Search

The order Piroplasmida, including the genera Babesia, Cytauxzoon, and Theileria is often referred to as piroplasmids and comprises of dixenous hemoprotozoans transmitted by ticks to a mammalian or avian host. Although piroplasmid infections are usually asymptomatic in wild animals, in domestic animals, they cause serious or life-threatening consequences resulting in fatalities. Piroplasmids are particularly notorious for the enormous economic loss they cause worldwide in livestock production, the restrictions they pose on horse trade, and the negative health impact they have on dogs and cats. Furthermore, an increasing number of reported human babesiosis cases are of growing concern. Considerable international research and epidemiological studies are done to identify existing parasite species, reveal their phylogenetic relationships, and develop improved or new drugs and vaccines to mitigate their impact. In this review, we present a compilation of all piroplasmid species, isolates, and species complexes that infect domestic mammals and which have been well defined by molecular phylogenetic markers. Altogether, 57 taxonomic piroplasmid entities were compiled, comprising of 43 piroplasmid species, 12 well-defined isolates awaiting formal species description, and two species complexes that possibly mask additional species. The extrapolation of the finding of at least 57 piroplasmid species in only six domestic mammalian groups (cattle, sheep, goat, horse, dog, and cat) allows us to predict that a substantially higher number of piroplasmid parasites than vertebrate host species exist. Accordingly, the infection of a vertebrate host species by multiple piroplasmid species from the same and/or different phylogenetic lineages is commonly observed. Molecular phylogeny using 18S rRNA genes of piroplasmids infecting domestic mammals results in the formation of six clades, which emerge due to an anthropocentric research scope, but not due to a possibly assumed biological priority position. Scrutinizing the topology of inferred trees reveals stunning insights into some evolutionary patterns exhibited by this intriguing group of parasites. Contrary to expectations, diversification of parasite species appears to be dominated by host-parasite cospeciation (Fahrenholz's rule), and, except for piroplasmids that segregate into Clade VI, host switching is rarely observed. When only domestic mammalian hosts are taken into account, Babesia sensu lato (s.l.) parasites of Clades I and II infect only dogs and cats, respectively, Cytauxzoon spp. placed into Clade III only infect cats, Theileria placed into Clade IV exclusively infect horses, wheras Theileria sensu stricto (s.s.) of Clade V infects only cattle and small ruminants. In contrast, Babesia s.s. parasites of Clade VI infect all farm and companion animal species. We outline how the unique ability of transovarial transmission of Babesia s.s. piroplasmids of Clade VI facilitates species diversification by host switching to other host vertebrate species. Finally, a deterioration of sequence fidelity in databases is observed which will likely lead to an increased risk of artifactual research in this area. Possible measures to reverse and/or avoid this threat are discussed. [ABSTRACT FROM AUTHOR]

This study reports the occurrence of parasites belonging to the Hepatozoon genus in fish Hoplias aimara from the Eastern Amazon. Fish (n = 54) were sampled from the Falsino River, located in the Amapá National Forest (FLONA), in the state of Amapá, northern Brazil. Fresh liver preparations were examined in the field between a slide and a coverslip under a light microscope. Cysts containing Hepatozoon cystozoites were observed in the liver of 5 (9%) out of 54 H. aimara individuals. The cysts were ovoid (mean dimensions 10.28 × 9.8 μm), presenting up to four elongated cystozoites (mean dimensions 11.04 × 1.68 μm), containing 1 to 4 residual bodies of different sizes. A single liver sample containing cysts was submitted to DNA extraction and PCR analyses based on a fragment of the 18S rRNA gene. The sequencing revealed a 465 bp fragment exhibiting 99% query coverage, 0.0 E-value, and 98.7% identity with Hepatozoon caimani (MF322538 and MF322539), detected in caimans (Caiman yacare) from Brazil. This is the first report of the occurrence of cysts containing Hepatozoon cystozoites in free-living fishes. [ABSTRACT FROM AUTHOR]

