Structural and Biochemical Features of Eimeria tenella Dihydroorotate Dehydrogenase, a Potential Drug Target.

Dihydroorotate dehydrogenase (DHODH) is a mitochondrial monotopic membrane protein that plays an essential role in the pyrimidine de novo biosynthesis and electron transport chain pathways. In Eimeria tenella, an intracellular apicomplexan parasite that causes the most severe form of chicken coccidiosis, the activity of pyrimidine salvage pathway at the intracellular stage is negligible and it relies on the pyrimidine de novo biosynthesis pathway. Therefore, the enzymes of the de novo pathway are considered potential drug target candidates for the design of compounds with activity against this parasite. Although, DHODHs from E. tenella (EtDHODH), Plasmodium falciparum (PfDHODH), and human (HsDHODH) show distinct sensitivities to classical DHODH inhibitors, in this paper, we identify ferulenol as a potent inhibitor of both EtDHODH and HsDHODH. Additionally, we report the crystal structures of EtDHODH and HsDHODH in the absence and presence of ferulenol. Comparison of these enzymes showed that despite similar overall structures, the EtDHODH has a long insertion in the N-terminal helix region that assumes a disordered configuration. In addition, the crystal structures revealed that the ferulenol binding pocket of EtDHODH is larger than that of HsDHODH. These differences can be explored to accelerate structure-based design of inhibitors specifically targeting EtDHODH.

Experimental infection by Neospora caninum in gerbil reduces activity of enzymes involved in energy metabolism.

Neospora caninum is a protozoan that has tropism for the central nervous system. The aim of this study was to determine whether experimental infection of gerbils would interfere with activity of enzymes associated with energy metabolism. We randomized 20 gerbils into two groups (ten animals per group): the control group (healthy animals; uninfected) and the infected group (experimentally infected with dose 7.8 × 102 tachyzoites of N. caninum per gerbil). On day six and twelve post-infection (PI), brain and spleen tissues were collected for biochemical and histopathological analyses. No histopathological lesions were observed in the brains of infected animals; however, inflammatory infiltrates were found in the spleen. Significantly greater levels of reactive oxygen species (ROS) were observed in the brain and spleen of infected gerbils than in the control group at 12 days PI. Cytosolic creatine kinase (CK-CYT), mitochondrial creatine kinase (CK-MIT), and pyruvate kinase (PK) activities were lower in the brains of infected gerbils than in those of the control group on day 12 PI. There was significantly less CK-CYT activity in the spleens of infected gerbils on day 6 and 12 PI. Finally, there was significantly less sodium-potassium ion pump (Na+/K+ ATPase) activity in the brains and spleens of infected gerbils on day 12 PI. These data suggest that experimental infection with N. caninum interfered with energy metabolism associated with ATP homeostasis in the brain and spleen, directly or indirectly, apparently mediated by ROS overproduction, contributing to inhibition of Na+/K+ ATPase activity.

Inducible Nitric Oxide Synthase is required for parasite restriction and inflammatory modulation during Neospora caninum infection.

Neospora caninum infection is an important cause of neuromuscular disease in dogs and abortion in cattle, leading to significant economic losses in beef and dairy industries. The protective immunity against apicomplexan parasites, specifically Toxoplasma gondii and N. caninum, is typically achieved by inducing an IL-12-driven Th1 immune response. IL-12 stimulates IFN-γ production, which activates Inducible Nitric Oxide Synthase (iNOS) and promotes consequent Nitric Oxide (NO) synthesis, classically described as one of the main effector mechanisms for parasite elimination. Here, we aimed to evaluate the role played by iNOS during N. caninum infection. Our results show that N. caninum infection in C57BL/6 wild type (WT) mice induce NO production in vivo and in vitro. In agreement, iNOS deficient mice, as well as WT mice treated with iNOS inhibitor aminoguanidine, succumbed during acute infection with a dose lethal to 50 % of the WT mice, and presented significant increase in parasite load when submitted to sub-lethal infection protocols. Interestingly, the lack of control of parasite proliferation observed in iNOS-/- mice was associated with notable CNS inflammation and increased production of the main systemic proinflammatory cytokines (IL-12, IFN-γ, IL-6, TNF and IL-17A). Taken together, our findings show that iNOS plays an important role in restricting N. caninum replication, while also modulates the inflammatory process induced by the infection.

