Bioinformatics analysis and characterization of a secretory cystatin from Thelohanellus kitauei.

Thelohanellus kitauei, is a member of obligate parasitic myxozoans, which causes intestinal giant-cystic disease of common carp (Cyprinus carpio) and has resulted in significant economic losses in carp farms. Cystatin secreted by parasites can regulate the immune response of host to facilitate parasite's survival. In this study, the secretory TK-cystatin gene, encoding a protein of 120 amino acid residues (13.65 kDa), was cloned from T. kitauei genome. Phylogenetic analysis showed that TK-cystatin gene is closely related to the cystatin-A from Hydra vulgaris. Multiple sequence alignment revealed that TK-cystatin had three conserved motifs: N-terminal G19G20, Q73VVAG77, and C-terminal L102P103. Molecular docking between TK-cystatin and three cysteine proteases showed a lower binding energy (- 13 KJ/mol) with cathepsin L whereas a higher binding energy (- 8.6 KJ/mol) with cathepsin B. TK-cystatin gene was expressed in Escherichia coli. Activity assays revealed that TK-cystatin has stronger inhibitory activity on endopeptidases (papain and cathepsin L) and weaker inhibitory activity on exopeptidase (cathepsin B). TK-cystatin was stable under the condition of acidity or alkalinity or below 57 °C. This study laid a foundation for the design and development of the anti-T. kitauei vaccine in carp culture in the future.

The life cycle of Thelohanellus kitauei (Myxozoa: Myxosporea) infecting common carp (Cyprinus carpio) involves aurantiactinomyxon in Branchiura sowerbyi.

Thelohanellus kitauei is a freshwater myxosporean parasite causing intestinal giant cystic disease of common carp. To clarify the life cycle of T. kitauei, we investigated the oligochaete populations in China and Hungary. This study confirms two distinct aurantiactinomyxon morphotypes (Aurantiactinomyxon type 1 and Aurantiactinomyxon type 2) from Branchiura sowerbyi as developmental stages of the life cycle of T. kitauei. The morphological characteristics and DNA sequences of these two types are described here. Based on 18S rDNA sequence analysis, Aurantiactinomyxon type 1 (2048 bp) and Aurantiactinomyxon type 2 (2031 bp) share 99.2-99.4 %, 99.8-100 % similarity to the published sequences of T. kitauei, respectively. The 18S rDNA sequences of these two aurantiactinomyxon morphotypes share 99.4 % similarity, suggesting intraspecific variation within the taxon, possibly due to geographic origin. Phylogenetic analyses demonstrate the two aurantiactinomyxon types clustered with T. kitauei. Regardless, based on 18S rDNA synonymy, it is likely that Aurantiactinomyxon type 1 and 2 are conspecific with T. kitauei. This is the fourth elucidated two-host life cycle of Thelohanellus species and the first record of T. kitauei in Europe.

New type of pathogenicity of Thelohanellus kitauei Egusa & Nakajima, 1981 infecting the skin of common carp Cyprinus carpio L.

Thelohanellus kitauei Egusa & Nakajima, 1981 is a common parasite infecting the intestine of common carp Cyprinus carpio L., resulting in mass mortality or loss of economic value of cultured carp. In the present study, T. kitauei infecting host skin was detected. The morphological, molecular and histological data of this parasite in the new organ record are presented. Morphological analysis showed the current specimen morphologically similar to T. kitauei from the intestine. Despite the spore length and polar capsule length of the current specimen larger than those of T. kitauei from the intestine, ranges of dimensions overlap, which is more suggestive of intraspecific variation than distinct species. BLAST search revealed that the present small subunit ribosomal DNA gene sequence is identical to those of T. kitauei. Histologically, most of spores distributed in the stratum spongiosum of dermis, and some spores in the strata compactum of host skin were also observed. Above all, both morphology and molecular analysis indicated that the current species from the skin of common carp is conspecific with T. kitauei from the intestine of carp and organ habitats transfer of T. kitauei from host intestine to skin may have occurred.

New phylogenomic and comparative analyses provide corroborating evidence that Myxozoa is Cnidaria.

