Observations on Haemogregarina balli sp. n. from the Common Snapping Turtle,Chelydra serpentina*

SYNOPSIS. Haemogregarina balli sp. n. is described from the blood and organs of the common snapping turtle Chelydra serpentina serpentina and from the gastric and intestinal ceca of the presumed invertebrate hosts, the leeches Placobdella parasitica and Placobdella ornata. In the peripheral blood of the turtle, male and female gametocytes and immature erythrocytic schizonts are found within erythrocytes. The maturation of erythrocytic schizonts containing 6–8 merozoites is recorded from liver imprints. Schizonts with 13–25 merozoites are found in various cells of the liver, lung and spleen. In the gastric ceca of the leeches the host erythrocytes are digested, releasing the gametocytes and immature erythrocytic schizonts. Immature erythrocytic schizonts degenerate. Association of the gametocytes occurs in the intestinal ceca. The microgametocyte apparently gives rise to 4 nonmotile microgametes, one of which fertilizes the macrogamete while the other remain as condensed, residual nuclei on the periphery of the developing oocyst. The oocyst increases in size with maturity. A mature oocyst produces 8 sporozoites from a single germinal center. Sporozoites liberated from the oocyst are found in the tissues of the leech. Transovarial transmission of the parasite does not occur in the turtle. Attempts at experimental transmission failed. Previously unfed (control) leeches were negative for the parasite. Haemogregarina balli is compared with other haemogregarines described from C. serpentina. Features of species of Haemogregarina and Hepatozoon as well as the taxonomy of these genera are discussed.     

Development of First-Generation Schizonts of Eimeria bovis in Cultured Bovine Cells

SYNOPSIS. Monolayer primary and secondary cultures of embryonic bovine kidney, spleen, intestinal and testicle cells, and secondary cultures of embryonic bovine thymus, maintained in lactalbumin hydrolysate, Earle's balanced salt solution and ovine serum were observed for a maximum of 21 days after inoculation of E. bovis sporozoites. The sporozoites entered the cells in all of these cultures but underwent development only in primary cultures of kidney and intestinal cells and in secondary cultures of kidney, spleen, thymus, intestinal, and testicle cells. In acellular media, the sporozoites retained motility no longer than 21 hr. In the cell cultures, free motile sporozoites were seen for as long as 18 days after inoculation. Sporozoites entered cells anterior end first; the process of penetration required a few seconds to about a minute. Sporozoites were also observed leaving host cells. Intracellular sporozoites were first seen 3 min after inoculation; they were observed at various intervals up to 18 days after inoculation. In transformation of sporozoites into trophozoites a marked change in size and appearance of the nucleus took place before the change in shape of the body occurred. Trophozoites were first found 7 days after inoculation, multinucleate schizonts after 8 days, and schizonts with merozoites after 14 days. Schizonts containing merozoites were seen only in kidney, spleen, and thymus cells. The mature schizonts were smaller and represented a much lower proportion of the total number than in comparable stages of infections in calves. Schizonts with many nuclei occurred in intestinal cells; the most advanced stage seen in testicle cells was the binucleate schizont. Nuclear and cytoplasmic changes were observed in the infected cells.     

The haemosporidian parasites of bats with description of Sprattiella alecto gen. nov., sp. nov

Four species of Haemoproteidae were found in Pteropus alecto Temminck, 1837 in Queensland, Australia: i) Johnsprentia copemani, Landau et al., 2012; ii) Sprattiella alecto gen. nov., sp. nov., characterised by schizonts in the renal vessels; iii) Hepatocystis levinei, Landau et al., 1985, originally described from Pteropus poliocephalus Temminck, 1825 and, experimentally from Culicoides nubeculosus and found in this new host and for which features of the hepatic schizonts are reported; iv) gametocytes of Hepatocystis sp. which are illustrated but cannot be assigned to a known species. A tentative interpretation of phylogenetic characters of haemosporidians of bats is provided from the morphology of the gametocytes and localisation of the tissue stages with respect to recent data on the phylogeny of bats.     