Modulation of cis- and trans- Golgi and the Rab9A-GTPase during infection by Besnoitia besnoiti, Toxoplasma gondii and Neospora caninum.

Like most intracellular pathogens, the apicomplexan parasites Besnoitia besnoiti, Toxoplasma gondii and Neospora caninum scavenge metabolites from their host cells. Recruitment of the Golgi complex to the vicinity of the parasitophorous vacuole (PV) is likely to aid in this process. In this work, we comparatively assessed B. besnoiti, T. gondii and N. caninum infected human retinal pigmented epithelial (hTERT-RPE-1) cells at 24 h post-infection and used antibodies to confirm Golgi ribbon compaction in B. besnoiti, and Golgi ribbon dispersion in T. gondii, while no alteration in Golgi morphology was seen in N. caninum infected cells. In either case, the Golgi stacks of infected cells contained both cis- (GM130) and trans- (TGN46) Golgi proteins. The localization of Rab9A, an important regulator of endosomal trafficking, was also studied. GFP-tagged Rab9A was recruited to the vicinity of the PV of all three parasites. Toxoplasma-infected cells exhibited increased expression of Rab9A in comparison to non-infected cells. However, Rab9A expression levels remained unaltered upon infection with N. caninum and B. besnoiti tachyzoites. In contrast to Rab9A, a GFP-tagged dominant negative mutant form of Rab9A (Rab9A DN), was not recruited to the PV, and the expression of Rab9A DN did not affect host cell invasion nor replication by all three parasites. Thus, B. besnoiti, T. gondii and N. caninum show similarities but also differences in how they affect constituents of the endosomal/secretory pathways.

Expression of digestive enzymes and nutrient transporters in Eimeria-challenged broilers.

Avian coccidiosis is a disease caused by the intestinal protozoa Eimeria. The site of invasion and lesions in the intestine is species-specific, for example E. acervulina affects the duodenum, E. maxima the jejunum, and E. tenella the ceca. Lesions in the intestinal mucosa cause reduced feed efficiency and body weight gain. The growth reduction may be due to changes in expression of digestive enzymes and nutrient transporters in the intestine. The objective of this study was to compare the expression of digestive enzymes, nutrient transporters and an antimicrobial peptide in broilers challenged with either E. acervulina, E. maxima or E. tenella. The genes examined included digestive enzymes (APN and SI), peptide and amino acid transporters (PepT1, ASCT1, b(0,+)AT/rBAT, B(0)AT, CAT1, CAT2, EAAT3, LAT1, y(+)LAT1 and y(+)LAT2), sugar transporters (GLUT1, GLUT2, GLUT5 and SGLT1), zinc transporter (ZnT1) and an antimicrobial peptide (LEAP2). Duodenum, jejunum, ileum and ceca were collected 7 days post challenge. E. acervulina challenge resulted in downregulation of various nutrient transporters or LEAP2 in the duodenum and ceca, but not the jejunum or ileum. E. maxima challenge produced both downregulation and upregulation of nutrient transporters and LEAP2 in all three segments of the small intestine and ceca. E. tenella challenge resulted in the downregulation and upregulation of nutrient transporters and LEAP2 in the jejunum, ileum and ceca, but not the duodenum. At the respective target tissue, E. acervulina, E. maxima and E. tenella infection caused common downregulation of APN, b(0,+)AT, rBAT, EAAT3, SI, GLUT2, GLUT5, ZnT1 and LEAP2. The downregulation of nutrient transporters would result in a decrease in the efficiency of protein and polysaccharide digestion and uptake, which may partially explain the weight loss. The downregulation of nutrient transporters may also be a cellular response to reduced expression of the host defense protein LEAP2, which would diminish intracellular pools of nutrients and inhibit pathogen replication.

Neospora caninum: Activity of cholinesterases during the acute and chronic phases of an experimental infection in gerbils.

