Light and electron microscopical study of Nosema eurytremae

A nuclear-polyhedrosis virus (NPV) of the silkworm, Bombyx mori, which forms an icosahedral inclusion body, was transmitted to larvae of the rice stem borer, Chilo suppressalis. Serial passages of Bombyx NPV in the alternate host by injecting the supernatant of diseased hemolymph produced inclusion bodies with cuboidal and other shapes that differed from the original shape formed in Bombyx. These different shapes increased with times of passages, and after the twelfth passage, only cuboidal inclusion bodies were formed. The icosahedral inclusion bodies in B. mori and the cuboidal inclusion bodies in C. suppressalis occluded singly enveloped virions of the same size (350 × 75 nm), but the cuboidal inclusion bodies contained only a few virions and a large number of membraneous spherical structures. The formation process of the cuboidal inclusion body differed from that of the icosahedral. At first, irregularly branched inclusion bodies containing “vacant” spaces appeared in the infected nuclei. The bodies grew larger with the deposition of protein in the spaces between the branches, and this was accompanied with the occlusion of a large number of membraneous structures formed in the vicinity of the inclusion bodies, which became cuboidal in shape.     

Red Blood Cells Preconditioned with Hemin Are Less Permissive to Plasmodium Invasion In Vivo and In Vitro

Malaria is a parasitic disease that causes severe hemolytic anemia in Plasmodium-infected hosts, which results in the release and accumulation of oxidized heme (hemin). Although hemin impairs the establishment of Plasmodium immunity in vitro and in vivo, mice preconditioned with hemin develop lower parasitemia when challenged with Plasmodium chabaudi adami blood stage parasites. In order to understand the mechanism accounting for this resistance as well as the impact of hemin on eryptosis and plasma levels of scavenging hemopexin, red blood cells were labeled with biotin prior to hemin treatment and P. c. adami infection. This strategy allowed discriminating hemin-treated from de novo generated red blood cells and to follow the infection within these two populations of cells. Fluorescence microscopy analysis of biotinylated-red blood cells revealed increased P. c. adami red blood cells selectivity and a decreased permissibility of hemin-conditioned red blood cells for parasite invasion. These effects were also apparent in in vitro P. falciparum cultures using hemin-preconditioned human red blood cells. Interestingly, hemin did not alter the turnover of red blood cells nor their replenishment during in vivo infection. Our results assign a function for hemin as a protective agent against high parasitemia, and suggest that the hemolytic nature of blood stage human malaria may be beneficial for the infected host.     

A study into the sexual dimorphisms of the Ampullae of Lorenzini in the lesser-spotted catshark, Scyliorhinus canicula (Linnaeus, 1758)

The Ampullae of Lorenzini can vary in their size, shape and distribution patterns among elasmobranch species. However, no study has compared the ampullary characteristics between the sexes within a species. The present study found a sexual dimorphism in the Ampullae of Lorenzini of the lesser-spotted catshark, Scyliorhinus canicula. Male S. canicula were found to possess longer ampullae and alveoli, greater numbers of alveolar bulbs, larger sensory epithelial surface areas and greater numbers of sensory receptor cells in the ampullae than female S. canicula. Greater lengths of both ampullae and alveoli, numbers of alveoli, larger sensory epithelial surface areas and greater numbers of sensory receptor cells in male S. canicula could increase the capability of adult male S. canicula in detecting females. The presence of the sexual dimorphism in the alveoli of the Ampullae of Lorenzini could be directly related to reproductive behaviour and/or reflect the sexual segregation patterns of adult S. canicula.     

