Flotillin-1 localization on sporozoites oF Eimeria tenella.

In an attempt to identify parasite surface components involved in the interaction with the host cell, the present research focuses on the rafts of Eimeria tenella that might be involved in the host cell invasion process. To that end, this study was undertaken to investigate the expression of flotillin-1, which is an important component and marker of lipid rafts at the plasma membrane of sporozoites of E. tenella. The expression of this plasma membrane protein was identified by an antibody that specifically reacts with flotillin- and was studied by electron microscopy. Flotillin-1 was found to occur in patches on the surface of E. tenella sporozoites. Immunoblot analysis of the total proteins of the sporozoites showed only 1 band of approximately 48 kDa. This indicates that the antibody exclusively recognized the molecules of flotillin-1 expressed on the surface of E. tenella sporozoites. The presence of flotillin-1 on the cellular membrane of sporozoites predominantly at the apical tip suggests that flotillin-1 belongs to the invasion machinery of E. tenella.

Differences in Hsp70 expression in the sporozoites of the original strain and precocious lines of Eimeria tenella.

The levels of expression of Heat shock protein 70 (Hsp 70) in sporozoites of a wild-type parent strain and 2 precocious lines of Eimeria tenella, were compared to investigate the relationship between the heat shock proteins expressed by the parasite and virulence of the strain. Hsp70 expression was analyzed in sporozoites by immunohistochemical techniques, immunoblot, and flow cytometric analyses. One band of 70 kDa was identified and the variation of the Hsp70 expression levels was quantified by optical densitometric analyses. The results showed a significant gradual decrease in the Hsp70 expression in sporozoites of E. tenella as attenuation progressed, suggesting that the Hsp70 expressed in the excysted sporozoites of E. tenella might be involved in parasite pathogenicity. In addition, the cytoplasmic distribution of the Hsp70, which was observed in the entire sporozoites of the wild strain, was reduced to the anterior portion in the precocious lines.

Expression of anti-apoptotic factors in cells parasitized by second-generation schizonts of Eimeria tenella and Eimeria necatrix.

Intracellular infections by parasites require a functional anti-apoptotic mechanism for parasite survival within the host cell. The intracellular cycle of Eimeria tenella and Eimeria necatrix in chicken intestinal cells involves the maturation of schizonts within the epithelial cells lining the crypt lumen of the ceca (E. tenella) and jejunum (E. necatrix). After invasion, these cells detach from the epithelial layer and migrate into the underlying connective tissue, where maturation of second-generation schizonts takes place. However, the detached epithelial cells that harbour the parasite and localize in the lamina propia do not undergo apoptosis despite the fact that they are parasitized cells and are located in an inappropriate microenvironment. In this study we consider the hypothesis that E. tenella and E. necatrix may inhibit the host cell apoptosis that accompanies parasite-mediated transformation during late schizogony. To that end, the expression of both NF-kappaB, a transcriptional factor that blocks parasite-induced apoptosis, and bcl-xL, an anti-apoptotic protein induced by NF-kappaB, were studied in the host cell during the maturation of second-generation schizonts. In addition, the expression of the phosphorylated inhibitor of NF-kappaB, p-IkBalpha, was also studied to further confirm NF-kappaB activation. Immunocytochemical techniques, flow cytometric and blott analysis were applied by using polyclonal antibodies that specifically react with bcl-xL, p-IkBalpha, and NF-kappaB to detect these anti-apoptotic proteins in the parasitized cell. Our results offer evidence that both these coccidial species first induce NF-kappaB activation to protect the transformed parasitized cells from apoptosis, allowing the second-generation schizonts to mature, and later, after complete schizonts maturation, cause NF-kappaB inhibition to trigger host cell apoptosis in order to facilitate the escape of merozoites. To determine whether inhibition of the NF-kappaB pathway would induce apoptosis of the host cell, a protease inhibitor (TPCK), which induces apoptosis by mediating inhibition of IkB phosphorylation, was administered to parasitized chickens.

Prevalence and analysis of potential risk factors for Cryptosporidium parvum infection in lambs in Zaragoza (northeastern Spain).

