Haemogregarina bigemina (Protozoa: Apicomplexa: Adeleorina)--past, present and future

This paper reviews past, current and likely future research on the fish haemogregarine, Haemogregarina bigemina Laveran et Mesnil, 1901. Recorded from 96 species of fishes, across 70 genera and 34 families, this broad distribution for H. bigemina is questioned. In its type hosts and other fishes, the parasite undergoes intraerythrocytic binary fission, finally forming mature paired gamonts. An intraleukocytic phase is also reported, but not from the type hosts. This paper asks whether stages from the white cell series are truly H. bigemina. A future aim should be to compare the molecular constitution of so-called H. bigemina from a number of locations to determine whether all represent the same species. The transmission of H. bigemina between fishes is also considered. Past studies show that young fish acquire the haemogregarine when close to metamorphosis, but vertical and faecal-oral transmission seem unlikely. Some fish haemogregarines are leech-transmitted, but where fish populations with H. bigemina have been studied, these annelids are largely absent. However, haematophagous larval gnathiid isopods occur on such fishes and may be readily eaten by them. Sequential squashes of gnathiids from fishes with H. bigemina have demonstrated development of the haemogregarine in these isopods. Examination of histological sections through gnathiids is now underway to determine the precise development sites of the haemogregarine, particularly whether merozoites finally invade the salivary glands. To assist in this procedure and to clarify the internal anatomy of gnathiids, 3D visualisation of stacked, serial histological sections is being undertaken. Biological transmission experiments should follow these processes.     

A new fish haemogregarine from South Africa and its suspected dual transmission with trypanosomes by a marine leech

Twenty two percent (22/98) of intertidal fishes of 10 species captured in South Africa at Koppie Alleen, De Hoop Nature Reserve (south coast) and Mouille Point, Cape Town (west coast), harboured single or combined infections of haemogregarines, trypanosomes and an intraerythrocytic parasite resembling a Haemohormidium sp. The haemogregarines included the known species Haemogregarina (sensu lato) bigemina (Laveran et Mesnil, 1901) Siddall, 1995 and Haemogregarina (sensu lato) koppiensis Smit et Davies, 2001, while Haemogregarina (sensu lato) curvata sp. n. was observed in Clinus cottoides Valenciennes and Parablennius cornutus (L.) at Koppie Alleen. This last haemogregarine is characterised particularly by its distinctly curved gamonts. Also at Koppie Alleen, squash and histological preparations of 9/10 leeches, Zeylanicobdella arugamensis De Silva, 1963, taken from infected C. cottoides and P. cornutus contained developmental stages of H. curvata and/or trypanosomes, but these were absent from haematophagous gnathiid isopods (Gnathia africana Barnard, 1914) taken from infected fishes. It is suspected that Z. arugamensis transmits the haemogregarine and trypanosomes simultaneously between fishes, a double event unreported previously from the marine environment.     

Haematozoans from deep water fishes trawled off the Cape Verde Islands and over the Porcupine Seabight, with a revision of species within the genus Desseria (Adeleorina: Haemogregarinidae)

