Merogonic Stages Research Articles

Developmental stages and molecular phylogeny of Hepatozoon fitzsimonsi (Dias 1953) (Adeleorina: Hepatozoidae) in tortoises Stigmochelys pardalis (Cryptodira: Testudinidae) and ticks of the genus Amblyomma (Acari: Ixodidae) from South Africa

BackgroundHepatozoon fitzsimonsi (Dias, 1953) is a frequently found haemogregarine of southern African tortoises. At the time of this species’ reassignment from the genus Haemogregarina to Hepatozoon, developmental stages such as sporocysts and sporozoites were observed in ticks associated with H. fitzsimonsi parasitised and non-parasitised tortoises. It was thus suggested that ticks may act as the potential vectors for this parasite. However, this earlier research was unable to confirm the identity of these sporogonic stages using molecular markers. In a separate study aimed at identifying tick species parasitising South African reptiles and molecularly screening these for the presence of Hepatozoon, that study identified H. fitzsimonsi in tortoise-associated ticks. Thus, the present study aimed to revisit the potential of ticks to act as vectors for H. fitzsimonsi in tortoises using a combined microscopy and molecular approach.MethodsSpecimens of Kinixys natalensis, Kinixys spekii, Kinixys zombensis and Stigmochelys pardalis were collected from Bonamanzi and Ndumo Game Reserve, South Africa. Upon capture, animals were examined for ticks, and these were collected along with blood and other tissues. Adult ticks were dissected and visceral impression slides were prepared along with thin blood and tissue smears on clean microscope slides. Smears and impression slides were stained with Giemsa, screened and micrographs of parasites were captured. Two primer sets were employed to target fragments of the 18S rRNA gene of parasites found in both tortoises and ticks and the resulting sequences were then compared with other known H. fitzsimonsi and haemogregarine sequences from the GenBank database.ResultsPeripheral blood gamont and liver merogonic stages were observed in S. pardalis, while the sporogonic stages were observed in the haemocoel of Amblyomma ticks. Gamont and sporocyst stages compared morphologically with previous descriptions of H. fitzsimonsi, identifying them as this species. Phylogenetic analysis revealed that the blood and tick sequences obtained in this study clustered in a monophyletic clade comprising known H. fitzsimonsi.ConclusionsThe present study provides further support for ticks acting as the vectors of H. fitzsimonsi by molecularly identifying and linking observed developmental stages in tortoises (S. pardalis) with those in the invertebrate host (Amblyomma spp.).Graphical

Co-infecting Haemoproteus species (Haemosporida, Apicomplexa) show different host tissue tropism during exo-erythrocytic development in Fringilla coelebs (Fringillidae)

Avian haemosporidians of the genera Plasmodium, Haemoproteus, and Leucocytozoon are common blood parasites in wild birds all over the world. Despite their importance as pathogens potentially compromising host fitness and health, little is known about the exo-erythrocytic development of these parasites, particularly during co-infections which predominate in wildlife. This study aimed to address this issue using Haemoproteus parasites of Fringilla coelebs, a common bird species of the Western Palearctic and host to a variety of haemosporidian parasite lineages. Blood and tissue samples of 20 F. coelebs, positive for haemosporidians by blood film microscopy, were analysed by PCR and sequencing to determine cytochrome b lineages of the parasites. Tissue sections were examined for exo-erythrocytic stages by histology and in situ hybridization applying genus-, species-, and lineage-specific probes which target the 18S rRNA of the parasites. In addition, laser microdissection of tissue stages was performed to identify parasite lineages. Combined molecular results of PCR, laser microdissection, and in situ hybridization showed a high rate of co-infections, with Haemoproteus lineages dominating. Exo-erythrocytic meronts of five Haemoproteus spp. were described for the first known time, including Haemoproteus magnus hCCF6, Haemoproteus fringillae hCCF3, Haemoproteus majoris hCCF5, Haemoproteus sp. hROFI1, and Haemoproteus sp. hCCF2. Merogonic stages were observed in the vascular system, presenting a formerly unknown mode of exo-erythrocytic development in Haemoproteus parasites. Meronts and megalomeronts of these species were distinct regarding their morphology and organ distribution, indicating species-specific patterns of merogony and different host tissue tropism. New pathological aspects of haemoproteosis were reported. Furthermore, phylogenetic analysis of Haemoproteus spp. with regard to their exo-erythrocytic stages points towards separation of non-megalomeront-forming species from megalomeront-forming species, calling for further studies on exo-erythrocytic development of haemosporidian parasites to explore the phylogenetic character of this trait.

