Description of three new species of <Emphasis Type="Italic">Gyrodactylus</Emphasis> von Nordmann, 1832 (Monogenea) parasitising <Emphasis Type="Italic">Oreochromis niloticus niloticus</Emphasis> (L.) and <Emphasis Type="Italic">O. mossambicus</Emphasis> (Peters) (Cichlidae)

Three new species of Gyrodactylus are described from two species of Oreochromis (Cichlidae): Gyrodactylus hildae sp. nov. from the Nile tilapia, Oreochromis niloticus niloticus, and from an unconfirmed cichlid in Ethiopia; Gyrodactylus ulinganisus sp. nov. from a South African population of Mozambique tilapia, Oreochromis mossambicus; and, Gyrodactylus yacatli sp. nov. from O. n. niloticus reared in Mexico. The hamuli and marginal hooks of G. hildae sp. nov. and G. yacatli sp. nov. differ notably from G. cichlidarum, a species commonly found on O. n. niloticus. The hooks of G. ulinganisus sp. nov., however, are morphologically similar to those of G. cichlidarum, but the two species were found to differ by 42 nucleotide substitutions (24 within the 342 bp long ITS1; 18 within the 303 bp long ITS2) and by 1 insertion/deletion. This study confirms that Nile and Mozambique tilapia harbour a number of different species of Gyrodactylus, with G. cichlidarum being the most frequently encountered and being associated with mortalities of juvenile O. n. niloticus. This study discusses the host specificity of gyrodactylids on commercial cichlid species and the potential repercussions of their movement on stocks of fish into new environments where cichlids are already present.     

Genetic features of DNA of <Emphasis Type="Italic">Borrelia miyamotoi</Emphasis> transmitted by <Emphasis Type="Italic">Ixodes persulcatus</Emphasis>

16S rRNA, p66 and glpQ gene fragments in Borrelia miyamotoi transmitted by Ixodes persulcatus are determined and analyzed. Specific nucleotide sequences of every locus have been shown to be identical. The highest homology level for nucleotide sequences of three loci has been shown for the sequences of strains isolated earlier in Japan. An analysis of the P66 protein amino-acid sequence has shown that the locus corresponding to the surface-exposed domain differs considerably from both Borrelia hermsii, a typical member of tick-borne relapsing fever and Borrelia lonestari, the closest related species. Three genetic variants of Borrelia miyamotoi P66 protein is characterized not only by amino-acid substitutions, but also by deletions.     

Polymerase chain reaction-based identification of <Emphasis Type="Italic">Plasmodium</Emphasis> (<Emphasis Type="Italic">Huffia</Emphasis>) <Emphasis Type="Italic">elongatum</Emphasis>, with remarks on species identity of haemosporidian lineages deposited in GenBank

Numerous lineages of avian malaria parasites of the genus Plasmodium have been deposited in GenBank. However, only 11 morphospecies of Plasmodium have been linked to these lineages. Such linking is important because it provides opportunities to combine the existing knowledge of traditional parasitology with novel genetic information of these parasites obtained by molecular techniques. This study linked one mitochondrial cytochrome b (cyt b) gene lineage with morphospecies Plasmodium (Huffia) elongatum, a cosmopolitan avian malaria parasite which causes lethal disease in some birds. One species of Plasmodium (mitochondrial cyt b gene lineage P-GRW6) was isolated from naturally infected adult great reed warblers (Acrocephalus arundinaceus) and inoculated to one naive juvenile individual of the same host species. Heavy parasitaemia developed in the subinoculated bird, which enabled identification of the morphospecies and deposition of its voucher specimens. The parasite of this lineage belongs to P. elongatum. Illustrations of blood stages of this parasite are given. Other lineages closely related to P. elongatum were identified. The validity of the subgenus Huffia is supported by phylogenetic analysis. Mitochondrial cyt b gene lineages, with GenBank accession nos. AF069611 and AY733088, belong to Plasmodium cathemerium and P. elongatum, respectively; these lineages have been formerly attributed to P. elongatum and P. relictum, respectively. Some other incorrect species identifications of avian haematozoa in GenBank have been identified. We propose a strategy to minimise the number of such mistakes in GenBank in the future.     