An epidemiologic study was carried out to investigate the prevalence of and to identify factors associated with the risk of Cryptosporidium infection in sheep in Zaragoza (northeastern Spain). Faecal samples from 583 lambs aged from 1 day to 3 months and 205 ewes older than 1 year were collected at 89 farms in the two regions of the province of Zaragoza with the highest sheep population (Zaragoza and Ejea de los Caballeros). In every sheep farm, data of the factors potentially associated with the likelihood of C. parvum infection were analysed: geographical location, season, size of herd, number of lambs in the herd at sampling time, lambing period, cleaning of lambing area and presence of diarrhoeic lambs in the farm. C. parvum oocysts were identified by using the Ziehl-Neelsen technique in 344 lambs (59%) from 75 farms (84.4%). Infected lambs ranged from less than 7 days to 90 days of age, although the percentage of animals shedding oocysts peaked at 8-14 days of age (76.2%). Statistical analysis showed that infection rates were significantly higher in lambs aged between 1 and 21 days (66.4%) than in those aged between 22 and 90 days (23%) (P<0.0001, chi(2)). Analysis of correlation between excretion of oocysts and diarrhoea revealed a relationship in all age groups and the probability of presenting diarrhoea was significantly higher for lambs shedding oocysts (86.3%) than for those which did not excrete the parasite (32.2%) (P<0.0001, chi(2)). Similarly, cryptosporidial infection rates were significantly higher in diarrhoeic (79.4%) than in non-diarrhoeic lambs (22.4%). Furthermore, infection intensity was correlated with the presence of clinical symptoms. Presence of diarrhoeic lambs in the farm was the only factor significantly associated with an increased risk of infection since the percentage of herds testing positive was significantly higher in farms with diarrhoeic lambs (91.3%) than in those without cases of neonatal diarrhoea (12.5%) (P<0.0001, chi(2)). Factors associated with a decreased risk of C. parvum infection in lambs included low numbers of lambs in the farm and cleaning of the lambing area. Additionally, lambs 8-14 days of age were less likely to be infected at the first lambing period and in spring/autumn. Cryptosporidial infection was also detected in 16 ewes (7.8%) which excreted few oocysts and without diarrhoea.

Eimeria tenella: hsp70 expression during sporogony.

There is an increasing interest in identifying the parasite components involved in the maturation, development, and infectivity of intracellular protozoan parasites. In the present study, a heat shock protein (hsp) of the family of 70 kDa hsp (hsp70), which play important roles in the stage conversion and virulence of these parasites, was examined. Whereas hsp70 expression has been examined in Eimeria tenella within host tissues, in the present study, oocysts of E. tenella were used to investigate the expression of hsp70 during sporulation without interference from the host; hsp70 expression during excystation was induced by incubating sporulated oocysts under various experimental conditions to produce the stimuli necessary for sporozoites to become active and to excyst in vitro. Hsp70 was detected by immunohistochemical techniques; quantitative flow cytometric analysis was also been carried out using specific monoclonal antibodies against hsp70. Hsp70 was expressed during sporulation but was not found in sporulated oocysts after the completion of sporulation. Oocysts re-expressed hsp70 when excystation was induced. The presence of hsp70 prior to infection may preadapt the parasite for additional stress in the host and may be involved in the formation of sporozoites.

Eimeria necatrix virus: intracellular localisation of viral particles and proteins.

The presence of the Eimeria necatrix virus was investigated in the following life cycle stages: sporocysts, sporozoites, merozoites, and macrogametes. Electron microscopy revealed virus-like particles (VLPs) in sporozoites, which were purified from sporozoite extracts and used to raise polyclonal antibodies. Viral proteins were identified as RNA polymerase (95 kDa) and the major capsid protein (80 kDa). Polyclonal antibody was used to detect the intracellular localisation of VLPs and proteins. Immunoelectron microscopy and immunohistochemistry identified a viral protein of 95 kDa in all the E. necatrix stages studied, whereas the 80 kDa protein was found only in sporocysts and sporozoites. In addition, no VLPs were found in sporocysts. These results indicate that the synthesis of viral capsid proteins takes place during the early events of sporulation, and is then packaged into novel viruses during the late events. No VLPs were seen and no capsid proteins were found in the merozoites and macrogametes, whereas the 95 kDa RNA polymerase was present in both these stages. In addition, no VLPs or proteins were detected in chicken tissues.

Treatment with beta-cyclodextrin of natural Cryptosporidium parvum infections in lambs under field conditions.

Following the unexpected activity of the excipient beta-cyclodextrin against experimental infection by Cryptosporidium parvum in suckling mice, its efficacy in the prevention and treatment of natural infections in lambs was evaluated under field conditions. Fifty-three crossbred neonatal lambs were randomly selected for the study. Treatment consisted of oral administration of an aqueous suspension of beta-cyclodextrin at a dose of 500 mg/kg of body weight. To test prophylactic efficacy, the suspension was administered at 1, 2 and 3 days of age. To evaluate therapeutic efficacy, the suspension was administered on each of the 3 days following onset of diarrhoea. Infection was monitored by daily examination of faecal samples, from birth to 30 days. The criteria studied in evaluating efficacy were: oocyst shedding, the presence of diarrhoea, and weight gain at 15 and 30 days. In the group that received prophylactic treatment with beta-cyclodextrin, there were no mortalities and, compared with control lambs, there was a decrease in the number of animals infected, a longer prepatent period and notable reduction in the patent period and the duration of diarrhoea. Therapeutic treatment also reduced the patent period and the severity of diarrhoea. beta-cyclodextrin was well tolerated by all of the treated animals.

