Molecular and light microscopy evidence for the transfer of Myxobolus honghuensis from Carassius auratus gibelio broodfish to progeny.

Myxozoans usually have a complex life cycle involving indirect transmission between vertebrate and invertebrate hosts. The vertical transmission of these parasites in vertebrate hosts has not been documented so far. Here, we assessed whether the myxozoan parasite Myxobolus honghuensis is vertically transmitted in naturally infected allogynogenetic gibel carp Carassius auratus gibelio (Bloch). M. honghuensis infection of broodfish, fertilized eggs and laboratory-cultured progeny was monitored in 2018 and 2019. The presporogonic stage was microscopically observed in the pharynx of broodfish and their progeny. In situ hybridization confirmed the presence of M. honghuensis presporogonic stage in the pharynx of broodfish and progeny. Nested PCR results showed that M. honghuensis was present in tissues and eggs of broodfish, fertilized eggs and their corresponding progeny. The sequences obtained from broodfish and progeny showed 98.0-99.8% similarity with ITS-5.8S rDNA of M. honghuensis. This study provides molecular and light microscopy evidence for the transfer of M. honghuensis from broodfish to progeny via the eggs, but it is insufficient to assert that M. honghuensis can transmit vertically in naturally infected allogynogenetic gibel carp. This is the first record about vertical transfer of myxozoan in the vertebrate host.

Myxobolus okamurae sp. nov. (Myxosporea: Myxozoa) causing severe gill myxoboliosis in the cyprinid Labeo bata in a cold water wetland, Punjab (India).

During the present study on myxozoan parasites infecting gills of cyprinid carps inhabiting Ranjit Sagar Wetland, a new parasite, Myxobolus okamurae sp. nov. infecting gills of Labeo bata has been described based on morphology, histopathology and partial 18S rDNA sequencing. For M. okamurae sp. nov., hundred fish specimens were examined, out of which thirty-three had large cylindrical to round, white plasmodia in gills, each plasmodium measured 0.9-3.0 mm in diameter. The myxospore body was pyriform in shape, measuring 12.25 × 4.93 µm, with a small intercapsular process at the anterior end. The polar capsules were equal and pyriform in shape, measuring 6.06 × 1.45 µm having polar filaments forming coils up to 13-14 in number. The intensity of infection was recorded to be heavy as indicated by gill plasmodium index (GPI = 3). Sequence analysis showed that M. okamurae sp. nov. is 91% similar with M. catlae infecting gills of Catla catla from India followed by M. intimus infecting gills of Leuciscus idus from Hungary. The phylogenetic tree based on the final edited alignment (403 bp) with Maximum-Likelihood showed the high bootstrap value of 75 and formed two major clades involving M. okamurae sp. nov. with M. pendula M. catlae and M. dispar in one clade with a low bootstrap value of 23 and the rest of the species in a separate clade. The plasmodium was located in the gill lamella and typed as "intralamellar vascular type, LV3".

Myxozoans and Our Dinner Table: Pathogenicity Studies of Myxobolus honghuensis (Myxosporea: Bivalvulida) Using a Suckling Mice Model.

Myxobolus honghuensis parasitizes the pharynx of the allogynogenetic gibel carp Carassius auratus gibelio (Bloch), an economically important food fish with large consumption rates in China. The fact that it is hard to detect the cysts formed by M. honghuensis without euthanizing the fish and the spores can reach the consumers has prompted us to investigate the effect of M. honghuensis on conventional, immunocompetent BALB/c suckling mice, in order to consider the possible pathological effects in general immunocompetent consumers by the ingestion of Myxobolus-infected fish. BALB/c suckling mice were inoculated with serial dilutions of the spore suspension and then hematological and histological studies were performed. Tests of suckling mice showed no statistically difference of fluid accumulation ratio between mice inoculated with 1.65 × 10(6) M. honghuensis spores and negative control after 8-h incubation. Spores did not disrupt intestinal histology, and no abnormal bowel movements were observed within 20 h postinoculation. No anomalous hematology parameters were documented. These results, taken together, reveal that M. honghuensis spores do not cause pathology (diarrhea and elevated fluid accumulation) in BALB/c suckling mice. However, an extrapolation is not currently feasible because of lack of a highly quantifiable risk extrapolation model, inaccessibility to data for the effect of M. honghuensis on humans, and lack of consideration of the immunosuppressed population. Our study extends the results of the effect of myxozoan on suckling mice and serves as baseline for future experiment using the same model in evaluating the myxozoan-caused foodborne disease.