<Emphasis Type="Italic">Sarcocystis</Emphasis> sp. from the herring gull (<Emphasis Type="Italic">Larus argentatus</Emphasis>) identity to <Emphasis Type="Italic">Sarcocystis wobeseri</Emphasis> based on cyst morphology and DNA results

Having studied 11 herring gulls (Larus argentatus) Sarcocystis cysts were found in neck and leg muscles of 4 birds. One type of sarcocysts (cyst type I) that have a thin (∼1.0 μm), smooth, or slightly wavy cyst wall without clearly visible protrusions and small (6.0–8.0 μm) lancet- or banana-shaped cystozoites was identified by the light microscopy. Sarcocysts extracted from one herring gull were used for electron microscopy and DNA analysis. Ultrastructurally, Sarcocystis sp. from the herring gull had the same tissue cyst wall type-1 as S. calchasi, S. columbae, and S. wobeseri parasitizing in birds. According to first internal transcribed spacer (ITS-1) region, 18S rRNA and 28S rRNA gene sequences, Sarcocystis sp. from the herring gull belongs to S. wobeseri. Nevertheless, without evidences of cross-transmission experiment sarcocysts extracted from herring gull at present time are named as S. wobeseri-like.     

Genetic characterization of avian malaria (Protozoa) in the endangered lesser kestrel, <Emphasis Type="Italic">Falco naumanni</Emphasis>

We genetically analyzed avian malaria (Protozoa) isolated from lesser kestrels (Falco naumanni) breeding in La Mancha, Central Spain. A total of 586 adult individuals were screened for blood parasites using a very efficient polymerase chain reaction approach that amplifies a partial segment (498 bp) of the cytochrome b gene of avian malaria of the genera Haemoproteus and Plasmodium. The prevalence of Plasmodium was 8.2%, and the prevalence of Haemoproteus was 4.1%. Sequence analyses revealed six unique lineages of avian malaria, three Plasmodium (LK5, LK6, RTSR1) and three Haemoproteus (LK2, LK3, LK4). According to sequence divergence, these lineages seem to correspond to at least three different species, although all recovered lineages could be independent evolutionary units. The third most common lineage (RTSR1) has been previously retrieved from two other avian host species, including a resident African bird species and a trans-Saharan migrant passerine, suggesting that lesser kestrels could acquire this Plasmodium lineage at their winter quarters in Africa.     

Gyrodactylids (Gyrodactylidae,Monogenea) infecting <Emphasis Type="Italic">Oreochromis niloticus niloticus</Emphasis> (L.) and <Emphasis Type="Italic">O. mossambicus</Emphasis> (Peters) (Cichlidae): A pan-global survey

Gyrodactylus infections in intensively-reared populations of Nile tilapia, Oreochromis niloticus niloticus, have been associated world-wide with high mortalities of juvenile fish. In this study, 26 populations of Gyrodactylus parasitising either O. n. niloticus or Mozambique tilapia, Oreochromis mossambicus, were sampled from fourteen countries and compared with type material of Gyrodactylus cichlidarum Paperna, 1968, Gyrodactylus niloticus (syn. of G. cichlidarum) and Gyrodactylus shariffi Cone, Arthur et Bondad-Reantaso, 1995. Representative specimens from each population were bisected, each half being used for morphological and molecular analyses. Principal component analyses (PCA) identified five distinct clusters: (1) a cluster representing G. cichlidarum collected from O. n. niloticus from 13 countries; (2) the G. shariffi paratype; (3) three specimens with pronounced ventral bar processes collected from two populations of Mexican O. n. niloticus (Gyrodactylus sp. 1); (4) four specimens collected from an Ethiopian population nominally identified as O. n. niloticus (Gyrodactylus sp. 2); (5) nine gyrodactylids from South African O. mossambicus (Gyrodactylus sp. 3). Molecular analyses comparing the sequence of the ribosomal transcribed spacer regions (ITS 1 and 2) and the 5.8S gene from the non-hook bearing half of worms representative for each population and for each cluster of parasites, confirmed the presence of G. cichlidarum in most samples analysed. Molecular data also confirmed that the DNA sequence of Gyrodactylus sp. 2 and Gyrodactylus sp. 3 (the morphologically-cryptic group of South African specimens from O. mossambicus) differed from that of G. cichlidarum and therefore represent new species; no sequences were obtained from Gyrodactylus sp. 1. The current study demonstrates that G. cichlidarum is the dominant species infecting O. n. niloticus, being found in 13 of the 15 countries sampled.     