Adeleorid apicomplexan parasites of the genus Hepatozoon Miller, 1908 are broadly distributed among the rodents. Broader molecular data on Hepatozoon from Palaearctic squirrels are necessary for evaluation of diversity and origin of Hepatozoon in Eurasian red squirrel Sciurus vulgaris populations, considering ongoing invasion by Gray squirrel S. carolinensis. Our report brings a set of molecular data from a population of S. vulgaris in the Czech Republic, non-invaded by any invasive squirrel species. Cadavers of 41 Eurasian red squirrels were examined using nested PCR targeting 18S rRNA gene; 30 animals tested positive for the presence of Hepatozoon spp. DNA in at least one tissue. Phylogenetic analysis of obtained sequence types revealed relatedness to sequences of Hepatozoon sp. from S. vulgaris from Spain and the Netherlands, forming a sister clade to Hepatozoon isolates from other European rodents. The fact that all available 18S rRNA gene sequences form a monophyletic clade is interpreted as a presence of a single Hepatozoon species in S. vulgaris in continental Europe, most probably Hepatozoon sciuri. The presented molecular data on the Hepatozoon from European squirrels provides a basis for future studies on possible exchange of Hepatozoon species between Eurasian red and gray squirrels. [ABSTRACT FROM AUTHOR]

In the present study, we provided the first 18S rRNA gene sequence data of two Tripartiella species, Tripartiella macrosoma Basson and Van As, 1987 and Tripartiella obtusa Ergens and Lom, 1970, which were isolated from Tachysurus fulvidraco (Richardson, 1846) and Hemibarbus maculatus Bleeker, 1871 in Chongqing, China, respectively. Morphologically, both species fall within the morphometry range of the original descriptions and are very similar to the original populations in the overall appearance of the adhesive disc. Tripartiella macrosoma can be easily distinguished from the other Tripartiella species by possessing the denticle with a long strip and conspicuously inclined backward blade and a robust and short ray. Tripartiella obtusa is mainly characterized by a broad blade and a relatively long ray. Phylogenetically, T. macrosoma clustered with Trichodinella myakkae (Mueller, 1937) Raabe, 1950 and further with Trichodinella sp., which was sister to a group that includes four populations of Trichodinella epizootica (Raabe, 1950) Šrámek-Hušek, 1953; finally, they formed a small clade with T. obtusa. This result suggested that T. macrosoma had a closer relationship with Trichodinella spp. than with T. obtusa and T. obtusa diverged earlier than T. macrosoma and Trichodinella spp. By combining morphological and molecular data, the polyphyletic characteristics of Tripartiella and Trichodinella were further analyzed, and the results revealed that the validity of the genus Tripartiella is doubtful. [ABSTRACT FROM AUTHOR]

Haemogregarina species are apicomplexan blood parasites infecting vertebrates such as fish, lizards, and turtles. Due to the high morphological similarity of the erythrocytic stages infecting host species, it has always been a challenge to identify the true diversity of these parasites. Therefore, taxonomic studies are presently based on the combination of morphological and molecular data. In Tunisia, two species of Haemogregarina have been reported within the freshwater turtle Mauremys leprosa (Geoemydidae) for more than 40 years. Since M. leprosa occurs in the same aquatic environments as Emys orbicularis (Emydidae) in Tunisia, our objectives were to assess parasite diversity and specificity on the basis of both morphological and molecular approaches. The turtles were surveyed and sampled across six aquatic areas of Tunisia. Among the 39 specimens of M. leprosa and seven of E. orbicularis that were trapped and investigated, the presence of haemogregarines was detected in the blood of turtles only at sites where leeches were observed. Three 18S variants were identified, which corresponded to three distinct Haemogregarina species, among which one was identified as Haemogregarina stepanowi. The two other species that were detected are likely new to science. Because we show the occurrence of more than one blood parasite species within a single host specimen, our study provides the first report of coinfection with molecularly distinct Haemogregarina spp. [ABSTRACT FROM AUTHOR]

The first study reporting the morphological characterization of Sarcocystis sybillensis was performed in 1983; however, without any molecular analysis. Sarcocystis nipponi has been recently described as a species synonymic to S. sybillensis. We reconfirmed the presence of S. sybillensis in Japanese sika deer (Cervus nippon) captured in its native territory; and performed its molecular and phylogenetic characterization. The morphological characteristics of the sarcocysts were consistent with those of S. nipponi and S. sybillensis described in the first report. However, the nucleotide sequence of 18S rRNA gene of S. sybillensis showed only 91.9% identity to that of S. nipponi, suggesting low homology among the concerned Sarcocystis spp. Accordingly, S. sybillensis was found to occupy a clade distinct from that of S. nipponi in a phylogenetic tree of Sarcocystis. Therefore, the present study provides essential information on 18S rRNA-based molecular characterization of S. sybillensis and disproves the existing notion of morphology-based species synonymity of S. sibillensis and S. nipponi. These results also suggest that S. sybillensis belongs to type 2 Sarcocystis. [ABSTRACT FROM AUTHOR]

