Managing the Welfare of Zebrafish in Laboratory: Overview of Prevailing Diseases and Health Issues

Abstract

The zebrafish (Danio rerio) is a freshwater species native to South Asia belonging to the Cyprinidae family. Due to its easy housing and breeding, high fecundity, and rapid development, it has become a popular laboratory animal. Furthermore, zebrafish is an increasingly successful and widely used vertebrate model organism in scientific research, e.g., in drug discovery, particularly preclinical development, mainly because of its immune response and well-known genome. Nevertheless, zebrafish health in laboratory facilities is crucial. Both the water quality and pathogens control are directly related to the zebrafish welfare, which, under inappropriate conditions, may confound experimental findings, leading to irreproducible outcomes and invalid or misinterpreted results. Infections by Aeromonas and mycobacterium, for example, interfere with the results of experiments by altering physiological parameters. Likewise, infection of the nervous system by Pseudoloma neurophilia causes behavioral changes in zebrafish, leading to misinterpretation in behavioral studies. In this chapter, we seek to provide valuable contributions about zebrafish housing and husbandry conditions known to influence animal health, drawing attention to the most common diseases and pathogens that eventually may affect zebrafish in the laboratory.

1. Introduction

Research in several areas using the vertebrate zebrafish (Danio rerio) confirms its relevance as an alternative model. Zebrafish are used in forefront science due to a large number of factors including, but not limited to, their similarities to humans, fast and external development, transparency of the embryos, versatility, and genome-editing capabilities. Recent breakthroughs allow researchers to analyze the specificities of the immune system at the cellular level and explore the interactions between immune responses and pathogens, benefiting from the knowledge offered by the sequenced genome and conserved immune response [1].

Maintaining zebrafish health in experimental environments is vital, and providing adequate water and food following ideal standards will ensure stability, immunity, and well-being. In these conditions of well-being, the probability of infections being manifested will be low [2]. On the contrary, a low sanitary aspect in zebrafish housing can induce changes in physiology, behavior, and immune response, detectable through altered characteristics and clinical signs, such as lethargy, opercular enlargement, swallowing air at the water surface, loss of floatability control, eating less or not eating, and signs in their appearance like ulcer, injuries, hemorrhage, changes in color or hydropsy [3].

Given the increasing growth of zebrafish research facilities worldwide, these organisms are housed within a spectrum of settings ranging from modest setups involving a few tanks to large collaborative core facilities boasting thousands of aquaria. In these scenarios, the typical configuration consists of numerous compact aquaria integrated into a recirculation framework using commercially designed “racks”, encompassing approximately 30 tanks to large systems supporting hundreds of tanks. Irrespective of scale, the fundamental layout involves the recirculation of effluent water through both mechanical and biological filtration stages, succeeded by ultraviolet sterilization procedures. Supplementary water is commonly sourced from reverse osmosis outlets to which salts are introduced to control conductivity level.

Moreover, the technologies applied to create mutants or transgenic lines have boosted the use of zebrafish in research. More often than not, these lines might suffer from the same diseases that wild-type animals, although depending on the type of gene edition, it could impair their health vulnerability and needs.

Understanding the specific requirements of zebrafish and comprehending the nuances of their immune system is paramount for maintaining this species in laboratory environments that ensure their optimal well-being and health. Zebrafish, as a model organism, offer valuable insights into various biological processes, but their intricate needs, both in terms of habitat and immune responses, must be met to uphold their viability as research subjects.

Adequate knowledge of their demands, such as optimal water quality, appropriate diet, and suitable housing conditions, directly influences their behavior, growth rates, immunity, reproductive success, survival, and life expectancy. Moreover, delving into their immune system intricacies equips researchers with the tools to address potential disease outbreaks anticipatively, promote disease resistance, and reduce mortality. By refining our understanding of zebrafish requirements and immune dynamics, we fortify the foundation for productive research while upholding the ethical responsibility to ensure their comfort and health within laboratory settings.