Two new <Emphasis Type="Italic">Isospora</Emphasis> species from the saffron finch, <Emphasis Type="Italic">Sicalis flaveola</Emphasis> in Brazil

Two new coccidian species (Protozoa, Apicomplexa, Eimeriidae) are reported from the saffron finch Sicalis flaveola Linnaeus, 1766, a very common species in South America. Isospora cetasiensis sp. nov. oocysts are subspherical to ellipsoidal, 23.1 × 21.6 μm, with smooth, bilayered wall, ∼1.0 μm. Micropyle, oocyst residuum and polar granule are absent. Sporocysts are ovoidal, 15.1 × 10.9 μm. Stieda body is knob-like and substieda body is rounded. Sporocyst residuum is composed of many scattered granules and spherules of different sizes. Sporozoites are vermiform with one refractile body and a nucleus. Isospora sicalisi sp. nov. oocysts are subspherical to ellipsoidal, 27.5 × 25.2 μm, with a smooth, bilayered wall, ∼1.1 μm. Micropyle, oocyst residuum and polar granule are absent. Sporocysts are ellipsoidal, 17.2 × 11.7 μm. Stieda body is knob-like and substieda body is trapezoidal. Sporocyst residuum is composed of scattered granules and spherules of different sizes. Sporozoites are vermiform with one refractile body and a nucleus.     

Phylogeny of sheep and goat<Emphasis Type="Italic"> Theileria</Emphasis> and<Emphasis Type="Italic"> Babesia</Emphasis> parasites

The phylogenetic relationship of Theileria and Babesia species infecting sheep and goats on the basis of their 18S RNA gene structure was addressed in the present study. For this purpose, the complete sequences of the small ribosomal RNA genes of a panel of sheep and goat piroplasm isolates, including T. lestoquardi, T. ovis, T. separata, B. ovis, B. motasi, B. crassa and several novel species, were sequenced and compared. The classification based on the established phylogenetic tree corresponded with traditional systematics and revealed that sheep/goat piroplasm species are of polyphyletic origin. The independent evolution of almost all sheep/goat piroplasms suggests that speciation may have occurred after transfer of the piroplasm-transmitting tick from a primal wild ruminant host to domestic sheep and goats. In accordance with recent reports, our study confirms the existence of at least two additional sheep/goat piroplasm species, designated Theileria sp. 1 (China) and Theileria sp. 2 (China). The recently reported pathogenic sheep/goat Theileria sp. 1 (China) seems to be identical with a Theileria sp. isolated from Japanese serow. Furthermore, our results suggest that T. ovis represents a single species.     

<Emphasis Type="Italic">Trypanosoma avium</Emphasis>: experimental transmission from black flies to canaries

Trypanosomes identified as Trypanosoma avium were found in the ornithophilic black flies (Eusimulium latipes) attacking buzzard nestlings (Buteo buteo). Parasites formed plugs and rosettes in the hindgut of the vector and were attached on the cuticular lining of the black fly anterior intestine (ileum) by hemidesmosome-like plaques. Hindgut stages from infected black flies were experimentally transmitted into canaries (Serinus canaria) by ingestion of vectors and by contamination of host conjunctiva. This is the first clear evidence of such transmission in avian trypanosomes. Parasites survived in peripheral blood of birds at the least 10 months. In contrast to the direct inoculation of insect stages, parasites from culture failed to produce infection in experimental birds; this has consequences for laboratory studies of host susceptibility and transmission.     

