Enzyme cytochemical observations on the tissue stages of the life cycle of Eimeria acervulina and Eimeria necatrix

Cytochemical studies concerning the presence and distribution of various enzymes in the tissue stages of the life cycle of Eimeria acervulina and E. necatrix have been undertaken.Acid and alkaline phosphatases as well as ATPase (pH 7·2) were found to be present in all the stages of the life cycles examined, the latter enzyme being very strong in the plastic granules of the macrogametocytes and in the inner membrane of the oocyst wall. The reaction for all three enzymes was observed in the cytoplasm but not the nucleus. Non-specific esterase and glucose-6-phosphatase were also found in all stages examined, the latter occurring in greater amounts in stages where deposition of glycogen (amylopectin) was pronounced. Succinic dehydrogenase occurred only in the second generation schizonts and merozoites of E. necatrix and in the formed oocysts of both species. No reaction for β-glucuronidase or leucine naphthylamidase was apparent in any stage, although a slight reaction for leucine naphthlyamidase was seen in the second generation merozoites of E. necatrix lying free in the intestinal lumen.The presence and distribution of these enzymes and their possible function were discussed.     

Cytochemical study of the different stages in the life cycle of Toxoplasma gondii. IX. The polysaccharides and lipids in the parasites at developmental stages from the cat intestine]

Amylopectin was detected in all the stages examined. In the oval stages the minute granules of PAS-positive material were seen in the cytoplasm when examined on fresh-frozen sections. In merozoites, amylopectin was more conspicuous with maturation. The residual body of microgametocytes contain large amounts of amylopectin; no polysaccharide was visualized in microgamete bodies. Amylopectin was most abundant in macrogametocytes and zygotes. However, no peripheral position of PAS-positive "plastic granules" (wall-forming bodies), so characteristic of other coccidia and revealed by the electron microscopy for T. gondii macrogametocytes, was seen. Acid mucopolysaccharides in the macrogametocyte were detected in the central zone, leaving the periphery of the cell unstained. Very small, if any, amounts of lipids were detected in asexual stages of T. gondii. Unlike, large accumulation of lipid droplets were seen in growing macrogametocytes suggesting the involvement of lipids along with amylopectin in the metabolism of oocysts later discharged from the host body.     

Montanide™ ISA 71 VG adjuvant enhances antibody and cell-mediated immune responses to profilin subunit antigen vaccination and promotes protection against Eimeria acervulina and Eimeria tenella

The present study was conducted to investigate the immunoenhancing effects of Montanide™ ISA 71 VG adjuvant on profilin subunit antigen vaccination. Broiler chickens were immunized subcutaneously with a purified Eimeria acervulina recombinant profilin protein, either alone or mixed with ISA 71 VG, and host immune responses were evaluated. After secondary immunization, antigen-specific antibody and T-cell responses were higher in the group which received profilin plus ISA 71 VG compared with the other groups. Furthermore, body weight gains and fecal oocyst shedding were evaluated following oral challenge infection with live E. acervulina or Eimeria tenella oocysts. Vaccination with profilin plus ISA 71 VG reduced oocyst shedding compared with animals immunized with profilin alone. These results demonstrate that the recombinant profilin subunit vaccine, when given in combination with Montanide™ ISA 71 VG, augments protective immunity against E. acervulina and E. tenella.     

Eimeria acervulina, E. brunetti, and E. maxima: pathogenic effects of single or mixed infections with low doses of oocysts in chickens.

The pathogenic effects of E. acervulina, E. brunetti, and E. maxima were modified when chickens received mixed infections with these species.Six-week-old chickens were inoculated with doses of 20,000 oocysts of E. acervulina, 1250 oocysts of E. brunetti, and 5000 oocysts of E. maxima given as a single or mixed infection.Typical signs of coccidiosis were apparent in chickens infected with a single Eimeria sp. When birds were given infections composed of two species, the weight loss was greater than that due to either given alone but when three species were given, weight loss was slightly less than that due to infection will E. brunetti alone. Oocyst production due to E. acervulina tended to be similar in birds given this species alone or with E. brunetti. Output fell to less than 50% when E. acervulina was administered with E. maxima. Oocyst production due to E. brunetti and E. maxima decreased when these species were inoculated together and when they were administered with E. acervulina. Lesions of E. acervulina and E. brunetti were superimposed on those of E. maxima in birds given mixed infections.Growth retardation was not evident in chickens inoculated with E. acervulina alone, although weight loss increased when this species was administered with either E. brunetti or E. maxima.     