Myxozoa, a diverse group of morphologically simplified endoparasites, are well known fish parasites causing substantial economic losses in aquaculture. Despite active research, the phylogenetic position of Myxozoa remains ambiguous. After obtaining the genome and transcriptome data of the myxozoan Thelohanellus kitauei, we examined the phylogenetic position of Myxozoa from three different perspectives. First, phylogenomic analyses with the newly sequenced genomic data strongly supported the monophyly of Myxozoa and that Myxozoa is sister to Medusozoa within Cnidaria. Second, we detected two homologs to cnidarian-specific minicollagens in the T. kitauei genome with molecular characteristics similar to cnidarian-specific minicollagens, suggesting that the minicollagen homologs in T. kitauei may have functions similar to those in Cnidaria and that Myxozoa is Cnidaria. Additionally, phylogenetic analyses revealed that the minicollagens in myxozoans and medusozoans have a common ancestor. Third, we detected 11 of the 19 proto-mesodermalgenes in the T. kitauei genome, which were also present in the cnidarian Hydra magnipapillata, indicating Myxozoa is within Cnidaria. Thus, our results robustly support Myxozoa as a derived cnidarian taxon with an affinity to Medusozoa, helping to understand the diversity of the morphology, development and life cycle of Cnidaria and its evolution.

Discrimination and simultaneous detection of two myxozoan parasites belonging to genus Thelohanellus by multiplex polymerase chain reaction.

Thelohanellus kitauei and Thelohanellus hovorkai are myxozoan parasites pathogenic in cyprinid fish especially adult Israel carp and common carp. In the present study, the complete 18S rRNA-ITS1-5.8S rRNA-ITS2 sequences of these two Thelohanellus species were cloned with primers designed from information from Genbank and previous studies. The results revealed that ITS1 and ITS2 sequences of T. kitauei and T. hovorkai were clearly distinguished although the 18S rRNA regions of the two parasites were highly conserved. Based on these sequences, new primer sets were designed for specific identification of these two parasites by multiplex PCR. Both single and multiplex PCR methods using these primers could identify these two myxozoan parasites from mixed DNA samples successfully. Our findings provide a powerful tool for the differentiation of the highly similar pathogenic Thelohanellus species for specific detection for the early diagnosis of diseases.

The phylogenetic study on Thelohanellus species (Myxosporea) in relation to host specificity and infection site tropism.

Thelohanellus kitauei (Myxobolidae) infects cyprinid fish. The evolution of species derived from common ancestors results in the sharing of biological features. To reveal the origin of T. kitauei biological features, the correlation between phylogeny and biological features of Myxobolidae was investigated by Bayesian inference tree and Bayesian tip association significance testing. The results demonstrated that host specificity and infection site tropism were correlated with the phylogeny of Myxobolidae, and that the biological features of T. kitauei originated from the ancient Myxobolidae as exhibited by the non-specific infection site tropism and the ability to infect cyprinids.

Molecular identification and real-time quantitative PCR (qPCR) for rapid detection of Thelohanellus kitauei, a Myxozoan parasite causing intestinal giant cystic disease in the Israel carp.

Intestinal giant-cystic disease (IGCD) of the Israel carp (Cyprinus carpio nudus) has been recognized as one of the most serious diseases afflicting inland farmed fish in the Republic of Korea, and Thelohanellus kitauei has been identified as the causative agent of the disease. Until now, studies concerning IGCD caused by T. kitauei in the Israel carp have been limited to morphological and histopathological examinations. However, these types of diagnostic examinations are relatively time-consuming, and the infection frequently cannot be detected in its early stages. In this study, we cloned the full-length 18S rRNA gene of T. kitauei isolated from diseased Israel carps, and carried out molecular identification by comparing the sequence with those of other myxosporeans. Moreover, conventional PCR and real-time quantitative PCR (qPCR) using oligonucleotide primers for the amplification of 18S rRNA gene fragment were established for further use as methods for rapid diagnosis of IGCD. Our results demonstrated that both the conventional PCR and real-time quantitative PCR systems applied herein are effective for rapid detection of T. kitauei spores in fish tissues and environmental water.

Molecular Identification and Real-time Quantitative PCR (qPCR) for Rapid Detection of Thelohanellus kitauei, a Myxozoan Parasite Causing Intestinal Giant Cystic Disease in the Israel Carp

Intestinal giant-cystic disease (IGCD) of the Israel carp (Cyprinus carpio nudus) has been recognized as one of the most serious diseases afflicting inland farmed fish in the Republic of Korea, and Thelohanellus kitauei has been identified as the causative agent of the disease. Until now, studies concerning IGCD caused by T. kitauei in the Israel carp have been limited to morphological and histopathological examinations. However, these types of diagnostic examinations are relatively time-consuming, and the infection frequently cannot be detected in its early stages. In this study, we cloned the full-length 18S rRNA gene of T. kitauei isolated from diseased Israel carps, and carried out molecular identification by comparing the sequence with those of other myxosporeans. Moreover, conventional PCR and real-time quantitative PCR (qPCR) using oligonucleotide primers for the amplification of 18S rRNA gene fragment were established for further use as methods for rapid diagnosis of IGCD. Our results demonstrated that both the conventional PCR and real-time quantitative PCR systems applied herein are effective for rapid detection of T. kitauei spores in fish tissues and environmental water.