Transmission of Haemogregarina balli from painted turtles to snapping turtles through the leech Placobdella ornata.

Six leeches (Placobdella ornata) were allowed to feed on a painted turtle (Chrysemys picta marginata) infected with Haemogregarina balli and subjected to a period of diapause before being allowed to feed on 2 laboratory-reared snapping turtles (Chelydra serpentina). Weekly examination of thin blood films revealed infections of the turtles at 130 days postfeeding. These observations provide support for broad host specificity of hemogregarine parasites of chelonians.

Recent advances in our knowledge of the Myxozoa.

In the last few years two factors have helped to significantly advance our understanding of the Myxozoa. First, the phenomenal increase in fin fish aquaculture in the 1990s has lead to the increased importance of these parasites; in turn this has lead to intensified research efforts, which have increased knowledge of the development, diagnosis. and pathogenesis of myxozoans. The hallmark discovery in the 1980s that the life cycle of Myxobolus cerebralis requires development of an actinosporean stage in the oligochaete. Tubifex tubifex, led to the elucidation of the life cycles of several other myxozoans. Also, the life cycle and taxonomy of the enigmatic PKX myxozoan has been resolved: it is the alternate stage of the unusual myxozoan, Tetracapsula bryosalmonae, from bryozoans. The 18S rDNA gene of many species has been sequenced, and here we add 22 new sequences to the data set. Phylogenetic analyses using all these sequences indicate that: 1) the Myxozoa are closely related to Cnidaria (also supported by morphological data); 2) marine taxa at the genus level branch separately from genera that usually infect freshwater fishes; 3) taxa cluster more by development and tissue location than by spore morphology; 4) the tetracapsulids branched off early in myxozoan evolution, perhaps reflected by their having bryozoan, rather than annelid hosts; 5) the morphology of actinosporeans offers little information for determining their myxosporean counterparts (assuming that they exist); and 6) the marine actinosporeans from Australia appear to form a clade within the platysporinid myxosporeans. Ribosomal DNA sequences have also enabled development of diagnostic tests for myxozoans. PCR and in situ hybridisation tests based on rDNA sequences have been developed for Myxobolus cerebralis, Ceratomyxa shasta, Kudoa spp., and Tetracapsula bryosalmonae (PKX). Lectin-based and antibody tests have also been developed for certain myxozoans, such as PKX and C. shasta. We also review important diseases caused by myxozoans, which are emerging or re-emerging. Epizootics of whirling disease in wild rainbow trout (Oncorhynchus mykiss) have recently been reported throughout the Rocky Mountain states of the USA. With a dramatic increase in aquaculture of fishes using marine netpens, several marine myxozoans have been recognized or elevated in status as pathological agents. Kudoa thyrsites infections have caused severe post-harvest myoliquefaction in pen-reared Atlantic salmon (Salmo salar), and Ceratomyxa spp., Sphaerospora spp., and Myxidium leei cause disease in pen-reared sea bass (Dicentrarchus labrax) and sea bream species (family Sparidae) in Mediterranean countries.

The blood parasites of anurans from Costa Rica with reflections on the taxonomy of their trypanosomes.

During May 1997, specimens of 7 species of anurans, that included 5 Phrynohyas venulosa Laurenti, 5 Rana forreri Boulenger, 7 Rana vaillanti Brucchi, 6 Eleutherodactylus fitzingeri Schimdt, 4 Smilisca baudinii Duméril and Bibron, 1 Leptodactylus melanonotus, and 3 Bufo marinus Linneaus, from the Guanacaste Conservation Area, Costa Rica were examined for blood parasites. Their hematozoan fauna included intraerythrocytic and intraleukocytic icosahedral viruses, a rickettsia (Aegyptianella sp.), 2 species of Hepatozoon, Lankesterella minima, 2 unknown species of apicomplexans, 9 morphologically distinct types of trypanosomes, and 2 species of microfilariae. Rana vaillanti, the most aquatic species of frog, harbored the most species of parasites. Recent evidence indicates that morphological changes in the highly pleomorphic trypanosomes of anurans from different geographical regions have not kept pace with biochemical (isozyme) and molecular (DNA sequence) changes. Describing new species based solely on bloodstream trypomastigotes is discouraged. Additional criteria described herein should be applied when naming new species of anuran trypanosomes.

