Biomedical subjects

Mansour El-Matbouli

Publications and source records attributed to Mansour El-Matbouli.

14 recordsLinked to original sources

PubMed2006

Myxozoan parasites disseminated via oligochaete worms as live food for aquarium fishes: descriptions of aurantiactinomyxon and raabeia actinospore types.

A total of 7 samples of live freshwater oligochaetes (mixed species), sold as 'tubifex' worms as food for aquarium fishes, were purchased over a 1 yr period from several pet shops in Munich, Germany, and screened for parasitic infections of myxozoans. The water associated with 5 samples contained actinospores at the time of purchase; 6 samples subsequently released spores in the laboratory. In all, 12 types of actinospores (Myxozoa: Myxosporea) from 4 collective groups were released by the oligochaetes. In the current study we provide descriptions of 2 aurantiactinomyxons (Myxobolus intimus Zaika, 1965 and type 1 nov.) and 3 raabeias (type 1 and 2 nov., Raabeia type 1 of Oumouna et al., 2003); descriptions of the 5 triactinomyxon and 2 hexactinomyxon types have been published previously. We include both raabeia and echinactinomyxon types in differential diagnoses of our raabeia forms because a clear distinction between these groups no longer exists in the literature. Comparison of 18S rDNA sequence data revealed that 1 of the novel aurantiactinomyxons was Myxobolus intimus. The sale of worms hundreds of km away from their point of origin is a means of dissemination of myxozoan parasites.

Animal Feed↗

PubMed2006

The effect of cohabitation of Tubifex tubifex (Oligochaeta: Tubificidae) populations on infections to Myxobolus cerebralis (Myxozoa: Myxobolidae).

The competitive interactions between susceptible and resistant Tubifex tubifex (Oligochaeta: Tubificidae) exposed to Myxobolus cerebralis (Myxozoa: Myxobolidae) infections were investigated in two laboratory trials. Competition was assessed by the total parasite production over the course of the trials in mixed and pure cultures of M. cerebralis exposed worms, and by the genetic analyses of worms from the control and experimental groups at the beginning and end of the experiments. Mixed cultures of resistant and susceptible worms showed a 70% reduction in production of parasites released when compared with pure cultures of susceptible worms. In studies with laboratory and field-collected oligochaetes the mixed cultures at the end of the cohabitation experiments were dominated by resistant Tubifex from lineage V (HB strain) this strain of Tubifex has a competitive advantage over worms from other lineages. The results of this study suggest that certain species of Tubifex may be dead-end hosts to M. cerebralis by absorbing or inactivating the parasite and may also show greater survival compared to susceptible oligochaetes in certain whirling disease enzootic habitats.

Animals↗

PubMed2005

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

BACKGROUND: Koi 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. RESULTS: A 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 degrees 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. CONCLUSION: This 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.

Animals↗

PubMed2005

Characterisation of carbohydrate-binding sites in developmental stages of Myxobolus cerebralis.

Glycans and lectins (carbohydrate-binding molecules) form a mutual recognition system, which enables parasitic organisms to attach themselves to the host cells and/or take part in the migration of their developmental stages into the target tissue. The aim of the present study was to identify and characterise the potential binding activity of glycoconjugates in different developmental stages of Myxobolus cerebralis, the causative agent of whirling disease in salmonids. The binding patterns of 13 biotinylated neoglycoconjugates were histochemically examined in thin-sections of infected rainbow trout (Oncorhynchus mykiss) and oligochaetes (Tubifex tubifex), as well as isolated waterborne triactinomyxon spores. A distinct structure-selective and developmental stage-regulated expression of certain classes of carbohydrate binding was observed. In triactinomyxon spores, the expression of carbohydrate binding activity for alpha-l-Fuc-BSA-biotin, alpha-d-GalNAc-BSA-biotin, beta-d-GlcNAc-BSA-biotin, Lac-BSA-biotin and ASF-biotin was up-regulated in the polar capsules; the shell valves showed no activity. In the gut of T. tubifex, polar capsules of the parasite showed strong positive reaction only for beta-d-GlcNAc-BSA-biotin. In fish cartilage, polar capsules were negative, but the spore shell valves showed a broad range of carbohydrate binding activity. No activity was detected for either alpha6- or alpha3-linked N-acetyl-d-neuraminic acid to galactose. An adhesion assay was performed on GlycoWell plates and Myxobolus spores were found to specifically adhere to matrices containing residues of lactose, fucose, galactose, N-acetyl-d-galactosamine and N-acetyl-d-glucosamine. This is the first study to identify lectin activity in a myxozoan parasite; activity that is likely to play a role in the recognition systems involved in host specificity and the processes of spore attachment and invasion.