Gyrodactylus nebulosus Kritsky and Mizelle, 1968 is reported for the first time from brown bullhead Ameiurus nebulosus (Siluriformes; Ictaluridae) in Nova Scotia. The study results from a screening of parasites with the potential to disrupt commercial rearing of wild-caught young-of-the-year (YOY) brown bullhead. Infected YOY were collected July 30, 2018 and estimated to be 3 weeks old. Eight of 10 fish were infected. Mean intensity was 3.1 ± 3.5 with a range of 1–10. The parasite occurred all over the body surface, but particularly on the ventral regions of the head including the base of the maxillary barbels. Diagnostically important features of the anchors, ventral bar/shield, marginal hooks, and male copulatory organ are described. A partial sequence of the 18S rRNA gene (432 bp) is included and represents the first confirmed molecular data for this species. Molecular analysis revealed a high similarity (99.3%) to a Gyrodactylus sp. reported from the same host, A. nebulosus, in Ontario and the next closest similarity (96.9%) to Gyrodactylus fairporti Van Cleave, 1921 from Ameiurus melas in Wisconsin. The report extends the known distribution of G. nebulosus from North Dakota, Iowa, and Ontario to Nova Scotia. The study concludes that any commercial venture to harvest local YOY brown bullhead from the wild for intense grow-out in captivity should include appropriate quarantine and therapeutic treatments for G. nebulosus as part of the operation. [ABSTRACT FROM AUTHOR]

The major aim of the present study was to determine by molecular methods whether the wide and narrow types of macroscopic sarcocysts in Spanish sheep belonged to different species, that is, Sarcocystis gigantea and Sarcocystis medusiformis, respectively. Additionally, we wanted to identify and characterize molecularly the species forming microscopic sarcocysts and determine the phylogenetic placement of all species found. Portions of the oesophagus, diaphragm and hind legs containing macroscopic sarcocysts were collected from slaughtered culled ewes at an abattoir in the Province of Madrid, Central Spain, but both macroscopic and microscopic sarcocysts were isolated for molecular examination. Genomic DNA from 63 sarcocysts (21 macroscopic, 42 microscopic) were examined at the cytochrome c oxidase subunit I gene (cox1), while selected isolates of each species found were further examined at the 18S and 28S ribosomal RNA (rRNA) genes. The 63 sarcocysts comprised five cox1 sequence types, each corresponding to a particular sarcocyst type, and thus represented five Sarcocystis spp. The slender fusiform and thick macrocysts belonged to S. medusiformis and S. gigantea, respectively. The microscopic sarcocysts belonged to Sarcocystis arieticanis, Sarcocystis tenella and a Sarcocystis mihoensis-like species with slanting thorn-like cyst wall protrusions, which was characterised molecularly for the first time. Based on its phylogenetic position, the S. mihoensis-like species probably uses corvids as definitive hosts. [ABSTRACT FROM AUTHOR]

Based on morphology and morphometry of gametocytes in blood and molecular phylogenetic analysis, we described a new species of hemoparasite from the genus Haemogregarina isolated from Lepidosiren paradoxa in the eastern Amazon region. Haemogregarina daviesensis sp. nov. is characterized by monomorphic gametocytes of varying maturity stage and their dimensions were 16 ± 0.12 μm (range 13–18) in length and 6 ± 0.97 μm (range 5–8) in width. The morphological and morphometric data were not identical with other haemogregarine species from fish. All specimens of L. paradoxa analyzed were infected by H. daviesensis sp. nov. and the parasitemia level was moderate (1–28/2000 blood erythrocytes). Two sequences were obtained from L. paradoxa, and these constituted a monophyletic sister clade to the Haemogregarina species. In addition, H. daviesensis sp. nov. detected here grouped with Haemogregarina sp. sequences isolated from chelonian Macrochelys temminckii, with 99% bootstrap support. This study provides the first data on the molecular phylogeny of an intraerythrocytic haemogregarine of freshwater fish and highlights the importance of obtaining additional information on aspects of the general biology of these hemoparasites in fish populations, in order to achieve correct taxonomic classification. [ABSTRACT FROM AUTHOR]

