Testing Susceptibility to Diplomonad Infection: Development of a New Plasma Incubation Test
Diplomonad flagellates are commonly encountered in the digestive tract, and less commonly in other organs, of a wide variety of aquatic animals, including fish, amphibians and reptiles. In aquaculture, diplomonads are associated with morbidity and mortality, especially among salmonids and cichlids. Although metronidazole is effective, it is not permitted for use in some countries, and thus other approaches to managing the infections, including awareness of species susceptibilities, are important.
Diplomonads have two distinct stages in their life cycle. The swimming trophozoite stage, easily recognized in fresh smear preparations from fish, reproduces by longitudinal binary fission. The less commonly recognized resistant cyst, is assumed to be responsible for transmission of the parasite through the water to the next host. Although the life cycle is poorly understood in piscine diplomonads, it is assumed that infection is via oral ingestion of cysts, and upon reaching a suitable site in the intestine, excystment occurs, trophozoites are released, and a new infection is initiated.
With such a direct life cycle, all fish in a given geographic locality are assumed to be equally exposed to infection. However not all fish become infected; there are marked differences in susceptibility both within and between species. The epizootiological literature suggests some host specificity among piscine diplomonads, and experimental infections support this, with some species of fish being susceptible to a given species of diplomonad, whereas others are resistant to infection by this same species.
We suggest that successful establishment of diplomonad infection will depend upon many factors, including innate immunity, and we decided to utilize this phenomenon as the basis for development of a new test to predict susceptibility to infection. Previously, susceptibility of species of fish to diplomonad infection has been determined by experimental infections, which are time consuming and expensive to undertake, therefore a simpler test is needed.
We focus our studies on rainbow trout, Oncorhynchus mykiss, and the diplomonad Spironucleus salmonis (previous called Hexamita salmonis)2. A given cohort of trout will contain some fish that are uninfected, and among the infected fish, density of S. salmonis infection varies from light to heavy. Furthermore, as the rainbow trout get older, prevalence and density of infected decreased. We modified the plasma incubation test, previously used for hemoflagellates1, and used it to detect susceptibility to the enteric flagellate S. salmonis. The principle of the test is that the plasma of susceptible hosts is benign (or relatively benign) to the parasites, while the plasma of resistant hosts causes measurable lytic and/or cytotoxic damage to the parasites.
To date we have tested the plasma of three species of fish to determine between-species differences in susceptibility to S. salmonis: rainbow trout, a host susceptible to S. salmonis; common carp Cyprinus carpio, a host not susceptible to S. salmonis but to another species of piscine diplomonad; and Atlantic sturgeon, Acipenser sturio, a non-susceptible host.
We use a 384-well tissue culture plate for our tests. The wells are loaded with culture medium, then fresh plasma is added in serial dilution (10 dilutions), and finally known numbers of cultured S. salmonis trophozoites are added. Control wells contain culture medium and trophozoites, but no plasma. Experiments are conducted in triplicate. The inoculated plates are held at 10 °C for experiment, since this is close to the optimum temperature for S. salmonis trophozoites. The wells are examined at 5, 15, 60 and 120 mins after inoculation, and we record lytic and cytotoxic damage (assessed as killed or missing cells, and abnormal movement, respectively).
Our preliminary data shows 1) that the plasma incubation test originally used for hemoflagellates can be successfully modified for enteric flagellates, 2) there are between-species differences in lytic and cytotoxic effects. The plasma of sturgeon has strong lytic effect, killing all trophozoites within 15 minutes, even at the lowest concentration. In contrast, the plasma of carp and rainbow trout was less strongly lytic, and most trophozoites were alive after 15 mins. However, subsequently there were more trophozoites killed by the carp plasma than by the rainbow trout plasma. The lytic effect exponentially decreases with decreasing plasma concentration, and fits well to the Exponential Decay mathematical model; meaning that the decrease is at a rate proportional to its value.
Our preliminary data shows that the between-species differences in lytic titre and cytotoxicity reflect susceptibility to S. salmonis infection observed in epizootiological studies. Thus the test holds considerable promise for predicting host-parasite susceptibility to diplomonad infection. Ongoing studies are planned for (i) investigation of within-species differences of susceptibility to
S. salmonis in rainbow trout, by testing fish from the same source that are uninfected, or lightly, moderately, or heavily infected, (ii) detection of mechanisms of lysis and cytotoxicity by treating plasma (for example by heat inactivation, which inactivates the complement).
We are indebted to our colleagues Klaus Kohlmann and Joern Gessner for kindly allowing us to sample blood from their carp and sturgeon respectively. The authors gratefully acknowledge their financial support: Sarah Poynton was supported by a Mercator Visiting Professorship from the German Research Foundation (DFG), and Reza Saghari Fard is supported by a Nafög stipendium from Nachwuchsförderung (Nafög) des Landes Berlin.
1. Bower SM, Woo PTK. 1977. Cryptobia catastomi: incubation in plasma of susceptible and refractory fishes. Experimental Parasitology 43: 63-68.
2. Poynton SL, Saghari Fard MR, Jenkins J, Ferguson HW. 2004. Ultrastructure of Spironucleus salmonis n.comb. (formerly Octomitus salmonis sensu Moore 1922, Davis 1926, and Hexamita salmonis sensu Ferguson 1979), with a guide to Spironucleus species. Diseases of Aquatic Organisms 60: 49-64.