferation kinetics did not differ between BAECs and fibroblasts. BVDV infection favoured early Besnoitia invasion but there was no difference in tachyzoite yields observed in BVDV-BAECs compared to BAECs. CONCLUSIONS: We have generated and characterized two novel standardized in vitro models for Besnoitia besnoiti infection based on bovine primary target BAECs and fibroblasts, and have shown the relevance of BVDV coinfections, which should be considered in further studies with other cattle pathogens.

Cystoisospora belli infections in humans: the past 100 years.

Cystoisospora belli is a coccidian parasite of humans, with a direct fecal-oral transmission cycle. It is globally distributed, but mainly found in tropical and subtropical areas. Many cases of C. belli infections have been reported in patients with HIV, and in patients undergoing immunosuppressive therapy for organ transplants or those treated for tumours worldwide. Unsporulated or partially sporulated oocysts of C. belli are excreted in feces. When sporulated oocysts in contaminated water or food are ingested, asexual and sexual stages of C. belli are confined to the epithelium of intestines, bile ducts and gallbladder. Monozoic tissue cysts are present in extra-intestinal organs (lamina propria of the small and large intestine, lymph nodes, spleen, and liver) of immunosuppressed humans. However, a paratenic host has not been demonstrated. Cystoisospora belli infections can be persistent, lasting for months, and relapses are common; the mechanism of relapse is unknown. Recently, the endogenous stages of C. belli were re-examined and attention was drawn to cases of misidentification of non-protozoal structures in the gallbladder of patients as C. belli. Here, we review all aspects of the biology of C. belli, including morphology, endogenous stages, prevalence, epidemiology, symptoms, diagnosis and control.

Bumped kinase inhibitor 1369 is effective against Cystoisospora suis in vivo and in vitro.

Cystoisosporosis is a leading diarrheal disease in suckling piglets. With the confirmation of resistance against the only available drug toltrazuril, there is a substantial need for novel therapeutics to combat the infection and its negative effects on animal health. In closely related apicomplexan species, bumped kinase inhibitors (BKIs) targeting calcium-dependent protein kinase 1 (CDPK1) were shown to be effective in inhibiting host-cell invasion and parasite growth. Therefore, the gene coding for Cystoisospora suis CDPK1 (CsCDPK1) was identified and cloned to investigate activity and thermal stabilization of the recombinant CsCDPK1 enzyme by BKI 1369. In this comprehensive study, the efficacy, safety and pharmacokinetics of BKI 1369 in piglets experimentally infected with Cystoisospora suis (toltrazuril-sensitive, Wien-I and toltrazuril-resistant, Holland-I strains) were determined in vivo and in vitro using an established animal infection model and cell culture, respectively. BKI 1369 inhibited merozoite proliferation in intestinal porcine epithelial cells-1 (IPEC-1) by at least 50% at a concentration of 40 nM, and proliferation was almost completely inhibited (>95%) at 200 nM. Nonetheless, exposure of infected cultures to 200 nM BKI 1369 for five days did not induce structural alterations in surviving merozoites as confirmed by transmission electron microscopy. Five-day treatment with BKI 1369 (10 mg/kg BW twice a day) effectively suppressed oocyst excretion and diarrhea and improved body weight gains in treated piglets without obvious side effects for both toltrazuril-sensitive, Wien-I and resistant, Holland-I C. suis strains. The plasma concentration of BKI 1369 in piglets increased to 11.7 µM during treatment, suggesting constant drug accumulation and exposure of parasites to the drug. Therefore, oral applications of BKI 1369 could potentially be a therapeutic alternative against porcine cystoisosporosis. For use in pigs, future studies on BKI 1369 should be directed towards ease of drug handling and minimizing treatment frequencies.

New Observations Allowing the Differentiation of Late Asexual Stages of Cystoisospora canis from Developing Microgamonts in the Intestines of Experimentally Infected Dogs.