This paper is the first record describing the molecular analysis of Eimeria species occurring in capercaillie (Tetrao urogallus) and black grouse (Tetrao tetrix) which inhabit northern Eurasia and are species critically endangered of extinction. Actions undertaken to protect endangered species, such as breeding individuals in closed aviaries, could allow saving those birds, but they also pose risk of accidental healing of invasive diseases, like coccidiosis. Therefore, an investigation was conducted on fecal samples collected from the capercaillies and black grouse originating from the Kirov region (Russia) and breeding centers located in Poland. Results indicate that the average prevalence of Eimeria revealed 72% (average OPG = 3548) and 80% (average OPG = 5220) in capercaillies and black grouse respectively. Most of the Eimeria spp. oocysts were non-sporulated; however, two different morphological types were observed. The phylogenetic analysis of cox-1 and 18S rRNA genes revealed the analyzed Eimeria sequences to belong to two species. In addition, it showed some similarities between both analyzed genes. Most of the sequences obtained from both grouse species coccidia belonged to one species partially homologous to the Eimeria spp. isolated from ring-necked pheasant (approx. 94 and 96% for cox-1 and 18S rRNA genes, respectively). Two strains isolated from capercaillies imported from Russia were related to turkey coccidia: E. innocua and E. dispersa (97-99% homology) in the cox-1 gene analysis and only one of them was related to those Eimeria species in the 18S rRNA gene analysis (98-99% homology). [ABSTRACT FROM AUTHOR]

Tick-borne intracellular protozoan parasites of the Theileria genus infect a wide range of both domestic and wild animals. In the present study, we describe the first PCR detection of Theileria luwenshuni in the blood of goats in Myanmar. Nested PCR targeting the Theileria 18S rRNA gene resulted in seven positive goats in central and northern Myanmar. Nucleotide sequencing of the PCR products revealed that all seven sequences were identical and showed 100% identity with T. luwenshuni sequences in GenBank from goats and sheep in China. Since T. luwenshuni parasites have recently been discovered and shown to have nationwide distribution in China, they might have been introduced into Myanmar via transboundary movement of infected domestic small ruminants and/or wild animals from China. [ABSTRACT FROM AUTHOR]

Samples of muscle tissue from the diaphragm, oesophagus and/or heart of eight adult red deer ( Cervus elaphus hispanicus) from the Quintos de Mora Park in Toledo, Central Spain, were screened for sarcocysts by means of the compression method. From positive samples, individual sarcocysts were excised and examined in wet mounts under a light microscope (LM) in order to study their basic morphology before being preserved for molecular studies. In all red deer examined, only microscopic sarcocysts were found. Those in the diaphragm and oesophagus were spindle-shaped and about 1 × 0.1 mm in size, while those in cardiac muscle were sac-like and 500-800 × 80-180 μm. By LM, the sarcocysts either had densely packed, about 8-μm-long, hair-like protrusions (type 1), sparsely distributed indistinct projections (fuzzy outline; type 2) or no visible protrusions (smooth surface; type 3). In cardiac muscle, only sarcocysts without visible protrusions were found. One of the latter sarcocysts was examined by scanning electron microscopy (SEM) and found to possess thin ribbon-like protrusions. Forty-eight sarcocysts isolated from the diaphragm, oesophagus and heart of one red deer, as well as 55 sarcocysts from the heart of three other red deer, 103 sarcocysts in total, were characterized molecularly through PCR amplification and sequencing of the partial cytochrome c oxidase subunit I gene ( cox1) of the mitochondrial genome, revealing the presence of six major cox1 sequence types. Each type comprised either a single sequence (three types) or a collection of several identical or nearly identical sequences. From selected isolates possessing each of these cox1 sequence types, the complete 18S ribosomal RNA (rRNA) gene was amplified and sequenced directly and/or after cloning of the 5′ end half. Supported by the sequence data from the latter gene, as well as the morphology of the sarcocysts from which the sequences originated, the six cox1 sequence types were considered to represent six separate Sarcocystis spp. Two cox1 sequence types were identified as belonging to the previously characterized species Sarcocystis hjorti (one sequence/sarcocyst) and Sarcocystis linearis (38 sequences/sarcocysts), respectively, whereas four sequence types were new. One of the latter types was assigned to the previously named species Sarcocystis cervicanis from red deer, since this sequence type was obtained from 52 sarcocysts from cardiac muscle, which matched the original morphological description (smooth surface) and habitat of this species. The remaining three sequence types were assigned to the three new species Sarcocystis iberica (one sequence/sarcocyst) Sarcocystis venatoria (10 sequences/sarcocysts) and Sarcocystis morae (one sequence/sarcocyst), respectively. The two species S. iberica and S. venatoria shared the same sarcocyst morphology (type 1) and habitat (diaphragm) and had virtually identical 18S rRNA gene sequences, but differed by 4% at cox1, which was considered sufficient to regard them as separate species. The single sarcocyst of S. morae (from the oesophagus) examined by LM had a smooth wall and this species was therefore believed to have the same type of ribbon-like protrusions (ultrastructurally) as sarcocysts of S. cervicanis and S. linearis, which were also the species most closely related to S. morae at cox1. Thus, there seems to be three species with similar ribbon-like cyst wall protrusions in red deer ( S. cervicanis, S. linearis, S. morae), as well as three species with similar hair-like protrusions ( S. hjorti, S. iberica, S. venatoria). Sarcocysts of S. cervicanis were only identified in cardiac muscle, whereas sarcocysts of S. linearis were found mainly in the diaphragm and oesophagus and rarely in the heart. The relative number of cox1 haplotypes was greater among sequences/isolates of S. linearis (17/38) than among isolates of S. cervicanis (7/52). Four of the species examined ( S. cervicanis, S. linearis, S. iberica, S. venatoria) possessed considerable intra-isolate (intra-genomic) sequence variation (insertions/deletions, substitutions) in the 18S rRNA gene. [ABSTRACT FROM AUTHOR]