Serum and liver zinc,copper, and iron in chicks infected withEimeria acervulina orEimeria tenella

Two-wk-old broiler chicks were inoculated via crop intubation withEimeria acervulina at two doses: 10⁵ or 10⁶ sporulated oocysts/bird or withEimeria tenella at a dose of 10⁵ sporulated oocysts/bird. Serum and liver samples were collected on days 3 and 6 post-inoculation (PI). There were no significant changes in serum or liver zinc, copper, and iron concentrations in any of the infected groups by 3 d PI. However, on d 6, PI serum protein was significantly reduced in all of the infected groups compared to their pair-fed controls. The chicks infected withE. tennella had significantly reduced serum zinc (1.20 vs 1.77 μg/mL) and iron (0.44 vs 1.28 μg/mL) concentrations and significantly elevated serum copper (0.28 vs 0.17 μg/mL) and ceruloplasmin levels (20.33 vs 11.11 μg/mL) compared to their pair-fed counterparts. Those chicks infected withE. acervulina (10⁶ oocysts/bird) exhibited significantly reduced serum iron concentration by 6 days PI (0.90 vs 1.14 μg/mL). Liver zinc was significantly increased in the chicks infected withE. tenella (349 vs 113 μg/g dry liver wt), as was copper (24 vs 19 μg/g), whereas liver iron concentration was significantly reduced (172 vs 243 μg/g) compared to pair-fed controls. At both dose levels, the chicks infected withE. acervulina exhibited a significant reduction in liver iron by 6 d PI. Hepatic cytosol metals generally reflected whole tissue levels. Metallothionein (MT)-bound zinc was significantly elevated in the chicks infected withE. tenella. Iron bound to a high molecular weight, heat-stable protein fraction (presumably cytoplasmic ferritin) was significantly reduced in chicks infected withE. acervulina, as well as those infected withE. tenella. Collectively, the changes in serum zinc, copper, and iron concentrations, as well as the changes in hepatic zinc and MT-zinc concentrations in the chicks infected withE. tenella were similar to changes evoked during an acute phase response to infection. It is possible that a secondary bacterial infection or inflammation stemming from erosion of the lining of the cecum may play a role in the response of trace element metabolism to theE. tenella infection. Mentions of a trademarkr, proprietary product or specific equipment does not consitute a guarantee or warranty by the US Department of Agriculture and does not imply its approval to the exclusion of other products.     

Dynamic of apoptosis of cells in duodenal villi infected with <Emphasis Type="Italic">Eimeria acervulina</Emphasis> in broiler chickens

The aim of our study was to evaluate the parasite — host interactions at apoptosis level. We studied histopathological changes and time course of apoptosis in the duodenum during Eimeria acervulina infection. One-day-old broiler chicks were randomly allocated into two equal groups. At the age of two weeks the first group was experimentally infected with a pure suspension of sporulated E. acervulina oocysts. The second group served as a negative control. Tissue samples from the upper part of duodenum were obtained at 0.5, 1, 2, 3, 4, 5 and 6 days post infection. Biopsies of duodenum were studied immunohistochemically using DeadEnd™ Colometric TUNEL System for apoptosis detection in duodenal mucosa. Number of parasites in duodenal epithelium was also investigated. Our experimental results demonstrate: (i) macroscopic and histopathological changes in epithelium detected mainly in proximal segment of duodenum in infected groups; (ii) the number of developmental stages of E. acervulina (DSEA) during our trial increased, reaching the maximum 5 days post infection (dpi) (332.2 ± 16.12) (mean ± SEM), whereas the amount of DSEA declined significantly as late as 6 dpi (124.6 ± 3.91); (iii) the highest apoptosis level was recorded in initiatory 0.5 dpi (13.2 ± 1.02) and on the end of parasite development cycle after 5 dpi (12.6 ± 1.36). Finally, results showed that there was a period of inhibition of apoptosis during infection by E. acervulina.     

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.     

Effect of dietary zinc level on serum carotenoid levels,body and shank pigmentation of chickens after experimental infection with coccidia