Intraerythrocytic development of species of Hepatozoon infecting ranid frogs: evidence for convergence of life cycle characteristics among apicomplexans.

Intraerythrocytic development of the adeleorin apicomplexans Hepatozoon clamatae and Hepatozoon catesbianae were investigated in the bullfrog, Rana catesbeiana, the green frog, Rana clamitans melanota, and the Northern leopard frog, Rana pipiens. Merozoites emerging from hepatic meronts penetrated erythrocytes and underwent 1-3 rounds of binary fission to produce 2-8 merozoites. Following their release from infected erythrocytes, individual merozoites entered new cells and transformed into gamonts. Although this is the first report of intraerythrocytic development for a fully described species of Hepatozoon, a phylogenetic reanalysis of 11 species of Hepatozoon, 6 species representative of the 5 other hemogregarine taxa, 2 species of dactylosomatids, and 2 species of piroplasms, indicates that asexual reproduction of parasites within blood cells of vertebrates has arisen at least 3 times in the apicomplexan lineage that includes adeleorins and piroplasms. This method of asexual development, which is also observed in species of hemospororin genera such as Plasmodium, is discussed in the context of the evolution of apicomplexan life cycles. In addition to supporting the paraphyly of the genus Hepatozoon determined in an earlier study, this phylogenetic analysis featured a monophyletic group, consisting of the sister taxa Hemolivia and Karyolysus, that was the sister group to a clade consisting of the more derived hemogregarines, the dactylosomatids, and the piroplasms.

Descriptions and phylogenetic systematics of Myxobolus spp. from cyprinids in Algonquin Park, Ontario.

Eight species of Myxobolus were collected from four species of cyprinids in Algonquin Park, Ontario. On the basis of spore morphology, five of these species are described as new and two are redescribed. The evolutionary relationships among these eight species were studied using partial small subunit ribosomal DNA (ssu-rDNA) sequence data. The resulting cladograms, which were highly resolved and with strongly supported relationships, allowed for the evaluation of spore morphology, host specificity, and tissue tropism, criteria traditionally used in species identification. These criteria, recently criticized for creating artificial rather than natural taxonomic groupings, were evaluated for their reliability in the systematics of the species examined. The data showed that distantly related species often infect the same host and tissue, and that closely related species often occur in different hosts. Morphologically similar species are more closely related to each other and the taxonomy based on spore morphology is consistent with the relationships depicted in the phylogenies. These results suggest that spore morphology is better than host specificity and tissue tropism as a species character, as well as for determining evolutionary relationships among the species of Myxobolus examined.

Cladistic analysis of myxozoan species with known alternating life-cycles.

The phylogenetic relationships of 15 myxozoan taxa with known alternating life-cycles were investigated in order to provide insight into the puzzling matches between myxosporeans and actinosporeans of the myxozoan life-cycle data. Phylogenetic analyses were performed using two partitioned data-sets of life-cycle stages, myxosporean stage from fish hosts versus actinosporean stage from annelid hosts, and a combined data-set of myxosporean and actinosporean stages. A cnidarian parasite of fish, Polypodium hydriforme Ussov, 1885, was used as the outgroup. The supraspecific level grouping in the conventional classification of actinosporeans was not supported in the analysis of the partitioned data from the actinosporean phase, which yielded two equally parsimonious trees. Analysis of the partitioned data from the myxosporean phase provided 24 equally parsimonious trees and did not support the current classification of myxosporeans. The analyses of the partitioned data of myxozoans by life-cycle stage revealed a lack of taxonomic congruence between the two life-stage partitions. Two equally parsimonious trees were obtained from analysis of the combined data. The suborder Variisporina of the Myxozoa was not supported by the total evidence trees, while the monophyly of the species of Myxobolus Butschli, 1882 and of the Myxidiidae were supported. The cladograms from the combined data revealed that these myxozoan species formed four major monophyletic groups. Among them, two were supported by the partitioned data of the actinosporean phase. The phylogenetic signals and the better resolution reflected by the trees of combined data suggest that the phylogenetic total evidence approach should be employed in future studies of the systematics of myxozoans.

