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M El-Matbouli

Publications and source records attributed to M El-Matbouli.

16 recordsLinked to original sources
PubMed2006

Molecular diagnostic methods for detection of Thelohania contejeani (Microsporidia), the causative agent of porcelain disease in crayfish.

Diagnosis of Thelohania contejeani in the crayfish Astacus astacus is currently based on observation of gross clinical signs--opaque appearance of the abdomen and whitish colouration of the musculature--and confirmed by microscopic examination of histological sections of muscle. We have developed 2 molecular diagnostic methods for sensitive and rapid detection of porcelain disease in its early stages: PCR and loop-mediated isothermal amplification (LAMP). The PCR test utilises a primer based on the T. contejeani small subunit ssu ribosomal RNA (ssu rRNA) gene and amplified parasite DNA with high specificity and a detection limit of 10(-5) dilution. The LAMP assay involves incubation of the target DNA with a set of 6 primers and Bst DNA polymerase for 60 min at 65 degrees C in a water bath or heating block, followed by visualisation of the reaction products with the SYBR Green I stain; sensitivity of visual detection with SYBR Green I is equivalent to that with agarose gel electrophoresis. The LAMP assay can detect T. contejeani DNA to a dilution of 10(-7). The LAMP assay is 100 times more sensitive than the PCR test and is the method we recommend as an alternative to traditional means of diagnosing T. contejeani.

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PubMed2005

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

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 degrees C for only 1h, and required only a simple water bath or heating block to provide a constant temperature of 63 degrees 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 10(6). 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.

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PubMed2005

Polar filament discharge of Myxobolus cerebralis actinospores is triggered by combined non-specific mechanical and chemical cues.

This study presents initial evidence for the requirement of both chemical and mechanical stimuli to discharge polar capsules of Myxobolus cerebralis actinospores, the causative agent of salmonid whirling disease. The obligate need for combined discharge triggers was concluded from data obtained in a before/after experimental set-up carried out with individual locally immobilized actinospores. Homogenized rainbow trout mucus as chemostimulus and tangency of the apical region of the spores to achieve mechanical stimulation were applied subsequently. The actinospores showed discharged polar filaments exclusively when mucus substrate application was followed by touching the polar capsule-bearing region, but not when either stimulus was offered solely to the same individuals. We measured filament discharge rates to mucus preparations in a microscopic assay using supplementary vibration stimuli to ensure mechanical excitation. The actinospores responded similarly to different frequencies, which suggested a touch-sensitive recognition mechanism. Discharge specificity for salmonid mucus could not be confirmed, as mucus of common carp and bream could trigger similar filament expulsion rates. To a lesser extent homogenized frog epidermis and bovine submaxillary mucin could also stimulate the attachment reaction. In contrast, mucus of a pulmonate freshwater snail elicited no response.

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PubMed2005

The life cycle of Henneguya nuesslini Schuberg & Schroder, 1905 (Myxozoa) involves a triactinomyxon-type actinospore.

The life cycle of the histozoic myxozoan parasite Henneguya nuesslini was investigated in two salmonid host species. Naive brown trout, Salmo trutta, and brook trout, Salvelinus fontinalis, were experimentally infected in two trials by triactinomyxon type actinospores from naturally infected Tubifex tubifex. In exposed common carp, Cyprinus carpio, no myxospore production was detected. The parasite formed cysts with mature myxospores in the connective tissue of the fish 102 days post-exposure. The morphology of both actinosporean and myxosporean stages was described by light microscopy and a 1417-bp fragment of the 18S rDNA gene was sequenced. Sequence analysis confirmed the absolute congruence of the two developmental stages and assisted in determining species identity. Host range, tissue specificity and myxospore measurements provided sufficiently distinctive features to confirm species validity and were thus crucial for identification. The triactinomyxon spores had 16 secondary germ cells, unique dimensions, a very opaque sporoplasm matrix and three conspicuously protruding, pyriform polar capsules. This is the first record of a Henneguya sp. life cycle with a triactinomyxon-type actinospore, which suggests a close relationship with the Myxobolus group and a polyphyletic origin of the genus Henneguya.

