A new age in AquaMedicine: unconventional approach in studying aquatic diseases.

BACKGROUND: Marine and aquaculture industries are important sectors of the food production and global trade. Unfortunately, the fish food industry is challenged with a plethora of infectious pathogens. The freshwater and marine fish communities are rapidly incorporating novel and most up to date techniques for detection, characterization and treatment strategies. Rapid detection of infectious diseases is important in preventing large disease outbreaks. MAIN TEXT: One hundred forty-six articles including reviews papers were analyzed and their conclusions evaluated in the present paper. This allowed us to describe the most recent development research regarding the control of diseases in the aquatic environment as well as promising avenues that may result in beneficial developments. For the characterization of diseases, traditional sequencing and histological based methods have been augmented with transcriptional and proteomic studies. Recent studies have demonstrated that transcriptional based approaches using qPCR are often synergistic to expression based studies that rely on proteomic-based techniques to better understand pathogen-host interactions. Preventative therapies that rely on prophylactics such as vaccination with protein antigens or attenuated viruses are not always feasible and therefore, the development of therapies based on small nucleotide based medicine is on the horizon. Of those, RNAi or CRISPR/Cas- based therapies show great promise in combating various types of diseases caused by viral and parasitic agents that effect aquatic and fish medicine. CONCLUSIONS: In our modern times, when the marine industry has become so vital for feed and economic stability, even the most extreme alternative treatment strategies such as the use of small molecules or even the use of disease to control invasive species populations should be considered.

Megalocytivirus infection in cultured Nile tilapia Oreochromis niloticus.

Megalocytiviruses, such as infectious spleen and kidney necrosis virus (ISKNV), induce lethal systemic diseases in both ornamental and food fish species. In this study, we investigated an epizootic affecting Nile tilapia Oreochromis niloticus cultured in the US Midwest. Diseased fish displayed lethargy, gill pallor, and distension of the coelomic cavity due to ascites. Histopathological examination revealed a severe systemic abundance of intravascular megalocytes that were especially prominent in the gills, kidney, spleen, liver, and intestinal submucosa. Transmission electron microscopic examination revealed abundant intracytoplasmic polygonal virions consistent with iridovirus infection. Comparison of the full-length major capsid protein nucleotide sequences from a recent outbreak with a remarkably similar case that occurred at the same facility many years earlier revealed that both epizootics were caused by ISKNV. A comparison of this case with previous reports suggests that ISKNV may represent a greater threat to tilapia aquaculture than previously realized.

Interaction of Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease, with host proteins in the kidney of Salmo trutta.

Tetracapsuloides bryosalmonae (Myxozoa) is the causative agent of proliferative kidney disease in various species of salmonids which are found in Europe and North America. Less information about the interactions of T. bryosalmonae proteins with salmonid proteins during parasite development is known. In this study, anti-T. bryosalmonae monoclonal antibody-linked to N-hydroxysuccinimide-activated spin columns were used to purify parasite and host proteins from the kidneys of infected and non-infected brown trout (Salmo trutta) Linnaeus, 1758. The samples were next analyzed by electrospray ionization coupled to mass spectrometry to identify proteins that may be involved in the infection and proliferation of T. bryosalmonae within the brown trout host. A total of 6 parasite proteins and 40 different host proteins were identified in this analysis. The identified host proteins function in various processes, which include host defense, enzymatic, and structural components. In conjunction with modern molecular based tools, such siRNA, gene replacement, or gene disruption, this data can ultimately be used to develop novel control methods for T. bryosalmonae, based on the proteins or pathways identified in this study.

CyHV-3: the third cyprinid herpesvirus.