The coccidian parasite Cystoisospora canis (syn. Isospora canis) can cause clinical disease in dogs. Three generations of meronts are reported to occur in the small intestine of experimentally infected dogs before gametogony and oocyst formation. Oocyst excretion in the feces occurs at 9 to 11 days post-inoculation (PI). We examined the late asexual and sexual development of C. canis in 2 dogs necropsied 10 days after oral inoculation with 100,000 sporulated C. canis oocysts; both dogs had excreted oocysts 9 days PI. Asexual and sexual stages were seen in the lamina propria, throughout the small intestine in sections stained with hematoxylin and eosin from both dogs. In other studies of the C. canis life cycle, little attention has been given to distinguishing the last asexual generation of meronts and early microgamonts that can appear similar due to their stage of maturation and both having multiple nuclei. Here we report newly identified features of developing meronts and microgamonts and their distinction from each other by using sections processed using the periodic acid-Schiff (PAS) reaction. Using this method, we demonstrated that PAS-positive granules could be used to identify microgamonts and differentiate them from developing meront stages. These findings will aid pathologists and others in properly identifying coccidial parasites, in determining the cause of microscopic lesions in intestinal tissue, and in accurately identifying etiological agents.

Re-Evaluation of Asynchronous Asexual Development of Cystoisospora canis in Intestines of Dogs.

The coccidian parasite Cystoisospora canis (syn. Isospora canis) can cause clinical disease in dogs. Three generation of schizonts have been reported in the small intestine of dogs before oocysts are excreted 9-11 days post inoculation (PI). Here, we re-evaluated asexual development of C. canis in 2 dogs necropsied 10 days after oral inoculation with 100,000 C. canis oocysts; both dogs had excreted oocysts 9 days PI. Asexual and sexual stages were seen in the lamina propria throughout the small intestine. Merozoites of different sizes were present, often in the same vacuole. They were arranged singly, in pairs, and many within a single parasitophorous vacuole. The maximum number of nuclei within developing merozoites in a group was 8, but it could not be discerned if they were individual nuclei or parts of merozoites. Findings of abundant asexual stages 1 day after dogs had started excreting oocysts indicated continued asexual multiplication beyond the prepatent period. The stages found resemble the 3 generations reported previously. The mode of division of the asexual generations remains unclear. The results of the present study indicate that there are many generations that are difficult to determine because of the multiplication of merozoites in the original host cell without leaving it to enter new host cells. From the literature, it is evident that cat and dog coccidia ( Cystoisospora spp.) divide by more than 1 type of division, including endodyogeny. In the past, the schizont/meront groups containing more than 1 generation have been called "cysts." However, cyst is not an accurate term because it is best used for an orally infective stage of coccidia; monozoic tissue cysts of C. canis can occur in paratenic hosts in extraintestinal organs. We recommend the term "types" as originally proposed for intestinal stages of Toxoplasma gondii and used for the original description of the life cycle of C. suis of swine when describing endogenous stages of the Sarcocystidae. Ultrastructural studies are needed to determine the precise form of multiplication of canine Cystoisospora species.

Re-evaluation of merogony of a Cystoisospora ohioensis-like coccidian and its distinction from gametogony in the intestine of a naturally infected dog.

Four species of Cystoisospora, C. canis, C. ohioensis, C. neorivolta and C. burrowsi are described from feces of dogs. Of these, the oocysts of C. canis are the largest and easily distinguished from the remaining three species. Oocysts of C. ohioensis, C. neorivolta and C. burrowsi are difficult to distinguish because of overlap in their sizes. However, based on endogenous developmental stages, C. ohioensis is distinct from C. neorivolta and C. burrowsi because its endogenous stages are confined to surface epithelium of intestine whereas endogenous stages of C. neorivolta and C. burrowsi are predominantly in the lamina propria. There are uncertainties regarding the endogenous stages of C. neorivolta and C. burrowsi and there is no way now to determine whether C. burrowsi and C. neorivolta are different parasites; therefore, these are referred as C. ohioensis-like organisms. Additionally, mode of division of asexual stages of coccidia of dogs is largely unknown and ultrastructural studies are lacking. In the present study, development of asexual and sexual stages of a C. ohioensis-like organism in a naturally infected dog is described by light microscopy and by transmission electron microscopy. Merozoites divided by endodyogeny/merogony. Meronts were crescent/merozoite-shaped and contained a maximum of eight nuclei. A distinctive feature of merozoites was the presence of many PAS-positive amylopectin granules that were absent or rare in immature microgamonts making it possible to distinguish them.

Dissection of the in vitro developmental program of Hammondia hammondi reveals a link between stress sensitivity and life cycle flexibility in Toxoplasma gondii.

