Eimeria tenella,Eimeria necatrix, and Eimeria adenoeides: Peripheral blood leukocyte response of chickens and turkeys to strains adapted to the turkey embryo

The peripheral blood leukocyte responses of chickens and turkeys inoculated with one of three strains of a chicken Eimeria species adapted to the turkey embryo with their respective parent lines, or with E. adenoeides of the turkey were studied. The adapted lines tended to cause hematological changes in chickens and turkeys similar to those caused by E. adenoeides. These parasites caused the most significant increases in large mononuclear white blood cells = (monocytes) in both chickens and turkeys. These results provide further evidence for a monocyte/macrophage effector mechanism in the rejection of heterologous species of Eimeria from a nonspecific host. The results also agree with previous studies that show that increases in mononuclear white blood cells during parent E. tenella and E. necatrix infections in chickens occur during the periods of greatest tissue damage (3–4 days after inoculation). The generally unaffected lymphocyte numbers and increases in mononuclear white blood cells during infections with the adapted lines probably explain the reduced pathogenicity and the lack of immunogenicity seen previously in chickens inoculated with these three lines. Possibly, monocytes/macrophages play a role in the host specificity of the parasites.     

Cross immunity of DNA vaccine pVAX1-cSZ2-IL-2 to Eimeria tenella, E. necatrix and E. maxima

The study describes cross protection experiments with chimeric DNA vaccine pVAX1-cSZ2-IL-2 to determine its efficacy against four important Eimeria species. Seven-day-old chickens were randomly divided into nine groups; group 1 negative control, groups 2, 3, 4, 5 positive controls; and groups 6, 7, 8 and 9 experimental groups. On days 7 and 14, groups 1-5 were injected with TE buffer, and groups 6-9 with the vaccine. At 21 days of age, all chickens were inoculated with 5 × 10⁴ sporulated oocysts except for the negative control. Groups 2 and 6 were inoculated with Eimeria tenella, groups 3 and 7 with Eimerianecatrix, groups 4 and 8 with Eimeria acervulina and groups 5 and 9 with Eimeria maxima. Seven days later, all chickens were weighed and slaughtered to obtain intestinal samples. Efficacy of immunization was evaluated on the basis of oocyst decrease ratio, lesion score, body-weight gain and anti-coccidial index. The results indicated that the recombinant plasmid can induce host immune responses by alleviating intestinal lesions, body weight loss and oocyst ratio and imparting good protection against E. tenella and E.acervulina, medium protection against E. necatrix but little effect against E. maxima. It is concluded that the conserved antigen can provide cross protection and should be explored further.     

Eimeria dispersa, E. adenoeides, and E. meleagrimitis: intestinal mucosal disruption in turkeys as seen with scanning electron microscopy.

Tissues from the digestive tract of turkeys infected with Eimeria dispersa, E. adenoeides, or E. meleagrimitis were compared with tissues from uninfected controls as seen with light microscopy (LM) and scanning electron microscopy (SEM). Six regions were examined--duodenum, jejunum, ileum, cecal neck, cecal pouch, and large intestine. Although LM showed large numbers of E. dispersa in the epithelial cells, SEM usually showed little mucosal disruption. Occasionally the surface of duodenum and jejunum, as seen with SEM, was convoluted and disrupted. In one bird, some parasite-induced damage was found with either LM or SEM in the duodenum and jejunum of turkeys given E. adenoeides oocysts. Although LM showed parasites in the rest of the digestive tract of all E. adenoeides infected birds, SEM showed only a localized sloughing of the mucosa in the cecal pouch. The most extensive damage to the villar surface was caused by E. meleagrimitis. Infections often disrupted the villar tips, especially in the small intestine and cecal neck. Localized areas of pitting were often found on individual villi. All 3 species produced oocyst extrusion sites, especially in the ileum and the ceca.     