Common carp (including ornamental koi carp) Cyprinus carpio L. are ecologically and economically important freshwater fish in Europe and Asia. C. carpio have recently been endangered by a third cyprinid herpesvirus, known as cyprinid herpesvirus-3 (CyHV-3), the etiological agent of koi herpesvirus disease (KHVD), which causes significant morbidity and mortality in koi and common carp. Clinical and pathological signs include epidermal abrasions, excess mucus production, necrosis of gill and internal organs, and lethargy. KHVD has decimated major carp populations in Israel, Indonesia, Taiwan, Japan, Germany, Canada, and the USA, and has been listed as a notifiable disease in Germany since 2005, and by the World Organisation for Animal Health since 2007. KHVD is exacerbated in aquaculture because of the relatively high host stocking density, and CyHV-3 may be concentrated by filter-feeding aquatic organisms. CyHV-3 is taxonomically grouped within the family Alloherpesviridae, can be propagated in a number of cell lines, and is active at a temperature range of 15 to 28°C. Three isolates originating from Japan (KHV-J), USA (KHV-U), and Israel (KHV-I) have been sequenced. CyHV-3 has a 295 kb genome with 156 unique open reading frames and replicates in the cell nucleus, and mature viral particles are 170 to 200 nm in diameter. CyHV-3 can be detected by multiple PCR-based methods and by enzyme-linked immunosorbent assay. Several modes of immunization have been developed for KHVD; however, fish immunized with either vaccine or wild-type virus may become carriers for CyHV-3. There is no current treatment for KHVD.

Using a Hand-Held Gene Gun for Genetic Transformation of Tetrahymena thermophila.

Biolistic bombardment is widely used as a means of delivering vector-coated microparticles into microorganisms, cultured cells, and tissues. The first particle delivery system contained a helium propulsion unit (the gun) mounted in a vacuum-controlled chamber. In contrast, the hand-held gene gun does not operate within a chamber. It is completely hand-held, easy, and efficient to use, and it requires minimal space on the laboratory bench top. This chapter describes protocols for using a hand-held gene gun to deliver transformation vectors for overexpression of genes or gene replacement into the macronucleus of Tetrahymena thermophila. The protocols provide helpful information for preparing Tetrahymena for biolistic bombardment, preparation of vector-coated microcarriers, and basic gene gun operating procedures.

Purification of Cytoskeletal Proteins by Fast Protein Liquid Chromatography (FPLC) Using an ÄKTA Start System.

Actin, myosin, and tubulin are ubiquitous components of the fibrous network known as the cytoskeleton. Cytoskeletal proteins are involved in a plethora of intracellular processes such as maintenance of cellular organization, organelle translocation, and various nuclear roles including chromosome separation during mitosis. Early methods for protein extraction primarily relied on the salting-out method which was performed in conjunction with biochemical assays. Since the advent of recombinant molecular biology, protein tagging has been coupled with chromatography to obtain highly purified proteins required for sensitive assays. This chapter provides a general standard operating procedure (SOP) for using the ÄKTA™ Start System controlled by UNICORN software for fast protein liquid chromatography (FPLC) of 6× his-tagged cytoskeletal proteins. The protocol can readily be modified for affinity and non-affinity purification techniques using the various ÄKTA™ Chromatography Systems.

Using a Handheld Gene Gun for Genetic Transformation of Tetrahymena thermophila.

This chapter describes protocols for using a handheld gene gun to deliver transformation vectors for overexpression of genes or gene replacement in the macronucleus of Tetrahymena thermophila. The protocols provide helpful information for preparing Tetrahymena for biolistic bombardment, preparation of vector-coated microcarriers, and basic gene gun operating procedures.

Proteomic Analysis of Cytoskeleton Proteins in Fish.

In this chapter, we describe laboratory protocols for rearing fish and a simple and efficient method of extracting and identifying pathogen and host proteins that may be involved in entry and replication of commercially important fish viruses. We have used the common carp (Cyprinus carpio L.) and goldfish (Cyprinus auratus) as a model system for studies of proteins involved in viral entry and replication. The chapter describes detailed protocols for maintenance of carp, cell culture, antibody purification of proteins, and use of electrospray-ionization mass spectrometry analysis to screen and identify cytoskeleton and other proteins that may be involved in viral infection and propagation in fish.

In vitro inhibition of Cyprinid herpesvirus-3 replication by RNAi.

