Standardization of In Situ Hybridization Techniques and Electron Microscopy for the Diagnosis of Aquatic Organism Diseases

1.1 In Situ Hybridization: This technique was first described in 1969 (Gal, 1969), but it only garnered the interest of the medical community in the 1980s. The appearance of cells and their architectural arrangement within a morphologically complex tissue represents only a fraction of the information within a histological section. These tissues contain all cellular proteins and express genes that will determine the biological behavior of the cell, and even provide clues about the origin and pathogenesis of diseases and their varying degrees (Murakami, 2001).

From this perspective, Jin & Lloyd (2001) assert that DNA or RNA analysis techniques will become increasingly commonplace, shedding light on unresolved questions. The undeniable benefits arising from the integration of this more refined technology with conventional Pathology are contributing to a remarkable increase in our knowledge of certain diseases. The primary allure of the in situ hybridization reaction lies in its ability to precisely locate a specific gene or its transcripts within paraffin-embedded or frozen tissue. While PCR can detect mRNA or DNA in tissue extracts, it does not allow us to observe the distribution of transcripts or DNA within a specific population of cells or in areas of adult or developing tissue (Young, 1989). 1.2 Nucleic Acid Probes: In in situ hybridization, probes are utilized to locate specific nucleic acid sequences at a subcellular level. Biotin is commonly employed in probes for non-radioactive detection. Biotin can be visualized through numerous methods, utilizing either avidin or streptavidin, both of which exhibit high affinity for this amino acid. Standard biotin detection employs enzymatic conjugates of streptavidin, producing a precipitation product that signals chromogenic enzyme substrates. Following the initial binding of biotinylated probes with streptavidin-peroxidase, peroxidase catalyzes the oxidation of biotinyl-tyramide, and this reaction deposits a significant amount of biotin at the hybridization site. This free biotin captures more streptavidin-peroxidase, amplifying the cycle further until the reaction reaches saturation. This signal is ultimately revealed using the chromogenic indicator diaminobenzidine (DAB), which is oxidized by peroxidase, yielding a dark brown precipitate at the hybridization site (Braissant and Wahli, 1998).

A probe is a known segment of DNA or RNA obtained through molecular cloning or chemical synthesis, which is complementary to a target sequence of interest and contains a label that enables selective visualization. DNA probes function similarly to antibodies used in immunocytochemistry in that they bind to a target and carry a signal. However, DNA hybridization probes offer certain advantages over immunodiagnostics because DNA is much more stable and easily preserved than most proteins. RNA is readily degraded by ribonucleases. The optimal probe size is around 200-500 base pairs for improved tissue penetration (although larger probes can be designed). The optimal hybridization temperature typically ranges from 15 to 25°C below the melting temperature (Warford and Lauder, 1991). 1.3 Application: This technique can primarily be used to detect RNA or DNA from microorganisms and differentiate productive from non-productive infections. It provides morphological information and allows for the observation of gene expression (mRNA), especially when protein expression is low or when it is rapidly exported from the cell, making it challenging to detect through Immunohistochemistry. Additionally, it also verifies the possibility of post-transcriptional control mechanisms (for example, the hybridization of viral mRNA in the liver has helped in understanding the complexity of hepatitis B infection) (McNicol and Fraquharson, 1997). 1.4 Electron Microscopy Negative Staining Technique: The negative staining technique involves a quick and easy preparation, providing results within minutes, making it the most productive approach in electron microscopy in terms of the number of samples. Particles from a suspension are adsorbed onto the surface of a specimen support, stabilized, and typically contrasted with drops of heavy metals. With this approach, particles can be visualized down to subnanometer sizes and categorized based on their morphology. The original term "negative staining" was introduced by Brenner and Horne in 1959.