Fresh (frozen/thawed) muscle samples from four 2-12-year-old roe deer ( Capreolus capreolus) from the Sondrio province in north-eastern Italy were examined under a dissecting microscope, and about 180 sarcocysts were isolated and identified to morphological type in wet mounts by light microscopy (LM). Seventy-seven of these sarcocysts were subsequently examined by molecular methods, comprising polymerase chain reaction (PCR) amplification and sequencing of the partial cytochrome c oxidase subunit I gene ( cox1) of all isolates, as well as PCR amplification, cloning and sequencing of the complete18S ribosomal RNA (rRNA) gene of two isolates of each species found. By LM, three major sarcocyst types were recognised: spindle-shaped sarcocysts, 0.5-3 mm long, either with no clearly recognisable protrusions (thin-walled) or with finger-like protrusions (thick-walled); and slender, thread-like sarcocysts, 2-3 mm long, with hair-like protrusions. Sequencing of cox1 revealed that the sarcocysts belonged to four different species. Those with no visible protrusions either belonged to Sarcocystis gracilis ( n = 24) or to a Sarcocystis taeniata-like species ( n = 19), whereas those with finger- and hair-like protrusions belonged to Sarcocystis silva ( n = 27) and Sarcocystis capreolicanis ( n = 7), respectively. The 19 cox1 sequences of the S. taeniata-like species, comprising five haplotypes, differed from each other at 0-16 of 1038 nucleotide positions (98.5-100% identity). They differed from 25 previous cox1 sequences of S. taeniata from moose and sika deer (with 98.0-100% intraspecific identity), at 33-43 nucleotide positions (95.9-96.8% interspecific identity), and there were 20 fixed nucleotide differences between the two populations. In the phylogenetic analysis based on cox1 sequences, the two populations formed two separate monophyletic clusters. The S. taeniata-like species in roe deer was therefore considered to represent a separate species, which was named Sarcocystis linearis n. sp. At the 18S rRNA gene, however, the two species could not be clearly separated from each other. Thus, there was considerable intraspecific sequence variation in the 18S rRNA gene of S. linearis (98.1-99.9% identity between 24 sequences), which was similar both in magnitude and nature to the variation previously found in this gene of S. taeniata. The new 18S rRNA gene sequences of S. linearis shared an identity of 97.9-99.6% with those of S. taeniata (overlap between intra- and interspecific identity), and in the phylogenetic tree, sequences of the two species were interspersed. By scanning electron microscopy (SEM), the sarcocysts of S. linearis were found to possess regularly spaced, thin and narrow ribbon-like cyst wall protrusions (about 2.8-3.2 μm long, 0.3-0.4 μm wide and about 0.02-0.03 μm thick), terminating in a plate-like structure of the same thickness but with an elliptic outline (about 0.3-0.4 μm wide and 0.7-0.9 μm long). The terminal plates were connected in the middle with the band-like portion of the protrusions like the board of a seesaw (tilting board). The terminal plates of adjacent protrusions were neatly arranged in a hexagonal pattern resembling tiles on a roof. Together, they formed an outer roof-like layer facing the surrounding cytoplasm of the host cell and completely covering the band-like proximal portion of the protrusions, which overlapped and were stacked in three to four layers close to the cyst surface. The sarcocyst morphology of S. linearis was consistent with that of an unnamed Sarcocystis sp. in roe deer previously found by transmission electron microscopy in several countries, including Italy. A few sarcocysts of S. gracilis and S. silva were also examined by SEM, confirming the presence of regularly distributed, short knob-like protrusions in S. gracilis (as seen in previous SEM studies) and revealing tightly packed, erect 6-7-μm-long villus-like protrusions having regularly distributed round depressions on their surface in S. silva. The sequencing of cox1 of 7, 24 and 27 new isolates of S. capreolicanis, S. gracilis and S. silva, respectively, recovered 7, 11 and 10 new haplotypes from each of the three species and expanded our knowledge on the intraspecific sequence variation at this marker. Similarly, the study revealed a more extensive intragenomic sequence variation at the 18S rRNA gene of S. capreolicanis and S. silva than known from previous studies and confirmed a near absence of such variation in the 18S rRNA gene of S. gracilis. [ABSTRACT FROM AUTHOR]