Neosporosis is an infectious disease primarily of dogs and cattle which has been found in many countries around the world. Neospora caninum causes an important immune response (cellular and humoral) in animals that it infects. Since the participation of the cholinergic system in the immune response is well documented, the aim of this study was to evaluate the relationship between N. caninum infection and activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) during the acute and chronic phase of infection. For that, tachyzoites of N. caninum (Nc-1 strain) were inoculated intraperitoneally in gerbils (Meriones unguiculatus), which were separated in two experiments, I and II, differing in infective doses of tachyzoites, aiming to reach an acute phase as well as chronic phase, respectively. Samples were collected on day 7 post infection (PI) for Experiment I and on days 15 and 30 PI for Experiment II. AChE activity was evaluated on whole blood and brain, while BChE was evaluated in plasma. On day 7 a reduction of AChE in total blood and brain was observed, along with reduction of BChE in plasma of infected animals when compared with non-infected. In Experiment II, AChE activity increased in total blood on day 30 PI; however, maintaining, during the same period, the AChE activity has a reduced in brain tissue. BChE activity was significantly increased on day 30 PI. Based on the results obtained, it was possible to observe a response of the cholinergic system, providing different grades of AChE and BChE activities, in response to the acute and chronic infection of gerbils experimentally infected with N. caninum. These results will serve as initial points to further studies of our research group about the relationship between the infection/disease and the cholinergic system.

The interactive effect of phytase and coccidia on the gross lesions as well as the absorption capacity of intestine in broilers fed with diets low in calcium and available phosphorous.

In an experiment with 2x2 factorial design, the influence of dietary phytase on the intestinal lesions as well as the absorption capacity of intestine for D-xylose in broiler chickens provided with a diet low in calcium (Ca) and available phosphorus (aP) and challenged with Eimeria oocysts, was evaluated. Four groups of 20 1-day-old male broiler were provided with diets low in total Ca and aP (8 and 3g/kg instead of 10 and 5g/kg of Ca and aP in the diet, respectively). On day 10, 10 chicks from each group were randomly kept in individual raised floor wire cages to adopt environmental conditions. The experimental groups were as follows, Group 1: received no Eimeria oocysts (negative control), Group 2: received oocysts of mixed Eimeria species on day 15 to create an experimental coccidiosis (positive control), Group 3: negative control received phytase enzyme in their diet, from the first day of life, and Group 4: positive control received phytase enzyme in the diet. On day 20, after 12h fasting, the D-xylose absorption test was performed and immediately after that, the intestinal lesion scoring was carried out. The results showed that coccidiosis in Groups 2 and 4 produced progressive lesions in intestinal tract and reduced the concentration of plasma D-xylose in Group 2 when compared to Groups 1 and 3. Dietary phytase had no influence on the concentration of plasma D-xylose in un-infected birds. The enzyme had no influence on the intestinal lesions caused by coccidiosis as well. However, it increased the plasma D-xylose concentration of Group 4 to the level that it was comparable with Groups 1 and 3, at 45 and 90min post-ingestion of the solution. It was concluded that the addition of phytase enzyme to the low Ca and aP diet, increased indirectly the absorption capacity of intestine for D-xylose in infected chickens most probably through the improvement of mechanisms involved in the absorption and transport of D-xylose.

Synthesis and biological activity of anticoccidial agents: 2,3-diarylindoles.

Novel 2,3-diarylindoles bearing an amine substituent at the indole 5- and 6-positions have been synthesized and evaluated as anticoccidial agents in both in vitro and in vivo assays. Both subnanomolar in vitro activity and broad spectrum in vivo potency were detected for several compounds, particularly compound 27.

Pathogenic effects of the coccidium Eimeria ninakohlyakimovae in goats.

Twenty-four coccidia-free goats were reared artificially in indoor cages and allocated to 6 groups of 4 animals each. At 20 days of age, goats in groups 1-3 received 10(4),10(5) and 10(6) sporulated oocysts of Eimeria ninakohlyakimovae per goat, respectively, each as a single dose. Goats in group 4 received daily doses increasing over a 3-week period, starting with 100/day for the first week, followed by 1000, and 10,000/day in weeks 2, 3, respectively. Goats in group 5 received 10(4) oocysts following a challenge dose of 10(6) oocysts on day 32. Goats in group 6 were kept as uninoculated controls. Infected animals showed diarrhoea and weight loss. Goats in group 4 showed longer periods of diarrhoea and patency than other infected goats. Goats in group 5 showed the same severe clinical signs as those in group 3 but produced very low oocyst output after a challenge dose. The diarrhoea was associated with a reduction in alkaline phosphatase activity and increases in packed cell volume and haemoglobin. No significant differences were found in serum aspartate aminotransferase, alanine aminotransferase, total protein, albumin, globulin, Na+, K+,Cl- between groups during 48 days after inoculation. There were no serum enzyme indications of damage to the liver. Histological examination performed 100 days after inoculation revealed that inoculated goats had mild subacute to chronic proliferative enteritis in the lower small intestine and the large intestine, and the mesenteric lymph nodes, gallbladders and livers also showed slight histological lesions. The results showed that E. ninakohlyakimovae was highly pathogenic.