Abstract

Two experiments were conducted to test the effects of a dietary zinc amino acid complex (Zn-AA) and an anticoccidial drug on Eimeria acervulina or Eimeria tenella infections. In each experiment, 288 day-old Three-Yellow-Chickens were used in a 2 × 3 factorial experimental design. Six groups were arranged randomly to receive three levels of Zn-AA (0, 40, or 80 mg/kg) alone or with salinomycin (60 mg/kg). Additionally an uninfected group was set as negative control. At the age of 21 days birds in Exp. 1 were inoculated with 3 · 10⁴ sporulated E. acervulina oocysts, while birds in Exp. 2 were inoculated with 1.5 · 10⁴ sporulated E. tenella oocysts. In Exp. 1, E. acervulina did not suppress growth performance significantly, but in groups without salinomycin it significantly reduced serum carotenoid levels on day 7 after inoculation and body and shank pigmentation on day 42. Salinomycin medication maintained serum carotenoids and visual colour of inoculated birds, but Zn-AA did not influence these parameters. In Exp. 2, growth performances of infected and uninfected chickens were similar. Infection decreased to only serum carotenoid levels on day 14 after infection, and colour scores on day 42 in the inoculated group without salinomycin and Zn-AA supplementation. The birds that received Zn-AA had significantly higher serum carotenoid levels and colour scores than those that did not. Although supplementation of Zn-AA cannot avoid coccidial damage of caecum, it prevents the reduction of serum carotenoids and pigmentation of Three-Yellow-Chicken infected with E. tenella, but not after infection with E. avervulina. The interactive effects between Zn-AA and salinomycin on growth performance and pigmentation were not significant.     

Effects of Carbon Dioxide on Coccidian Oocysts from 6 Host Species*

SYNOPSIS Activation of sporozoites in oocysts of Eimeria acervulina (chicken), E. intricata (sheep), and E. scabra (swine) occurred after pretreatment in aqueous 0.02 M cysteine hydrochloride under an atmosphere of CO₂, followed by incubation in a trypsin-bile mixture. Sporozoites of E. stiedae (rabbit), E. bilamellata (squirrel), and Isospora canis (dog) became activated when incubated in trypsin and bile with or without prior CO₂-pretreatment of oocysts; however, when CO₂-pretreatment was used, activation of these species in trypsin and bile was greatly enhanced. For E. acervulina, 12% of the oocysts were activated after 4 hr CO₂-pretreatment and 10 hr incubation in trypsin and bile at 43 C; higher temperatures or longer pretreatment times did not cause greater activation. Eimeria intricata oocysts became activated after 1 hr pretreatment and 10 hr incubation in trypsin and bile at 37, 39 or 41 C, respectively. The highest activation (31%) occurred after 20 hr pretreatment and 10 hr incubation in trypsin and bile at 41 C. Ninety percent of E. scabra oocysts contained active sporozoites after 1 hr CO₂-pretreatment and 10 hr incubation in trypsin and bile at 37 C. At 39 or 41 C, 100% activation occurred with this species after similar pretreatment and treatment periods. With E. bilamellata, 64% activation occurred in nonpretreated oocysts incubated 10 hr in trypsin and bile at 41 C, whereas 100% activation occurred if oocysts were pretreated with CO₂ for 1 hr before treatment with trypsin and bile. Thirty-one, 35, and 36% of CO₂-pretreated E. stiedae oocysts were activated after 1 hr incubation in trypsin and bile at 37, 39 or 41 C, respectively, whereas 1, 2, and 20% activation occurred in nonpretreated oocysts incubated at the same temperatures. Sporozoites in 99-100% of I. canis oocysts were activated after 10 hr treatment in trypsin and bile with or without 1 hr CO₂-pretreatment at 23, 37, 39 or 41 C.     

Eimeria acervulina, E. brunetti, and E. maxima: immunity in chickens with low multiple doses of mixed oocysts.

Young chickens inoculated with multiple low doses of mixed oocysts of Eimeria acervulina, E. brunetti, and E. maxima had a high level of resistance to reinfection with a mixed challenge dose on Day 28, Day 84, or Day 140. Immunity was enhanced when the number of immunizing doses was increased from three to four. Resistance was also high in birds maintained on a proprietary mixture of amprolium, ethopabate, and sulphaquinoxaline (Pancoxin-Merck, Sharp and Dohme Ltd.) during immunization, although immunity to E. acervulina was lower in these birds. Oocyst production was lower in birds given mixed infections as compared with that of birds given pure infections with similar doses of oocysts. Competition between species did not inhibit the development of immunity in birds given low doses of mixed oocysts.     

Positive periodic acid-Schiff staining of acid mucopolysaccharides

Synopsis Evidence is presented to demonstrate that acid mucopolysaccharides will stain with the periodic acid-Schiff technique following prolonged oxidation with periodic acid. Smears of purified acid mucopolysaccharides begin to stain slightly with the Schiff reagent after 4 hr of periodate oxidation and reach an optimal staining intensity some time between 7 and 16 hr. The acid mucopolysaccharides in umbilical cord and cock's comb sections begin to stain at about 7 hr, reaching an optimum between 16 and 24 hr. It is suggested that the mechanism of staining of acid mucopolysaccharides in the PAS technique appears to derive from cleavage oftrans groups of the hexuronic acid fraction which require prolonged oxidation since the theoretical more yield is reached with difficulty. Moreover, it would appear that the mechanism of staining with glycogen under the usual conditions of PAS staining perhaps derives from end-group oxidation and subsequent Schiff staining of the engendered aldehydes since glycogen is almost entirely in thetrans configuration. Acid mucopolysaccharides, on the other hand, will not stain uncer the usual conditions as they appear to have a negligible proportion of end groups.     