<Emphasis Type="Italic">Pepo aphid</Emphasis>-<Emphasis Type="Italic">borne yellows virus</Emphasis>: a new species in the genus <Emphasis Type="Italic">Polerovirus</Emphasis>

Pepo aphid-borne yellows virus (PABYV) has been proposed as a putative representative of a new species in the genus Polerovirus in the family Luteoviridae. The genomes of two South African (SA) isolates of cucurbit-infecting PABYV were described in this record. Total RNA, extracted from a pattypan (Cucurbita pepo L.) and a baby marrow (C. pepo L.) leaf samples, was subjected to next-generation sequencing (NGS) on the HiSeq Illumina platform. Sanger sequencing was subsequently used to authenticate the integrity of PABYV’s genome generated from de novo assembly of the NGS data. PABYV genome of SA isolates consists of 5813 nucleotides and displays an organisation typical of poleroviruses. Genome sequence comparisons of the SA PABYV isolates to other poleroviruses support the classification of PABYV as a new species in the genus Polerovirus. Recombination analyses showed that PABYV and Cucurbit aphid-borne yellows virus (CABYV) shared the same ancestor for the genome part situated between breaking points. Phylogenetic analyses of the RNA-dependent RNA polymerase and the coat protein genes showed that SA PABYV isolates shared distant relationship with CABYV and Suakwa aphid-borne yellows virus. Based on our results, we propose that PABYV is a distinct species in the genus Polerovirus.     

Phylogenetic analysis of<Emphasis Type="Italic"> Theileria</Emphasis> species transmitted by<Emphasis Type="Italic"> Haemaphysalis qinghaiensis</Emphasis>

The phylogenetic relationships between six isolates of Theileria spp. infective to small ruminants, and two isolates of Theileria spp. infective to yak, all transmitted by Haemaphysalis qinghaiensis, together with the Theileria orientalis/sergenti/buffeli group and T. sinensis, were analyzed using the 18S ssrRNA gene sequence. The target DNA segment was amplified by polymerase chain reaction (PCR). The PCR product was used either for direct sequencing or was ligated to the PCR II vector for sequencing. The length of the 18S ssrRNA gene of all Theileria spp. involved in this study was around 1,740 bp. Two phylogenetic trees were inferred based on the 18S ssrRNA gene sequence of the Chinese isolates only, and Chinese isolates and other species of Theileria available in GenBank. In the first tree, the Theileria sp. infective to yaks was found to be T. sinensis. The Theileria sp. infective to small ruminants was found to be composed of two separate species of Theileria. Theileria sp. from Qinghai, Madang, Ningxian and Lintan, which was identical to the unidentified Theileria sp. described previously, is designated Theileria sp (China 1). The Theileria sp. from Longde, Zhangjiachuan and Lintan, which has not been described previously, is designated Theileria sp. (China 2) in order to avoid confusion. In the second tree, Theileria sp. (China 1) was closely related to benign Theileria, such as T. buffeli and T. sergenti, while Theileria sp. (China 2) was separated from other Theileria spp. The results indicate that H. qinghaiensis transmit at least three species of Theileria, two which are infective to sheep and goats, but not yak and one which is infective to yaks and cattle, but not to sheep and goats.     

Complete nucleotide sequences of the mitochondrial genomes of two avian malaria protozoa, <Emphasis Type="Italic">Plasmodium gallinaceum</Emphasis> and <Emphasis Type="Italic">Plasmodium juxtanucleare</Emphasis>

We analyzed mitochondrial genomes of two avian malaria protozoa, Plasmodium gallinaceum and Plasmodium juxtanucleare. Both mitochondrial genomes were estimated to be 6,002 and 6,014 bp in length, respectively, and to have the identical gene organization and contents to that of other Plasmodium species previously analyzed; three functional genes for cytochrome c oxidase subunit I, III, and cytochrome (cyt b), with following sets of discontinuous and scrambled 15 ribosomal subunit RNA (rRNA) genes. Similarities of the three protein-coding genes showed closer relationship within avian malaria protozoa rather than mammalian Plasmodium species. In addition, we showed the tandem repeated structure of each mitochondrial genome of both P. gallinaceum and P. juxtanucleare as well as previously found in mammalian Plasmodium species. This study revealed the complete sequences and structure of the mitochondrial genomes of avian malaria protozoa for the first time.     