Pathogenicity of Leucocytozoon caulleryi for specific pathogen-free laying hens.

The pathogenicity of Leucocytozoon caulleryi against specific-pathogen-free laying hens was investigated. Many large schizonts (second-generation schizonts) of L. caulleryi were seen in the ovary and oviducts of chickens. Edema and pressure atrophy of the adjacent tissues were associated with these schizonts. The eggshell-secreting portion of the uterus exhibited the most severe damage in the oviduct. This experiment reconfirms that L. caulleryi may stop egg production in laying hens, presumably as a result of damage to ovaries and oviducts.     

A Malaria Parasite, Plasmodium aurulentum sp. nov. from the Neotropical Forest Gecko Thecadactylus rapicaudus

SYNOPSIS. Plasmodium aurulentum sp. nov. from the neotropical forest gecko Thecadactylus rapicaudus in Panama is characterized by oval or round to lenticular gametocytes, 6–22 nuclei in crudely fan-shaped schizonts, and light golden pigment masses. A prominent, pinkish red-staining mass, present in older schizonts, disappears by the time schizonts reach maturation.     

Schizogonic development of Leucocytozoon smithi.

The schizogonic development of Leucocytozoon smithi in the liver of experimentally infected turkey poults was examined by electron microscopy. Following intraperitoneal injection, sporozoites migrated to the liver and entered hepatic cells to become intracellular trophozoites. Three to four days post inoculation (PI), trophozoites underwent asexual multiple fission known as merogony or schizogony. Two generations of schizonts were observed. The primary or first generation schizonts, abundant on day 4 PI, appeared as interconnected cytoplasmic masses (pseudocytomeres). Each pseudocytomere was enclosed by a membranous vacuole and contained varying numbers of nuclei. As nuclear division and growth of the schizonts continued, larger discrete cytoplasmic masses or cytomeres were formed with rhoptries and multiple nuclei in various stages of division. Synchronous multiple cytoplasmic cleavage of the schizont resulted in the formation of numerous uninucleate merozoites. Second generation schizonts, which developed from hepatic merozoites released from primary schizonts, were abundant in hepatocytes on day 6 PI. Although tissue samples from liver, lung, spleen, kidney, intestine, brain, blood vessels and lymph nodes were examined, schizogonous forms were observed in liver only. No megaloschizonts were detected in any host tissue examined. Schizogonic development was completed by day 7 PI as merozoites developed into gametocytes within mononuclear phagocytes.     

Simultaneous purification of merozoites and schizonts of Eimeria tenella (Apicomplexa) by Percoll flotation and assessment of cell viability with a double fluorescent dye assay.

The asynchronous development of Eimeria tenella in orally infected chickens makes it possible to purify second generation merozoites (meros) and shizonts from a single mucosal homogenate. After centrifugation in 30% Percoll in phosphate-buffered saline (Percoll-PBS), debris, villi, and schizonts float, whereas meros and erythrocytes are pelleted. Erythrocytes are lysed by a mild hypotonic shock; meros are filtered through a cotton wool plug and collected by centrifugation. The 30% Percoll-PBS supernatant fraction is diluted to 25% Percoll-PBS and centrifuged to sediment mature schizonts. By repeated slow-speed centrifugation, schizonts are separated from nuclei and small-sized debris. In less than 3 hr, 8.8 +/- 2.3 x 10(8) meros and 7.2 +/- 3.9 x 10(6) schizonts are collected from 10 infected chickens. Contamination with host material is 2% for meros but variable for schizonts. For the assessment of cell viability, ethidium bromide (EB) and acridine orange (AO) have been used as markers for dead and living cells, respectively, in a single step method. More than 95% of the schizonts and meros accumulate AO and no EB, whereas lysed erythrocytes and all cells hosting a schizont are permeable to EB. After incubation of meros and schizonts in synthetic media with [5,6- 3H]uracil, label accumulates in the perchloric acid-soluble and -insoluble fractions, indicating transport, salvage, and incorporation of the pyrimidine precursor in nucleic acids. If stored on ice, meros and schizonts retain metabolic activity for at least 5 hr, but metabolism declines rapidly during incubation at 41 C.     

