Does the fish-infecting Trypanosoma micropteri belong to Trypanosoma carassii?

Recently, based on a limited morphological characterisation and partial 18S rRNA gene sequence, Jiang et al. (2019) described Trypanosoma micropteri Jiang, Lu, Du, Wang, Hu, Su et Li, 2019 as a new pathogen of farmed fish. Here we provide evidence based on the expanded sequence dataset, morphology and experimental infections that this trypanosome does not warrant the establishment as a new species, because it is conspecific with the long-term known Trypanosoma carassii Mitrophanow, 1883, a common haemoflagellate parasite of freshwater fish. The former taxon thus becomes a new junior synonym of T. carassii.

Nile tilapia (Oreochromis niloticus) can be experimentally infected with both marine and freshwater fish trypanosomes.

Trypanosomes are haemoflagellates found in vertebrate species and many of them can cause death in infected hosts including fish and humans. With the development of high-density farming in marine and freshwater fish aquaculture systems, severe disease or death, caused by trypanosomiasis, has been frequently reported. However, due to the lack of a model system, particularly for marine fish trypanosomes, and a paucity in the understanding of the biology and pathogenesis of these parasites, effective treatment for fish trypanosomiasis is significantly hampered. The goldfish is the common model system for freshwater fish trypanosomes, mainly of the species Trypanosoma carassii, while a similar model for marine fish trypanosomes has not yet been established. To address this issue, we found that Nile tilapia (Oreochromis niloticus) could be easily infected with a marine fish trypanosome, Trypanosoma epinepheli isolated from Lates calcarifer. Obvious clinical symptoms, associated with a high parasitemia (>108/ml), were found in the infected tilapias and more than 70% mortality was recorded in individuals within 20 days of infection. Interestingly, we also found that the Nile tilapia could also be infected with a freshwater fish trypanosome isolated from the largemouth bass (Micropterus salmoides) and caused significant death (more than 13%) in infected fish. This system not only provides an economical and effective laboratory model to study the biology and pathogenesis of marine and freshwater fish trypanosomes, but also provides a useful platform to develop vaccines and screen compounds for the protection and treatment of fish trypanosomiasis.

A new species of mammalian trypanosome, Trypanosoma (Megatrypanum) bubalisi sp. nov., found in the freshwater leech Hirudinaria manillensis.

Leeches have long been considered potential vectors for the aquatic lineage of trypanosomes, while bloodsucking insects are generally considered as the vectors for the terrestrial lineage of trypanosomes. The freshwater leech, Hirudinaria manillensis, is a widely distributed species in southern China and could potentially act as the vector for trypanosomes. Prior to this study, no trypanosomes had been reported from this leech. However, in this study, leeches were collected from three different places in Guangdong province, China, and a large number of flagellates were isolated and successfully cultured in vitro. Based on morphology, these flagellates looked like a typical trypanosome species. Analysis was carried out on the molecular sequences of the 18S rRNA gene and the glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene. To our surprise, these flagellates were identified as likely to be a mammalian trypanosome belonging to the clade containing Trypanosoma (Megatrypanum) theileri but they are significantly different from the typical TthI and TthII stocks. Analyses of blood composition indicated that the source of the blood meal in these leeches was from the water buffalo (Bubalus bubalis). To further test if this flagellate from the freshwater leech was indeed a mammalian trypanosome, we transferred the trypanosomes cultured at 27-37 °C and they were able to successfully adapt to this mammalian body temperature, providing further supporting evidence. Due to the significant genetic differences from other related trypanosomes in the subgenus Megatrypanum, we propose that this flagellate, isolated from H. manillensis, is a new species and have named it Trypanosoma bubalisi. Our results indicate that freshwater leeches may be a potential vector of this new mammalian trypanosome.

Nitric oxide blocks the development of the human parasite Schistosoma japonicum.

Human schistosomiasis, caused by Schistosoma species, is a major public health problem affecting more than 700 million people in 78 countries, with over 40 mammalian host reservoir species complicating the transmission ecosystem. The primary cause of morbidity is considered to be granulomas induced by fertilized eggs of schistosomes in the liver and intestines. Some host species, like rats (Rattus norvegicus), are naturally intolerant to Schistosoma japonicum infection, and do not produce granulomas or pose a threat to transmission, while others, like mice and hamsters, are highly susceptible. The reasons behind these differences are still a mystery. Using inducible nitric oxide synthase knockout (iNOS-/-) Sprague-Dawley rats, we found that inherent high expression levels of iNOS in wild-type (WT) rats play an important role in blocking growth, reproductive organ formation, and egg development in S. japonicum, resulting in production of nonfertilized eggs. Granuloma formation, induced by fertilized eggs in the liver, was considerably exacerbated in the iNOS-/- rats compared with the WT rats. This inhibition by nitric oxide acts by affecting mitochondrial respiration and energy production in the parasite. Our work not only elucidates the innate mechanism that blocks the development and production of fertilized eggs in S. japonicum but also offers insights into a better understanding of host-parasite interactions and drug development strategies against schistosomiasis.