Pathology and Development of the Grasshopper Inclusion Body Virus in Melanoplus sanguinipes

Grasshoppers, Melanoplus sanguinipes (F.), infected with the grasshopper inclusion body virus (GIBV) showed a general torpor, took longer to develop, and had abnormally high rates of mortality. Infection was found only in the fat body, and developing viruses and inclusion bodies were observed in the nuclei and cytoplasm of infected cells. Although the size of the inclusion bodies in cells varied at different stages of infection, the inclusion bodies appeared to grow during the infection. Electron microscopic investigations of viral replication showed that at about 8 days after inoculation presumptive viral particles had developed as buds or protrusions from precursor granular masses; thereafter, these particles underwent internal differentiation and were incorporated into developing inclusion bodies. The GIBV was similar to insect viruses in the genus Vagoiavirus Weiser and to pox viruses, particularly vaccinia.     

A new virus disease in the housefly, Musca domestica (Diptera)

Reovirus particles were isolated from adults in laboratory colonies of the housefly, Musca domestica. These particles were spherical in outline, 57–76 nm in diameter, and were found only in hemocyte cytoplasm, where virions have been disclosed by a new technique. Virions were present in large numbers, and viral inclusion bodies were identified. The virus particles had pentagonal and hexagonal shapes resembling a simple icosahedral structure. The virus was shown to be infectious and pathogenic to adult flies through injection or by feeding them suspensions from flies that had died of the virus. Electron micrographs of midgut sections from infected flies showed that the midgut cells were packed with dark undulating threads which were not present in uninfected flies. However, no virus particles or inclusion bodies could be seen in these cells. On the basis of their association with infected flies, and the similarity to results from other studies on reoviruses and insect viruses, it is suggested that these threads are an alternative replicative form of the reovirus. When the virus suspensions from heavily infected flies were dialyzed against weak alkaline solutions, the threads showed an inner component of coiled material, 12 nm in diameter, inside an envelope with a diameter of 50–83 nm, mean 60.3 ± 7.5, composed of subunits 7–8 nm long and 7–8 nm across.     

Babesia rodhaini, Babesia bovis, and Babesia bigemina: analysis and sorting of red cells from infected mouse or calf blood by flow fluorimetry using 33258 Hoechst.

The DNA of Babesia spp. parasites within host intact red blood cells was labeled using the fluorescent bisbenzimidazole dye 33258 Hoechst. The labeled cells were sorted on a fluorescence activated cell sorter on the basis of cell fluorescence (proportional to DNA content) and the intensity of light scattered from the cells at low angles (related to cell size). The optimal conditions for dye uptake were established for the murine parasite Babesia rodhaini and the bovine parasites B. bovis and B. bigemina. Uninfected cells were nonfluorescent after incubation with the dye and could be completely separated from infected fluorescent cells. The fluorescence of cells infected with B. rodhaini was proportional to the number of parasite nuclei per cell. With saturation levels of dye, samples infected with B. bovis or B. bigemina in which erythrocytes contained one or two parasites, both exhibited only one fluorescent cell peak. Cell sorting did not eliminate the infectivity of B. rodhaini. The method may be used to separate populations of uninfected blood cells and cells infected with Babesia spp. for biochemical and immunochemical experiments.     

Trypanosoma evansi: Levels of copper, iron and zinc in the bloodstream of infected cats

The aim of this study was to evaluate the concentrations of copper, iron and zinc in blood serum of cats experimentally infected with Trypanosoma evansi. Animals were divided into two groups: control and infected with T. evansi. The animals were infected with 10⁸ trypomastigotes each and parasitemia was estimated daily for 56 days by microscopic examination of smears. Hematological and biochemical parameters were evaluated for monitoring of the disease. Serum metal levels were determined in blood samples collected at days 7, 28 and 56 of the experiment. Inductively coupled plasma optical emission spectrometry was used to measure the levels of copper, iron and zinc. Significant differences were observed among groups (P < 0.05). Increased levels of copper and decreased iron and zinc levels were observed. A decrease in the number of red blood cells was also observed 7 days after inoculation. Biochemical parameters were not altered. Therefore, the infection by T. evansi might alter the serum metal levels, causing metabolic disturbances in cats.     