Morphometric and molecular characterisation of Hepatozoon bashtari n. sp. in painted saw-scaled viper, Echis coloratus (Ophidia, Viperidae).

Hepatozoon species are the most widely known haemogregarines infecting a wide range of vertebrates, although predominately snakes. Herein, Hepatozoon bashtari n. sp., originally infecting the painted saw-scaled viper, Echis coloratus, in Saudi Arabia is described using both morphological features and molecular data from 18S rDNA sequences. The overall prevalence of infection was 60% (9/15) with parasitaemia ranging from 52 to 60%. Gamonts were entirely intraerythrocytic and were observed to cause considerable hypertrophy within the host cell. The mean size of mature gamonts was 15.4 × 3.3 μm. Merogonic stages were confined to the lung endothelial cells with monomorphic meronts. The average size of mature meronts was 32 × 12 μm and they were estimated to produce 13-16 merozoites each. The phylogenetic tree generated from SSU rDNA sequences revealed that Hepatozoon bashtari sp. n. clusters with the vast majority of other Hepatozoon species infecting snakes, lizards and geckos in various regions of the world, which would appear to support the hypothesis of prey-predator transmission of the genus Hepatozoon. Through a combination of morphological comparison with closely related Hepatozoon spp. and 18S rRNA gene sequence analysis, it is possible to confirm Hepatozoon bashtari sp. n. as a new species.

Light Microscopic Study of Caryospora sp. (Apicomplexa: Eimeriidae) from Savigny’s Agama Trapelus savignii (Reptilia: Agamidae) in Egypt

Developmental stages of a suggested new speciesof CaryosporaLeger, 1904 were described from the agamid lizards Trapelus savigniicaptured during summer 2019 from the Eastern Desert in Egypt. Overall recorded prevalence is 11.65% (12/103) and sporulation is endogenous. Sporulated oocysts are subspherical (70%) to spherical (30%) measuring 22.29–35.74 ×20.91–32.89 μm (length × width). The oocyst wall is bilayered, smooth, and measures 1.46–2.00 μm. Distinctive features of most oocysts include the presence of a sutured edge-like extension on one side of the oocyst wall and the ability of sporocyst contents to form a condensed curved mass within the sporocyst cavity. One or rarely two polar granules are present. Micropyle and oocyst residuum are absent. Sporocysts are ovoid to ellipsoidal measuring 17.17–22.76 × 12.86–16.76 μm. The morphometric descriptions and measurements of Stieda, sub-Stieda bodies, and sporocyst residuum are determined. Sporozoites are sausage-shaped and measure 5.69–8.59 × 2.08–3.29 μm. Merogonic stages and microgamonts occur mainly in the enterocytes of the small intestine. Endogenous stages are seen mostly with clear large parasitophorous vacuoles. Mature meronts measure 3.95–7.60 × 3.37–7.24μm and yield 3–11 merozoites with the formation of a residual body.Merozoites are banana-shaped measuring 2.51–3.29 × 0.72–1.20μm. A microgamont is noticed measuring in 9.53 × 8.42 μm.

Blood and Merogonic Stages of Haemogregarina Species Naturally Infecting The Nile Monitor Varanus niloticus, Egypt

