# COMPARATIVE IMMUNOLOGY OF MARINE MAMMALS

EDITED BY : Giovanni Di Guardo, Michael F. Criscitiello, Eva Sierra and Sandro Mazzariol PUBLISHED IN : Frontiers in Immunology

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# COMPARATIVE IMMUNOLOGY OF MARINE MAMMALS

Topic Editors:

Giovanni Di Guardo, University of Teramo, Italy Michael F. Criscitiello, Texas A&M University, United States of America Eva Sierra, University of Las Palmas de Gran Canaria, Spain Sandro Mazzariol, University of Padova, Italy

Marine mammals (MMs) are regarded as valuable bioindicators with tremendous potential for public health. However, many aspects of their immune system remain poorly understood. Monitoring immune responses of MMs is pivotal for the health assessment of both individuals and populations, as well as providing the scientific basis for analyzing the anthropogenic environmental impact on marine ecosystems and marine-terrestrial interphases. For instance, the increasing susceptibility of Mediterranean whale and dolphin populations to various diseases has been linked to a possible negative influence of multiple environmental factors on the immune system of MMs.

The currently limited knowledge on MM immunology has mainly centered on: (i) lymphocyte transformation assays; (ii) natural killer cell activity; (iii) phagocytic activity and respiratory burst; (iv) humoral immune responses; (v) cytokines and (vi) acute phase immune responses. Therefore, further research is essential for deepening our understanding of the specificity of the host immune response in MMs, with a particular emphasis on the genesis and dynamics of (i) cytokine 'networks' or 'signatures'; (ii) transcriptional regulation of immune cells and (iii) major immunomodulators. High-throughput molecular techniques, such as transcriptomic analysis and RNA sequencing, may enable the characterization of immune gene responses at the transcriptomic level. This integrative and holistic approach requires sophisticated tools and methods capable of unveiling the diversity of immune cells and immunologically relevant molecules that orchestrate environmental adaptation and immune protection against pathogens in MMs.

This Research Topic aims to provide a comprehensive overview of the current knowledge of MM immunology with a particular emphasis on structural and functional studies at the protein and cellular level. We wish to encourage and coordinate studies and investigations in order to fill gaps of knowledge in this field. This article collection aims to help gain more data regarding: a) The characterization of the immune system in several species of MMs, i.e. cetaceans, pinnipeds and sirenians; b) The interplay between the host immune system and the most relevant pathogens, e.g., Morbillivirus, Brucella, Toxoplasma gondii and c) The possible interplay between the immune system and contaminants.

Citation: Di Guardo, G., Criscitiello, M. F., Sierra, E., Mazzariol, S., eds. (2019). Comparative Immunology of Marine Mammals. Lausanne: Frontiers Media. doi: 10.3389/978-2-88963-203-9

# Table of Contents

Ignacio Peñín, Mónica E. Figueroa-Cabañas, Fabiola Guerrero-de la Rosa, Luis A. Soto-García, Roberto Álvarez-Martínez, Adriana Flores-Morán and Karina Acevedo-Whitehouse

*24 Comparative Immunopathology of* Cetacean morbillivirus *Infection in Free-Ranging Dolphins From Western Mediterranean, Northeast-Central, and Southwestern Atlantic*

Josué Díaz-Delgado, Kátia R. Groch, Rodrigo Ressio, Isis P. J. Riskallah, Eva Sierra, Simona Sacchini, Óscar Quesada-Canales, Manuel Arbelo, Antonio Fernández, Elitieri Santos-Neto, Joana Ikeda, Rafael Ramos de Carvalho, Alexandre de Freitas Azevedo, Jose Lailson-Brito Jr., Leonardo Flach, Cristina T. Kanamura, Natália C. C. A. Fernandes, Bruno Cogliati, Cinzia Centelleghe, Sandro Mazzariol, Ludovica Di Renzo, Gabriella Di Francesco, Giovanni Di Guardo and José Luiz Catão-Dias

*43 The Marine Mammal Class II Major Histocompatibility Complex Organization*

André Luiz Alves de Sá, Breanna Breaux, Tibério Cesar Tortola Burlamaqui, Thaddeus Charles Deiss, Leonardo Sena, Michael Frederick Criscitiello and Maria Paula Cruz Schneider

*57 Insights Into Dolphins' Immunology: Immuno-Phenotypic Study on Mediterranean and Atlantic Stranded Cetaceans*

Cinzia Centelleghe, Laura Da Dalt, Letizia Marsili, Rossella Zanetti, Antonio Fernandez, Manuel Arbelo, Eva Sierra, Massimo Castagnaro, Giovanni Di Guardo and Sandro Mazzariol

*68 Acute Phase Proteins in Marine Mammals: State of Art, Perspectives and Challenges*

Maria Elena Gelain and Federico Bonsembiante

*76 Insights Into Cetacean Immunology: Do Ecological and Biological Factors Make the Difference?*

Letizia Marsili, Giovanni Di Guardo, Sandro Mazzariol and Silvia Casini

### *87 T Helper Cell Subsets and Their Functions in Common Bottlenose Dolphins (*Tursiops truncatus*)*

Sylvain De Guise, Milton Levin, Lindsay Jasperse, Guillermo Risatti and Randall S. Wells

# Editorial: Comparative Immunology of Marine Mammals

#### Giovanni Di Guardo1 \*, Michael Frederick Criscitiello2 , Eva Sierra3 and Sandro Mazzariol 4

*1 Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy, 2 Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, United States, 3 Faculty of Veterinary Medicine, Instituto Universitario de Sanidad Animal y Seguridad Alimentaria, University of Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain, 4 Department of Comparative Biomedicine and Food Science, University of Padua, Padua, Italy*

Keywords: comparative immunology, innate immunity, acquired immunity, host–pathogen interaction, immunotoxic contaminants, marine mammals, cetaceans, pinnipeds

**Editorial on the Research Topic**

### **Comparative Immunology of Marine Mammals**

Marine mammals rank among the most charismatic creatures inhabiting our planet. Yet despite their long evolutionary history, we have a limited knowledge of their biology, ecology, and behavior, including their morphofunctional adaptations to sea life. This is also frustrating for translational studies, as gas and fat embolic syndrome, a pathologic condition of Ziphiidae (deep-diving cetaceans), mimics decompression sickness (DCS) in human divers (1). Indeed, the development of a DCS-like condition in animals living exclusively in a marine environment for such a long evolutionary history was totally unpredictable!

Marine mammals, comprising cetaceans, pinnipeds, manatees, sea otters, and polar bears, are all included in the International Union for the Conservation of Nature (IUCN) Red List, being increasingly threatened worldwide by several anthropogenic and natural factors, often acting synergistically with each other, as in the case of infectious agents and immunotoxic environmental pollutants (2), high levels of which may be found in tissues from stranded cetaceans, especially dolphins, given their recognized role of "apex predators" (3). Despite the increased efforts made by the scientific community, a considerable knowledge gap still exists on many aspects of marine mammals' biology. This is especially true regarding their immune response(s) at both individual and population levels, an absolute prerequisite to understand their immunopathological counterparts (i.e., immunodeficiencies, hypersensitivity reactions, and autoimmunity), impaired by a substantial amount of data coming from studies performed on stranded animals, which are "not normal" by definition. Within such context, primary fibroblast cell cultures from skin biopsies of free-living pinnipeds and cetaceans have marked a significant progress in our capability of monitoring the effects of a wide range of persistent environmental pollutants on marine mammals' health, including their stress and immune response genetic control (4).

Still noteworthy, aquatic mammals are increasingly threatened by several infectious noxae like morbilliviruses, herpesviruses, Brucella ceti, B. pinnipedialis, and Toxoplasma gondii, just to cite a few (2). Indeed, cetacean morbillivirus (CeMV), phocine distemper virus (PDV), and canine distemper virus (CDV) have recently caused several mass die-offs among dolphin, whale, and seal populations across the globe (5, 6). Consequently, characterizing the host- and the virus-related factors driving the occurrence, the behavior and persistence of morbilliviral infection(s) inside a given marine mammal species and/or population are of paramount importance (7), together with the study of host innate and acquired antiviral immunity.

Edited and reviewed by: *Brian Dixon, University of Waterloo, Canada*

> \*Correspondence: *Giovanni Di Guardo<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Comparative Immunology, a section of the journal Frontiers in Immunology*

Received: *22 July 2019* Accepted: *11 September 2019* Published: *01 October 2019*

#### Citation:

*Di Guardo G, Criscitiello MF, Sierra E and Mazzariol S (2019) Editorial: Comparative Immunology of Marine Mammals. Front. Immunol. 10:2300. doi: 10.3389/fimmu.2019.02300* No information is available, for instance, on the pathogenetic evolution of CeMV, PDV, and CDV infections in Th1-dominant vs. Th2-dominant individuals within susceptible dolphin, whale, and seal species (8). In this respect, ad hoc investigations on human immunodeficiency virus have clearly shown that Th2 dominant patients are much more prone than Th1-dominant individuals to develop full-blown AIDS (9). Studying the host's antiviral immunity could also shed light on the "transmission barrier's jump," which has been repeatedly observed across the Western Mediterranean, in recent years, from CeMV-infected "donor" species (like striped dolphins, Stenella coeruleoalba) to "recipient" species like fin whales (Balaenoptera physalus) (10), sperm whales (Physeter macrocephalus) (11), Cuvier's beaked whales (Ziphius cavirostris) (12), and even common seals (Phoca vitulina) (13) and Eurasian otters (Lutra lutra) (14). Within such context, the possibility that CeMV infection may also spread transplacentally is of concern, given the less efficient immune response during pregnancy (15). A defective functioning of innate and acquired immunity could also represent a key factor underlying viral persistence inside the host's brain, exemplified by the peculiar "brain-only" forms of morbilliviral disease reported in CeMV-infected striped dolphins, which share neuropathologic similarities with subacute sclerosing panencephalitis in measles virus-infected humans and with old dog encephalitis in CDV-infected canines (8). Deepening our knowledge on the comparative immunology of marine mammals may additionally provide valuable insights into the interplay between the infectious pathogens of concern to and the microbiota of pinnipeds, cetaceans, and sea carnivores, on one side, and a wide range of immunotoxic pollutants, which may heavily accumulate inside their tissues, on the other (2, 3). These high contaminant tissue loads could also exert a celltransforming activity, as clearly shown in the beluga whale (Delphinapterus leucas) population residing in the St. Lawrence River Estuary, Canada, in which neoplastic disease was found in association with high tissue burdens of organochlorines, heavy metals, and benzo-a-pyrene. A direct and an indirect carcinogenic activity could have both been involved in the

### REFERENCES

development of the aforementioned neoplasms, together with an antitumor immune surveillance deficiency (16).

A critical component of all studies on the comparative immunology of marine mammals is that the vast majority of the laboratory reagents available on the market have been developed and "validated" only in "conventional" species. Therefore, ad hoc immunologic tools, specifically developed and/or validated also for marine mammals, are urgently needed.

The aforementioned issues represent the focus, along with several others, of the present Research Topic on the comparative immunology of marine mammals, hosting eight original contributions from leading scientists and scientific groups worldwide. In more detail, the monograph's contents address Thelper lymphocytes and other cellular and molecular effectors of cetaceans' immune response, acute-phase proteins and class II major histocompatibility complex in marine mammals, along with antioncogenic virus immune surveillance in California sea lions (Zalophus californianus), cetacean host–pathogen interaction(s), and the comparative immunopathology of CeMV infection in free-ranging dolphins. In this respect, we would like to express our most sincere feelings of appreciation and gratitude to Prof. Miki Nakao, chief editor of Frontiers in Immunology, who enthusiastically accepted our proposal to host this monographic collection in such an outstanding and prestigious Journal, as well as to Dr. Tara Sugrue, Research Topic team leader, Dr. Carmen E. Flores Nakandakare, and all the eminent scientists, associate editors, and reviewers who provided their valuable contributions for the present Research Topic.

### AUTHOR CONTRIBUTIONS

GD wrote the first draft of the present Guest Editorial, which was subsequently revised by MC as well as by ES and SM, who provided their valuable and precious comments, remarks, and suggestions. The final Editorial's draft and the current Editorial's text were entirely agreed and approved by all the 4 Guest co-Editors of the present Research Topic, dealing with the Comparative Immunology of Marine Mammals.

16. De Guise S, Lagacé A, Béland P. Tumors in St. Lawrence beluga whales (Delphinapterus leucas). Vet Pathol. (1994) 31:444–9.

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Di Guardo, Criscitiello, Sierra and Mazzariol. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Cetacean Host-Pathogen Interaction(s): Critical Knowledge Gaps

Giovanni Di Guardo1 \*, Cinzia Centelleghe2 and Sandro Mazzariol 2

1 Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy, 2 Department of Comparative Biomedicine and Food Science, University of Padua, Padova, Italy

Keywords: cetaceans, Cetacean Morbillivirus, Herpesvirus, Brucella ceti, Toxoplasma gondii, host-pathogen interaction, cell receptors, immunotoxic pollutants

Within the broad range of viral and non-viral pathogens infecting cetaceans, Cetacean Morbillivirus (CeMV), Herpesvirus (HV), Brucella ceti, and Toxoplasma gondii are of special concern, due to their impact(s) on the health and conservation of free-ranging cetacean populations worldwide (1). The most "paradigmatic" example in this direction is represented by CeMV, which throughout the last 3 decades has caused more than 10 mass mortality outbreaks among different cetacean species and populations across the globe (2, 3).

