Early stages of E. leei were seldom found, and their detection at light microscope level is compromised by the difficulty of identification when using only morphological characteristics. In addition, myxosporean stages, differing morphologically from typical E. leei stages, were detected in epi-epithelial location in several fish. To get an accurate identification of the true E. leei stages and confirm the nature of suspicious early stages, a protocol or ISH detection was developed. The strategy used consisted of the design and synthesis of two DIG-labelled oligonucleotides and the optimization of conditions allowing the hybridization of the probes with specific complementary targets located in E. leei nuclear rDNA and cytoplasmic rRNA. Using in situ hybridisation (ISH), we could conclude that stages located on the mucosa epithelium surface of the intestine of some recipient fish do not belong to E. leei but to a different myxozoan species. Scarce early or suspicious E. leei stages were confirmed outside of the target organs using ISH, and usually in fish with confirmed infections in the intestine. Thus, putative extra-intestinal stages, responsible for the penetration in the fish and colonization of intestine would be very rare in these experimental infections, although they could be have more significance in spontaneous infections.
Humoral responses. A contrary pattern of variation in serum complement activity and serum peroxidase level in recipient (both levamisole treated un untreated) fish respect to control (both treated and untreated) fish was found, with complement values being lower and peroxidase values higher in recipient fish than in controls. No clear differences were detected in levamisole treated fish in respect to non treated fish. Cellular responses. The leucocyte respiratory burst was lower in recipient (both levamisole treated un untreated) fish than in control fish, whereas the phagocytosis and cytotoxic activities were generally higher in recipient than in untreated control fish. Unfortunately, infection levels obtained in the cohabitation experiment were very low. Although some changes in immune factors were observed, mainly in recipient and treated fish, no clear pattern in relation to treatment and/or infection could be established. Thus, the possible role of levamisole as preventive treatment for this enteromyxosis could not be determined.
Knowledge on the pathogenic effect of E. leei infection in Sparus aurata, including the histopathological damage
The histopathological study demonstrated a minor effect of the parasites in mildly infected guts, while in severe infections parasites occupied extended areas of the gut mucosa with subsequent disruption of both absorptive and osmoregulatory intestinal functions. A cachectic condition may appear in chronic infections, eventually leading to the death of affected fish. Mortality was not very high, and bacterial secondary infection may be involved in the final stages. The inflammatory response to E. leei was usually more evident in the pyloric caeca, and involved infiltration of lymphocytes, granulocytes, eosinophilic granular cells and macrophages (eventually having engulfed degenerated stages). An increase in the number of rodlet cells was also noticed. Foci of necrosis as well as an intense inflammatory reaction with haemorrhages and desquamation of the gut mucosa were sometimes observed in severely infected fish.
The following management measures are recommended to prevent and contain the disease: Sparus aurata - The first preventive measures must be addressed to avoid the entry of infective stages into the cultures: -- A strict screening of fish for enteromyxosis before introduction in the culture facilities is mandatory. The non lethal PCR assay (NL-PCR), not requiring the sacrifice of fish, is recommended to check the situation of a representative sample of suspicious stocks. -- This strict control measure should be applied particularly when fish are to be transported between different countries and facilities. The NL-PCR assay is a rapid and accurate method and by far the most reliable developed to date for this purpose. When the fish size does not allow NL-sampling (or more accurate results are necessary), lethal sampling is recommended. -- Water supply must be surveyed as it is an alternative access route of the parasite into the cultures. As the risk is especially high in pond facilities, the control of water quality is particularly recommended in these systems. As filtration is usually unaffordable, well water or sea intake sites from rocky bottom, as far from the coast as possible, are recommended. -- Sea cages must be placed in non risky sites, far from land, enzootic farms and avoiding mud bottoms and coastal zones with abundant susceptible wild fish which have been proven to act as reservoirs. - Subsequent measures deal with the adequate management to avoid the disease enhancement and spreading if infection is detected in the cultures. -- A periodic monitoring of fish stocks is necessary to detect the presence of the infection from the very beginning. The NL-PCR diagnostic test is the recommended diagnostic routine method (when the fish are very small or a more accurate diagnosis is needed, lethal PCR should be used). -- Sustained year-round temperatures over 20ºC should be avoided, mainly in the high-risk pond facilities. -- Necrophagy must be reduced to a minimum, through methodical culling of symptomatic fish and removal of dead fish. -- As prevalence generally increases with fish size, marketing of fish is preferably sooner in infected facilities. Diplodus puntazzo - All the general recommendations for gilthead sea bream S. aurata to avoid the entry and dispersion of the parasite are also applicable to sharpsnout sea bream D. puntazzo. The emphasis must be put in this case on surveillance of juveniles, as fish at the usual size of introduction into the sea-cages are especially susceptible to the disease.
