Immunological and therapeutic strategies against salmonid cryptobiosis

Abstract

Salmonid cryptobiosis is caused by the haemoflagellate, Cryptobia salmositica. Clinical signs of the disease in salmon (Oncorhynchus spp.) include exophthalmia, general oedema, abdominal distension with ascites, anaemia, and anorexia. The disease-causing factor is a metalloprotease and the monoclonal antibody (mAb-001) against it is therapeutic. MAb-001 does not fix complement but agglutinates the parasite. Some brook charr, Salvelinus fontinalis cannot be infected (Cryptobia-resistant); this resistance is controlled by a dominant Mendelian locus and is inherited. In Cryptobia-resistant charr the pathogen is lysed via the Alternative Pathway of Complement Activation. However, some charr can be infected and they have high parasitaemias with no disease (Cryptobia-tolerant). In infected Cryptobia-tolerant charr the metalloprotease is neutralized by a natural antiprotease, alpha2 macroglobulin. Two vaccines have been developed. A single dose of the attenuated vaccine protects 100% of salmonids (juveniles and adults) for at least 24 months. Complement fixing antibody production and cell-mediated response in vaccinated fish rise significantly after challenge. Fish injected with the DNA vaccine initially have slight anaemias but they recover and have agglutinating antibodies. On challenge, DNA-vaccinated fish have lower parasitaemias, delayed peak parasitaemias and faster recoveries. Isometamidium chloride is therapeutic against the pathogen and its effectiveness is increased after conjugation to antibodies.

Figure 1

Cryptobia salmositica with red cell from an experimentally infected rainbow trout (reproduced from Woo [5]).

Figure 2

Exophthalmia in rainbow trout with an acute experimental cryptobiosis (reproduced from Woo [5]).

Figure 3

Positive antiglobulin reaction; red blood cells from an experimentally infected rainbow trout (reproduced from Woo [5]).

Figure 4

Purification of cysteine protease and metalloprotease from Cryptobia salmositica. Lane A: crude cell lysate; lane B: partially purified cysteine protease from a DEAE-agarose column; lane C: partially purified metalloprotease from a DEAE-agarose column; lane D: a single band of purified metalloprotease from a Sephacryl S-300 column; lane M: molecular markers (kDa) (reproduced from Zuo & Woo [18]).

Figure 5

In vitro proteolytic degradation of collagen type V by purified metalloprotease from Cryptobia salmositica. Lanes A–E: collagen incubated with metalloprotease for 0, 2, 4, 6, and 8 hours respectively; lane F: collagen incubated under same conditions as lanes A–E, but without metalloprotease (control); lane M: molecular markers (reproduced from Zuo and Woo [18]).

Figure 6

Immunoblot using mAb-001 on Cryptobia salmositica lysate. Lane A: mAb-001 after immunoabsorption with live parasites; lane B: mAb-001 without immunoabsorption; lane C: hybridoma culture medium. The numbers on the left are molecular mass (kDa) of protein standards.

Figure 7

Peritoneal macrophage in the ascites of an experimentally infected rainbow trout, Cryptobia salmositica in the process of being ingested (reproduced from Woo [7]).

Figure 8

Ultrastructural lesions in Cryptobia salmositica after in vitro exposure to isometamidium chloride. (a) Parasite kinetoplast (K) not exposed to the drug; (b) condensation of kinetoplast DNA after exposure to the drug; (c) vacuole (V) formation after drug exposure; (d) swelling of mitochondrial cristae (C) after drug exposure; (e) vacuole formation in cytoplasm after drug exposure (reproduced from Ardelli and Woo [44]).

Figure 9

Phase contrast and fluorescent microscopy of Cryptobia salmositica after exposure to isometamidium chloride. (a): Exposure to drug only, note accumulation of drug (in red) in the kinetoplast; (b): exposure to drug conjugated to polyclonal antibodies from a recovered fish, note that the drug (in red) is throughout the organism (reproduced from Ardelli and Woo [59]).