Cetaceans live permanently in the marine environment, a peculiar feature differentiating them from pinnipeds, that are also susceptible to morbilliviral infections. This has been clearly shown, for instance, by the dramatic Phocine/Phocid Distemper Virus (PDV) and Canine Distemper Virus (CDV) epidemics among North Sea common seals (Phoca vitulina) and Lake Bajkal seals (Pusa siberica) as well as Caspian seals (Pusa caspica), respectively (4). Due to their "in-water-only" life, stranded cetaceans play a key role as "sentinels" (potentially) able to "recapitulate" the "natural history, evolution, ecology, epidemiology, and encounters" of infectious noxae, on one side, and cetacean hosts, on the other. Consequently, based upon their crucial relevance as "health and conservation biomonitors" for their increasingly threatened "conspecifics and heterospecifics" living in the open sea, a detailed post-mortem examination of stranded cetaceans on behalf of specifically trained veterinarians is mandatory!

These concepts are nicely exemplified by the motto "Hic est locus ubi mors gaudet succurrere vitae." This phrase, written in the nineteenth century by Luciano Armanni—the co-discoverer of "Armanni-Ebstein Diabetic Nephropathy" (5) -, stands at the entrance of the autopsy room of "Ospedale degli Incurabili" in Naples, Italy. Literally translated, it means "This is the place where death is pleased to support life."

Taking into consideration all the above, we believe there are still a number of critical "knowledge gaps" regarding "cetacean host(s)-pathogen(s) interaction(s)," with special emphasis on the 4 herein dealt infectious noxae and, most likely, also in relation to any other pathogen infecting wild cetaceans.

These "knowledge-deficient areas" may be identified as follows: (1) characterization of the cell receptor(s) allowing infection; (2) interaction(s) and effects of chemical pollutants on the expression levels of the aforementioned cell receptors; (3) pathogenetic evolution of the concerned infections in T helper 1 (Th1)-dominant versus (vs.) Th2-dominant cetacean individuals; (4) effects of pregnancy-associated immune status on the infectious potential of the herein dealt pathogens; (5) usefulness of cetaceans and their pathogens as models for human disease.

The present Opinion Article, after a brief introduction on these 5 issues, will critically address each of the aforementioned knowledge gaps.

#### Edited by:

Fabrizio Ceciliani, Università degli Studi di Milano, Italy

### Reviewed by:

Francisco Javier Aznar, University of Valencia, Spain

### \*Correspondence:

Giovanni Di Guardo<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Comparative Immunology, a section of the journal Frontiers in Immunology

Received: 14 September 2018 Accepted: 14 November 2018 Published: 28 November 2018

#### Citation:

Di Guardo G, Centelleghe C and Mazzariol S (2018) Cetacean Host-Pathogen Interaction(s): Critical Knowledge Gaps. Front. Immunol. 9:2815. doi: 10.3389/fimmu.2018.02815

**7**

As far as issue (1) is specifically concerned, CeMV, HV, B. ceti, and T. gondii may infect several cetacean hosts, with the latter 2 agents also carrying a zoonotic potential (1). Since different tissues from susceptible cetaceans may be colonized by the 4 herein dealt pathogens (1, 2), a detailed characterization of the cell receptor(s) allowing their entry into (and subsequent shedding from) host's tissues would be of paramount relevance. In this respect, as with other animal and human morbilliviruses, the lymphotropic behavior typically displayed by CeMV is specified by the lymphoid cell receptor "Signaling Lymphocyte Activation Molecule" (SLAM/CD150) (2). Notwithstanding the above, the cell receptor(s) targeted by this lympho-epithelioneurotropic virus within the central nervous system (CNS) from susceptible cetacean hosts is/are still unknown (6, 7).

Noteworthy, striped dolphins (Stenella coeruleoalba) infected by Dolphin Morbillivirus (DMV, a CeMV strain) may occasionally develop a peculiar, "brain-only" form of infection sharing neuropathologic similarities with "Subacute Sclerosing Panencephalitis" (SSPE) and "Old Dog Encephalitis" (ODE) in Measles Virus (MeV)-infected humans and CDVinfected canines, respectively (2, 8–10). Despite the recent characterization of the neuronal and non-neuronal cell populations residing in the brain from striped dolphins affected by such neuropathy (11), the receptor(s) allowing viral persistence and spread within their brains—as well as in those from SSPE-afflicted patients and ODE-affected dogs—is/are also undetermined (6, 7, 12).

Identical considerations apply to HV, B. ceti, and T. gondii, provided that the cell receptor(s) allowing their entry into and subsequent dissemination throughout cetacean hosts' tissues have not been yet identified, to the best of our knowledge. Making specific reference to B. ceti, the host's cellular prion protein (PrPC) has been recently hypothesized to serve as a neuronal cell receptor for this zoonotic microorganism causing fatal neurobrucellosis in striped dolphins (13). Indeed, PrPC had been previously recognized as a receptor for B. abortus heat shock protein (HSP)60—a member of the GroEL family of chaperonins—on murine macrophages, thereby allowing bacterial internalization and establishment of B. abortus infection inside these cells (14).

As far as concerns issue (2), it is well established that a wide range of persistent environmental pollutants may heavily accumulate in cetacean tissues, especially in those of "top predators" like dolphins and other Odontocetes, with simultaneous "biomagnification" processes additionally occurring in most cases (15). Many of these pollutants, as in the case of lipophilic polychlorinated biphenyls (PCBs), dioxins and dioxin-like substances, along with methylmercury (MeHg), have also been shown to exert powerful immunotoxic effects (16). Notwithstanding the above, we are not aware of any study investigating the relationship(s), if any, between pollutant-related immunotoxicity, on one side, and the tissue expression profiles, on the other, of given cell receptors (e.g., SLAM/CD150) for highly immunosuppressive agents like CeMV (6). This should be regarded as another critical knowledge gap within the general framework of "cetacean host(s)-pathogen(s) interaction dynamics." Within such context, the growing concerns over the exponentially increasing plastic pollution of oceans and seas across the entire globe should be also taken into account. As a matter of fact, relevant health- and conservation-related issues arise for fish, birds and aquatic mammals, due to their prolonged exposure to micro-nanoplastics through the marine food web(s). Furthermore, the documented roles of "plastic debris" as an "attractor and concentrator" for many persistent pollutants like PCBs, dioxins and other organochlorine (OC) and non-OC compounds (15), as well as for a huge number of invertebrate organisms (17), would deserve special consideration. In this respect, plastics/micronanoplastics-based "rafts" have been recently hypothesized to play a role also in the ecology and epidemiology of T. gondii infection (18). This could be of interest, provided that the Scientific Community has not yet clarified by which modalities and dynamics striped dolphins and other typically "pelagic" or "offshore" cetaceans may acquire an "oro-faecally transmitted infection" characterized by a "land-to-sea flow," as in the case of that caused by the zoonotic protozoan T. gondii (19).

As far as issue (3) is specifically concerned, among the many lessons provided by natural history of Human Immunodeficiency Virus (HIV) infection in mankind, we have learned that Th2-dominant patients are much more prone than their Th1-dominant "counterparts" to develop "full-blown AIDS" ("Acquired Immunodeficiency Syndrome") in the time course of HIV infection (20). In this respect, while in recent years we have also learned quite a bit on the pathogenetic evolution of other human and animal viral and non-viral infections in Th1 dominant vs. Th2-dominant individuals, we are unaware, on the contrary, of any published reports dealing with the pathogenetic behavior of CeMV infection—as well as of HV, T. gondii, and B. ceti infections—among Th1-dominant vs. Th2-dominant cetacean hosts.

This could of relevance also in relation to issue (4), given that a reduced efficiency of host's immune response is physiologically observed during pregnancy (21). Indeed, several cases of DMV infection have been recently described both in newborns and in cetacean fetuses (6, 22–24). This concern and its related knowledge gap are additionally amplified by the documented occurrence of cases of DMV infection in cetacean species mainly from the Western Mediterranean Sea—into which the virus has apparently "jumped," most likely as the result of recent "spillovers" from DMV-infected striped dolphins (22–24).

Finally, with specific reference to the last of the 5 herein dealt issues, the potential role of cetaceans as "models for human disease" should be also taken into account (25). In fact, as recently reported for Alzheimer's disease-related neuropathology in striped and bottlenose dolphins (Tursiops truncatus) (26, 27), and as previously described for the aforementioned "brainonly" forms of DMV infection among striped dolphins (6, 8– 10), cetaceans and—more in general—aquatic mammals could serve as valuable "models" also in "Comparative Immunology and Immunopathology."

This could apply, in parallel with the herein dealt "host(s) pathogen(s) interaction dynamics," also to the "ontogeny" and "evolutionary phylogeny" of cetaceans' immune response as well as to further key issues like "immune tolerance," "autoimmunity," and "immune surveillance against neoplasia," just to cite a few.

In conclusion, deepening our understanding of "host(s)-pathogen(s) interaction(s)" in cetaceans and, more broadly, in marine mammals, may provide not only a very useful and insightful "set of tools" to monitor and protect their increasingly threatened health and conservation, but also a reliable and precious source of knowledge highlighting the simultaneous role of cetaceans as putative "models for human (and animal)

### REFERENCES

disease" as well as for "Comparative Immunology and Immunopathology."

### AUTHOR CONTRIBUTIONS

GDG wrote the first draft as well as the first revision's draft of the present Opinion Article, to be included in the Research Topic Comparative Immunology of Marine Mammals, with CC and SM subsequently integrating and providing a thorough and critical revision of both the aforementioned manuscript's drafts.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Di Guardo, Centelleghe and Mazzariol. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Transcriptional Profiles of California Sea Lion Peripheral NK and CD+8 T Cells Reflect Ecological Regionalization and Infection by Oncogenic Viruses

Ignacio Peñín1 , Mónica E. Figueroa-Cabañas 1 , Fabiola Guerrero-de la Rosa1 , Luis A. Soto-García1 , Roberto Álvarez-Martínez 1 , Adriana Flores-Morán1 and Karina Acevedo-Whitehouse1,2 \*

1 Unit for Basic and Applied Microbiology, School of Natural Sciences, Autonomous University of Queretaro, Santiago de Queretaro, Mexico, 2 The Marine Mammal Center, Sausalito, CA, United States

#### Edited by:

Mike Criscitiello, Texas A&M University, United States

#### Reviewed by:

Jorge Galindo-Villegas, Nord University, Norway Javier Santander, Memorial University of Newfoundland, Canada

\*Correspondence:

Karina Acevedo-Whitehouse<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Comparative Immunology, a section of the journal Frontiers in Immunology

Received: 13 October 2018 Accepted: 15 February 2019 Published: 12 March 2019

#### Citation:

Peñín I, Figueroa-Cabañas ME, Guerrero-de la Rosa F, Soto-García LA, Álvarez-Martínez R, Flores-Morán A and Acevedo-Whitehouse K (2019) Transcriptional Profiles of California Sea Lion Peripheral NK and CD+8 T Cells Reflect Ecological Regionalization and Infection by Oncogenic Viruses. Front. Immunol. 10:413. doi: 10.3389/fimmu.2019.00413 The California sea lion is one of the few wild mammals prone to develop cancer, particularly urogenital carcinoma (UGC), whose prevalence is currently estimated at 25% of dead adult sea lions stranded along the California coastline. Genetic factors, viruses and organochlorines have been identified as factors that increase the risk of occurrence of this pathology. Given that no cases of UGC have as yet been reported for the species along its distribution in Mexican waters, the potential relevance of contaminants for the development of urogenital carcinoma is highlighted even more as blubber levels of organochlorines are more than two orders of magnitude lower in the Gulf of California and Mexican Pacific than in California. In vitro studies have shown that organochlorines can modulate anti-viral and tumor-surveillance activities of NK and cytotoxic T-cells of marine mammals, but little is known about the activity of these effectors in live, free-living sea lions. Here, we examine leukocyte transcriptional profiles of free-ranging adult California sea lions for eight genes (Eomes, Granzyme B, Perforin, Ly49, STAT1, Tbx21, GATA3, and FoxP3) selected for their key role in anti-viral and tumor-surveillance, and investigate patterns of transcription that could be indicative of differences in ecological variables and exposure to two oncogenic viruses: sea lion type one gammaherpesvirus (OtHV-1) and sea lion papillomavirus type 1 (ZcPV-1) and systemic inflammation. We observed regional differences in the expression of genes related to Th1 responses and immune modulation, and detected clear patterns of differential regulation of gene expression in sea lions infected by genital papillomavirus compared to those infected by genital gammaherpesvirus or for simultaneous infections, similar to what is known about herpesvirus and papillomavirus infections in humans. Our study is a first approach to profile the transcriptional patterns of key immune effectors of free-ranging California sea lions and their association with ecological regions and oncogenic viruses. The observed results add insight to our understanding of immune competence of marine mammals, and may help elucidate the marked difference in the number of cases of urogenital carcinoma in sea lions from US waters and other areas of their distribution.

Keywords: California sea lion, cytotoxicity, NK, CD8+ T cells, otarine gammaherpesvirus, sea lion papillomavirus

## INTRODUCTION

The California sea lion (Zalophus californianus) is one of the few wild mammals prone to develop cancer under natural conditions. Since the initial report in the early 80's, the prevalence of urogenital carcinoma has remained consistently high, with post mortem examination of dead individuals revealing a prevalence of up to 25% in adult sea lions necropsied after stranding along the California coast (1). The high incidence of such an aggressive and fatal pathology in a long-lived top predator of the coastal marine ecosystem warrants studies to increase our understanding of the factors that contribute to its occurrence.

As is the case for most cancers, sea lion urogenital carcinoma appears to be multifactorial, and various risk factors have been identified. These factors include an oncogenic genital gammaherpesvirus, named OtHV-1 (2, 3) and genetic components (4–6). Furthermore, high concentrations of organochlorines have been detected in the blubber of sea lions with urogenital carcinoma (7). This latter association is particularly relevant as studies conducted in laboratory animals have shown that organochlorines can induce carcinogenesis, either directly at high concentrations (8) or indirectly by modulating immune responses, particularly when exposure is low (9–11). In vitro experiments with different marine mammal cells have shown that organochlorines modulate NK and cytotoxic T-cell activity (12–14). Based on their known anti-viral and tumor surveillance activity (15–17), and the evidence of organochlorine-induced modulation, it is parsimonious to speculate that NK and cytotoxic T-cells play an important role in preventing the development of urogenital carcinoma in the California sea lion, and that these immune effectors are sensitive to extrinsic and intrinsic factors.

