Molecular genetic comparison of Onchocerca sp. infecting dogs in Europe with other spirurid nematodes including Onchocerca lienalis

In the past 15 years, subconjunctival onchocercosis has been reported from 63 dogs in south-western United States (Arizona, California, Utah) and Southern and Central Europe (Germany, Greece, Hungary, Portugal, Switzerland). To reveal the taxonomic status of the parasite responsible for these infections, fragments of the mitochondrial cytochrome oxidase subunit I (COI) and NADH dehydrogenase subunit 5 (ND5) genes of three European strains of canine Onchocerca sp. and the 16S ribosomal RNA (16S rRNA) gene of their Wolbachia endosymbionts were sequenced and compared to the homologous sequences of other spirurid nematodes. The evolutionary divergence between COI and ND5 gene sequences of Greek, Hungarian and Portuguese strains of canine Onchocerca sp. were similar in magnitude to that seen within Thelazia callipaeda or Onchocerca lienalis. The evolutionary divergence between the sequences of canine Onchocerca sp. and other Onchocerca spp. including O. lienalis were similar or higher in magnitude to that seen between other Onchocerca spp. The results of the current and earlier phylogenetic analyses indicate that canine Onchocerca sp. separated from other Onchocerca spp. early in the evolution. Based on the similar clinical pictures, the identical morphology of nematodes and the sequence analyses of COI and ND5 genes of the worms and 16S rRNA gene of their wolbachiae, the Onchocerca worms isolated from European dogs appear to belong to the same species. The results support the earlier biological and morphological arguments that a distinct species, most likely O. lupi originally described from the subconjunctival tissues of a Caucasian wolf is responsible for canine ocular onchocercosis in Europe.     

Electron microscopic and molecular identification of Wolbachia endosymbionts from Onchocerca lupi: implications for therapy

It was recently demonstrated that Wolbachia intracellular bacteria (alpha 2 proteobacteria, Rickettsiales) living in filarial nematodes are obligatory symbionts of their hosts. Herein, we report the electron microscopic and 16S ribosomal DNA-based (16S rDNA) identification of the endobacteria harboring in Onchocerca lupi. The worm nodules containing the nematodes were removed from three Hungarian dogs naturally infected with O. lupi. Wolbachia-like endobacteria were detected by electron microscopy in the lateral chords of both adult worms and microfilariae. The endosymbionts in O. lupi resemble in location, size, and morphology the wolbachiae found in other filariae. The presence of wolbachiae in O. lupi was also confirmed by PCR amplification of the 16S rDNA of the bacteria. The 16S rDNA-based phylogenetic analysis revealed that the endosymbionts of O. lupi infecting dogs belong to the supergroup C of Wolbachia pipientis and are not identical with those of other Onchocerca spp. sequenced so far. Since intermittent treatment with oxytetracycline has adulticid and microfilaricid activity by depletion of Wolbachia endobacteria, this antibiotic treatment regimen may offer an alternative of ivermectin or diethylcarbamazine in the suppression of postoperative microfilaridermia in Onchocerca-infected dogs and may prevent relapse.     

Canine onchocercosis in Greece: report of further 20 cases and molecular characterization of the parasite and its Wolbachia endosymbiont

Recently, sporadic cases of subconjunctival Onchocerca infection have been reported in dogs in Greece and Hungary. Herein we report further cases from Greece and the results of the molecular analysis of Onchocerca sp. removed from Greek dogs and its Wolbachia endosymbionts. Twenty dogs of various breeds, 1-11 years of age with subconjunctival onchocercosis (4 cases each in right or left eye, 12 cases in both eyes) were presented having similar manifestations. Periorbital swelling, exophthalmos, lacrimation, discharge, photophobia, conjunctival congestion, corneal edema, protrusion of the nictitating membrane, and subconjunctival granuloma or cyst formation were the most important clinical signs. After surgical excision of the periocular masses containing the worms, all animals recovered fully from onchocercosis. Based on the similarities of the clinical picture of the Greek and Hungarian cases, the similar morphology of the Greek and Hungarian isolates, and the identical sequences of the cytochrome oxidase gene of the filarial parasites and that of the 16S ribosomal RNA gene from their Wolbachia endosymbionts, the Onchocerca sp. isolated from dogs in Greece and Hungary appears to belong to the same species.     

