Two, morphologically indistinguishable myxosporean species, Myxobolus elegans Kashkovsky, 1966 and M. hungaricus Jaczó, 1940 were differentiated using molecular biological methods. Polymerase chain reaction (PCR) with primers specific for the family Myxobolidae was used to amplify an approximately 1600 base pairs (bp) long fragment of the 18S ribosomal RNA gene. In restriction fragment length polymorphism (RFLP) study with HinfI, MspI and TaqI enzymes, the two parasite species were easily distinguishable. The genetic distinctness was also confirmed by the DNA sequence of their PCR products. Although M. elegans and M. hungaricus are morphologically very similar, based on the results of the PCR-RFLP and the DNA sequences, we concluded that they are valid species.
The genetic relatedness of two kidney-parasitic Sphaerospora species was studied. Although S. renicola, the causative agent of swimbladder inflammation of common carp fingerlings (Cyprinus carpio), and Sphaerospora sp. originating from goldfish (Carassius auratus auratus) were indistinguishable on the basis of spore morphology, they were found to be genetically different as their 18S rDNA sequences shared only 71.9% identical nucleotides. In the phylogenetic trees, Sphaerospora sp. from goldfish grouped with Myxidium truttae (AJ582061) within the clade of the coelozoic freshwater species. Sphaerospora renicola clustered with S. molnari (AF378345) within the group of myxosporeans histozoic in gills. The topology of the six Sphaerospora species on the phylogenetic trees implied that myxospore morphology does not correlate with the genetic relationships, and the genus seems to be polyphyletic.
Here, we experimentally studied the site preference of Myxobolus cerebralis, one of the most pathogenic myxozoan (Cnidaria, Myxozoa) fish parasites, which causes whirling disease in salmonids. Parasite invasion was examined in three fish species with various susceptibility levels: the type host brown trout, the highly susceptible rainbow trout, and the non-susceptible gibel carp, in which parasite spores do not develop. We investigated the first two hours of fish invasion, and measured the site preference of triactinomyxons (TAMs) during attachment and penetration of fish in three body parts (gills, fins, skin). Infection prevalence and intensity were estimated using a species-specific nested PCR, optimised in the present study. The highest infection prevalence was detected in the most susceptible fish species, rainbow trout. Interestingly, higher prevalence was observed in gibel carp than in the type host, brown trout (95.2% vs. 85.7%). Considering body locations, remarkable differences were detected in infection intensities. The highest intensity was observed in fins, whereas skin was the least infected body part in every fish species examined. Infection prevalence and intensity did not differ significantly among fish species. Thus, we confirmed that M. cerebralis TAMs cannot discern fish species. Furthermore, we proved experimentally that fish fin is significantly more attractive to fish-invading parasite TAMs than gills or skin.
By a broad-range PCR, we detected a novel herpesvirus (HV) in the specimen of a wels catfish (Silurus glanis) presenting disseminated, carp pox-like dermal lesions all over its body. The sequence analysis of the 463-bp PCR product from the viral DNA polymerase gene indicated the presence of a hitherto unknown virus, a putative member of the family Alloherpesviridae in the sample. Another PCR, targeting the terminase gene of fish HVs, provided an additional genomic fragment of over 1,000 bp. Surprisingly, the sequence of a co-amplified, off-target PCR product revealed its origin from a putative gene homologous to ORF87 and ORF45 of cyprinid HVs and anguillid herpesvirus 1 (AngHV-1), respectively. With specific primers, designed according to the genomic maps of the cyprinid and anguillid HVs, a genomic fragment of 15 kb was also amplified and sequenced by primer walking. In phylogeny inferences, based on several genes, the putative wels catfish HV clustered closest to various cyprinid HVs or to AngHV-1. The novel virus, named as silurid herpesvirus 2, represents a distinct species in the genus Cyprinivirus. However, its association with the skin disease remains unclear.
In in vivo infection trials, rainbow trout eyed eggs were exposed to three Saprolegnia parasitica (Oomycota) isolates, which differed in biological and genetic characteristics. Infection prevalence, hatching rate, hatching dynamics of fish eggs were quantified, and the study was complemented with histopathology and phylogenetic analyses. We experimentally detected intraspecific differences in the pathogenicity of S. parasitica on rainbow trout eggs. The isolate from rainbow trout eggs was the most virulent to eggs of the same host, whereas isolates from carp skin and fry did not cause as much damage to the eggs. Comparing the outcome of two experimental settings, we confirmed that invasion of fish eggs is more effective by hyphae growth than by the actively moving zoospores. In addition, our findings highlighted that S. parasitica isolates with 100% identical ITS DNA sequences, could differ significantly in virulence. These isolates can be clearly distinguished based on a 650-bp DNA fragment of the DNA-directed RNA polymerase II subunit (RPB2) gene.
One of the main obstacles in freshwater aquaculture is the parasitic ciliate Ichthyophthirius multifiliis (Ich), the causative agent of white spot disease. The use of immunostimulants as feed additives may be a promising approach to control Ich infection. In the present study, we tested the prophylactic effect of orally administered β-1,3/1,6-glucan and propolis extract E50 against Ich infection in common carp. In total, 122 fish were separated into three experimental groups fed with a control, 3% β-glucan and 1% propolis diet for 40 consecutive days, respectively. On day 40, 16 fish per group were individually exposed to Ich theronts and the number of trophonts was counted 5 days post exposure. Relative gene expression of interleukin 1-β (IL-1-β) in common carp liver was examined by qPCR. Compared to control, the mean infection intensity was lower in the β-glucan- and propolis-fed groups; however, the difference was not statistically significant. The relative expression of IL-1-β significantly decreased in the propolis-fed group at day 10. In the β-glucan-fed group, a significant IL-1-β decrease was detected at day 15 compared to control. Although the Ich infection intensity was slightly decreased in both treated groups, and IL-1-β was moderately down-regulated in the liver of common carp, our results suggest that the applied feeding regime is insufficient to prevent Ich outbreaks in common carp.