The blood parasites of the spiny pocket mouse Liomys salvini (Thomas, 1893) from Costa Rica.

A survey of the blood parasites of 20 Liomys salvini revealed 3 types of parasites. Sixty percent of the mice harbored Trypanosoma zeledoni, 5% an unnamed species of Hepatozoon, and 20% of the mice were infected with a species of Haemobartonella.

The longevity of actinosporean spores from oligochaetes of Lake Sasajewun, Algonquin Park, Ontario, and their reaction to fish mucus.

The longevity of 7 forms of actinosporean spores and the reaction of 6 forms of actinosporeans to fish mucus were investigated. The maximum longevity of actinosporean spores kept at ambient laboratory temperatures was 14 days. Spore longevity ranged from 11 to 14 days among actinosporeans. The reaction of spores to fish mucus varied among the actinosporeans. Triactinomyxon F of Xiao and Desser, 1998 reacted only to the mucus of the common shiner Luxilus cornutus, and golden shiner Notemigonus crysoleucas, whereas the aurantiactinomyxon form of Xiao and Desser, 1998, and raabeia B of Xiao and Desser, 1998 reacted readily to mucus of all fish species tested. The differences in reaction to fish mucus among actinosporeans may indicate their different host range. These results indicate that actinosporean spores are short-lived and that actinosporeans respond to their hosts by chemodetection.

Molecular characterization of myxozoan parasites from Lake Sasajewun, Algonquin Park, Ontario, by riboprinting.

The small subunit-rRNA genes of 18 myxozoans from Lake Sasajewun, Algonquin Park were amplified and digested with restriction endonucleases for riboprinting analysis. Identical riboprints were not found between the myxosporeans and the actinosporeans. The distinct riboprinting patterns observed among these myxozoans indicate considerable genetic diversity within this group. Identical riboprints were found between Myxobolus pendula and Myxobolus pellicides, and between triactinomyxon 'C' and Triactinomyxon ignotum. Parsimony analysis of the riboprints demonstrated that neither the myxosporeans nor the actinosporeans formed a monophyletic group. Some species of Myxobolus are more closely related to forms of triactinomyxon, echinactinomyxon or raabeia than to other Myxobolus species. These results are consistent with the two-host life cycle hypothesis of myxozoans that myxosporeans and actinosporeans are alternating stages of the same organisms.

Phylogenetic relationships among Hepatozoon species from snakes, frogs and mosquitoes of Ontario, Canada, determined by ITS-1 nucleotide sequences and life-cycle, morphological and developmental characteristics.

The molecular biological characteristics of Hepatozoon species infecting various species of snakes, frogs and mosquitoes were investigated by determining the nucleotide sequences of the first internal transcribed spacer region. A phylogenetic analysis was performed on seven isolates of Hepatozoon infecting snakes, including Hepatozoon sipedon and four morphologically similar but not identical forms, and two isolates of Hepatozoon catesbianae infecting Green frogs (Rana clamitans melanota). This analysis, which utilised data from first internal transcribed spacer nucleotide sequences, morphological and morphometric features of gamonts, oocysts and sporocysts, and previously determined life-cycle and host-specificity characteristics, revealed that H. sipedon is a polymorphic species with a wide host and geographic range. Four synapomorphies. including two nucleotide substitutions and two morphological character state changes, supported a monophyletic group of six isolates of H. sipedon from the central region of Ontario which was the sister group for an isolate (HW1) from the southern part of the province. Based on the results of this study, an evaluation of which criteria are useful for describing species of Hepatozoon is presented, with the intent of curtailing the practice of naming species based on morphological features of gamonts or on incomplete life-cycle data.

Actinosporean stages of myxozoan parasites of oligochaetes from Lake Sasajewun, Algonquin Park, Ontario: new forms of triactinomyxon and raabeia.