A method for the sequential study of eimerian chromosomes by light and electron microscopy.

In the present study, the authors describe a simple method to isolate chromosomes from eimerian oocysts and to submit them to sequential study by light and electron microscopy. This method includes a reliable and reproducible technique for transferring eimerian chromosomes from slides to grid that fulfills the essential requirements for generalized use in cytogenetics. In addition, this method overcomes the difficulty of the resistance of protozoan oocysts to disruption and permits the release of intact meiotic chromosomes. The observation by the authors of synaptonemal complexes in meiotic chromosomes of different Eimeria species by applying the above-mentioned method to oocysts revealed its importance to future applications.

Immunohistochemical study of the cyst of Besnoitia besnoiti.

The present study has been undertaken in order to provide information on the molecular structure of the cysts of Besnoitia besnoiti. To that end, immunohistochemical techniques have been used to investigate the expression of several enzymes and proteins implicated in the cellular membrane permeability of bradyzoites. Paraffin and frozen sections, which were obtained from subcutaneous tissue samples taken from naturally infected cattle (coming from northeast Spain), were treated with a panel of antibodies. These were specific for Na(+), K(+)-ATPase, alkaline phosphatase, calmodulin, S100 protein, heat shock proteins, hsp60, and hsp70. Positive-cysts for the said antibodies were found in 23.3% of the cows studied. Bradyzoites showed a positive immunoreaction in every positive cyst with respect to all these antibodies. In addition to the low percentage of positive animals, it is worth noting that positive and unstained cysts were observed in the same tissue section. These results suggest that bradyzoites may pass through both active and dormant metabolic phases.

Field trial on the therapeutic efficacy of paromomycin on natural Cryptosporidium parvum infections in lambs.

The objective of this study was to evaluate the therapeutic efficacy of paromomycin against cryptosporidiosis in naturally infected lambs under field conditions. The 36 cross-bred neonatal lambs, 3-10 days old, were used. On the first day that lambs showed diarrhea (Day 1) they were randomly divided into three groups. The infected control group (14 lambs) remained unmedicated whereas the two other groups were orally medicated with paromomycin solution (Humatin((R)), Parke Davis, France): 12 lambs (Group A) at 100mg/kg per day for three consecutive days (Days 1-3) and 10 lambs (Group B) at 200mg/kg per day for two days (Days 1 and 2). Drug efficacy was assessed by evaluating the presence of diarrhea, oocyst shedding and weight gains from Days 1 to 23. The results show the efficacy of paromomycin in reducing both cryptosporidial oocyst output and severity of clinical signs. On Day 4, all unmedicated lambs remained infected and excreted large numbers of cryptosporidial oocysts (mean score: 2.5) whereas oocyst output had stopped in most medicated lambs (>60%) and low numbers of oocysts were excreted in the remaining lambs (mean score: 0.45 in Group A and 1 in Group B). Mean oocyst excretion was significantly reduced in medicated lambs from Days 2 to 5 (P<0.05). Treatment also reduced, but not completely prevented, clinical symptoms although diarrhea stopped in most medicated lambs just after drug withdrawal. The mean weight gains of Group A lambs were higher than that of unmedicated lambs throughout the study and statistically significant differences were found from Days 1 to 11 (1.99+/-0.81 versus 1.47+/-0.53) (P<0.05). By contrast, the growth rate of Group B lambs from Days 11 to 23 was impaired when compared with the two other groups (P<0.05) although no significant differences were found at the end of the study (Days 1-23).

Immunohistochemical study of S100-like protein in Eimeria brunetti and Eimeria acervulina.