Antioxidant status during the course of Eimeria tenella infection in broiler chickens.

The antioxidant status of broiler chickens (Cobb 500 hybrids) infected with Eimeria tenella was monitored by determining blood plasma malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT). The results of the experiment showed an increase in MDA - a marker of radical-induced damage of E. tenella-infected birds, compared to healthy chickens (3.01 micro mol/L vs. 2.55 micro mol/L, P<0.05). Correspondingly, a decreased SOD activity was observed in infected birds compared to controls (2429.0 U/g Hb vs. 3044.6 U/g Hb, P<0.05). Furthermore, CAT activity in infected birds was higher than in healthy ones (2242.2 U/g Hb vs. 1367.0 U/g Hb, P<0.001). The observed enzyme changes suggest an impaired antioxidant status of chickens during the course of an E. tenella infection and the occurrence of oxidative stress following infection. Alterations in the caecum, oocyst production, weight gain and feed conversion ratio were indicative of a severe infection involving pathogenic oxidative stress and impaired ecological oxidative balance.

Responses of adipose and muscle lipoprotein lipase to chronic infection and subsequent acute lipopolysaccharide challenge.

Infection of male Swiss Webster mice with Toxoplasma gondii or Neospora caninum leads to long-term alterations in energy balance. Following an initial 20 to 30% weight loss in all T. gondii-infected mice, half of the animals regain most of the lost weight (gainers), whereas the others maintain their low body weight (nongainers). Infection with N. caninum does not elicit weight loss. Lipoprotein lipase (LPL), the enzyme responsible for plasma triglyceride (TG) clearance and partitioning among tissues, is under tissue-specific modulation associated with energy balance. It is also a major determinant of infection-induced hypertriglyceridemia. This study aimed to assess the long-term modulation of adipose and muscle LPL activity in mice infected with T. gondii or N. caninum, to evaluate the effects of subsequent acute lipopolysaccharide (LPS) administration, and to relate LPL modulation in these conditions with infection-related changes in body weight gain. Twenty-eight days after infection, LPL activity in muscle of both gainer and nongainer T. gondii-infected mice was reduced by 40 to 50% compared with the levels in controls and N. caninum-infected mice, whereas LPL activity in adipose depots remained unchanged in all infected groups compared to the level in controls. LPS (from Escherichia coli, 100 ng/kg) injection induced a global reduction in adipose LPL in all groups, as assessed 90 min later. In both T. gondii-infected subgroups, muscle LPL was not further reduced by LPS treatment, whereas it was decreased by 40 to 50% in muscles of control and N. caninum-infected mice. Pre-LPS TG levels in plasma were similar in all groups. LPS greatly increased TG levels in plasma in both control and N. caninum-infected animals, whereas it did not alter those of T. gondii-infected gainer or nongainer animals. These results show that (i) independently of the extent of postinfection weight gain, long-term infection with T. gondii chronically reduces muscle LPL, which becomes unresponsive to acute endotoxemia; (ii) modulation of tissue LPL activity during chronic T. gondii infection favors TG partitioning towards adipose tissue; and (iii) skeletal muscle LPL is a key determinant of the acute response of triglyceridemia to LPS.

Lymphocyte depletion in ileal Peyer's patch follicles in lambs infected with Eimeria ovinoidalis.