桑天牛卵啮小蜂孤雌生殖及体内沃尔巴克氏体(Wolbachia)的检测

桑天牛卵啮小蜂(Aprostocetus prolixus)有孤雌生殖现象,其后代几乎为雄性。沃尔巴克氏体(Wolbachia)是广泛分布于节肢动物体内的一类共生菌,参与多种调控寄主的生殖活动机制。通过对外膜蛋白基因(wsp)、细菌细胞分裂蛋白基因(ftsZ)和核糖体16S rDNA基因的特异性扩增,在米蛾(Corcyra cephalonica Stainton)的DNA中分别扩增出约600、1000和900 bp的片断,验证了米蛾体内已感染了Wolbachia;而在桑天牛卵啮小蜂的DNA中未扩增出任何片断,表明Wolbachia在桑天牛卵啮小蜂体内未被感染或感染率极低。     

昆虫共生菌沃尔巴克氏体(Wolbachia)的wsp基因密码子使用模式分析

沃尔巴克氏体(Wolbachia)主要感染昆虫等节肢动物,是一类革兰阴性共生细菌。为探讨昆虫纲共生菌Wolbachia的系统发育,研究Wolbachia基因组中进化速度较快的外膜蛋白基因wsp的密码子使用模式及影响因素,统计分析该基因的GC含量(GCall、GC1、GC2、GC3、GC3s)、有效密码子数(ENc)、相对同义密码子使用度(RSCU)等指标。结果显示:昆虫纲共生菌Wolbachia的wsp基因密码子偏好性均不强,昆虫纲不同目间共生菌Wolbachia的wsp基因碱基组成及ENc值差异较小;多数基因数据点沿标准曲线或在其附近分布,突变对碱基组成的影响较弱;18种由多个密码子编码的氨基酸中有11种氨基酸的偏好性密码子在昆虫纲7个目及对照蛛形纲1个目间均相同,有2种氨基酸的偏好性密码子在7个目间相同,这些密码子均以A/U结尾;供分析的140个wsp基因中仅编码6个半胱氨酸(Cys);对应分析中第1、第2向量轴贡献率均不高,分别为13.53%和12.49%,均与碱基组成(GC1、GC2、GC3)显著相关。综合各项分析认为,Wolbachia的wsp基因密码子偏好性不具有宿主分类特异性,密码子使用模式主要受碱基组成影响,而碱基组成主要受选择影响。     

Mitochondrial cytochrome c oxidase subunit 1 (cox1) gene sequence of Spirocerca lupi (Nematoda, Spirurida): avenues for potential implications

Canine spirocercosis is a life-threatening parasitosis caused by Spirocerca lupi (Nematoda, Spirurida) that is presently emerging in several countries. This study characterised an informative region within the mitochondrial (mtDNA) gene encoding for the cytochrome c oxidase subunit 1 (cox1) of S. lupi by Polymerase Chain Reaction (PCR)-coupled sequencing. Specimens from five different countries in Europe, Asia and Africa were examined and two different sequence variants of cox1 (i.e. haplotypes) were determined, displaying nucleotidic variation at 6 of 689 positions. All of these positions were invariable among all the parasite individuals from Europe (haplotype 1) and among the African and Asian individuals (haplotype 2), but differed between Europe and Asia/Africa. The S. lupi cox1 sequences were consistent with those of other common Spirurida previously reported at both nucleotidic and phylogenetic levels. This study provides molecular information essential for identification of the nematode, irrespective of its life cycle stage. Crucial implications for the specific molecular diagnosis of clinical spirocercosis and investigation of the evolution, population genetics, ecology and epidemiology of S. lupi are discussed.     