Linkage between mitochondrial cytochrome <Emphasis Type="Italic">b</Emphasis> lineages and morphospecies of two avian malaria parasites<Emphasis Type="Italic">,</Emphasis> with a description of <Emphasis Type="Italic">Plasmodium (Novyella) ashfordi</Emphasis> sp. nov

Numerous lineages of avian malaria parasites of the genus Plasmodium have been deposited in GenBank. However, only seven morphospecies have been linked to these lineages. This study linked two molecular sequences with morphospecies of malaria parasites. Two species of Plasmodium (mitochondrial cytochrome b gene lineages P-GRW2 and P-GRW4) were isolated from naturally infected adult great reed warblers (Acrocephalus arundinaceus) and inoculated to naive juvenile individuals of the same host species. Heavy parasitemia developed in the subinoculated birds, which enable identification of the species and deposition of their voucher specimens. Parasites of the lineage P-GRW2 were described as a new species, Plasmodium (Novyella) ashfordi, which is characterized primarily by the fan-like mature erythrocytic meronts containing seven to eight merozoites and the terminal position of clumped pigment granules in the gametocytes. Illustrations of the blood stages of the new species and Plasmodium (Haemamoeba) relictum (lineage P-GRW4) are given. The parasites of both lineages are transmitted in Africa and probably not in northern Europe. Other lineages closely related to P. ashfordi and P. relictum are identified. This study establishes the value of PCR-based identification of avian malaria parasites.     

Identification of<Emphasis Type="Italic"> Borrelia burgdorferi</Emphasis> sensu lato,<Emphasis Type="Italic"> Anaplasma</Emphasis> and<Emphasis Type="Italic"> Ehrlichia</Emphasis> Species,and Spotted Fever Group Rickettsiae in Ticks from Southeastern Europe

Prevalence data for tick-borne pathogens are used to assess the risk for human health. In this study the presence and identity of Borrelia burgdorferi sensu lato, Ehrlichia, Anaplasma, and Rickettsia species in Bulgarian Ixodes ricinus ticks and in non-Ixodes ticks from Turkey and Albania was determined by polymerase chain reaction (PCR) and reverse line blot hybridization. In the adult Bulgarian ticks, the prevalence of Borrelia burgdorferi sensu lato infection was approximately 40%, while Borrelia afzelii was the predominant species, representing more than half of all Borrelia-positive ticks. Ehrlichia and Anaplasma species were detected in 35% of the adult Ixodes ricinus ticks and in 10% of the nymphs. Sequence analysis of PCR products reacting with the Anaplasma phagocytophila probe revealed a 16S rRNA gene identical to that of the Anaplasma phagocytophila prototype strain. Ehrlichia and Anaplasma species were found in approximately 7% of the non-Ixodes ticks. Sequence analysis of some of these samples revealed the presence of Anaplasma ovis, Ehrlichia canis, and a species closely resembling Ehrlichia chaffeensis. About half of all adult ticks examined and approximately 20% of all nymphs were infected with Rickettsia species. In Ixodes ricinus ticks, Rickettsia helvetica and a Rickettsia species designated as IRS3 were found in high prevalence. Rickettsia conorii was found in virtually all non-Ixodes tick species from Albania and Turkey. The results of this study show that many tick-borne diseases are most probably endemic in the Balkan area. Furthermore, the results suggest that there is a considerable chance for simultaneous transmission of tick-borne pathogens to human beings.     