Despite its high prevalence in California, urogenital carcinoma has not been observed in sea lions inhabiting the Gulf of California, in spite of systematic surveys of the breeding colonies by researchers and park managers. However, precancerous transformation of the genital epithelium, including binucleation and koilocytes, do appear to be relatively common in California sea lions from the Gulf of California (18). In humans, the presence of these cellular phenotypes is considered the first step toward carcinogenesis if the abnormal cells are not promptly detected and destroyed by tumor-surveillant and cytotoxic immune cells (19). Interestingly, compared to values reported for sea lions in California (7), blubber PCB levels are three orders of magnitude lower (20) in sea lions from the Gulf of California, and two orders of magnitude lower in sea lions from the Mexican North Pacific (21). This implies that there could be differences in NK and cytotoxic T-cell activity (12–14), which could, in turn, result in differences in oncogenesis.

Within the Gulf of California, 13 sea lion breeding colonies are spread along 177,000 km2 , from the northernmost colony, Rocas Consag, located at less than 100 km from the Colorado River Delta, to the southernmost colony, Islotes, 29 km from the city of La Paz, in the tip of the peninsula of Baja California. Oceanographic and ecological differences among zones have led to regionalization of the Gulf of California, and colonies are grouped in four main regions (22), largely defined by upwelling and phytoplankton profiles (23) that influence the availability of resources (24, 25). Sea lion colonies vary per region in terms of population trends (26), genetic substructure (27, 28) and pathogen exposure (29). In terms of pollutants, there is a marked latitudinal gradient, with the northern region being more polluted than the southern region due to deposition from the Colorado River Delta (30).

Based on the species' genetic substructure in the Gulf of California, and on spatial differences in oceanographic and ecological factors, it is plausible to assume that California sea lions experience intra- and interregional differences in terms of their exposure to organochlorine pollutants and other extrinsic factors that could impact their tumor surveillance and cytotoxic capability, as well as induce chronic inflammation. Furthermore, as disposable energy in a region is limited by its productivity, prey availability, and feeding range (25), it is unlikely that a sea lion's investment of resources for immune activities will be independent of its environment. Some evidence for this phenomenon has already been reported for this species (31).

Lymphocyte development and activation are characterized by marked changes in gene expression (32). Thus, we hypothesized that transcription of key genes relevant to immune surveillance of tumors and cytotoxic responses of California sea lions differs spatially and is related to genital infection by two oncogenic viruses, sea lion type one gammaherpesvirus (OtHV-1) and sea lion papillomavirus type 1 (ZcPV-1), and systemic inflammation. To challenge our hypotheses, we examined transcription patterns of key genes expressed by NK, CD8+ T cells and by T regulatory cells in the blood of apparently healthy adult females sampled at breeding colonies within the Gulf of California and the Mexican North Pacific.

## MATERIALS AND METHODS

### Collection of Samples

During the summer of 2016 we visited 12 California sea lion breeding colonies in the Gulf of California. Based on the ecological regionalization proposed (22), we sampled sea lions in three colonies in the Northern region (Rocas Consag, San Jorge, and Lobos), three in the North-Central (Midriff) region (Granito, Cantiles, and Los Machos) five in the Central region (Partido, El Rasito, San Esteban, San Pedro Mártir, and San Pedro Nolasco), and one (Los Islotes) in the far South region (**Figure 1**).

Samples of peripheral blood were collected from 54 apparently healthy adult female California sea lions, which had been captured using hoop nets, and manually restrained before using inhaled anesthesia (Isoflurane). Once anesthetized, a trained veterinarian examined the sea lions in order to determine their general health, and to monitor their heart rate and respiratory frequency throughout the procedure, which lasted 10–12 min. All sea lions were considered to be in good body condition, with at least 20 mm of lateral blubber depth (skin fold thickness measured with calipers). Two 7-ml blood samples were collected from the caudal gluteal vein of each sea lion with vacuum tubes (Vacutainer), one of them coated with sodium heparin and one with EDTA. Genital epithelial swabs were also collected. Briefly, a sterile genital speculum was introduced to visualize the cervix

and the cervical mucosa was scraped with a sterile cytobrush. The swabs were stored in a vial containing 96% ethanol and were protected from sunlight until staining in the laboratory. The blood samples were centrifuged using a clinical centrifuge (Clay Adams compact II, Daigger Scientific, USA) for 10 min at a fixed speed of 3,200 rpm within 3 h after collection. The buffy coat was separated using a sterile Pasteur pipette and stored immediately in cryogenic tubes prior to snap-freezing in liquid nitrogen, where kept until processing. EDTA-preserved blood was used to total and differential leukocyte counts. Based on the hematological information, we determined the neutrophil to lymphocyte ratio (NLR) as a measure of chronic systemic inflammation and stress (33), which has been shown to be a useful marker in the California sea lion (34). We also had equivalent samples from nine adult female sea lions, which had been captured during the summer of 2014 at a colony situated in the Archipelago of San Benito, in the Mexican North Pacific (see **Figure 1**). Sampling and processing of these samples was performed as described above. Adult males were not included in our study due to the difficulty and extremely high-risk of capturing and restraining these individuals in the field.

All procedures were approved by the Bioethics committee of the Universidad Autónoma de Queretaro (Mexico), and were conducted under permits SGPA/DGVS/11744/13 (for samples collected in 2014) and SGPA/DGVS/09004/15 (for samples collected in 2016) issued by the Secretariá de Medio Ambiente y Recursos Naturales through the Direccion General de Vida ´ Silvestre in Mexico.

### Relative Quantification of Gene Expression in Lymphoid Subpopulations

We extracted total RNA from the buffy coat samples using Trizol (Sigma-Aldrich, USA) as per the manufacturer's instructions. We treated the solubilized RNA with DNA-freeTM DNA removal kit (Thermo Fisher Scientific, USA) as per the manufacturer's instructions. RNA integrity was assessed by electrophoresis in a 1% agarose gel stained with ethidium bromide and purity was determined by Nanodrop spectrophotometry (Qiagen, USA). Good quality samples were those that had no evidence of RNA degradation, showed clear 28S and 18S rRNA bands (see **Figure S1**), and whose A260/A280 ratio was between 1.75 and 2.10. Five of the RNA samples did not clearly show the two rRNA bands despite having a good A260/A280 ratio. In these cases, we re-extracted the RNA and reassessed the quality. In all of these cases, we were able to confirm that quality was adequate to proceed with reverse transcription (see **Supplementary Material** for more details).

Reverse transcription was performed for each sample with a QuantiTect Reverse Transcription Kit (Qiagen, USA) using 200 ng of RNA in 20 µL reactions. As per the manufacturer's instructions, the procedure included a 2 min. incubation at 42◦C with gDNA wipeout buffer to further ensure the elimination of genomic DNA contamination. cDNA was frozen in aliquots to avoid repeated freeze-thaw cycles.

The levels of transcription of eight genes were assessed by real time quantitative PCR (RT-qPCR). The genes were selected a priori to represent the activity of different lymphocyte subpopulations and immune activities relevant to antiviral and antitumor activities (see **Table 1** for genes and primers). RPS5 and HpRT were used as reference (housekeeping) genes as they are expressed in all nucleated cells, and we have previously shown their transcription levels to be stable in California sea lion peripheral white blood cells (35). In order to comply with the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (36), we used a subset of 10 samples to run RT-qPCR to evaluate expression stability. This allowed us to confirm that both genes were appropriate to use as reference genes (see details and **Figure S2**). All primers were designed to span exon-exon junctions. All primer pairs were evaluated for their efficiency. The efficiency of the primers ranged between 90 and 112%, and the coefficient of determination (R 2 ) was >0.95 (E and R 2 results for all the primers used for RT-qPCR can be seen as **Figures S3, S4**).

RT-qPCR reactions contained 4 microliters of cDNA (1:4 to 1:16 of template generated by retrotranscription of 10 ng/µL of RNA), 0.15 µL of each primer (at 0.2µM each), 5 µL SYBR R Green master mix (Thermo Fisher), and 0.7 µL of water to reach 10 µL of final volume. The reactions were run on a CFX ConnectTM Real-Time PCR Detection System (BioRad, USA) as follows: 95◦C for 15 min, followed by 40 cycles of 15 s at 95◦C, 1 min at 55C (during which the plate was read) and 72◦C for 1 min. The ending cycle was kept at 95◦C for 15 s and a final step for the melting curve at 60 to 90◦C (0.5◦C increase and 15 s of wave length measurement for each temperature). Reaction specificity was monitored by melting curve analysis using a final data acquisition phase of 60 cycles of 65◦C for 30 s. The threshold was established manually after amplification take-off. Optimal results were achieved when using 1:2–1:8 of cDNA. For each primer pair, a reaction mixture containing water, but no cDNA, was used as a non-template control (NTC) to monitor contamination and primer dimer formation; a noreverse transcriptase (-RT) mixture was included as a control to monitor DNA contamination. Gene expression levels were determined by relative quantification (i.e., transcription of the target gene relative to the average of the reference genes) as per the 1Ct method (37). The data were considered reliable if the difference between replicates was below one cycle. If the reliability of the reference genes failed, the samples were rerun for all the primer pairs of a plate. Optimal results were achieved when using 1:2–1:8 of cDNA.

### Molecular Detection of Oncogenic Viruses

Genomic DNA was extracted from the ethanol-preserved genital swabs using a routine proteinase K digestion followed by a phenol-chloroform protocol and isopropanol precipitation. DNA was quantified and quality was assessed using a spectrophotometer (Nanodrop 2000, Thermo Fisher Scientific, USA). We amplified a 210 bp fragment of the DNA polymerase (Dpol) gene of OtVH-1 in genital swabs. Primers used were Dpol 697 5′ - GCGGGAACGCAACTATATCCT and Dpol 65 5′ -TCT TCGTCCAGTATCATTG; (38). For ZcPV-1, we used GenBank sequence NC\_015325 (Zalophus californianus papillomavirus 1) as a reference to design a pair of primers that amplified a 450 bp fragment of gene L2 (ZcPV\_F5957-ATACAGGACGGGGACA TGG, ZcPV\_R6495-TCATATTCCTCAGCGTGCCT).

All 12.5 µl PCR reactions were performed on an ABI 3100 thermal cycler (Applied Biosystems, Inc.) and were conducted in duplicate. Cycling conditions were 95◦C for 15 min, 30 cycles of 94◦C for 40 s, 52◦C (OtHV-1) or 53◦C (ZcPV-1) for 30 s and 72◦C for 40 s, and a final extension step at 72◦C for 10 min. A template free (template free reaction) was included with every PCR. Amplified products were electrophoresed on an ethidium bromide stained 1.8% agarose gel and visualized on a UV transilluminator. To ensure the amplified products were not the result of non-specific amplification, for each pathogen, two bands selected at random were gel excised, column purified (QiaQuick, Qiagen, USA), cloned and bi-directionally sequenced for confirmation. Each sequence was visually inspected and compared to those reported in GenBank (http://www.ncbi.nlm. nih.gov/genbank/).

### Statistical Analyses

Before beginning the analyses, we examined the normality and homoscedasticity of the relative level of transcription of each target gene with Shapiro and Bartlett tests, respectively. None of the gene expression levels showed deviation of normality or heteroscedasticity. We used Spearman correlations to identify relationships between gene transcripts. The GATA3 to Tbx21 (hereafter Ga/Tb) ratio, a measure of Th1/Th2 profile (39) deviated from expectations of normality and, based on a Cullen and Frey graph, appeared to follow a beta distribution. Goodness of fit was examined with a Kolmogorov test.

To challenge our hypotheses, we first built generalized linear models (GLM) for each of the genes, and the Ga/Tb ratio defining the error family as per the distribution of the error (40), and indicating the region where each sea lion was sampled as the explanatory variable. We next used independent GLMs to examine whether the transcription level of each gene and the Ga/Tb ratio was affected by the presence of OtHV-1, ZcPV-1, or simultaneous infection by both viruses in the genital epithelium, and whether gene transcription levels were influenced by systemic inflammation, as assessed by the NLR of the sea lions. For genes whose transcription levels varied among regions, we included this variable in the respective models. We used a top-down strategy to determine which variables explained a significant fraction of the data (40), and we examined the residual distribution by inspecting Q-Q plots, Cook distance and plots of the adjusted residuals vs. the obtained residuals to validate the model. As lower 1Ct values represent higher levels of expression, interpretation was made easier by using a negative transformation of the response variable (–log 1Ct). To account for differences in prevalence of OtVH-1, ZcPV-1 and simultaneous infection among regions, we built contingency

tables and ran Fisher exact tests. In all cases we considered results statistically significant if the p-value was less than 0.05.

Our second approach was to analyze gene transcription profiles with no pre-defined regionalization to allow natural clustering of immune profiles. For this, data were subjected to hierarchical clustering to generate heat maps and dendrograms of the gene transcripts according to the degree of similarity of the transcriptional profiles. Multiple imputation chain equation was used to account for missing values. We first used the full set of target genes, as the "complete profile" of each individual. Next, we used the relative expression of genes representative of responses relevant to this study: (i) NK and CD8+ activity and differentiation (Ly49, perforin, granzyme, STAT-1, Tbx21, and Eomes), (ii) Th2 and immune modulation (GATA3 and FoxP3), and (iii) Th1/Th2 ratio (Tbx21 and GATA3). The number of natural clusters was determined according to a rarefaction curve and two dendrograms were initially created for each set of genes. Algorithms used were Ward.D2 and Average (41). Goodness of fit of the models was based on the cophenetic correlation coefficient, with an optimality value of ≥0.8. As values were lower than 0.8 for all models, we used an Euclidean approach to build the final dendrogram (42). All statistical analyses were conducted in R v3.4.2 (43) using core packages as well as cluster, dendextend, factoextra, fitdistrplus, mice, prevalence and ggplot2.