Haemogregarines are haemoparasites (Apicomplexa: Adeleiorina) of all class of vertebrates nearly, worldwide. The present study was aimed to establish and describe the developmental stages of a Haemogregarina species infecting Nile monitor (Varanus niloticus). The captured Nile monitors were three animals with 80-100 cm in length, they captured from Qena province, Southern Egypt, and thin blood films were prepared and microscopically examined. Also, small pieces of the liver and the lungs of the infected animals were separated and prepared for sectioning, which stained and examined. The recovered parasites have rod-like bodies, infecting the erythrocytes only, with a single stage, in a parasitophorous vacuole and have no karyolitic effect. The parasitaemia level was found up to 5010,000 counted erythrocytes. Only gamonts have been recognized in blood smears, and they were found in two forms; the first form is short, thick (called short gamont) and measured 11.02 × 2.65 µm. The second form was found long and thin (called long gamont), with a subterminal nucleus, measured 14.18 × 2.45 µm. Both forms cause either slightly or strong displacement of the host cell nucleus. The infected erythrocytes had either slightly or markedly hypertrophied in presence of short and long gamonts. The infected erythrocyte measured 18.05 × 8.87 µm, but the uninfected erythrocyte was 16.52 × 8.77 µm in length and width, respectively. Schizogony phase was observed in the lungs only of the infected Nile monitor, where the meronts were formed within parasitophorous vacuoles. The parasite nucleus divided many times, and the produced nuclei are peripherally arranged in the meront, forming merozoites. After separation of the merozoites, residual bodies formed. The merogonic stages were differentiated into two forms, micromeront that measured 15.57×14.29 µm, and produces a few numbers of macromerozoites, meanwhile the macromeront measured 19×15.9 µm and produces up to six micromerozoites.

Hepatozoon pyramidumi sp. n. (Apicomplexa: Adeleorina) from the blood of Echis pyramidum: morphology and SSU rDNA sequence.

Hepatozoon pyramidumi sp. n. is described from the blood of the Egyptian saw-scaled viper, Echis pyramidum, captured from Saudi Arabia. Five out of ten viper specimens examined (50%) were found infected with Hepatozoon pyramidumi sp. n. with parasitaemia level ranged from 20-30%. The infection was restricted only to the erythrocytes. Two morphologically different forms of intraerythrocytic stages were observed; small and mature gamonts. The small ganomt with average size of 10.7 × 3.5 μm. Mature gamont was sausage-shaped with recurved poles measuring 16.3 × 4.2 μm in average size. Infected erythrocytes were hypertrophied; their nuclei were deformed and sometimes displaced from their central position in the normal uninfected cell. Merogonic stages were observed in the lung endothelial cell and the liver parenchyma cells. Mature meront was 17.8 × 13.6 µm and contained banana-shaped merozoites with average size of ~15 × 2 µm. Phylogenetic analysis based on the SSU rDNA sequence clustered Hepatozoon pyramidumi sp. n with previously sequenced Hepatozoon spp., most of them infected reptilian hosts without geographic consideration. The morphological and molecular comparison with closely related species proved the taxonomic uniqueness and novelty of the present form.

A new species, Dactylosoma piperis n. sp. (Apicomplexa, Dactylosomatidae), from the pepper frog Leptodactylus labyrinthicus (Anura, Leptodactylidae) from Mato Grosso State, Brazil.

The Dactylosomatidae Jakowska and Negrelli, 1955 are one of four families belonging to adeleorinid coccidia and comprise the genera Babesiosoma Jakowska and Nigrelli, 1956 and Dactylosoma Labbé, 1894. These blood protozoa occur in peripheral blood of lower vertebrates, and are commonly reported parasitising amphibians. The present study describes Dactylosoma piperis n. sp. from the pepper frog Leptodactylus labyrinthicus (Spix, 1824) (Anura: Leptodactylidae), collected in 2018 at the municipality of Araguaiana, Mato Grosso State, Brazil, based on morphology of intra-erythrocytic trophozoite, primary and secondary merogonic stages and a molecular analysis (partial 18S rDNA). Dactylosoma piperis n. sp. forms a well-supported clade with other Dactylosomatidae. This is the first molecular characterization of a species of Dactylosoma from a Brazilian anuran.

Molecular characterisation of Hepatozoon aegypti Bashtar, Boulos & Mehlhorn, 1984 parasitising the blood of Spalerosophis diadema (Serpentes: Colubridae).