Animals↗

PubMed2005

Life cycle studies of Myxobolus parviformis sp. n. (Myxozoa: Myxobolidae) from bream.

We experimentally followed the life cycle of Myxobolus parviformis sp. n., a myxozoan parasite from the gills of common bream Abramis brama L. Establishing the development of both stages (myxospore and actinospore) in 2 consecutive, full transmission trials, we were able to separate plasmodia of a common genotype from sympatric Myxobolus spp. occurring in naturally infected gill lamellae. Therefore, isolated gill plasmodia representing individual myxosporean 'clones' were used for subsequent infection of oligochaetes after molecular and morphological identification. The plasmodia of this species are located in median to distal regions of the primary gill filaments, forming small spherical pseudocysts. The comparatively small myxospores share some uniform characteristics: they taper posteriorly, have 2 inward inclining polar capsules that occupy half of the spores' interior space, and usually show 4 posterior sutural edge markings. The corresponding actinosporean has already been described morphologically and molecularly. The 18S rDNA sequence of the actinosporean stage was identical in all our samples, including myxospores. The triactinomyxon had a stout style, 32 sporozoites and short tapering caudal processes, and was shed by the tubificid oligochaete Limnodrilus hoffmeisteri. The ellipsoid sporoplasm was covered by a soft sheath which was emitted after valve shell opening upon stimulation by agitation or fish mucus. The molecular data (unique restriction fragment length polymorphism pattern and a 1586 bp 18S rDNA sequence) clearly differ from those for similar species and, together with the morphological data, justify the description of this parasite as a new species.

Animals↗

PubMed2005

Lectin blot studies on proteins of Myxobolus cerebralis, the causative agent of whirling disease.

It is known that Myxobolus cerebralis antigens, both surficial and secreted, are key modulators for, or targets of, host immune system compounds. We undertook SDS-PAGE glycoprotein characterisation of M. cerebralis developmental stages isolated from infected rainbow trout and Western blot analyses using selected biotin-labelled plant lectins (GSA-I, PHA-E, SJA, GSA-II) and anti-triactinomyxon polyclonal antibodies. Glycoproteins were isolated with lectin-affinity chromatography, and prominent bands were characterised by matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI/MS). We identified glycoproteins of M. cerebralis myxospores that contained carbohydrate motifs reactive with Phaseolus vulgaris erythroagglutinin (proteins 20 to 209 kDa, PHA-E), Sophora japonica agglutinin (proteins 7 to 70 kDa, SJA), Griffonia simplicifolia Agglutinin I (proteins 10 to 209 kDa, GSA-I) and G. simplicifolia Agglutinin II (proteins 5 to 40 kDa, GSA-II). Mcgp33, a glycoprotein isolated by lectin-affinity chromatography, was reactive with SJA (about 33 kDa). Antiserum produced against M. cerebralis triactinomyxons was found to have differences in the antigenicity of isolated glycoproteins from both M. cerebralis myxospores and actinospores. We also demonstrated modified antigen expression, especially involving the glycoprotein Mcgp33, in different developmental stages of M. cerebralis.

Animals↗

PubMed2005

Rapid diagnosis of Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease (PKD) in salmonid fish by a novel DNA amplification method, loop-mediated isothermal amplification (LAMP).