We have investigated the expression of a calcium-binding protein, the S100 protein, in Eimeria brunetti and Eimeria acervulina stages. For this purpose, paraffin sections of distal ileum and bursa of Fabricius or duodenum from experimentally infected chickens were treated with anti-alpha-S100 (anti-alpha subunit of S100 protein) and anti-beta-S100 (anti-beta subunit of S100 protein) monoclonal antibodies and anti-S100 whole molecule polyclonal antibody. The avidin-biotin peroxidase method was used to demonstrate immunoreactivity. In the ileum, our results reveal a positive immunoreaction for the beta subunit and S100 whole molecule within the macrogametes of E. brunetti, whereas they were devoid of immunostaining after treatment of the paraffin sections with the anti-alpha-S100 antiserum. Schizonts and oocysts of E. brunetti and all the E. acervulina stages gave a negative reaction after treatment with any of the three antiserum used in the study. This result indicated that the S100 protein molecules within these stages were not recognized by the antibodies, suggesting that these molecules are different from those identified in macrogametes of E. brunetti. By contrast, in the epithelial cells, lining the lumen of the bursa of Fabricius, macrogametes of E. brunetti were stained by the three antibodies used. These results may indicate the existence of metabolic adaptations that enable the parasite to invade tissue sites different from those where the parasite usually develops.

Immunohistochemical identification of the cells parasitized by second-generation schizonts of Eimeria tenella.

Conflicting reports exist in the literature concerning the type of cells within the lamina propria of the ceca that harbor second-generation schizonts of Eimeria tenella. Most of the previous studies concerning these cells have been performed using routine light or electron microscopy. Consequently, difficulties are evident in precise definition of the type of these cells using normal morphological criteria, since growth of the schizonts of E. tenella alters the morphology of the parasitized cell, making it difficult to recognize the cell type. This has led us to investigate the possibility of precisely identifying the subepithelial cells that are parasitized by mature schizonts. For this purpose we used cytoskeletal markers, namely, keratin and vimentin intermediate filaments, which allow the discrimination between epithelial and mesenchymal cells. Localization of keratin and vimentin on frozen cecal sections was studied immunohistochemically using specific monoclonal antibodies. Sites of antigenicity were detected by the avidin-biotin complex (ABC) immunoperoxidase technique and visualized by the deposition of diaminobenzidine. The identity of the cells was confirmed by the immunodetection of keratin intermediate filaments in the cytoplasm of the cells. Immunoreactivity for vimentin was absent in the parasitized cells. Therefore, we conclude that the development of second-generation schizonts of E. tenella takes place in epithelial cells within the lamina propria, which are presumably crypt epithelial cells that leave the crypts and enter the lamina propria after infection by first-generation merozoites.

Expression and localization of an S100 protein-like molecule in Eimeria tenella.

We investigated the expression of a calcium-binding protein, the S100 protein, in Eimeria tenella. Cecal paraffin sections from experimentally infected chickens were treated with anti-alpha-S100 (anti-alpha subunit of S100 protein) and anti-beta-S100 (anti-beta subunit of S100 protein) monoclonal antibodies and anti-S100 whole molecule polyclonal antibody. The avidin-biotin peroxidase method was used for immunocytochemical staining. Our results demonstrated a positive immunoreaction within the schizonts, macrogametes, and oocysts. These stages were all beta subunit and S100 whole-molecule positive. Immunoblot studies of the total proteins of E. tenella merozoites and sporozoites of the original strain and 2 precocious lines have demonstrated that precocious attenuation produced different S100 protein isotypes.

Identification of avian dendritic cells in the spleen using a monoclonal antibody specific for chicken follicular dendritic cells.

BACKGROUND: In the chicken, circulating antigens enter the splenic white pulp via the Schweigger-Seidel sheaths (ellipsoids), where they are bound by cells, the ellipsoid associated cells (EAC), which are located on the periphery of the ellipsoid. There is an increasing body of evidence that these antigen-binding cells move through the PALS, to be finally located within the germinal centers, where these antigen-transporting EAC function as follicular dendritic cells (FDC). The aim of the current study was to further study the relationship between the EAC, the FDC, and the antigen-bearing EAC which migrate through the splenic white pulp. METHODS: In order to identify the splenic FDC and their presumed migrating EAC precursors in the chicken, we used a monoclonal antibody produced against chicken FDC and an antiserum anti-S-100 protein which identifies chicken dendritic cells in lymphoid organs. RESULTS: Cells reacting with the 74.3 monoclonal antibody, which identifies FDC, were found within the germinal center, around the penicilliform capillary, in the periellipsoidal white pulp, and in the periarteriolar lymphatic sheaths (PALS). S-100+ cells were found in these same locations. CONCLUSIONS: A comparison between the staining patterns obtained with both antibodies strongly suggested that the intrasplenic distribution of 74.3+ cells was identical with that of FDC, EAC, and antigen-binding EAC migrating in the PALS. Therefore, the 74.3 monoclonal antibody identified not only FDC but also the splenic precursor cells of FDC, in accordance with the hypothesis of the migration of the EAC through the white pulp. S-100+ cells were more numerous than 74.3+ cells, which is in accordance with the fact that S-100 protein antibody stains both FDC and interdigitating dendritic cells (ID). This has allowed us to suggest that 74.3- EAC may represent precursors of ID. The current findings reinforce previous investigations, which provided evidence supporting the migration of EAC through the PALS and further supported the hypothesis which considers EAC precursors of FDC.