Hepatozoon aegypti Bashtar, Boulos & Mehlhorn, 1984 was first described from the blood of the diadem snake (Spalerosophis diadema) in Egypt. During an investigation of the diversity of reptilian haemogregarines in Saudi Arabia, seven diadem snakes (100% of the sample) were found to be highly parasitised by H. aegypti, with an average parasitaemia of 37% per 500 counted erythrocytes. A complete characterisation of this species with morphometrics and 18S rDNA sequence data is therefore presented here. The infection was found to be restricted to the erythrocytes with, frequently, single and, sometimes, double infections. Mature gamonts were sausage-shaped with round posterior and anterior extremities and measured 14 (13-17) × 3.5 (3-5) μm. The infected erythrocytes were hypertrophied with a faintly stained cytoplasm and longitudinally stretched nuclei. The merogonic stages occurred only in the endothelial cells of the snakes' lungs, and no stages were found in other organs. Mature meronts were round in shape, measured 18 (17-21) μm in diameter and were estimated to produce between 9 and 15 merozoites. Phylogenetic analysis based on the partial 18S small subunit ribosomal DNA sequences indicates that Hepatozoon aegypti cluster within a mixed clade of Hepatozoon species parasitising snakes, geckos and rodents from various geographic areas. Our results might reinforce the theory of prey-predator transmission in respect to the relationships of snake-host Hepatozoon species.

Life cycle of Hepatozoon affluomaloti sp. n. (Apicomplexa: Haemogregarinidae) in crag lizards (Sauria: Cordylidae) and in culicine mosquitoes from South Africa.

A new haemogregarine species Hepatozoon affluomaloti sp. n. is described from erythrocytes in the peripheral blood of crag lizards Pseudocordylus melanotus (Smith) and Pseudocordylus subviridis (Smith) (Sauria: Cordylidae) from mountainous regions in the Eastern Free State, South Africa. This species can be distinguished from all other congeners based on its large size, staining properties and life cycle development in its vector, Culex (Afroculex) lineata (Theobald) (Diptera: Culicidae). Mature gamonts stain mostly uniformly pinkish-purple with Giemsa, sometimes containing darker azurophilic granules anterior and posterior to the nucleus. The reflexed posterior extremity of the gamont stage sometimes stains slightly deeper purple and the nucleus is dense and placed in the posterior third of the parasite body. Merogonic stages of this haemogregarine occur in the liver tissues of P. melanotus with dizoic meronts. Macromeronts contains 2-7 macromerozoites and micromeronts contains 9-24 micromerozoites. Sporogonic developmental stages found in the proposed final host and vector, C. lineata, include large oocysts, measuring 54 × 48 µm on average. Sporulating oocysts with 8 nuclei are present in mosquitoes 6-7 days post-feeding on infected lizards. Sporocysts with mature sporozoites measure 31.0 × 21.8 µm on average and each contains 2-8 large sporozoites. It is suggested that transmission of infective sporozoites is achieved through predation of lizards on mosquitoes.

A description of Haemogregarina species naturally infecting white-spotted gecko (Tarentola annularis) in Qena, Egypt.

The present study describes the developmental stages of Haemogregarina species in the blood and tissues of naturally infecting white-spotted gecko Tarentola annularis collected from Qena, Egypt. Different parasite's forms were observed infecting the erythrocytes. The gamonts enclosed within parasitophorous vacuole and seems to have no clear effect on the host cell especially in case of immature parasite forms. But in the presence of mature gamonts the host cell nucleus displaced. The parasitaemia level is up to 280 per 10,000 erythrocytes counted. Trophozoites and gamonts have been recognized in the blood smears. The rounded trophozoite diameter is 3.84 ±0.87 μm, while the elongated trophozoite measured 4.42 ± 0.69 x 2.8 ± 0.56 μm. The mature gamonts were differentiated into two forms; short gamont measuring 10.82 ± 0.82 x 3.30 ± 0.73 μm (range: 10-12.1 x 2.2-4.4 μm) and the long gamont measured 14.67 ± 0.83 x 3.96 ± 0.77 μm (range: 14.1-16.5 x 3.3-5.5 μm). Merogony carried out only in the endothelial cells of the blood capillaries in the lung. Different merogonic stages have been recognized and differentiated in two forms; micromeront measured 13.25 ± 0.50 x 12 ± 0.0 μm and produces a few number of large merozoites, macromeront measured 19.75+0.87x13.25+0.50 im and produces more small sized merozoites. The gamonts and merozoites have the general characteristic ultrastructures of the Apicomplexa containing components of the apical complex, e.g. pellicle, micronemes, rhoptries, and few dense bodies and subpellicular microtubules.