Proliferation and resistance difference of a liver-parasitized myxosporean in two different gynogenetic clones of gibel carp.

Some myxosporeans have been demonstrated to be harmful to worldwide aquaculture. However, the proliferation information has remained unclear in the fish hosts. In this study, we utilized the mix-culturing equality to reveal significant difference in disease assistance between two different clones of gibel carp, in which clone D had been cultured for nearly 40 years, whereas clone A(+) was a newly created clone. According to morphological and genetic analysis of isolated spores, the diseasing pathogen was identified as Myxobolus wulii of the genus Myxobolus in Myxosporea. Subsequently, a polyclonal antibody specific to soluble proteins of the purified spores was generated. Using the antibody, we performed immunofluorescence observation of the liver lump sections sampled from the heavily diseased clone D individuals, and found that the liver lumps were completely composed of numerous honeycomb-like cysts, full of maturing and mature myxosporean spores, and almost all of liver tissues were destroyed. Comparative co-localization detection revealed a significantly inducing expression of apo-14 protein around the infected myxosporean sporoplasms and plasmodia, and the inducing level was much stronger in clone A(+) than in clone D. Furthermore, a primarily screening of 15 different major histocompatibility complex class Iα variants also excavated major variants that respectively belong to clones D and A(+). Therefore, these data provide significant information for differences in myxosporean proliferation and disease resistance in fish clone hosts with different genetic background. Further studies on myxosporean development and the mechanism for disease resistance will be very important for preventing and controlling the parasitic myxosporean disease.

The susceptibility of diverse species of cultured oligochaetes to the fish parasite Myxobolus pseudodispar Gorbunova (Myxozoa).

This study provides detailed information on the invertebrate hosts of Myxobolus pseudodispar (Myxozoa) and explores the susceptibility range of several species and analyses the relevance of the species composition of an oligochaete population. Our findings demonstrate that the oligochaete host range of M. pseudodispar is similarly wide as the number of vertebrate host species. Besides Tubifex tubifex and Limnodrilus hoffmeisteri, Psammoryctides barbatus and Psammoryctides moravicus were also found to be susceptible invertebrate hosts. The genetic characterization of the mitochondrial 16S rDNA of T. tubifex sensu lato revealed that lineages I, II and III are susceptible to M. pseudodispar, whereas T. tubifex lineage VI seems to be non-susceptible. T. tubifex lineage V and L. hoffmeisteri specimens were positive in a M. pseudodispar-specific PCR, but in most cases, the release of mature actinospores could not be detected. Hence, these non-susceptible oligochaetes likely serve as `biological filters` as they remove myxospores from the sediment without producing actinospores. Together with the phylogenetic analysis of the susceptible and non-susceptible oligochaete hosts on the basis of mt 16S rDNA sequences, the route of the development of M. pseudodispar in the oligochaete hosts was tracked by in situ hybridization. According to our findings, the gut epithelia seem to be a portal of entry of the sporoplasms, where the development of the parasite also takes place. The basal lamina seems to be involved in the migration of the parasite, and the worm's cellular immune response is activated by the infection.

Myxobolus honghuensis n. sp. (Myxosporea: Bivalvulida) parasitizing the pharynx of allogynogenetic gibel carp Carassius auratus gibelio (Bloch) from Honghu Lake, China.

Myxobolus honghuensis n. sp. is described from allogynogenetic gibel carp Carassius auratus gibelio (Bloch), during a survey of myxosporean parasites in Honghu Lake, Hubei Province, China. It is characterized by the presence of round plasmodia of 5-12 mm in diameter in the pharynx of host. Mature spores of M. honghuensis were pyriform in frontal view and anterior pointed with bluntly round posterior, they measured 16.9 ± 0.5 (15.1-19.5) µm long, 10.4 ± 0.4 (9.0-11.3) µm wide, and 8.4 ± 0.4 (7.9-9.1) µm thick. Two polar capsules were pyriform and slightly unequal with larger polar capsule 8.4 ± 0.4 (7.6-10.2) µm × 3.9 ± 0.2 (3.0-4.5) µm and smaller capsule 7.9 ± 0.2 (7.0-9.3) µm × 3.7 ± 0.3 (2.8-4.1) µm. Polar filaments coiled with seven to eight turns. Both morphology and DNA sequence data revealed that M. honghuensis n. sp. was distinct from other described Myxobolus species. Phylogenetic analysis placed M. honghuensis n. sp. in a clade of gill-infecting myxobolids.

Myxobolus myleus n. sp. infecting the bile of the Amazonian freshwater fish Myleus rubripinnis (Teleostei: Serrasalmidae): morphology and pathology.

