Myxobolus cerebralis , is the causative agent of salmonid whirling disease which results in higher economic losses. In our investigation , we studied the effects of Amprolium (Amp.), Salinomycin and Amproulium (Sal/Amp), chitosan nanoparticles (CNs), Amprolium -loaded chitosan nanoparticles (Amp- CNs), and Salinomycin + Amprolium -loaded chitosan nanoparticles (Sal/Amp- CNs) on the infectivity of myxospores of Myxobolus celebralis in vitro and in vivo. Preparation of the chitosan nanoparticles has been done by using the ionic gelation method and load ed with different drugs.
In the in vitro study, Myxospores were obtained from 60 clinically diseased rainbow trout. The myxospores have been divided into 6 groups each of them incubated with 3 different concentrations of the treatments at the 3-time interval (12,24,48 hr ) except the un-treated group which serves as a control . Each group of spores has been analyzed using a light microscop e. The strongest effect was found to be after 48hr. The percentage of dead and defective spores were 15±8.6% normal,61± 9% amp.,61.7±2.9% Sal/Amp, 65.3±0.6%CNs, 63±2.6%CNs-Amp, and 66±2.6%CNs-Sal/Amp. We tested the cytotoxicity effect of the chosen concentration of each treatment using the Alamar blue cytotoxicity essay on two different types of cell lines (EPC and BF2) for 72hr. Treatments were found to be safe for both cell lines. The effect of different treatments for the chosen concentration and incubation time has been evaluated using a fluorescent microscope (FDA and PI staining) and Transmission Electron Microscope (10000 spore/group for each technique). Sal/Amp-CNs and Amp-CNs showed the strongest effect regarding the alteration in spores’ morphology and dead spores counting. Followed by CNs- treated group, then Sal/Amp and Amp. While the non-treated group shows normal morphology and dead spores counting .
On the other hand, in the in vivo study , Tubifex tubifex were infected with four different groups of myxospores (Sal/Amp CNs -treated spores, CNs-treated spores, Sal/Amp -treated spores, non-treated spores). The worm was sampled for Conventional PCR and qPCR at 3-time intervals (1st month, 2nd month, 3rd-month post-infection). All groups were found to be infected with light infection after the 2nd month with slight differences between the 2nd and 1st months.
The 3rd- month PCR and the final filtration are in progress.