Concisely, during early-life stages, zebrafish rely on their innate immune response, which involves innate molecules, barriers (skin, gills, gastrointestinal - GI tract), phagocytic cells, and soluble mediators, such as cytokines, pore-forming toxins, and lectins, which regulate immune cell interactions and activities [4, 5, 6]. On the other hand, juvenile and adult zebrafish possess a well-developed complement system and an adaptive immune response similar to other vertebrates. Surface barriers like skin, gills, and GI tract are coated by mucus, offering both physical and chemical protection against pathogen microorganisms or aggressors, besides promoting osmotic balance. Further, zebrafish surfaces are provided with phagocytic macrophages and neutrophils. Also, GI tract secretions produce adverse environments for exogenous microorganisms [7].

This book chapter intends to provide valuable contributions about zebrafish housing and husbandry conditions known to influence animal welfare, drawing attention to the most common diseases and pathogens that might affect zebrafish in laboratories.

2. Biotic and abiotic factors and the zebrafish health

Zebrafish are subject to water quality concerns similar to those of other fish reared in intensive recirculating systems. The zebrafish housing facility acts as a life support system, similar to natural habitats, and alterations or poor water quality compromise several biological processes in zebrafish, such as growth, metabolism, immune function, behavior, and stress [8]. Thus, although zebrafish are quite tolerant to water quality parameter fluctuations, the standardization of husbandry procedures must be pursued to optimize experimental techniques and reach reliable results [9].

Therefore, physical, chemical, and biological parameters must be fulfilled to achieve conditions, where the fish perform their best once inadequate water quality produces important subclinical and clinical manifestations with severe morbidity and mortality [10].

Zebrafish are naturally found in South Asia, inhabiting slow-flowing water bodies in different ranges of water parameters, temperatures varying from 10 to 40°C, pH between 6 and 10, and up to 60 cm of water column [9]. According to Schaefer & Ryan (2006), thermal tolerance varies between individuals, and for zebrafish, the optimum range is between 24 and 28°C [11]. Temperature also interferes with other phenomena like sex determination, where masculinization is related to high rearing temperatures (reviewed in [8]). Higher temperatures also decrease the saturation of dissolved oxygen (DO), which leads to an imbalance of total ammonia nitrogen (TAN) and enhances the prevalence of unionized ammonia (NH₃), increasing the NH₃ toxicity [12].

The TAN balance is also influenced by pH concentration. Unionized ammonia and ammonium ions (NH₃ and NH₄⁺, respectively) rely on pH and must be regulated in order to prevent disbalance. Ammonia is oxidized to nitrite (NO₂⁻), which is also toxic by oxidizing hemoglobin and converting it to meta-hemoglobin. Thus, the respiratory process is affected, leading to potential asphyxiation and death. The conversion of nitrite to nitrate (NO₃⁻) is essential once this molecule presents minor toxicity, and levels up to 200 mg.L⁻¹ are considered safe for zebrafish [13].

Water quality diseases caused by ammonia toxicity generally lead to behavioral alterations, like hyperexcitability and anorexia, which can be observed in the aquarium, and lesions observed in additional exams, such as gill hyperplasia and hypertrophy (reviewed in [2]). Sporadically, mats of bacteria are associated with hyperplastic gill lesions, although this is also most often secondary to suboptimal water quality and not a primary bacterial problem [14].

pH values reflect the concentration of hydrogen ions present in the solution and characterize the water as acidic, alkaline, or neutral when around pH 7. The production of TAN in the creation system acidifies the water (pH <7); thus, neutralization with buffer solutions is necessary [12]. The tolerable pH range for freshwater fish is between 6.5 and 9, and for zebrafish, it stands around 6.2 and 8.5, with no behavioral or physiological alterations (reviewed in [8]). Acidification or alkalinization of water has effects on fish, resulting in several physiological alterations in gills that are in direct contact with water, but also in gas exchange and electrolyte balance [15].

Moreover, pH alterations cause massive mucus production, besides behavioral changes [16], glycemia, a decrease in red blood cells (RBC), and an increase in white blood cells (WBC) [17]. Additionally, any disturbance in the osmotic state leads to physiological stress and disease (reviewed in [8]).