Cholecystokinin and anorexia in sheep infected by the intestinal nematode Trichostrongylus colubriformis

Symons L. E. A. and Hennessy D. R. 1981. Cholecystokinin and anorexia in sheep infected by the intestinal nematode Trichostrongylus colubriformis. Internationaljournal for Parasitology11: 55–58. It was postulated that there is a correlation between anorexia in intestinal nematode infection and the plasma concentration of cholecystokinin (CCK). In the first experiment plasma concentration of CCK rose as food consumption fell until, when sheep infected with Trichostrongylus colubriformis were almost completely anorexic, it had increased by 65%. Plasma CCK and food consumption returned to pre-infection levels in from four to six days after administration of an anthelmintic. In the second experiment food consumption by uninfected sheep was reduced in the first ten minutes after intravenous infusion of 150–300 μg of the octapeptide of CCK. It was concluded that anorexia in these infections may be due to or be mediated by higher concentrations of CCK.     

Interactions between Trichinella spiralis and Hymenolepis nana in the intestine of the mouse

Ferretti G., Gabriele F., Palmas C. and Wakelin D. 1984. Interactions between Trichinella spiralis and Hymenolepis nana in the intestine of the mouse. International Journal for Parasitology14: 29–33. The rejection of the intestinal phase of Trichinella spiralis in outbred CD₁ mice is associated with an inflammatory response. The effects of this inflammation were studied in mice concurrently infected with an unrelated parasite, Hymenolepis nana, which stimulates a slight inflammation but is also immunologically rejected by this strain of mice. Moreover though Cd(In)1 mice seem to be ‘slow responders’, in concurrent infections with T. spiralis and H. nana there was a marked effect upon the cesode survival. At the highest level of T. spiralis infection, H. nana failed to establish at all. On the other hand, if 300 T. spiralis were administered 10 weeks earlier, H. nana become established in all mice and the distribution of parasites suggests that immunodepression is involved. An accelerated loss of the nematode, when H. nana was present, was also demonstrated. There was no evidence of any reciprocal or specific cross-immunity between the two parasites.     

Eimeria pragensis infection alters the gut microenvironment to favor extrinsic shiga toxin-producing Escherichia coli O157:H7 colonization in mice

We examined the effects of Eimeria pragensis infection on intestinal peristalsis, goblet cell proliferation and intestinal flora in C57BL/6 mice. Intestinal peristalsis was evaluated by radiography using barium at 7 days post-infection (p.i.). The intestinal peristalsis of E. pragensis-infected mice was significantly suppressed compared with uninfected control mice. Twenty-three mice were divided into 5 groups of 4 or 5 mice each; 2 groups of mice were infected with E. pragensis and the others were kept uninfected. At 7 days p.i., E. pragensis-infected and -uninfected mice were sacrificed to examine goblet cell numbers in the intestines, and significant decreases were observed only in the infected mice. Shiga toxin-producing Escherichia coli (STEC) O157:H7 was inoculated orally in mice both infected and uninfected with E. pragensis at 7 days p.i., with the remaining mice used as uninoculated controls. When mice were sacrificed at 2 days after STEC inoculation, STEC was only detected in the intestines of E. pragensis-infected mice. Colonization of STEC was also confirmed by immunohistochemistry on the surface of epithelial cells in concurrently infected/inoculated mice. Also, an overgrowth of residential E. coli was observed only in E. pragensis-infected mice. These results suggest that E. pragensis induces the suppression of intestinal peristalsis and modifies the intestinal environment to facilitate artificially introduced STEC colonization and multiplication, in addition to residential E. coli overgrowth.     

Eimeria tenella: Effects of diclazuril treatment on microneme genes expression in second-generation merozoites and pathological changes of caeca in parasitized chickens

The effects of diclazuril on mRNA expression levels of invasion-related microneme genes were examined in second-generation merozoites of Eimeria tenella (E. tenella) by quantitative real-time (QRT) PCR. Diclazruil treatment of infected chickens significantly decreased the number of second-generation merozoites by 65.13%, and resulted in downregulation of EtMIC genes: EtMIC1 by 65.63%, EtMIC2 by 64.12%, EtMIC3 by 56.82%, EtMIC4 by 73.48%, and EtMIC5 by 78.17%. SEM images of caecum tissue from uninfected chickens showed regular intestinal villus structure. In infected chickens, a distinct loss of the superficial epithelium, with a flattened mucosa and large-area necrosis and anabrosis, was evident. In diclazruil-treated chickens, a decrease in merozoite number and a visibly improved appearance of the caeca were noted. These improvements appeared to be mediated in part by downregulation of the expression of invasion-related EtMIC genes in response to diclazuril.     