A total of 14 lambs were experimentally infected with Eimeria ovinoidalis in two separate experiments in two consecutive years. Nine lambs served as uninoculated controls. Material was collected from the ileum 2 weeks after infection in eight lambs and 3 weeks after infection in six lambs. Lambs examined 2 weeks after infection had normal follicles. After three weeks, the follicle-associated epithelium covering the lymphoid follicles of the ileal Peyer's patches showed fusions with adjacent absorptive epithelium, focal hyperplasia, and occasionally necrosis. Macrogametes, microgamonts, and oocysts were often found in the follicle-associated epithelium and the dome region. Various degrees of lymphocyte depletion were present in the ileal lymphoid follicles in all six infected lambs 3 weeks after infection, and four lambs had decreased follicle size. Reduced staining for leukocyte common antigen (CD45), B-cell markers, and the proliferation marker Ki-67 was present in these lambs. Application of the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling method for apoptotic cells revealed decreased staining in the ileal lymphoid follicles 3 weeks after infection. A marker of follicular dendritic cells, 5'- nucleotidase, showed increased reactivity, probably due to condensation of reticular cells following loss of follicle lymphocytes. Reduced staining for carbonic anhydrase in the follicle-associated epithelium and the domes was present in all six lambs examined 3 weeks after infection, indicating decreased production of carbonic anhydrase-reactive 50-nm particles and a decreased lymphoproliferative stimulus. In conclusion, the present study shows that severe E. ovinoidalis infection in lambs causes lesions of the follicle-associated epithelium and may result in lymphocyte depletion and atrophy of the ileal Peyer's patch follicles.

The cathepsin-D expression in the cyto-invasive stages of coccidia in intestinal mucosa of chickens, and the relevance of this proteinase for intracellular penetration and spreading of coccidia in host tissues.

The Cathepsin-D proteinase was demonstrated immunohistochemically in cases of intestinal coccidiosis in chickens; its expression concerned almost the cyto-invasive stages of reproductive cycle of several strains of coccidia (merozoites of the II-nd generation and also microgametes). The authors consider Cathepsin-D as a potentional "penetration/spreading factor", being significant for intracellular invasion and spreading of coccidia into host cells. In diagnostic practice, an immunohistochemical demonstration of Cathepsin-D is a relatively simple and sensitive method, which brings information on the extent of parasitic lesions, and on participation of particular evolutive forms of coccidial life cycle; it may also be useful in proving isolated coccidia in the intestinal epithelium, poorly recognized in routine HE preparation. Some Cathepsin-D protease reactivity in intestinal Cryptosporidiosis is also briefly mentioned. The examined cases of cerebral toxoplasmosis and primary acanthamoebic meningoencephalitis (PAME) showed in above mentioned methods negative results.

Three enzymes newly identified from the genus Eimeria and two more newly identified from E. maxima, leading to the discovery of some aliphatic acids with activity against coccidia of the domesticated fowl.

Nine enzymes were detected in sporulated oocysts of Eimeria tenella and E. maxima, parasites of the domesticated fowl (Gallus gallus). Three enzymes, hydroxybutyrate dehydrogenase, alanine aminotransferase and gamma-glutamyltransferase, all identified for the first time in Eimeria of fowl, occurred both in E. tenella and in E. maxima. The remaining enzymes assayed had previously been found in various Eimeria species of fowl, although creatine kinase and glutamate dehydrogenase were hitherto unknown from E. maxima. The three enzymes newly recorded from Eimeria of fowl are of interest as potential genetic markers, and also as potential chemotherapeutic targets. The discovery of hydroxybutyrate dehydrogenase led to the demonstration of anticoccidial activity by some aliphatic acids. The paper also includes a list of the enzymes detected in Eimeria of fowl in previous studies.

The hepatic and duodenal activities of some drug metabolizing enzymes in chickens: influence of infection with Escherichia coli endotoxin and coccidiosis.

The activities of the drug metabolizing enzymes aminopyrine-N-demethylase, aniline hydroxylase and UDP-glucuronyl transferase, together with protein concentration, were measured in liver microsomes and duodenal mucosa from healthy chickens and chickens experimentally infected with Escherichia coli endotoxin, or naturally infected with Eimeria necatrix and Eimeria tenella (clinically classified as slight, moderate or severe infections). E. coli (2 mg/kg, 3 days) and severe coccidiosis significantly decreased the activities of the three enzymes in the liver and duodenum. However, infection by the E. coli endotoxin at lower doses (0.5 and 1 mg/kg, 3 days) and moderate or slight coccidial infections had no significant effect on the activities of these enzymes. Neither E. coli nor coccidial infections significantly affected the liver/body weight ratio. However, infection with the E. coli endotoxin (2 mg/kg, 3 days) and the moderate or severe coccidial infections significantly reduced microsomal protein concentration in the liver.