Redescriptions of Eimeria irresidua Kessel & Jankiewicz, 1931 and E. flavescens Marotel & Guilhon, 1941 from the domestic rabbit

Eimeria flavescens and E. irresidua from the domestic rabbit are redescribed. The relatively smaller ovoidal oocysts of E. flavescens which measure on average 31.7 X 21.4 micrometer, possess a wide micropyle at the broad end. First-generation schizonts of this species develop deep in the glands of the lower small intestine. Merozoites migrate to the caecum and colon where second, third and fourth-generation schizonts develop in the superficial epithelium followed by the fifth-generation schizonts and gametocytes which form in the glands. In young Dutch rabbits E. flavescens is very pathogenic; low doses of oocysts produce a severe enteritis with high mortality and morbidity. The larger, broadly ellipsoidal oocysts of E. irresidua measure on average 38.4 X 23.2 micrometer and often possess a very small cryptic oocyst residuum. The endogenous stages develop in the small intestine only; first-generation schizonts in the glands and second-generation schizonts in the lamina propria whilst third and fourth-generation schizonts and gametocytes develop in the villous epithelium. E. irresidua is not pathogenic in young Dutch rabbits; even heavy infections produce only a transient pause in weight gain.     

Isolates of Sarcocystis falcatula-like organisms from South American opossums Didelphis marsupialis and Didelphis albiventris from S?o Paulo, Brazil.

Isolates of Sarcocystis falcatula-like organisms from South American opossums were characterized based on biological and morphological criteria. Sporocysts from intestinal scrapings of 1 Didelphis marsupialis and 8 Didelphis albiventris from S?o Paulo, Brazil, were fed to captive budgerigars (Melopsittacus undulatus). Budgerigars fed sporocysts from all 9 isolates became ill and S. falcatula-like schizonts were identified in sections of their lungs by immunohistochemical staining. Sarcocystis falcatula-like organisms were cultured from lungs of budgerigars fed sporocysts from D. marsupialis and from lungs of budgerigars fed sporocysts from 3 of 8 D. albiventris. The 33/54 locus amplified by polymerase chain reaction from culture-derived merozoites contained both a HinfI endonuclease recognition site previously suggested to diagnose S. falcatula and a DraI site thought to diagnosed S. neurona. Development of the isolate from D. marsupialis was studied in cell culture; its schizonts divided by endopolygeny, leaving a residual body. Morphological and genetic variation differentiated this Sarcocystis isolate originating in D. marsupialis from the Cornell I isolate of S. falcatula. This is the first report of a S. falcatula infection in the South American opossum, D. marsupialis.     

Clinical Toxoplasma gondii, Hammondia heydorni, and Sarcocystis spp. infections in dogs

Concurrent infections with coccidians Toxoplasma gondii, Sarcocystis spp., and a Hammondia heydorni-like parasite were identified in tissues of three littermate pups on a Kelpie dog breeding farm in Australia. In total, 20 pups in four litters had died following vaccination with an attenuated distemper virus vaccine. Toxoplasma gondii tachyzoites were identified immunohistochemically in tissues of two dogs. Sarcocystis sp. sporocysts were seen in the intestinal lamina propria of two dogs. Asexual and sexual stages of H. heydorni-like parasite were found in enterocytes of the small intestine of two dogs. Ultrastructural development of schizonts and gamonts of this parasite is described. None of the protozoa in these dogs reacted with antibodies to Neospora caninum. Feeding of uncooked tissue of sheep was considered to be the likely source of infection for these coccidians in dogs.     