Biological processes like neuronal signaling, muscle contraction, and osmoregulation depend on essential ions—calcium, magnesium, and ferrous iron, among others—that are absorbed from rearing water; these ions’ concentration is represented by water hardness [18]. According to Chen et al. (2003), hardness >75 ppm (parts per million) is adequate for freshwater fish [19].

The concentration of DO is affected by external factors like water temperature, feeding rates, and fish density in an inversely proportional manner. Oxygen concentration between 6 and 8 ppm is suitable for zebrafish culture [12]. Meanwhile, high concentrations of carbon dioxide affect fish by diminishing hemoglobin affinity to oxygen and acidifying blood. Thus, it is imperative to keep lower concentrations of carbon dioxide. Besides DO and carbon dioxide, water nitrogen levels interfere with fish physiology. When water nitrogen supersaturation occurs, fish develop a syndrome known as gas bubble disease (Figure 1).

Figure 1.

Illustration of gas bubble disease; lateral view (left) and dorsal view (right).

In housing systems, leaks on suction water pumps or intensive air injection on water can cause water gas supersaturation. Also, abrupt temperature alters gas concentration stability. Supersaturated water produces emboli (free gas bubbles) in the vascular system that spread through other tissues, mainly subcutaneous tissue [8], which is particularly problematic. Local inflammation and behavioral abnormalities can occur in affected zebrafish. Also, air emboli in the vascular system may cause obstruction of blood vessels, leading to local ischemia and tissue damage, and the severity of the disease depends on the intensity of the formed emboli. Necrosis areas derived from disease can suffer from secondary bacterial infections. Eyeballs are frequently injured by tissue gas bubbles, and clinical signs, such as exophthalmos (Figure 2), eye inflammation, cataracts, and blindness may occur [14]. Chronically affected fish can present emaciation, buoyancy defects, and reduced activity.

Figure 2.

Illustration exophthalmia; healthy (left) and sick (right) animals in lateral and dorsal view.

Likewise, nephrocalcinosis is also triggered by rough water quality. A range of nephrocalcinosis lesions in zebrafish can be assessed by histology, most of which are diagnosed in clinically normal fish. Calcium deposits in renal tubules and ducts may occur as a consequence of high dissolved CO₂ [14].

Abnormal behaviors, such as frequent aggression, lethargy, and erratic swimming, are reliable indicators of welfare disruption, as well as specific clinical signs indicating punctual situations, such as opercular flaring (Figure 3), reflecting respiratory distress.

Figure 3.

Illustration of opercular flaring.

3. Stress and zebrafish

As highly evidenced, among all the critical modulating factors on health and equilibrium, stress has a lot to do with many biological responses as a state of threatened homeostasis. Stress is the consequence of external or internal factors (stressors) implicated in changes in biological equilibrium. It might initiate an adaptive response to restore baseline physiology, seen as a change in physiology, psychological state, or behavior.

In laboratory zebrafish, stress can induce alterations notably in four broad physiologic categories: behavior, the autonomic nervous system (ANS), the neuroendocrine system, and immune function [20]. Consequent modifications in these functions can be assessed by evaluating behavioral patterns, e.g., appetite, displacement, aggression, and, eventually, response to handling; physio-morphological alterations, e.g., respiration intensity, weight loss, growth rate, color change, blood cell count and cell structure, cardiac output and blood flow; and biochemical alterations, such as through hormone levels; and reproductive performance impairment, which can manifest in zebrafish as decreased spermatozoa quality, reduced spermatozoa cell motility [21], accelerated ovulation, reduction in the number of embryos and their survival rates. Overall, threats to homeostasis activate a complex pattern of behavioral changes and responses in the central and autonomic nervous and endocrine systems; if the stress system is chronically activated, these effects may lead to severe pathology.

Lastly, zebrafish nutrition is an additional important factor for their health and proper development. Some diseases that affect zebrafish are caused by a nutritional deficiency. In this regard, for example, vitamin E or selenium deficiency might cause muscle degeneration, vitamin C deficiency can lead to scoliosis, and pantothenic acid deficiency is likely to cause gill disease.