Preliminary findings of alterations in serum alkaline phosphatase activity in chickens during coccidial infections.

Two-week-old broiler chickens were inoculated orally with various doses of avian Eimeria that infect different sections of the intestine. The coccidia used were E. acervulina (duodenum), E. maxima (jejunum), E. brunetti (ileum) and E. tenella (caecum). The measurements evaluated were weight gains, intestinal lesion scores and changes in serum alkaline phosphatase (SAP) activity 6 days after infection. SAP activity was inversely proportional to both oocyst dose and gross intestinal lesion score, and directly proportional to weight gains with infections of E. acervulina, E. maxima and E. tenella. However, no significant changes in the SAP activity were found in those birds infected with E. brunetti. Taken together, the data suggest that SAP activity may be a sensitive marker of the pathogenesis of certain coccidial infections, especially in the duodenum, jejunum and caecum, that may be used as an additional factor in evaluating coccidial control measures.

[Synthesis of nonspecific esterase in small intestine enterocytes during experimental coccidiosis in suckling pigs]. / Syntéza nespecifické esterázy enterocytû tenkého streva behem exprimentální kokcidiózy sajících selat.

The activity of nonspecific esterase (EC. 3.1.1.1.) was evaluated in the small intestine mucosa of 21 conventional piglets infected on day 5 after parturition (DAP) with oocysts of the Eimeria debliecki coccidium (infection dose of 200,000 oocysts) for this evaluation a microdensitometric analysis at the level of enterocytes was used. The same examination was also performed in the small intestine mucosa of four control conventional piglets at the age of 2-5 days (Tab. I). The synthesis of nonspecific esterase in the experimentally infected piglets was followed on day 1 to day 10 after infection (DAI). The activity of nonspecific esterase in the small intestine mucosa was found to decrease in a direction from duodenum absorption cells (D mean 34.15) to caudal ones (Fig. 1); ileum enterocytes have the optical density of the enzyme by 8.2% lower (D mean 31.38). The deposition of nonspecific esterase is localized mainly in the supranuclear zone of enterocytes while in the para- and infranuclear zones of absorption cells its concentration is only minute. In the experimentally infected piglets a marked increase in the optical density of nonspecific esterase of enterocytes was observed as soon as on day 1 after infection when the enzyme concentration increased by 19.4% (Tab. II). The maximum increase in the activity of nonspecific esterase of absorption cells was recorded on DAI 9 when the enzyme D mean value was higher by 165% in comparison with the activity of nonspecific esterase demonstrated in the control piglets (Fig. 2, 3, 4). But at the end of experimental infection (DAI 10) the total density of nonspecific esterase of enterocytes decreased by 38.2%.(ABSTRACT TRUNCATED AT 250 WORDS)

[Dehydrogenase activity in small intestine mucosa in experimental coccidiosis in suckling pigs]. / Aktivita dehydrogenáz ve sliznici tenkého streva pri experimentální kokcidióze sajících selat.

The activities of 3-beta-hydroxybutyrate dehydrogenase (EC. 1.1.1.30.; HBD) and isocitrate dehydrogenase (EC. 1.1.1.41; ICD) were evaluated microdensitometrically in the mucosa of duodenum, jejunum and ileum of 19 conventional piglets infected on the first day after parturition (DAP) with oocysts of Eimeria debliecki coccidium (infection dose of 200,000 oocysts). The two investigated enzymes are deposited in mitochondria which are dispersed in the supra-, para- and infranuclear region of absorption cells (Fig. 1). The synthesis of the two dehydrogenases was investigated in the small intestine mucosa in the period of 1st to 10th day after infection (DAI). The HBD and ICD activities were also followed in the small intestine of four control conventional piglets at the age of 2-5 days (Tab. I). The two dehydrogenases could be characterized by a topographic gradient; it means that their activity was increasing in the small intestine mucosa through duodenum in an aboral direction. The ICD activity is higher in the intestinal mucosa of healthy piglets (Figs. 2 and 3), where its topic concentration was more marked while the HBD activity is dispersed in enterocytes (Fig. 4). In infected piglets the density of the two enzymes was demonstrated to decrease already in the starting period of experimental infection, and it reaches the lowest values for the first time on DAI 5-6 (Fig. 5, Tab. II), then on DAIs 9 (HBD; Fig. 6, graph 11)) or 8 (ICD, Fig. 7 and 10). In the period of experimental infection no statistically significant predisposition to the hypoactivity of target dehydrogenases nor its marked shift were observed. Somewhat rapid resumption of synthesis was demonstrated as soon as on DAI 8 in ICD (Fig. 8); its activity on DAI 10 in the intestinal mucosa corresponded to the 93% activity of this dehydrogenase recorded in the small intestine of control piglets. The density of HBD to the same day (DAI 10) reached in the intestinal mucosa of infected piglets the values making only 44.7% of those demonstrated in the intestinal mucosa of the control group of animals.