### RESULTS

## NK and CD8+ Activity

Most of the transcription levels of the six genes involved with NK and CD8+ activity were significantly correlated to each other (**Table 2**), with correlation coefficients ranging from 0.75 to 0.31. The exceptions were Ly49 and perforin, both of which were not correlated to STAT-1.

Transcription levels of Ly49, perforin and granzyme B did not vary significantly among regions [GLM; Ly49: F(4, 49) = 0.21, p =

TABLE 2 | Spearman's correlation coefficients of genes expressed by NK and CD8+ T cells.

The bottom half of the table shows p-values.

0.932, **Figure 2C**; perforin: F(4, 31) = 1.44, p = 0.244, **Figure 2B**; granzyme B: F(4, 34) = 1.26, p = 0.304; **Figure 2A**]. In nearly all sea lions, granzyme B was upregulated more than 2-fold with respect to the reference genes, while perforin showed the inverse pattern, being downregulated on average 2.73-fold; Ly49 was slightly downregulated in all but a few individuals, with only a few sea lions showing upregulation.

Transcription of the three transcription factors involved with Th1 development varied among regions [GLM; Eomes: F(4, 30) = 2.64, p = 0.049, **Figure 2D**; Tbx21: F(4, 49) = 6.22, p = 0.016, **Figure 2E**; STAT-1: F(4, 49) = 3.58, p = 0.012; **Figure 2F**]. Eomes was slightly downregulated (average: 0.93-fold) with respect to the reference genes in the majority of sea lions from colonies within the midriff and central regions, and upregulated in the northern and southern regions, as well as in the Mexican North Pacific (average: 0.38-fold). Tbx21 was mostly downregulated, and levels increased across samples collected from north to south within the Gulf of California, reaching the highest values in animals from the Mexican North Pacific (Adj. R 2 = 0.09, df = 52, p = 0.016; **Figure 2E**). In contrast to the other genes indicative of Th1 development, STAT-1 was generally upregulated, and levels of transcription also exhibited a north to south trend within the Gulf of California, reaching the highest values in the sea lions

FIGURE 2 | Transcription levels of selected genes in California sea lion blood sampled at different regions. (A) Granzyme B, (B) Perforin, (C) Ly49, (D) Eomes, (E) Tbx21, (F) STAT-1, (G) GATA3, (H) FoxP3. Ly49, perforin, granzyme B and FoxP3 did not vary significantly among regions. Eomes was slightly downregulated in sea lions from the midriff and central regions, and upregulated in the northern and southern regions, and the Mexican North Pacific. Tbx21, STAT-1 and GATA3 increased from north to south within the Gulf of California and reached highest levels in the Mexican North Pacific. The plot shows the median (thick line), first and third quartile (box), and 95% confidence interval of the median (whiskers).

from the Mexican North Pacific (Adj. R 2 = 0.11, df = 52, p = 0.007; **Figure 2F**).

### Th2 and Immune Modulation

GATA3 and FoxP3 were significantly correlated (r = 0.39, p = 0.006). Transcription of GATA3 varied significantly among regions [GLM; F(4, 49) = 3.19, 0.017], with a north-to-south increasing gradient within the Gulf of California, and showing the highest levels of transcription in sea lions from the Mexican North Pacific (Adj r 2 = 0.12, df = 52, p = 0.006; **Figure 2G**). FoxP3 was consistently downregulated (average: 5.29-fold), with no significant differences among regions [GLM; F(4, 44) = 1.35 p = 0.268; **Figure 2H**].

## Th1/Th2 Ratio

There was a wide variation in Ga/Tb, with values ranging to 45.29 to −1.49 (mean = 1.64), and there was no evidence of differences among regions [GLM; F(4, 49) = 0.81, p = 0.528].

## Patterns of Gene Transcription Levels Using no Pre-defined Regionalization

When considering transcriptional patterns of all target genes together, no clear grouping emerged (see **Figure S5**). A few sea lions had a distinct pattern, and each belonged to a different region. The transcriptional profile related to the NK and CD8+ cells (considering Ly49, perforin, granzyme, STAT-1, Tbx21, and Eomes transcription levels) showed some heterogeneity and clustering among sea lions (**Figure 3A**), and the best dendrogram separated the samples in three main branches (**Figure 3B**). One of the branches harbored 92% (12/13) of the sea lions sampled in the northern region, and 90.1% (10/11) of the sea lions sampled in the midriff region. Furthermore, sea lions from these regions were grouped into smaller clusters, separating them from samples from the central and southern regions, as well as from the Mexican North Pacific. The second main branch grouped only one sea lion from the northern Gulf of California, two from the midriff region, and a mixture of sea lions from southern and central regions, and from the Mexican North Pacific. The third main branch separated a single sea lion sampled in the southern Gulf of California.

The clustering analysis of Th2 and immune modulation profiles revealed some heterogeneity and clustering (**Figure 4A**), and the dendrogram separated the samples in three main branches (**Figure 4B**); the first branch grouped all of the sea lions from the northern region, 91% (10/11) of the sea lions from the midriff region and a few sea lions from the other regions; namely, four from the central region, one from the southern region and two the Mexican North Pacific. The second branch harbored sea lions from the southern (7/8) and central (8/11) regions, and from the Mexican North Pacific (8/11). One sea lion from the central region was separated from the rest in its own branch.

Clustering patterns of Th1/Th2 profiles were less clear than observed for the other profiles (**Figure 5A**). The dendrogram separated the samples in four main branches (**Figure 5B**); one grouping 92% (12/13) of the sea lions sampled in the northern region, 91% (10/11) of the sea lions from the midriff region, 42% (5/12) of the sea lions sampled in the central region, and 18% (2/11) of the sea lions from the Mexican North Pacific. The second and third branches grouped a mixture of sea lions from the central and south regions, and from the Mexican North Pacific. The fourth main branch separated a single sea lion sampled in the central region.

### Gene Transcription and Genital Infection by Oncogenic Viruses

The prevalence of genital viral infections varied significantly across regions (**Figure 6**). Genital ZcPV-1 infections were highest in the Mexican North Pacific followed by the midriff and southern Gulf of California (Pearson's Chi2 = 11.32, df = 4, p = 0.042), while OtHV-1 infections were highest in the central region, followed by the northern and midriff regions (Pearson's Chi2 = 26.39, df = 4, p = 2.71 × 10−06). The prevalence of simultaneous infections by both viruses also varied among regions, with a similar pattern to that observed for OtHV-1 (Chi2 = 11.757, df = 4, p = 0.0231).

Infection status impacted transcription of four genes related to cytotoxicity, even when accounting for spatial differences [GLM; Eomes: F(3, 29) = 4.21, p = 0.015, **Figure 7A**; perforin: F(3, 30) = 4.743, p = 0.008, **Figure 7B**; Tbx21: F(3, 48) = 4.16, p = 0.012, **Figure 7C**] granzyme B: F(3,33) = 3.39, p =

0.036, **Figure 7D**. The pattern was consistent and contrasting between viruses. Namely, sea lions infected by OtHV-1 had transcription levels similar to or lower than those of noninfected sea lions and sea lions simultaneously infected by both viruses. In contrast, sea lions infected by ZcPV-1 had significantly higher transcription levels. None of the other target genes were significantly influenced by genital infection status. In order to examine whether the expression levels of Tbx21 and Eomes in sea lions with single infections (either ZcPV-1 or OtHV-1) or with concomitant infections were associated with the up-regulation of inhibitory receptors or suggested a skewed maturation phenotype, we examined the relationship of transcription levels of these genes with those of the inhibitory receptor Ly49. We found no correlation for sea lions infected

the northern and midriff regions, and concurrent infections by both viruses varied significantly among regions, following the pattern observed for OtHV-1. Bars = ± s.e.

simultaneously by both viruses (r 2 = 0.04, p = 0.52), nor was there a significant relationship for sea lions that were infected only by OtHV-1 (r 2 = 9.11, p = 0.43) or only by papillomavirus (r 2 = 0.12, p = 0.26; see **Figure S6**).

### Gene Transcription and Inflammation

The marker of chronic systemic inflammation and stress here used, NLR, did not vary significantly across regions. However, the variance differed significantly [F-test; F(39, 55) = 0.37, p = 0.002], being larger for sea lions from the northern and midriff regions (**Figure 8A**). Eight percent of the variation in transcription levels of Ly49 was influenced by the NLR, and higher NLR values were associated with lower levels of transcription [F(1, 38) = 4.19, p = 0.048, **Figure 8B**]. None of the other target genes were significantly impacted by the NLR of the sea lions sampled.

## DISCUSSION

Our study examined the transcriptional levels of key genes related to cytotoxic responses to viruses and tumor cells, as well as those that are involved with immune modulation in free ranging California sea lions. We found indication of regional differences in the expression of genes related to Th1 responses and immune modulation, and detected different transcription levels in sea lions infected by genital papillomavirus compared to those infected by genital gammaherpesvirus. Furthermore, when analyzing the gene expression profiles according to functional groupings, a north to south division became apparent, where sea lions from the northern and midriff regions were clustered together, while most sea lions from the central and southern regions, as well as from the Mexican North Pacific, clustered together in another group. As is unavoidably the case when studying free-ranging species, our results are correlative, and impede the inference of direct causation. Furthermore, given that we focused on gene transcription rather than on protein expression, we cannot make inferences regarding the expression of the gene products themselves. Nonetheless, the patterns observed were consistent with what is known for herpesvirus and papillomavirus infections in humans and model species, and the relationships in gene expression levels between the markers selected are strongly suggestive of differential ecological impacts on immune effectors. Owing to the paucity of information regarding pinniped immunity, we will discuss our findings in light of what is known for humans and model animals.

Transcription of two genes, granzyme B and perforin, directly involved with cytotoxic responses did not vary amongst ecological regions. Granzyme B and perforin are expressed by NK and CD8+ T cells, and are crucial to induce apoptosis of transformed or virus-infected cells (44). Both genes are induced progressively upon activation by antigen-presenting cells. Granzyme B encodes a serine protease (granzyme B) that is released together with perforin, a glycoprotein encoded by the perforin gene. Perforin forms a pore in the membrane of the target cell, allowing granzyme B to enter the target cell and trigger apoptosis via the activation of various caspases, generation of mitochondrial reactive oxygen species, and DNA fragmentation (45). The nearly 5-fold difference in transcription levels between both genes implies that individual sea lion NK and CD8+ T cells transcribe different combinations of perforin and granzyme B, and that each gene is differentially regulated at the single cell level (46). It is possible that, as has been reported for humans (47), granzyme B could exhibit some perforin-independent activity in the California sea lion, Alternatively, it is also plausible that the results indicate that for every transcribed mRNA of perforin, more granzyme B transcription is required.

We also selected Ly49 as a marker of NK function, and found its expression to be slightly downregulated with respect to the reference genes in nearly all of the sea lions. Ly49 encodes functional inhibitory NK cell transmembrane receptors in the California sea lion (48). Inhibitory NK receptors are essential for detecting missing or altered MHC class I molecules in target cells (49). The low levels of expression of Ly49 could reflect that the adult sea lions have a small population of peripheral mature NK cells that are undergoing Ly49 gene activation. If so, most of the granzyme B and perforin would have been transcribed by peripheral CD8+ cells, as they tend to be more abundant than NK cells in blood (50). This possibility is strengthened by the loose correlation observed between the transcription levels of Ly49 and those of granzyme B and perforin. Future studies should aim to explore the functional role of other NK cell transmembrane receptors, such as the killer cell lectin like receptor 1 gene (nkg2d), whose role for modulating cytotoxicity has been associated with susceptibility to papillomavirus-related cancers in humans (51).

FIGURE 7 | Gene transcription levels varied according to genital infection status. (A) Eomes, (B) Perforin, (C) Tbx21, (D) Granzyme. The plot shows the median (thick line), first and third quartile (box), and 95% confidence interval of the median (whiskers).

Relative expression levels of Eomes, Tbx21, and STAT-1, genes also expressed by NK and CD8+ T cells (52), was closely correlated, as would be expected in a Th1 (pro-inflammatory) environment (53, 54). Furthermore, these genes exhibited regional variation in transcription. Eomes was downregulated in most sea lions from the midriff and central regions, but upregulated in sea lions from the northern and southern regions, as well as in the Mexican North Pacific, while Tbx21 and STAT-1 transcription levels showed a clear north-to-south increase within the Gulf of California and reached highest levels in the Mexican North Pacific. When considering the transcriptional profile of NK and CD8+ cells, the hierarchal clustering analysis revealed two main profiles. Sea lions from the northern and midriff regions exhibited one of them, while most sea lions from the southern region and the Mexican North Pacific exhibited the other. Sea lions from the central region exhibited one or the other profile, and a few sea lions from the southern region and the Mexican North Pacific exhibited a more "northern" transcriptional profile. This suggests a spatial difference in the prevalence of intracellular pathogens or differential exposure to other factors that could cause cellular insults.

