Klaus Knopf

Differences in susceptibility of the European eel (Anguilla anguilla) and the Japanese eel (Anguilla japonica) to the swim-bladder nematode Anguillicola crassus.

The swim-bladder nematode Anguillicola crassus originates from the Far East where it is a parasite of the Japanese eel (Anguilla japonica). After A. crassus was introduced to Europe, it became a predominant parasite of the European eel (Anguilla anguilla). A study performed with experimentally infected eels (98 days, 23 degrees C) revealed significant differences in the susceptibility of the two eel species to this parasite. The recovery rate of 30 administered infective A. crassus larvae (L3) from A. japonica was less than half of that from A. anguilla (33.2% and 13.8%, respectively). Almost 60% of the worms recovered from A. japonica were found as dead, encapsulated and necrotic larvae in the swimbladder wall. In contrast, no dead larvae were found in A. anguilla. Additionally, the development of the worms was shown to be significantly slower in A. japonica compared with A. anguilla. The lower survival rate of the worms, together with their slower development, resulted in a significantly lower adult worm burden (11 and 428 mg wet weight, respectively) and in a decreased reproductive success in A. japonica compared with A. anguilla. These results demonstrate that the original host, A. japonica, possesses more effective defence mechanisms against A. crassus than does the non-adapted host, A. anguilla.

Induction of stress by the swimbladder nematode Anguillicola crassus in European eels, Anguilla anguilla , after repeated experimental infection

The purpose of this study was to determine under laboratory conditions over a period of 311 days if infection with the nematode Anguillicola crassus induces stress in European eels (Anguilla anguilla), and stimulates the endocrine stress axis as measured by serum cortisol levels. Eels were experimentally infected with 3rd-stage larvae (L3) in different doses to simulate natural conditions with varying infection pressures. Blood samples were drawn from the caudal vein every 2 weeks and serum cortisol concentrations were determined by radioimmunoassay (RIA). The results showed that the application of L3 resulted in a significant increase in the cortisol levels. The period of time at which elevated cortisol values were observed was consistent with the time of larval development and the appearance of adult A. crassus. Thus, there is a stress response to the larval and young adult stages, but no chronic response to older adults. Therefore, it is likely that infection of eels under natural conditions especially with a high number of larvae may be a considerable stressor, in combination with different environmental factors like water temperature, pH, oxygen concentration, pollution and interindividual relationships.

Vaccination of eels (Anguilla japonica and Anguilla anguilla) against Anguillicola crassus with irradiated L3.

The original host of the swimbladder nematode Anguillicola crassus, the Japanese eel (Anguilla japonica) and the recently colonized European eel (Anguilla anguilla) were immunized with 40 irradiated (500 Gy) 3rd-stage larvae (L3) of this parasite and challenged with an infection of 40 normal L3. The immunization induced a significant reduction of the number of adult worms developing from the challenge infection in A. japonica, but not in A. anguilla. The induced resistance (calculated using the relation of the number of adult worms in immunized eels and in non-immunized control eels) in A. japonica was 87.3%+/-30.4%. Following a single infection, the percentage of adult worms found in A. japonica was lower as compared to A. anguilla, and the few adult worms were much smaller, revealing a lower susceptibility of A. japonica to A. crassus in comparison to A. anguilla. Both eel species developed an antibody response against A. crassus, but the level of antibody responses was not positively correlated with the protection against infection, suggesting that the antibody response is not a key element in resistance of eels against A. crassus. This study suggests that the original host of A. crassus is able to mount efficient protective immune responses against its parasite, whereas the newly acquired host seems to lack this ability.

Low frequency ultrasound and UV-C for elimination of pathogens in recirculating aquaculture systems

Low frequency ultrasound (LFUS) was evaluated as a novel disinfection technique within recirculating aquaculture systems both individually and combined with UV-C. Dose-dependent inactivation rates were determined for the total viable counts and model organisms representing different taxa of common fish parasites: the ciliate Paramecium sp., second larval stage (L2) of the nematode Anguillicola crassus and metanauplii of Artemia sp. Application of LFUS up to 19 kJ/L did not reduce the number of colony forming units (CFU), whilst UV-C irradiation was highly effective. Pre-treatment with LFUS reduced the mean size of suspended solids in aquaculture water and thus increased the germicidal effect of UV-C by up to 0.6 log units. LFUS was effective against the eukaryotic organisms, and the dose-dependent inactivation could be well described by functions of an exponential decay. However, the efficiency of LFUS differed greatly between species. A LFUS dose of 1.9 kJ/L (consumed energy) was sufficient to inactivate Artemia by 99%, but a ten times higher dose was necessary to inactivate 95% and 81% of Paramecium and Anguillicola larvae, respectively. In clear water, the energetic efficiency of UV-C (emitted by a low pressure lamp) against Paramecium and Anguillicola larvae was higher compared to LFUS, but LFUS was more efficient against Artemia. However, the efficiency of LFUS against ciliates or nematode larvae would be similar or even higher than UV-C in highly turbid water or if less efficient medium pressure lamps are used. This study shows that LFUS can be applied safely at energy densities that are effective against a wide range of parasites like ciliates, nematodes and crustaceans. The combination of LFUS and UV-C could provide an appropriate water treatment with regards to all relevant pathogens in recirculating aquaculture systems.