4. Bacterial infections

Clinical or subclinical infections may distract experimental findings, leading to irreproducible outcomes and invalid or misinterpreted results [22]. In general, local lesions are noticeable and may indicate bacterial, fungal, or protozoal infections located on the skin and fins, with hemorrhagic areas or ulcerative lesions. These clinical signs vary in severity according to the intensity of infection and are the base for correct diagnosis, along with microorganism culture [23].

In fish, skin infections are caused by bacteria such as Cytophaga, Flavobacterium, and Flexibacteria, while Aeromonas salmonicida, Pseudomonas spp., Edwardsiella spp., Streptococcus spp., and Mycobacterium spp. are mostly responsible for systemic infections (reviewed in [23]). Moreover, species like Flavobacterium columnare are prone to causing bacterial gill disease (Figure 4) or fin rot (Figure 5).

Figure 4.

Illustration of gill inflammation in zebrafish.

Figure 5.

Illustration of zebrafish fins and tail with frayed edges.

4.1 Columnariosis

The infection by the opportunistic bacteria F. columnare generates a disease called columnariosis. The disease can clinically manifest in different forms, including skin, gills, and fins, and has a high rate of morbidity and mortality. The infection lesions appear as white or grayish lesions, exposing muscles in the more eroded wounds [24]. Clinical signals of distress may occur, such as increased breathing rate and opercular flaring, and this is due to intensive infections of the gills.

Zebrafish with compromised immune system are especially vulnerable to F. columnare infection and can transmit the disease through direct contact between infected and healthy fish and also through contaminated water or equipment. Poor water quality and other stressors, such as overcrowding and inadequate handling, can contribute to the onset of the disease, while open wounds can serve as bacteria entry [25].

As an infectious disease, the affected fish must be isolated from healthy individuals and treated with antibiotics—oxytetracycline or florfenicol. Improving water quality and general handling, as well as quarantine adoption of new fishes, tend to be effective against the F. columnare infection.

4.2 Edwardsiellosis

The gram-negative bacterium Edwardsiella ictaluri is the etiologic agent causative of Edwardsiellosis and the enteric septicemia of catfish. It has been reported to infect other species and, regarding zebrafish, is a disease that must be monitored to prevent outbreaks between zebrafish colonies. The infection in zebrafish is uncommon; however, the disease has a high mortality rate [26]. Zebrafish liver and spleen are the most affected organs, while skin may be stricken with ulcerations, along with intestine, heart, and nervous tissue [27, 28]. Behavioral alteration can also appear before or during the intense infection.

Diffuse erythema on the skin and necrosis in intern organs are common clinical findings, and the olfactory neural pathway is also a target for the infection [29]. Quarantine is key to prevention, once infected fishes rapidly evolve the characteristic lesions, approximately two weeks, antibiotics are the usual treatment for infected fishes.

Besides local signs, infections in zebrafish also have a systemic character, with clinical manifestations such as peritonitis and generalized edema. The edematous condition of skin and abdominal distension is called dropsy (Figure 6) and is derived from fluid accumulation into the elomatic cavity caused by systemic infections [23, 30].

Figure 6.

Illustration of dropsy zebrafish with fluid accumulation in the coelomic cavity (top) and scales protrusion (bottom).

According to Pullium et al. (1999) [31], housing situations like high density, poor water quality, and inadequate husbandry (i.e., stress, abrupt temperature changes) favor systemic infections, especially with Aeromonas hydrophila and Streptococcus sp. [2]. In these infections, symptoms such as lethargy and petechial hemorrhage are observed. These bacteria are gram-negative and usually found in the mucosa and internal organs of healthy fish. Thus, the infection is considered opportunistic [32, 33]. One of the differences between A. hydrophila infection and mycobacteriosis, for example, is that the mortality rate is acute.

4.3 Mycobacteriosis

Bacterial infections usually cause notable symptoms, provoking mortality or health decline in the zebrafish population. However, subclinical infections lead to uncontrollable biases in experimental protocols [34], so in laboratory conditions, the fish must be observed, and good practices should be taken to prevent these infections.