Cyprinid herpesvirus-3 (CyHV-3) is an etiological agent of a notifiable disease that causes high mortality rates affecting both the common and koi carp Cyprinus carpio L. There is no current treatment strategy to save CyHV-3 infected fish. RNA mediated interference (RNAi) is an emerging strategy used for understanding gene function and is a promising method in developing novel therapeutics and antiviral medications. For this study, the possibility of activating the RNAi pathway by the use of small interfering (si)RNAs was tested to inhibit in vitro viral replication of CyHV-3 in common carp brain (CCB) cells. The siRNAs were designed to target either thymidine kinase (TK) or DNA polymerase (DP) genes, which both code for transcripts involved in DNA replication. The inhibition of viral replication caused by the siRNAs was measured by a reporter gene, termed ORF81. Treatment with siRNA targeting either TK or DP genes reduced the release of viral particles from infected CCB cells. However, siRNA targeting DP was most effective at reducing viral release as measured by qPCR.

MyTH4, independent of its companion FERM domain, affects the organization of an intramacronuclear microtubule array and is involved in elongation of the macronucleus in Tetrahymena thermophila.

Myo1 is a class XIV Tetrahymena myosin involved in amitotic elongation and constriction of the macronucleus into two subnuclei at cell division. Elongation of the macronucleus is accompanied by elongation of an intramacronuclear microtubule array, which is oriented parallel to the axis of nuclear elongation. Elongation of the macronucleus often fails to occur or is only partially completed in a MYO1 knockout, and division of the macronucleus is frequently uncoupled from cytokinesis. Myo1 contains a myosin tail homology 4 (MyTH4) and a band 4.1, ezrin, radixin, moesin homology (FERM) domain. Recently, we used green fluorescent protein (GFP) fusions to demonstrate that the entire FERM domain, independent of MyTH4, is essential for localization of FERM to the cytoskeleton and does not appear to directly affect nuclear division. Antiactin coprecipitates GFP-FERM, tubulin, actin, and Myo1. The immunoprecipitated GFP-FERM cosediments with either exogenous F-actin or exogenous microtubules. Here, we show that overexpressed GFP-MyTH4 colocalized with antitubulin to intramacronuclear microtubules. Ninety percent of overexpressing cells assembled intramacronuclear microtubules that did not become organized into a parallel array. Amitosis did not advance in the absence of the parallel array of intramacronuclear microtubules. Five percent of overexpressing cells organized the parallel array, but the microtubules and the macronucleus did not achieve full elongation. Partially elongated macronuclei constricted without cytokinesis. Antiactin coprecipitated GFP-MyTH4, tubulin, and actin. AntiGFP pulled down GFP-MyTH4, tubulin, and actin. GFP-MyTH4 cosedimented with either exogenous microtubules or exogenous F-actin. A novel finding from this study is that MyTH4 and FERM have overlapping and distinct roles in the function of a myosin.

A FERM domain in a class XIV myosin interacts with actin and tubulin and localizes to the cytoskeleton, phagosomes, and nucleus in Tetrahymena thermophila.

Previous studies have shown that Myo1(myosin class XIV) localizes to the cytoskeleton and is involved in amitosis of the macronucleus and trafficking of phagosomes. Myo1 contains a FERM domain that could be a site for interaction between Myo1 and the cytoskeleton. Here, we explore the function of FERM by investigating its cytoskeleton binding partners and involvement in localization of Myo1. Alignment of Myo1 FERM with a talin actin-binding sequence, a MAP-2 tubulin-binding sequence, the radixin FERM dimerization motif, and the SV40 nuclear localization sequence (NLS) revealed putative actin- and tubulin-binding sequences, a putative FERM dimerization motif, and NLS-like sequences in both the N-terminal and C-terminal regions of Myo1 FERM. Alignment of Myo1 with an ERM C-terminal motif revealed a similar sequence in the Myo1 motor domain. GFP-FERM and two truncated FERM domains were separately expressed in Tetrahymena. GFP-FERM contained the entire Myo1 FERM. Truncated Myo1 FERM domains contained either the N-terminal or the C-terminal region of FERM and one putative sequence for actin-binding, one for tubulin-binding, a putative dimerization motif, and a NLS-like sequence. Actin antibody coprecipitated GFP-fusion polypeptides and tubulin from lysate of cells expressing GFP-fusions. Cosedimentation assays performed with either whole cell extracts or anti-actin immunoprecipitation pellets revealed that F-actin (independent of ATP) and microtubules cosedimented with GFP-fusion polypeptides. GFP-FERM localized to the cytoskeleton, phagosomes, and nucleus. Truncated GFP-FERM domains localized to phagosomes but not to the cytoskeleton or nucleus.