Second-generation Merozoites Research Articles

Eimeria tenella: immunogenicity of the first generation of schizogony.

The life-cycle of a precocious and attenuated line (WisF96) of Eimeria tenella, derived from the Wisconsin (Wis) strain, contained only the first of the three generations of schizogony undergone by the parent strain. The reproductive capacity of WisF96 was less than that of the parent strain by a factor of about 2000, but inoculation of chickens on two occasions with a large number of its oocysts induced resistance against challenge with oocysts, or with second-generation merozoites of Wis. The immunizing abilities of the attenuated line and its parent were compared by priming groups of chickens with numbers of oocysts of WisF96 or Wis, designed to produce infections of equal magnitude in terms of oocysts production (standard inocula), and then challenging with oocysts of Wis. The results suggested that an equivalent parasite mass of WisF96 was more immunogenic than Wis. This was confirmed by the finding that, even when the priming dose of Wis was increased 10-fold, the standard inoculum of WisF96 still produced the greater protective effect. These results indicate that the first generation of schizogony of E. tenella is highly immunogenic.

Morphological effects of arprinocid on developmental stages of Eimeria tenella and E. brunetti.

Medication of chicks with 70 p.p.m. arprinocid in the food, starting 2 days prior to inoculation with Eimeria tenella, resulted in decreased oocyst production and oocyst sporulation. The main morphological alteration seen during the development of the second-generation schizont was a reduction in the number of second-generation merozoites due to incomplete merogony, resulting in large masses of vacuolated residual cytoplasm. In places around the developing macrogamete the integrity of the parasitophorous vacuole was lost by either the host cell limiting membrane making contact with the parasite pellicle, or the disruption of one or both of these surfaces apparently brought about by the intervention of the host cell mitochondria. Thirty p.p.m. arprinocid reduced sporulation of the oocysts of E. brunetti and caused enlargement of the perinuclear space in the late merozoites and swelling of the endoplasmic reticulum around wall-forming bodies II.

Immune Complexes Induce the Formation of an Unusual Micropore in Second-Generation Merozoites of Eimeria tenella

kept at 4 C for approximately 40 hr to eliminate the sharp interfaces between the layers. Afterwards 2 ml of the suspension having sheath fragments, exsheathed as well as few sheathed mf, as obtained earlier, was applied over the gradients. After centrifugation at room temperature (1,000 g for 20 min), 2 distinct bands were visualized. Each band was removed using an Eppendorf pipette. Spans of bands, expressed in terms of the ml graduations on the centifugation tube were 1.6 to 1.70 and 1.3 to 1.5 respectively. The exsheathed mf along with a few intact mf were t at 4 C for ap roximately 40 hr to eliminate sharp interfaces between the layers. Afters 2 ml of the suspension having sheath fragretained at the bottom while the sheath fragm nts were at the top. The method presented offers a simple and rapid means for collecting the sheath in a reasonably pure form. The yield obtainable is approximately 20.0-50.0 microgram dry weight per 106 mf. This investigation received financial support from the Indian Council of Medical Research, New Delhi, India. The author gratefully acknowledges the contribution of Dr. P. K. Murthy, and wishes to thank Dr. S. Ghatak and Dr. A. B. Sen for their interest in this work.

The asexual pre-cyst development of Sarcocystis tenella in experimentally infected specific-pathogen-free lambs

Twenty-two lambs raised under sporozoa-free conditions were infected at weaning with 0.25–2.0 million Sarcocystis tenella sporocysts obtained from dogs and then necropsied at intervals between 1.3 h and 41 days post-inoculation (dpi). Fixed and frozen sections from 47 different tissues from each lamb were examined respectively by a variety of histochemical stains and by indirect fluorescent-antibody staining. The remnants of excysted sporocysts were found within the gastro-intestinal tract between 1.3 h and 3 dpi. No enteric proliferative stage of the parasite was detected. First-generation meronts (schizonts) were detected from 6 to 19 dpi within the endothelial cells of arterioles in most organs and tissues. The meronts were undifferentiated from 6 to 9 dpi but were mature in appearance from 12 to 19 dpi (measuring 22.3 × 13.4 μm and containing 18–28 merozoites). Second-generation meronts were detected from 21 to 34 dpi within the endothelial cells of capillaries throughout the entire body. They were undifferentiated from 21 to 23 dpi but appeared mature from 25 to 34 dpi (measuring 15.2 × 10.6 μm and containing 18–38 merozoites). Several smaller meronts were then detected at 36 dpi within cells of the mononuclear phagocytic system. They were all mature in appearance (measuring 7.4 × 5.1 μm and containing 6–9 merozoites) and are thought to have formed facultatively following the incorporation of some second-generation merozoites into phagocytic cells. Only developing cysts were then detected at 41 dpi throughout the cardiac and skeletal musculature. Elements in both first- and second-generation merozoites stained markedly with haematoxylin and eosin, periodic acid-Schiff's, alkaline Giemsa-colophonium and basic fuchsin stains. However, only second-generation meronts and developing cysts exhibited strong specific reactions with the fluorescent-antibody stain.