Mycobacteria are ubiquitous, naturally occurring gram-positive, acid-fast, and aerobic bacteria. They are found in both environmental and laboratory water besides soil, and several species have been described to infect zebrafish, like Mycobacterium abscessus, M. chelonae, M. fortuitum, M. haemophilum, M. marinum, and M. peregrinum(reviewed in [2]), and approximately 40% of zebrafish facilities present mycobacterium occurrence [35]. According to Whipps et al. (2012), due to its large distribution and its environmental occurrence, mycobacteria may be considered pathogenic or opportunist in zebrafish [36].

Poor water quality and stressed animals are the pre-existing factors for this organism to establish itself. The clinical presentation varies greatly. Some lesions observed in the fish will be discoloration (Figure 7), skin ulcerations, or granulomas (nodules) in the abdominal cavity (Figure 8). However, lethargy may be another sign of infection. This is a chronic disease, and if not properly treated, it can lead to death. Mortality and morbidity of Mycobacteriosis vary according to species, where M. marinum and M. haemophilum are the most severe infections. Inflammatory granulomas in hematopoietic organs and occasional chronic inflammation are commonly described in infected zebrafish (reviewed in [37]).

Figure 7.

Illustration of zebrafish showing epithelial discoloration.

Figure 8.

Illustration of zebrafish with skin ulcerations.

Zebrafish facilities affected by Mycobacteriosis must adopt rigorous protocols to eliminate the contamination. Preventive measures, such as quarantine of newly introduced fish, embryo disinfection, effective sterilization of system water, and satisfactory sanitation of rearing tanks, must be taken in every zebrafish facility [36].

Mycobacteria are oligotrophic organisms and, thus, can survive and proliferate in a plethora of environments. In zebrafish aquaria, when sanitation is not efficient, biofilms are formed, which are adequate environments for the survival of mycobacteria despite the cleanness of water [37].

Chang, Lewis, and Whipps (2019) reported that M. chelonae can be transmitted by infected fishes to aquaria biofilms, suggesting that resident fishes could naturally acquire M. chelonae infections from tank biofilms. The authors also suggested that other species of mycobacteria could be transmitted in the same way and emphasized the importance of safety control in zebrafish facilities [37].

Research programs with zebrafish can be severely affected by bacterial outbreaks in the facilities. Generally, mycobacteria are opportunistic pathogens; however, two species are of special relevance: M. marinum and M. haemophilum. These species produce severe and persistent infections, and M. marinum is associated with high mortality rates (Reviewed in [38]).

The gastrointestinal system is also considered a route for mycobacteria infection. There have been reports of salmonids and medaka infected through feed, and mycobacteria were identified in the zebrafish intestinal tract (reviewed in [39]). Studies pointed out that transmission through other organisms could maximize contamination of Mycobacteriosis since Japanese medaka were more infected by mycobacteria-contaminated mosquitoes, and infected protozoan Paramecium caudatum transmitted the infection to zebrafish [40], as well as Chang, Benedict, & Whipps (2019) demonstrated the transmission of mycobacteria through live feed [39].

Mycobacteriosis is considered a zoonotic disease, and non-tuberculous mycobacteria (NTM)-induced cutaneous infections have been described in dermatological care clinics. Most of the cases were caused by M. marinum, although other species have also been identified in some cases. Thus, besides ensuring the zebrafish health, sanitation measures also prevent contamination of staff members (Reviewed in [38]).

5. Fungal infections

Fungal infections generally present a cotton or wool-like form that can derive from necrotic lesions, although species like Aphanomyces sp. produce primarily necrotic ulcerative lesions. The most common fungal infection in freshwater fish is Saprolegniasis, also referred to as molds or water molds, which can affect fish and their eggs. It is important not to overfeed or leave dead fish in your tanks, because Saprolegnia will establish itself and grow extremely fast on the animals or the excess food. Lecythophora mutabilis is another water mold that creates a biofilm around the fish head and then establishes itself in the biofilm. Then, the cause of death usually happens due to starvation and asphyxiation of the animals. The fungal identification is usually made by skin scraping, fungal hyphae and spores visualization, and posterior fungal culture to confirm [29].