Scanning Electron Microscopy of Schizogony in Eimeria tenella

SYNOPSIS. Developing 2nd- and 3rd-generation schizonts of Eimeria tenella were found in the ceca of chicks infected orally with sporulated oocysts. Several free 2nd-generation schizonts, which varied in diameter from 11 to 21.6 μm, were found on the epithelial surface of the cecum. Some schizonts appeared to have lost merozoites. Other schizonts were intact, one of which was surrounded by an unbroken membrane that followed the contours of the merozoites. Third-generation schizonts, much smaller than 2nd-generation schizonts and with fewer merozoites, were found only on cut or fractured surfaces of the cecal tissue. Third-generation merozoites appeared shorter and thicker than those of the 2nd-generation and were attached to the schizont residuum. A form with conical protuberances and another with 4 triangular segments were found; they were believed to be developing stages 3rd-generation schizonts.     

A malarial parasite of Australian skinks, Plasmodium mackerrasae sp. n.

Plasmodium mackerrasae sp. n. parasitizes the Australian lizards Egernia cunninghami and E. striolata (Sauria: Scincidae). Described from an experimental host, E. whitei, it produces mature schizonts containing 6--12 nuclei arranged peripherally as a rosette, and round to oval gametocytes which are equal to or slightly smaller than host cell nuclei. Both schizonts and gametocytes parasitize all cells in the erythrocyte series. Presence of pigment in both asexual and sexual stages is correlated with maturity of the host cell. Asexual forms contain a single large vacuole, whereas mature gametocytes may show 1--4 vacuoles. Plasmodium mackerrasae resembles most closely P. sasai of Japan and P. tropiduri of tropical America. It differs from P. sasai by lacking fan-shaped schizonts and by having less heavily pigmented gametocytes, and from P. tropiduri by less variability in shape and greater vacuolation of the gametocytes. Host and geographic differences further support its distinction.     

A new hemogregarine from marine fishes.

Haemogregarina uncinata sp. n. is described from the blood of 2 marine eelpouts, Lycodes lavalaei and Lycodes vahlii (Perciformes: Zoarcidae). Erythrocytic schizogony occurred in peripheral and cardiac blood, but mature schizonts were restricted to the latter site. Mature and rupturing schizonts contained 10 to 30 merozoites, which were short and thick in small schizonts while slender and long in larger schizonts. Gametocytes developed in mature erythrocytes and displayed morphologic and morphometric characters that distinguished them from other species described. Syzygy and gamete formation occurred in the gut of a leech, Johanssonia sp. Each microgametocyte produced up to 4 apparently nonflagellated gametes. Oocysts developed intracellularly in the epithelial wall of the intestine and at maturity produced under 100 sporozoites from (apparently) several germinal centers. Sporozoites subsequently migrated to the probosces of the leeches. The failure to transmit the parasite to a sculpin (Myoxocephalus octodecemspinosus) and 3 Atlantic cod (Gadus morhua) via regurgitation by the leeches might be indicative of host specificity.     

Development of Eimeria callospermophili and E. bilamellata from the Uinta Ground Squirrel Spermophilus armatus in Cultured Cells*

SYNOPSIS. Cell lines or established cell lines of bovine, ovine or human origin and primary cells from whole embryos of groundsquirrels were used in a study of the in vitro development of Eimeria callospermophili and E. bilamellata from the Uinta ground squirrel, Spermophilus armatus. Monolayers in Leighton tube cultures were inoculated with sporozoites of either of these 2 species and examined with phase-contrast microscopy at various intervals. After such examination, coverslips were fixed in Schaudinn's or Zenker's fluid and variously stained. E. callospermophi sporozoites penetrated cells and underwent development to mature 1st generation schizonts in most cell types. At different times after inoculation, both species formed sporozoite-shaped schizonts, which later became spheroidal. Intracellular movements of sporo zoite-shaped schizonts of E. callospermophili were observed and such schizonts penetrated cells when freed by mechanical disintegration of the host cells. Merozoites were formed at the periphery of the schizont in both species. Mature 1st generation schizonts of E. callospermophili, with 6–14 merozoites, were first seen 15 hr after inoculation; the corresponding values for E. bilamellata were 12–27 merozoites and 4 days. Merozoites of both had anterior and posterior refractile bodies. Exposure to a trypsin-bile solution stimulated motility in merozoites of E. callospermophili. Second generation trophozoites and immature schizonts of E. callospermophili were seen in cultures of primary cells of whole ground-squirrel embryos 20–24 hr and 44–48 hr, respectively, after inoculation of sporozoites.     