Identification of a fibronectin-like molecule on Eimeria tenella.

The attachment of Eimeria tenella to its target cells as an obligatory intracellular pathogen is essential for the development of disease. Previous reports have established that other intracellular protozoa parasites have either fibronectin, an adhesion protein, or fibronectin receptors, both of which are involved in the interaction with the host cells. In this current research, studies have been undertaken to visualize a surface component that may be involved in the attachment of E. tenella to host cells. For this purpose, monoclonal antibodies, both anti-chicken and anti-human fibronectin, and also anti-chicken integrin were used. Our results show a fibronectin-like molecule with an apparent molecular weight of 110 kDa in mature schizonts and microgametes. Staining with serum directed against chicken integrin revealed immunoreactivity within mature schizonts. Both the fibronectin-like molecule and the integrin may play an important role in the parasite stage-cell interaction and the promotion of parasite uptake.

Comparison of an acid-fast stain and a monoclonal antibody-based immunofluorescence reagent for the detection of Cryptosporidium oocysts in faecal specimens from cattle and pigs.

A commercially available direct immunofluorescence (IF) assay with monoclonal antibodies (Monofluo Kit Cryptosporidium, Diagnostics Pasteur, France) was compared with the modified Ziehl-Neelsen (MZN) acid-fast technique for the detection of Cryptosporidium oocysts in faecal samples from cattle and pigs. Stool specimens individually collected from 108 bovines and 90 pigs were examined in a blind test. The results of the two procedures corresponded (both positive or negative) in 102 (94.4%) cattle samples and 80 (88.9%) pig faecal samples. However, the remaining six (5.5%) cattle specimens and 10 (11.1%) pig stool samples, all of them harboring few oocysts (0-1 oocysts per 20 x field), were negative by MZN and positive by IF. False-negative results of the acid-fast stain occurred in suckling (17.2% of discrepant results) and weaned calves (2.9%) as well as weaned piglets (43.7%) and fattening pigs (10%). Stool specimens from the remaining age groups were negative by both techniques. The MacNemar's chi-square test showed that differences between both methods were statistically significant (P < 0.05). Compared with immunofluorescence procedure, the sensitivity of MZN technique in samples from cattle and pigs was 79.3% and 67.7% and the negative predictive value was 92.9% and 85.5% respectively. The specificity and positive predictive values of the acid-fast stain were 100% in both animal species. It is concluded that the monoclonal antibody-based immunofluorescence reagent evaluated is more efficient that the MZN technique, especially for detecting a low number of Cryptosporidium oocysts, in faecal specimens from both cattle and pigs.

Prevalence of Cryptosporidium infections in pigs in Aragón (northeastern Spain).

Faecal samples from 620 pigs randomly selected from 27 farms throughout Aragón were examined to determine the prevalence of Cryptosporidium infections. Detection of oocysts was performed using the ethyl-acetate stool concentration method and the modified Ziehl-Neelsen technique. Cryptosporidium parvum oocysts were identified in 136 (21.9%) pigs from 21 (77.8%) farms. Infected animals ranged from 1 to 6 months old and oocysts were not detected in suckling piglets or adults. Infection rates were significantly higher in weaned, 1-2 month old piglets (59.2%) than in fattening, 2-6 month old pigs (34.3%) (P < 0.001). Cryptosporidial infections were asymptomatic in most of the pigs (90.4%) and usually of low intensity, since 92.6% of the infected pigs excreted few oocysts (0-1 oocysts per field at x 200 magnifications). Although 24.1% of weaned and 5.6% of fattening pigs infected by C. parvum had diarrhoea, it was not found to be statistically associated with infection. In fact, infection rates were higher in non-diarrhoeic than in diarrhoeic pigs, in both weaned (64.7% and 46.7%, respectively) and fattening pigs (34.3% and 33.3%).

Identification of a fibronectin-like molecule on the surface of Leishmania amastigotes.

The major surface glycoprotein of Leishmania gp63, a fibronectin-like molecule, plays a key role in parasite-macrophage interaction. In this article, we describe a cross-reactivity between an anti-fibronectin monoclonal antibody and the amastigote gp63 by means of immunoelectron microscopy and immunoblot. Immunoreactivity was found on the amastigote membrane and in the flagellar pocket. We suggest that gp63 may play a role in the protection and/or nutrition process of the parasite in the phagolysosomes of the macrophage.