Due to its ease of use and comparatively high yield, negative staining is often employed for quality assurance, such as testing virus cultures. Various types of samples can be easily transferred into a suspension without disrupting the structure of viral particles. Efficiency in terms of preparation speed is a critical factor in transmission electron microscopy diagnostics, making it a frontline method in this field (Curry et al., 2006). Furthermore, the open view of electron microscopy provides direct information about all nanoparticles present in a sample. Virus particles are identified based on morphological parameters such as size, shape, surface structure, and peculiarities. Considering that the morphology of a virus is relatively stable throughout evolution, diagnosis is easily achieved even if nucleic acids have undergone significant mutation, making identification through other methods more challenging. Therefore, diagnostic electron microscopy is valuable for identifying viruses in emerging infectious diseases or suspected bioterrorism cases. In veterinary medicine, diagnostic electron microscopy plays an even more crucial role because other diagnostic tools are often unavailable (Laue, 2010). To combine structural information with molecular data, negative staining can be paired with immunolabeling. This immunonegative staining can enhance specificity in diagnostic electron microscopy or provide insights into the molecular topology of viruses (Biel and Gelderblom, 1999; Biel and Madeley, 2001). 1.5 Electron Immunomicroscopy: Electron immunomicroscopy (IEM) is employed when the number of viral particles in a sample is very low, when virions are pleomorphic and difficult to identify due to the absence of typical viral morphology such as defined symmetry, shape, spikes, particle size, or capsomer number and arrangement or when samples are very electron-dense, and the aggregates generated by the technique can aid in identification (Lavazza et al., 2015). It allows for virus identification through specific antigen-antibody reactions, relying on the morphological characteristics. It is also used to serotype morphologically similar (but antigenically distinct) particles (Katz Kohn, 1984; Fields et al., 1996).

Various variations of the method, such as immune agglutination or direct immunoelectron microscopy (DIEM) (Anderson et al., 1973) or immune aggregation electron microscopy (IAEM) (Lavazza et al., 2015), solid-phase immunoelectron microscopy (SPIEM) (Derrick, 1973) have been employed. Hyperimmune sera, monoclonal antibodies, or convalescent sera can be used in performing the technique (Hazelton et al., 2003; Lavazza et al., 2015). SPIEM has been used to detect most viruses causing gastroenteritis, such as bovine rotavirus, swine rotavirus, equine rotavirus, canine parvovirus, and bovine viral diarrhea virus (BVDV).

IAEM has been used to detect porcine rotavirus (PoRV), porcine torovirus (PoToV), and porcine epidemic diarrhea virus (PEDV) in pigs with enteritis using convalescent serum (Lavazza et al., 2015). 1.6 Immunogold Colloidal Particle Labeling in Negative Staining Technique : In this technique, the antigen-antibody reaction is enhanced by labeling the antigen with colloidal gold particles associated with protein A, using specific antibodies for the type and genus. This method also allows for the detection and identification of virus-induced antigen structures and their location in infected cells, serotyping viral strains (Kjeldsberg, 1986), and determining antigenic variants in isolated strains (Patterson & Oxford, 1986).

This technique has been used to label porcine epidemic gastroenteritis virus (TGEV) particles in feces and small intestine fragments of infected pigs (Martins et al., 2013), type A rotavirus and coronavirus in fecal samples from diarrheic calves and winter dysentery in cattle (Kooijman et al., 2016), the simultaneous presence of coronavirus and rotavirus in the feces of calves with diarrhea (Catroxo et al., 2007), and BVDV in the feces of cattle with diarrhea (Catroxo et al., 2007). 1.7 Resin Fragment Inclusion Technique: The resin embedding technique, followed by ultrathin sectioning, is especially important for revealing fine details of the ultrastructure of all types of cells and tissues (Martins et al., 2013). In an infectious process, it allows the observation of infection pathogenesis and the identification of the agent (Fields et al., 1996). Ultrathin sections have the advantage of allowing the observation of virus-cell interaction, revealing the site of viral replication and maturation within host cells, which is pertinent information for the identification of unknown viruses (Fong, 1989). The overall ultrastructural details not only determine infection but also the course of disease in populations (Catroxo & Martins, 2015). The resin embedding technique has allowed the study of various ultrastructural aspects of the intracellular behavior of TGEV in intestinal fragments of infected pigs (Martins et al., 2013) and parvovirus in intestinal fragments of neonatal dogs with diarrhea (Catroxo & Martins, 2015). This technique also enables the study of the effectiveness of vaccines based on non-infectious virus-like particles (RVLPs) produced in situ (Meier et al., 2017).

In this work, we aimed to review the standardizations and protocols of in situ hybridization techniques in optical or photonic microscopy and immunoelectron microscopy (IEM), immunocytochemistry with immunogold colloid particle labeling (IMCG), and resin fragment embedding in transmission electron microscopy to enhance the efficiency of diagnosing diseases in aquatic organisms.