Developing strategies for the control of Ichthyophthirius multifiliis Fouquet, 1876 (Ciliophora)

The intensification of freshwater aquaculture worldwide has facilitated the propagation of the parasitic ciliate protozoan Ichthyophthirius multifiliis Fouquet, 1876 commonly known as “fish white spot” or “Ich”. Ichthyophthirius multifiliis infections lead to high mortalities, generating significant economic losses in most cultured freshwater fish species worldwide. Until recently, malachite green was the chemical treatment traditionally used to control I. multifiliis infections. Its reclassification as carcinogenic to humans and its subsequent ban for use in food fish has left the industry without any suitable treatments. Currently, in-bath formaldehyde and sodium chloride treatments are the most common option used in farm systems to control I. multifliis infections. Given their low efficacy, however, they are not considered as sustainable long–term options. There is, therefore, an urgent necessity to find efficacious alternatives for controlling I. multifliis infections. The general aim of this research project was to improve the management of I. multifiliis infections in order to develop more comprehensive, environmentally friendly and sustainable therapeutic strategies for use in freshwater food fish aquaculture. The present PhD-thesis present first a literature review chapter providing an overview and critical assessment of chemotherapeutants and physical interventions tested within the last 30 years against I. multifiliis infections. The experimental worked consisted of a number of in vitro and in vivo trials were conducted using experimental scale flow-through, static tank systems and commercial scale raceways within a rainbow trout hatchery, in addition to molecular work on different isolates of the parasite. The results of this research are organised into three experimental chapters which describe the testing of chemical and non-chemical treatments against I. multifiliis infections and work undertaken to determine the most suitable molecular markers to identify I. multifiliis isolates. In the first experimental chapters, the possibility of efficiently controlling I. multifliis infections through the administration of novel environmentally-friendly chemical treatments (e.g. bronopol and peracetic acid-based products) was investigated. The results clearly showed that bronopol and peractic acid-based products have a strong biocidal/cytotoxic effect against all free-living stages of I. multifiliis (e.g. tomonts, cysts and theronts). The administration of high concentrations of bronopol (e.g. 20, 50 and 100 mg L-1) over short periods of exposure (e.g. 30 min) significantly reduced the survival of tomonts, cysts and theronts and delayed the development of I. multifiliis tomonts and cysts. Prolonged low concentrations of bronopol (e.g. 1 mg L-1) greatly reduced the survival of infective theronts, although such treatment did not affect the ability of surviving theronts to subsequently infect a host. When tested in vivo, the continuous prolonged exposure (e.g. 27 days) of low concentrations of bronopol (e.g. 2 and 5 mg L-1) had an impact on the population dynamics of I. multifiliis, this being demonstrated by a significant reduction in the number of trophonts developing within the fish. Low concentrations of bronopol (e.g. 2 mg L-1) administrated as a preventive treatment prior to infection also proved to be very successful at reducing the colonisation success of I. multifiliis. Peracetic acid administrated at low concentrations (e.g. 8, 12 and 15 mg L-1) over a short window of exposure (e.g. 1 h) displayed a strong biocidal effect against all the free-living stages of I. multifiliis (e.g. tomonts, cysts and theronts). The bronopol and peracetic acid-based products tested here both appear to be capable of disrupting the development of the cyst stage of I. multifiliis which is seldom reported for chemotherapeutants currently used against this parasite. These results suggest that bronopol and peracetic acid-based products have a place in the arsenal of treatment options for controlling I. multifiliis infections in commercial aquaculture systems. The use of a mechanical device or a biological control agent to remove the cyst stage of I. multifiliis and the impact of such control on the population dynamics and the levels of infection of fish were also investigated. The results revealed that tomonts preferentially settle and encyst on the base of culture systems and on biofilm–covered substrates. The survival of the tomont stage is greatly affected by the composition of the substrate upon which it settles and is significantly lower on polypropylene-based plastic. The lining of raceways in a commercial rainbow trout hatchery with a low-adhesion polymer created a smooth surface facilitating the dislodgement and elimination of the cyst stage of I. multifilis by natural flushing or brushing. The physical removal of the cyst stage alone, through the use of a mechanical device or substrate detrivorous/algae feeder as a biological control agent, significantly reduced the propagation of I. multifiliis to a low level of infection without the need to deploy an additional chemical treatment. These studies demonstrate that the cyst is a key stage in the dynamics of I. multifiliis infection and its removal from the fish culture systems could constitute an effective and simple mean of managing I. multifiliis infections. The third experimental chapter explores the utilisation of molecular marker to characterise different isolates of I. multifiliis. The results highlight the unsuitability of the rDNA region (ITS-1 and ITS-2) and the strong potential of the mtDNA (COI) as molecular markers to discriminate isolates of I. multifiliis from distant geographical locations. It is suggested that genetic “barcoding” using mtDNA is the most effective method to identify I. multifiliis isolates. Importantly, genetic “barcoding” could allow associating I. multifiliis strains or geographical isolates with particular properties as regards their ecophysiology, pathogenicity and sensitivity to treatment, in order to improve the management of I. multifiliis infections according to the specific genetic isolate encountered. This research project demonstrates the efficacy of a range of new approaches against the propagation of I. multifiliis. Together, our findings contribute towards the development of a more effective and integrated system for managing I. multifliis infections in farm systems. The utilisation of physical methods and of environmentally friendly chemotherapeutants holds great potential for the control of I. multifiliis infections in organic fish production and in a broader context to any freshwater food fish farms affected by I. multifiliis.