Development of Eimeria meleagrimitis Tyzzer from sporozoites and merozoites in turkey kidney cell cultures.

Sporozoites and 1st-, 2nd-, and 3rd-generation merozoites of Eimeria meleagrimitis were inoculated into primary cultures of turkey kidney cells. In vitro-excysted sporozoites developed into mature macrogamonts in 8 days; in vivo-excysted sporozoites developed into 2nd- or 3rd-generation schizonts within 5 to 7 days. First-generation merozoites obtained from infected turkeys produced mature 2nd-generation schizonts within 24 h. Second-generation merozoites from turkeys produced mature macrogamonts and oocysts within 72 h, whereas 3rd-generation merozoites produced these stages within 48 h. The oocysts that developed from 3rd-generation merozoites sporulated at 25 C and were infective for turkeys. The timing of the early stages and the intervals between schizogonic generations in cultures were comparable with those in turkeys. Morphologic parameters, however, indicated that some differences existed between in vitro and in vivo development. Second- and 3rd-generation schizonts and gamonts that developed after inoculation of cultures with merozoites were similar to stages in turkeys. Oocysts, however, were significantly smaller (P less than 0.05) in cultures. All stages that developed after inoculation of cultures with sporozoites were smaller (P less than 0.05) than their in vivo counter parts.

Preparation and Purification of Merozoites of Eimeria tenella

Second-generation merozoites of Eimeria tenella were obtained from both infected cecal tissue and infected chorioallantoic membranes of embryonated eggs. The merozoites were harvested from the tissue by incubation with hyaluronidase, yielding approximately 4 times 10(7) merozoites per cecum and 3 times 10(6) merozoites per chorioallantoic membrane. Subsequent purification of the merozoites by density centrifugation and glass bead filtration resulted in a 54% overall yield and a final preparation of approximately 95% purity. The viability of such preparations was established by inoculation of the merozoites to the ceca of chickens, resulting in oocyst production by 48 hr. This purification procedure allows for a rapid preparation of E. tenella during its second asexual stage in sufficient quantity and purity for biochemical study.

Ultrastructural development of first- to second-generation merozoites in Eimeria contorta Haberkorn, 1971.

The development of first-generation merozoites to second-generation schizonts and merozoites of Eimeria contorta in one of its natural hosts, the mouse, was investigated with the electron microscope. Merozoites inside a host cell show a marked U-shape and a degeneration of the inner-pellicular membrane complex prior to transformation into schizonts. These processes closely resemble those seen in transforming sporozoites. In young schizonts with about 3-5 nuclei, the Golgi-adjuncts (structures of unknown function) form a large interconnected network. Nuclear divisions in growing schizonts involve the formation of a centrocône, which develops in a pocket-like indentation of the nuclear envelope. At least one centriole is present immediately adjacent to this indentation. In a later stage, the centrocône forms a conical nuclear protrusion directed towards a merozoite-anlage. This developing merozoite contains anlagen of a conoid, of rhoptries, and of micronemes and a refractile body in addition to the nucleus, centrioles, and a Golgi-adjunct. The merozoite-anlage is limited by a triple unit membrane complex. Schizonts give rise to 8-15 second-generation merozoites. Interesting features of these merozoites are the high number of micronemes, the finding of one single large mitochondrion per merozoite, and the occurrence of 26 subpellicular microtubules, i.e. the same number as in sporozoites of E. contorta. At the end of their development, merozoites come into direct contact with the host cell cytoplasm as the parasitophorous vacuole breaks down.

Fine structure of the first and second generation merozoites of Eimeria necatrix.

The fine structure of the first- and second-generation merozoites ofEimeria necatrix was studied in experimentally infected chickens. The outer membrane and inner membrane complex were separated by a space of approximately 0.019 μm. The anterior polar ring (0.49 μm), the posterior pore (0.33 μm), subpellicular microtubules (22 in number), conoid (0.34 μm) and anterior annuli were similar to those found in most eimerian merozoites. In the region of the conoid, the subpellicular microtubules were in spaces formed by the fluted inner membrane. A fine osmiophilic “canopy” was seen over the anterior end of the merozoite. The rhoptries, numerous micronemes, 1 to 5 electron-dense refractile bodies, large elongate mitochondria, and polysaccharide granules were found anterior to the nucleus. Cisternae of rough endoplasmic reticulum, 1 or 2 elongate mitochondria and polysaccharide granules were seen posterior to the nucleus.