Life Cycle of Isospora canis Nemeséri, 1959 in the Dog*

The life cycle of I. canis Nemeséri, 1959 was studied in experimentally infected dogs. Freshly sporulated oocysts were ovoid and 34–40 × 28–32 μm. The endogenous stages were found directly beneath the epithelium of the distal portion of the small intestinal villi. Most of the endogenous stages were in the lower 1/3 of the small intestine, but occasionally they were found in other portions of the small intestine. Three asexual generations were present. First-generation schizonts were 16–38 × 11–23 μm and contained 4–24 merozoites; mature 1st-generation merozoites were 8–11 × 3–5 μm. First-generation schizogony lasted up to 7 days after inoculation. Second-generation schizonts were 12–18 × 8–13 μm and contained up to 12 merozoites which were 11–13 × 3–5 μm. Second-generation schizogony was present on postinoculation days 6 and 7. Third-generation schizonts were formed by nuclear division of 2nd-generation merozoites. Most 2nd-generation merozoites underwent nuclear division without leaving the parasitophorous vacuole of the 2nd-generation schizont. Mature 3rd-generation schizonts were 13–38 × 8–24 μm and contained 6–72 merozoites. Third-generation merozoites were 8–13 × 1–3 μm. Third-generation schizogony was present on days 6–8 after inoculation. Mature macrogametes were 22–29 × 14–23 μm. Mature microgametocytes were 20–38 × 14–26 μm. Gametes were present on postinoculation days 7–10. Oocysts were present in tissue sections on postinoculation days 8–10 and 12. The prepatent period was 9–11 days.     

The fine structure of the endogenous stages of Eimeria labbeana. 3. Feeding Organelles.

The fine structure of the feeding organelles of the endogenous developmental stages of Eimeria labbeana from the ileal mucosa of the common Pigeon, Columba livia, is described and compared with similar structures of other species of Eimeria. Intra-cytoplasmic, membrane-bound vesicles of varying shapes and dimensions, and pinocytotic vesicle, were seen in association with cup-shaped or v-shaped invaginations in early schizonts, early macrogamonts, and macrogametes. Deep invaginations, averaging 1.7 x 0.5 mu in size, and found on the surface of early schizonts, early and young macrogamonts, and developing microgamonts, apparently function as organelles of ingestion and breakdown of host-cell cytoplasm. Micropores were rarely seen in schizonts and never in microgametes. The merozoite had one typical micropore (850 x 680 A) and a number of micropore-like invaginations. Micropores of the microgamonts averaged 610 x 580A, and those of macrogamonts and macrogametes averaged 1,220 x 780 A. A typical micropore was observed in an early oocyst. Intravacuolar tubules, each 580 A in diameter and composed of nine microtubule-like subunits, were observed only in about 1 per cent of the more than 4,000 macrogametes studied. This paper establishes that E. labbeana is a species that possesses all the known organelles associated with feeding, expect the intravacuolar folds.     

The life cycle of Eimeria falciformis var. pragensis (Sporozoa: Coccidia) in the mouse, Mus musculus