About 200 individual sarcocysts were excised from 12 samples of cattle beef from five countries (Argentina, Brazil, Germany, New Zealand, Uruguay) and tentatively identified to species or cyst type on the basis of their size and shape and cyst wall morphology. Genomic DNA was extracted from 147 of these sarcocysts and used initially for PCR amplification and sequencing of the partial mitochondrial cytochrome c oxidase subunit I gene ( cox1) in order to identify the sarcocysts to species and/or sequence type. In addition, seven Sarcocystis sinensis-like sarcocysts collected from the oesophagus of water buffaloes in Egypt were examined at cox1 for comparative purposes. Based on the results from the cox1 marker, selected sarcocyst isolates from both hosts were further characterised at one to three regions of the nuclear ribosomal (r) DNA unit, i.e. the complete 18S rRNA gene, the complete internal transcribed spacer 1 (ITS1) region and the partial 28S rRNA gene. This was done in order to compare the results with previous molecular identifications based on 18S rRNA gene sequences and to evaluate the utility of these regions for species delimitations and phylogenetic inferences. On the basis of sarcocyst morphology and molecular data, primarily the cox1 sequences, four Sarcocystis spp. were identified in the samples of cattle beef. Twenty-two microscopic sarcocysts (1 × 0.1 mm) with hair-like protrusions were assigned to Sarcocystis cruzi, 56 macroscopic sarcocysts (3-8 × 0.5 mm) with finger-like protrusions were assigned to Sarcocystis hirsuta and 45 and 24 microscopic sarcocysts (1-3 × 0.1-0.2 mm) with finger-like protrusions were assigned to Sarcocystis bovifelis and Sarcocystis bovini n. sp., respectively. Sarcocysts of S. cruzi were identified in samples of beef from Argentina and Uruguay; sarcocysts of S. hirsuta in samples from Argentina, Brazil, Germany and New Zealand; sarcocysts of S. bovifelis in samples from Argentina and Germany; and sarcocysts of S. bovini in samples from Argentina and New Zealand. The microscopic sarcocysts from water buffaloes were confirmed to belong to S. sinensis. The cox1 sequences of S. bovifelis and S. bovini, respectively, shared an identity of 93-94 % with each other, and these sequences shared an identity of 89-90 % with cox1 of S. sinensis. In contrast, the intraspecific sequence identity was 98.4-100 % ( n = 45), 99.3-100 % ( n = 24) and 99.5-100 % ( n = 7) for sequences of S. bovifelis, S. bovini and S. sinensis, respectively. In each of the latter three species, an aberrant type of cox1 sequences was also identified, which was only 91-92 % identical with the predominant cox1 type of the same species and about 98 % identical with the aberrant types of the two other species. These aberrant cox1 sequences are believed to represent non-functional nuclear copies of the mitochondrial genes (numts or pseudogenes). They might be used as additional markers to separate the three species from each other. Sequencing of a considerable number of clones of S. bovifelis, S. bovini and S. sinensis from each of the three regions of the rDNA unit revealed intraspecific sequence variation in all loci in all species and particularly in the ITS1 locus (78-100 % identity). As regards the 18S rRNA gene, it was possible to separate the three species from each other on the basis of a few consistent nucleotide differences in the less variable 3′ end half of the gene. A comparison of the new sequences with GenBank sequences obtained from S. sinensis-like sarcocysts in cattle in other studies indicated that previous sequences derived from cattle in Germany and Austria belonged to S. bovifelis, whereas those derived from cattle in China belonged to S. bovini. On the basis of the new 28S rRNA sequences, it was possible to separate S. sinensis from S. bovifelis and S. bovini, whereas the latter two species could not be separated from each other. Based on ITS1 sequences, the three species were indistinguishable. Phylogenetic analysis using maximum parsimony placed with fairly high support cox1 sequences of S. bovifelis, S. bovini and S. sinensis, respectively, into three monophyletic clusters, with S. bovifelis and S. bovini being a sister group to S. sinensis. In contrast, phylogenies based on each of the three regions of the rDNA unit did not separate sequences of the three species completely from each other. Characterisation of cox1 of 56 isolates of S. hirsuta from four countries revealed only 13 haplotypes and an intraspecific sequence identity of 99.3-100 %. In the three regions of the rDNA unit, there was more extensive sequence variation, particularly in the ITS1 region. The 22 cox1 sequences of S. cruzi displayed a moderate intraspecific variation (98.6-100 %), whereas there was no variation at the 18S rRNA gene among 10 sequenced isolates. Sequencing of 16 clones of the partial 28S rRNA gene of S. cruzi yielded two markedly different sequence types, having an overall sequence identity of 95-100 %. [ABSTRACT FROM AUTHOR]

