Mansour El-Matbouli

Recent advances in our knowledge of the Myxozoa

Abstract In the last few years two factors have helped to significantly advance our understanding of the Myxozoa. First, the phenomenal increase in fin fish aquaculture in the 1990s has lead to the increased importance of these parasites; in turn this has lead to intensified research efforts, which have increased knowledge of the development, diagnosis, and pathogenesis of myxozoans. The hallmark discovery in the 1980s that the life cycle of Myxobolus cerebralis requires development of an actinosporean stage in the oligochaete, Tubifex tubifex, led to the elucidation of the life cycles of several other myxozoans. Also, the life cycle and taxonomy of the enigmatic PKX myxozoan has been resolved: it is the alternate stage of the unusual myxozoan, Tetracapsula bryosalmonae, from bryozoans. The 18S rDNA gene of many species has been sequenced, and here we add 22 new sequences to the data set. Phylogenetic analyses using all these sequences indicate that:1) the Myxozoa are closely related to Cnidaria (also supported by morphological data); 2) marine taxa at the genus level branch separately from genera that usually infect freshwater fishes; 3) taxa cluster more by development and tissue location than by spore morphology; 4) the tetracapsulids branched off early in myxozoan evolution, perhaps reflected by their having bryozoan, rather than annelid hosts; 5) the morphology of actinosporeans offers little information for determining their myxosporean counterparts (assuming that they exist); and 6) the marine actinosporeans from Australia appear to form a clade within the platysporinid myxosporeans. Ribosomal DNA sequences have also enabled development of diagnostic tests for myxozoans. PCR and in situ hybridisation tests based on rDNA sequences have been developed for Myxobolus cerebralis, Ceratomyxa shasta, Kudoa spp., and Tetracapsula bryosalmonae (PKX). Lectin-based and antibody tests have also been developed for certain myxozoans, such as PKX and C. shasta. We also review important diseases caused by myxozoans, which are emerging or re-emerging. Epizootics of whirling disease in wild rainbow trout (Oncorhynchus mykiss) have recently been reported throughout the Rocky Mountain states of the USA. With a dramatic increase in aquaculture of fishes using marine netpens, several marine myxozoans have been recognized or elevated in status as pathological agents. Kudoa thyrsites infections have caused severe post-harvest myoliquefaction in pen-reared Atlantic salmon (Salmo salar), and Ceratomyxa spp., Sphaerospora spp., and Myxidium leei cause disease in pen-reared sea bass (Dicentrarchus labrax) and sea bream species (family Sparidae) in Mediterranean countries.

Experimental transmission of twoMyxobolus spp. developing bisporogeny via tubificid worms

Spores ofMyxobolus cotti El-Matbouli and Hoffmann 1987 andM. cerebralis Hofer 1903 (fresh or after 5 months in mud) are taken in by tubifex worms and develop in their gut epithelium cells into actinosporea of the genusTriactinomyxon. Triactinomyxon deriving fromM. cotti differ distinctly from those derived fromM. cerebralis in morphology and the number of sporozoites in the epispore. It could be shown thatTriactinomyxon spores infect fish either via the water (bullhead, rainbow trout) or by feeding of infected tubifex (rainbow trout), developing intoMyxobolus spores in central nervous tissue (bullhead) or cartilage (rainbow trout). Fresh or ripened spores ofM. cotti andM. cerebralis were not infectious for bullheads or rainbow trout, respectively. The results of our experiments confirm the hypothesis that the life cycle ofM. cotti andM. cerebralis includes an intermediate host and a metamorphosis into actinosporea of the genusTriactinomyxon.

Light and electron microscopic studies on the chronological development of Myxobolus cerebralis to the actinosporean stage in Tubifex tubifex

