Immunosuppression is the reduction of the activity or efficiency of the immune system (REZENDE, 2011). In cetaceans, infection with morbillivirus (HEANEY et al., 2002), contamination with toxic chemicals (ROSS, 2002; BENNETT et al., 2001), and stress-induced conditions such as concomitant diseases (parasitism) and anthropogenic activities (accidental capture and habitat degradation) are the major causes of immunosuppression (Figure 1) (LEONARD, 2005; BEINEKE et al., 2005).

Figure 1
Response to stressors in cetaceans through the release of glucocorticoid hormones and consequent lymphoid depletion.

Viruses of the genus Morbillivirus are one of the leading causes of immunosuppression in cetaceans. Morbillivirus are RNA viruses of the Paramyxoviridae family that infect several species of mammals (DI GUARDO et al., 2005). The genus includes different species, whose names refer to the animal from which the virus was isolated, including canine distemper virus (CDV, initially described in canids), rinderpest virus (RPV, in bovids), and phocine distemper virus (PDV, in the seal Phoca vitulina). In cetaceans, cetacean Morbillivirus (CeMV) includes three well-characterized strains-dolphin morbillivirus (DMV, described in the striped dolphin, Stenella coeruleoalba), porpoise morbillivirus (PMV, described in the harbour porpoise, Phocoena phocoena), and pilot whale morbillivirus (PWMV, described in the long-finned pilot whale, Globicephala melas) (HALL, 1995; KENNEDY, 1998)-and three other strains, including the new strain from the Guyana dolphin, Sotalia guianensis (GD-CeMV) (GROCH et al., 2018).

In marine mammals, Morbillivirus PDV and CeMV were detected in 1988 in seals and cetaceans, respectively (VAN BRESSEM et al., 2014). PVD caused the death of approximately 18,000 seals (P. vitulina) and hundreds of gray seals (Halichoerus grypus) along the coast of Europe (COSBY et al., 1988). CeMV infection is characterized by histological changes similar to the distemper reported in specimens of P. phocoena stranded on the coast of Ireland and later identified in dead cetaceans on the coast of England, Scotland, and the Netherlands (KENNEDY et al., 1988; GROCH et al., 2018). Since then, several cases of mass mortality have been reported in cetaceans.

The occurrence of CeMV in Brazil was reported in two species of cetaceans: Fraser’s dolphins (Lagenodelphis hosei), detected by specific antibodies (VAN BRESSEM et al., 2001), and S. guianensis. The first case in S. guianensis was reported in the state of Espírito Santo in the southeast region of Brazil, and the diagnosis was made by immunohistochemistry and confirmed by polymerase chain reaction, PCR (GROCH et al., 2014). In addition, three cases of Morbillivirus were reported in S. guianensis in the state of Paraná and detected by immunohistochemistry (DOMICIANO et al., 2016). However, the discovery of the new GD-CeMV (Guiana dolphin-CeMV) strain specific to S. guianensis was confirmed by compatible molecular and immunohistological analyses, and this strain caused the mass mortality of dolphin populations in Rio de Janeiro (GROCH et al., 2018).

Information on clinical signs of Morbillivirus is scarce in cetaceans. However, neurological and behavioral changes were observed, including disorientation and cachexia (DOMINGO et al., 1992; KENNEDY, 1998), and were predominant during the epidemic in Rio de January in 2017/2018 (Azevedo, A., personal communication). The pathological findings observed macroscopically include severe bilateral pneumonia with congestion, edema, in addition to interlobular, subpleural, and mediastinal emphysema (KENNEDY, 1998). Pulmonary abscesses associated with parasitic granulomas and enlarged and edematous lymph nodes with multifocal necrosis are frequent in the lungs. Some animals present ulcerations of the upper gastrointestinal tract and genitals, subcutaneous edema, hemorrhagic necrotizing encephalitis, and congestion of hepatic vessels (KENNEDY, 1998; GROCH et al., 2014).

The most significant microscopic changes involved the respiratory tract, central nervous system, and lymphoid tissues. The lungs presented interstitial bronchopneumonia, edema, fibroplasia, and inflammatory infiltrate with syncytial cells; these cells were usually found in the bronchial, bronchiolar, and alveolar lumen, and less frequently in the alveolar interstitium. Morbillivirus-infected lungs are often affected by secondary bacterial and parasitic infections, which are common sequelae of strong immunosuppression caused by Morbillivirus infections (KENNEDY, 1998).