Archived blood smears from 32 of 113 fishes in 18 families and 12 orders, trawled from deep North Atlantic waters off the Cape Verde Islands in 1999 and over the Porcupine Seabight in 2001 were found to harbour haematozoans. These included four species of haemogregarines (Adeleorina, Haemogregarinidae) and a species of trypanosome (Trypanosomatina, Trypanosomatidae) located in Porcupine Seabight fishes. Also present were Haemohormidium-like structures of uncertain status found in samples from this location and from the Cape Verde Islands. Although material was limited, two of the haemogregarines were provisionally named Desseria harriottae sp. n. from Harriotta raleighana Goode et Bean (Chimaeriformes, Rhinochimaeridae), and Haemogregarina bathysauri sp. n. from Bathysaurus ferox Günther (Aulopiformes, Bathysauridae). The two remaining haemogregarines were identified as Desseria marshalllairdi (Khan, Threlfall et Whitty, 1992) from Halosauropsis macrochir (Günther) (Notacanthiformes, Halosauridae), and Haemogregarina michaeljohnstoni (Davies et Merrett, 2000) from Cataetyx laticeps Koefoed (Ophidiformes, Bythitidae). The name H. michaeljohnstoni was proposed to replace Haemogregarinajohnstoni Davies et Merrett, 2000 from C. laticeps and to avoid confusion with Hepatozoon johnstoni (Mackerras, 1961) Smith, 1996 from varanid lizards, originally named Haemogregarina johnstoni Mackerras, 1961. The trypanosome formed a mixed parasitaemia with D. harriottae in H. raleighana and was provisionally named Trypanosoma harriottae sp. n. No blood parasites had been described previously from cartilaginous fishes of the Holocephali, making the finds in H. raleighana unique. Haemohormidium-like structures were located in erythrocytes in one fish, Coryphaenoides armatus (Hector), among the Cape Verde Islands samples and in 12 species of fishes from the Porcupine Seabight; all these hosts were bony fishes. Finally, the haemogregarine species listed in the genus Desseria Siddall, 1995 were reassessed. Of the original list of 41 species, 30 were retained and 5 species added, including D. harriottae, so that the genus now contains 35 species.     

Life cycle of Eimeria coecicola Cheissin, 1947

Life cycle of Eimeria coecicola was studied in experimentally infected rabbits by light microscopy and by transmission and scanning electron microscopy. First and second generation meronts developed in the vermiform appendix; third and fourth generation meronts were located in the epithelium of the ileum. Gametogony developed again in the vermiform appendix. The prepatent period was 9 days. New data were obtained by the study of asexual reproduction. First generation meronts were first observed 4 days post infection (DPI), which is relatively late in comparison with other species of rabbit coccidia. Sporozoites were found in lymphatic follicles of the vermiform appendix at 4 DPI by transmission electron microscopy. This suggests, together with selective location of first generation meronts in the epithelium adjacent to these follicles, that major part of sporozoites enter the epithelium cells through lymphatic follicles and not through the lumen of the vermiform appendix. The process of development of first generation merozoites is similar to endodyogeny. The differences are in formation of apical parts of daughter merozoites which is not coincidental with nuclear division and in formation of the outer membrane of pellicle which arises within the mother cell. Some first generation merozoites have 2-3 nuclei, second and fourth generation merozoites are only uninucleate, while third generation merozoites are only multinucleate. We found that further merozoites are formed in multinucleate third generation merozoites by endopolygeny.     

Intraerythrocytic merogony in Haemogregarina koppiensis (Apicomplexa: Adeleorina: Haemogregarinidae)

During October 2003, a specimen of Amblyrhynchotes honckenii (Bloch, 1795) was captured at low tide, with a hand net, in a rock pool at Koppie Alleen, De Hoop Nature Reserve, South Africa. This fish was heavily parasitized by unidentified gnathiid praniza larvae, caligid copepods identified as Caligus tetrodontis Barnard, 1948, cymothoid isopods identified as Cinusa tetrodontis (Schioedte et Meinert, 1884), and the blood protozoan Haemogregarina koppiensis Smit et Davies, 2001. Giemsa-stained blood smears from this fish revealed new and unusual stages of merogony for H. koppiensis that included small, rounded, likely intraerythrocytic merozoites arranged in circles of eight around the host nucleus. Host cells appeared ghost-like and enlarged compared with normal erythrocytes. Identical merozoites, usually in clusters of up to 16, were also observed free of host cells. The pattern of merogony seen in H. koppiensis is unusual for a fish haemogregarine.     

The life cycle of Eimeria danailovi from ducks.