A new molecular diagnostic assay was developed for detection of Tetracapsuloides bryosalmonae the causative agent of proliferative kidney disease (PKD) in salmonid fish using a loop-mediated isothermal amplification method (LAMP). The PKD-LAMP assay amplifies the T. bryosalmonae DNA extracted from infected kidney, under constant temperature of 65 degrees C within 1 h. The required equipment for DNA amplification is only a water bath. The amplification products were detected visually by using SYBR green I dye, which turns green in the presence of amplified products and remains orange in its absence, and by electrophoresis without any difference in the sensitivity of both methods. The developed PKD-LAMP assay demonstrated an exceptionally higher sensitivity than the conventional PCR. PKD-LAMP assay was found to be 100-fold more sensitive than the PCR assay. The developed assay is simple, rapid, cost-effective, specific and highly sensitive. The assay is also characterized by its field applicability, as it does not require the use of sophisticated equipment or skilled personnel.

Animals↗

PubMed2005

Dissemination of triactinomyxons (Myxozoa) via oligochaetes used as live food for aquarium fishes.

Freshwater 'tubifex' oligochaetes sold as live food for aquarium fishes were purchased from several pet shops in Munich, Germany, over a 1 yr period (March 2001 to February 2002). These samples were screened for parasitic infections of actinosporean myxozoans to gauge the possibility of parasite dispersal via this route. Of 7 samples, 6 contained infected oligochaetes; waterborne spores were present in 5 samples at the time of purchase. In the laboratory, 12 different types of actinosporeans were released by the oligochaetes. These could be assigned to 4 collective groups: triactinomyxon, aurantiactinomyxon, raabeia and hexactinomyxon; 4 novel triactinomyxons are described herein, a fifth triactinomyxon has been described earlier. Phenotypic descriptions of the spores are accompanied by molecular sequence data (18S rDNA). Descriptions of the other actinosporean types appear elsewhere. The worms sold as 'tubifex' originated from eastern European countries and were identified as a mix of Tubifex tubifex, Limnodrilus hoffmeisteri and L. udekemianus. Sale of live worms (and their accompanying parasite load) has clearly the potential to facilitate introduction both of parasites and suitable hosts to new areas.

Animals↗

PubMed2004

Myxobolus cerebralis internal transcribed spacer 1 (ITS-1) sequences support recent spread of the parasite to North America and within Europe.

Molecular approaches for resolving relationships among the Myxozoa have relied mainly on small subunit (SSU) ribosomal DNA (rDNA) sequence analysis. This region of the gene is generally used for higher phylogenetic studies, and the conservative nature of this gene may make it inadequate for intraspecific comparisons. Previous intraspecific studies of Myxobolus cerebralis based on molecular analyses reported that the sequence of SSU rDNA and the internal transcribed spacer (ITS) were highly conserved in representatives of the parasite from North America and Europe. Considering that the ITS is usually a more variable region than the SSU, we reanalyzed available sequences on GenBank and obtained sequences from other M. cerebralis representatives from the states of California and West Virginia in the USA and from Germany and Russia. With the exception of 7 base pairs, most of the sequence designated as ITS-1 in GenBank was a highly conserved portion of the rDNA near the 3-prime end of the SSU region. Nonetheless, the additional ITS-1 sequences obtained from the available geographic representatives were well conserved. It is unlikely that we would have observed virtually identical ITS-1 sequences between European and American M. cerebralis samples had it spread naturally over time, particularly when compared to the variation seen between isolates of another myxozoan (Kudoa thyrsites) that has most likely spread naturally. These data further support the hypothesis that the current distribution of M. cerebralis in North America is a result of recent introductions followed by dispersal via anthropogenic means, largely through the stocking of infected trout for sport fishing.

Animals↗

PubMed2004

Molecular methods clarify morphometric variation in triactinomyxon spores (Myxozoa) released from different oligochaete hosts.