Merogonic stages of Theileria cervi in mule deer (Odocoileus hemionus)

In February 2012, 12 farmed mule deer (Odocoileus hemionus) were moved from a facility in southwestern Oklahoma to a facility in southeastern Oklahoma that housed 100 farmed white-tailed deer (Odocoileus virginianus). Between the third and fifth weeks, 9 of the 12 mule deer had died, 4 of which were submitted for necropsy. The deer were heavily infested with Amblyomma americanum (lone star ticks). Hematologic data from 1 deer revealed severe anemia, leukocytosis, and intraerythrocytic hemoparasites consistent with Theileria spp. Microscopically, the liver, lymph nodes, and spleen contained multifocally distributed, enlarged monocytic cells whose cytoplasm was replaced by developing meronts in various stages of merogony. It appears that, upon arrival, the Theileria cervi-naïve mule deer became infested with large numbers of Theileria-infected lone star ticks leading to massive exposure of the mule deer to sporozoites of the protozoan, resulting in an acute hemolytic crisis and fatalities. The merogonic stages of T. cervi are also described. The lack of earlier reports of merogony may be due to the fact that only a single, short-lived, merogonic cycle follows exposure to sporozoites and thus merogonic stages are demonstrable for only a short period. Polymerase chain reaction testing of paraffin-embedded tissue yielded a 507-bp amplicon sequence that was 100% identical with the sequence of T. cervi previously reported from white-tailed deer in Oklahoma and from elk in Wisconsin and Indiana.

A redescription of Haemogregarina damiettae Ramadan et al. 1996 naturally infecting the Acanthodactylus boskianus from Egypt, with new merognic data

Haemogregarina damiettae was previously described form Acanthodactylus boskianus in Egypt, and the description was base only on the blood stages with no data on the tissue stages. In the present study, both blood and tissue stages were encountered. The prevalence of infection was found to be 60% (30/50). The blood stages were found to be confined to the erythrocytes and none in the leucocytes. The erythrocytic stages could be differentiated into small and large forms. The small form, trophozoite, measured 10 × 3μm, while the large form, mature gamontocyte, measured 18 × 5μm. The cytoplasm of the mature gamontocyte was finely granular and faintly stained with Giemsa, while the nucleus was relatively large and occupied about one third of the body length and was formed of a network of chromatin filaments. Infected erythrocytes were hypertrophied and their nuclei either longitudinally stretched or split into two fragments. The tissue stages were observed in the lungs and hearts of the infected lizards. The merogonic stages were evident as multinucleated ovoid to subspherical bodies enclosed by parasitophorous vacuoles. Mature meronts measured 16 × 12μm and estimated to produce 15-25 merozoites.

An ultrastructural study on the merogonic stages of Goussia senegalensis (Faye, 1988) Diouf and Toguebaye, 1993 (Apicomplexa, Coccidia) from the liver of Pagellus bellottii (Pisces, Teleostei)

Little is known about the merogony of coccidians from fishes in Africa. To our knowledge, including both freshwater and marine fishes, developmental stages have been recorded for only Goussia cichlidarum, Cryptosporidium sp. parasitizing the stomachs of juvenile cichlid fish, and Eimeria (s. l.) vanasi. A survey of coccidians parasitizing marine fishes from the coast of Senegal (West Africa) was done between 1988 and 1992 and samples of infected organs were collected for further transmission electron microscopy observations. Merogonic stages were observed. Goussia senegalensis undergoes merogony in the hepatic cells of Pagellus bellottii as follows: meronts develop inside the cytoplasm of the hepatocytes and merozoites develop from them through endodyogeny and endopolygeny. Early meronts are uninucleate, but for most advanced meronts the single large nucleus is divided into 2 or more nuclei as the pellicle precursors of new merozoites arise. In this study, those associated with invaginations of the inner membrane were then arranged around portions of the cytoplasm, each including one nucleus. Mature meronts contained either 2 or several merozoites. At least 2 generations of merozoites developed.