The life cycle of Eimeria danailovi Gr?fner, Graubmann et Betke in experimentally infected ducks was studied by optical microscopy. The asexual generation developed in the posterior part of jejunum and in the whole ileum. The sexual stages occurred in jejunum and ileum, and, in addition, in cecum and colon. All endogenous stages were localized in the cytoplasm of epithelial cells in the apical and basal parts of villi. Two generations of meronts were found to develop, differing from one another in the number of merozoites. The meronts of the first generation were observed 2 days post infection (DPI) and contained 10-14 (on the average 11) merozoites. The second generation of meronts, containing 12-22 (on the average 16) merozoites, developed on 3 DPI. The sexual stages were found in histological preparations on 5 and 6 DPI. They appeared in the faces of experimentally infected ducks first on 5 DPI and they were shed for three days. Oocyst sporulation at room temperature lasted 2-3 days.     

Genetic characterization of the Golovinomyces cichoracearum complex in Australia

To characterize the evolutionary lineages of the Golovinomyces cichoracearum complex on introduced plants in Australia, the rDNA ITS regions from 47 herbarium specimens were compared by RFLPs and sequencing. Six RFLP groups were found, each corresponding to a previously reported evolutionary lineage in the complex. The largest of these groups (Group 1) contained 15 specimens infecting a range of tribes in the Asteraceae, despite a previous report that this lineage is largely restricted to the Tribe Heliantheae (Asteraceae). This group contained the only specimens with ascomata. Curved foot cells were formed by two lineages; one (Groups 5 & 6) containing specimens from a range of host families including the Asteraceae (Tribe Lactucae only), the other (Groups 2 & 3) containing members of the Asteraceae (Tribe Anthemidae only). Group 5 may represent G. orontii sensu stricto , while Group 6 is currently unnamed. Golovinomyces cichoracearum sensu stricto (specimens from Scorzonera ) formed a distinct group that did not contain any specimens from Australia. Suggestions are made for future species delimitation in the complex.     

Molecular characterization of <Emphasis Type="Italic">Ditylenchus dipsaci</Emphasis> on Onion in Turkey

Ditylenchus dipsaci is a species complex including diploid and polyploid individuals. The onion race of D. dipsaci is a sensu stricto group and has a wide range of host spectrum. Identification of the D. dipsaci onion race is difficult using morphological and morphometrical methods. Species specific primers are mostly used in molecular approaches for identification of D. dipsaci populations. Fifty one morphologically selected Ditylenchus spp. populations from onion production areas in Turkey were subjected to molecular identification using four D. dipsaci species specific primer sets (PF1-PR1, PF2-PR2, DdpS1-rDNA2, DitNF1- rDNA2, H05-H06) targeting 5.8S and 18S rDNA, ITS1 and flanking ITS regions. Thirty nine percent of the nematode samples were positive with four primers tested, while four of the nematode samples gave specific bands with H05-H06 primers. Ditylenchus dipsaci sensu stricto was identified with specific primer sets in Adana, Hatay, Tekirdag, Bursa, Aksaray, Karaman, Eskisehir and Ankara provinces in Mediterranean, Trace, Aegean and Central Regions in Turkey.     

Isospora carliae sp. n. (Apicomplexa: Eimeriidae) from the skink Carlia rhomboidalis (Peters) from Daintree Forest, North Queensland, Australia: description and fine-structural account of endogenous development

Isospora carliae sp. n. is described from the blue-throated rainbow skink Carlia rhomboidalis (Peters), from Daintree Forest, North Queensland, Australia. Oocysts are ellipsoidal, 16.8-21.0 x 12.6-15.4 microm in size, with their two sporocysts, 9.0-14.0 x 7.0-9.24 microm in size, positioned along the wide axis. Sporozoites contain a distinct refractile body and are accompanied by a residuum. All endogenous development occurs within the host-cell nucleus. Nuclei are sometimes invaded by several merozoites, but only infections by a single parasite persist. Nuclei lodging meronts, mature microgamonts and premature macrogamonts have an elongate shape. Some meronts exhibit a membrane-bound cytoplasmic inclusion that contains many micronemes.     