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.     

Haemogregarina pseudemydis n. sp. (Apicomplexa: Haemogregarinidae) and Pirhemocyton chelonarum n. sp. in Turtles from Louisiana*

SYNOPSIS. The blood of each of 95 turtles (8 species) collected from southeastern Louisiana was infected with some or all of the merogonic stages and gametocyte stage of Haemogregarina pseudemydis n. sp. Five species of turtles harbored Pirhemocyton chelonarum n. sp. Turtle Haemogregarina and Pirhemocyton are locality records for Louisiana. Pirhemocyton is reported for the first time in turtles and in the continental U.S.A.     

An enzootic nuclear polyhedrosis virus of pink shrimp: Ultrastructure,prevalence, and enhancement

A nuclear polyhedrosis virus exists in pink shrimp, Penaeus duorarum, from waters of the northern Gulf of Mexico. This virus is rod-shaped, 269 nm long, and possesses an outer envelope surrounding its nucleocapsid. The nucleocapsid is 50 nm in diameter. The virus occurs in nuclei of host hepatopancreatic and midgut cells, and is both free in the nucleus and occluded within pyramidal-shaped polyhedral inclusion bodies (PIB's). Histochemically and ultrastructurally, the shrimp PIB's appear to be ribonucleoprotein and in fine structure bear close resemblance to polyhedral inclusion bodies of Baculovirus species from insects. However, the lattice line-to-line spacing is greater than that usually reported for insect PIB's. Crowding and chemical stress of shrimp in aquaria may enhance and increase the virus infection and prevalence. In limited experiments, shrimp fed heavily infected hepatopancreatic tissues had much higher mortality than controls fed only fish. The virus appears to be enzootic in pink shrimp in nature. Cytopathological changes in infected cells of shrimp appear similar to those in insects infected with certain species of Baculovirus. The name Baculovirus penaei n.sp. is proposed for the shrimp virus.     

Plasmodium berghei: modification of sialic acid on red cells from infected mouse blood

The surface membrane glycoproteins of normal mouse erythrocytes can be labeled by oxidation with either periodate or galactose oxidase in the presence of neuraminidase, followed by reduction with NaB³H₄. Without neuraminidase there is little galactose oxidase-catalyzed labeling of protein. Analysis of labeled proteins by SDS-polyacrylamide gel electrophoresis showed that both methods labeled the same set of glycoproteins. Plasmodium berghei infection dramatically reduced the sialoglycoprotein labeling of red blood cells from infected blood using the periodate/NaB³H₄ method. Provided neuraminidase was present, labeling by the galactose oxidase method gave identical results to normal erythrocytes. We conclude that the glycoprotein sialic acid of uninfected as well as infected red cells is modified during infection such that it is refractory to periodate oxidation. Acylation of the exocyclic hydroxyls of sialic acid is suggested to account for this. Lectin binding and cell agglutination experiments using Limulin, soybean and wheatgerm lectins, and concanavalin A confirmed and extended these observations. The possible implications of these results with regard to anemia induced by malaria are briefly discussed.     

A picornavirus-like pathogen of Cotylogaster occidentalis (Trematoda: Aspidogastrea), an intestinal parasite of freshwater mollusks

Nonoccluded, icosahedral picornavirus-like (PVL) particles, 23 nm in diameter, forming paracrystalline arrays were seen in the cytoplasm of various cells in Cotylogaster occidentalis. Viral inclusions were visible in live specimens and in sections prepared for light and electron microscopy. All worms examined over a 2-year period were found to be infected. Infections were naturally acquired and susceptibility was not associated with any particular developmental stages. Development of viral inclusions involved an increase in the inclusion volume, progressive accumulation and condensation of materials into the interior of the inclusions, and formation of multilamellar membrane networks. Virus particles were observed in the stroma of the inclusions in association with multilamellar spherical bodies. Mature PVL particles aggregated into polygonally shaped paracrystalline arrays. When such arrays occurred in the surface tegument, local disruption of the tegumentary membrane may liberate these particles into the environment. PVL particle production did not exhaust glycogen content of infected cells and did not appear to affect short-term survival of the parasite outside the molluscan host.     