The PCR assays for the diagnosis of Enteromyxum leei in gilthead se bream were developed and validated. High sensitivity and specificity were obtained for both lethal (L-PCR) and non-lethal (NL-PCR) tests. The superior performance of PCR assays respect to gold standard diagnosis is remarkable, especially for L-PCR on posterior intestine and NL-PCR. Considering these results, NL-PCR is recommended as routine diagnostic method to monitor the status of fish for enteromyxosis. The validation studies evidenced the difficulties of obtaining an accurate diagnosis using routine histology, which enhances the usefulness of PCR tests.
General view of the epidemiological situation of enteromyxosis in cultured sparids in the studied countries
A general view of the situation of enteromyxosis in cultured sparids in the studied countries was obtained in the epidemiological study. The utility of PCR assays, and particularly of the NL-PCR test to screen farm stocks for E. leei was demonstrated. In Sparus aurata, enteromyxosis was detected in the five countries, France, Greece, Italy, Spain and Israel, in geographical locations from North Atlantic (English Channel), South Atlantic (Canary Islands), Mediterranean (French, Greek, Spanish and Israel coasts) and Read Sea. Nine out of 12 sampled farms were found infected. Land-based systems had significantly higher infection levels and mortalities than sea cage farms. Temperatures over 20 ºC and poor water exchange may have contributed to enhance enteromyxosis in pond systems. In sea cages, prevalences were generally low, except in some Red Sea farms. Usually, a minimum exposure time to enzootic water is necessary for the infection to be detected. In Diplodus puntazzo (examined only in two Greek farms), the highest prevalences were observed in juvenile fish, in which high mortality also occurred, whereas most fish were negative at harvesting size.
Knowledge on the factors involved in the parasite transmission and course of infection in Sparus aurata
The direct fish-to-fish transmission of E. leei by the three assayed routes, cohabitation (CH), oral (OR) and contaminated effluent (EF) was confirmed in the present project, although differences in the onset and course of infection were observed. Several factors, such as parasite availability, entry route, differences in fish susceptibility and temperature could be involved. According to the obtained results, the main entry route in CH and EF transmission [which reproduce the mode of contagion in natural (spontaneous) infections], is probably the anus, although the oral port of entry might also play an important role, considering the success of OR infections. The rectum and pyloric caeca are the main target organs in CH and OR, respectively. Parasite stages were only found in the different parts of the intestinal tract. Other parts of the digestive (stomach, gall bladder), haematopoietic organs, liver and blood were never found infected. The earliest stage observed was a primary (P) cell harbouring one secondary (S) cell. As the infection progressed, parasite maturation also occurred, and proliferative and sporogonic stages were present in fish with mild or severe infections. Parasites were mainly found in small patches, in which initial stages were usually located in the periphery and more mature stages (sporoblasts and spores) in the central area. These observations were confirmed by ultrastructural studies.
Standardized procedure to maintain Enteromyxum leei in vivo, in Sparus aurata and alternative fish species
Enteromyxum leei infection was successfully maintained in experimental facilities, in Sparus aurata and other fish. The utility of some freshwater species susceptible to infection and easy to culture (mainly Puntius tetrazona, Astronotus ocellatus and Danio rerio) to maintain infection in laboratory was demonstrated. Differences in susceptibility between fish species were detected
In the epidemiological survey of wild fish, 10 sites in the Mediterranean (Spain, France, Italy and Israel), Ionian (Greece) and Red Sea (Israel) were sampled. Amongst 2260 fish examined, 55 specimens belonging to eight families (Apogonidae, Carangidae, Chaetodontidae, Lethrinidae, Mugilidae, Pomacentridae, Siganidae, Sparidae) were positive in the PCR-L assay. Parasitized fish belonged mainly to the familes Mugilidae (Mediterranean, Italy and Israel), Siganidae and Carangidae (Mediterranean and Red Sea, Israel). The high proportion of PCR-positive individuals caught at enzootic farms as compared to the other sites indicated that close proximity of wild fish to infected farm stocks created feral infection reservoirs.