The use of pectic enzymes in the characterization of Armillaria isolates from Africa

Isozyme analysis has been used to compare 26 tropical African and 26 European Armillaria isolates from a wide range of geographic regions and host plants. Pectic enzymes from culture filtrates, especially pectin lyase (PL) and pectin methylesterase (PME), were particularly useful in grouping the isolates. Isolates from Africa previously classified as either A. heimii or A. mellea were compared with three Zimbabwean groups, previously distinguished by their morphological and biochemical properties. Group I isolates from Zimbabwe resembled A. heimii isolates, while groups II and III from Zimbabwe, and A. mellea from Kenya, formed distinct groups. The African A. mellea group was absent from isolates collected in Zimbabwe. Five European Armillaria species ( A. tabescens A. mellea A. gallica A. ostoyae A. cepistipes ) also showed species-specific pectic enzyme patterns. Thirteen isolates of A. mellea sensu stricto collected from four European countries showed almost identical PL and PME patterns, but these patterns were quite distinct from those of isolates from Africa previously referred to as A. mellea , indicating that this species is not identical to A. mellea sensu stricto. These observations confirm the potential use of pectic enzymes in grouping Armillaria species.     

Haemogregarines of the genus Hepatozoon (Apicomplexa: Adeleina) in rodents from northern Europe

We studied the prevalence and distribution of Hepatozoon infections in small rodents from Finland and other areas in northern Europe. Hepatozoon infections were more common in voles (Arvicolinae) than mice (Murinae) and more prevalent in voles of the genus Clethrionomys than in voles of the genus Microtus. Transmission electron microscopical examination of Hepatozoon erhardovae Krampitz, 1964 from bank voles Clethrionomys glareolus (Schreber) showed that intracellular lung meronts were located in alveolar septa. Meronts consisted of varying numbers of merozoites packed with amylopectin vacuoles inside electron-lucent parasitophorous vacuole. The size of the meronts was approximately 19 x 14 microm. Monozoic or dizoic cysts were frequent findings in the lung alveoles; the size of cysts was approximately 10 x 6 microm. Gametocytes were found inside eosinophilic granulocytes in the capillaries of lung tissue. Ultrastructurally, micronemes, microtubules, mitochondria, nuclei and lipid droplets were visible.     

An antigen from Eimeria tenella merozoites for the indirect fluorescence antibody test.

The suitability of the antigen prepared from second generation merozoites of Eimeria tenella for the IFAT was confirmed. In comparison with other two antigens used until now in routine work, i. e., that prepared from histological sections of infected organs or that from in vitro released sporozoites, the merozoite antigen has the advantage of easy preparation in large quantities.     

Ultrastructural study of meronts and gamonts of Choleoeimeria rochalimai (Apicomplexa: Eimeriidae) developing in the gall bladder of the gecko Hemidactylus mabouia from Brazil

Endogenous development of Choleoeimeria rochalimai (Carini et Pinto, 1926) Lainson et Paperna, 1999 in the gall bladder of Hemidactylus mabouia (Moreau de Jonnes, 1818) from Belém, Brazil is reported at the fine structural level. Meronts and gamonts develop in the epithelial cells of the gall bladder. Infected cells become enlarged and displaced above the epithelial layer. Developing merozoites, dividing meronts and succession of developing microgamonts from initial nuclear division up to final microgamete differentiation are described. In addition towall forming bodies, mature macrogamonts possess a large inclusion or cisterna with fine granular contents.     

Molecular Diagnostics, Taxonomy, and Phylogeny of the Stem Nematode Ditylenchus dipsaci Species Complex Based on the Sequences of the Internal Transcribed Spacer-rDNA