Myxobolus myleus n. sp. is described from the gall-bladder of the freshwater fish Myleus rubripinnis collected near the city of Oriximiná in the Amazon System, Brazil. The spores obtained from the bile contained two equal symmetrical and smooth valves, each forming the spore wall. The spores were large, with a cone-like form, a semi spherical basal contour and measured (in µm) 19.3 ± 0.5 (n = 25) × 8.3 ± 0.5 (n = 25) × 4.0 ± 0.3 (n = 15). The apical end of the spores contained two elongate, equal and pointed conical polar capsules measuring 13.2 ± 0.4 µm (n = 25) in length and 3.0 ± 0.3 µm (n = 15) in width, each having a slightly tapering polar filament with 19 to 21 turns. The polar capsules were extended below at about 4/5 of the total length of the spores. The sporoplasm was binucleate and contained some sporoplasmosomes. All infected fish presented hypertrophy of the gall-bladder due to presence of the brownish parasite floating in the bile. In this paper we describe this new species of myxosporean based on light and ultrastructural observations, together with its associated pathology.

A synopsis of the species of Myxobolus Bütschli, 1882 (Myxozoa: Bivalvulida) parasitising Indian fishes and a revised dichotomous key to myxosporean genera.

A synopsis of 131 nominal species of Myxobolus Bütschli, 1882 (Myxozoa: Myxosporea: Myxobolidae) reported from India is presented. For each species, the relevant morphometric and morphological data are indicated, as well as the host(s), site(s) of infection within the host and locality data. A revised dichotomous key of 59 genera of the class Myxosporea has also been included. This key incorporates 10 additional genera to that proposed in 1991 by Lom & Dyková.

Cutaneus myxosporidiasis in the Australian green tree frog (Litoria caerulea).

This case is reported with the intention of highlighting the presentation of cutaneous myxosporidiasis in Australian tree frog (Litoria caerulea) caused by genus Myxobolus. The morphology and morphometric characteristic of the spores were determined using light microscopy and differential interference contrast microscopy. Spores were pyriform in shape in frontal view and oval in lateral view, and the average size was respectively 11.4 × 6.0 × 4.5 µm (12.1 - 9.5 × 6.3 - 5.4 × 5.0 - 4.1 µm). To the best of our knowledge, this is the second case of skin invasion caused by myxosporeans in amphibians.

Redescription and molecular analysis of Myxobolus shantungensis Hu, 1965 (Myxozoa: Myxosporea) infecting common carp Cyprinus carpio haematopterus.

Myxobolus shantungensis Hu, 1965 infects gill arches of common carp Cyprinus carpio haematopterus (Temminck and Schlegel), causing serious pathological effects on host fish. An inadequate original description and absence of molecular data make accurate early diagnosis challenging. To augment the original description, M. shantungensis is redescribed here using morphological and molecular biological methods. Mature spores of M. shantungensis were ellipsoidal or apple shaped in frontal view and lemon shaped in lateral view, averaging 8.2 ± 0.3 µm (8.0-9.0 µm) × 10.1 ± 0.5 µm (9.2-11.1 µm) × 6.9 ± 0.3 µm (6.0-7.4 µm). Some spores had three to four "V"-shaped valve edge markings on the posterior of the spore. The two equal polar capsules were oval, measuring 4.3 ± 0.3 µm (3.8-5.0 µm) × 3.2 ± 0.2 µm (2.8-3.5 µm), situated at the anterior extremity of the spore. Polar filaments coiled with six to seven turns. Scanning electron microscopy revealed that the surface of mature spores of M. shantungensis was generally pitted with a number of irregular ridges in shape. M. shantungensis is also characterized on the molecular level using the sequence of the small subunit ribosomal RNA gene. A BLAST search revealed that this sequence did not match any available sequences in GenBank.

Myxobolus harikensis sp. nov. (Myxozoa: Myxobolidae) infecting fins of Cirrhina mrigala (Ham.)--an Indian major carp in Harike Wetland, Punjab (India).