R

The encoded products of Eomes, Tbx21, and STAT-1 (Eomesodermin, T-bet, and STAT-1, respectively) are essential to Th1 cell differentiation, and are consequently important for downstream anti-viral and anti-tumoral responses (16). Eomes gene expression can be upregulated by type I interferon signaling in CD8+ T cells (55), while Tbx21 is upregulated in response to stimulation by antigens and macrophage-derived cytokines (56). STAT-1 regulates Tbx21 transcription, and in turn, high levels of Tbx21 influence the transcription of perforin and granzyme B in antigen-stimulated cells (57). Furthermore, T-bet promotes and sustains virus-specific CD8+ responses, particularly during chronic infections (58). The relatively higher transcription levels observed in sea lions from the southern Gulf of California and Northern Pacific could indicate increased exposure to intracellular pathogens or anti-tumoral activity. Two results add support for this proposed explanation. First, sea lions with genital infections by papillomavirus had increased expression of Eomes, Tbx21, and perforin, and second, papillomavirus infections were significantly higher in the southern Gulf of California and Mexican North Pacific. Research on human papillomavirus has shown that the oncogenic proteins E6 and E7 can induce Tcell responses in individuals with a functional immune system. The responses, characterized by higher densities of T cells that express eomesodermin (16), T-bet (17), and perforin, among other markers help clear, or avoid, the occurrence of cervical neoplasia (17). Based on our results, we suggest that ZcPV-1 induces systemic immune responses in otherwise healthy California sea lions.

An opposite pattern was observed for OtHV-1. Here, genetic expression of Eomes, Tbx21, and perforin was lower in infected sea lions. In marked contrast to papillomavirus, whose tropism is restricted to the basal layer of the epithelium (59), gammaherpesviruses have a wider tropism that includes epithelial cells and B-lymphocytes (60). In humans, active gammaherpesvirus infections are most often associated with severe immunosuppression, and the viruses can switch to latency, establishing lifelong persistent infections, which can then lead to lymphomas and carcinomas, as well as lymphoproliferative disorders of NK and T cells (61). Immune control of gammaherpesviruses infections is complicated, as they have evolved various immune evasion strategies (62). Unfortunately, knowledge about OtHV-1 pathogenesis is currently limited, but based on our results, it is tempting to speculate that detectable infections in the genital tract will be much more common in sea lions with a suboptimal immune status, and that infections will, in turn, avoid cytotoxic immune responses, further increasing the risk of malignant transformation of infected cells. This hypothesis is strengthened by two findings: one, a relative decrease in lymphocyte counts was observed in herpesvirusinfected sea lions compared to sea lions with papillomavirus or those with no apparent infections (see **Figure S7**); and two, when comparing levels of transcription of genes related to cytotoxicity among apparently uninfected sea lions, sea lions infected by OtHV-1, ZcPV-1, and by both viruses simulaneously, levels of Eomes, perforin, and tbx21 of sea lions infected by OtHV-1 were equal to or lower than uninfected sea lions. Interestingly, simultaneous infection of the genital epithelium by OtHV-1 and ZcPV-1 resulted in a pattern of transcription similar to that of OtHV-1 alone. This was unexpected, and implies that coinfections might be an important risk factor for transformation of the genital epithelium, owing to a reduction in anti-viral and anti-tumor activity. A recent study reported coinfection by herpesviruses and papillomaviruses in genital tumors of Atlantic bottlenose dolphins, Tursiops truncatus (63) and it has already been shown that co-infections by oncogenic papillomaviruses and herpesviruses increase the risk of cervical carcinoma in humans (64). Based on our results, we believe that the potential role of papillomavirus as a co-factor in for sea lion urogenital carcinoma should be reconsidered [see (3)].

Transcription levels of the two genes selected as markers of Th2 responses and immune modulation varied among regions, once again with a north to south gradient. GATA3 was downregulated in sea lions from the northern and midriff Gulf of California, and upregulated in sea lions from the central and southern Gulf of California and Mexican North Pacific, where levels were the highest. FoxP3 mirrored the spatial pattern, although transcription levels of this gene were, on average 4.5 fold lower than that of GATA3. Both genes were correlated, as would be expected since GATA3 modulates FoxP3 expression and activity (65), however, GATA3 only explained 13% of the variation in FoxP3 levels, and a number of individuals deviated greatly from the predicted regression. GATA3 is a transcription factor expressed by mature CD8+ T cells, and it plays a key role in leading to the differentiation of Th2 cells, regulating T cell development, proliferation, metabolism, and maintenance. In particular, it promotes development of anti-inflammatory Th2 and Th9 cells, and suppresses pro-inflammatory Th1 and Th17 differentiation as well as B cell development (65). In turn, FoxP3 is highly expressed by CD4+ T regulatory (Treg) cells, and it is essential to ensure immune homeostasis by suppressing (or destroying) active leukocytes via stimulating direct cytotoxicity, depleting growth factors in the extracellular environment, and secreting anti-inflammatory cytokines and co-inhibitory molecules that can modulate the activity of antigen-presenting cells (65). Expression of FoxP3 is particularly important to control excessive anti-viral responses and limit the extent of immunopathology (66). Based on the fact that (i) several of the genes involved with promoting pro-inflammatory and cytotoxic responses were upregulated in sea lions from the central and southern Gulf of California and Mexican North Pacific, and (ii) these are the regions where transcription of GATA3 was increased, and that expression of GATA3 is known to be tightly regulated by TCR and pro-inflammatory cytokine stimulation, which in turn responds to active CD8+ and NK activity (67), it is possible that sea lions in these regions are experiencing active responses to one or more viruses, and are, at the same time, experiencing some level of immunomodulation to avoid damage associated with these responses. The hierarchal analysis showed some support for this explanation, as the dendrogram revealed two large groups that separated sea lions from the northern and midriff region from those from the central and southern Gulf of California, and the Mexican North Pacific. Thus, it is likely that differences in viral exposure, as well as other as yet unidentified environmental and ecological variables are impacting sea lion immune modulation in addition to their cytotoxic immune activity. Having found that the NLR was higher in sea lions from the northern and midriff region implies that these individuals are undergoing chronic inflammation. However, as our study focused exclusively on assessing gene transcription levels, rather than proteins, this possibility will need to be examined in more detail in the future by quantifying cytokine levels in the blood.

The ratio of GATA3 to Tbx21 (here Ga/Tb) is an indirect measure of the Th1/Th2 profile of an individual (39). Both transcription factors cross-regulate each other, as T-bet modulates GATA-3 function and Th2 cytokines block Th1 differentiation (66). The interrelationship of these molecules and the effector responses that they regulate define the host response and, therefore, influence the outcome of a given infection. While mean Ga/Tb did not vary among regions, when analyzing the Th1/Th2 profile by hierarchal clustering, a north to south pattern emerged once again, with one group comprised by sea lions from the midriff, northern, and one comprised by sea lions from the southern region. As observed for transcriptional markers of cytotoxicity, sea lions from the central region exhibited either one the responses.

The observed patterns in the transcriptional profiles of genes related to cytotoxicity, Th2 and immunomodulation, and Th1/Th2 balance were congruent. In all cases there appeared to be a distinction between patterns exhibited by sea lions from the northern and midriff regions and those exhibited from the southern region and the Mexican North Pacific. While differences in transcription levels do not necessarily indicate differences in the synthesis of the proteins of interest due to transcriptional and post-transcriptional regulation (68), the congruence in the transcriptional profiles observed cannot be ignored. In particular, these profiles appear to be related to genital infection by two oncogenic viruses, and are likely to reflect other ecological and environmental factors, such as differences in contaminant levels (7, 20, 21) and availability of energetic resources (25) to implement immune responses (32). These possibilities will need to be addressed in future studies. Given the role of NK and CD8+ cells for tumor surveillance and anti-viral responses (15–17), the significance of OtHV-1 in the development of sea lion urogenital carcinoma (2, 3), the potential importance of ZcPV-1 in oncogenesis (63), and the stark difference in the prevalence of urogenital carcinoma between sea lions from the US and Mexican waters (18), the patterns observed are likely to be biologically significant.

Marine mammals, as apex predators, are continuously exposed to xenobiotics that can impact their immune competence. This is particularly problematic, given the growing number of emerging pathogens in the marine environment and the rise in disease conditions and unusual mortality events in the past decades. In this context, it is relevant and timely to increase our understanding of the factors that can hinder different immune effectors of these species (69), particularly of those that are prone to develop chronic and deadly diseases, such

### REFERENCES

1. Deming AC, Colegrove KM, Duignan PJ, Hall AJ, Wellehan JFX, Gulland FMD. Prevalence of urogenital carcinoma in stranded california sea lions as urogenital carcinoma, which could be considered sentinels of immune competence (70). Our study is a first approach to profile the transcriptional patterns of key immune effectors of free-ranging California sea lions, and their association with ecological regions and infection by oncogenic viruses. The observed results and suggested patterns add insight to our understanding of immune competence of marine mammals, and may help elucidate the marked difference in the number of cases of urogenital carcinoma in sea lions from US waters and other areas of their distribution.

### DATA AVAILABILITY

The datasets generated for this study are available on request to the corresponding author.

### AUTHOR CONTRIBUTIONS

KA-W conceived, designed and coordinated the study. IP conducted gene expression assays, statistical analyses, and drafted the manuscript. AF-M conducted hematological analysis; MF-C and RÁ-M assisted with statistical design, analysis and interpretation of data. FG-dlR and LS-G performed pathogen detection assays. All authors read and commented the final draft of the manuscript and gave final approval for publication.

### FUNDING

This study was funded by grant CONACYT-Fronteras de la Ciencia 446 awarded to KA-W. IP was funded by a CONACYT PhD studentship (298065). AF-M was funded by a CONACYT PhD studentship (287579).

### ACKNOWLEDGMENTS

We thank Marina Banuet-Martínez, Daniela Bárcenas, Cecilia Barragán-Vargas, Rafael Chávez, Carlos Domínguez-Sánchez, Fernando Elorriaga-Verplancken, and Manuel Vargas for their help with animal capture and restraint and processing of samples in the field. Diego Ruiz and The Museo de la Ballena staff kindly provided logistical support during fieldwork while aboard the Narval ship. Erika Vargas Perusquia greatly helped with grant administration. We are grateful to Frances Gulland for reading an early draft of the manuscript and providing feedback that greatly improved the paper.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.00413/full#supplementary-material

(Zalophus californianus) from 2005–2015. J Wildl Dis. (2018). 54:581–6. doi: 10.7589/2017-08-208

2. Lipscomb TP, Scott DP, Garber RL, Krafft AE, Tsai MM, Lichy JH, et al. Common metastatic carcinoma of California sea lions (Zalophus californianus): evidence of genital origin and association with novel gammaherpesvirus. Vet Pathol. (2000) 37:609–17. doi: 10.1354/vp.37-6-609

californianus californianus) from the Gulf of California, México. Arch Environ Contam Toxicol. (2009) 56:350–9. doi: 10.1007/s00244-008-9181-y

predominant role of GATA-3. J Immunol Methods. (2003) 278:157–169. doi: 10.1016/S0022-1759(03)00200-X

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Peñín, Figueroa-Cabañas, Guerrero-de la Rosa, Soto-García, Álvarez-Martínez, Flores-Morán and Acevedo-Whitehouse. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Comparative Immunopathology of Cetacean morbillivirus Infection in Free-Ranging Dolphins From Western Mediterranean, Northeast-Central, and Southwestern Atlantic

#### Edited by:

Brian Dixon, University of Waterloo, Canada

### Reviewed by:

John Reif, Colorado State University, United States Sylvain De Guise, University of Connecticut, United States

#### \*Correspondence:

Josué Díaz-Delgado<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Comparative Immunology, a section of the journal Frontiers in Immunology

Received: 13 December 2018 Accepted: 22 February 2019 Published: 18 March 2019

#### Citation:

Díaz-Delgado J, Groch KR, Ressio R, Riskallah IPJ, Sierra E, Sacchini S, Quesada-Canales Ó, Arbelo M, Fernández A, Santos-Neto E, Ikeda J, Carvalho RR, Azevedo AF, Lailson-Brito J Jr, Flach L, Kanamura CT, Fernandes NCCA, Cogliati B, Centelleghe C, Mazzariol S, Di Renzo L, Di Francesco G, Di Guardo G and Catão-Dias JL (2019) Comparative Immunopathology of Cetacean morbillivirus Infection in Free-Ranging Dolphins From Western Mediterranean, Northeast-Central, and Southwestern Atlantic. Front. Immunol. 10:485. doi: 10.3389/fimmu.2019.00485 Josué Díaz-Delgado1 \*, Kátia R. Groch1 , Rodrigo Ressio2 , Isis P. J. Riskallah2,3 , Eva Sierra4 , Simona Sacchini 4 , Óscar Quesada-Canales 4 , Manuel Arbelo4 , Antonio Fernández 4 , Elitieri Santos-Neto5 , Joana Ikeda5 , Rafael Ramos de Carvalho5 , Alexandre de Freitas Azevedo5 , Jose Lailson-Brito Jr. 5 , Leonardo Flach6 , Cristina T. Kanamura2 , Natália C. C. A. Fernandes 2 , Bruno Cogliati 3 , Cinzia Centelleghe7 , Sandro Mazzariol 7 , Ludovica Di Renzo8 , Gabriella Di Francesco8 , Giovanni Di Guardo9 and José Luiz Catão-Dias 1

1 Laboratory of Wildlife Comparative Pathology, Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil, 2 Pathology Center, Adolfo Lutz Institute, São Paulo, Brazil, 3 Laboratory of Morphologic and Molecular Pathology, Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil, 4 School of Veterinary Medicine, Institute for Animal Health and Food Safety, University of Las Palmas of Gran Canaria, Arucas, Spain, 5 Laboratory of Aquatic Mammals and Bioindicators: Profa Izabel M. G. do N. Gurgel' (MAQUA), Faculty of Oceanography, Rio de Janeiro State University, Rio de Janeiro, Brazil, 6 Instituto Boto Cinza, Mangaratiba, Rio de Janeiro, Brazil, 7 Department of Comparative Biomedicine and Food Hygiene (BCA), University of Padova, Legnaro, Italy, 8 Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise "G. Caporale", Teramo, Italy, 9 Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy

Cetacean morbillivirus (CeMV; Paramyxoviridae) causes epizootic and interepizootic fatalities in odontocetes and mysticetes worldwide. Studies suggest there is different species-specific susceptibility to CeMV infection, with striped dolphins (Stenella coeruleoalba), bottlenose dolphins (Tursiops truncatus), and Guiana dolphins (Sotalia guianensis) ranking among the most susceptible cetacean hosts. The pathogenesis of CeMV infection is not fully resolved. Since no previous studies have evaluated the organ-specific immunopathogenetic features of CeMV infection in tissues from infected dolphins, this study was aimed at characterizing and comparing immunophenotypic profiles of local immune responses in lymphoid organs (lymph nodes, spleen), lung and CNS in CeMV-molecularly (RT-PCR)-positive cetaceans from Western Mediterranean, Northeast-Central, and Southwestern Atlantic. Immunohistochemical (IHC) analyses targeted molecules of immunologic interest: caspase 3, CD3, CD20, CD57, CD68, FoxP3, MHCII, Iba1, IFNγ, IgG, IL4, IL10, lysozyme, TGFβ, and PAX5. We detected consistent CeMV-associated inflammatory response patterns. Within CNS, inflammation was dominated by CD3+ (T cells), and CD20+ and PAX5+ (B cells) lymphocytes, accompanied by fewer Iba1+, CD68+, and lysozyme+ histiocytes, mainly in striped dolphins and bottlenose dolphins. Multicentric lymphoid depletion was characterized by reduced numbers of T cells and B cells, more pronounced in Guiana dolphins. Striped dolphins and bottlenose dolphins often had hyperplastic (regenerative) phenomena involving the aforementioned cell populations, particularly chronically infected animals. In the lung, there was mild to moderate increase in T cells, B cells, and histiocytes. Additionally, there was a generalized increased expression of caspase 3 in lymphoid, lung, and CNS tissues. Apoptosis, therefore, is believed to play a major role in generalized lymphoid depletion and likely overt immunosuppression during CeMV infection. No differences were detected regarding cytokine immunoreactivity in lymph nodes, spleen, and lung from infected and non-infected dolphins by semiquantitative analysis; however, there was striking immunoreactivity for IFNγ in the CNS of infected dolphins. These novel results set the basis for tissue-specific immunophenotypic responses during CeMV infection in three highly susceptible delphinid species. They also suggest a complex interplay between viral and host's immune factors, thereby contributing to gain valuable insights into similarities, and differences of CeMV infection's immunopathogenesis in relation to body tissues, CeMV strains, and cetacean hosts.

Keywords: Cetacean morbillivirus, immunopathogenesis, neuroimmunopathology, lymphocytes, histiocytes, apoptosis, cytokines

## INTRODUCTION

Cetacean morbillivirus (CeMV; genus Morbillivirus, family Paramyxoviridae) has caused multiple outbreaks of lethal disease in odontocetes and mysticetes worldwide. Interepizootic or endemic morbidity and mortality is also recorded (1). There are three well characterized CeMV strains (porpoise MV, dolphin [D]-MV, and pilot whale MV) mainly in northern hemisphere, and three novel strains, one of them detected in Brazil, i.e., Guiana dolphin (GD)-MV, which is also considered the first reported example of CeMV infection among cetaceans from South America (2, 3). Studies suggest there is different species-specific susceptibility to CeMV infection with bottlenose dolphins (Tursiops truncatus), striped dolphins (Stenella coeruleoalba), and Guiana dolphins (Sotalia guianensis) ranking among the most susceptible cetacean hosts, with fatal epizootics (1, 3, 4). CeMV may cause severe lymphoid, respiratory, and neurologic disease in susceptible species, leading to stranding and death. Four major presentations of CeMV-associated pathology (CeMV-AP) are currently recognized, which bear resemblance to the pathologic features of measles virus (MeV) and canine distemper virus (CDV) infections, the major morbilliviral diseases in humans and dogs, respectively (5, 6).

The pathogenesis of infections by terrestrial morbilliviruses (TMVs) involve initial replication in lymphoid tissues, followed by viral dissemination in infected lymphocytes through the lymphatics and blood stream ("leukocyte trafficking"), and eventual spread to epithelial and nervous cells (6, 7). Immunohistochemical (IHC) studies on naturally occurring CeMV infections in cetaceans support the above pathogenesis, with a predominant aerogenous transmission (1). Several studies have focused on the immunophenotypic characterization of local inflammatory responses (LIRs) in TMV infections, especially in measles (in both humans and non-human primates) and distemper (in canids and susceptible non-canid carnivore species), with major emphasis on lymphoid tissues, lung, and central nervous system (CNS) (8, 9). In measles and distemper, various lymphocytic, histiocytic, and cytokine patterns have been shown to vary depending on disease chronology and other factors. Furthermore, cytokine imbalance in Th1 and Th2 immune responses (early and advanced disease stages, respectively) plays a major role in disease susceptibility and progression in MeV- and CDV-infected individuals (5, 8, 9). Nevertheless, there are no published studies focused on the pathogenetic evolution of CeMV infection in Th1-dominant vs. Th2-dominant cetacean hosts (10). Immunophenotypic studies on LIR during CeMV infection are also lacking, except for a previous study focused on peripheral blood leukocytes (PBLs) in a set of bottlenose dolphins with subclinical infection (11). In order to partially fill in this knowledge gap, the present study was aimed at characterizing and comparing the immunophenotypic profiles of CeMV-associated LIR in lymphoid, lung and CNS tissues of infected cetaceans from Western Mediterranean (Italy), Northeast-Central (Canary Islands), and Southwestern Atlantic (Brazil).

## MATERIAL AND METHODS

### Data and Sample Collection

The marine mammal databases and tissue banks of collaborative research institutions, namely the "Laboratory of Wildlife Comparative Pathology—LAPCOM" (São Paulo, Brazil), the "Laboratory of Aquatic Mammals and Bioindicators Profa. Izabel M. G. do N. Gurgel—MAQUA" (Rio de Janeiro, Brazil), the "Institute for Animal Health and Food Safety—IUSA" (Canary Islands, Spain), the "Department of Comparative Biomedicine and Food Science of the Faculty of Veterinary Díaz-Delgado et al. The Immunopathology of Cetacean morbillivirosis

Medicine of the University of Padua (Legnaro, Italy)," the "Laboratories of Histopathology and Immunohistochemistry of Istituto Zooprofilattico Sperimentale dell'Abruzzo e Molise G. Caporale," and the "Faculty of Veterinary Medicine of the University of Teramo (Teramo, Italy)," were queried based upon the following criteria: "Sotalia guianensis," "Stenella coeruleoalba," "Tursiops truncatus," "CeMV reverse transcription polymerase chain reaction (RT-PCR)-positive" "Toxoplasma gondii PCR-negative." Only individuals in a "fresh" (code 2) post mortem preservation status, or in a "moderate post mortem autolysis" (code 3) condition (12), which could also warrant a sufficient amount of formalin-fixed, paraffinembedded (FFPE), and frozen tissues for extensive analysis, including immunohistochemical (IHC) and cytokine gene expression investigations (parallel manuscript) on target organs (lymph nodes, spleen, lung, brain), were included. Additionally, tissues from three CeMV-negative dolphins including one striped dolphin (Canary Islands), one bottlenose dolphin (Italy), and one Guiana dolphin (Brazil) that were fresh, in good body condition and died as result of bycatch and/or traumatic interaction(s) and lacked morphological and molecular evidence of any infectious etiology were used as "controls" for IHC comparison purposes. The tissue samples came from complete standard necropsies. Epidemiologic and biologic data, along with necropsy reports, photographic material, and ancillary diagnostic techniques were retrieved and further analyzed. Required permissions for the management of tissues from cetaceans found stranded along the coasts of Brazil, the Canarian archipelago, and Italy were issued by the respective official authorities. All dolphins had spontaneous naturally occurring CeMV infection and no experiments were performed on live animals. Detailed comparative histopathologic investigations and viral IHC results for these animals will be published elsewhere.

### Immunohistochemistry

Selected FFPE tissues including lymph nodes (mediastinal/tracheobronchial, pulmonary, mesenteric, prescapular) and spleen, lung, and CNS (cerebrum, cerebellum, brain stem, spinal cord) were subjected to IHC using the following primary antibodies (pAbs): cleaved caspase 3 (CAS3) [final apoptosis pathway], cluster of differentiation (CD)-3 [T cell], CD20 [B cell], CD57 [natural killer cell], CD68 [histiocyte], Forkhead Box (Fox)-P3 [regulatory T cell], human leukocyte antigen (HLA-DRα; syn. major histocompatibility complex II, MHCII) [antigen presenting cell], ionized calcium binding adaptor molecule 1 (Iba1) [histiocyte/microglia], interferon gamma (IFNγ), immunoglobulin (Ig)-G, interleukin (IL)-4, IL10, lysozyme, transforming growth factor beta (TGFβ), and paired box protein (PAX)-5 [B-cell]. Most of this IHC panel was standardized with successful cross-reactivity in cetacean tissues (13). Further details on the IHC protocol are recorded in **Table 1**. Briefly, serial sections at 3 µm-thick were cut and collected onto coated slides. Antigen retrieval was followed by endogenous peroxidase blocking and nonspecific binding blocking with normal serum of same species where pAbs were raised. PAbs were incubated overnight (18 h, 4C◦ ). Amplification and visualization was achieved by the HiDef DetectionTM HRP Polymer System (Cell Marque, Rocklin, California, USA) followed by diaminobenzidine (DAB D-5637; Sigma, St. Louis, Missouri, USA) chromogen and counterstaining with Harris' haematoxylin. Normal human, mouse and franciscana (Pontoporia blainvillei) lymph node, spleen, lung, and brain were used as positive controls (13). Tissue sections in which the pAbs were replaced by non-immune homologous serum served as negative controls.

For immunophenotypic characterization of local inflammatory cell populations, the number of immunopositive cells was semiquantitatively evaluated for each marker in lymphoid (lymph nodes, spleen), lung, and CNS tissues according to following score: –, no; +, <10%; ++, 10–50%; + + +, 51–90%, and + + ++, >90% immunopositive cells, in 10 high-power (400x) fields. The histo-anatomical compartments analyzed were: lymph nodes (primary and secondary follicles, paracortex, medullary cords and cortical, paracortical, and medullary sinuses; afferent/efferent lymphatics); spleen (follicles, perifollicular zone, periarteriolar lymphoid sheath [PALS], red pulp sinuses, and cords); lung (bronchial/bronchiolar mucosa and submucosa including glands and associated lymphoid tissue [BALT]; alveolar spaces and septa; interstitium; pleura; vasculature including lymphatics); brain (cerebral cortex, cerebellum, brain stem, spinal cord) (13, 14). Additionally, IHC expression intensity was subjectively scored (increasing intensity) as 1, 2, and 3. The results were compared between CeMV-infected and non-infected "control" dolphins.

### RESULTS

Twenty-seven CeMV-positive dolphins, including 13 striped dolphins, 11 Guiana dolphins, and 3 bottlenose dolphins met the inclusion criteria. Guiana dolphins were infected by GD-CeMV (2, 3), while striped dolphins and bottlenose dolphins were infected by DMV (15–18). Epidemiologic and biologic data as well as CeMV-RT-PCR-positive tissues are recorded in **Table 2**. CeMV-positive animals included: calves (n = 2), juveniles (n = 11), and adults (n = 14). All CeMV-negative dolphins were calves. Detailed gross and microscopic pathologic findings with most probable cause(s) of stranding and/or death for CeMV-positive are published elsewhere and for CeMVnegative dolphins are recorded in **Supplemental Table S1**.

Consistent immunolabeling (with variations according to LIR) was detected for CAS3, CD3, CD20, CD57, CD68, FoxP3, MHCII, Iba1, IFNγ, IgG, IL4, IL10, lysozyme, TGFβ, and PAX5 in all organs/tissue sections examined. The following alterations in immunophenotypic profiles of LIRs are referred as to comparisons to "control" animals. "Normal" or "physiologic" antigen cell distribution and intensity for the pAbs aforementioned in control animals (cases 28, 29, and 30) were similar to those previously reported (13) (**Supplemental Figure**).

### Lymphoid Tissues: Lymph Nodes, Spleen

CeMV-LIR often overlapped focally with verminous lymphadenitis-associated LIR, especially in Guiana dolphins. The following changes had somewhat similar distribution patterns in all lymph nodes (mediastinal/tracheobronchial, pulmonary, TABLE 1 | Tested antigen, clone, species of origin, clonality, antigen retrieval methods, working dilutions for primary antibodies and amplification and visualization method for immunohistochemistry of selected immune components in formalin-fixed, paraffin-embedded lymphoid tissues of striped dolphins (Stenella coeruleoalba), bottlenose dolphins (Tursiops truncatus), and Guiana dolphins (Sotalia guianensis).

