Effects of long duration, low dose bronopol exposure on the control of Ichthyophthirius multifiliis (Ciliophora), parasitising rainbow trout (Oncorhynchus mykiss Walbaum).

Ichthyophthirius multifiliis Fouquet, 1876 infections on intensively reared fish stocks can increase rapidly, which if left unmanaged, can result in the heavy loss of stock. The present study explores the efficacy of long duration, low dose (1, 2 and 5 mg L(-1)) treatments of bronopol (marketed as Pyceze™, Novartis Ltd.) in reducing the number of trophonts establishing on juvenile Oncorhynchus mykiss held under small scale culture conditions. The effect of bronopol on the colonisation success of infective theronts was also investigated by adding 2 mg L(-1) bronopol to the water prior and during the infection process. The number of parasites surviving on fish treated this way was compared to groups of fish that only received treatment after infection had occurred. The effect of bronopol on exiting trophonts throughout their external development to the point of theront release was also assessed through the delivery of 1 mg L(-1), 2 mg L(-1) and 5 mg L(-1) bronopol for up to 27 days consecutively (days 9-36 post-infection). The trial showed that a nominal dose of 2 mg L(-1) bronopol administered prior to infection significantly reduced the number of theronts surviving in the water column at the time of the initial challenge by 35-40% (P<0.05). Similarly, doses of 2 and 5 mg L(-1) bronopol administered as the first wave of mature I. multifiliis trophonts exited fish (i.e. day 11 onwards) to develop externally, reduced the number of trophonts establishing on fish as the second cycle of infection by 52-83%. Continuous application of 2 and 5 mg L(-1) bronopol throughout the second and third cycles of I. multifiliis infection gave further reductions of between 90 and 98%. The number of trophonts on the fish in the control tanks and those treated with 1 mg L(-1) and the 2 mg L(-1) dose at the time of initial infection, by comparison, were observed to increase with successive cycles of infection. From these small scale tank trials, this study demonstrates that the strategic, long duration, low dose delivery of drugs like bronopol can significantly reduce the number of trophonts establishing on fish suggesting the potential of this drug at managing I. multifiliis infections.

The anti-protozoal activity of bronopol on the key life-stages of Ichthyophthirius multifiliis Fouquet, 1876 (Ciliophora).

Pyceze™ (Novartis Animal Vaccines Ltd.) is licensed as a veterinary medicine to treat fungal infections in salmon, trout and their eggs. The active ingredient is bronopol, which due to its broad-spectrum activity has the potential to be an effective treatment against other important aquatic pathogens. In this study the efficacy of bronopol against Ichthyophthirius multifiliis was tested both in vitro and in vivo. In vitro trials demonstrated a 30 min exposure to 100 mg L(-1) bronopol killed 51.7% of the infective theronts. In vitro exposure of the protomonts to bronopol (0, 20, 50 and 100 mg L(-1)) for 30 min was observed to kill 0%, 76.2%, 97.2% and 100% respectively. Protomonts surviving treatment, demonstrated delayed development with the time taken from protomont until the release of theronts ranging from 28.3h for 0 mg L(-1) exposure, to 70 h for parasites in 20 and 50 mg L(-1) exposure groups. These concentrations also caused asymmetric cell division of the encysted tomonts. Exposure of encysted tomonts (min. 8 cell stage) to 100 mg L(-1) bronopol for 30 min, killed 50% within this period, with the remainder dying within the subsequent 42 h post exposure. Lower doses of bronopol were less effective in killing encysted tomonts than the higher doses (3.3% of parasites were killed in 20 mg L(-1); 10% in 50 mg L(-1)), but they still delayed theront release significantly (25.7 h for 0 mg L(-1) to 46.2h for parasites exposed to 20-50 mg L(-1)). Long, low dose (1 mg L(-1)) exposure to bronopol was also efficacious against theronts. Survival after 12h was 29% (c.f. 100% in control parasites), and <1% after 24 h exposure (c.f. 74% in control parasites). Theronts surviving these exposures demonstrated reduced infection success compared to control theronts. The findings of this study demonstrate that bronopol (Pyceze™) affects the survival of all free-living stages of I. multifiliis (protomonts, tomont and theronts), thus suggesting that bronopol may serve a useful role in the control of I. multifiliis infections.

