Co-infection of bacterial and parasitic pathogens including the brain myxosporean Zschokkella auratis in farmed striped murrel Channa striata (Bloch, 1793) causing large scale mortality

Co-infections in fish by the wide array of pathogens have been little researched, although such infections are commonly noticed in nature. A murrel farm recorded 100% mortality in adults of Channa striata within 3-4 days of infection during winter. The clinical signs included cloudy eyes, excess mucus secretion, haemorrhages on the body, lethargy and anorexia in C. striata before mortality. Heavy infection with Ichthyophthirius multifiliis on the body surface and Zschokkella auratis, a new myxosporean to this country, in the brain tissue were detected by wet mount microscopy and 18s rDNA sequencing, respectively. Histological sections of the brain also demonstrated the presence of myxosporean cyst. Examination of eye fluid and eye tissue revealed the presence of bacterium Acinetobacter baumannii and blood samples containing Aeromonas sobria and Klebsiella pneumoniae were detected under 16s rDNA and species-specific PCRs followed by sequencing. Interestingly, samples collected from infected fishes also revealed multiple parasitic pathogens viz., Epistylis sp., Trichodina sp., Argulus siamensis and Ergasilus sp. Thus, it was delineated that prolonged low water temperature might be playing one of the influential roles in creating stress in fish, thus making the fish susceptible to complicated or mixed bacterial and parasitic infections.

Selection of candidate reference genes for RT-qPCR analysis in Argulus siamensis and their validation through screening of drugs and drug targets

Argulus spp. are economically important fish ectoparasites. The development of antiparasitic drugs is thus important and real time PCR is an indispensable tool in drug development. The analytical potential of RT-PCR depends upon accurate normalisation by the use of stable reference genes. Here, we identified stable reference genes of Argulus siamensis for validation of efficacy of drugs and drug targets. Seven candidate genes were evaluated by evaluating their expression under different states of Argulus using the RefFinder tool. The four algorithms together generated a comprehensive ranking with elongation factor-1 alpha (EF-1α) being the most stable and 18S ribosomal protein (18S) the least stable gene. Taking EF-1α and 18S genes as references, the effectiveness of six anti-parasitic compounds against Argulus was evaluated by studying their effect on the expression pattern of few ion channel genes; this was to understand their mode of action, besides validating the reference genes. EF-1α was found to be the most stable gene in the validation. Collectively, this study is the first report to validate the optimal reference genes of A. siamensis for normalisation, and the potential of the ion channel genes for evaluating effective drug targets in parasite control.

Transcriptional analysis of immune-relevant genes in the mucus of Labeo rohita, experimentally infected with Argulus siamensis

The knowledge of mucosa-associated molecular events that occur during infections is scarce despite the well-established importance of mucus in fish immunity. Using qRT-PCR, we analyzed the immune gene expression patterns in mucus of Labeo rohita experimentally infected with an ectoparasite Argulus siamensis. Mucus samples were collected at 0 h, 12 h, 24 h, 3 d, 7 d, 15 d, and 30 d post challenge of L. rohita with metanauplii of A. siamensis. All interleukins studied herein (IL 6, IL 15, and IL 1β) showed significant upregulation of expression levels in mucus of A. siamensis-infected fish compared to control samples. Further, the expression levels of molecules involved in pathogen recognition, toll like receptor 22, and pathogen presentation, β2 microglobulin, were found to be significantly upregulated in experimental samples until 7 d post challenge compared to control samples. The upregulated expression of lysozyme G at all time points post infection indicated the early activation of acute phase responses in mucus of infected L. rohita. Moreover, the expression levels of natural killer cell enhancing factor B were found to be higher in infected fish than they were in the control fish. The early upregulation of the immune genes observed herein reinforces the role of mucus as the first line of defense against pathogenic assault; furthermore, it expands our understanding of mucosal-immune responses to A. siamensis infection, which can aid development of immunological interventions.

Evaluation of ribosomal P0 peptide as a vaccine candidate against Argulus siamensis in Labeo rohita

Argulusspp. are important ectoparasites of fish, and the current approach of their control using chemical pesticides has numerous drawbacks. Vaccination is a promising alternative but identification of protective antigens is a limiting step. The ribosomal protein P0, essential for protein synthesis, has been studied as a vaccine candidate. We generated sequence information of the P0 protein of the ectoparasiteArgulus siamensisand the hostLabeo rohita. The region of the parasite P0 protein with less sequence similarity with that of the host P0 protein and high predicted antigenicity was used for peptide synthesis. The peptide was conjugated with keyhole limpet hemocyanin (KLH) for immunization of rohu at a dose of 1.5 μg/g body weight. Dot blot assays confirmed production of antibodies against pP0-KLH in immunized fish. We evaluated the efficiency of pP0-KLH as a vaccine antigen by challenge of the immunized fish withA.siamensis. Although there was no significant difference in parasite load between both groups, a reduced and delayed mortality of 59% (15 days post-infection) in immunized group was noticed as compared to 75% mortality (within 7–15 days post-infection) in control group. The partial protection observed indicated the need for further optimization of this molecule to develop it into a vaccine candidate.