[Synthesis of glucose-6-phosphatase in small intestine mucosa during experimental coccidiosis in suckling piglets]. / Syntéza glukózo-6-fosfatázy sliznice tenkého streva behem experimentální kokcidiózy sajících selat.

Glucoso-6-phosphatase activity (EC. 3.1.3.9.) was evaluated microdensitometrically in the small intestine mucosa of 19 conventional piglets infected with Eimeria debliecki coccidium oocysts (infection dose of 200,000 oocysts) on day 1 after delivery (DAD). The synthesis of the observed hydrolase was followed within days 1 to 10 after infection (DAI). The activity of the same enzyme was also determined in four control conventional piglets at the age of two to five days. The small intestine mucosa of healthy piglets was found to have relatively balanced glucoso-6-phosphatase concentrations in all observed sections (duodenum, middle and posterior jejunum and ileum). The topographic gradient failed to be demonstrated in control piglets. In piglets experimentally infected with E. debliecki coccidium the glucoso-6-phosphatase activity was decreasing during infection already since DAI 1. The enzyme hypoactivity was still lower in the following days and reached the minimum value at the end of the target period (DAI 10), when its value made only 33% of the concentration recorded in the intestine mucosa of control piglets. The topographic predisposition to the lower activity of glucoso-6-phosphatase was not found to be high. The only exception are DAI 6 and 7, when the lowest enzyme concentration was observed in the duodenum mucosa (52% on DAI 6; 54% on DAI 7). The activity of the observed enzyme was higher in the other sections of small intestine (57% on DAI 6; 62% on DAI 7).

[beta-D-glucosidase in the microvillous zone of small intestine enterocytes in experimental coccidiosis in suckling piglets]. / beta-D-glukosidáza mikrovilózní zóny enterocytu tenkého streva pri experimentální kokcidióze sajících selat.

In the small intestine mucosa of 24 gnotobiotic farrows experimentally infected with the oocysts of coccidiosis of Isospora suis (infection administration--100,000 oocysts) on the first day after the delivery, we carried out the microdensitometric evaluation of the activity of beta-D-glucosidase (phlorizin-hydrolase; hetero-beta-galactosidase; lactase-beta-glucosidase complex; EC. 3.2.1.21). Great attention was paid to the topochemistry of enzyme, deposited in a microvillous zone of enterocytes. We studied likewise the activity of beta-D-glucosidase in the striped fringe of enterocytes of the four control gnotobiotic farrows, in the age from 2 to 5 days. We found out that in healthy farrows the reaction product of studied disaccharidase is located in high concentrations in the microvillous zone of absorptive cells of the whole small intestine. We proved a topographic gradient at which the beta-D-glucosidase activity decreases in control farrows the duodenum mucosa in the aboral direction. When using the choice substrate for beta-D-glucosidase (5-Br-4-Cl-beta-indolyl-3-D-glucoside) we did not prove the enzyme deposition in the small intestine wall. The negative enteral effect of coccidiosis I. suis was provable in the farrows experimentally infected already on the first day after the infection (DPI) when the beta-D-glucosidase activity decreased within the whole small intestine by 15% (ileum) and even by 23% (middle jejunum). The activity reduction had been deepening since the first after the infection and it reached its maximum on the 9th day after the infection when the enzyme concentration in the microvillous zone of absorptive cells reached only 11% of the activity level found in control farrows. On the 10th and 11th day after the infection we registered the increase of the density of beta-D-glucosidase reaction product, however the microvillous zone was even in that final stage of experimental infection significantly deficient (31% of intestine mucosa activity of control farrows).