Diagnosis, epidemiology and control of an enteric myxosporosis of commercial Mediterranean fish MyxFishControl

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1 Diagnosis, epidemiology and control of an enteric myxosporosis of commercial Mediterranean fish MyxFishControl (QLRT ) October 2002-December 2005 QoL Strategies to identify and control infectious diseases Overall aim: Prevention and control of Enteromyxum leei infections Enteromyxum leei (formerly Myxidium leei) background Virulent myxosporean parasite Invades the intestinal tract and produces severe chronic enteritis due to epithelial destruction, extensive necrosis of the gut Disease signs: anorexia, weight loss, anaemia, emaciation (knife-like fish), mortality 1

2 More about E. leei Wide host range: cultured and wild Sparidae, Mugílidae, Labridae, etc. (>25 species). Wide geographical distribution :Greece, Turkey, Malta, France, Italy, Israel, Spain High economic impact in maricultured and aquarium fish: Epizootics (100 % mortalities in some stocks): on-growing animals The disease is not included in Council Directive 91/67 EEC lists Direct fish-to-fish transmission Unknown life cycle Proliferation: temperature-dependant No efficacious treatment were available Preventive measures very important No efficacious diagnostic tools were available Instituto de Acuicultura de Torre de la Sal (CSIC) Dr. P. Alvarez-Pellitero Universitat Autònoma de Barcelona (UAB) Dr. S. Crespo Universidad de Murcia (UMU) Dr. J. Meseguer Université Montpellier II (USTL) Dr. A. Marques University of Thessalia (UTH) Dr. F. Athanassopoulou Istituto Zooprofilattico Sperimentale dell Umbria e delle Marche (IZSUM) Dr. C. Ghittino National Center for Mariculture (NCM) Dr. A. Diamant Several private farms & consulting companies collaborate in samplings and have expressed their interest as end-users of the project results 2

3 OBJECTIVES 1. Validation of a PCR-based assay for the specific diagnosis of the parasite, including non-lethal sampling methods (WP2) 2. Application of this PCR test in field epidemiological studies (WP3) 3. Use of this PCR test to evaluate the role of wild fish and invertebrates as reservoirs of the disease (WP3) 4. Study of the transmission, pathogenesis and infectious process in sparid fish, in the course of experimental infections (WP4) 5. Study of relevant aspects of the innate and adaptive immune responses to the disease. Application of immunoprophylactic measures (WP5) 3

4 PCR diagnosis of E. leei Specific primers targeting SSU rdna gene Optimised reaction parameters Straightforward sampling and DNA preparation methods Objective 1:Validation of a PCR-based diagnostic test for the specific diagnosis of the parasite, including non-lethal sampling (WP2) Fish Sampling: Non-lethal swabs (PCR NL) Small pieces of intestine Entire digestive for histology Blinding of samples: random mixing in lots Procesing and analyses of the samples in a blind fashion. PCR of samples Routine microscopy (two labs) In-depth histopathological study (Goldstandard by consensus of two labs) Validated test(s) 4

5 Objective 1:Validation of a PCR-based diagnostic test (WP2) PCR assays have been definitively validated, with excellent accuracy respect to gold standard diagnosis Remarkable good performance of PCR NL (advisable for routine diagnosis) D2: Validated PCR test for the diagnosis of E. leei infections in fish (Manuscript in press) M2: Availability of reliable PCR diagnostics for further studies Objectives 2 & 3: Application of PCR test for field epidemiological studies in the participating countries (WP3) Survey of sparid cultures in the different countries with regard to E. leei. Sampling at two points during the production cycle of fish stocks, and several additional samplings. Study of the role of wild fish inhabiting the neighbouring of sparid culture facilities as reservoirs of the parasite Study of the role of invertebrates in the facilities and bordering areas as feral infection reservoirs or true alternate hosts. ACTAGTACCGATG ACT--GTAC--GATG ACTGGTAC--GATG ACT--GTACCGTTG ACTGGTAC--GATG 5