5.1 Saprolegnia

Saprolegniosis is an infectious disease caused by the fungi of the Saprolegnia genus, notably S. ferax, S. diclina, and S. parasitica. The infection is known as “water mold” since the skin lesions produce cotton-like growths that start as small lesions and develop into more aggressive ones. A wide range of aquatic species, including zebrafish, can be affected by the fungus. As the genus Saprolegnia is usually found in aquatic environments, the disease generally occurs when the fish’s immune system is compromised, making them susceptible to infection [41].

The affected fish can present lesions on the skin, gills, and fins, showing a “fuzzy” appearance, and with the disease aggravation, a significant portion of the body is affected [42]. The affected surface area, which may be composed of large areas, predicts the severity of the disease. Thus, destruction of the epidermis and respiratory collapse due to infection of the gills cause behavioral changes, with lethargy leading to a high risk of predation. The impairment of physiological processes such as osmoregulation and the eventual entrance of fungal hyphae in internal organs in advanced stages can lead to organ failure.

Pre-existent skin lesions and infection by other pathogens could represent a serious risk for contracting Saprolegnia infections. Moreover, physiological stressors such as poor water quality, high density, and sudden temperature change have an important impact on zebrafish immune homeostasis, increasing the susceptibility to opportunistic infections, such as Saprolegniosis. Improving water quality, avoiding stressing factors, quarantining newly introduced fish, and regularly cleaning tanks and equipment are key actions to prevent fungal occurrence [43].

Infected fishes must be isolated from the experimental aquaria, along with antifungal treatments in order to preserve sanitary conditions of the experimental set and prevent further infections. Also, proper monitoring of fish is essential to recognize and take immediate action for treatment.

6. Parasitic infections

6.1 Ichthyophthiriasis

Ichthyophthiriasis is a parasitic infection caused by the protozoan Ichthyophthirius multifiliis. This disease is commonly known as “white spot disease” due to the clinical signs of infected fish: small, white, raised spots in skin, fins, and gills. The protozoan I. multifiliis occurs in aquatic environments and is prevalent in both natural habitats and experimental sets, and its ability to spread rapidly is of concern to zebrafish laboratories [44].

The Ichthyophthiriasis characteristic white spots consist in the trophont stage of the parasite, which is the stage when the parasite adheres to fish skin. The skin infection leads to behavior alteration, and the fish starts to rub against objects’ surfaces, increasing mucus production. This behavior represents a visible irritation and discomfort of infected fishes, and the stressful situation provokes immunodepression, making them more susceptible to I. multifiliis and other opportunity pathogens, such as bacteria and fungus. In advanced stages, the disease can have a significant impact on the zebrafish population [45].

Containment measures like regular monitoring of fish behavior, avoiding high density, and keeping water quality seem to be effective in avoiding the parasite. Meanwhile, infected fish must be isolated for therapeutic intervention and to avoid the spread of the disease [46]. The treatment can be executed through two approaches: the interruption of the parasite’s life cycle, with a gradual elevation of salinity and temperature, or with chemical substances, such as formalin or copper-based medication.

6.2 Pseudoloma neurophilia

Microsporidia are obligate intracellular spore-forming parasites that can infect approximately 150 fish species, as well as terrestrial animals such as rodents, rabbits, and primates including humans [47]. Microsporidiosis is a common disease in laboratory fish that affects the central nervous system and can cause emaciation and spinal deformity [48, 49]. Also, infection by microsporidia is characterized by xenomas, a cellular growth formed by hypertrophied, unorganized, and unstructured polyploid cells due to parasite proliferation [50].

In zebrafish, microsporidia infection was described by de Kinkelin (1980) [51], and the most common parasite was identified as Pseudoloma neurophilia [48]. This parasite is associated with multiple xenomas in hindbrain, spinal cord, and nerve roots, and free spores can be found within phagocytes in other areas, causing myositis, meningitis, and encephalitis, and can be transmitted vertically and horizontally. In laboratory zebrafish, P. neurophilia infection is asymptomatic, only detected when clinical signs turn perceptible, like spinal deformation (Figure 9) or emaciation (Figure 10) [48, 49].

Figure 9.

Illustration of zebrafish with abnormal morphology, spinal deformity (top), and kyphosis (bottom).