Binding and Cleavage of E. coli HUβ by the E. coli Lon Protease

The Escherichia coli Lon protease degrades the E. coli DNA-binding protein HUβ, but not the related protein HUα. Here we show that the Lon protease binds to both HUβ and HUα, but selectively degrades only HUβ in the presence of ATP. Mass spectrometry of HUβ peptide fragments revealed that region K18-G22 is the preferred cleavage site, followed in preference by L36-K37. The preferred cleavage site was further refined to A20-A21 by constructing and testing mutant proteins; Lon degraded HUβ-A20Q and HUβ-A20D more slowly than HUβ. We used optical tweezers to measure the rupture force between HU proteins and Lon; HUα, HUβ, and HUβ-A20D can bind to Lon, and in the presence of ATP, the rupture force between each of these proteins and Lon became weaker. Our results support a mechanism of Lon protease cleavage of HU proteins in at least three stages: binding of Lon with the HU protein (HUβ, HUα, or HUβ-A20D); hydrolysis of ATP by Lon to provide energy to loosen the binding to the HU protein and to allow an induced-fit conformational change; and specific cleavage of only HUβ.     

Plasmodium berghei: II. Immunobiological properties of fractions of a soluble extract

A soluble extract of Plasmodium berghei was prepared from parasites freed from infected erythrocytes by saponin lysis. The extract was separated into 12 fractions by preparative disc electrophoresis, and the fractions were employed (1) to seek precipitins in hyperimmune rat serum, and (2) in the vaccination of rats. Species-specific antigens were identified in some of them.Two regions of the disc-electrophoretic column (Fractions 4–5 and 7–8) were regularly the most reactive in all systems tested. Thus, they reacted most frequently by precipitating with hyperimmune rat sera containing protective antibody, while other fractions were nonreactive or only rarely reactive. Antigens in these disc-electrophoretic regions were among those inducing precipitins in rats, though they were not the only ones to do so. These regions contained species-specific antigens, not shared with the noncross-protecting Plasmodium vinckei. Finally, fractions from these regions employed as vaccines were capable of inducing immunobiological effects: enhancement or protection in varying limited degrees.     

Impacts of an endoparasitic copepod, Ismaila belciki, on the reproduction,growth and survivorship of its nudibranch host, Janolus fuscus

Copepods from the genus Ismaila are large endoparasites that inhabit the main body cavity and/or cerata of opisthobranch molluscs. These parasites exhibit many life history characteristics typically found in parasitic castrators, yet the actual impact of infection on reproduction, growth or survivorship of the hosts are unknown. On the Oregon (USA) coast, Ismaila belciki can infect over 80% of their hermaphroditic hosts, Janolus fuscus. In laboratory mating experiments, we compared the reproductive output (egg mass weight, number of egg capsules, number of viable embryos) and the gonadal somatic index of infected versus uninfected J. fuscus. Infected J. fuscus could produce viable sperm and copulate. Mating with an infected individual did not limit a sea slug’s reproductive output. However, infected J. fuscus had significantly lower reproductive output (by 34–54%), producing smaller egg masses with fewer capsules and viable embryos. Infected hosts had significantly lower gonadal somatic index than their uninfected counterparts, although there was no significant difference in gonadal somatic index between hosts with single and double infections. By collecting the egg sacs produced by the copepod parasite during experiments, we estimated that 25–34% of the host’s reproductive output is usurped by the parasite and re-directed to the parasite’s own reproduction. In the laboratory, infection did not alter growth in J. fuscus. However, infection significantly decreased survivorship in mature (but not immature) nudibranch hosts. These results suggest that I. belciki is not a true castrator, but it does reduce the reproductive output of its host and may therefore limit the natural population size of J. fuscus.     