Most eukaryotic parasites are obligately heteroxenous, requiring sequential infection of different host species in order to survive. Toxoplasma gondii is a rare exception to this rule, having a uniquely facultative heteroxenous life cycle. To understand the origins of this phenomenon, we compared development and stress responses in T. gondii to those of its its obligately heteroxenous relative, Hammondia hammondi and have identified multiple H. hammondi growth states that are distinct from those in T. gondii. Of these, the most dramatic difference was that H. hammondi was refractory to stressors that robustly induce cyst formation in T. gondii, and this was reflected most dramatically in its unchanging transcriptome after stress exposure. We also found that H. hammondi could be propagated in vitro for up to 8 days post-excystation, and we exploited this to generate the first ever transgenic H. hammondi line. Overall our data show that H. hammondi zoites grow as stringently regulated, unique life stages that are distinct from T. gondii tachyzoites, and implicate stress sensitivity as a potential developmental innovation that increased the flexibility of the T. gondii life cycle.

In vitro efficacy of bumped kinase inhibitors against Besnoitia besnoiti tachyzoites.

Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a chronic and debilitating disease that causes systemic and skin manifestations and sterility in bulls. Neither treatments nor vaccines are currently available. In the search for therapeutic candidates, calcium-dependent protein kinases have arisen as promising drug targets in other apicomplexans (e.g. Neospora caninum, Toxoplasma gondii, Plasmodium spp. and Eimeria spp.) and are effectively targeted by bumped kinase inhibitors. In this study, we identified and cloned the gene coding for BbCDPK1. The impact of a library of nine bumped kinase inhibitor analogues on the activity of recombinant BbCDPK1 was assessed by luciferase assay. Afterwards, those were further screened for efficacy against Besnoitiabesnoiti tachyzoites grown in Marc-145 cells. Primary tests at 5µM revealed that eight compounds exhibited more than 90% inhibition of invasion and proliferation. The compounds BKI 1294, 1517, 1553 and 1571 were further characterised, and EC99 (1294: 2.38µM; 1517: 2.20µM; 1553: 3.34µM; 1571: 2.78µM) were determined by quantitative real-time polymerase chain reaction in 3-day proliferation assays. Exposure of infected cultures with EC99 concentrations of these drugs for up to 48h was not parasiticidal. The lack of parasiticidal action was confirmed by transmission electron microscopy, which showed that bumped kinase inhibitor treatment interfered with cell cycle regulation and non-disjunction of tachyzoites, resulting in the formation of large multi-nucleated complexes which co-existed with viable parasites within the parasitophorous vacuole. However, it is possible that, in the face of an active immune response, parasite clearance may occur. In summary, bumped kinase inhibitors may be effective drug candidates to control Besnoitiabesnoiti infection. Further in vivo experiments should be planned, as attainment and maintenance of therapeutic blood plasma levels in calves, without toxicity, has been demonstrated for BKIs 1294, 1517 and 1553.

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.

In vitro cultivation of Hammondia heydorni: Generation of tachyzoites, stage conversion into bradyzoites, and evaluation of serologic cross-reaction with Neospora caninum.

Hammondia heydorni was in vitro isolated from oocysts shed by three dogs using a finite cell line from embryonal bovine heart (KH-R). The oocysts were purified and suspended in 2% potassium dichromate or 2% sulphuric acid for sporulation for 2-5 days at room temperature. The parasites were confirmed as H. heydorni by PCR using specific primers (JS4/JS5) and by negative reaction for Neospora caninum employing the primers Np6+/Np21+. H. heydorni sporulated oocysts (1 × 10(6)) from each dog were initially treated with sodium hypochlorite. For excystation of sporozoites, oocysts from one dog were lysed by ultrasound followed by incubation with 0.75% taurocholate. Excystation of sporozoites from the other two dogs was achieved by oocyst fragmentation with glass beads with no further chemical treatment. Tachyzoites were clearly seen in the cultures at three days post inoculation (dpi). Bradyzoite conversion and cyst formation were evaluated at different time points by using a polyclonal rabbit serum against a bradyzoite-specific antigen (anti-BAG1), and a rat monoclonal antibody (mAbCC2) against a cyst wall protein. Bradyzoites were firstly detected at 7 dpi. Between 18 and 21 dpi most of cultured parasites consisted of encysted bradyzoites. The H. heydorni cysts increased in size during cultivation and reached a length of up to 135 µm. The parasite was maintained in the bovine heart cells up to 4.5months. Sera from mice and sheep experimentally infected with H. heydorni oocysts reacted with H. heydorni by IFAT, but did not cross-react with N. caninum antigens using IFAT or immunoblot. These findings suggest that serological cross-reactivity between H. heydorni and N. caninum seems to be of minor importance.