The primary changes detected in the central nervous system were meningoencephalitis, demyelination, and perivascular cuffs, degenerative and necrotic neuronal changes, microgliosis, and neuronophagia. Syncytial cells were also observed in the nervous system (KENNEDY, 1998). Severe lymphoid depletion occurred in lymphoid organs (lymph nodes, spleen, thymus, and gut-associated lymphoid tissue). The presence of syncytial cells was reported in the lymph nodes and spleen (DOMINGO et al., 1992; KENNEDY, 1998).

The transmission of Morbillivirus to cetaceans is predominantly horizontal because of the social behavior of these animals (CARTER et al., 1992). However, large amounts of the PDV antigen were detected by immunocytochemistry in the lactating mammary glands of a female specimen of the striped dolphin Stenella coeruleoalba, suggesting the possibility of transmission to the baby via milk (DOMINGO et al., 1992).

Lymphoid depletion due to infection with Morbillivirus allows the development of opportunistic diseases, and some diseases are more severe and rare under normal conditions. The first case of co-infection between Morbillivirus and Herpesvirus in cetaceans was reported in a specimen of S. coeruleoalba stranded on the Mediterranean coast during the peak of the second epizootic outbreak in 2007 (SOTO et al., 2012). The most prominent lesions were found in lymphoid tissues with marked lymphocyte depletion. Intranuclear basophilic inclusions not-suggestive of DMV were observed and investigated. Using PCR and sequencing techniques, the samples were identified as belonging to the cetacean Alphaherpesvirus group, and sequencing demonstrated a degree of similarity of 99% and 98% base pair (bp) to two herpesviruses previously identified in Ziphius cavirostris and Stenella coeruleoalba, respectively. In fin whales (Balaenoptera physalus), co-infections of Morbillivirus and Toxoplasma gondii (DI GUARDO et al., 2013; MAZZARIOL et al., 2012) with parasites (DUIGNAN et al., 1992; TAUBENBERGER et al., 2000), bacteria and fungi (LIPSCOMB et al., 1994) were reported.

Stress is defined as a response to a situation that induces a disturbance in homeostasis (LEONARD, 2005). In cetaceans, stress agents range from natural situations such as the search for prey and reproductive periods to anthropogenic activities. Anthropogenic activities may include accidental catches, collision with vessels, pollution by chemical substances and domestic and industrial effluents, noise disturbance (particularly in port areas), and reduction of the food supply by overfishing (BRUHN et al., 1999; SIEBERT et al., 1999; BEINEKE et al., 2010). The response of cetaceans to stress is similar to that described in other mammals (CURRY, 1999) and stress adaptation reactions may involve the mobilization of different physiological responses, including immunological responses (DOHMS; METZ, 1991).

The response to stressors is mediated by different systems, particularly the autonomic nervous system (ANS), hypothalamic-pituitary-adrenal (HPA) axis, and complementary systems involving neuropeptides and neurotransmitters (GRIFFIN, 1989). The immediate response is mediated by ANS, in which sympathetic activation induces the release of catecholamines (adrenaline and noradrenaline) and increases heart rate and blood pressure, preparing the body for a fight or flight response. This response to stress is short-lived because of the parasympathetic reflex (ULRICH-LAI; HERMAN, 2009).

After activation of the ANS, stress activates the HPA axis, releasing glucocorticoids, including cortisol. Cortisol production is regulated by the adrenocorticotrophic hormone (ACTH) produced in the pituitary gland (GRIFFIN, 1989). The release of ACTH, in turn, is controlled by the corticotrophin-releasing hormone (CRH), synthesized in the hypothalamus, or by the antidiuretic hormone (ADH) (GRIFFIN, 1989). In contrast to the ANS-mediated response, in which stress responses occur in seconds, the mechanism of activation of the HPA axis is slow, and the peak glucocorticoid level occurs minutes after the stimulus (ULRICH-LAI; HERMAN, 2009). It is important to emphasize that the objective of the stress responses is immediate survival and does not necessarily preserve the functioning of the organism in the future (WRIGHT et al., 2009).

During stress responses, cortisol produces the following: i. mobilizes carbohydrates in metabolic pathways to increase the levels of energy substrates, causing hyperglycemia; ii. induces catecholamines to act in metabolic pathways and blood vascularization; and iii. induces protective immunological reactions, including inflammation, to minimize cellular and tissue damage (MUNCK et al., 1984; BREAZILE, 1988).

Cortisol also promotes immunosuppression, characterized by lymphopenia and eosinopenia (BUSH, 2004), and insulin resistance, with potential diabetogenic action (DAMIANI et al., 2001). Therefore, during chronic stress, immune adaptation is impaired, leading to changes due to hypercortisolism (ARBORELIUS et al., 1999). Venn-Watson et al. (2013) compared a group of T. truncatus dolphins maintained in captivity and a free-living group and observed that captive dolphins were more susceptible to the development of insulin resistance and metabolic syndrome than free-living animals. These authors indicated that stress was a primary contributor to the induction of these changes because of the diabetogenic effect of cortisol.