Eight forms of triactinomyxon and 6 forms of raabeia of oligochaetes from Lake Sasajewun, Algonquin Park are described. Of the 8 forms of triactinomyxon, 2 designated as triactinomyxon 'B' and 'E' were found in both Limnodrilus hoffmeisteri and Tubifex tubifex. Three additional forms, designated as triactinomyxon 'A,' 'C,' and 'D,' occurred in L. hoffmeisteri. One form infecting L. hoffmeisteri was identified as Triactinomyxon ignotum Stolc, 1899, and another infecting T. tubifex was tentatively identified as Triactinomyxon dubium Granata, 1924. One form infecting L. hoffmeisteri and Rhyacodrilus coccineus was designated as triactinomyxon 'F.' Of the 6 forms of raabeia, 4 designated as raabeia 'A,' 'B,' 'C,' and 'F' were found in L. hoffmeisteri, and 2 designated as raabeia 'D' and 'E' were recorded in T. tubifex. The sporoplasmic mass of each form of triactinomyxon usually moved posteriorly within the cavity of the spore axis and was released through its posterior end, whereas the sporoplasmic mass of each form of raabeia was released through the anterior end of the epispore. An increase in the number of germ cells was observed in the floating spore stage of triactinomyxon 'B,' 'D,' 'E,' and raabeia 'B.'

Actinosporean stages of myxozoan parasites of oligochaetes from Lake Sasajewun, Algonquin Park, Ontario: new forms of echinactinomyxon, neoactinomyxum, aurantiactinomyxon, guyenotia, synactinomyxon and antonactinomyxon.

Six forms (5 of which are new) of echinactinomyxon and 5 new forms of the collective groups neoactinomyxum, aurantiactinomyxon, guyenotia, synactinomyxon, and antonactinomyxon of oligochaetes in Lake Sasajewun, Algonquin Park are described. Five forms of echinactinomyxon designated as echinactinomyxon 'A,' 'B,' 'C,' 'D,' and 'E,' a form of neoactinomyxum, and a form of aurantiactinomyxon were found in Limnodrilus hoffmeisteri. Echinactinomyxon radiatum, a form of synactinomyxon, and a form of antonactinomyxon were recorded from Tubifex tubifex. A form of guyenotia was found in Lumbriculus variegatus. The sporoplasmic mass of these actinosporeans was released through the anterior end of the epispore. An increase in the number of germ cells was observed in the floating spore stage of all forms except echinactinomyxon 'C' and the form of antonactinomyxon.

The oligochaetes and their actinosporean parasites in Lake Sasajewun, Algonquin Park, Ontario.

There is little ecological information on actinosporean parasites and their oligochaete hosts. Between 1995 and 1997, about 14,100 oligochaetes belonging to 19 species were collected from Lake Sasajewun. The oligochaete fauna consisted of 5 tubificid, 10 naidid, 3 enchytraeid, and 1 lumbriculid species. The most widely distributed species in the lake were Limnodrilus hoffmeisteri, Dero nivea, and Stylaria lacustris, with L. hoffmeisteri being the most prevalent. The diversity and abundance of worms decreased with increased water depth. Four species, L. hoffmeisteri, Tubifex tubifex, Rhyacodrilus coccineus, and Lumbriculus variegatus, harbored actinosporean parasites and were distributed along shallow areas of the lake shore where the sediment was comprised mainly of clay-mud, with the shoregrass, Littorella americana. The actinosporean parasites of the oligochaetes belong to 25 forms of 8 collective groups. Triactinomyxon 'F' was the most prevalent form, whereas triactinomyxon, raabeia, and echinactinomyxon were the most speciose groups. The overall prevalence of actinosporeans was about 1%. Waterborne spores of different actinosporean forms showed distinct temporal patterns. Spores of triactinomyxon 'F' occurred throughout the sampling season, whereas spores of other forms occurred only at certain times. The presence of waterborne spores peaked from late June to August when water temperatures ranged from 18 to 24 C and coincided with the feeding and growing season of larval fish.

The life history and host specificity of Hepatozoon clamatae (Apicomplexa: Adeleorina) and ITS-1 nucleotide sequence variation of Hepatozoon species of frogs and mosquitoes from Ontario.

