Project
title / Immunology of PKD in fish- identification of protective antigens and host immunity
/ DEFRA
project code / FC1111

Department for Environment, Food and Rural Affairs CSG 15

Research and Development

Final Project Report

(Not to be used for LINK projects)

Two hard copies of this form should be returned to:
Research Policy and International Division, Final Reports Unit
DEFRA, Area 301
Cromwell House, Dean Stanley Street, London, SW1P 3JH.
An electronic version should be e-mailed to
Project title / Immunology of PKD in fish- identification of protective antigens and host immunity
DEFRA project code / FC1111
Contractor organisation and location / Institute of Aquaculture,
University of Stirling
Total DEFRA project costs / £ 205,060
Project start date / 01/10/01 / Project end date / 30/09/04
Executive summary (maximum 2 sides A4)
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CSG 15 (Rev. 6/02) 3

Project
title / Immunology of PKD in fish- identification of protective antigens and host immunity
/ DEFRA
project code / FC1111

The aims of the project were to develop in vitro and in vivo culture methods for the Myxozoan parasite Tetracapsuloides bryosalmonae, and to examine aspects of the humoral immune response. These aims were met during the project.

An in vivo culture was developed for the culture of T. bryosalmonae infected bryozoa. The diet was selected following an examination of the ability of bryozoa to ingest/ digest a panel of 82 species of algae and protozoa. The culture system developed relied on a specific diet composed of easily cultivable algae and protozoa as follows; 40% Cryptomonas ovata; 40% Synechoccus leopoliensis; 10% Colpidium striatum; 10% Pediastrum boryanum. It allowed for culture of infected bryozoa over a period of months.

Using this method, infected bryozoa collected from the field were cultured in a closed system. Novel observations were made on the development of the T. bryosalmonae infecting the bryozoa. Spore release was noted from bryozoa as was the development of the sac stages. A transmission experiment to rainbow trout determined that exposure to one spore was enough to result in clinical PKD in the fish, while small portions of infected bryozoa were capable of generating thousands of spores. Observations on the sister taxon to T. bryosalmonae, Buddenbrockia plumatellae were also made and developmental comparisons made between the two species.

In vitro culture of extrasporogonic T. bryosalmonae was attempted. Empirical studies were conducted on a wide range of culture media and conditions. From this an in vitro culture method was developed that resulted in the replication of parasites from anterior renal explants from fish with +1 kidney grade swelling. The culture media used was as follows; L-15, ITS supplement (1%), Pen/strep (0.5%), L-glutamine (1%), Glucose (0.3% w/v), BSA fraction V (0.1% w/v), Non essential amino acids (1%), Foetal calf sera (3%), Naïve trout sera (1%). The parasites formed clumps within this media and survived for several weeks until the explant became melanised and fibroblastic. This is the first time an in vitro technique has been developed for a Myxozoan parasite that encourages replication of the parasite an important step in developing sustainable cultures.

Purification of parasite stages was attempted using immunomagnetics with limited success. Lectins were investigated for their potential in purification studies. Through this work it was determined that the two lectins used for T. bryosalmonae diagnosis, GS I and SBA reacted with intra-cellular sugar residues. It was further determined that there are changes in the glycosylation of the parasite, particularly when phagocytes adhere to it and intimated the presence of a distinct blood form of the parasite. The most reliable method of purification of parasite remains centrifugation.

The effect of exposing spores to naïve and exposed fish mucus was investigated. No difference could be ascertained between mucus and the effect on sporoplasm release. This demonstrates that immunity to T. bryosalmonae is likely to have an immunological basis rather than through changes in mucosal composition and spore recognition.

Examination of the humoral antibody response of T. bryosalmonae indicated that fish produced antibodies to a wide range of antigens during the disease. There was inter-fish variation regarding which antigens were recognised. Sera antibody titres between fish with similar levels of renal swelling also had a wide level of variation, but were notably all raised when compared to control fish. Efforts to purify antigens resulted in the development of an immunomagnetic technique whereby sera antibodies were coupled to magnetic beads and used to extract antigens from homogenised kidney. The antigens thus collected were analysed by MALDI- TOF mass spectroscopy. Actin was found to be co-purified during the procedure but none of the other derived peptide fingerprints were identified from the databases suggesting that they are of parasite origin.