Developmental biology of Cystoisospora (Apicomplexa: Sarcocystidae) monozoic tissue cysts.

Tissue cyst stages are an intriguing aspect of the developmental cycle and transmission of species of Sarcocystidae. Tissue-cyst stages of Toxoplasma, Hammondia, Neospora, Besnoitia, and Sarcocystis contain many infectious stages (bradyzoites). The tissue cyst stage of Cystoisospora (syn. Isospora) possesses only 1 infectious stage (zoite), and is therefore referred to as a monozoic tissue cyst (MZTC). No tissue cyst stages are presently known for members of Nephroisospora. The present report examines the developmental biology of MZTC stages of Cystoisospora Frenkel, 1977 . These parasites cause intestinal coccidiosis in cats, dogs, pigs, and humans. The MZTC stages of C. belli are believed to be associated with reoccurrence of clinical disease in humans.

Hammondia hammondi, an avirulent relative of Toxoplasma gondii, has functional orthologs of known T. gondii virulence genes.

Toxoplasma gondii is a ubiquitous protozoan parasite capable of infecting all warm-blooded animals, including humans. Its closest extant relative, Hammondia hammondi, has never been found to infect humans and, in contrast to T. gondii, is highly attenuated in mice. To better understand the genetic bases for these phenotypic differences, we sequenced the genome of a H. hammondi isolate (HhCatGer041) and found the genomic synteny between H. hammondi and T. gondii to be >95%. We used this genome to determine the H. hammondi primary sequence of two major T. gondii mouse virulence genes, TgROP5 and TgROP18. When we expressed these genes in T. gondii, we found that H. hammondi orthologs of TgROP5 and TgROP18 were functional. Similar to T. gondii, the HhROP5 locus is expanded, and two distinct HhROP5 paralogs increased the virulence of a T. gondii TgROP5 knockout strain. We also identified a 107 base pair promoter region, absent only in type III TgROP18, which is necessary for TgROP18 expression. This result indicates that the ROP18 promoter was active in the most recent common ancestor of these two species and that it was subsequently inactivated in progenitors of the type III lineage. Overall, these data suggest that the virulence differences between these species are not solely due to the functionality of these key virulence factors. This study provides evidence that other mechanisms, such as differences in gene expression or the lack of currently uncharacterized virulence factors, may underlie the phenotypic differences between these species.

Identification of Besnoitia besnoiti proteins that showed differences in abundance between tachyzoite and bradyzoite stages by difference gel electrophoresis.

Bovine besnoitiosis is a chronic and debilitating disease, caused by the apicomplexan parasite Besnoitia besnoiti. Infection of cattle by B. besnoiti is governed by the tachyzoite stage, which is related to acute infection, and the bradyzoite stage gathered into macroscopic cysts located in subcutaneous tissue in the skin, mucosal membranes and sclera conjunctiva and related to persistence and chronic infection. However, the entire life cycle of this parasite and the molecular mechanisms underlying tachyzoite-to-bradyzoite conversion remain unknown. In this context, a different antigenic pattern has been observed between tachyzoite and bradyzoite extracts. Thus, to identify stage-specific proteins, a difference gel electrophoresis (DIGE) approach was used on tachyzoite and bradyzoite extracts followed by mass spectrometry (MS) analysis. A total of 130 and 132 spots were differentially expressed in bradyzoites and tachyzoites, respectively (average ratio ± 1.5, P<0.05 in t-test). Furthermore, 25 differentially expressed spots were selected and analysed by MALDI-TOF/MS. As a result, 5 up-regulated bradyzoite proteins (GAPDH, ENO1, LDH, SOD and RNA polymerase) and 5 up-regulated tachyzoite proteins (ENO2; LDH; ATP synthase; HSP70 and PDI) were identified. The present results set the basis for the identification of new proteins as drug targets. Moreover, the role of these proteins in tachyzoite-to-bradyzoite conversion and the role of the host cell environment should be a subject of further research.