Eimeria nieschulzi and Trichinella spiralis: Analysis of concurrent infection in the rat

The effects of concurrent primary infection of the rat with Eimeria nieschulzi and Trichinella spiralis on the number of oocysts of E. nieschulzi shed by the host and on the number, distribution, and fecundity of adult T. spiralis were analyzed. When rats were initially infected with E. nieschulzi followed 9 days later by infection with T. spiralis there occurred a significant decrease in the total numbers of adult worms in the small intestine, a significant shift in the position of these worms along the length of the small gut, a decrease in the fecundity of adult female worms, and a decrease in muscle parasitism when compared with rats infected with T. spiralis alone. When rats were initially infected with T. spiralis, followed 9 days later by infection with E. nieschulzi, there occurred a significant decrease in the numbers of oocysts shed over 24 hr on Days 7, 9, and 11 postinfection below that seen with rats infected only with Eimeria. These changes are discussed in terms of the enteropathophysiologic lesions and enteric inflammation known to occur during infections with these two parasites.     

Purification and properties of d-galactose-binding lectins from some erythrina species: Comparison of properties of lectins from E. indica,E. arborescens,E. suberosa, and E. lithosperma

The lectins of the seeds of four species of the genus Erythrina, namely E. indica, E. arborescens, E. lithosperma, and E. suberosa were isolated by affinity chromatography on acid-treated ECD-Sepharose 6B. The lectins were found homogeneous in polyacrylamide gel electrophoresis and immunochemical tests. In SDS-gel electrophoresis, E. indica and E. lithosperma lectins each gave two bands with subunit molecular weights of 30,000 and 33,000 in the case of the former and 26,000 and 28,000 in the case of the latter. E. arborescens and E. suberosa gave single bands corresponding to polypetide chain molecular weight of 28,000. The lectins were found to be glycoproteins with their neutral sugar contents ranging from 4–9%. In carbohydrate specificity all the lectins were d-galactose specific. Their close similarity was also demonstrated by their homologous cross-reaction against the antiserum to E. indica lectin. In hemagglutinating activity toward human erythrocytes, E. indica and E. suberosa lectins showed higher activity toward the O group and E. arborescens toward the B group. The results show the similarity of the lectins derived from different species of the same genus in respect of immunochemical properties and carbohydrate specificity. In studies on E. indica lectin, the protein was found homogeneous by electrophoretic, immunochemical, and sedimentation experiments. Its molecular weight of 68,000 determined from sedimentation and diffusion data indicated that the molecule was a dimer of two noncovalently bound unequal subunits whose SDS-gel electrophoretic molecular weights are noted above. The lectin was devoid of cysteine and methionine and contained valine as its N-terminal amino acid. It had 9% neutral sugars and 1.5% glucosamine. Equilibrium dialysis studies with lactose showed that the values of the association constant K at different temperatures were of similar orders of magnitude to other lectins and the dimeric molecule possessed two noninteracting binding sites.     

Rye Affects Bacterial Translocation,Intestinal Viscosity,Microbiota Composition and Bone Mineralization in Turkey Poults

Previously, we have reported that rye significantly increased both viscosity and Clostridium perfringens proliferation when compared with corn in an in vitro digestive model. Two independent trials were conducted to evaluate the effect of rye as a source of energy on bacterial translocation, intestinal viscosity, gut microbiota composition, and bone mineralization, when compared with corn in turkey poults. In each experiment, day-of-hatch, turkey poults were randomly assigned to either a corn or a rye diet (n = 0 /group). At 10 d of age, in both experiments, 12 birds/group were given an oral gavage dose of fluorescein isothiocyanate dextran (FITC-d). After 2.5 h of oral gavage, blood and liver samples were collected to evaluate the passage of FITC-d and bacterial translocation (BT) respectively. Duodenum, ileum and cecum gut sections were collected to evaluate intestinal viscosity and to enumerate gut microbiota. Tibias were collected for observation of bone parameters. Broilers fed with a rye diet showed increased (p<0.05) intestinal viscosity, BT, and serum FITC-d. Bacterial enumeration revealed that turkey poults fed with rye had increased the number of total lactic acid bacteria (LAB) in all three sections of the gastrointestinal tract evaluated when compared to turkey poults fed with corn. Turkey poults fed with rye also had significantly higher coliforms in duodenum and ileum but not in the ceca, whereas the total number of anaerobes increased only in duodenum. A significant reduction in bone strength and bone mineralization was observed in turkey poults fed with rye when compared with corn fed turkey poults. In conclusion, rye evoked mucosal damage in turkey poults that increased intestinal viscosity, increased leakage through the intestinal tract, and altered the microbiota composition and bone mineralization. Studies to evaluate dietary inclusion of selected Direct-Fed Microbial (DFM) candidates that produce exogenous enzymes in rye fed turkey poults are currently being evaluated.     