Phylogeography of Angiostrongylus cantonensis (Nematoda: Angiostrongylidae) in southern China and some surrounding areas.

Angiostrongylus cantonensis is of increasing public health importance as the main zoonotic pathogen causing eosinophilic meningitis or meningoencephalitis, which has been documented all over the world. However, there are very limited studies about its phylogeography and spread pattern. In the present study, the phylogeography of A. cantonensis in southern China (including Taiwan) and partial areas of Southeast Asia were studied based on the sequences of complete mitochondrial cytochrome b (Cytb) gene. A total of 520 individuals of A. cantonensis obtained from 13 localities were sequenced for the analyses and grouped into 42 defined haplotypes. The phylogenetic tree (NJ tree and BI tree) revealed a characteristic distribution pattern of the four main lineages, with detectable geographic structure. Genetic differentiation among populations was significant, but demographic expansion could not be detected by either neutrality tests or mismatch distribution analysis, which implied a low gene flow among the local populations in different regions where the samples were collected. Two unique lineages of the A. cantonensis population in Taiwan were detected, which suggests its multiple origin in the island. Populations in Hekou (China) and Laos showed the highest genetic diversities, which were supported by both genetic diversity indices and AMOVA. These results together infer that the area around Thailand or Hekou in Yunnan province, China are the most likely origins of Angiostrongylus cantonensis.

Comparative phylogeography of two monogenean species (Mazocraeidae) on the host of chub mackerel, Scomber japonicus, along the coast of China.

In the present paper, the phylogeographies of two monogenean species, Pseudokuhnia minor and Kuhnia scombri, on the same species of host, Scomber japonicus, were studied. Fragments of the mitochondrial cytochrome c oxidase subunit 1 gene were sequenced for 264 individuals of P. minor and 224 individuals of K. scombri collected from 10 localities along the coast of China. Genetic diversity of K. scombri was higher than that of P. minor, which may imply that P. minor has a lower evolution rate and/or is a younger species. The neighbour-joining (NJ) trees of both parasites were comprised of two clades without association to sample sites, which is the signature of remixing populations following past division. Analyses of molecular variance and pairwise fixation index revealed different genetic structures for the populations of these two closely related species along the coast of China: P. minor without significant genetic structure, while K. scombri has some genetic differentiation. Both neutrality tests and mismatch distribution suggested that the populations of these two species of parasites experienced population expansion in the late Pleistocene era due to the glacial-interglacial cycles induced by climatic oscillations.

Phylogeography of Tetrancistrum nebulosi (Monogenea, Dactylogyridae) on the host of mottled spinefoot (Siganus fuscescens) in the South China Sea, inferred from mitochondrial COI and ND2 genes.

To examine the phylogeographical pattern of Tetrancistrum nebulosi (Monogenea, Dactylogyridae) in the South China Sea, fragments of mitochondrial cytochrome c oxidase subunit I and NADH dehydrogenase subunit 2 genes were obtained for 220 individuals collected from 8 localities along the southeast coast of China and 1 locality in Terengganu, Malaysia. Based on these two genes, two and three distinct clades with geographic signals were revealed on the phylogenetic trees respectively. The divergence between these clades was estimated to occur in the late Pleistocene. Analysis of molecular variance and pairwise FST suggested a high rate of gene flow among individuals sampled from the Chinese coast, but with obvious genetic differentiation from the Malaysian population. Mismatch distribution and neutrality tests indicated that the T. nebulosi population experienced expansion in Pleistocene low sea level periods. Vicariance was considered to account for the genetic divergence between Chinese and Malaysian populations, while sea level fluctuations and mainland-island connections during glacial cycles were associated with the slight genetic divergence between the populations along the mainland coast of China and those off Sanya. On the contrary, oceanographic circulations and host migration could lead to genetic homogeneity of populations distributed along the mainland coast of China.

