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Concept: Tetracapsuloides bryosalmonae

169

The freshwater bryozoan Fredericella sultana (Blumenbach) is the most common invertebrate host of the myxozoan parasite Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease in salmonid fish. Culture media play an important role in hatching of statoblasts and maintaining clean bryozoan colonies for Malacosporea research. We developed a novel culture medium, Bryozoan Medium C (BMC), for the cultivation and maintenance of F. sultana under laboratory conditions. Statoblasts of F. sultana were successfully hatched to produce transparent-walled, specific pathogen-free (SPF) colonies that were maintained >12 months in BMC at pH 6.65. Tetracapsuloides bryosalmonae was successfully transmitted from infected brown trout, Salmo trutta L., to newly hatched F. sultana colonies in BMC, then from the infected bryozoan to SPF brown trout. This study demonstrated the utility of BMC (pH 6.65) for hatching statoblasts, long-term cultivation of clean and transparent bryozoan colonies and maintenance of the Tetracapsuloides bryosalmonae life cycle in the laboratory for molecular genetic research and other studies such as host-parasiteinteraction.

Concepts: Fish, Salmo, Rainbow trout, Brown trout, Brook trout, Myxozoa, Tetracapsuloides bryosalmonae, Myxobolus cerebralis

144

In the 1990s, the Tubifex tubifex aquatic oligochaete species complex was parsed into 6 separate lineages differing in susceptibility to Myxobolus cerebralis, the myxozoan parasite that can cause whirling disease (WD). Lineage III T. tubifex oligochaetes are highly susceptible to M. cerebralis infection. Lineage I, IV, V and VI oligochaetes are highly resistant or refractory to infection and may function as biological filters by deactivating M. cerebralis myxospores. We designed a 2-phased laboratory experiment using triactinomyxon (TAM) production as the response variable to test that hypothesis. A separate study conducted concurrently demonstrated that M. cerebralis myxospores held in sand and water at temperatures ≤15°C degrade rapidly, becoming almost completely non-viable after 180 d. Those results provided the baseline to assess deactivation of M. cerebralis myxospores by replicates of mixed lineage (I, III, V and VI) and refractory lineage (V and VI) oligochaetes. TAM production was zero among 7 of 8 Lineage V and Lineage VI T. tubifex oligochaete groups exposed to 12500 M. cerebralis myxospores for 15, 45, 90 and 135 d. Among 4 mixed lineage exposure groups, TAM production averaged 14641 compared with 2202495 among 12 groups of Lineage III oligochaetes. Among the 6 unexposed Lineage III experimental groups seeded into original Phase 1 substrates for the 45, 90 and 135 d treatments during the Phase 2 portion of the study, TAM production was reduced by 98.9, 99.9 and 99.9%, respectively, compared with the average for the 15 d exposure groups. These results are congruent with the hypothesis that Lineage V and Lineage VI T. tubifex oligochaetes can deactivate and destroy M. cerebralis myxospores.

Concepts: Experiment, Myxozoa, Tetracapsuloides bryosalmonae, Myxobolus cerebralis, Myxobolus, Lineage III, Myxosporea

10

Proliferative kidney disease (PKD) is a major threat to wild and farmed salmonid populations because of its lethal effect at high water temperatures. Its causative agent, the myxozoan Tetracapsuloides bryosalmonae, has a complex lifecycle exploiting freshwater bryozoans as primary hosts and salmonids as secondary hosts. We carried out an integrated study of PKD in a prealpine Swiss river (the Wigger). During a 3-year period, data on fish abundance, disease prevalence, concentration of primary hosts' DNA in environmental samples [environmental DNA (eDNA)], hydrological variables, and water temperatures gathered at various locations within the catchment were integrated into a newly developed metacommunity model, which includes ecological and epidemiological dynamics of fish and bryozoans, connectivity effects, and hydrothermal drivers. Infection dynamics were captured well by the epidemiological model, especially with regard to the spatial prevalence patterns. PKD prevalence in the sampled sites for both young-of-the-year (YOY) and adult brown trout attained 100% at the end of summer, while seasonal population decay was higher in YOY than in adults. We introduce a method based on decay distance of eDNA signal predicting local species' density, accounting for variation in environmental drivers (such as morphology and geology). The model provides a whole-network overview of the disease prevalence. In this study, we show how spatial and environmental characteristics of river networks can be used to study epidemiology and disease dynamics of waterborne diseases.

Concepts: Epidemiology, Disease, Infectious disease, Biology, Demography, Prevalence, Myxozoa, Tetracapsuloides bryosalmonae

1

Myxozoan parasites pose emerging health issues for wild and farmed salmonid fish. Rainbow trout (Oncorhynchus mykiss) is a particularly susceptible species to Tetracapsuloides bryosalmonae (Malacosporea), the etiological agent of Proliferative Kidney Disease (PKD), and to Myxobolus cerebralis (Myxosporea), the etiological agent of Whirling Disease (WD). The objective of this study was to investigate the impact of myxozoan co-infections on the pathogenesis of PKD and WD in the rainbow trout.