During a study of myxosporean parasites of freshwater fishes of Punjab Wetlands, India, one new myxosporean species, Myxobolus harikensis sp. nov. was recorded from the caudal fin of Cirrhina mrigala (Ham.) (Cypriniformes: Cyprinidae). Plasmodia are small, white, spherical-to-rounded and are present on the caudal fin (in between the fin rays) having two to five spores per plasmodium. Spores of M. harikensis measure 10.1 × 8.5 µm in size, pyriform in valvular view and lenticular in sutural view pointing in the direction to the plane of slightly bent sutural line. Shell valves are thick, smooth, asymmetric and flattened at the sutural plane. Parietal folds are absent. Polar capsules are two, prominently unequal, broadly pyriform-to-rounded in shape and lie at different levels in the spore body cavity. The larger polar capsule is 5.0 × 3.1 µm in size, placed anteriorly on the sutural plane and opening to the exterior anteriolaterally. The smaller polar capsule measures 1.7 × 1.4 µm in size, placed posteriorly, perpendicular to the sutural plane and opening to the exterior mediolaterally. Spores of M. harikensis closely resembles to that of Myxobolus africanus in having polar capsules placed at different levels in the spore body cavity, however, are unique in having unequal polar capsules both discharging laterally.

Differentially expressed parasite genes involved in host recognition and invasion of the triactinomyxon stage of Myxobolus cerebralis (Myxozoa).

The host recognition and invasion process of Myxobolus cerebralis actinospores (triactinomyxon, TAM) was studied on a genetic level. A small-scale in vitro assay was developed to activate a large number of TAMs simultaneously, and to monitor the host invasion in the absence of live fish. The transcriptomes of non-activated and in vitro-activated TAMs were compared by suppressive subtractive hybridization (SSH) to identify parasite genes involved in the host invasion process. Differential screening and a subsequent BLAST search revealed 15 of 452 SSH-library clones expressed differently in activated TAMs. None of the 15 transcripts obtained has previously been identified from M. cerebralis. Quantitative real-time PCR was used to examine the relative expression profile of 8 selected transcripts upon TAM activation and after penetration of the host. Four of these were found to be up-regulated in activated TAMs, while expression was relatively low in non-activated TAMs and in infected fish tissue, indicating that they are relevant genes during host recognition or subsequent host invasion of M. cerebralis TAMs.

Myxobolus mauriensis n. sp. Infecting Rib Cartilage of Young-of-the-Year River Herring in New Jersey: Notes on Pathology, Prevalence, and Genetics.

: River herring populations, including Alosa pseudoharengus and Alosa aestivalis , have significantly declined as a result of anthropogenic factors throughout their range in eastern North America. To better understand the health of the species, parasite surveys were conducted in several New Jersey rivers. A novel myxozoan parasite, Myxobolus mauriensis n. sp., is described infecting the cartilage of pleural ribs in young-of-the-year fish. The parasite forms large polysporic plasmodia forming pseudocysts within the ribs, which extend into the musculature. Pathology associated with infection includes costochondritis, breaks in the rib bones, and deformed bone growth. Rupture of large pseudocysts and release of mature spores are associated with myositis, dermatitis, and peritonitis. Phylogenetic analysis reveals that M. mauriensis n. sp. occurs in a long-branching clade basal to other myxobolids, grouping with several Myxobolus species from marine fish (Myxobolus groenlandicus, Myxobolus aeglefini, and Myxobolus albi). The closest identity is to M. groenlandicus, with 83% identity based on a 1,762-bp sequence of the SSU 18S rDNA. Similarly, spore morphology, tropism for cartilage, and association with marine/brackish environments are shared in these 4 species. Mature spores of M. mauriensis n. sp. are similar to other reported myxobolids, though spores are slightly wider (12.1 ± 0.44 µm) than long (11.4 ± 0.44 µm), with a length:width relationship of 0.94 (±0.04), a feature not commonly described for other species of Myxobolus. Prevalence was studied by histology and fresh observation. In both Blueback Herring and Alewife, the highest infection prevalence occurred in the Maurice River at around 20%, and lower prevalence was found in the Great Egg Harbor River at around 5%. In the Delaware River, prevalence was about 2% in Blueback Herring, while the parasite was not detected in Alewife samples.

Counter-insurgents of the blue revolution? Parasites and diseases affecting aquaculture and science.

Aquaculture is the fastest-growing segment of food production and is expected to supply a growing portion of animal protein for consumption by humans. Because industrial aquaculture developed only recently compared to industrial agriculture, its development occurred within the context of a growing environmental awareness and acknowledgment of environmental issues associated with industrial farming. As such, parasites and diseases have become central criticisms of commercial aquaculture. This focus on parasites and diseases, however, has created a nexus of opportunities for research that has facilitated considerable scientific advances in the fields of parasitology and aquaculture. This paper reviews Myxobolus cerebralis , Lepeophtheirus salmonis , white spot syndrome virus, and assorted flatworms as select marquee aquaculture pathogens, summarizes the status of the diseases caused by each and their impacts on aquaculture, and highlights some of the significant contributions these pathogens have made to the science of parasitology and aquaculture.