Cetaceans maintained in captivity are usually more severely affected by stressors than free-living animals. Stressful situations include reduced habitat space, overcrowding, and changes in habitat structure. Consequently, opportunistic diseases are common in captivity, indicating immunosuppression or inadequate management of the animals (BUCK, 1980). The pathogens identified in captive cetaceans included Candida albicans, which was the most common (SWEENEY; RIDGWAY, 1975); Giardia sp., and Cryptosporidium spp. (REBOREDO-FERNÁNDEZ et al., 2014), and Cryptococcus laurentii (MARTINS et al., 2002), among others.

Stress may lead to severe injuries when animals are handled for containment or transport, particularly wild animals (terrestrial and aquatic). The most common example is capture myopathy caused by excessive release of catecholamines (PACHALY et al., 1993). Capture myopathy is a degenerative muscular disease caused by tissue hypoxia and change in pH, inducing the necrosis of cardiac muscle fibers and release of potassium, myoglobin, and lactate. Death is a consequence of heart failure. However, another sign of the disease is acute renal failure due to myoglobinemia (DIAS, 1993). Injuries suggestive of capture myopathy, including myocardial contraction band necrosis and ischemia-reperfusion injuries in the intestine and kidneys were identified in several stranded cetaceans (TURNBULL; COWAN, 1998; COWAN; CURRY, 2008). Although capture myopathy is common in many mammals, it is more pronounced in cetaceans, which hampers and even prevents attempts at rehabilitation (CLARK et al., 2006).

Noise pollution causes stress to cetaceans, and the response to these stressors may be immediate or delayed. The most common noises are those generated by different types of vessels, including whale-watching, fishing, and icebreaking boats, and sounds coming from marine construction such as drilling, dredging, explosions, maritime seismic activity, sonars, and even aircraft. Coastal cetaceans are exposed to the synergistic and cumulative effects of noise pollution. However, isolated events such as acoustic pulses emitted by seismic activity may cause irreversible injuries to these animals, and environments free from these impacts are not available to cetaceans (NOWACEK et al., 2007).

In seawater, sound travels almost five times faster than through the air, spreading over hundreds of miles and affecting organisms in large areas (URICK, 1983). The behavioral changes observed in several species included the abandonment of habitats and essential activities such as food foraging (NOWACEK et al., 2007). In the USA, the reduction of the marine fleet led to a significant reduction of noise produced by ships, and glucocorticoid levels were decreased substantially in whales of the species Eubalaena glacialis (ROLLAND et al., 2012). In small cetaceans from Babitonga Bay, Santa Catarina state, Brazil, a population of S. guianensis temporarily abandoned its habitat near a port during the months of expansion of the coast (CREMER et al., 2009), potentially impairing food foraging and increasing exposure to other impacts, including catches in fishing nets. The reported effects are evident because these noises are strong stressors to cetaceans by limiting animal communication, search for prey, and the capacity of displacement and assessment of the environment.

Mass-mortality events in marine mammals are associated with toxins produced during algal blooms (diatoms and dinoflagellates). These events are caused by four classes of toxins: saxitoxins, brevetoxins, domoic acid, and ciguatoxins. These substances are neurotoxins, but their chemical structures are different. Furthermore, the symptom, pathological, and epidemiological characteristics are different in animals contaminated with these substances (VAN DOLAH et al., 2003). Walsh et al. (2005) evaluated lymphocyte proliferation in Florida manatees exposed to red tide (excessive multiplication of Karenia brevis) and cold temperatures (stressors) and compared with free-living healthy animals. These authors observed that the lymphocyte proliferation of the first group was approximately one third that of healthy animals and that the direct effects of the toxin were evaluated by co-culturing the lymphocytes of stressed manatees with K. brevis extracts, and further indicated that the proliferative response was further reduced, suggesting that red tides impaired the immune function.

The lymphoid system of cetaceans is similar to that of other mammals and is composed of the thymus, spleen, lymph nodes, mucosa-associated lymphoid tissues (tonsils and appendix), bone marrow, blood, and lymph (COWAN; SMITH, 1999).