Only 5 out of 350 invertebrate samples (France, Israel, Greece) yielded a positive result in the PCR assay. All of the PCR-positive organisms were collected from sites in the neighbourhood of enzootic farms and belonged to Anthozoa, Polychaeta and Crustacea. Thus, some invertebrates from the neighbourhoods of enzootic farms were positive for E. leei. Therefore, although the involvement of an intermediate invertebrate host cannot be ruled out, there is no direct evidence supporting the presence of an actinosporean stage, and the scarcity of positive samples points to a limited significance of these possible intermediate hosts in the culture systems.
A protocol for isolation and purification of spores was specifically developed by IATS, and implemented at IOLR. However, the paucity of spores in the Israeli donor fish made it necessary to modify the method, using a potassium dichromate treatment to allow for obtaining samples containing viable spores in which proliferative stages had been destroyed. In a specifically designed experiment, it was demonstrated that infection could not be transmitted by purified spores, whereas several fish became infected after oral administration of mucosal scrapings containing different parasite stages. These results provide additional support to the hypothesis that the direct fish-to fish transmission occurs through vegetative stages and that spores are not involved in the process.
Knowledge on the innate and adaptative immune factors involved in the response of Sparus aurata to enteromyxosis
Innate immune factors. Cellular response. In head kidney (HK), the percentage of acidophilic granulocytes, and the leucocyte peroxidases content, phagocytosis and respiratory burst activity decreased significantly at different sampling times, whereas the cytotoxic activity was greatly enhanced (up to 2.3 times) in recipient respect to control fish. In donor fish, peroxidase content was higher and respiratory burst was significantly lower in the gut associated lymphoid tissue (GALT) than in the HK leucocytes. Cytotoxic activity was higher in HK than in GALT leucocytes, and also higher in parasitized versus non-parasitized fish.Humoral factors. Alternative complement activity and peroxidase content in serum were lower, higher or similar in recipient fish respect to controls depending on the sampling time. At day 10 p.e., recipient fish showed a higher peroxidase content and a lower complement activity than those of controls. Cytokine expression. A contrary effect of the exposure to the parasite on pro-inflammatory cytokine mRNA expression in HK was demonstrated. Il-1b expression was enhanced and TNFa depleted in recipient fish respect to control fish. Adaptative immune factors. Immunoassays were applied to determine the presence of specific anti-E. leei antibodies in the sera of gilthead sea bream exposed to infection. The presence of specific antibodies was finally analysed using immunohistochemistry (IHQ), as the developed ELISA assay lacked specificity. The presence of specific anti-E. leei antibodies was confirmed in the sera of infected fish and those which had overcome infections. In cohabitation experiments, positive fish were observed from 52 days post-exposure and onwards. The percentage of positive fish was slightly higher in exposed levamisole-treated (RT) than in exposed untreated (RC) fish. Specific antibodies were more frequently found in non-parasitized than in parasitized fish. In conclusion, several immune factors of gilthead sea bream are affected by enteromyxosis, and both innate and adaptative immunity are involved in the response to infection. Leucocyte cytotoxic activity might play an important role, whereas specific antibodies were detected after a minimum of about 50 days post-exposure. The observed immune response in this fish host could contribute to keep the infection controlled.
Knowledge on the course of infection, histopathology and innate immune response in Diplodus puntazzo
In cohabitation (CH) transmission, enteromyxosis was detected earlier and with higher infection levels in sharpsnout sea bream (Diplodus puntazzo) than in gilthead sea bream (Sparus aurata). Different parasite stages were found in the gut epithelium, but not in kidney or spleen. Increased infiltration of eosinophilic granular cells (EGC), lymphocytes and macrophages in the intestinal submucosa was observed in infected fish. Infections progressed faster and with higher pathological manifestations in the D. puntazzo than in S. aurata, which is in line with previous observations in field and epidemiological studies. Amongst the three humoral factors studied, peroxidase content, and antiproteases and antitumoral activities, only peroxidase content increased in response to infection in all exposed fish at all sampling points, when compared to controls. Antiproteases activity was higher in recipient fish than in controls only on days 12 and 19 p.e., whereas variations in antitumoral activity were slight. Cellular response was evaluated in histological sections, which demonstrated a remarkable abundance of eosinophilic granular cells.