Paratrichosoma-associated helminthiasis has been identified in saltwater crocodiles under intensive farming conditions. The development of sustainable integrated management practices is dependent on a detailed understanding of Paratrichosoma population genetics and infection dynamics. This study investigated the genetic relationships of Paratrichosoma sp in a population of commercially farmed saltwater crocodiles, Crocodylus porosus, in northern Australia. 18S ribosomal RNA gene sequence data were obtained from Paratrichosoma sp eggs present in the epidermis of infected animals. A high level of genetic diversity was distributed within the Paratrichosoma sp population (241 variable positions in the 1094 bp alignment), indicating an accelerated rate of nucleotide base-pair substitutions in this genus of nematodes. Several possible environmental correlates of the incidence and intensity of helminthiasis, including season, rainfall, and mean monthly temperature, were investigated by visual inspection of crocodile skins. Stepwise logistic regression revealed a significant negative linear relationship ( P = 0.011, R = 32.69 %) between mean monthly rainfall and the incidence of monthly Paratrichosoma-associated helminthiasis. Variation in the severity of Paratrichosoma-associated helminthiasis could not be explained by any of the independent environmental variables included within an ordinal regression analysis. The large genetic diversity in these nematodes indicates a high probability of anthelmintic resistant alleles occurring in the population. We discuss how the spread of these alleles may be mitigated by adopting targeted treatment protocols. [ABSTRACT FROM AUTHOR]

The free-living amoebae, Acanthamoeba, can act as opportunistic parasites on a wide range of vertebrates and are becoming a serious threat to human health due to the resistance of their cysts to harsh environmental conditions, disinfectants, some water treatment practices, and their ubiquitous distribution. Subgenus classification based on morphology is being replaced by a classification based on the sequences of the 18S rRNA gene with a total of 18 different genotypes (T1-T18). A new environmental strain of Acanthamoeba isolated from a waste water treatment plant is presented in this study as a candidate for the description of the novel genotype T19 after phylogenetic analysis. [ABSTRACT FROM AUTHOR]

Individual sarcocysts were isolated from fresh or alcohol-fixed muscle samples of two moose from Alberta, Canada, and examined by light (LM) and scanning electron microscopy (SEM) and molecular methods, comprising polymerase chain reaction (PCR) amplification and sequencing of the complete18S rRNA gene and the partial cytochrome c oxidase subunit I gene ( cox1). By LM, four sarcocyst types were recognized, and the sequencing results showed that each type represented a distinct species, i.e. Sarcocystis alces, Sarcocystis alceslatrans, Sarcocystis ovalis and Sarcocystis taeniata n. sp. The finding of S. alceslatrans and S. ovalis has been reported briefly previously, but further details are provided here, including the ultrastructure of sarcoysts of S. alceslatrans as seen by SEM. The species S. alces was found for the first time in Canadian moose, whereas the finding of S. taeniata is the first record of this species in any host. The sarcocysts of S. taeniata were sac-like and about 1,000-1,100 × 60-80 μm in size. By LM, the cysts had a thin and smooth wall with no visible protrusions, whereas SEM revealed that the cyst surface had sparsely but regularly distributed, thin ribbon-like protrusions, about 2 μm long and 0.2 μm wide, lying flat against the surface and leaving most of the cyst surface naked. Similar protrusions have previously been reported from Sarcocystis grueneri in reindeer, which was found by sequence comparisons and phylogenetic analyses to be the species most closely related to S. taeniata. The phylogenetic analyses further suggested that S. taeniata, like S. alces and S. alceslatrans, use canids as definitive hosts, whereas corvid birds are known definitive hosts for S. ovalis. In contrast to the three other species found, S. taeniata displayed considerable intra-specific and intra-isolate sequence variation (substitutions, insertions/deletions) in certain regions of the 18S rRNA gene. [ABSTRACT FROM AUTHOR]