The life cycle of an intraerythrocytic hemogregarine, Hepatozoon clamatae, was studied in green frogs (Rana clamitans melanota), bullfrogs (Rana catesbeiana), northern leopard frogs (Rana pipiens), and in the mosquito, Culex territans. Gametogenesis, fertilization, and sporogony occurred within cells of the Malpighian tubules of laboratory-reared Cx. territans that had fed on naturally infected frogs. Mature oocysts containing hundreds of sporocysts were observed in mosquitoes 30 days postfeeding. Each sporocyst enclosed 4 sporozoites. Merozoites appeared in the peripheral circulation of laboratory-reared bullfrogs, green frogs and leopard frogs that had been fed sporocysts 35-70 days previously. Attempts to infect American toads (Bufo americanus) and blue-spotted salamanders (Ambystoma laterale) were not successful. Gamonts of this parasite induced nuclear fragmentation or segmentation in host erythrocytes. The life cycle, morphological, and morphometric features of H. clamatae are compared with H. catesbianae, a similar species that also infects ranids. Nucleotide sequence analysis of the internal transcribed spacer region (ITS-1) of these sympatric species revealed that only 6 nucleotide sites of the 129 base pairs of this region were variable among 4 isolates of H. clamatae and 2 isolates of H. catesbianae. A redescription of H. clamatae is presented based on data from this study and from the original description by Stebbins in 1905.

Ultrastructural features of cystic and merogonic stages of Hepatozoon sipedon (Apicomplexa: Adeleorina) in northern leopard frogs (Rana pipiens) and northern water snakes (Nerodia sipedon) from Ontario, Canada.

The cystic and merogonic stages of the haemogregarine Hepatozoon sipedon, infecting Northern water snakes (Nerodia sipedon sipedon) and Northern leopard frogs (Rana pipiens), respectively, in Ontario, Canada, were investigated by transmission electron microscopy. Cysts, which were observed in the liver of Northern leopard frogs (Rana pipiens) after these anurans ingested mosquitoes (Culex pipiens) containing oocysts of the parasite, harboured two cystozoites, each of which contained a large crystalloid inclusion anterior to the nucleus. Two types of meronts were observed in snakes that were fed the liver of infected frogs. Macromeronts, which matured in endothelial cells of the liver approximately 16 d after snakes ingested infected frogs, contained about 50 large macromerozoites. Macromerozoites emerged from macromeronts, entered the bloodstream of the snake, and reinfected endothelial cells. Micromeronts, which matured about 34 d post-inoculation, contained about 150 micromerozoites that infected erythrocytes and transformed into gamonts. The ultrastructural features of micromeronts and macromeronts differed only slightly: immature macromeronts and macromerozoites contained numerous amylopectin and lipid inclusions, whereas immature micromeronts and micromerozoites did not contain amylopectin inclusions and featured fewer, smaller lipid inclusions. A comparison of cystic stages among Hepatozoon species in different groups of vertebrates is presented with respect to their structure and evolutionary significance.

The distribution, prevalence, and morphological features of the cystic stage of an apicomplexan parasite of native littleneck clams (Protothaca staminea) in British Columbia.

Sixty-nine of 98 native littleneck clams (Protothaca staminea) collected from Cooper's Cove, Sooke Basin, British Columbia during November 1995 contained apicomplexan cysts. The cysts, which measured 20-150 microns in diameter occurred in several tissues, particularly in the kidney and in connective tissue surrounding the intestine and contained closely packed, banana-shaped zoites that measured about 25 x 4 microns. A pronounced fibrillar layer underlain by labyrinthine structures separated the host tissues from the cyst wall. The apical region of the zoites was reinforced and appeared as an electron-dense, caplike structure. The cysts probably represent a stage of a heteroxenous coccidian life cycle, with a predator of clams serving as the definitive host in which gamogony and sporogony occur.

Margolisiella kabatai gen. et sp. n. (Apicomplexa: Eimeriidae), a parasite of native littleneck clams, Protothaca staminea, from British Columbia, Canada, with a taxonomic revision of the coccidian parasites of bivalves (Mollusca: Bivalvia).