Complete mitochondrial genome of the giant African snail, Achatina fulica (Mollusca: Achatinidae): a novel location of putative control regions (CR) in the mitogenome within Pulmonate species.

The whole sequence (15,057 bp) of the mitochondrial DNA (mtDNA) of the terrestrial snail Achatina fulica (order Stylommatophora) was determined. The mitogenome, as the typical metazoan mtDNA, contains 13 protein-coding genes (PCG), 2 ribosomal RNA genes (rRNA) and 22 transfer RNA genes (tRNA). The tRNA genes include two trnS without standard secondary structure. Interestingly, among the known mitogenomes of Pulmonata species, we firstly characterized an unassigned lengthy sequence (551 bp) between the cox1 and the trnV which may be the CR for the sake of its AT bases usage bias (65.70%) and potential hairpin structure.

Resistance to normal human serum reveals Trypanosoma lewisi as an underestimated human pathogen.

Human-infectious trypanosomes such as Trypanosoma cruzi, T. brucei rhodesiense, and T. b. gambiense can be discriminated from those only infecting animals by their resistance to normal human serum (NHS). These parasites are naturally resistant to trypanolysis induced by the human-specific pore-forming serum protein apolipoprotein L1 (ApoL-1). T. lewisi, a worldwide distributed parasite, has been considered as rat-specific and non-pathogenic to the natural hosts. Here we provide evidence that 19 tested T. lewisi isolates from Thailand and China share resistance to NHS. Further investigation on one selected isolate CPO02 showed that it could resist at least 90% NHS or 30 µg/ml recombinant human ApoL-1 (rhApoL-1) in vitro, in contrast to T. b. brucei which could not survive in 0.0001% NHS and 0.1 µg/ml rhApoL-1. In vivo tests in rats also demonstrated that this parasite is fully resistant to lysis by NHS. Together with recent reports of atypical human infection by T. lewisi, these data allow the conclusion that T. lewisi is potentially an underestimated and thus a neglected human pathogen.

Phylogeography and demographic history of Gotocotyla sawara (Monogenea: Gotocotylidae) on Japanese Spanish mackerel (Scomberomorus niphonius) along the Coast of China.

Recent studies indicate that the northwestern Pacific Ocean is an ideal system in which to study and understand the roles of the Pleistocene sea-level fluctuations and ocean currents in shaping phylogeographic patterns of species, but most of these investigations have been concerned with vertebrates, and only a few have focused on invertebrates. In the present study, we examined the genetic population structure and historic demography of a platyhelminth species, Gotocotyla sawara (Monogenea, Gotocotylidae), a gill parasite of Japanese Spanish mackerel, Scomberomorus niphonius , along the coast of China. A fragment of the mitochondrial cytochrome oxidase I (COI) gene for 169 individuals and the internal transcribed spacers for 24 individuals were sequenced from specimens representing 8 populations of this parasite along the coast of China. High levels of COI haplotype diversity (0.9994) and nucleotide diversity (0.015805) were detected for G. sawara. Phylogenetic analysis revealed no phylogeographical pattern for G. sawara in the sample area. Analysis of molecular variance (AMOVA) revealed no significant differences at all hierarchical levels, and pairwise FST analysis demonstrated a high rate of gene flow of this parasite among different populations in coastal Chinese waters. Moreover, the exact test of differentiation supported the null hypothesis that G. sawara along the coast of China constitutes a panmictic population. Both neutrality tests and mismatch distribution revealed that G. sawara underwent population expansion in the late Pleistocene era. Recent range expansion after the last glacial maximum and insufficient time to attain migration-drift equilibrium may account in part for the lack of genetic structure in the geographic areas considered in this study. Dispersal of parasite eggs and larvae along ocean currents, coupled with the long-distance migrations of host fishes, could also be responsible for genetic homogeneity of this parasite. It is also possible that other hosts of this monogean, such as the ridged-eye flounder, Pleruonichthys cornutus, may contribute to the genetic mixing of Gotocotyla sawara populations.

The apple snail Pomacea canaliculata, a novel vector of the rat lungworm, Angiostrongylus cantonensis: its introduction, spread, and control in China.

The freshwater apple snail Pomacea canaliculata was introduced to Taiwan then to mainland China in the early 1980s from Argentina, its native region, for the purpose of aquaculture. Because of the lack of natural enemies and its tolerance of a wide range of environmental conditions, both its abundance and distribution have dramatically increased and it has become a harmful species to local agriculture and other native species in many areas of China. Unfortunately, the snail also acts as an intermediate host of Angiostrongylus cantonensis, and has been implicated in transfer of the parasite to people, resulting in angiostrongyliasis manifested as eosinophilic meningitis. Efforts to prevent its further spread and population expansion were initiated many years ago, including the use of chemicals and biological control agents to control the snail.