Samples of Gyrodactylus fairporti Van Cleave, 1921 from young-of-the-year black bullhead (Ameiurus melas) stranded in riverside pools of the Black River (La Crosse County,) WI, USA, are used to supplement the species diagnosis, including new details on the marginal hook sickles, the male copulatory organ (MCO), and 18S rRNA gene sequence data. The anchors of G. fairporti are relatively long and thin, 58.2 ± 1.2 μm in length; roots 15.5 ± 1.0 μm; shaft 38.1 ± 1.5 μm; point 31.3 ± 1.5 μm. The ventral bar is 19.4 ± 0.4 μm wide and 5.7 ± 0.9 μm long, with small anterolateral processes, 2.0 ± 0.6 μm long, and an almost rectangular posterior shield 15.5 ± 1.1 μm in length. The marginal hooks are 29.2 ± 1.0 μm long, with the handle 23.9 ± 1.2 μm in length. These measurements are similar to those reported from Iowa and Alabama, with the additional observation of the anchor point bending outwardly halfway along its length. The marginal hook sickle blade leaves the base angled ventrally away from the longitudinal axis; the sickle point is short; the toe has a rounded shelf and the heel is small, thin, and rounded. The MCO has eight small spines, two large ones laterally and the others of various lengths, with two of the smallest spines being slightly out-of-line compared with all the others. The taxonomy of G. fairporti is compared to Gyrodactylus ictaluri Rogers, 1967 and G. nebulosus Kritsky & Mizelle, 1967, the other two species known from captive and wild ictalurids endemic to North America. The three species all have a relatively compact ventral bar with short anterolateral processes, a short almost rectangular ventral bar membrane, an MCO with up to eight small spines of varying length, and a hook sickle angled ventrally. Diagnostically, the species are readily identified by the total length and shape of the anchors. G. fairporti bears the longest (53-65 μm) and most slender anchors of the trio, G. nebulosus intermediate (49-51 μm) and G. ictaluri with the shortest and stoutest (40-45 μm) of these species. A BLAST search of a partial (413 bp) 18S rRNA gene showed the highest similarity with Gyrodactylus sp. reported from Ameiurus nebulosus (Siluriformes) in Ontario.

Tropical theileriosis is a serious animal disease transmitted by tick vectors. The agents of theileriosis are obligate intracellular parasites that cause mild to severe disease in the mammalian host. Tropical theileriosis has been recognized as a burden to the development of the dairy industry in Sudan and causes major economic losses. However, knowledge about the distribution of Theileria spp. in Sudan and the extent of sequence variation within the 18S rRNA gene is currently unknown. The aim of this study was to determine the diversity of Theileria spp. using 18S rRNA-based PCR to detect parasites in cattle followed by cloning and sequencing. We observed an overall prevalence rate of 63% hemoparasite infection in cattle from Sennar state. A subset of samples was used for cloning and sequencing of the 18S rRNA gene. Nineteen of 44 animals were co-infected with more than one species of Theilera. Phylogenetic analysis revealed three Theileria spp. that were predominant in cattle including pathogenic T. annulata and apathogenic T. velifera and T. mutans. The present study provides information regarding the prevalence of theileriosis in Sudan and will help to design strategies to control it. Additionally, more study is needed to determine tick vector competence and degree of coinfection with multiple Theileria spp. in Sudan. This represents the first molecular phylogeny report to identify Theileria spp. in cattle from Sudan.

Ligictaluridus michaelalicea n. sp. (Monogenea: Dactylogyridae, Ancyrocephalinae) is described from the gills of Pylodictis olivaris (Siluriformes: Ictaluridae) from Wisconsin and Iowa portions of the upper Mississippi River. Diagnostic features include a relatively large, strongly curved tubular cirrus with minor terminal flare; an accessory piece with a prominent basal lobe and a simple, thick terminal limb featuring a thin lateral flange and blunt distal tip devoid of recurved hooks. The sinistral vagina, two prostatic reservoirs, and a terminal seminal vesicle of the vas deferens are prominent. The anchors, ventral and dorsal bars, and hooks are similar in overall form to those of other members of the genus. The description includes sequence data for the 18S rRNA gene, which aligned most closely with species of ancyrocephaline monogeneans, with the highest similarity being with Ligictaluridus pricei (Mueller, 1936). Other monogenean species identified from the flathead catfish examined included L. pricei and Ligictaluridus mirabilis (Mueller, 1937). L. michaelalicea n. sp. is the fourth species to be described from P. olivaris within its natural range in central and eastern North America. Implications resulting from taxonomic name changes, including species of Ligictaluridus, and United States Food and Drug Administration treatment regulations are discussed. An updated key to species of the genus Ligictaluridus is presented.