Diplomonad flagellates are enteric and/or systemic parasites of shellfish, finfish, amphibians, and reptiles. In aquaculture and aquaria, diplomonads can cause acute and chronic loss of stock, especially in salmonids and cichlids. Metronidazole treatment is effective against the swimming trophozoite stage in the vertebrate host, but is not permitted in some countries. Diplomonads have a direct life cycle with pyriform trophozoites, 10-15 µm long with 8 flagella, and resistant cysts. Trophozoites are easily recognized in the intestinal lumen, cysts are only rarely reported from the posterior intestine. Cysts are presumed to be responsible for direct transmission through the water to another host by oral ingestion. No treatment targets the cysts in the water. The transmission of cysts and adaptions to enhance infectivity remain little known in piscine diplomonads, in contrast to avian and mammalian diplomonads.
We hypothesise that piscine diplomonads have adaptions for transforming, from trophozoites in fish to cysts in water, that ensure the transmission of infection; such adaptions can be new targets for management and treatment.
We established a primary culture of S. salmonis by inoculating contents from the pyloric region from 1-3 heavily infected juvenile rainbow trout Oncorhynchus mykiss into 45 ml of Minimum Essential Media (MEM) supplemented with 10% newborn calf serum and antibiotics (penicillin, gentamycin, nystatin) in a 50 ml culture flask. After 4 days, cell density had doubled, and a 1:1 dilution subculture was made. All cultures were incubated at 10°C in the dark. After 4 days in subculture, we observed novel stages of attachment and colony formation preceding encystment, and cultures were fixed in 3% glutaraldehyde to facilitate photography, and scanning and transmission electron microscopy investigation.
Trophozoites attached by the tip of their posterior flagella to food debris and/or to the posterior flagella of other trophozoites, thus the posterior flagella tip had become adhesive. Attaching trophozoites aggregated, forming colonies of up to 50 individuals. After attachment, the trophozoites changed from pyriform to sub-spherical, posterior flagella shortened, anterior flagella were inactive, and surface blebs produced a refractile cyst wall. Sub-spherical cysts measured 3.0-4.0 x 2.5-4.0 µm.
Our observations appear to be the first to document attachment by the posterior flagella in diplomonads, although flagellar attachment is known in ectozoic kinetoplastids. The subsequent formation of cyst colonies in vivo would provide an effective mechanism to enhance transmission through the water. Gas bubbles and plant debris associated with colonies would help to maintain buoyancy, and hence transmission. Colonies would ensure the simultaneous ingestion of numerous cysts, presumably exceeding the maximum effective dose for initiating a new infection. Although minimum infective oral dose has not been established for piscine diplomonads, the number of individual diplomonads in the colonies we observed (approximately 50) lies within the range for minimum infective dose for avian and mammalian diplomonads.
We suggest that transmission of the life cycle can be disrupted by mechanical and/or chemical breakup of the adhesive colonies. Our observations thus open up a new approach for development of management and treatments approaches targeted to the cyst stage of the life cycle.