Whirling disease caused by Myxobolus cerebralis has become the most widely known disease of salmonids in the 1990s. In the last 5 years we have studied many aspects regarding the host-pathogen relationship of this parasite. The parasites histozoic development causes significant damage to cartilage and induces CNS symptoms by pressure on the brain and spinal cord. Myxobolus cerebralis has a two-host life-cycle involving a salmonid fish and a tubificid oligochaete. Two different stages of sporogony occur, one in each host. Early developmental stages in the fish can be found multiplying in the epidermis and peripheral and central nervous systems. The presporogenic stages then migrate to vertebral and cranial cartilages, where the first sporogonic phase occurs. Mature M. cerebralis spores found in fish cartilage are infectious for T. tubifex when ingested by the oligochaete after destruction of the infected fish. In the gut lumen of the tubificid, the spores extrude their polar capsules and attach to the gut epithelium by polar filaments. The shell valves then open along the suture line and the sporoplasm penetrates between the gut epithelial cells. The binucleate sporoplasm multiplies by schizogony, producing many one-cell stages which begin gamogonic development. As a result of the multiplication process, the intercellular space of the epithelial cells in more than 10 neighbouring worm segments may become infected. At this time (60-90 days p.i.), pansporocysts with eight zygotes start the sporogonic phase. The final stage of this development is a pansporocyst containing eight folded triactinomyxon spores. Shortly afterwards, the spores are liberated into the gut lumen. The spores reach the water either by egestion or following the death of the infected tubificids. Infected tubificids can release triactinomyxons for at least 1 year. The ultrastructure of all four phases, schizogony, gametogony, gametogamy and sporogony, is demonstrated and discussed.

Present knowledge on the life cycle, taxonomy, pathology, and therapy of some Myxosporea spp. important for freshwater fish.

Abstract In this review some important myxosporean species of fish are depicted and described, with special reference to Myxobolus cerebralis (causing whirling disease of salmonids), Sphaerospora renicola (causing Swimbladder inflammation of common carp), Hoferellus carassii (causing kidney enlargement disease of goldfish and proliferative kidney disease [PKD] of salmonids). Detailed information on the life cycles of M. cerebralis, M. cotti, and M. pavlovskii is presented proving that these myxosporeans develop in an intermediate host, the oligochaete worm, Tubifex tubifex to actinosporeans which represent the only infective stage for susceptible fish. These findings imply that the present taxonomy for the phylum Myxozoa has to be revised. Trends for treatment of myxosporean associated diseases are summarized with special emphasis on the antibiotic Fumagillin DCH.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of viral hemorrhagic septicaemia virus (VHS).

Abstract A one step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detection of viral hemorrhagic septicaemia virus (VHS). A set of six primers were designed, based on the G-protein sequence of the VHS virus serotypes (He, F1, 23.75, Klapmolle and Rindsholm). The assay was optimised to amplify VHS RNA by incubation at 63°C for only 1h, and required only a simple water bath or heating block to provide a constant temperature of 63°C. RT-LAMP amplification products were detected by visual inspection using SYBR Green I stain and had a ladder-like appearance when electrophoresed on an agarose gel. The detection limit of the RT-LAMP assay was found to be similar to the commonly used RT-PCR method: both methods detected VHS RNA at a dilution of 106. The assay was evaluated using clinical samples and the results indicated the suitability and simplicity of the test as a rapid, field diagnostic tool for VHS virus.

Transmission of Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea) to Fredericella sultana (Bryozoa: Phylactolaemata) by various fish species

Tetracapsuloides bryosalmonae is a myxozoan parasite of salmonids and freshwater bryozoans, which causes proliferative kidney disease (PKD) in the fish host. To test which fish species are able to transmit T. bryosalmonae to bryozoans, an infection experiment was conducted with 5 PKD-sensitive fish species from different genera. Rainbow trout Oncorhynchus mykiss, brown trout Salmo trutta, brook trout Salvelinus fontinalis, grayling Thymallus thymallus and northern pike Esox lucius were cohabitated with T. bryosalmonae-infected Fredericella sultana colonies and then subsequently cohabitated with statoblast-reared parasite free Bryozoa. Statoblasts from infected colonies were tested by PCR to detect cryptic stages of T. bryosalmonae, which may indicate vertical transmission of the parasite. In this study, brown trout and brook trout were able to infect Bryozoa, while there was no evidence that rainbow trout and grayling were able to do so. Few interstitial kidney stages of the parasite were detected by immunohistochemistry in brown trout and brook trout, while rainbow trout and grayling showed marked proliferation of renal interstitial tissue and macrophages with numerous parasite cells. Intraluminal stages in the kidney tubules were only detected in brown trout and rainbow trout. In contrast to previous observations, pike was not susceptible to PKD in these trials according to the results of T. bryosalmonae-specific PCR. No DNA of T. bryosalmonae was detected in any statoblast.