Experimental infections of Atlantic salmon Salmo salar with Spironucleus barkhanus.

Atlantic salmon Salmo salar L. (Salmonidae) were experimentally infected with Spironucleus barkhanus (Diplomonadida: Hexamitidae). Parasites were found in the blood 1 to 8 wk after infection, after which they disappeared from the blood and were found mainly in the internal organs (e.g. spleen and liver), eye socket or muscles. Mortality (38 out of 40 infected fish) occurred when fish had lesions in internal organs and/or on the body surface. Uninfected fish cohabiting with infected fish became infected after 4 wk, indicating direct transmission. There was no difference in susceptibility to spironucleosis between 3 different families of Atlantic salmon. All families developed the disease with a similar pattern of parasitaemia in the blood, similar clinical signs and gross pathology, and with very high mortality (29 out of 30). Clinical signs of systemic spironucleosis may include anemia, skin blisters, muscle ulcerations or unilateral exophthalmia. Gross pathologies include hemorrhaging of internal organs, splenomegaly or deformed (globulated) spleen, or granulomatous lesions in the spleen and liver.

Detection and quantification of Spironucleus barkhanus in experimentally infected Atlantic salmon Salmo salar.

The course of Spironucleus barkhanus (Diplomonadida: Hexamitidae) infection in Atlantic salmon Salmo salar L. (Salmonidae) has 2 distinct phases, a blood phase and a tissue phase. To detect and quantify an infection, 3 parasitological techniques, namely Wet Mount Examination (WME), Hematocrit Centrifuge Technique (HCT) and the Hemocytometer (HCM) were used. In addition, 1 immunological technique, enzyme-linked immunosorbent assay (ELISA), was developed to detect specific antibodies against S. barkhanus. This technique would be particularly useful for epidemiological studies where large numbers of fish had to be examined. It would also be a good technique to detect infection during the tissue phase of the disease when there were no or a low number of parasites in the blood.

Effect of Cryptobia salmositica-induced anorexia on feeding behavior and immune response in juvenile rainbow trout Oncorhynchus mykiss.

At 10 degrees C, rainbow trout Oncorhynchus mykiss (n = 13 per group) infected with Cryptobia salmositica Katz, 1951 became anorexic at 3 wk post-infection (w.p.i.), with feed-intake decreasing significantly from 1.33 to 0.94% body weight (b.w.). Anorexia was most severe at 4 w.p.i. (0.80% b.w.), coinciding with peak parasitemia (9.2 x 10(6) parasites ml blood(-1)) and anemia. At 8 w.p.i., fish had recovered their appetite although they still had contained detectable parasites (6.8 x 10(5) parasites ml(-1)) and were anemic (pack cell volume, PCV, of 24.4%). However at 5 degrees C, anorexia occurred at 5 w.p.i. (0.81% b.w.), and was most severe at 7 w.p.i. (0.40% b.w.). At 8 w.p.i. (0.43% b.w.), fish displayed high parasitemia (4.6 x 10(6) parasites ml(-1)) and low PCV (10.8%). Fish at 5 degrees C had lower gastric evacuation (GE) rates (GE48h) than 10 degrees C fish, however there were no differences between infected and naive fish at both temperatures. Before anorexia, there was no significant correlation between mean share of meal (MSM, a measure of how food was partitioned within a group) and coefficient of variation in feeding but this became significant during anorexia (p = 0.02 and p = 0.0002 at 10 and 5 degrees C respectively). Significant correlations were detected between b.w. and MSM before onset of anorexia at 10 degrees C (p = 0.005) and 5 degrees C (p = 0.02); this was maintained at 10 degrees C (p = 0.001) but not at 5 degrees C (p = 0.98). Fish on an anorexic diet (0.93% b.w.) responded well at 10 degrees C to a live C. salmositica vaccine; this could partly be due to constant antigenic stimulation by the live vaccine.