Two expression libraries were constructed from infected kidneys and probed using fish sera. These libraries were 5x105 and 1x106 in size respectively and therefore were considered representative. No colonies could be obtained from these libraries that reacted with immune fish sera. This was considered to be due to the dilution of the antibody immune response mentioned above and the use of HRP in antigen detection. Future probing of expression libraries with fish sera should use Biotin/Streptavidin amplification for detection. Fish sera should be pooled and the same pool used for the entirety of the colony selection.

Due to the lack of colonies from the expression libraries monoclonal antibodies to specific antigens could not be made. Passive immunisation trials however, determined that one available anti- T. bryosalmonae antibody (mAb B4) did have an effect on parasite replication. This effect was similar to that observed when immune fish sera was used. Expression of the mAb B4 antigen occurred during sporogenesis in the bryozoan and the fish host. The antibody also detected related antigens in a number of Myxozoan spp. indicating that it is conserved across some members of the Myxozoa. Western blotting, immunomagnetic separation and SDS PAGE demonstrated that the B4 antigen was of a molecular weight between 100 and 150kDa and was composed of at least 2 protein sub-units of 60 and 40kDa. Immunohistochemistry using mAb B4 and immune fish sera suggested that the antigen is recognised by the fishes humoral immune response.

As stated above there was a significant elevation in antibody titres during the clinical disease although wide inter fish variability was observed. The action of the sera antibodies was examined when added to the in vitro culture material and appeared to have an agglutinating effect. Sera lysozyme levels were also measured but no significant differences between the disease states could be identified.

During the project regular communication was maintained with other UK researchers examining PKD in the form of annual meetings and email correspondence. This allowed for collaboration and the sharing of ideas and experience. From this project there have been 27 research outputs to date.

CSG 15 (Rev. 6/02) 3

Project
title / Immunology of PKD in fish- identification of protective antigens and host immunity
/ DEFRA
project code / FC1111
Scientific report (maximum 20 sides A4)
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CSG 15 (Rev. 6/02) 3

Project
title / Immunology of PKD in fish- identification of protective antigens and host immunity
/ DEFRA
project code / FC1111

Background to project

Tetracapsuloides bryosalmonae is a Myxozoan parasite that causes Proliferative kidney disease (PKD) in salmonid fish. This disease is an important constraint on the rainbow trout industry and is estimated to cost this industry in excess of £1.8 million per annum. The effect of the disease on wild salmonid populations is unknown, however it is likely to have a significant impact. Usually 100% of fingerling fish on a farm site are infected with mortalities attributed to the disease of around 20%. There are currently no licensed treatments for the disease with farmers having to rely on husbandry measures to reduce stressors during the clinical disease

The life cycle of the parasite has not been completed. However, it is known that T. bryosalmonae infects a range of freshwater bryozoa (colonial filter-feeding invertebrates) that release spores into the river water. These spores transmit the parasite to the salmonid, where they migrate to the kidney and start to replicate. The replicating parasites (referred to as extrasporogonic stages) become the focus of an intense inflammatory reaction. The extrasporogonic stages endogenously form sporogonic stages and migrate to the renal tubules. Here the sporogonic stages enter the lumens of the tubules and in some species form spores. The role of these spores (if any) in the lifecycle is currently unknown. Fish that recover from the clinical disease are resistant to further exposure to the parasite. This acquired resistance and the high antibody titres associated with the disease have suggested that the resistance may be antibody mediated. The resistance also suggests that vaccination as a control strategy may be possible.

The disease is distinctly seasonal occurring during the summer months. It is believed that this is linked to seasonality of the bryozoan host with fish recovering faster during the cooler autumnal months.

Although there have been significant developments in our understanding of PKD much remains unknown. A major limitation has been the inability to obtain an experimental supply of parasite material throughout the year and the development of standardised methodologies.

The project

Project FC1111 had 8 main objectives. The project initially was to develop standardised methods for obtaining parasite material and then to examine the humoral immune response in more detail. The objectives are listed below.