CD, cluster of differentiation; FoxP3, Forkhead Box P3; HLA, human leukocyte antigen (synonym major histocompatibility complex); IFN, interferon; Ig, immunoglobulin; IL, interleukin; TGF, transforming growth factor; Pax-5, paired box 5; Mon, monoclonal; Pol, polyclonal; PK, proteinase K; NE: not evaluated; PoH; HiDef DetectionTM Polymer System; DAB, Diaminobenzidine. Best antigen retrieval and primary antibody concentrations are indicated in bold. pH6, citrate; pH9, EDTA, Ethylenediamine tetraacetic acid. aCell signaling (Danvers, MA, USA), bDako-Agilent (Santa Clara, CA, USA), cThermo (Fremont, CA, USA), dNovocastra-Leica (Nussloch, Germany), eWako Pure Chemical (Richmond, VA, USA), fBio-Rad (Hercules, CA, USA), gProteintech (Rosemont, IL, USA), hBiocare (Pacheco, CA, USA), ICell Marque (Rocklin, CA, USA), jDAB, 3,3′ -Diaminobenzidine (D-5637; Sigma, St. Louis, Missouri, USA).

mesenteric, prescapular) and spleens evaluated (**Table 3**). There was an overall increased CAS3 expression in mononuclear cells (MNCs), including lymphocytes and histiocytes (often with engulfed apoptotic debris) in the cortex and paracortex, and histiocytes of subcortical sinuses and medullary cords (**Figure 1A**) in lymph nodes of CeMV-infected dolphins. While there was a consistently decreased number of CD3+ (**Figure 2A**), CD20+ (**Figure 3A**), and PAX5+ (**Figure 4A**) lymphocytes (hereafter, T cells, and B cells, respectively) in lymph nodes of most Guiana dolphins, striped dolphins, and bottlenose dolphins often had cortical, paracortical, and medullary cord expansion of T cells and B cells in addition to milder multifocal depletion phenomena. MHCII expression varied between CeMV-infected and uninfected animals. Approximately half of the dolphins, more predominantly in Guiana dolphins, infected by CeMV had reduced MHCII expression associated with diminished B-cells; however, there were increased MHCII+ histiocytes (see below) influx (**Figure 5A**). Fewer MHCII+ MNCs were detected in the paracortex and subcortical medullary cords. Iba1+ histiocytes (macrophages, monocytes, dendritic cells) (**Figure 6A**) correlated with CD68+ (**Figure 7A**) and lysozyme+ (**Figure 8A**) histiocytes; however, Iba1+ tended to label a greater number of histiocytic cells. Increased histiocytes (dendritic cells and circulating monocytes/macrophages) were common in depleted follicles, paracortex, and sinuses of infected dolphins. Occasional syncytia were Iba1+ and CD68+. CD57+ MNCs in lymph nodes varied slightly among CeMV-infected dolphins but overall did not differ considerably from "control" dolphins, with only few scattered immunoreactive cells in cortex and paracortex. Furthermore, the number of IgG+ lymphocytes varied among CeMV-infected dolphins; however, they tended to be slightly more numerous than in "control" animals, being mostly detected in germinal centers (**Figure 9A**) and paracortex and to a lesser extent in medullary cords and occasionally within sinuses. Scattered FoxP3+ lymphocytes were seen in the cortex, paracortex, and medullary cords of CeMV-infected and control dolphins with no apparent differences from "control" dolphins.

In the spleen, there was increased CAS3 expression in MNCs of white pulp and to a lesser extent in red pulp (**Figure 1B**). There was a consistently reduced number of T cells (**Figure 2B**) and B cells (**Figures 3B**, **4B**), especially prominent in Guiana dolphins; however, striped dolphins and bottlenose dolphins occasionally presented "reactive (regenerative) hyperplasia" in addition to multifocal lymphoid depletion. As observed in lymph nodes, infected dolphins tended to have reduced expression of MHCII in B cell areas (**Figure 5B**); however, increased MHCII+ histiocytes contributed to overall similar semiquantitative results for MHCII expression in spleen of "control" animals. Iba1+ (**Figure 6B**) histiocytes correlated to CD68+ (**Figure 7B**) and lysozyme+ (**Figure 8B**) histiocytes, and were more numerous in the red pulp. Rare CD57+ MNCs were detected in the white pulp with no evident difference regarding "control" dolphins. IgG+ lymphocytes were more common in the white pulp and to a lesser extent in the red pulp (**Figure 9B**) of infected dolphins, particularly in striped dolphins from the Canary Islands and Italy, in contrast to Guiana dolphins. No differences were detected regarding FoxP3+ lymphocytes in CeMV-infected and uninfected "control" dolphins.

aCeMV-positive; bCeMV-negative; NR, not recorded; Ca, calf; Ju, juvenile; Ad, adult; Fe, female; Ma, male; NS, nutritional status; Po, poor; Mo, moderate; G, good; DC, decomposition code (2, fresh; 3, moderate autolysis); SC, stranding condition (A: alive; D: dead). AS, acute systemic; SS, subacute systemic, CS, chronic systemic; BOFDI, brain only form of DMV infection.

Approximately a third of the infected dolphins had increased IFNγ immunoreactivity in lymph nodes and/or spleen tissue compared to uninfected dolphins. IFNγ was variably expressed in MNCs (lymphocytes, macrophages), rare syncytia and extracellularly in lymph nodes, and spleen (**Figures 10A,B**). Except for few dolphins, most infected dolphins showed no immunohistochemically evident difference regarding TGFβ, IL4, and IL10 immunoreactivity in lymph nodes and spleen. Occasional MNCs expressed TGFβ, rarely expressed also by syncytia (case 25). Scattered IL4+ and IL10+ MNCs along with occasional extracellular labeling were noted in cortex and paracortex (lymph nodes) and white pulp (spleen), and to a lesser extent in lymph node and spleen sinuses.

### Lung

CeMV-LIR often overlapped focally with verminous pneumoniaassociated LIR, especially in Guiana dolphins. IHC results in lung tissue are recorded in **Table 4**. There was increased CAS3 expression in circulating, infiltrating, and exocytosing/exudating MNCs as well as luminal cell debris (**Figure 1C**). There was a mildly to moderately increased number of T cells (**Figure 2C**) and B cells (**Figures 3C**, **4C**) in alveolar septa, interstitium, and bronchial/bronchiolar mucosa/submucosa including BALT. Lung from "control" dolphins had scattered detectable T cells and B cells. Case 25 (with concomitant septicemia by Staphylococcus aureus) had abundant T cell and B cell infiltrates around bronchi/bronchioles, and alveoli (**Figure 2C**). Overall, the number and intensity of CAS3+ and T cells and B cells was greater in parasitic inflammatory foci. MHCII immunoexpression was consistently increased and involved presumed resident and circulating MNCs, including putative pulmonary intravascular macrophages (PIVMs), and infiltrating inflammatory MNCs (**Figure 5C**), being more abundant in foci of parasitic injury. Histiocytes were increased

 NE, not evaluated. Semiquantitative analysis of immunopositive cells: –, no; +, <10%; ++, 10–50%; + + +, 50–90%, and + + ++, >90% immunopositive cells. Subjective labeling intensity score of immunopositive cells: 0, 1,and 3.

 2,

> aMultisystemichyphatemycosis(lung,kidney).

5;

 bHyphatemycosis(lung).

 cSuspectBrucellacoinfection(primarily

 CNS lesions). dSepsisbyStaphylococcusaureus(particularlysevere CNS and lung lesions).

 eMultisystemic hyphate mycosis (lung, cerebrum).

FIGURE 1 | Caspase 3 (CAS3) immunohistochemical findings. (A) Lymph node (case 22). Increased expression of CAS3 throughout the cortex. 100×. Inset: lymph node (case 22). Detail of increased CAS3 expression in mononuclear cells (MNCs) of mantle and paracortex. 400×. (B) Spleen (case 21). Increased expression of CAS3 throughout the white and red pulp. 100×. Inset: spleen (case 21). Detail of increased expression of CAS3 in white and red pulp. 400×. (C) Lung (case 18). Increased expression of CAS3 in proliferative and necrotizing bronchiolitis. 100×. Inset: lung (case 21). Detail of CAS3+ circulating MNCs in alveolar capillaries. 200×. (D) Cerebrum (case 24). Increased CAS3 expression in inflammatory cell infiltrates in Virchow-Robins space and adjacent neuroparenchyma. 400×. Inset: cerebrum (case 26). Detail of CAS3+ neurons, neuroglia, and infiltrating MNCs. 400×.

FIGURE 2 | Cluster of differentiation (CD)-3 immunohistochemical findings. (A) Lymph node (case 4). Markedly depleted CD3+ lymphocytes of cortex and paracortex. 100×. Inset: lymph node (case 22). CD3+ lymphocyte hyperplasia in paracortex (interfollicular). 400×. (B) Spleen (case 24). Reduced CD3+ lymphocytes through white pulp. 100×. Inset: spleen (case 17) CD3+ hyperplasia in white pulp and red pulp districts. 200×. (C) Lung (case 17). Mild, multifocal circulating and infiltrating CD3+ lymphocytes in alveolar walls and interstitium. 200×. Inset: lung (case 25). Marked CD3+ lymphocyte bronchiolar hyperplasia. 100×. (D) Cerebrum (case 24). Multiple Virchow-Robin spaces are expanded by CD3+ lymphocytes, which also infiltrate the adjacent parenchyma. 40×. Inset: cerebrum (case 24). Detail of abundant CD3+ lymphocytes perivascular and neuroparenchymal infiltration. 400×.

FIGURE 3 | Cluster of differentiation (CD)-20 immunohistochemical findings. (A) Lymph node (case 18). Moderate depletion of CD20 lymphocytes in cortical areas. 40×. Upper inset: lymph node (case 22). CD20+ lymphocyte hyperplasia. 40×. Lower inset: lymph node (case 18). CD20+ lymphocyte hyperplasia in medullary cords. 400×. (B) Spleen (case 21). Mild to moderate multifocal CD20-expressing lymphocyte depletion in white pulp. 40×. Inset: spleen (case 21). Detail of relatively normal CD20+ lymphocyte number in follicle. (C) Lung (case 13). Multifocal parabronchiolar, interstitial/alveolar septa CD20+ lymphocytic infiltrates. 100×. Inset: lung (case 25). Marked, multifocal peribronchiolar CD20+ lymphocytic infiltrates. 100×. (D) Spinal cord (case 13). Marked CD20+ leptomeningeal infiltration. 40×. Inset: cerebrum (case 26). Subpial CD20 perivascular and leptomeningeal CD20+ infiltrates. 400×.

in CeMV-infected dolphins. Iba1+ histiocytes were seen circulating as well as infiltrating the bronchial/bronchiolar mucosa/submucosa, BALT, interstitium, and exocytosing and filling the alveolar lumina (**Figure 6C**). CD68+ (**Figure 7C**) and lysozyme+ (**Figure 8C**) histiocytes paralleled Iba-expressing cells yet they were often less numerous. Histiocytes and lysozyme+ neutrophils were overall more abundant in parasitic inflammatory foci. Hyperplastic (type II) pneumocytes and alveolar exudates expressed lysozyme in many animals, including parasitic and fungal pneumonia cases. CD57+ MNCs were

FIGURE 4 | Paired box protein (PAX)-5 immunohistochemical findings. (A) Lymph node (case 17). Mild to moderate PAX5+ lymphocyte depletion in cortical area. 100×. Left inset: lymph node (case 17). Detail of mild PAX5+ lymphocyte depletion in follicle. 400×. Right inset: lymph node (case 18). PAX5+ lymphocyte hyperplasia in medullary cord. 400×. (B) Spleen (case 17). Mild PAX5+ lymphocyte follicular depletion. 200×. (C) Lung (case 18). Increased numbers of PAX5+ lymphocytes in lung interstitium. 400×. (D) Cerebrum (case 13). Moderate infiltration of PAX5 lymphocytes in leptomeninges. 200×. Inset: cerebrum (case 13). Detail of leptomeningeal infiltrating PAX5+ lymphocytes. 400×.

FIGURE 5 | Major histocompatibility (MHC) class II immunohistochemical findings. (A) Lymph node (case 12). MHCII expression in cortical cell elements mainly B-cell areas and sinuses. 100×. (B) Spleen (case 2). MHCII expression mostly in white pulp cell elements and red pulp. 100×. (C) Lung (case 9). Increased expression of MHCII in inflamed alveolus and exudative cells including reactive and binucleated histiocytes and syncytia. 400×. Inset: lung (case 12). Abundant MHCII-expressing inflammatory cells in inflamed distal bronchus. 100×. (D) Spinal cord (case 13). Overexpression of MHCII in spinal nerve root cell elements and infiltrating inflammatory cells. 200×. Inset: cerebrum (case 18). Overexpression of MHCII in perivascular inflammatory cells and vascular and neuroparenchymal cell elements. 200×.

FIGURE 6 | Ionized calcium binding adaptor molecule (Iba)-1 immunohistochemical findings. (A) Lymph node (case 17). Small to moderate amounts of Iba1-expressing histiocytes (including intra- and interfollicular dendritic cells, and sinus histiocytes) in cortex/paracortex interface and medullary cords. 100×. Upper inset: lymph node (case 17). Detail of Iba1+ follicular dendritic cells. 400×. Lower inset: lymph node (case 18). Detail of Iba1-expressing sinus histiocytes and hyperplastic medullary cords. 400×. (B) Spleen (case 15). Abundant expression of Iba1+ in histiocytic populations in red and white pulp. 100×. Inset: spleen (case 15). Detail of Iba1-expressing histiocytic cells in follicle and perifollicular zone. 400×. (C) Lung (case 19). Striking alveolar Iba1+ histiocytic inflammatory response including reactive, binucleated, and multinucleate macrophages/syncytia. 400×. (D) Cerebellum (case 25). Marked infiltration of Iba1+ histiocytes in interfoliar leptomeninges. 100×. Upper inset: cerebellum (case 25). Detail of Iba+ meningeal inflammatory infiltrate. 400×. Lower inset: prominent perivascular histiocytic infiltration including macrophages and microgliosis. 400×.

rare in the lung; no differences were noted compared to control animals. IgG+ lymphocytes were increased in CeMVinfected dolphins and were detected mainly in BALT (**Figure 9C**) and to a lesser extent in alveolar septa and interstitium; they were especially prominent in parasitic LIR. Intra-alveolar syncytia were generally Iba1+. Rare FoxP3+ lymphocytes were seen in lung of some CeMV-infected dolphins; by contrast, no FoxP3-expressing lymphocytes were seen in control dolphins.