A new cytoplasmic polyhedrosis virus from the salt-marsh mosquito, Aedes cantator (Diptera: Culicidae)

The ultrastructure, development, and histopathology of a new cytoplasmic polyhedrosis virus of Aedes cantator are described. Virus particles measure 70 nm in diameter, are icosahedral in shape, and consist of a central electron-dense core surrounded by a capsid with six projections. Occlusion bodies are irregular in size (0.5–3.0 μm) and shape and contain several virus particles. Virus particles are assembled within an interconnecting network of fine filaments and are occluded by the deposition of a proteinaceous crystal around groups of mature virus particles within a virogenic stroma. Infections are confined to cells of the cardia, gastric ceca, and posterior portion of the midgut, which hypertrophy and frequently lyse. Infected larvae die during the fourth larval instar or as pupae. The prevalence of infection in natural field populations is less than 1%.     

An exported kinase (FIKK4.2) that mediates virulence-associated changes in Plasmodium falciparum-infected red blood cells

Alteration of the adhesive and mechanical properties of red blood cells caused by infection with the malaria parasite Plasmodium falciparum underpin both its survival and extreme pathogenicity. A unique family of parasite putative exported kinases, collectively called FIKK (Phenylalanine (F) – Isoleucine (I) – Lysine (K) – Lysine (K)), has recently been implicated in these pathophysiological processes, however, their precise function in P. falciparum-infected red blood cells or their likely role in malaria pathogenesis remain unknown. Here, for the first time, we demonstrate that one member of the FIKK family, FIKK4.2, can function as an active kinase and is localised in a novel and distinct compartment of the parasite-infected red blood cell which we have called K-dots. Notably, targeted disruption of the gene encoding FIKK4.2 (fikk4.2) dramatically alters the parasite’s ability to modify and remodel the red blood cells in which it multiplies. Specifically, red blood cells infected with fikk4.2 knockout parasites were significantly less rigid and less adhesive when compared with red blood cells infected with normal parasites from which the transgenic clones had been derived, despite expressing similar levels of the major cytoadhesion ligand, PfEMP1, on the red blood cell surface. Notably, these changes were accompanied by dramatically altered knob-structures on infected red blood cells that play a key role in cytoadhesion which is responsible for much of the pathogenesis associated with falciparum malaria. Taken together, our data identifies FIKK4.2 as an important kinase in the pathogenesis of P. falciparum malaria and strengthens the attractiveness of FIKK kinases as targets for the development of novel next-generation anti-malaria drugs.     

Ultrastructural investigation on a strain of a cytoplasmic-polyhedrosis virus with nuclear inclusions

A strain of a cytoplasmic-polyhedrosis virus causes the formation of crystalline inclusions almost entirely in the nucleus, and only rarely in the cytoplasm, of the midgut epithelial cells of the silkworm Bombyx mori. It also differs from the typical strain in causing the hypertrophy of the nucleoli and the formation of dense reticulum and spherical bodies in the nucleus. The virus particles and the virogenic stromata of the new strain resemble those of the typical strain. The cytoplasmic inclusions contain virus particles, while the nuclear inclusions do not. When the infected larvae are kept at 30°C for 15 hr or at 35°C for 3–15 hr, the nuclear inclusions break up into particles of 70–250 nm in diameter. The particles are dispersed in the cells but not present in the spaces previously occupied by the decomposed inclusions.     