Spotted fever group <Emphasis Type="Italic">Rickettsia</Emphasis> in brown dog ticks <Emphasis Type="Italic">Rhipicephalus sanguineus</Emphasis> in southwestern Spain

A total of 2,229 adults ticks (1,428 males and 801 females) belonging to the brown dog tick, Rhipicephalus sanguineus Latreille, 1806, collected from dogs in Seville province (Andalusia), distributed in 500 lots ranging from one to eight specimens per lot, were examined for the presence of rickettsiae by molecular techniques. Specific rickettsiae DNA were detected in 90 lots (18%) of ticks tested. Sequence analysis of amplicons revealed that R. sanguineus ticks were infected exclusively with Rickettsia massiliae (including the strain Bar-29). The results of this study extend the knowledge of the geographic distribution and prevalence of these spotted fever group (SFG) rickettsiae and indicate that at least two of them, with yet uncertain pathogenicity to humans, are present in brown dog ticks in south western Spain. Although Mediterranean spotted fever (MSF) is an endemic disease in Andalusia, Rickettsia conorii was not found, whereas R. massiliae, recently described as a pathogenic species, was highly prevalent in this area. Our data suggest that in Andalusia a number of MSF or MSF-like cases attributed to R. conorii could have been actually caused by other SFG rickettsia present in R. sanguineus, particularly, R. massiliae.     

<Emphasis Type="Italic">Sarcocystis</Emphasis> sp. from the goldeneye (<Emphasis Type="Italic">Bucephala clangula</Emphasis>) and the mallard (<Emphasis Type="Italic">Anas platyrhynchos</Emphasis>): cyst morphology and ribosomal DNA analysis

By light microscopy, cysts of Sarcocystis sp. (cyst type I) from the goldeneye (Bucephala clangula) seemed filamentous with a smooth and thin (<1 μm) cyst wall. Ultrastructurally, the cyst wall surface was irregular with minute undulations of the primary cyst wall. These sarcocysts had type-1 cyst wall. Cystozoites were banana-shaped and measured 7.0–8.5 μm in length. By light microscopy, cysts of Sarcocystis sp. (cyst type II) from the mallard (Anas platyrhynchos) were ribbon-shaped, very long, and thin. On the surface of the wall (up to 1.5 μm), they had palisade-like villar protrusions closely crowded together. Electron micrographs showed villar protrusions (up to 1.3 μm in length) different in size and shape. The latter had short microprojections especially obvious in the oblique sections. Cystozoites were slightly bent with blunt ends, broader at one end, and measured 13.0–16.1 × 1.8–2.5 μm. Phylogenetic analysis based on the comparison of partial 28S rRNR gene sequences of Sarcocystis sp. (cyst type II) derived from the mallard, Sarcocystis sp. (cyst type I) and Sarcocystis sp. (cyst type III) both derived from the white-fronted goose (Anser albifrons) suggested that these sequences belonged to separate Sarcocystis species.     

<Emphasis Type="Italic">Macroderoides texanus</Emphasis> n. sp. (Digenea: Macroderoididae) from alligator gar, <Emphasis Type="Italic">Atractosteus spatula</Emphasis> in Texas

Macroderoides texanus n. sp. is described based on 16 specimens collected from the intestine of the North American alligator gar, Atractosteus spatula. Of the five established species of Macroderoides, the new species is morphologically most similar to Macroderoides spiniferus and Macroderoides trilobatus. M. texanus n. sp. differs from M. spiniferus by having the ovary situated immediately posterior to the cirrus sac rather than at mid-way between the cirrus sac and anterior testis, the ventral sucker situated further posteriorly, and the vitelline fields extending somewhat posterior to posterior testis rather than to the middle of posterior testis. M. texanus n. sp. differs from M. trilobatus by having the ovary positioned immediately adjacent to, or overlapping the cirrus sac rather than at some distance posterior to it, and by having significantly larger eggs. Additionally, the new species has two distinctive rows of spines on the postero-ventral surface of the oral sucker that are lacking in M. spiniferus and M. trilobatus. Comparison of approximately 2,700-base-pair sequences of nuclear rDNA (partial 18S, complete ITS region and partial 28S) from M. texanus n. sp., M. spiniferus and M. trilobatus, strongly supports the status of M. texanus n. sp. as a new species.