Lower expression of inducible nitric oxide synthase and higher expression of arginase in rat alveolar macrophages are linked to their susceptibility to Toxoplasma gondii infection.

Rats are naturally resistant to Toxoplasma gondii infection, particularly the RH strain, while mice are not. Previous studies have demonstrated that inducible nitric oxide synthase (iNOS) and arginase-1 of rodent peritoneal macrophages are linked to the mechanism of resistance. As an increasing number of studies on human and animal infections are showing that pulmonary toxoplasmosis is one of the most severe clinical signs from T. gondii infection, we are interested to know whether T. gondii infection in alveolar macrophages of rats is also linked to the levels of iNOS and arginase-1 activity. Our results demonstrate that T. gondii could grow and proliferate in rat alveolar macrophages, both in vitro and in vivo, at levels higher than resistant rat peritoneal macrophages and at comparable levels to sensitive mouse peritoneal macrophages. Lower activity and expression levels of iNOS and higher activity and expression levels of arginase-1 in rat alveolar macrophages were found to be linked to the susceptibility of T. gondii infection in these cells. These novel findings could aid a better understanding of the pathogenesis of clinical pulmonary toxoplasmosis in humans and domestic animals.

Workshop on research priorities for management and treatment of angiostrongyliasis(1).

In a concluding session of the workshop, the participants developed a list of 115 research and outreach needs, outlining the top 5-7 needs in each of 8 areas (Table). For complete information, including presenter details and abstracts, visit the workshop website at www.hawaii.edu/cowielab/Angio%20website%20home.htm.

Cathepsin L in the orange-spotted grouper, Epinephelus coioides: molecular cloning and gene expression after a Vibrio anguillarum challenge.

The orange-spotted grouper, Epinephelus coioides, is an important fish maricultured in many Asian countries. In the present study, the full-length cDNA of cathepsin L, an immunity related gene of fishes, was isolated from E. coioides using rapid amplification of cDNA ends (RACE). It is 1,443 bp in length, including an open reading frame (ORF) of 1,011 bp. The open reading frame encoded a preproprotein of 336 amino acids (aa), which consisted of a signal peptide of 16 aa, a proregion peptide of 98 aa and a mature peptide of 222 aa. The preproprotein contained an oxyanion hole (Gln), a catalytic triad formed by Cys, His and Asn, and the conserved ERWNIN, GNFD and GCNGG motifs, all characteristic of cathepsin L. Homology analysis revealed that the deduced amino acid sequence of E. coioides cathepsin L shared 80.1-94.8 % identity with those of reported fishes. Tissue-dependent mRNA expression analysis showed that the cathepsin L transcript was expressed in all the examined tissues of the healthy E. coioides, being highest in the liver and moderate in the heart, gonad and intestine. After Vibrio anguillarum stimulation, the mRNA expression of cathepsin L in E. coioides was significantly increased in the skin, fin, gills, liver, blood, spleen, head kidney and intestine, with the highest observed in the spleen (10.6-fold) at 12 h post-injection and the next in blood (7.5-fold) at 8 h post-injection. These results provided initial information for further studies on the physiological and immunological roles of the cathepsin L gene in the orange-spotted grouper.

Differences in iNOS and arginase expression and activity in the macrophages of rats are responsible for the resistance against T. gondii infection.

Toxoplasma gondii infects humans and warm blooded animals causing devastating disease worldwide. It has long been a mystery as to why the peritoneal macrophages of rats are naturally resistant to T. gondii infection while those of mice are not. Here, we report that high expression levels and activity of inducible nitric oxide synthase (iNOS) and low levels of arginase-1 (Arg 1) activity in the peritoneal macrophages of rats are responsible for their resistance against T. gondii infection, due to high nitric oxide and low polyamines within these cells. The opposite situation was observed in the peritoneal macrophages of mice. This discovery of the opposing functions of iNOS and Arg 1 in rodent peritoneal macrophages may lead to a better understanding of the resistance mechanisms of mammals, particularly humans and livestock, against T. gondii and other intracellular pathogens.

Detection of Trypanosoma lewisi from wild rats in Southern China and its genetic diversity based on the ITS1 and ITS2 sequences.