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PubMed2005

Development of a rapid assay for the diagnosis of Myxobolus cerebralis in fish and oligochaetes using loop-mediated isothermal amplification.

A loop-mediated isothermal amplification assay was developed for the rapid detection of Myxobolus cerebralis in both fish and oligochaete hosts. The assay was optimized to amplify parasitic DNA by incubation with Bst DNA polymerase and a set of six specially constructed primers at 65 degrees C for 60 min. The amplification products were detected visually using SYBR Green I dye which gave identical results to gel electrophoresis analysis. Parasite DNA was detected from infected oligochaetes, and from the anal fin, caudal fin, dorsal fin and operculum of clinically infected fish. This 'Myxo-LAMP' assay has a detection limit similar to that of a polymerase chain reaction assay (10(-6)), but is more rapid and only requires a water bath for amplification and is therefore practical for simple and rapid diagnosis of infected tissue.

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PubMed2002

Seasonal occurrence of actinosporeans (Myxozoa) and oligochaetes (Annelida) at a trout hatchery in Bavaria, Germany.

A systematic inventory of actinosporeans and oligochaetes conducted over 3 years at a trout fish farm in Bavaria, Germany, allowed the identification of 12 actinosporeans from five collective groups: four Triactinomyxon (Triactinomyxon nov. types 1-4), two Raabeia (Raabeia nov. types 1, 2), two Echinactinomyxon ( E. radiatum, Echinactinomyxon nov. type 1), two Aurantiactinomyxon ( A. pavinsis, Aurantiactinomyxon nov. type 1) and two Neoactinomyxum (Neoactinomyxum nov. types 1, 2). Nine forms are novel but can be placed within existing collective groups. All 12 forms were detected in the laboratory in aquarium water associated with farm sediment. However, only four of these could be linked with an oligochaete host. Three families of oligochaetes were identified from the sediment: Tubificidae, represented by eight species, Lumbriculidae with one species and Naididae with two species. Only tubificid oligochaetes were found to host actinosporeans.

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PubMed2002

Early developmental stages of two actinosporeans, Raabeia and Aurantiactinomyxon (Myxozoa), as detected by light and electron microscopy.

The development of actinosporeans in their oligochaete host proceeding pansporocyst formation is relatively well documented, however, phases preceding it are not as well known. The initial stages in the development of two actinosporeans, Raabeia type 1 of Oumouna et al. [Parasitol. Res. 2002] and Aurantiactinomyxon pavinsis (Ormières, 1968) Marquès [Languedoc, Universite des Sciences et Techniques, Dissertation, 1984] from schizogony to gametogony and sporogony are described. Both actinosporeans begin their development as multinucleate stages near the basal lamina of the oligochaete intestine. Proximal to these stages and between the host epithelium cells are uninucleate cells whose nuclei divide to produce binucleate cells. These divide mitotically to produce cells with four nuclei which then undergo plasmotomy to yield a tetracellular stage and the first phase in pansporocyst formation. From the uninucleate stage to the tetranucleate stage, the cell membrane of the parasite is associated closely via finger-like projections with the intestinal epithelial and glandular cells of the host.

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PubMed2000

Electron microscopic study of a new microsporean Microsporidium epithelialis sp. n. infecting Tubifex sp. (Oligochaeta).

The cytology of a new microsporean parasite Microsporidium epithelialis sp. n. from the intestinal epithelial cells of the freshwater oligochaete Tubifex sp. (Tubificidae) is described. The microsporean occurred together with an actinosporean of the genus Triactinomyxon, which was found between the epithelial cells. The merogonic and sporogonic stages (mature spores included) of the microsporean parasite are monokaryotic. An individual sporophorous vesicle surrounds each spore. The fixed and stained spore has an average dimension of 1.9-2.5 x 0.9-1.2 microm. The spores are oval with a characteristic surface layer, showing ornamentation-like projections, which are in close contact to the exospore. A short polar filament forming three to four coils traverses the polaroplast with two lamellar layers. The ultrastructure and other characteristic features of this microsporean parasite are distinct from those of the microsporean species described so far from oligochaetes.

