Antigenic and biochemical study of PKX, the myxosporean causative agent of proliferative kidney disease of salmonid fish

Abstract

This work aimed at developing techniques to enable further investigations on the epidemiology and immunology of proliferative kidney disease (PKD) and of its causative agent, PKX, a probable myxosporean parasite. An improved PKX cell concentration technique resulted in preparation of an ennched proliferative kidney cell (EPKC) antigen (Ag) that offered a suitable material for rabbit and mouse immunizations, electrophoretical analysis and antigenicity studies of parasitic materials. Rabbit sera and a panel of 11 monoclonal antibodies (MAbs) specific for PKX were obtained. They reacted in ELISA, immunofluorescence tests, immunohistochemistry, immunodot blot and Western blot with EPKC Ag, live or fixed parasitic cells and infected kidney extracts, depending on the Abs and i.mmunological test used. These MAbs were divided into 4 groups according to their reactivity in the above tests and led to the study of the possible localization of putatlve epitopes on parasite cells. At least 4 major parasitic proteins were visualized by SDS-PAGE electrophoresis and a fifth one, with a relative mobility of 13 kDa, was immunostained in Western blot by at least 2 MAbs and rabbit antiserum. The membrane fluorescence reactivity of the epitope suggested this protein was external and could be of immunological importance The antigenicity detected by histoimmunochemistry was that of primary cells and was apparently specific for PKX since the screening of kidney tissue of 9 nonsalmonid fish species harbouring developmental stages of 3 myxosporean genera remained negative. EPKC Ag was also antigenic for trout undergoing experimental PKD, thus providing evidence for a humoral response to this infection. Our investigations result in the establishment of a novel set of immunological probes suitable for monitoring the course of PKD, checking antigenicity of actinosporeans, purifying parasitic proteins and screening a PKX expression library.

Infection dynamics of two renal myxozoans in hatchery reared fry and juvenile Atlantic cod Gadus morhua L.

“…This coincides with our findings that only half of the 6-month-old cod presented G. atlantica blood stages and that blood stages had disappeared in 1+ cod. Resistance to re-infection, based on acquired immunity and the production of specific antibodies, has been reported for various other myxozoans, including T. bryosalmonae (Saulnier and de Kinkelin, 1996), Enteromyxum spp. (Munoz et al 2000(Munoz et al , 2007Sitja-Bobadilla et al 2004, Myxobolus cerebralis Hofer, 1903 (Hedrick et al 1998) and Myxobolus artus (Furuta et al 1993).…”

Fish immune response to Myxozoan parasites

“…However, nowadays, the presence of specific antibodies has been unambiguously reported in fish infected by the myxosporeans Myxobolus cerebralis (Hedrick et al, 1998), Myxobolus artus (Furuta et al, 1993), Tetracapsuloides bryosalmonae (Saulnier & Kinkelin, 1996), Ceratomyxa shasta (Bartholomew, 2001) and Enteromyxum scophthalmi (Sitjà-Bobadilla et al, 2004). The speed of antibody production is relatively low in comparison with available data for other fish immunized or vaccinated with different parasites.…”

Malacosporean-like spores in urine of rainbow trout react with antibody and DNA probes to Tetracapsuloides bryosalmonae

“…The number of spores observed at any one time was insufficient to conduct detailed electron microscopic studies. Both a monoclonal antibody and polyclonal antibodies prepared to purified T. bryosalmonae (Saulnier and de Kinkelin 1996) reacted with the extrasporogonic stages of T. bryosalmonae from the kidney of fish with PKD and the spores found in the urine of rainbow trout (Fig. 2).…”

“…Indirect fluorescent antibody tests on cytospin preparations from urine to detect T. bryosalmonae followed the protocol described by Saulnier and de Kinkelin (1996). Imprints made from the kidney tissues of rainbow trout infected with T. bryosalmonae that were fixed with acetone:alcohol were also subjected to the same staining procedure and served as known positive controls.…”

“…8) and polyclonal rabbit serum (3093 ep 98). Both antibodies were prepared in the Fish Pathology Laboratory at INRA, Jouy en Josas, France (Saulnier and de Kinkelin 1996). Slides with fixed preparations from the cytospin or with imprints were flooded with 10% normal goat serum in 0.01 M phosphate buffered saline pH 8 (PBS) for 30 min at 37°C as a blocking step.…”