An inexpensive and rapid diagnostic method of Koi Herpesvirus (KHV) infection by loop-mediated isothermal amplification

BackgroundKoi Herpesvirus (KHV) affects both juvenile and adult common carp and koi, and is especially lethal to fry. The high mortalities caused by the disease have had a negative impact on the international koi trade. Different diagnostic techniques have been used to detect KHV, including: isolation of the virus in cell culture, electron microscopy, several PCR tests, ELISA and in situ hybridisation. All of these methods are time consuming, laborious and require specialised equipment.ResultsA rapid field diagnosis of KHV in common and koi carp was developed using loop-mediated isothermal amplification (LAMP). The LAMP reaction rapidly amplified nucleic acid with high specificity and efficiency under isothermal conditions using a simple water bath. Two methods of extracting DNA from host tissue were compared: extraction by boiling and by using a commercial extraction kit. A set of six primers – two inner primers, two outer primers and two loop primers – was designed from a KHV amplicon. The reaction conditions were optimised for detection of KHV in 60 min at 65°C using Bst (Bacillus stearothermophilus) DNA polymerase. When visualised by gel electrophoresis, the products of the KHV LAMP assay appeared as a ladder pattern, with many bands of different sizes from 50 base-pairs (bp) up to the loading well. The KHV LAMP product could also be simply detected visually by adding SYBR Green I to the reaction tube and observing a colour change from orange to green. All samples positive for KHV by visual detection were confirmed positive by gel electrophoresis. The KHV LAMP had the same sensitivity as a standard PCR assay for the detection of KHV.ConclusionThis paper describes an accelerated LAMP assay for diagnosis of KHV. The entire procedure took only 90 minutes to produce a result: 15 minutes for DNA extraction; 60 min for the LAMP reaction; 2 min for visual detection using SYBR Green I. The test can be used under field conditions because the only equipment it requires is a water bath.

Comparison of 18S and ITS-1 rDNA sequences of selected geographic isolates of Myxobolus cerebralis.

Myxobolus cerebralis, the myxosporean parasite-causing salmonid whirling disease, was first reported among rainbow trout (Oncorhynchus mykiss) in Germany in 1903. The parasite was reported for the first time in North America in 1958 among hatchery-reared trout in the eastern USA, presumably arriving with frozen trout shipments from Europe. A comparison of 18S and ITS-1 ribosomal DNA sequences was conducted to identify potential strain differences between selected geographic isolates of this parasite from Europe and North America. Only fourteen of 1700 base pairs were different in the 18S rRNA gene from isolates obtained from California and West Virginia in the USA, and the Federal German Republic. No evidence for strain differences was obtained from ITS-1 sequences that were found to be identical among all parasite isolates. This finding is consistent with the hypothesis that the parasite was recently introduced to the USA from Europe.

Agent of whirling disease meets orphan worm: phylogenomic analyses firmly place Myxozoa in Cnidaria.

Myxozoa are microscopic obligate endoparasites with complex live cycles. Representatives are Myxobolus cerebralis, the causative agent of whirling disease in salmonids, and the enigmatic “orphan worm” Buddenbrockia plumatellae parasitizing in Bryozoa. Originally, Myxozoa were classified as protists, but later several metazoan characteristics were reported. However, their phylogenetic relationships remained doubtful. Some molecular phylogenetic analyses placed them as sister group to or even within Bilateria, whereas the possession of polar capsules that are similar to nematocysts of Cnidaria and of minicollagen genes suggest a close relationship between Myxozoa and Cnidaria. EST data of Buddenbrockia also indicated a cnidarian origin of Myxozoa, but were not sufficient to reject a closer relationship to bilaterians. Phylogenomic analyses of new genomic sequences of Myxobolus cerebralis firmly place Myxozoa as sister group to Medusozoa within Cnidaria. Based on the new dataset, the alternative hypothesis that Myxozoa form a clade with Bilateria can be rejected using topology tests. Sensitivity analyses indicate that this result is not affected by long branch attraction artifacts or compositional bias.

The role of tubificid worms as an intermediate host in the life cycle of Myxobolus pavlovskii (Akhmerov, 1954).

Myxobolus pavlovskii from the gills of silver carp (Hypophthalmichthys molitrix) were used in attempts to transmit the infection under laboratory conditions. Spores ofM. pavlovskii were placed in aquaria that contained sterilized sand and had been filled with tap water at 15–16°C. Furthermore, oligochaetes (90%Tubifex tubifex) were added and examined by wet mounts and histology,Hexactinomyxon spores developed after about 3 months only inT. tubifex that had been exposed toM. pavlovskii. Myxosporea-free silver carp developed cysts in their gills that containedM. pavlovskii at 120 days after contact withHexactinomyxon spores, proving thatM. pavlovskii also needs an oligochaete intermediate host for its development.