An electron microscopal study of Eimeria tenella grown in the liver of the chick embryo

Lee D. L. and Long P. L. 1972. An electron microscope study of Eimeria tenella grown in the liver of the chick embryo. International Journal for Parasitology, 2: 55–58. Chick embryos were inoculated intravenously with sporozoites of Eimeria tenella on the 11th day of incubation. Dexamethasone was inoculated into the allantoic cavity when the embryos were 10 days old. The liver was removed from these chick embryos 109, 160 and 164 h after inoculation of the sporozoites into the embryos and fixed for ultrastructural work. Early schizonts of the second generation were usually found in fibrocytes of the connective tissue of the liver and, less frequently, in endothelial cells lining the sinusoids and in hepatocytes. Schizonts were similar in appearance to those found in infections in the bird. Gamonts were found in hepatocytes but were never numerous. Second-generation merozoites were detected in the sinusoids in the space of Disse, in endothelial cells, in hepatocytes, in fibrocytes and in macrophages. Those merozoites which were inside macrophages appeared to be in the process of being destroyed. These results are consistent with the known behaviour of the parasite in tissue culture and in the normal site. They also suggest affinities with other sporozoa.

A fine structural study on the merozoite of Eimeria tenella with special reference to the conoid apparatus.

The ultrastructure of the merozoite of Eimeria tenella has been studied by means of electron microscopy. The first-generation merozoite is approximately 3·4 μm in length, and 1·2 μm in width, while the second-generation merozoite is approximately 10·5 μm in length and 1·5 μm in width.The cell wall of the merozoite consists of a double membrane, but at the anterior extremity the existence of a fluted collar gives the appearance of two narrow double membranes separated by a zone of less electron-dense material. Twenty-four surface fibrils are distributed around the periphery; they extend along the entire length of the organism and lie beneath the double limiting membrane.The anterior end of the merozoite is distinguished by a conoid apparatus which includes several components. A bulbiform outer annulus is invested by a fluted collar, and itself encloses an extrusible papilla. Two osmiophilic fibrils, the paired organelles, arise within the extrusible papilla and extend longitudinally into the cytoplasm. Twenty-four smaller fibrils, or toxonemes, also arise within the conoid and pass back into the main body of the organism.The cytoplasm of the merozoite includes mitochondria, glycogen, dense elliptical granules and endoplasmic reticulum, together with a definite Golgi complex. A nucleus is located in the posterior third of the organism and is enclosed by a perforated double membrane. At the posterior extremity the double membrane which bounds the organism is broken by a pore 700 A in diameter.Our sincere thanks are due to Mr P. Richmond for technical assistance, and we are grateful to the Central Veterinary Research Laboratory, Weybridge, for supplying the strain of Eimeria tenella.

Eimeria duodenalis sp.nov. from English covert pheasants (Phasianus sp.)

A new species of coccidium E. duodenalis sp.nov., has been described, from the pheasant. It is characterized by the subspherical shape of the oocyst, the lack of polar granules and the presence of large sporocystic residual bodies. Endogenous stages are found in the epithelial cells lining the villi, and occur chiefly in the duodenum and upper intestine. Second-generation merozoites form gametocytes and/or third-generation schizonts. The prepatent period is 5 days.E. duodenalis is moderately pathogenic, doses of 50 000 oocysts causing a 30% mortality and a significant weight loss in the survivors. Treatment with a range of sulphonamides in the drinking water was successful as was preventive treatment with Sulphaquinoxaline mixed with the food.Acknowledgements are due to Mr S. F. M. Davies for initiating this programme and carrying out a number of the animal inoculations, to Dr Helen Hein for help and advice in the design of the experiments and to Dr L. P. Joyner for advice in the preparation of the manuscript. The author also wishes to thank Miss C. N. Hebert for the statistical analysis of weight gains and Mr M. A. Peirce, Mr G. L. Choon, Miss J. Saddington and Miss M. Blore for technical assistance.