Figure 10.

Illustration of zebrafish with emaciation.

The spinal cord is associated with locomotor ability and motor function, and areas of the hindbrain control neural pathways associated with behaviors like anxiety, fear, and learning. Thus, it has been suggested that lesions caused by P. neurophilia in these areas could alter zebrafish neural signaling, directly interfering with outcomes of experiments in several fields, such as neurobiology, toxicology, and pharmacology [52].

According to Spagnoli, Sanders, & Kent [53], zebrafish infected with P. neurophilia presented altered shoal behavior and startle response, as well as inflammation on the spinal cord, meninges, and muscles, which could be caused by spore stage [54]. As reviewed by Midttun et al. [52], several model organisms present altered behaviors and biological processes due to intra- and extracellular parasites, e.g., Drosophila spp. colonies presenting altered circadian rhythm and rodents with activated cytokine signaling. Thus, behavioral phenotypes such as stress and anxiety-like behavior may be a result of P. neurophilia infection despite experimental treatment per se [53].

7. Viral infections

Health management in zebrafish laboratories is mandatory since this model system becomes strikingly important to biomedical sciences in both larvae and adult stages [55]. Although several mammalian viral infections are modeled in zebrafish [56], naturally occurring viral infections are scarcely studied in zebrafish [57].

As the importance of zebrafish in science rises, aquatic laboratory facilities also start to emerge in research centers, and the utilization of shared systems exacerbates the possibilities for transmission of undiagnosed viral infections [58, 59]. The implementation of different mutant lines, such as immunocompromised fish, to studies in the immunology field contributes to the introduction of new diseases in the population, including viral infections (reviewed in [57]).

Bermúdez et al. (2018) described for the first time a natural occurrence of Megalocytivirus infection in zebrafish research facilities [60]. The author reported the contagion by the infectious spleen and kidney necrosis virus (ISKNV), an iridovirus. Iridoviridae is a virus family that infects poikilothermic vertebrates, and the authors identified the virus by genetic alignment with known Megalocytivirus sequences. The clinical observations included altered swimming and behavior, hyperemic gills, respiratory distress, and hemorrhage. The diagnosis was based on genetic correspondence and pathological findings once hypertrophied cells, besides necrotic and degenerative areas, were found primarily in the kidney and spleen.

Picornaviridae is a virus family known to infect vertebrates through cell surface receptors of intestinal, respiratory, or hepatic tissue. Altan et al. described a natural infection of a divergent picornavirus in zebrafish without marked clinical signs [61]. According to Kent, Harper, and Wolf [62], the occurrence of asymptomatic diseases impacts the results of research using zebrafish as they impose additional variants on experimental data.

8. Concluding remarks

Zebrafish have been considered an outstanding model for biomedical research. One of many advantages of the zebrafish is the possibility to screen small molecules to identify potential therapeutics with precision. Moreover, the conservation of immune mechanisms among vertebrates strengthens the translational character of zebrafish as a model system.

However, like other animal models, zebrafish are subject to suffering from disorders, including being contaminated and transmitting infectious diseases. P. neurophilia is a common infectious agent that produces important effects on zebrafish, leading to behavioral alterations, which cause misinterpretation of data, invalidating experimental protocols. Natural viral infections have been described in zebrafish, and although several studies model mammal viruses in zebrafish and explore unique insights, the occurrence of natural viruses includes unknown biases in protocols, generating uncertainties in the experiment.

The study of natural diseases that can occur in zebrafish, mainly in fish kept in a scientific experimentation environment, may provide the opportunity to directly visualize the pathogen-host dynamics and to carry out chemical screenings, which facilitates the development and testing of new therapies, ensuring animal welfare.

Acknowledgments

We thank the São Paulo Research Foundation (FAPESP) for the support—notably through the Center for Toxins, Immune Response, and Cell Signaling (CeTICS). All illustrations presented here were made exclusively for this chapter by Daniela Bená.

This work was supported by the São Paulo Research Foundation—FAPESP (#2019/27677-7; #2021/08891-8 and #2013/07467-1). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflict of interest

The authors declare no conflict of interest.