The life cycle of Eimeria falciformis var. pragensis, established from a single oocyst, is described in experimentally infected mice (Mus musculus). The coccidium had a prepatent period of 7 days and a patent period of 10--16 days. Oocysts were spherical to ellipsoidal in shape and measured 21.2 x 18.3 micron. Sporulation time was 3 to 3.5 days. Sporocysts measured 12.2 x 7.2 micron and contained a circular to avoid granular sporocyst residuum measuring 5.5 X 5.0 micron. One, 2 or 3 circular to rectangular polar granules were observed within each sporulated oocyst. The endogenous stages developed primarily in the cecum and colon and only occasionally in the lower ileum. Four generations of schizonts were found. Mature 1st-generation schizonts, first observed 48 hr postinfection (PI), measured 17.8 x 12.3 micron and had 12 merozoites that measured 13.3 x 2.0 micron. Mature 2nd-generation schizonts appeared 78 hr PI. They measured 10.2 x 9.3 micron and had 8 merozoites measuring 5.0 x 1.6 micron. Mature 3rd-generation schizonts appeared first at 114 hr PI and measured 17.5 x 10.2 micron and had 10 merozoites that measured 12.4 x 1.8 micron. Mature 4th-generation schizonts appeared first at 144 hr PI. They measured 18.2 x 15.3 micron and had 18 merozoites. The merozoites of the 4th-generation schizont were 4.5 x 1.2 micron. Mature macrogamonts and microgamonts developed simultaneously appearing at 156 hr PI. Macrogamonts measured 16 x 14.5 micron and microgamonts were 18.2 x 15.3 micron. In experimentally infected rats (Rattus norvegicus), development of E. falciformis var. pragensis progressed only as far as mature 1st-generation schizonts.     

贝氏隐孢子虫在北京鸭体内发育的超微结构研究

贝氏隐孢子虫各期虫体均位于宿主粘膜上皮细胞的带虫空泡中。在虫体与上皮细胞接触处,虫体表膜反复折迭形成营养器。子孢子或裂殖子与粘膜上皮细胞接触后,逐步过渡为球形的滋养体;滋养体经2—3次核分裂、产生含4或8个裂殖子的两代裂殖体,裂殖体以外出芽方式产生裂殖子;裂殖子无微孔,顶端表皮形成3—4个环嵴,裂殖子进一步发育成为配子体;大配子体含有两种类型的成囊体。小配子呈楔形,无鞭毛和顶体,有一个致密的长椭圆形细胞核,小配子表膜内侧有9根膜下微管;孢子化卵囊内含四个裸露的子孢子和一个大残体。本文是有关鸭体内隐孢子虫超微结构的首次报导。     

Discovery of the pre-erythrocytic stages of a saurian malaria parasite, hypnozoites, and a possible mechanism for the maintenance of chronic infections throughout the life of the host

Re-examination of tissue sections from four Takydromus tachydromoides (Sauria: Lacertidae) naturally infected with Plasmodium sasai found liver parenchymal cells, containing uninucleate parasites which may correspond to the hypnozoite stage of primate malaria parasites, schizonts and segmenters in parenchymal cells, and hepatic macrophages which contained numerous schizonts. Following destaining of the original H&E and prolonged restaining with warm Giemsa stain, encysted schizonts, protected by a hyaline wall, were discovered in the connective tissue or capillary endothelium of lung, liver, brain, heart, pancreas, kidney, intestine wall, testis, and both intra- and intermuscularly in the femoral muscles. Unencysted schizonts in the pulmonary endothelium apparently represent the phanerozoic stages, which, following encystment in the various tissues, are recognized as a new stage in the life cycle of reptilian malarial parasites, the chronozoic schizonts. A hypothesis is presented to describe the life cycle of P. sasai, which may be characteristic of other saurian malaria parasites. It interprets the sequence of pre-erythrocytic stages found as follows: sporozoites enter hepatic parenchymal cells where some may become dormant as hypnozoites, and others form cryptozoic schizonts. The cryptozoites parasitize hepatic macrophages and form metacryptozoic schizonts. Metacryptozoites produce phanerozoic schizonts in the capillary endothelium and connective tissue of the lung and other organs. Phanerozoites and possibly metacryptozoites then invade the erythrocytes to begin the erythrocytic cycle. Some of the phanerozoites in endothelium, connective tissue and skeletal muscle become encysted as chronozoic schizonts, and their progeny, chronozoites, renew the erythrocytic cycle throughout the life of the host and produce seasonal relapses of gametocytemia, in spring, at the end of hibernation by the lizard.