Serum Lysins in Chickens Infected with Eimeria tenella

Sera from fowls infected with Eimeria tenella acquired the capacity to lyse second-generation merozoites in vitro. Serum became positive for lysins approximately 8 days following a moderate oral inoculation of oocysts although the time of appearance and the intensity of the lytic reaction were influenced by the size of the oocyst dose. Surgically cecectomized birds produced lysins, presumably in response to an active infection in the large intestine. The lytic response in bursectomized chickens was weaker than in nonbursectomized controls. First-generation merozoites formed approximately 72 hr after infection probably provided a major stimulus to lysin production. The lytic effect was diminished in heated immune serum. Dialysis against 0.85% NaCl caused no diminution of the lytic activity. Fractionation by gel filtration and separation by disc electrophoresis showed that the behavior of the lytic component was that which would be expected of globulins of intermediate size. Sporozoites, unlike merozoites, were lysed in normal serum and this effect was enhanced following an infection. Evidence for the presence of an antibody response explaining the acquired resistance to cecal coccidiosis has been sought by many investigators. Indirect evidence for protective humoral resistance was given by Burns and Challey (1959) and by Horton-Smith, Beattie, and Long (1961). In both studies, protection was induced in one cecal pouch through an immunizing infection in the other. Direct evidence has also appeared that an antibody response is elicited during infection of the chicken with Eimeria tenella. McDermott and Stauber (1954) reported the agglutination of second-generation merozoites by sera of chickens recovered from cecal coccidiosis. Rose and Long (1962) demonstrated precipitins in immune sera, but showed that their presence was not required for a solid resistance to infection. Further evidence for the lack of protective humoral antibody was indicated by the inability of Pierce, Long, and Horton-Smith (1963) to passively transfer immunity to E. tenella with hyperimmune serum. Long, Rose, and Pierce (1963) and Rose (1963) reported the in vitro lysis of E. tenella sporozoites and merozoites by sera from resistant chickens. The lytic activity resembled a typical complement-requiring antibody reaction. The presence of serum lysins was evidently not required for immunity since birds immunized by injections of parasite extracts Received for publication 21 September 1964. * This research was supported by research grant AI-04101 from NIAID, U. S. Public Health Service and in part by the Graduate School Research Committee, University of Wisconsin. yielded lytic sera yet remained susceptible to oral infection with oocysts. This paper confirms the presence of lysins in the serum of infected chickens and provides some additional information on factors influencing their appearance and activity. We have als made a preliminary characterization of the lytic factor from immune serum. MATERIALS AND METHODS White Leghorn cockerels were obtained as dayold chicks from local hatcheries and were fed an all mash ration containing no antibiotics or coccidiostats. The birds were kept in isolation until needed for experimental use. Periodic fecal examinations for oocysts were made to assure freedom from accidental infections. Fresh oocyst cultures were prepared routinely from the ceca of donor birds 7 days after inoculation with 2 to 5 X 103 oocysts. Second-generation merozoites were obtained from scrapings of the cecal mucosa of birds killed 90 hr after inoculation of 1 to 2 X 10? oocysts. The scrapings were homogenized in 80 ml of 0.85% saline in a micro-Waring blendor and the mixture was poured into two 50-ml centrifuge tubes. After standing for about 10 min the homogenate became stratified into a lower liquid portion, a flocculent layer above it, and a foamy layer at the top. The liquid portion was removed with a pipette and centrifuged. The sediment contained large numbers of merozoites, host erythrocytes, and tissue debris. After two washings with saline the merozoites were resuspended in 8.0 ml of saline. Suspensions of first-generation merozoites were prepared in the same fashion from chicks infected 72 hr previously. Much cleaner merozoite suspensions were obtained by passing homogenates prepared as above through a column (2.0 by 4.0 cm) composed of Whatman ashless cellulose powder equilibrated against 0.85% saline. This removed much of the

The Effect of Cecal Coccidiosis on the Blood Cells of the Domestic Fowl: I. A Comparison of the Changes in the Erythrocyte Count Resulting from Hemorrhage in Infected and Mechanically Bled Birds. The use of the Hematocrit Value as an Index of the Severity of the Hemorrhage Resulting from the Infection

INTRODUCTIONONE of the more noticeable manifestations of cecal coccidiosis is the hemorrhage that results when chickens become severely infected with the disease. Because the disease has such pronounced effect on the chickens, various studies have been carried out to explain the physiological changes that occur in the infected animal. Edgar (1944) in his study on the life cycle of the sporozoan parasite, Eimeria tenella, the causal organism, substantiated the findings of Tyzzer (1929) and others who observed that the hemorrhage begins in the infected cecal mucosa when the second-generation merozoites approach maturity. Edgar (1944) suggests that the hemorrhage is the result of the pinching off of mucosal capillaries by pressure resulting from the enlargement of epithelial cells in which the intracellular parasitic development (schizogony) has been taking place. Late in the fourth day and during the fifth day of infection, the second-generation merozoites are discharged from the infected epithelial …