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PubMed1999

Detection of early developmental stages of Myxobolus cerebralis in fish and tubificid oligochaete hosts by in situ hybridization.

The myxosporean and actinosporean spores of Myxobolus cerebralis develop through many stages in their respective hosts, salmonid fishes and a tubificid oligochaete. Using a modified, non-radioactive in situ hybridization protocol, the parasite, which exhibits radically different structural forms during its development in each host, could be specifically detected in paraffin-embedded tissues of both fish and oligochaetes. Our study aims to demonstrate the application of the technique for detection of early stages of M. cerebralis in both hosts.

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PubMed1999

Effect of water temperature on the development, release and survival of the triactinomyxon stage of Myxobolus cerebralis in its oligochaete host.

The development of the triactinomyxon stage of Myxobolus cerebralis and release of mature spores from Tubifex tubifex were shown to be temperature dependent. In the present work, the effect of temperature over a range of 5-30 degrees C on the development and release of the triactinomyxon stages of M. cerebralis was studied. Infected T. tubifex stopped releasing triactinomyxon spores 4 days after transfer from 15 degrees C to 25 degrees C or 30 degrees C. Transmission electron microscopic examinations of the tubificids held at 25 degrees C and 30 degrees C for 3 days showed that all developmental stages degenerated and transformed to electron-dense clusters between the gut epithelial cells of T. tubifex. In contrast, tubificid worms held at 5 degrees C and 10 degrees C examined at the same time were heavily infected with many early developmental stages of triactinomyxon. At 15 degrees C, the optimal temperature for development, maturing and mature stages of the parasite were evident. Infected T. tubifex transferred from 15 degrees C to 20 degrees C stopped producing triactinomyxon spores after 15 days. However, 15 days at 20 degrees C was not sufficient to destroy all developmental stages of the parasite. When the tubificid worms were returned to 15 degrees C, the one-cell stages and the binucleate-cell stages resumed normal growth. It was also demonstrated that T. tubifex cured of infection by holding at 30 degrees C for 3 weeks and shifted to 15 degrees C could be re-infected with M. cerebralis spores. The waterborne triactinomyxon spores of M. cerebralis did not appear to be as short-lived as previously reported. More than 60% of experimentally produced waterborne triactinomyxon spores survived and maintained their infectivity for rainbow trout for 15 days at water temperatures up to 15 degrees C. In natural aquatic systems, the triactinomyxon spores may survive and keep their infectivity for periods even longer than 15 days.

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PubMed1998

Determination of nuclear DNA concentration in cells of Myxobolus cerebralis and triactinomyxon spores, the causative agent of whirling disease.

Myxobolus cerebralis (Myxozoa: Myxosporea) has a complex two-host life cycle, which begins when waterborne triactinomyxon spores released from the infected oligochaete Tubifex tubifex contact a susceptible trout. Upon contact the triactinomyxon spores attach to the fish and release their sporoplasm cells into the epidermis. At approximately 50 days postinfection, sporogenesis begins, resulting in a large number of M. cerebralis spores in the cartilage of infected fish 6 weeks later. The spores of M. cerebralis can be released from infected fish only after the fish die or are eaten by predators. In both cases, spores released into the aquatic environment can be ingested by oligochaete worms of the species T. tubifex and then develop into the actinosporean triactinomyxon stage in the intestine within about 3 months. The triactinomyxon is the only stage infectious for salmonid fish. We determined the DNA concentration in sporoplasm cells, capsulogenic cells, and valvogenic cells of M. cerebralis spore stages from the trout and of triactinomyxon spore stages from T. tubifex. DNA was visualized using the DNA-specific fluorescent stain DAPI. Our results demonstrate that meiosis occurs only once in the developmental cycle of M. cerebralis in contrast to the previously published hypothesis. This takes place within the pansporocyst found in T. tubifex. Thereafter, the sporoplasm cells of the triactinomyxon spores in T. tubifex and M. cerebralis in trout are diploid.

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PubMed1998

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 parasite's 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.

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PubMedDate not supplied

Recent advances in our knowledge of the Myxozoa.

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.

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