家蚕核糖体18S RNA基因的序列分析及分子系统学研究

为研究家蚕18S DNA基因的特点及分子进化,以家蚕丝腺为材料提取家蚕基因组DNA,通过PCR扩增、测序鳞翅目家蚕(Bombyx mori)核糖体小亚基18S RNA基因(18S rDNA)的全序列,将该序列与等翅目、直翅目、衤责翅目、鞘翅目、膜翅目、双翅目、捻翅目、弹尾目、蜉蝣目各一种昆虫的18S rDNA进行了比较。用DNAstav软件分析并进行序列比对,结果表明,昆虫18S rDNA有4段序列较为保守,以18S rDNA比对的第2保守区段构建的分子系统发育树表明鳞翅目和双翅目、膜翅目、鞘翅目进化关系最近,等翅目、直翅目、衤责翅目进化上较为接近,捻翅目昆虫与弹尾目昆虫的亲缘关系较为接近,捻翅目在分类上应是一种古老的昆虫。     

Molecular evidence for a novel encapsulated genotype of Trichinella from Patagonia, Argentina

At present, Trichinella spiralis is the only species of this genus reported from South America. Herein, we detail a molecular analysis of a new encapsulated isolate of muscle larvae of Trichinella, found in a mountain lion (Puma concolor) coming from the Patagonia, Argentina. We studied three DNA regions previously probed to be useful for the identification of all eleven recognized Trichinella genotypes: expansion segment 5 (ES5), cytochrome c-oxidase subunit I (COI) and 5S ribosomal DNA intergenic spacer region (5S ISR). BLAST searches with these DNA sequences showed that the mitochondrial and nuclear ribosomal regions most closely resemble other Trichinella sequences available in GenBank. However, they did not exactly match any of the eleven recognized genotypes. The phylogenetic analysis from COI and 5S ISR sequences showed that the mountain lion isolate is grouped with encapsulated members, in concordance with morphological data. Furthermore, this new isolate was located at the base of the encapsulated genotypes, signifying that it is an old genotype that could have emerged earliest in this group. These data strongly suggest that this isolate from the Patagonia represents the twelfth genotype (T12) described in the genus Trichinella. Nevertheless, further studies are necessary to adequately establish this isolate as a unique genotype.     

湖南省猬迭宫绦虫的线粒体nad5和rrnS基因的序列测定及种系发育分析

本研究旨在阐明猬迭宫绦虫湖南分离株线粒体烟酰胺腺嘌呤二核苷酸（NADH）脱氢酶亚单位5基因（nad5）部分序列（pnad5）和核糖体小亚基基因（rrnS）部分序列（prrnS）的遗传变异情况,并用pnad5和prrnS序列重构猬迭宫绦虫与其他绦虫的种群遗传关系。利用聚合酶链反应（PCR）扩增猬迭宫绦虫的pnad5和prrnS,应用ClustalX 1.81程序对序列进行比对,再用Phylip3.67程序MP法和Mega4.0程序NJ法绘制种系发育树,并用Puzzle5.2程序构建最大似然树。结果显示,所获得的pnad5和prrnS序列与预期（片段的）长度一致,分别为531bp和328bp。种系发育树表明,湖南分离株与已知猬迭宫绦虫位于同一分枝。结果表明,由于猬迭宫绦虫pnad5和prrnS序列种内相对保守,种间差异较大,均可作为种间遗传变异研究的标记。     

Dicyema sphyrocephalum (Phylum Dicyemida: Dicyemidae) isolated from Korean common octopus Callistoctopus minor in Korea