First 2-DE approach towards characterising the proteome and immunome of Besnoitia besnoiti in the tachyzoite stage.

Bovine besnoitiosis is caused by the cyst-forming apicomplexan parasite Besnoitia besnoiti. It is considered to be a re-emergent disease in Europe and is also present in Africa and Asia. Due to the chronic and debilitating course of the disease, bovine besnoitiosis is responsible for severe economic losses. However, many aspects of the disease and parasite biology remain unknown. Proteomics studies could help to investigate relevant biological processes as well as host immune response associated with parasite infection. Both the proteome and immunome of the tachyzoite stage of B. besnoiti of the Bb-Spain1 isolate are described herein for the first time. Tachyzoite protein extracts were first separated by 2-DE SDS-PAGE using pH 3-10 NL IPG strips for Coomassie Brilliant Blue-stained gels and immunoblots. Eighty-five out of 265 spots visualised on Coomassie-stained gels were immunogenic when pooled serum from naturally infected cattle was used, and the distribution of immunogenic spots correlated with the 1-DE IDA pattern. Because most spots were found in the acidic range of the pH gradient, pH 3-6 L IPG strips were used next, and 58 out of 123 visualised spots proved to be immunogenic. Twenty-seven spots were identified by MALDI TOF/TOF to be 20 different proteins due to the presence of protein species. All proteins identified corresponded to highly conserved proteins among eukaryotes. Six proteins identified are related to energy metabolism, 3 are heat shock proteins, 4 proteins are related to host cell invasion processes, and 2 proteins are involved in cell redox homeostasis. A tryptophanyl tRNA synthetase, a putative gbp1p, nucleoredoxin, a putative receptor for activated C kinase, and a nuclear movement domain-containing protein were also identified. Among these proteins, fructose-1,6-bisphosphate aldolase, lactate dehydrogenase, pyruvate kinase, enolase, HSP60, HSP70, HSP90, actin and profilin proved to be immunogenic, and 5 were cross-reactive antigens between B. besnoiti and N. caninum. This first proteomic approach carried out in B. besnoiti should be followed by other studies to identify more specific parasite proteins.

Development of early tissue cysts and associated pathology of Besnoitia besnoiti in a naturally infected bull (Bos taurus) from South Africa.

Besnoitia besnoiti is an apicomplexan that causes serious economic loss in cattle in many countries and the disease is now spreading in Europe. At least 2 phases of bovine besnoitiosis are recognized clinically. An acute febrile phase characterized by anasarca and necrosis of skin is associated with multiplication of tachyzoites in vascular endothelium; this phase is short-lived and rarely diagnosed. Chronic besnoitiosis characterized by dermal lesions is associated with the presence of macroscopic tissue cysts and is easily diagnosed. Here we report the development of early B. besnoiti tissue cysts in a naturally infected Hugenoot bull from South Africa. Tissue cysts were 10-70 µm in diameter, contained 1-12 bradyzoites, and were most numerous in the dermis, testicles, and pampiniform venous plexus. Amylopectin granules in bradyzoites stained red with periodic acid Schiff (PAS) reaction. Bradyzoites varied in size and in the intensity of PAS reaction (some were PAS-negative), some were plump, and others were slender. With immunohistochemical staining with Besnoitia oryctofelisi and bradyzoite-specific antibodies (BAG-1 made against Toxoplasma gondii bradyzoites), the staining was confined to parasites, and all intracystic organisms were BAG-1 positive. With Gomori's silver stain only bradyzoites were stained very faintly whereas the rest of the tissue cyst was unstained. Ultrastructurally many tissue cysts contained dead bradyzoites and some appeared empty. Unlike bradyzoites from mature cysts, bradyzoites in the present case contained few or no micronemes. These findings are of diagnostic significance. Ultrastructually host cyst cells had features of myofibroblasts, and immunohistochemistry using antibodies against MAC387, lysozyme, vimentin, Von Willebrand factor, and smooth muscle actin confirmed this. Polymerase chain reaction on DNA extracted from lymph node of the bull confirmed B. besnoiti diagnosis. Associated clinical findings, lesions, and histopathology are briefly presented. The bull died of nephrotic syndrome; anasarca in acute besnoitiosis due to protein-losing glomerulopathy is a finding not previously reported in cattle.