6 Objectives 2 & 3: Application of PCR test for field epidemiological studies (WP3) Cultured fish (Sparus aurata and Diplodus puntazzo) Sea cages (8 farms) and land based facilities (3 farms) Positive fish in all countries and systems. Highest prevalence in land farms Wild fish Five sampling sites in Mediterranean, Ionian an Red seas More than 3600 specimens belonging to 34 families tested Positive fish in six families, mainly Mugilidae, Siganidae and Carangidae Invertebrates 25 positive samples out of 350 (Anthozoa, Polychaeta, Crustacea) Deliverables and milestones (Palenzuela et al Applicability of PCR screening for the monitoring Enteromuxum leei (Myxozoa) infection in the Mediterranean aquaculture: an epidemiologic survey in sparid facilities. In: Biotecjnologies for quality. S. Adams & J. A. Olafsen (Comp.). Aquaculture Europe, 2004, Barcelona, Spain, Oct Europ.Aquac.Soc. Spec. Publ. Nº 34, pp Other manuscripts on epidemiology are in preparation D4, M3: Reports on epidemiology of cultured fish D5, M4, M5: Reports on epidemiology of feral fish and invertebrates D6: Management recommendations based on epidemiological risk Applicability of tests The designed sampling methods are usable in field conditions. Results with non-lethal swabs comparable with those produced with tissues, but samples are obtained in an easier fashion and with higher throughput. Results are more reliable than routine histopathology in early infections, and typically equivalent to an in-depth examination of the entire intestine. Early diagnosis and management of infected stocks is so far the only approach to control spread and cronification of enteromyxosis. Increased specificity and sensitivity, speed, and cost of PCR testing allow epidemiological screening not affordable by other methods. 6

7 Objective 4: Study of the transmission, pathogenesis, host/parasite interactions and infectious process in sparid fish (WP4) Experimental infections of Sparus aurata and Diplodus puntazo. Spontaneous infections. Study of the host/parasite interactions, routes of invasion used by the parasite and pathogenesis (histology, histochemistry, electron microscopy, In situ hybridization- ISH) Deliverables and milestones D7: Protocol for obtaining an inoculum with viable spores free of proliferative stages D8, M6: Protocol for ISH hybridisation D9, M7: Report on the pathogenic effect, P/H interaction and course of infection Objective 4: Transmission, pathogenesis, H/P interactions and infectious process in sparid fish (WP4) D8, M6: ISH D9, M7: Histopathology 12 th EAFP Conf.: - P G. Albinyana et al. (2005). Enteromyxum leei experimental infections in Sparus aurata (Sparidae).: Histopathological observations - P M. Cuadrado et al. (2005). Studies on the ultrastructure of Enteromyxum leei (Myxozoa), an enteric parasite of gilthead sea bream (Sparus aurata L.) - P M. Cuadrado et al. (2005). Occurrence of an enigmatic coelozoic myxosporean in sea bream (Sparus aurata) intestine in the course of Enteromyxum leei experimental infections Other manuscripts on histopathology and infection process are in preparation 7

8 Objective 5: Knowledge of relevant aspects of the innate and adaptive immune responses to the disease, and potential immunomodulation (WP5) In experimentally infected gilthead sea bream: - Humoral innate response - Study and characterization of cellular responses - Specific antibodies in fish serum: ELISA, IHQ Comparative study of some immune parameters with experimentally infected sharpsnout sea bream (D. puntazo) Analysis of immune-related gene expression in fish exposed to the disease. Potential use of immunomodulator (levamisole) for the prevention of enteromyxosis. Objective 5: Summary of results, deliverables and milestones (provisional) (WP5) IL-1β mrna increased and TN-α gene expression decreased in HK of exposed fish respect to controls. No statistically significant effect on expression of HK IL-1β and TNF-α found in levamisole-treated fish. Data on characterisation of cellular and humoral immune response and comparison of S.aurata with D.puntazzo M8-M10: A. Cuesta et al. (2006). Gilthead sea bream (Sparus aurata L.) innate defence against the parasite Enteromyxum leei (Myxozoa). Parasitology, 132: ). A. Cuesta et al. (2006). Cell-mediated cytotoxicity is the main innate immune mechanism involved in the cellular defence og gilthead sea bream (Teleostei: Sparidae) against Enteromyxum leei (Myxozoa). Parasite Immunology (in press). Other manuscripts on immune response and levamisole effects are in preparation 8

9 Synergies with other Aquafunc Projects AQUAFIRST (CSIC coordinator: J. Pérez-Sánchez) Experimental model of sea bream parasite infection in which there is variable susceptibility to the disease with tools for monitoring the status of the fish nonlethally. Study of candidate genes expression in the course of infection by q-pcr Identification of new, immune-related genes by substractive RNA hybridization and generation of microarrays. Acknowledgements Partners of MyxFishControl Collaborating farms & consulting firms Partner s national supplementary funding 9