Invasion and initial replication of ultraviolet irradiated waterborne infective stages of Myxobolus cerebralis results in immunity to whirling disease in rainbow trout.

Myxobolus cerebralis is a microscopic metazoan parasite (Phylum Myxozoa: Myxosporea) associated with salmonid whirling disease. There are currently no vaccines to minimise the serious negative economical and ecological impacts of whirling disease among populations of salmonid fish worldwide. UV irradiation has been shown to effectively inactivate the waterborne infective stages or triactinomyxons of M. cerbralis in experimental and hatchery settings but the mechanisms by which the parasite is compromised are unknown. Treatments of triactinomyxons with UV irradiation at doses from 10 to 80 mJ/cm(2) either prevented (20-80 mJ/cm(2)) or significantly inhibited (10 mJ/cm(2)) completion of the parasite life cycle in experimentally exposed juvenile rainbow trout (Oncorhynchus mykiss). However, even the highest doses of UV irradiation examined (80 mJ/cm(2)) did not prevent key steps in the initiation of parasite infection, including attachment and penetration of the epidermis of juvenile rainbow trout as demonstrated by scanning electron and light microscopy. Furthermore, replication of UV-treated parasites within the first 24h following invasion of the caudal fin was suggested by the detection of concentrations of parasite DNA by quantitative PCR comparable to that among fish exposed to an equal concentration of untreated triactinomyxons. Subsequent development of parasites treated with an 80 mJ/cm(2) dose of UV irradiation however, was impaired as demonstrated by the decline and then lack of detection of parasite DNA; a trend beginning at 10 days and continuing thereafter until the end of the study at 46 days post parasite exposure. Treatments of triactinomyxons with a lower dose of UV irradiation (20 mJ/cm(2)) resulted in a more prolonged survival with parasite DNA detected, although at very low concentrations, in fish up to 49 days post parasite exposure. The successful invasion but only short-term survival of parasites treated with UV in rainbow trout resulted in a protective response to challenges with fully infective triactinomyxons. Prior treatments of juvenile rainbow trout with UV-treated triactinomyxons (10 and 20 mJ/cm(2)) resulted in a reduced prevalence of infection and significantly lower concentrations of cranial myxospores (two direct measures of the severity of whirling disease) compared with trout receiving no prior treatments when assessed 5 months post parasite exposure to fully infective triactinomyxons.

Potential for dissemination of the nonnative salmonid parasite Myxobolus cerebralis in Alaska.

Myxobolus cerebralis, the myxozoan parasite responsible for whirling disease in salmonids, was first introduced into the United States in 1958 and has since spread across the country, causing severe declines in wild trout populations in the intermountain western United States. The recent detection of the parasite in Alaska is further evidence of the species' capability to invade and colonize new habitat. This study qualitatively assesses the risk of further spread and establishment of M. cerebralis in Alaska. We examine four potential routes of dissemination: human movement of fish, natural dispersal by salmonid predators and straying salmon, recreational activities, and commercial seafood processing. Potential for establishment was evaluated by examining water temperatures, spatial and temporal overlap of hosts, and the distribution and genetic composition of the oligochaete host, Tubifex tubifex. The most likely pathway of M. cerebralis transport in Alaska is human movement of fish by stocking. The extent of M. cerebralis infection in Alaskan salmonid populations is unknown, but if the parasite becomes dispersed, conditions are appropriate for establishment and propagation of the parasite life cycle in areas of south-central Alaska. The probability of further establishment is greatest in Ship Creek, where the abundance of susceptible T. tubifex, the presence of susceptible rainbow trout Oncorhynchus mykiss, and the proximity of this system to the known area of infection make conditions particularly suitable for spread of the parasite.

Application of an aqueous acid-fast staining technique to detect pathogens of aquatic species.

Many bacterial and protozoan pathogens in fish and shellfish exhibit acid-fast staining characteristics that are important for pathogen identification and disease diagnosis. The classic acid-fast staining techniques for light microscopy use carbol-fuchsin; a major ingredient of this stain is caustic and hazardous phenol. A new technique using heated aqueous basic fuchsin can stain two acid-fast human pathogens, Mycobacterium tuberculosis and M. avium. This method is a potentially valuable and safer diagnostic tool for aquatic pathologists. M. marinum, a finfish pathogen, and various additional acid-fast parasitic pathogens of finfish and marine invertebrates were stained successfully using this heated aqueous basic fuchsin method.