Observations on Eimeria raillieti in the "Slow-worm", Anguis fragilis (Reptilia, Anguidae)

SYNOPSIS. The oocysts, sporulation process, and endogenous stages of Eimeria raillieti (Léger, 1899) Galli-Valerio, 1930 from the slow-worm, Anguis fragilis , in England are described. The oocysts average 18 × 15 μ. Schizonts, microgametocytes and macrogametocytes were found in the ileum, and macro-gametocytes alone in the duodenum.     

Electron Microscope Studies on Eimeria perforans (Sporozoa)

SYNOPSIS. By means of electron microscopy, a study has been made of the fine structure of the macrogametocytes, microgametocytes and oocysts of Eimeria perforans from the intestine of the wild rabbit (Oryctolagus cuniculus). The parasites lie in a vacuole within the host cell. The surface of the gametocytes is not plain, but displays irregular protrusions. A large intranuclear body can be detected within the macrogametocytes. Similar structures are also found within the cytoplasm. Within the latter there exists a large spread out reticulum, the channels and vesicles of which concentrate especially close to the nuclear membrane. Tubuli are seen in the numerous mitochondria, which often have a dumb-bell shape.
In most of the gametocytes irregular, strongly osmiophilic lipid inclusions are observed, which always are surrounded by the endoplasmic reticulum. Strange folded ovoid bodies are found within the cytoplasm of the oocysts. Nothing can be told with certainty of their nature and function. Probably they represent specific storage bodies.     

Failure of Development of the Sexual Phase of Eimeria intricata in Heavily Inoculated Sheep*

SYNOPSIS Each of 4 bottle-fed lambs received totals of 3500-8100 sporulated oocysts of Eimeria intricata in series of 3-6 inoculations during 4- to 14-day periods; all produced oocysts of the parasite. The periods of patency in some infections indicated that only the earliest inoculations of a series may have produced oocysts. Fifteen other bottle-fed lambs were inoculated, each with a single dose of 0.01-2 million sporulated oocysts, and necropsied at intervals of 1-2 days over 29 days. Parasites apparently resulting from the inoculations were found in only the lambs killed up to and including 17 days postinoculation; the most advanced stages were 2nd generation schizonts. It was concluded that a reaction occurred against the large numbers of asexual stages of E. intricata, which were evidently present, and the infections were terminated before formation of sexual stages.     

Eimeria cicaki sp. n. and Isospora thavari sp. n. from the House Lizard Gehyra mutilata Boulenger in Malaysia*

SYNOPSIS. Oocysts and endogenous stages of new species of Eimeria and Isospora from the house lizard, Gehyra mutilata, are described. The ellipsoid to subspherical 2-layered oocysts of E. cicaki averaged 24.0 × 21.0 μm. Polar granules are present. Micropyle and oocyst residuum are absent. Ellipsoid sporocysts average 12.2 × 9.0 μm. A sporocyst residuum is present, but the Stieda body is absent. Endogenous stages are in epithelial cells of the small intestine. The subspherical single-layered oocysts of I. thavari average 23.8 × 22.8 μm. The polar granule is present; micropyle and oocyst residuum are absent. Ellipsoid sporocysts average 12.8 × 9.4 μm. Stieda body and sporocyst residuum are present. There are endogenous stages in epithelial cells of the small intestine.     

Etude histochimique des Mucopolysaccharides de lépiderme et des organes récepteurs du système de la ligne latérale du gymnarque,Gymnarchus niloticus

Résumé Diverses réactions histochimiques (A.P.S. et bleu de toluidine) démontrent que l'épiderme du Gymnarchus niloticus est riche en mucopolysaccharides. Ils se rencontrent à la fois dans le cytoplasme des cellules superficielles de l'épiderme et au niveau des organes récepteurs cutanés de la ligne latérale. Les mucopolysaccharides dans les cellules épidermiques superficielles sont neutres.Les mucopolysaccharides des cavités intraépidermiques des organes de type B et C et du canal de l'organe de type A sont métachromatiques, La métachromasie est due à la présence d'un radical acide carboxylé. Les particularités dans la constitution et la répartition du mucus au niveau de ces trois organes résident dans le fait que l'invagination de la cellule sensorielle de l'organe de type A est occupé par du mucus neutre et que le cytoplasme de la petite cellule de l'organe de type C contient du mucus acide de même nature que celui de la cavité intraépidermique.Il faut admettre que l'origine des mucopolysaccharides réside dans les cellules de soutien qui entourent la cellule sensorielle.