Redescription of Cardiosporidium cionae (Van Gaver and Stephan, 1907) (Apicomplexa: Piroplasmida), a plasmodial parasite of ascidian haemocytes

Cardiosporidium cionae (Apicomplexa), from the ascidian Ciona intestinalis L., is redescribed with novel ultrastructural, phylogenetic and prevalence data. Ultrastructural analysis of specimens of C. intestinalis collected from the Gulf of Naples showed sporonts and plasmodia of C. cionae within the host pericardial body. Several merogonic stages and free merozoites were found in the pericardial body, together with sexual stages. All stages showed typical apicomplexan cell organelles, i.e. apicoplasts, rhoptries and subpellicular microtubules. Merogonic stages of C. cionae were also produced inside haemocytes. A fragment of the rSSU gene of C. cionae was amplified by PCR using DNA extracted from the pericardial bodies. The amplified product showed closest affinity with other apicomplexan representatives and a 66 bp unique insertion, specific for C. cionae, at position 1644. Neighbour-joining phylogenetic analysis placed C. cionae in a clade with other piroplasm genera, including Cytauxzoon, Babesia and Theileria spp. The parasite was found in different populations of C. intestinalis with highest prevalence in October–November. Ultrastructural and DNA data showed that the organism, described in 1907 from the same host but not illustrated in detail, is a member of a novel marine apicomplexan radiation of tunicate parasites.

The status of Haemogregarina mansoni Sambon and Seligmann from Zamenis flagelliformis Laurenti.

Haemogregarina mansoni Sambon and Seligmann 1907, a parasite of the coachwhip snake (Masticophis flagellum) is redescribed on the basis of sporogonic stages obtained in the experimental vector Aedes aegypti and designated as Hepatozoon mansoni from hosts collected in north Florida. Gamonts average 14.7 x 4.7 (13-16 x 4-6) and are not recurved, with nuclei situated in the second quarter of the gamont. Erythrocyte cytoplasm is often thin, appearing partially dehemoglobinized, or contracted into a central mass with infected erythrocytes always distorted, longer, and more slender than uninfected cells. Sporogony occurred within the head and thorax of A. aegypti. Oocysts were spherical to ovoid, 144.2 x 126.1 (79-198 x 69-178), containing on average 29.2 (7-64) sporocysts. Sporocysts were spherical to ovoid, 33.1 x 29.8 (19-48 x 18-44), with 20.2 (12-32) sporozoites contained within. Experimental infection in Tantilla relicta produced gamonts that did not differ from those in M. flagellum, but dehemoglobinization and cytoplasmic contraction of the host erythrocyte did not occur, and persistent merogonic stages were not present in the tissues.

Hepatozoon kisraen. sp. infecting the lizardAgama stelliois transmitted by the tickHyalommacf.aegyptium

Hepatozoon kisrae n. sp. was found infecting a starred lizard at a site in southeastern Samaria, Palestine. These lizards were also hosts to the ixodid tick Hyalomma cf. aegyptium, which was demonstrated to be the vector of this hemogregarine. Hepatozoon and tick infections occurred in lizards within a very restricted locality; at a second site, nearby, ticks occurred without Hepatozoon infection. Micro- and macromeronts occurred mainly in the lungs, while cyst-like merogonic stages, mainly dizoic, occurred in the liver. Mature intraerythrocytic gametocytes were stout and encapsulated. Development from oocysts to sporocysts took place in the tick hemocoel, and was examined by transmission electron microscopy. Lizards were successfully infected when fed on sporocyst-infected ticks or viscera of infected lizards. Ticks become infected when fed on infected lizards; sporogony was complete when the ticks reached adult stage, over 40 days after initial attachment.

Fine structure of erythrocytic stages of a Plasmodium tropiduri-like malaria parasite found in the lizard Kentropyx calcarata (Teiidae) from north Brazil.