All "control" dolphins had scattered MNCs immunoreactive and multifocal extracellular signal for IFNγ, IL4, IL10, and TGFβ in lung tissue. However, even though these cytokines appeared

FIGURE 7 | Cluster of differentiation (CD)-68 immunohistochemical findings. (A) Lymph node (case 25). Detail of CD68+ follicular and interfollicular dendritic (histiocytes) cells. 400×. (B) Spleen (case 25). Abundant sinus histiocytic cells expressing CD68. 100×. Inset: spleen (case 25). Detail of perifollicular CD68+ histiocytic cells. 400×. (C) Lung (case 25). Abundant intrabronchiolar and intraalveolar histiocytes express CD68. Inset: lung (case 25). Detail of intraalveolar histiocytes expressing CD68. 400×. (D) Cerebellum (case 25). Detail of CD68+ histiocytes infiltrating the leptomeninges. 400×.

FIGURE 8 | Lysozyme immunohistochemical findings. (A) Lymph node (case 25). Lysozyme+ histiocytic cells in cortical and paracortical area. 200×. Inset: lymph node (case 25). Abundant intrasinus lysozyme-expressing histiocytes. 200×. (B) Spleen (case 14). Lysozyme+ histiocytes through the red pulp. 40×. Inset: spleen (case 14). Detail of lysozyme histiocytes in red pulp. 400×. (C) Lung (case 11). Overexpression of lysozyme by inflammatory cells and exudate in inflamed bronchioles and alveoli associated with CeMV and concomitant verminous pneumonia. 40×. Upper inset: lung (case 13). Circulating MNCs (including presumed pulmonary intravascular macrophages) are lysozyme+. 400×. Lower inset: lung (case 21). Increased lysozyme+ (mainly histiocytes) cells within inflammatory focus. 400×. (D) Spinal cord (case 13). Moderate number of lysozyme+ histiocytes in spinal cord leptomeninges. 100×. Inset: spinal cord (case 13). Detail of lysozyme+ histiocytic meningeal infiltrate. 400×.

FIGURE 9 | IgG immunohistochemical findings. (A) Lymph node (case 18). Scattered IgG+ cells in follicular and parafollicular areas. 200×. Left inset: lymph node (case 15). Increased IgG-expressing cells in follicular and parafollicular areas. 200×. Right inset: lymph node (case 12). IgG+ cell hyperplasia in medullary cord. 400×. (B) Spleen (case 13). Scattered IgG+ cells at white/red pulp interface. 200×. Inset: spleen (case 17). Increased IgG+ cells in white/red pulp interface. 400×. (C) Lung (case 12). IgG+ cells in hyperplastic BALT of inflamed bronchiole. 400×. (D) Cerebrum (case 14). Scattered IgG+ cells in leptomeninges. 400×. Inset: cerebrum (case 14). Detail of IgG+ cells in meningeal infiltrates. 400×.

expressed by a slightly greater number of MNCs in lung of infected dolphins they represented <10% of the inflammatory cells (including BALT and interstitium); thus, no IHC evident differences were noted based on the proposed semiquantitative approach. IFNγ was mainly expressed by exudating MNCs including alveolar macrophages and syncytia (**Figure 10C**), and fewer infiltrating macrophages and lymphocytes, and occasional epithelial cells with an extracellular signal in inflammatory foci. Both, IL4 and IL10 were expressed by infiltrating and circulating MNCs compatible with histiocytes and lymphocytes, and extracellularly. TGFβ+ cells were less numerous and typically involved alveolar macrophages, bronchial/bronchiolar epithelia, exocytosing MNCs, and occasional mesenchymal cells.

Left inset: lymph node (case 17). Moderate number of IFNγ-expressing histiocytes and lymphocytes within paracortical/medullary sinuses. (B) Spleen (case 22). Scattered IFNγ-expressing cells in white and red pulp. Detail of IFNγ-expressing cells. 100×. Inset: 22. 400×. (C) Lung (case 23). IFNγ-expressing cells mainly include alveolar macrophages and scattered interstitial mononuclear cells. 100×. Inset: lung (case 23). Detail of IFNγ-expressing cells within perialveolar lymphocytic aggregate and rare alveolar macrophage. 400×. (D) Cerebrum (case 9). Meningeal and neuroparenchymal IFNγ-expressing cells. 400×.

### Central Nervous System

IHC results are recorded in **Table 5**. CAS3+ MNCs were seen in meningeal, parenchymal, Virchow-Robin spaces (VRSs), and neuroparenchymal inflammatory infiltrates as well as in circulating MNCs (**Figure 1D**). In areas of marked neurodegeneration, neurons and neuroglia occasionally expressed CAS3. In decreasing order, T cells (**Figure 2D**) and B cells (**Figures 3D**, **4D**) predominated in meningeal, VRSs, and neuroparenchymal inflammatory infiltrates in striped dolphins and bottlenose dolphins with varying degrees of CNS inflammation. B-cells were comparatively more numerous in case 13, a case with a suspect Brucella co-infection based on cellular inflammatory components and neuroanatomical distribution of lesions. Rare T cells were seen circulating and/or in meningeal perivascular spaces in three Guiana dolphins, likely representing early CNS inflammation. CAS3+ cells appeared to involve mostly T and B cells. MHCII was widely overexpressed, involving MNCs either circulating or expanding the VRS and/or infiltrating the neuroparenchyma, along with vascular endothelial cells (**Figure 5D**). Occasionally, neuroglia (microglia and astrocytes) expressed MHCII. Few animals additionally exhibited MHCII labeling in roots of spinal nerves and meningeal mesenchyme. Inflamed CNS tissues often harbored Iba1+ histiocytes, either circulating or located within the vessel walls, expanding the VRSs and infiltrating the neuroparenchyma (**Figure 6D**). Microglia was consistently labeled by Iba1. In few cases, degenerating neurons expressed Iba1+. Lysozyme+ MNCs were rare in the CNS, except for cases 13 and 25 (**Figure 7D**). CD68+ MNCs were rarely seen in CNS inflammatory foci (**Figure 8D**). CD57+ cells were not detected in CNS tissues examined. Small numbers of IgG+ lymphocytes were common in meningeal inflammatory infiltrates and VRS (**Figure 9D**) of striped dolphins. Case 13, a bottlenose dolphin with a suspect coinfection by Brucella sp. had greater numbers of IgG-expressing cells and histiocytes. Only cases 13 and 14 showed rare FoxP3+ cells intermingled with perivascular inflammatory infiltrates in CNS tissue sections examined.

Brain tissue from "control" dolphins had no immunoreactivity to any of the cytokines evaluated (IFNγ, IL4, IL10, TGFβ); however, CeMV-infected striped dolphins and bottlenose dolphins had consistent IFNγ (**Figure 10D**) and occasional IL4 immunoexpression in CNS tissue sections examined. Overall, there was a more consistent number of IFNγ+ cells in all these cases, but scattered IL4+ cells could be also observed. IFNγ was mainly expressed by MNCs and neuroparenchymal elements. IL4 was expressed by scattered circulating MNCs. Five striped dolphins and one bottlenose dolphin had detectable TGFβ and/or IL10 immunoexpression. The latter involved MNCs in inflamed choroid plexus of fourth ventricle (case 25).

## DISCUSSION

Comparative analyses of LIRs are of paramount relevance as they help unraveling local immunopathogenetic mechanisms of disease processes. While in humans and in given domestic and livestock animal species, as well as in laboratory animals there is a considerable body of knowledge, the immunopathogenetic bases of disease including LIRs in cetaceans remain largely unknown (11). We have herein characterized, by means of a set of lymphocytic, histiocytic, antigen-presenting cell-, cell deathrelated and cytokine immunomarkers, LIRs among CeMVinfected dolphins from the Western Mediterranean as well as from the Northeast-Central and the Southwestern Atlantic, with special emphasis on their lymphoid, pulmonary, and CNS tissues. There are no previous data on the LIRs in cetaceans infected by CeMV, except for a previous study focused on PBLs in a set of free-ranging bottlenose dolphins with subclinical infection (11). In that study, dolphins with subclinical CeMV infection had elevated lysozyme concentrations, marginally significant increases in monocytic phagocytosis, reduced mitogen-induced T lymphocyte proliferation responses, and marginally significant CD4+ decreases in PBLs compared to seronegative dolphins (11). We will discuss our results tissue by tissue and correlate them with the tissue-specific immunopathology and immunopathogenesis of MeV (6) and CDV infections (5) whenever appropriate. This IHC-based approach was coupled with inferred CeMV disease presentation forms for all animals included in this study (data published elsewhere).

 aMultisystemic

 hyphate mycosis (lung, kidney).

bHyphate mycosis (lung).

cSuspect Brucella coinfection (primarily CNS lesions).

dSepsis by eMultisystemic

 hyphate mycosis (lung, cerebrum).

Staphylococcus

 aureus (particularly severe CNS and lung lesions).

**35**

bHyphate mycosis (lung).

cSuspect Brucella coinfection (primarily CNS lesions).

dSepsis by eMultisystemic

 hyphate mycosis (lung, cerebrum).

Staphylococcus

 aureus (particularly severe CNS and lung lesions).

**36**

Humoral and cellular immune responses to MeV (6, 19) and CDV (20–23) are crucial for viral clearance and recovery, as well as for the establishment of a "lifelong" protective immunity. However, they are also the pathological basis of measles and distemper morbidity and mortality (5, 9). In both, early immunosuppression is associated with viremia and lysis of lymphocytes and macrophages (6, 24). B-cells, follicular dendritic cells, and T-cells, especially CD4+, and CD8+ cells, are initially targeted by MeV and CDV resulting in generalized lymphoid depletion in lymph nodes and spleen, mucosa-associated lymphoid tissue (MALT), and tonsils (5, 6). Hyperplasia of reticular cells (sinus histiocytosis) in the medullary region of lymph nodes typically accompanies this early phase of infection (24, 25). In this study, we observed consistently decreased numbers of T cells and B cells in Guiana dolphins, striped dolphins, and bottlenose dolphins; however, lymphoid cell depletion appeared to be more severe in Guiana dolphins and in acute/subacute cases from the Canary Islands (cases 13, 14, and 19) and Mediterranean Sea (cases 21, 25, 26, and 27). Additionally, we observed increased sinus histiocytosis (Iba1-, CD68-, and lysozyme-expressing cells) in these dolphins, recapitulating features observed in acute/subacute measles and distemper (5, 9).

CAS3 is a frequently activated death protease, catalyzing the specific cleavage of many key cellular proteins and playing a major role in apoptosis (26). In MeV and CDV infections, upregulation/overexpression of CAS3 is commonly observed in infected lymphocytes and uninfected lymphocytes, suggesting the existence of virus-dependent, and virus-independent mechanisms of apoptosis (27–29). We detected an overall increased CAS3 expression mainly in lymphocytes and histiocytes of cortex and paracortex, but to a lesser extent in histiocytes of subcortical sinuses and medullary cords in lymph nodes. These findings suggest apoptosis is a major cell death mechanism (30) also in the time course of CeMV infection, as it is in measles and distemper (27, 31, 32). Although in these cases viral-induced apoptosis may be the major triggering factor (27, 31, 33), the pathogenetic intricacies of such phenomenon, including viral-independent apoptosis pathways remain undetermined in CeMV infection. Altogether, the above findings concerning lymphocytic and histiocytic disarrangements and generalized immunoexpression of CAS3 in CeMV-infected dolphins provide compelling morphological and IHC evidence of a compromised immune response capacity in these animals.

If the host survives, incipient MeV and CDV infections are generally followed by regeneration of lymphoid organs (5, 6). Repopulation and germinal center formation in lymphoid tissues from persistently infected and convalescent hosts are common in this stage (5, 6). Detection of such "persistently infected" and/or "convalescent" dolphins is complicated; however, we observed similar regenerative findings in several cases from the Canary Islands and the Mediterranean Sea largely involving those animals with chronic systemic and chronic localized "brain only forms" of CeMV infection [supported by IHC and PCR results [data published elsewhere]] (1, 14). These changes were characterized by follicular, paracortical, and medullary cord expansion (reactive hyperplasia), typically encompassing distorted or poorly delineated lymphocytic growths including T cells and B cells accompanied by slightly increased numbers of IgG+ cells. Thus, we detected IHC evidence of a somewhat similar immune response progression in CeMV-infected dolphins compared to MeV- and CDV-infected individuals (5, 6). Nonetheless, further conclusions are limited by the lack of other means of infection's chronology surrogates, including serological profiling. Future studies addressing these limiting factors would allow more accurate comparisons with TMVs. Interestingly, morphologically intact appearing compartments after lymphoid repopulation and virus clearance from lymphoid tissues does not necessarily result in complete functional regeneration of the immune response (22, 34, 35). This may also apply to CeMV infection, so that apparently "normal" lymphocytic density and distribution patterns in lymphoid districts from CeMV-infected cetaceans may not necessarily be associated with appropriate immunological fitness. In chronic measles and distemper, often this multicentric repopulation is accompanied by CD4- and CD8-dominated inflammatory responses in the CNS (22, 36, 37). This remains to be evaluated in cetaceans.

Mammals differ in their expression of MHCII molecules (38). In this study, we observed MHCII expression was largely confined to MNCs consistent with B-lymphocytes, dendritic cells, and macrophages (especially sinus histiocytes) which are considered professional antigen-presenting cells (38). Also, there were small numbers of MHCII-expressing MNCs in the paracortex and subcortical medullary cords, diminishing centripetally. This distribution pattern is consistent with reports in other species, including rodents (38); however, more accurate inferences may be obtained by double immunolabeling analyses. We suspect certain T-cell lymphocytes could also express MHCII in CeMV-infected animals; however, greater T cell numbers, including resting ones, as reported in pigs, dogs, cats, mink, and horses (38), were not a feature in CeMV-infected or "control" animals. No significant alterations in MHCII immunoexpression were observed between infected and control animals on the basis of semiquantitative comparative analysis.