BackgroundDicyemids are parasites found in the renal sac of cephalopods. The first species of dicyemid was found from kidneys of the Korean common octopus Callistoctopus minor.ObjectivesThis study aimed to identify the dicyemid and investigate the effect on renal sac of host.MethodsIn this study, we compared the morphological characteristics of isolate to dicyemids (Dicyema sphyrocephalum, Dicyema clavatum, and Dicyema dolichocephalum) reported from C. minor in Japan. We compared the 18S ribosomal RNA (rDNA) and cytochrome c oxidase subunit I (COI) sequences of isolate to the sequences of D. shyrocephalum and D. clavatum. The infected octopuses renal tissues were histologically compared with the tissues of uninfected individuals.ResultsThe morphological characteristic of this isolated species corresponds to D. sphyrocephalum. The sequences similarities of 18S rDNA and COI gene of isolate are 99.7% and 98.1% with D. sphyrocephalum. We observed morphological changes in the epithelia folds of kidney at the dicyemids attached areas.ConclusionsThe present study identified the isolate as D. sphyrocephalum and this is the first report of dicyemid species from Republic of Korea. Further studies on the effects of dicyemids on growth and health status of cephalopods will be needed.     

Molecular identification of polydorid polychaetes (Annelida: Spionidae): is there a quick way to identify pest and alien species?

The early detection and correct identification of polydorid polychaete species is essential as they are often encountered as invasive alien pests in aquaculture facilities or the intertidal where they may modify the ecosystem. Accurate identification is, however, often hampered by high levels of morphological similarity among species. This taxon will therefore benefit from the development of a library of sequences, such as COI barcodes, to aid identification. However, the universal primers for the cytochrome c oxidase I (COI) DNA barcoding marker has failed to consistently amplify this gene for polydorids, greatly hampering the development of such a library. We describe the development of unique PCR primers for the COI gene that work across four genera and nine species of polydorids. We also compared its efficacy with sequence data for mitochondrial cytochrome b and nuclear 18S rRNA, and a concatenated dataset consisting of all three markers. The nuclear 18S rRNA gene showed the least variation both intra- (0.0–1.2%) and interspecifically (0.6–4.3%), and was the most accurate for species identifications among the three markers. Although COI was characterised by higher intraspecific variation compared with Cyt b (0.0–14.5% and 0.0–4.2%, respectively), Cyt b showed considerably higher levels of interspecific variation (16.6–30.2%) compared with COI (2.2–20.7%). Of the two mitochondrial DNA markers, COI was actually less accurate for species identifications, having suggested two species within Boccardia pseudonatrix that was not supported by the other markers. Overall, the concatenated dataset yielded the most consistent intraspecific groupings, suggesting that this is the most accurate means of identifying polydorids using DNA sequence data. Thus, there may not be a quick and easy way to identify these species accurately using only molecular data.     

Comparison and phylogenetic analysis of the heat shock protein 70 gene of Babesia parasites from dogs

The heat shock protein 70 (hsp70) genes of Babesia gibsoni, B. canis canis, B. canis vogeli, and B. canis rossi isolated from infected dogs were cloned by polymerase chain reaction (PCR) and sequenced. In the nucleotide sequence and the predicted amino acid sequence of the gene, the parasites were very similar to each other. The nucleotide sequences of the hsp70 gene had more variety than those of 18S nuclear subunit ribosomal DNA (18S rDNA). A phylogenetic analysis of these sequences and comparisons with sequences from other Babesia and Theileria species revealed that all canine babesial isolates analyzed in the present study were closely related to each other and formed one cluster. Additionally, a phylogenetic analysis of Babesia and Theileria species showed that these parasites could be divided into three groups: group A including canine babesial isolates, B. divergens, B. odocoilei, B. bovis, B. caballi, and B. ovis; group B including Theileria annulata, T. orientalis, and T. cervi; and group C including B. microti and B. rodhaini. These results suggested that a phylogenetic analysis of the hsp70 gene sequence might be helpful in classifying Babesia and Theileria species, and that canine babesial isolates might be closely related to each other, indicating their evolution from the same ancestry.     