Two of 98 native littleneck clams, Protothaca staminea Conrad, from Cooper's Cove, Sooke Basin were infected with an eimeriorin coccidian parasite. Merogonic gamontogonic and sporogonic development were observed in renal tubular epithelial cells. Sporulation of the oocysts occurred within the host. Mature oocysts were spherical mean 41 microns (range 30-44), and contained about 32 subspherical sporocysts (9 x 10 microns), each of which contained 4 sporozoites. Spherical 19 microns (18-20), cyst-like structures and smaller multinucleate bodies, some of which resembled sporocysts, were also seen. A review of the coccidian parasites of bivalves led to the erection of the new genus, Margolisiella (family Eimeriidae Minchin, 1903) to accommodate M. kabatai sp. n., the parasite in Protothaca staminea described herein. Four previously described monoxenous species (Pseudoklossia patellae Debaisieux, P. chitonis Debaisieux, P. tellinovum Buchanan and P. haliotis Friedman, Gardner, Hedrick, Stephenson, Cawthorn et Upton) were also transferred to the new genus. The 2 remaining possibly heteroxenous species (P. pelseneeri Léger and P. glomerata Léger et Duboscq) were retained in the genus Pseudoklossia Léger et Duboscq (family Aggregatidae Labbé, 1899).

Comparisons of molecular karyotype and RAPD patterns of anuran trypanosome isolates during long-term in vitro cultivation.

The patterns of random amplified fragments and molecular karyotypes of 12 isolates of anuran trypanosomes continuously cultured in vitro were compared by random amplified polymorphic DNA (RAPD) analysis and pulsed field gradient gel electrophoresis (PFGE). The time interval between preparation of two series of samples was one year. Changes were not observed in the number and size of sharp, amplified fragments of DNA samples from both series examined with the ten primers used. Likewise, changes in the molecular karyotypes were not detected between the two samples of these isolates. These results suggest that the molecular karyotype and the RAPD patterns of the anuran trypanosomes remain stable after being cultured continuously in vitro for one year.

Analysis of isolates within species of anuran trypanosomes using random amplified polymorphic DNA.

A total of 20 decamer primers were used to generate random applied polymorphic DNA (RAPD) markers from 5 isolates of Trypanosoma fallisi, 3 isolates of T. ranarum, 2 isolates of T. rotatorium, and 2 isolates of T. rotatorium-like trypanosomes in addition to 2 species from the American Type Culture Collection, T. chattoni (ATCC 50294) and Trypanosoma sp. (ATCC 50295). A slight polymorphism was observed among the four isolates of T. fallisi obtained form American toads, Bufo americanus, collected in Algonquin Park, Ontario, Canada, and an isolate obtained from the same species of host collected in Marquette, Michigan, United States, and produced similarity coefficients ranging from 80.7% to 96.9%. Pronounced polymorphism was recorded among the three isolates of T. ranarum from bullfrogs, Rana catesbeiana, collected in Ontario, Canada, and in Maryland, United States, and from a Northern leopard frog, R. pipiens, collected in Minnesota (USA). The similarity coefficients ranged from 54.7% to 59.5%, suggesting that alleles of these isolates were conserved over a wide geographic range. The high degree of polymorphism observed in two isolates of T. rotatorium from a bullfrog collected in Ontario and two isolates of a T. rotatorium-like parasite from the green frog R. clamitans, collected in Louisiana (USA) suggests that they are different species. These results reflect the high similarity among isolates from the same geographic location and the pronounced polymorphism apparent among isolates from distant geographic locations.

Karyotype analysis of anuran trypanosomes by pulsed-field gradient gel electrophoresis.

The chromosomes of 12 species and isolates of anuran trypanosomes were investigated by pulsed-field gradient gel electrophoresis. Twelve to 16 chromosomes ranging from 0.45 to 2.2 megabase pairs were found in each of these trypanosomes. Minichromosomes were not observed in any of the examined isolates. Results indicate that different species of anuran trypanosomes display distinct karyotype patterns, and that isolates from the same region are similar. Our findings also reveal that most chromosome profiles of these trypanosomes are in accordance with isoenzyme and random amplified polymorphic DNA analysis data.