Eimeria acervulina,E. brunetti,E. maxima, and E. necatrix: Low doses of oocysts to immunize young chickens

Resistance to reinfection varied with the species of Eimeria and with the number of oocysts in the inoculum. Chickens immunized with doses of 20,000 and 80,000 oocysts of E. acervulina, 312 and 1250 oocysts of E. brunetti or E. necatrix, or 1250 and 5000 oocysts of E. maxima at 2 and 4 weeks of age, respectively, were almost completely immune to a challenge dose at 6 weeks of age. Resistance was slightly less in chickens immunized with 1250 and 5000 oocysts of E. acervulina or 312 and 1250 oocysts of E. maxima. Birds given three immunizing infections of 1250, 5000, and 20,000 oocysts of E. maxima were completely immune 8 weeks after the last dose. Resistance was slightly less in birds immunized with similar doses of E. brunetti or E. necatrix. Doses of 20,000, 80,000, and 320,000 oocysts appeared necessary to confer a high level of immunity to E. acervulina. More than three low doses of oocysts appear necessary to induce a complete and enduring immunity against a high challenge for E. acervulina, E. brunetti, and E. necatrix. Higher immunizing doses would not be satisfactory due to the pathogenic effects of the coccidia after the initial infection.     

Developing an in vitro method for Eimeria tenella attachment to its preferred and non-preferred intestinal sites

A frozen section method utilising chicken intestinal tissue was developed to study the Eimeria tenella attachment ex vivo. In order to examine Eimeria-epithelial cell attachment, 10⁵E. tenella sporozoites were incubated with each caecal frozen section (6, 10 and 14 μm) for 1 h in 5% CO₂ incubator at 41 °C. E. tenella sporozoites attached successfully to enterocytes in 14 μm thick of caecal sections. Sporozoite attachment to caecal sections was shown to be dependent on the number of parasites added. To evaluate the method, E. tenella sporozoites were incubated to its preferred (caecum) and non-preferred (duodenum and jejunum) intestinal sites. The number of sporozoites attached to the caecal enterocytes was significantly greater (P < 0.0001) in comparison with the limited number of sporozoites attached to enterocytes of non-preferred intestinal sites. This method was shown to be able to reveal differences in binding capability and allows for comparison of intestinal site attachment.     

Pathophysiology of cestode infections: Effect of Hymenolepis diminuta on oxygen tensions,pH and gastrointestinal function

Studies on the normal and parasitized rat intestine were used to investigate the effect of the tapeworm, Hymenolepis diminuta, on in vivo intestinal lumenal oxygen tensions, acid-base balance and mucosal absorption and accumulation of fluid and glucose.The lumenal bulk aqueous phase is considerable, well mixed and aerobic with an oxygen tension of 40–50 mm Hg. Neither the unstirred layers adjacent to the brush border membrane nor the area adjacent to the mucosa (“paramucosal lumen”) are significant barriers to the diffusion of oxygen from the blood to the intestinal lumen. In the uninfected distal ileum and colon anoxic conditions may occur in the central lumen, but, in the parasitized intestine fluid absorption is reduced and anoxic conditions do not occur. Increased H⁺ ion concentration in the parasitized intestine plays a role in increasing the availability of oxygen to intestinal helminths. Concomitant with the lower pH, the pCO₂ in the lumen of the parasitized intestine was twice as high as that found in normal animals. The total CO₂ in the parasitized intestine steadily decreased over a 3-h perfusion period, while in the normal intestine the total CO₂ content increased after an initial fall during the first 30 min of perfusion. When the worms were removed, the ability of the intestine to restore normal acid-base balance was restored. Glucose and fluid absorption in both the infected and uninfected intestine were reduced by an increase in H⁺ ion concentration; both parameters were lower in the parasitized intestine than in the normal animals. Low pH increased fluid and glucose transport by H. diminuta.While the dry weights of both the parasitized and uninfected total small intestine and of the intestinal mucosa were the same, the wet weights were considerably different, indicating defective fluid balance in the infected intestine. Accumulation of glucose by the parasitized mucosa was greater than in control animals and decreased with an increase in H⁺ ion concentration. The glucose transport system in the parasitized gut was therefore affected at two levels, one at the brush border, where transport into the mucosa was decreased by lowering the pH, and secondly at the level of the basal and lateral membranes, where transport out of the mucosal tissue into the circulatory system was also reduced.The above results are discussed in terms of current widely accepted but erroneous concepts relating to the intestinal ‘microcosm’.     