Thirty-nine freshwater tubificid oligochaetes were isolated, each of which harboured a triactinomyxon infection. Spore characteristics include the typical triactinomyxon anchor shape, eight germ cells within the sporoplasm and three unequal (two long and one shorter) caudal processes with square tips. Despite morphological similarities between the spores from the different hosts, their morphometrical data varied considerably; significantly, the ranges of dimensions of the smallest and largest exemplars were mutually exclusive. In order to ascertain the true number of spore types present, molecular methods were employed. Samples of waterborne spores, including the smallest and largest representatives, were selected from 11 host oligochaetes (all Tubifex tubifex Müller) and a nested PCR-RFLP 'riboprint' analysis performed. The small subunit ribosomal DNA gene (18S rDNA) was targetted and amplified through two rounds of PCR, then digested with the restriction enzymes Dde I and Hha I. The resultant major cleavage patterns produced by both enzymes indicated a single triactinomyxon form; however, the pattern of several less intense bands varied between the samples. From a subset of five samples drawn from across the full spectrum of spore sizes, a 327 bp region near the 5' was sequenced and was identical for all five samples. Comparison of this 327 bp region with that of 12 other triactinomyxons in GenBank showed 68.7-96.9% similarity (at least 9 base differences). A further 469 bp generated for each of the smallest, largest and mid-range (= reference) spore samples was identical also. The reference sample was sequenced further to yield 1,554 bp of 18S rDNA (GenBank accession number AY162270); comparison with other Myxozoa indicated this sequence was novel. The morphometrics of our triactinomyxon did not correlate with any published description. The morphometrical variation exhibited by spores of the triactinomyxon type in this study raises questions about the validity of using morphometrical data to distinguish spore types and suggests that there could be taxonomic redundancy in the diversity of actinosporeans recorded in the literature. The additional information provided by molecular data in this study was pivotal in the clarification of morphometrical variation exhibited by morphologically similar triactinomyxon spores released from different oligochaete hosts.

Animals↗

PubMed2004

Extensive release of an antigen associated with the sporogonic stages of myxobolus cerebralis (Myxozoa: Myxosporea) is detected by a heterologous antibody raised to Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea).

Monoclonal antibody B4 (mAb B4) was previously developed to the myxozoan parasite Tetracapsuloides bryosalmonae Canning, Curry, Feist, Longshaw et Okamura, 1999, the causative agent of proliferative kidney disease of salmonids, Here we describe the reaction of mAb B4 against Myxobolus cerebralis Hofer, 1903, the parasite that causes 'whirling disease' in salmonids. Tissues examined were collected from experimentally infected rainbow trout Oncorhynchus mykiss (Walbaum) and the aquatic oligochaete Tubifex tubifex (O.F. Müller), the two hosts involved in the life cycle of M. cerebralis. Paraffin sections of infected rainbow trout taken at 4 h and 3, 10, 17 and 54 days post-exposure to infective M. cerebralis actinospores were immunohistochemically stained with mAb B4. Longitudinal sections through infected T. tubifex sampled 120 days post-exposure to M. cerebralis myxospores were also examined using this method. The only phase of the M. cerebralis life cycle that expressed the mAb B4 antigen was during sporogenesis in the salmonid host. The immunohistochemical staining demonstrated that the antigen was released into the tissues surrounding the spore and sporogonic stages of the parasite. The localisation of the antigen was diffuse in the fish, suggesting that the possible effect of M. cerebralis infection is extensive through the head tissues and not limited to areas of cartilage destruction as previously thought.

Animals↗

PubMed2003

Susceptibility of two strains of rainbow trout (one with suspected resistance to whirling disease) to Myxobolus cerebralis infection.

The susceptibility of 2 strains of rainbow trout Oncorhynchus mykiss, 1 from North America (TL) and 1 from Germany (GR), to Myxobolus cerebralis (the cause of salmonid whirling disease) was assessed following exposure to the infectious stages (triactinomyxons). Two laboratory experiments were conducted with age-matched rainbow trout of each strain. At the beginning of the study, the 2 trout strains were aged ca. 570 degree-days in Expt 1, and ca. 999 degree-days in Expt 2. In both experiments, replicate groups of each trout strain were exposed to 10, 100, 1000 or 10000 triactinomyxons (TAMs) fish(-1) for 2 h. The fish were then held in aquaria receiving 15 degrees C well-water. Severity of infection was evaluated 5 mo after exposure by presence of clinical signs (whirling and/or black tail), prevalence of infection, severity of microscopic lesions, and spore counts. Clinical signs of whirling disease were evident only in the younger fish exposed in Expt 1: These occurred first among TL rainbow trout at the highest dose at 6 to 7 wk post exposure and then 2 wk later in fish at the 1000 TAMs dose. Black tail was also observed among GR rainbow trout at the 10000 TAMs dose only, but in fewer fish. The prevalence of infection, spore numbers, and severity of microscopic lesions due to M. cerebralis among GR rainbow trout were less at all doses compared to TL rainbow trout. Risk of infection analyses showed that TL rainbow trout were more prone to infection at the lower doses than GR trout. Mean spore counts were consistently (10- to 100-fold) less in GR than TL trout at doses of 1000 TAMs or lower. Microscopic lesions increased with increasing dose in both strains of rainbow trout. The mechanisms underlying the greater resistance of the GR strain to M. cerebralis infections are unknown, but are under investigation as part of a long-term project to determine the basis for resistance and susceptibility of salmonid fishes to whirling disease.