Concepts: Salmon, Oncorhynchus, Salmonidae, Rainbow trout, Myxozoa, Tetracapsuloides bryosalmonae, Myxobolus cerebralis, Myxosporea

1

Fredericella sultana is an invertebrate host of Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease in salmonids. The bryozoan produces seed-like statoblasts to facilitate its persistence during unfavourable conditions. Statoblasts from infected bryozoans can harbor T. bryosalmonae and give rise to infected bryozoan colonies when conditions improve. We aimed in the present study to evaluate the integrity and viability of T. bryosalmonae-infected statoblasts after a range of harsh treatment conditions. We tested if statoblasts could survive ingestion by either brown trout or common carp. After ingestion, the fish faeces was collected at different time points. We also tested physical stressors: statoblasts collected from infected colonies were desiccated at room temperature, or frozen with and without Bryozoan Medium C (BMC). After treatments, statoblasts were assessed for physical integrity before being incubated on BMC to allow them to hatch. After 4 weeks, hatched and unhatched statoblasts were tested by PCR for the presence of the parasite. We found that statoblasts ingested by brown trout and those frozen in BMC were completely broken. In contrast, statoblasts ingested by common carp and those subjected to dry freezing were able to survive and hatch. T. bryosalmonae was detected by PCR in both hatched and unhatched infected statoblasts, but neither from broken nor uninfected statoblasts. Our results confirmed for the first time the ability of infected statoblasts to survive passage through a fish, and freezing. These findings suggest potential pathways for both persistence and spread of T. bryosalmonae-infected statoblasts in natural aquatic systems.

Concepts: Coprophagia, Animal, Salmo, Rainbow trout, Bryozoa, Myxozoa, Tetracapsuloides bryosalmonae, Myxobolus cerebralis

1

Myxozoa is a speciose group of endoparasitic cnidarians that can cause severe ecological and economic effects. Although highly reduced compared to free-living cnidarians, myxozoans have retained the phylum-defining stinging organelles, known as cnidae or polar capsules, which are essential to initiating host infection. To explore the adaptations of myxozoan polar capsules, we compared the structure, firing process and content release mechanism of polar tubules in myxospores of three Myxobolus species including M. cerebralis, the causative agent of whirling disease.

Concepts: Immune system, Animal, Cnidaria, Myxozoa, Tetracapsuloides bryosalmonae, Myxobolus cerebralis, Myxosporea, Radiata

0

Conventional PCR is an established method to detect Tetracapsuloides bryosalmonae DNA in fish tissues and to confirm diagnosis of proliferative kidney disease (PKD) caused by T. bryosalmonae. However, the commonly used PKX5f-6r primers were designed with the intention of obtaining sequence information and are suboptimal for determining parasite DNA presence. A new PCR assay to detect T. bryosalmonae 18s rDNA, PKX18s1266f-1426r, is presented that demonstrates specificity, repeatability, and enhanced sensitivity over the PKX5f-6r assay. The limit of detection of the PKX18s1266f-1426r assay at 95% confidence was 100 template copies and the new primers detected parasite DNA more consistently at template concentrations below 100 copies than PKX5f-6r. The PKX18s1266f-1426r also achieved 100% detection at sample DNA concentrations one order of magnitude lower than PKX5f-6r. Out of 127 salmonid fish with unknown T. bryosalmonae infection status, PKX5f-6r detected 35 positive samples while the new assay detected 43. The discrepancy in T. bryosalmonae detection between the two primer sets may be attributed to several differences between the assays, including oligonucleotide melting temperatures, the use of a touchdown PCR thermal cycle, and amplicon length. This article is protected by copyright. All rights reserved.

Concepts: Polymerase chain reaction, Molecular biology, DNA sequencing, DNA replication, All rights reserved, Kidney diseases, Copyright, Tetracapsuloides bryosalmonae

0

The myxozoan Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease (PKD), is responsible for considerable losses in farmed and wild fish populations in Europe and North America. Recently, T. bryosalmonae was detected in many European countries, and strategy to control the disease in the wild and farmed fish population is yet to be developed. Recombinase polymerase amplification (RPA) is a novel isothermal nucleic acid amplification technology that does not require any thermal cycling, and lateral flow dipstick (LFD) is a rapid, cost-effective, and easy-to-handle assay that enables stable detection.

Concepts: DNA, United States, Europe, Salmon, North America, Myxozoa, Tetracapsuloides bryosalmonae, Myxobolus cerebralis

0

A new species of myxozoan, Myxobolus imparfinis n. sp. is described based on material from the gills of Imparfinis mirini (Haseman) (Heptapteridae). Mature myxospores are round, measuring 7.1-8.4 (7.9 ± 0.3) μm in length, 4.5-6.2 (5.5 ± 0.5) μm in width and 3.1-4.2 (3.7 ± 0.3) μm in thickness. The polar capsules are of unequal size, the larger polar capsule measuring 3.4-4.5 (3.9 ± 0.3) μm in length and 1.4-2.0 (1.7 ± 0.1) μm in width and the smaller capsule measuring 3.1-3.8 (3.4 ± 0.2) μm in length and 1.2-1.8 (1.5 ± 0.2) μm in width. The polar filament presents 6-7 coils. Spores had a prevalence of infection of 75% (6/8). In histological analyses we detected the development site of spores in primary filaments, in afferent branchial artery, thus classifying the type of infection to the filamental type and vascular subtype. The phylogenetic analyses of a dataset including species Myxobolus Bütschli, 1882 and Henneguya Thélohan, 1892 from South America recovered M. imparfinis n. sp. as a sister species of Myxobolus flavus Carriero, Adriano, Silva, Ceccarelli & Maia, 2013. To our knowledge, this is the first record of a myxozoan species parasitising I. mirini.

Concepts: Phylogenetic tree, Phylogenetics, Units of measurement, Height, Myxozoa, Tetracapsuloides bryosalmonae, Myxobolus cerebralis, Myxosporea

0

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”.

Concepts: Phylogenetic nomenclature, Phylogenetics, Cladistics, Gill, Myxozoa, Tetracapsuloides bryosalmonae, Myxobolus cerebralis, Myxosporea