Trypanosoma lewisi has widely been considered as a non-pathogenic rat trypanosome. However, more and more cases of humans infected with T. lewisi have been reported around the world, indicating that it can infect humans in some undetermined circumstances. Quick and sensitive diagnosis of infection by T. lewisi is important for both treatment of patients and epidemiological studies of this parasite. In this paper, three methods i.e. wet blood smear (diagnosis by microscopy), PCR and LAMP were used to detect T. lewisi from 238 wild rats (Rattus norvegicus) collected from the field in Huadu, Guangdong province, China. Infection rates of these samples detected by the 3 methods was 6.7% (16/238), 12.6% (30/238), and 18.9% (45/238), respectively. LAMP could detect all samples shown positive by microscopical observation of wet smear and by single PCR indicating good potential for application in the detection of T. lewisi. So far as we know, this is the first report of the LAMP method being used to detect T. lewisi in wild rats. The specific T. lewisi LAMP primers were able to amplify the target fragment from the genomic DNA of 19 T. lewisi strains isolated from Huadu, Guangdong province (n=16), Changchun, Jilin province of China (n=1) and from Thailand (n=2). Based on the analyses of ITS1 (internal transcribed spacer 1) and ITS2 sequences, these 19 strains show a very close genetic relationship with over 96-97% similarity to the other corresponding sequences of T. lewisi published in Genbank. Phylogenetic trees of the species in the subgenus Herpetosoma were constructed, based on the ITS1 and ITS2 sequences, and these results also indicate that they are closely related and in the same clade.

Phylogeographical pattern of Mazocraeoides gonialosae (Monogenea, Mazocraeidae) on the dotted gizzard shad, Konosirus punctatus, along the coast of China.

In the present study, we examined the phylogeographical pattern of the monogenean, Mazocraeoides gonialosae, which parasitises the dotted gizzard shad (Konosirus punctatus) along the coast of China. Fragments of 756 bp of the mitochondrial cytochrome c oxidase subunit I gene were sequenced for 147 individuals from seven localities along the coast of China. Phylogenetic analysis revealed no significant genealogical clades of samples corresponding to sampling localities. Analyses of molecular variance and pairwise F(ST) suggested a high rate of gene flow and the lack of a predictable genetic structure between different populations of this parasite. Both neutrality tests and mismatch distribution analyses indicated a recent population expansion in M. gonialosae after the last glacial maximum. Gradually decreasing genetic diversity in more northerly populations implied a historical south-to-north expansion of this parasite. Dispersal of eggs and larvae with ocean currents was considered to be associated with the genetic homogeneity of this species. The limited time to accumulate genetic variation after the last glacial maximum may also account in part for the lack of phylogeographical structure in the studied region.

Haploporus mugilis sp. n. (Digenea: Haploporidae) from Valamugil engeli (Pisces: Mugilidae) in the Taiwan Straits, China.

Haploporus mugilis sp. n. (Digenea: Haploporidae) was obtained from the intestine of kandas, Valamugil engeli (Bleeker) (Mugilidae, Perciformes) in the Taiwan Straits, China. It most closely resembles Haploporus spinosus Machida, 1996 from the intestine of Crenimugil crenilabis in Japanese and adjacent waters in the intestinal bifurcation posterior to the acetabulum, in egg-size (0.039-0.044 x 0.019-0.022 in H. mugilis and 0.034-0.042 x 0.018-0.023 in H. spinosus) and in the hermaphroditic sac armed with spines. However, it differs from the latter in its hermaphroditic sac with two long and numerous small spines rather than with four long and six small spines and in the excretory vesicle with a single prominent refractory concretion instead of being empty.

Two new species of Pseudohapladena yamaguti, 1952 (Digenea: Waretrematidae) from mugilid fish from the Taiwan Straits, China.

Two new species of Pseudohapladena Yamaguti, 1952 (Digenea: Waretrematidae) are described. Pseudohapladena megoorchis n. sp. was obtained from the intestine of Mugil engeli (Bleeker) (Mugilidae), and Pseudohapladena lizae n. sp. from the intestine of Liza carinatus (Cuvier and Valenciennes) (Mugilidae). Pseudohapladena megaorchis is the most closely related in general morphology to Pseudohapladena scatophagi Yamaguti, 1952, but differs from the latter in the position of the hermaphroditic sac, and in the nature and distribution of the vitellaria. Features of P. lizae distinguishing it from other species of the genus include the shorter uterus, which contains only 1 egg, and the lesser extent of the vitellaria.