The fine structure is described of the merogonic stages and gametocytes of a Plasmodium tropiduri Aragão et Neiva, 1909-like parasite infecting the teiid lizard Kentropyx calcarata Spix from North Brazil. The trophozoites are bordered by two membranes, and with growth a pellicle is formed by the addition of an inner, thick double layer and fragmented membrane. The same type of inner membrane occurs in the pellicle of the merozoites differentiating from the meronts. Merozoites contained a large electron-dense body, sometimes seen to be embraced by a tubular mitochondrion with a dense matrix. Micro- and macrogametocytes are bounded by a double membrane, closely apposed by the detached wall of the parasitophorous vacuole. Both contain osmiophilic bodies. The microgametocyte contains an electron-dense aggregate, and the macrogametocyte has a large mitochondrion and a complex of tubuli and cisternae. These features are compared with those described in other malarial parasites.

Ultrastructure of developmental stages of Hemolivia mariae (Apicomplexa: Haemogregarinidae), natural parasite of the Australian sleepy lizard, in experimentally infected deviant hosts.

Mabuya vitatta (Olivier) (Scincidae) and Agama stellio (L.) (Agamidae) were infected with Hemolivia mariae Smallridge et Paperna, 1997 by ingestion of tick viscera from Amblyomma limbatum Neumann, fed as nymphs on naturally infected Australian sleepy lizards, Tiliqua rugosa Gray. The unnatural infection apparently interfered with the developmental schedule of the parasites. Transmission electron microscopic images of merogonic stages were obtained, as well as images of early developing gametocytes. Tissue and intraerythrocytic meronts were bound by a hardened wall. Intraerythrocytic gametocytes were lodged in a parasitophorous vacuole, which was filled with granular material, and were bound by a two-membrane wall. Small and large osmiophilic bodies were located in a sub-pellicular position. With differentiation, the wall membranes tightened with the parasitophorous vacuole wall, and the osmiophilic bodies disappeared. The outer parasite membrane consolidated into a thick encasing with distinct sutures. Late infection in A. stellio comprised gametocytes only.

Localization of the initial developmental stages of Loma salmonae in rainbow trout (Oncorhynchus mykiss).

The intracellular microsporidian parasite Loma salmonae affects salmonids of the genus Oncorhynchus and is a significant cause of economic losses in pen-reared Chinook salmon (O. tshawytscha) in British Columbia. Loma salmonae infection is easily recognized by the xenomas that form in the gills, but early stages of infection are difficult to detect in histologic sections. In situ hybridization (ISH), using an L. salmonae-specific digoxigenin-labeled single-stranded DNA probe, was used to detect the parasite during the early stages of infection. Loma salmonae was detected in the gut mucosal epithelium as early as 24 hours postexposure (PE), and it localized in the lamina propria of the intestine within 24 hours of infection. After the parasite was detected in the lamina propria, dividing merogonic stages in infected cells in the heart were detected by ISH as early as 2 days PE, providing the first evidence of parasitaemia and hematogenous distribution of this parasite in infected blood cells. The parasites inside the infected cells appeared to be undergoing merogony as they passed through the heart, indicating that proliferation may start at the site of infection, before the parasite arrives to the gills for their final developmental phase. This is the first time that L. salmonae passage through the intestinal wall and migration to the heart has been visualized; however, the identity of the cells harboring the parasite has yet to be determined.

In vitro efficacy of nikkomycin Z against the human isolate of the microsporidian species Encephalitozoon hellem.

Since 1985 microsporidia have been recognized as a cause of emerging infections in humans, mainly in immunocompromised human immunodeficiency virus-positive subjects. As chitin is a basic component of the microsporidian infective stage, the spore, we evaluated in vitro the susceptibility of a human-derived strain of Encephalitozoon hellem to nikkomycin Z, a peptide-nucleoside antibiotic known as a competitive inhibitor of chitin synthase enzymes. Transmission electron microscopy showed that this drug, at 25 microgram/ml, reduced the number of parasitic foci by about 35% +/- standard deviation after 7 days of culture (P < 0.0001) and induced cell damage of both mature and immature spores and also other sporogonic and merogonic stages. In particular, an irregular outline of the cell shape and an abnormally condensed cytoplasm in meronts and sporonts were documented. Also, the polar tubule and the polaroplast membranes appeared disarrayed in the sporoblast stage. The spore wall showed an enlarged endospore and delaminated exospore. Mature spores had a complete cytoplasmic disorganization and a swollen and delaminated cell wall. No ultrastructural cell damage was observed in uninfected control cultures treated with the drug.