ABSTRACT The stem nematode Ditylenchus dipsaci is of great economic importance worldwide as a parasite of agricultural crops and horticultural plants. The internal transcribed spacer (ITS) of rDNA from 23 populations of the D. dipsaci complex from various host plants were amplified and sequenced. Seven previously studied populations were also included in the study. The phylogenetic analysis of the full ITS and ITS2 sequence alignments using minimum evolution, maximum parsimony, and Bayesian inference under the complex model of DNA evolution revealed trees with two main clades: (i) D. dipsaci sensu stricto with diploid chromosome numbers and comprising most isolates from agricultural, ornamental, and several wild plants, and (ii) Ditylenchus spp. with polyploid chromosome numbers, reproductively isolated from diploid populations, and subdivided into six subclades ("giant race" from Vicia faba, Ditylenchus species parasitizing various Asteraceae, and a Ditylenchus sp. from Plantago maritima). Using the energy minimization approach and comparative sequence analysis, it has been found that the secondary structure of ditylenchid ITS2 is organized in three main domains. The importance of knowledge on the RNA structure for phylogenetic analysis is discussed. Conventional polymerase chain reaction (PCR) and real-time PCR with SYBR green dye I with a species specific primer have been developed for detection and quantification of D. dipsaci sensu stricto Validation tests revealed a rather high correlation between real numbers of fourth-stage juveniles of the stem nematodes in a sample and expected numbers detected by real-time PCR. Problems of accuracy of quantification are discussed.     

Effect of salivary gland extract from Ixodes ricinus ticks on the proliferation of Borrelia burgdorferi sensu stricto in vivo

Saliva-activated transmission (SAT) of Borrelia burgdorferi sensu stricto was demonstrated using real-time PCR and salivary gland extract (SGE) from partially fed Ixodes ricinus ticks. C3H/HeN mice were injected intradermally with 1.5 x 10(3) spirochetes mixed with 40 microg of SGE per mouse. The control group was inoculated with the same dose of spirochetes without SGE. The accelerating effect of SGE on spirochete proliferation was demonstrated on day 1 post infection, when a 4.2-fold increase in spirochetes was found in the skin and a 10-fold increase in the blood, compared with control mice. The data represent the first direct evidence of a SAT effect of I. ricinus SGE on infection with the Lyme disease agent B. burgdorferi.     

Amplified Fragment Length Polymorphism Analysis and Internal Transcribed Spacer and coxII Sequences Reveal a Species Boundary Within Pythium irregulare

ABSTRACT Pythium irregulare is a plant-pathogenic oomycete that causes significant damage to a variety of crops, including ornamentals and vegetables. Morphological as well as molecular studies have reported high levels of genetic diversity within P. irregulare sensu lato which has raised the question as to whether it is a single species or is actually a complex of morphologically similar (cryptic) species. In this study, we used amplified fragment length polymorphism (AFLP) fingerprinting and DNA sequence analysis of the internal transcribed spacer (ITS) region of the ribosomal genes (ITS region) and a portion of the mitochondrial cytochrome oxidase II gene and the spacer region between coxI and coxII to characterize 68 isolates of P. irregulare from the United States. The ITS sequence of a P. irregulare neotype at the CBS collection as well as ITS and coxII sequences for P. irregulare, P. spinosum, and P. sylvaticum from previous studies were included in our analysis. Cluster analysis identified a 19-isolate group (IR-II) that separated itself from the rest of the sample (IR-I). Population structure and sequence analyses supported the distinction of IR-I and IR-II and identified IR-II as P. irregulare sensu stricto. IR-I was designated Pythium sp. clade IR-I. Two insertion/deletion mutations and nine nucleotide substitutions in the ITS region and three in the sequence of coxII and the adjacent spacer region separated the two species. Additionally, they differed significantly (P > 0.01) in the frequency of 182 (77%) AFLP alleles. Gene flow results suggested that P. irregulare sensu stricto and Pythium sp. clade IR-I are cryptic species capable of exchanging favorable alleles (Nm = 0.72).     

The Entomophthorales of Israel and their arthropod hosts

Species of entomophthoraceous fungi attacking insects and mites are very well represented in Israel. In the last decade, 22 species ofEntomophthora sensu stricto, Triplosporium, Zoophthora, Conidiobolus, and others under the designationEntomophthora sensu lato, have been identified, of which three are new species:Entomophthora turbinata, Zoophthora erinacea andZ. orientalis. Fiftyfour host-pathogen combinations hitherto not recorded in Israel are listed, of which 19 insect species are new hosts of entomophthoraceous species anywhere.     

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 Arag?o 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.