In-depth validation of acridine orange staining for flow cytometric parasite and reticulocyte enumeration in an experimental model using Plasmodium berghei

Flow cytometry is potentially an effective method for counting malaria parasites, but inconsistent results have hampered its routine use in rodent models. A published two-channel method using acridine orange offers clear discrimination between the infected and uninfected erythrocytes. However, preliminary studies showed concerns when dealing with Plasmodium berghei-infected blood samples with high numbers of reticulocytes.In hyperparasitemic or chronic P. berghei infection, enhanced erythropoietic activity results in high numbers of circulating immature reticulocytes. We show that even though the protocol offered good discrimination in newly infected animals, discrimination between infected erythrocytes and uninfected reticulocytes became difficult in animals with hyperparasitemia or chronic infections maintained with subcurative treatment. Discrimination was especially hampered by increased nucleic acid content in immature uninfected reticulocytes. Our data confirms that though flow cytometry is a promising analytical tool in malaria research, care should still be taken when analysing samples from anemic or chronically infected animals.     

A standardizable protocol for infection of Rhodnius prolixus with Trypanosoma rangeli, which mimics natural infections and reveals physiological effects of infection upon the insect

Trypanosoma rangeli is a protozoan parasite that shares hosts - mammals and triatomines - with Trypanosoma cruzi, the etiological agent of Chagas disease. Although T. rangeli is customarily considered to be non-pathogenic to human hosts, it is able to produce pathologies in its invertebrate hosts. However, advances are hindered by a lack of standardization of infection procedures and these pathologies need documentation. To establish a suitable, and standardizable, infection protocol, the duration of the fourth instar was evaluated in nymphs infected by injection into the thorax with different concentrations of parasites, and compared with nymphs infected naturally (i.e. orally). We demonstrate that delays in moult were attributable to the presence of the parasite in the haemolymph (vs. the gut) and propose that the protocol presented here simulates closely natural infections. This methodology was then used for the evaluation of physiological parameters and several hitherto unreported effects of T. rangeli infection on Rhodnius prolixus were revealed. Haemolymph volume was greater in infected than uninfected nymphs but this alteration could not be attributed to water retention, since infected insects lost the same amount of water as controls. However, we found that lipid content and fat body weight were both increased in insects infected by T. rangeli. We propose that this is due to the parasite’s sequestration of host blood lipids and carrier proteins. With these findings, we have taken a few first steps to unravelling physiological details of the host-parasite interaction. We suggest future directions towards a fuller understanding of mechanistic and adaptive aspects of triatomine-trypanosomatid interactions.     

Endogenous stages of Eimeria sigmodontis (Protozoa: Eimeriidae) in the cotton rat, Sigmodon hispidus

Endogenous stages of Eimeria sigmodontis were studied in experimentally infected cotton rats, Sigmodon hispidus. The parasites were located in mucosal epithelial cells of the cecum and colon. E. sigmodontis had a typical coccidian endogenous cycle that consisted of three asexual schizogonous stages and sexual stages composed of the macrogametes and microgametes. The first-, second-, and third-generation schizonts contained 10–17, 5–8 and 10–21 merozoites, respectively. Only one type of wall-forming body was present in the macrogamete. The microgametocyte had a monocentric type of development.