Haematology of the australian eastern quoll,Dasyurus viverrinus

• 1.1. A variety of haematological parameters were determined in adult Dasyurus viverrinus.
• 2.2. Haemoglobin and red cell counts were high with a very low mean cell volume.
• 3.3. Basophils are absent but the eosinophils contain small numbers of basophilic granules which may indicate a dual role for this cell.
• 4.4. “Ring Form” leucocytes are present.
• 5.5. Three types of red cell picture could be identified, some animals showing large numbers of spherocytes, spicule cells, and inclusion bodies.
• 6.6. These cells resemble those found in some inherited human haemolytic anaemias but there was no evidence of haemolysis in the animals.
• 7.7. An alkali resistant haemoglobin component is present.

     

ISOLATION OF A NON‐PHAGE‐LIKE LYTIC VIRUS INFECTING AUREOCOCCUS ANOPHAGEFFERENS1

We have been working to characterize viruses that infect the HAB‐forming pelagophyte Aureococcus anophagefferens Hargraves et Sieburth. Field samples were collected during brown‐tide events in 2002 and tested for the presence of lytic agents. Here, we describe a recently isolated, lytic virus‐like particle (VLP) that is morphologically similar to particles observed in thin sections of infected A. anophagefferens cells from natural samples. TEM and SEM have revealed VLPs consistent with the morphological characteristics of previously described Phycodnaviridae. Large icosahedral particles (～140 nm) of similar shape and morphology dominate cell lysates and are accompanied by smaller phage‐like particles and heterotrophic prokaryotes that appear to be incurable from our cultures. To determine which of these particles interacts with the Aureococcus cells, we preserved cultures during the early stage of infection so that SEM could be used to visualize those particles that attach to the surface of naïve cultures. SEM revealed that 63% of the large icosahedral‐shaped particles attached to A. anophagefferens cells after only 30 min of exposure, while no significant frequency of attachment to the alga was observed for the phage‐like particles. The results of these observations are in contrast to previous studies, where phage‐like particles were reported to infect cells. When considered in conjunction with field observations, the results suggest that this newly isolated virus represents the dominant virus‐morphotype associated with bloom collapse and termination.     

A molecular and ecological study of Grillotia (Cestoda: Trypanorhyncha) larval infection in small to mid‐sized benthonic sharks in the Gulf of Naples,Mediterranean Sea

AimTrypanorhyncha cestodes comprise a wide range of heteroxenous parasites infecting elasmobranchs as definitive hosts. Limited data exist on the larval infection of these cestodes and the role of intermediate and paratenic hosts in the life cycle of these parasites. We investigated the factors that determine the occurrence and the level of infection of Grillotia plerocerci in the skeletal muscles of various benthonic sharks and analyzed the parasites through an integrative taxonomic approach.LocationMediterranean Sea.MethodsSharks obtained as bycatch of commercial trawling activities (i.e., Etmopterus spinax, Galeus melastomus, and Scyliorhinus canicula) were used in this study. Data from a limited number of Dalatias licha and Scyliorhinus stellaris were also included. Grillotia plerocerci were molecularly characterized using the partial 28S large subunit rDNA. Boosted regression trees were used to model the relationship between the abundance of infection with both morphological and physiological predictors in each host.ResultsPlerocerci of Grillotia were detected in all shark species except S. stellaris. Host species significantly differed in terms of parasite abundance, with the highest and lowest prevalence and abundance of infection detected in G. melastomus and E. spinax, respectively. The relative influence of the traits involved in explaining the parasite abundance was related to the host size in G. melastomus, while both morphology‐ and physiology‐related traits explained the patterns observed in E. spinax and S. canicula. The 28S rDNA sequences shared an identity of ∼99.40% with a Grillotia species previously found in the Mediterranean Sea. At intraspecific level, two different genotypes were found. A first type was retrieved only from D. licha, whereas a second type was found in G. melastomus, E. spinax, and S. canicula.Main conclusionsPresent results suggest that the two genotypes could be involved in different consumer‐resource systems and confirm most of the examined shark species as transport hosts of Grillotia species for unknown larger top predators.