Analysis of Kudoa thyrsites (Myxozoa: Myxosporea) spore antigens using monoclonal antibodies

Abstract

A method employing Percoll™ gradient centrifugation was developed to purify Kudoa thyrsites spores from somatic muscle tissue of Atlantic salmon Salmo salar. Highly purified spores were then used to immunize inbred BALB/c mice for derivation of hybridomas secreting Kudoaspecific monoclonal antibodies (mAbs). Analysis of mAbs by immunofluorescence microscopy and flow cytometry showed that several were specific for antigens on the surface of K. thyrsites spores whereas other mAbs reacted with polar capsules or with polar filaments of spores of K. thyrsites, K. paniformis and K. crumena. Immunoblots on spore lysates using the surface-binding mAbs showed a broad band of 46 to > 220 kDa, whereas mAbs specific for antigens of polar capsules and polar filaments detected sharper bands of various molecular masses, depending on the Kudoa species. The dominant epitope of the K. thyrsites spore surface antigen was shown to be carbohydrate as determined by its sensitivity to treatment with anhydrous trifluoromethane sulfonic acid and by its resistance to treatment with Proteinase K. Immunofluorescence microscopy using the K. thyrsites-specific mAbs on isolated, intact, permeabilized plasmodia and on thin sections of somatic muscle tissue containing plasmodia revealed intense labeling of spores both within the spore-producing plasmodia and in the flesh of infected Atlantic salmon. As few as 100 spores were detected by immunoblotting, indicating that these mAbs have potential for use in developing a field-based diagnostic test.

Epitopes associated with mature spores not recognized on Kudoa thyrsites from recently infected Atlantic salmon smolts

“…The monoclonal antibodies recognized epitopes associated with the polar capsule and filament (1H2, 3E8) and with the spore surface (2F4, 4H2) (Chase et al 2001). Furthermore, the surface epitope recognized by mAb 4H2 is associated with an abundant carbohydrate (Chase et al 2001). Reactivity with the high-titer polyclonal antibody further suggested that in addition to those that are sequentially expressed, other epitopes are consistently expressed throughout development.…”

“…It is unlikely that the differences in antibody reactivity were the result of the loss or masking of the epitopes, since samples from both 600 and 1600°D infections were fixed and assayed in an identical manner. The monoclonal antibodies recognized epitopes associated with the polar capsule and filament (1H2, 3E8) and with the spore surface (2F4, 4H2) (Chase et al 2001). Furthermore, the surface epitope recognized by mAb 4H2 is associated with an abundant carbohydrate (Chase et al 2001).…”

“…Serial sections (5 µm) were alternately assayed with either ISH or IHC, allowing direct comparison between locations on sequential slides. We tested 4 monoclonal antibodies (mAb: 2F4, 4H2, 1H2 and 3E8; Chase et al 2001) and 1 polyclonal antibody (pAb) raised in rabbits against mature Kudoa thyrsites spores as described in Chase et al (2003). Tissue sections were deparaffinized and rehydrated in phosphate-buffered saline (PBS) (pH 7.4), plus 0.15% Tween-20 (PBS/T).…”

<i>Kudoa septempunctata</i>-Induced Gastroenteritis in Humans after Flounder Consumption in Japan: a Case-Controlled Study

“…The pellet potentially containing Kudoa spores was re-suspended in 1 mL of PBS, and cells were counted using a hemocytometer. Spores were also purified from the suspension using Percoll density gradient centrifugation, as described previously (15).…”

Guide to the identification of fish protozoan and metazoan parasites in stained tissue sections

“…Kudoa species infect marine fish and are histozoic, mostly intracellular in muscles; exceptionally they are coelozoic. Immunofluorescence procedures have been used for detection and identification of Kudoa species in tissue sections (Yokoyama et al 2000, Chase et al 2001.…”