Enhanced Aeromonas salmonicida bacterin uptake and side effects caused by low frequency sonophoresis in rainbow trout (Oncorhynchus mykiss)

Low frequency sonophoresis (LFS) has been recognized as one of the most advanced technologies in transdermal delivery of substances, due to the modification of the stratum corneum lipid bilayer, in focal skin applications in mammals. Based on these findings, LFS has been suggested as a potential technology to be used for enhancement in immersion fish vaccination. In contrast to mammals where LFS is applied to discrete regions of the skin, in fish the whole individual needs to be exposed for practical purposes. The current study evaluated the impact of LFS at 37 kHz on the uptake of an Aeromonas salmonicida bacterin and side effects of the treatment in rainbow trout. Quantitative real time PCR (qPCR) and immunohistochemistry were used to examine the bacterin uptake into skin and gill tissue. Side effects were assessed by behavioural examination, histology and blood serum analysis. The sonication intensity of 171 mW/cm² was enough for increasing skin permeability, but caused heavy erratic swimming and gill haemorrhages. Sonication intensities as low as 105 mW/cm² did not modify skin permeability and enhanced the bacterin uptake into the gill tissue by factor 15 compared to conventional immersion. Following sonication, the gill permeability for the bacterin decreased after 20 min and 120 min by factor 3 and 2, respectively. However, during sonication, erratic swimming of the fish raised some concerns. Further reduction of the sonication intensity to 57 mW/cm² did not induce erratic swimming, and the bacterin uptake into the gill tissue was still increased by factor 3. In addition, a decreasing albumin-globulin ratio in the serum of the rainbow trout within 40 min revealed that LFS leads to an inflammatory response. Consequently, based on both increased bacterin uptake and the inflammatory response, low intensity LFS has the potential to enhance vaccine immunity without significant side effects.

The adjuvant effect of low frequency ultrasound when applied with an inactivated Aeromonas salmonicida vaccine to rainbow trout (Oncorhynchus mykiss)

Vaccine adjuvants are classified according to their properties of either inducing the persistence of antigens within the animal after immunisation and/or activation of the animals immune response. The adjuvant effect of low intensity low frequency sonophoresis (LFS) was tested in rainbow trout using an Aeromonas salmonicida bacterin vaccine administered by immersion vaccination using LFS at 37 kHz. The adjuvant effect obtained with LFS was compared with that of normal immersion or intraperitoneal injection vaccination. Quantitative PCR was used to measure bacterial DNA in vaccinated fish up to 35 days post-vaccination, while RT-qPCR was used to assess gene expression during the early and late immune response post-vaccination. Results showed that antigen uptake in the gills was significantly higher in the group exposed to low intensity LFS compared to the other two vaccination groups 15 min post-vaccination, but this initially high uptake did not persist over the rest of the experiment. In the kidney, by comparison, the vast majority of the samples analysed did not show the presence or persistence of the bacterin. Showing that the route of vaccine uptake using the A. salmonicida bacterin, does not influence the persistence of the bacterin in the gills or the kidney. On the other hand, LFS induced a higher inflammatory response and T-helper cell activation, characterized by a significant up-regulation of interleukin-8 (IL-8), IL-1ß and CD-4, respectively. The expression of Ig-M, Ig-T and Ig-D was up-regulated in gills (being significant for Ig-M), but not in the spleen and kidney of the sonicated group. Conversely, Ig-M was up-regulated in the spleen of the non-sonicated groups, but not in the sonicated group. This highlights the ability of ultrasound to enhance mucosal immunity. It remains to be established whether the up-regulation of Ig-M in gills would be sufficient to offer protection in fish infected with A. salmonicida.

Impact of trematode infections on periphyton grazing rates of freshwater snails

In freshwater ecosystems, snails can significantly influence the competition between primary producers through grazing of periphyton. This activity can potentially be modified by trematodes, a large group of parasites which mostly use molluscs as the first intermediate host. Available studies, however, show contradictory effects of trematodes on snail periphyton grazing. Here, we used four different freshwater snail–trematode systems to test whether a general pattern can be detected for the impact of trematode infections on snail periphyton grazing. In our experimental systems, mass-specific periphyton grazing rates of infected snails were higher, lower, or similar to rates of non-infected conspecifics, suggesting that no general pattern exists. The variation across studied snail–trematode systems may result from differences on how the parasite uses the resources of the snail and thus affects their energy budget. Trematode infections can significantly alter the grazing rate of snails, where, depending on the system, the mass-specific grazing rate can double or halve. This underlines both, the high ecological relevance of trematodes and the need for comprehensive studies at the species level to allow an integration of these parasite–host interactions into aquatic food web concepts.

The eye fluke Tylodelphys clavata affects prey detection and intraspecific competition of European perch (Perca fluviatilis)

Parasites that occupy the eyes of fish have the potential to affect visual perception and consequently alter the host’s behaviour, as these organs provide information about their surroundings. In an experimental study, the feeding behaviour of European perch (Perca fluviatilis) infested with the eye fluke Tylodelphys clavata was examined. The results showed that an individual’s ability to identify and approach food items was negatively affected by the infestation intensity of T. clavata. Additionally, the foraging success of an individual was reduced in competition with another, less heavily infested conspecific, when the same food resource was exploited. These alterations in the ability to locate food may have important consequences on the feeding strategy of the fish. Furthermore, the impaired feeding capability caused by T. clavata may also increase the predation risk as heavily infested fish need to spend more time foraging to attain a rate of food intake equivalent to less infested conspecifics.