1. To produce an in vivo culture method for T. bryosalmonae in bryozoa

2. To produce an in vitro culture method for T. bryosalmonae

3. To purify parasite stages

4. To examine effect of fish mucus on release of sporoplasms from spores

5. Examine expression of antigens by electrophoresis

6. Examine expression of antigens by cDNA expression libraries

7. To produce antibodies to immunologically important antigens

8. To examine aspects of the humoral response

These objectives were derived from a specific DEFRA call for examining culture methods for the parasite and examination of the humoral response. Progress to the objectives are detailed below.


Scientific progress with respect to the objectives.

Production of an in vivo culture method for T. bryosalmonae in bryozoa

Previous attempts at culturing bryozoa have relied on using a large aquarium containing goldfish and pond water that feeds smaller tanks containing bryozoa attached onto Petri dishes. This method, although effective, is not controlled and relies on a suitable site for obtaining pond water that is known parasite/ pathogen free and contains the right mix of protozoa/ algae for bryozoa maintenance.

Therefore, a new system was developed for culturing infected bryozoa collected from the wild in a controlled, regulated environment. For this a range of possible culture systems and putative bryozoa diets were examined. This work was performed in conjunction with Mr Charles McGurk at the Institute of Aquaculture.

Bryozoa (Plumatella sp.) were hatched onto Petri dishes and fed with a selected algae/ protozoan and the bryozoans ability to ingest it was assessed. To determine whether the algae/ protozoan had nutritional value to the bryozoan faecal pellets were collected, squashed under a coverslip and examined for whole or partially digested test organisms. In total 62 species of algae (including diatoms) and 20 species of protozoa were examined, obtained from a commercial supplier (Sciento, Manchester). From this work it was determined that bryozoa although ingesting most algal species could not digest many of them. Indeed some flagellate species (such as Chlamydomonas nivens) were observed swimming out of the pellets apparently unharmed. However, some algae appeared to be totally digested with little evidence of their occurrence in the pellets. Previous published examinations of bryozoa feeding had only examined faecal pellets and suggested that the identified (ie undigested) algae present in these pellets constituted the dietary needs of the organisms. Only one protozoa examined appeared to be detrimental to bryozoa, Actinophys sol. This species attached onto the tentacular crown (lophophore) of the bryozoan causing the tentacles to clump until eventually it was released. All protozoa that were ingested were digested including rotifers. Nematodes, accidentally introduced into one of the cultures were not found to be digested. These results suggest that far from having a cosmopolitan diet the dietary requirements of bryozoa are relatively restrictive. No one species of algae or protozoa was found to be suitable for culturing bryozoa. Instead a mixture of species has to be used. The following is a recommended diet for Plumatella spp.

40% Cryptomonas ovata,

40% Synechoccus leopoliensis,

10% Colpidium striatum

10% Pediastrum boryanum.

Synechoccus leopoliensis promoted growth in the bryozoa could be harmful if this culture was allowed to crash. Therefore, like all of the algaes, it is recommended to add them to the culture during the log phase of growth. Pediastrum boryanum was included in the diet even though it didn’t appear to be digested by the bryozoa. The motion of the bryozoa gut suggested effective digestion occurred with substantial mixing of the contents, therefore inclusion of this species was to aid in the effective breakdown of other species. Further species may be added to the diet including Haematococcus lacustris and Chlorogonium sp.. A wider diversity of food species appears to enhance growth.

Feeding experiments were also conducted on Fredericella sultana, however due to the erect colony form faecal pellets could not be examined. However, they were found to ingest the same types of algae and protozoa as the Plumatella sp. with few exceptions. The above mix of algae / protozoa was also found to be beneficial to this species.

Culture of bryozoa were initially conducted in sandwich boxes. However, this culture method is relatively intensive with media being changed daily so efforts were made to develop a less intensive system. The enhanced system involved a large lighted aquarium, containing algae being drip fed fresh algal media. Water from this tank was circulated to a small tank containing bryozoa attached to Petri dishes on a rack. The small tank was water-jacketed to maintain a constant temperature. The replacement of media in the system helped maintain the balance of algae and prevented overt blooming of a particular species. To prevent insect larvae from collected colonies overgrowing the system 50mm mesh was placed over the tanks and water inlets. Using this system it is possible to grow most types of bryozoa. However, Cristatella mucedo (an unusual motile species of bryozoa) is unable to grow using the diet/culture system and will require further dietary trials.