The thymus of cetaceans is similar to that of other mammals and regresses progressively with the advancement of age (COWAN, 1994). However, the exact age in which regression begins in cetaceans is unknown (COWAN; SMITH, 1999). In P. phocoena and T. truncatus, cysts were observed next to the thymus and were characterized as tumorous lesions (BEINEKE et al., 2010). However, microcysts are common in the thymus of P. phocoena and are related to age (BEINEKE et al., 2010). Macrocysts were observed in animals with impaired health and are caused by chronic diseases and accelerated thymic atrophy (WÜNSCHMANN et al., 1999).

The spleen of cetaceans is similar to that of other mammals. However, although the spleen is a single organ, multiple accessory spleens are common in whales and dolphins, in contrast to the spleen of other species (BLESSING et al., 1972). Multiple structures are also found in domestic animals (incidental findings) but are usually caused by splenic rupture following trauma or surgery (MAUÉS et al., 2013). The spleen of cetaceans is smaller than that of other mammals (its mass corresponds to approximately 0.2% of the total mass of the animal) (SLIJPER, 1958) and does not store blood. The hypothesis for the lack of ability to store blood is the lack of predators for most species, with no need for extra oxygen distribution throughout the body in fight or flight responses (HARTWIG; HARTWIG, 1985).

The lymph nodes of cetaceans are morphologically and histologically similar to those of other mammals and perform the same functions. It is a secondary lymphoid organ whose parenchyma can be divided into a cortical and a medullary region, covered by a collagen capsule of a dense connective tissue. Between these two regions, there is a deep cortical or paracortical region composed primarily of T lymphocytes and reticular cells, together with a few plasma cells and macrophages (JUNQUEIRA; CARNEIRO, 2008; SILVA et al., 2014). B lymphocytes are the predominant cell type in the cortical region and are organized in spherical or ovoid arrangements designated lymphoid follicles (JUNQUEIRA; CARNEIRO, 2008). When a lymph node is activated by antigens, B lymphocytes multiply, expand, and form the germinal centers where cell multiplication and somatic mutation occur. Somatic mutation is related to the rigidity and specificity of the interaction between antigens and immunoglobulins produced by B cells (TIZARD, 2009; ROMANO et al., 2002). However, lymph nodes in cetaceans rarely present germinal centers and are composed primarily of a paracortical and a medullary region (ROMANO et al., 1993). The medullary region, in turn, contains macrophages, B lymphocytes entangled in lymphatic vessels, blood vessels, and diffuse lymphoid tissue (ROMANO et al., 2002; JUNQUEIRA; CARNEIRO, 2008).

Silva et al. (2014) described the morphology and location of lymph nodes of seven Odontocetes species: Sotalia guianensis, S. fluviatilis, Stenella clymene, S. longirostris, Inia geoffrensis, Peponocephala electra, and Globicephala macrorhynchus, from the North and Northeast regions of Brazil. The following lymph node types were found according to their location in the organism: parotid, mandibular, hyoid, cervical (superficial and deep), periscapular, mediastinal, hilar respiratory, pulmonary, diaphragmatic, gastric, mesenteric, mesocolic/colic, hilar hepatic, pancreatic, hilar renal, pelvic, and genital (male and female). Of these, most were found in these seven species, except parotid, hyoid, periscapular, and pancreatic lymph nodes. In all species studied, smooth muscle fibers in the subcapsular zone were observed.

The arrangement of lymphoid follicles in cetaceans is controversial. Moskov et al. (1969) described the lymph nodes of Phocoena phocoena and Delphinus delphis with a central location of the lymphoid follicles, and this arrangement was similar to the inverse architecture described in swine. In other species, the arrangement follows the cortex-medulla pattern, common in most mammals (SILVA et al., 2014).

Lymphoid tissues (lymphoepithelial structures) such as oropharyngeal tonsils, which are similar to Waldeyer’s tonsillar ring in terrestrial mammals, and the lymphoepithelial gland in the larynx and anal tonsil were also observed in cetaceans. The probable function is antigen presentation at the site of contact with the external environment, as during breathing and diving (when there is rectal water reflux) (COWAN; SMITH, 1999; BEINEKE et al., 2010).

One of the histological findings that characterize immunosuppression in cetaceans is lymphoid depletion, which is the decreased number of lymphocytes in lymph nodes. Lymphoid depletion was reported in S. guianensis stranded on the coast of Paraná and infected with morbillivirus (DOMICIANO et al., 2016). Lymphoid depletion in the Guiana dolphin (S. guianensis) stranded on the coast of Paraná is illustrated in Figure 2.

Figure 2
(A) Normal lymph node of Sotalia guianensis. Observe lymph node (→) activated. (B) Sotalia guianensis lymph node with marked lymphoid depletion. Observe parenchyma presenting spaces not filled by lymphoid cells. Bar: 100µm, HE.

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