DNA条形码技术在野生哺乳动物物种鉴定中的可行性

为新疆野生哺乳动物物种鉴定找出最好的方法并建立新疆哺乳动物DNA条形码数据库提供资料,本研究选取新疆野生哺乳动物的肌肉、血液、粪便作为研究材料,运用DNA条形码技术检测野生哺乳动物线粒体DNA COI基因序列。共检测片段长度为642～729 bp的52条COI基因序列,52条COI基因序列通过BLAST网站同源性比较,序列同源性达到98%～100%,相似度达到90%～99%。全部COI基因序列中A、T、C和G的平均含量为26. 3%、30. 3%、26. 0%和17. 4%,A+T的含量(56. 6%)高于C+G含量(43. 4%),有明显的碱基偏倚性。种内遗传距离为0%～2%,种间遗传距离为15%～42%,二者分离度高。邻接法和最小进化法构建的系统发育树,两种方法所得的系统进化树拓扑结构高度一致,分支明显,都分配在单个支上。说明DNA条形码技术可用于新疆野生哺乳动物物种鉴定并弥补其他物种鉴定方法的不足和缺点,在新疆濒危野生动物的管理与保护中有重要意义。     

Onchocerca lupi causing ocular disease in two cats

Although canine ocular onchocerciasis has been well described as an emerging pathogen of dogs in Europe and North America over the past 20 years, there are no previous reports of feline ocular onchocerciasis. This report details the clinical, histopathologic, and molecular diagnosis of two domestic short hair cats residing in the United States infected with Onchocerca lupi causing episcleritis and orbital cellulitis. The results of this report suggest that O. lupi is a newly recognized disease of domestic cats.     

Molecular taxonomic relationships of Psoroptes and Chorioptes mites from China based on COI and 18S rDNA gene sequences

In this present study, the mitochondrial DNA gene cytochrome coxidase subunit I (COI) and the small subunit ribosomal RNA (18S rDNA) gene were used to determine the taxonomic relationships of Psoroptes and Chorioptes mites from China. The neighbor-joining and maximum-parasimony approach were used to evaluate the evolutionary relatedness among different hosts in the genera Psoroptes and Chorioptes. Phylogenetic analysis showed that Psoroptes cuniculi and Psoroptes natalensis may be two different species within the genus Psoroptes, and Chorioptes texanus and Chorioptes panda are different species within the genus Chorioptes.     

Unrecognized diversity in New Guinean crayfish species (Decapoda,Parastacidae): The evidence from molecular data

The phylogenetic relationships among imported ornamental crayfish belonging to the genus Cherax were inferred from a combined dataset of 3 mitochondrial genes (COI, 16S and 12S) and by comparison with available GenBank sequences of 14 Cherax species. Furthermore, the concordance of previously described species obtained from a wholesaler (Cherax boesemani, C. holthuisi and C. peknyi) with available GenBank sequences was verified based on COI with special respect to comparison with sequences assigned as Cherax species. Recently described species C. gherardiae, C. pulcher and C. subterigneus belong to the northern group of Cherax species. Comparison and analysis with other GenBank COI sequences show previously unreported diversity of New Guinean species, suggesting 5 putative new species. Surprisingly, species assigned to the subgenus Astaconephrops do not form a monophyletic clade; this subgenus should be reappraised relative to the purported typical morphological characteristic of the uncalcified patch on male chelae. Increasing importation of crayfish underscores the importance of accurate species identification. Use of basic molecular methods is a necessary requisite for documenting occurrence, abundance and population trends of target species. Consequently, it helps to support eventual conservation decision‐making by stakeholders.     

First evidence of previously undescribed trichomonad species in the intestine of pigs?