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Histochemical investigation of the mucopolysaccharides in the epidermis and lateral line sensory organs of gymnarchus niloticus

Summary The mucopolysaccharide content of different epidermal structures in Gymnarchus niloticus has been demonstrated by two histochemical methods, periodic acid Schiff (P.A.S.) and toluidine blue. Mucopolysaccharides are present in the cytoplasm of the superficial cells and in the three specific cutaneous sensory organs of the lateral line system, type A, B and C.The superficial epidermal cells contain only neutral mucopolysaccharide.The mucopolysaccharides of the intraepidermal cavities in the type B and C organs as well as in the pores of the type A organ are acid; the metachromatic reaction of the toluidine blue demonstrates the presence of an acid carboxylic radicle.Histochemical analysis of the pore contents of the type A organ reveals that the mucus in the pore is acid while that of the sensory cell invagination is neutral.Histochemical differences were determined between the two sensory cells of the organ type C: the cytoplasm of the small sensory cell contains acid mucopolysaccharides while that of the large sensory cell is free of it, although the cytoplasmic invagination (extracellular) of the latter is filled with acid mucus.The results suggest that the mucus in each organ type is produced by the supporting cells surrounding the sensory cells.

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Je tiens à remercier le Docteur T. Szabo, dont les conseils pour la présentation de ce travail m'ont été des plus précieux.

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Ce travail a été aidé par le contrat No. 659-535 de la D.R.M.E. conclu avec le Docteur T. Szabo, Maître de Recherche au C.N.R.S. Avec la participation de Madame L. Planque, technicienne au C.N.R.S.     

The Biology and Pathogenicity of a Recent Field Isolate of Eimeria praecox Johnson, 1930

A recent isolate of Eimeria praecox, strain G, was obtained from Georgia and purified. Studies of the life history, pathogenicity, and cross-immunity of the isolate were conducted to verify its identity. In inoculated three-week-old chickens, the occurrence of merogony and gametogony was limited to the superficial epithelium of the upper intestine. Oocysts, 23 × 19.5 äm, with a shape index of 1.17 were first observed 83 h after inoculation. Mortality and morbidity were not observed in any of the experimental birds. However, there was a positive correlation between dose of oocysts, reduced weight gain, and the incidence of exudative diathesis. These studies showed that E. praecox depresses weight gains in chickens and may be of economic importance. Although complete immunity to avian coccidiosis is believed to be species specific, chickens immune to E. praecox (G) or E. acervulina had a degree of cross-immunity to a heterologous challenge. Electrophoretic analysis of glucose phosphate isomerase and lactate dehydrogenase prepared from the European strain of E. praecox and E. praecox (G) showed no differences, confirming the identity of the isolate as E. praecox.     

Parental Development of Eimerian Coccidia in Sandhill and Whopping Cranes1

In contrast with isosporoid species of coccidia that have established extraintestinal phases of development, the eimeriids, except for a few species, generally have been considered inhabitants of the intestinal tract. Eimeria infection in sandhill cranes (Grus canadensis) and whooping cranes (G. americana) may result in disseminated visceral coccidiosis. Nodules were observed in the oral cavity of 33% (n = 95) of the G. canadensis at the Patuxent Wildlife Research Center (PWRC) in Laurel, MD. Necropsy of six of the afflicted cranes revealed granulomatous nodules in many tissues and organs. Histologic studies disclosed protozoan organisms morphologically resembling schizonts in the granulomas, and endogenous stages of coccidia were present in the intestines of four birds. Fecalysis of three of four sandhill cranes yielded oocysts of E. reichenowi and E. gruis. Only E. reichenowi-type oocysts were recovered from a dead whooping crane sample. Domestic broiler chicks each intubated with about 1 times 10⁶ pooled sporulated oocysts of E. reichenowi and E. gruis were not infected. Exposure of six incubator-hatched and hand-reared sandhill crane chicks to oocysts artificially (two chicks) and naturally (four chicks) resulted in typical infection of intestinal epithelium with invasion of subepithelial tissues extending to the muscular layer and widespread extraintestinal development. Asexual and sexual stages occurred primarily in macrophages in the liver, spleen, heart, and lung. In the lung, oocysts were found in bronchial exudate and epithelial lining cells. Six of ten G. canadensis chicks, one adult G. americana, and three of five G. americana chicks that died naturally at PWRC had disseminated visceral coccidiosis.