Age Factors↗

PubMed2003

Characterisation of two novel types of hexactinomyxon spores (Myxozoa) with subsidiary protrusions on their caudal processes.

Two types of hexactinomyxon spores, Hexactinomyxon type 1 nov. and Hexactinomyxon type 2 nov., are reported from freshwater tubificid oligochaetes, Limnodrilus hoffmeisteri and L. udekemianus. Spores are triradially symmetrical and comprise a spore body, style and 6 caudal processes. The caudal processes arise from the division of each of the 3 valve cells into an equal pair of projections at the base of the style. One of each pair is fused conspicuously to its nearest neighbour for the initial 1/5 to 1/4 of their total length. Distally, each process possesses subsidiary protrusions which are irregularly distributed and irregularly shaped extensions of the valve cell. Scanning electron microscopy of Hexactinomyxon type 2 nov. revealed that these protrusions are a seamless extension of the valve cell wall which branch distally, occasionally laterally, and terminate in a distinct bulbous structure; they also form the terminus of each process. The small subunit ribosomal DNA gene (18S) of both hexactinomyxon types was amplified through a nested PCR, then digested with the restriction enzymes Dde I and Hha I. The resultant cleavage patterns suggested the presence of 2 forms. Subsequent partial sequencing of 18S rDNA confirmed the identification of 2 novel types.

Animals↗

PubMed2002

Prevalence and susceptibility of infection to Myxobolus cerebralis, and genetic differences among populations of Tubifex tubifex.

The prevalence of infection and susceptibility of the aquatic oligochaete Tubifex tubifex to Myxobolus cerebralis, was examined in 2 studies on the upper Colorado River, Colorado, USA, where whirling disease occurs in wild trout populations. In the first study, the prevalence of infection ranged from 0.4 to 1.5%, as determined by counting the number of T. tubifex releasing triactinomyxons of M. cerebralis directly following their collection from the field. The susceptibility of those T. tubifex not releasing triactinomyxons was assessed by the number of these oligochaetes releasing triactinomyxons 3 mo following experimental exposures to spores of M. cerebralis. The prevalence of infection following experimental exposures of these T. tubifex ranged from 4.2 to 14.1%. In a second study, all T. tubifex collected at 2 different times directly from the 2 field sites in Colorado were exposed to spores of M. cerebralis. Individual oligochaetes representing those groups of T. tubifex releasing and those groups not releasing triactinomyxons at 3 mo were screened with molecular genetic markers. T. tubifex populations found at the 2 study sites consisted of 4 genetically distinct lineages that varied with respect to their susceptibility to experimental exposure to M. cerebralis. Lineages I and III contained the most oligochaetes susceptible to M. cerebralis and were the most prominent lineages at Windy Gap Reservoir, a site of high infectivity for wild rainbow trout on the upper Colorado River. In contrast, at the Breeze Bridge site which is below Windy Gap Reservoir and where M. cerebralis infections are less severe in wild trout, oligochaetes in lineages V and VI that are resistant to M. cerebralis were more prominent. These results suggest that certain habitats, such as Windy Gap Reservoir, are conducive to large and more homogenous populations of susceptible T. tubifex lineages that may serve as point sources of infection for M. cerebralis. Although not a direct objective of this study, there was no evidence of M. cerebralis infections among any oligochaetes other than those that would be classified as T. tubifex by standard morphological characteristics.

Animals↗