Three different parasites of the phylum Parabasala (Tritrichomonas foetus, Trichomitus rotunda and Tetratrichomonas buttreyi) have been described in pigs. In a previous study (Mostegl et al., 2011) approximately 47% of 91 paraffin wax-embedded intestinal samples of pigs which were Trichomonas-positive by in situ hybridization using a probe with a broad reactivity spectrum contained other species than T. foetus. Out of these, intestinal trichomonads from three pigs (pigs 1-3) were further analyzed by gene sequencing of a part of the 18S ribosomal RNA (rRNA) gene using primer walking. Subsequently, the partial sequences achieved by the different primer pairs were combined to a sequence of about 1000 bp for each trichomonad. In all three pigs unique sequences were acquired which showed only moderate similarities to sequences available in the GenBank. Alignments and the BLAST analysis showed a high degree of homology between sequences of trichomonads from pig 1 and pig 3 with only 1% difference. These sequences were found to be 92% similar to Hypotrichomonas acosta, a trichomonad isolated from squamate reptiles. The trichomonad sequence detected in the intestine of pig 2 showed about 10% nucleotide differences compared to pigs 1 and 3. This sequence was 97% similar to two Trichomitus batrachorum (a frog symbiont) sequences. A phylogenetic analysis using the neighbor-joining and maximum likelihood methods supported the data of the BLAST analysis. These results suggest the presence of at least two as yet undescribed trichomonad species in the intestinal contents of pigs.     

Morphological and molecular characterization of Pseudocohnilembus longisetus Thompson, 1965 from farmed black rockfish Sebastes schlegelii in Korea

The morphology, infraciliature, silverline system, and the small subunit ribosomal RNA (SSU rRNA) of the little-known marine scuticociliate Pseudocohnilembus longisetusThompson, 1965 from the diseased black rockfish Sebastes schlegelii in Korea were studied. This scuticociliate possessed the typical characteristics of the genus Pseudocohnilembus, but could be discriminated from Pseudocohnilembus hargisi, and Pseudocohnilembus persalinus in terms of the body size, shape, the number of somatic kineties and kinetids in somatic kinety 1, and the number/position of contractile vacuole pores. The SSU rRNA gene of P. longisetus was sequenced in order to gain a better understanding of appropriate phylogenetic classification. The SSU rRNA was 1754 bp and the sequence was deposited in GenBank under accession number FJ899594. The SSU rRNA gene sequences of P. longisetus had an identity of 98.1%, 96.8% and 95.3% with P. hargisi, P. persalinus, and Pseudocohnilembus marinus SSU rRNA sequences, respectively. Our population of P. longisetus belonged to the genus Pseudocohnilembus and was in an isolated position based on the SSU rRNA gene tree, which was consistent with the conclusions based on the morphological studies. However, further investigation is required to determine the pathogenicity of this species.     

Hemotrophic mycoplasmas (hemoplasmas): a review and new insights into pathogenic potential

Abstract: The red cell parasites formerly known as Haemobartomlla and Eperythrozoon spp have been reclassified as hemotrophic mycoplasmas (hemoplasmas) based on strong phylogenetic evidence and 16S ribosomal RNA gene sequences. The latter form the basis for polymerase chain reaction assays used to detect infection. Candidatus designation was given to incompletely characterized species. Like other mycoplasmas, hemoplasmas are small epicellular parasites that lack a cell wall and are susceptible to tetracyclines; their circular, double-stranded DNA encodes only those gene products essential for life. Diseases caused by infection with hemoplasmas range from overt life-threatening hemolytic anemia to subtle chronic anemia, ill-thrift, and infertility. In addition, the organisms may act as cofactors in the progression of retroviral, neoplastic, and immune-mediated diseases. Intimate contact of hemoplasma organisms with RBCs leads to cell injury through immune-mediated and other mechanisms that have not yet been defined. Despite an intense immune response and even with antibiotic treatment, infected animals probably remain chronic carriers after clinical signs have resolved.