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

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 \u201cparadigmatic\u201d 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)

The immune system of cetaceans and their interaction with dolphin morbillivirus

Immunology of marine mammals is a relatively new field of scientific studies and its monitoring plays an important role on the individual and group management of these animals, as well as an increasing value of environmental health indicator: cetaceans are viewed as environmental sentinels. The current knowledge about the immune system of cetaceans and its function is recognized as incomplete. Therefore this study aims to implement the knowledge on the immune response in normal conditions in cetaceans stranded along the Italian coastline in order to provide a base-line useful for assessing the immune status of bottlenose dolphin (Tursiops truncatus) and striped dolphin (Stenella coeruleoalba), the species most found in our seas and included in some international conventions such as species with high protection. The selection of cetaceans to be included in the study was based on the availability of samples of spleen, thymus and lymph node tissue of the animals, on the way (in formalin or frozen) and the state of preservation; They have in fact been preferred tissues of animals whose sampling occurred within 48 hours of death in order to reduce as much as possible post-mortem alterations. Thereafter, animals were divided into groups on the basis of information obtained from signaling, such as species, sex, age, and environment of origin, on the outcome of virological investigations, microbiological, parasitological and toxicological, if performed, and the cause and/or the death mechanism. On the lymph node slides obtained from the formalin-fix, paraffin-embedded samples it was made basic hematoxylin-eosin staining and immunohistochemical staining using the following antibodies: Monoclonal Mouse Anti-Human CD3 to identify T lymphocytes, Monoclonal Mouse Anti-Human CD20 for the identification of mature B lymphocytes and Monoclonal Mouse Anti-Human HLA-DR Antigen, Alpha-Chain for the identification of the major histocompatibility complex type II. It is also arranged to validate by means of the Western blotting technique antibodies used in immunohistochemistry (IHC). The use of these antibodies was valid only for humans and some domestic animals (dogs and cats), but not in the species of interest to us such as bottlenose and striped dolphins. With the antibodies mentioned above, also the lymphoid tissues of dolphins stranded along the coasts of the Canary Islands were tested and used as negative control since the death was probably due to collisions with boats/ships. Finally, a semi quantitative samples analysis was performed by acquisition of slides via slide scanner for digital pathology (D-SIGHT) and a manual count of the number of cells positive for each antibody in 10 fields to 40x magnification, considered to be representative of the entire organ, and these data were statistically analyzed using the T TEST method. Statistical analysis showed no significant correlation between the variables considered and the expression of the different lymphocyte populations. Future analysis should be aimed at analyzing the relationship between CD4+ and CD8+ cells in order to understand the effect of the major immunomodulatory pathogens, such as the dolphin morbillivirus, on sub-populations of T cells. It would also be important to compare the data obtained on the immune system with those obtained from the same samples as regards the search for environmental pollutants in order to understand the real role on the health of marine mammals present in our seas. The study was not focused only on the immune response of the guests to the different pathogens, but we concentrate our work also on the most important pathogen currently causing die-offs of marine mammals: the dolphin morbillivirus (DMV). In particular, from tissues of a positive DMV fin whale (Balaenoptera physalus) stranded along the Italian coastline in October 2013 it was possible to completely sequence the P / V / C gene (1520 bp), M (1007 bp) , N (1573 bp), F (1659 bp) and H (1814 bp) respectively coding for the phosphoprotein and two virulence factors (V and C), for the matrix protein, the nucleoprotein, the fusion protein and the hemagglutinin protein. The complete sequences of the mentioned genes were deposited in GenBank (GenBank provisional Acc. No. KU977449, KU977450, KU977451, KU977452 and KU977453). The isolation of the virus in the tissues was made possible by molecular techniques such as RT-PCR using primers designed ad hoc and cloning using plasmid vector. The nucleotide changes, and consequently the amino acid variations, of each individual gene were subsequently analyzed and compared with the viral genome of the preceding epidemics. It was then possible to carry out a study of the tertiary structure of the viruses to see how these single mutations had a role in the structural change of the proteins themselves. On the basis of this work we proceeded to identify an appropriate diagnostic technique usable even for large whales where correct sampling and appropriate samples storage is not always possible in field condition. We develop a nested RT-PCR which allows the unambiguous identification of a 200 bp fragment of the DMV genome corresponding to a highly conserved part of the gene H. This technique permits, if the viral genome is particularly fragmented because of the poor tissue conservation status, to verify the positivity to the DMV in tissue analysis, to isolate part of the virus, otherwise difficult to be isolated, and it can be sequenced. This technique was later used successfully for identification and sequencing of DMV in the tissues of three sperm stranded in Vasto beach in 2014 (GenBank Acc. No. KU886570). We can therefore say that the DMV is affecting species that until recently were considered species not susceptible to infection, such as fin whale and sperm whale. Individuals of these new species affected by the infection are mainly young animals or even pup and the virus isolation in a fetus organs confirms the possibility of vertical transmission. Given the species barrier carried out by the virus, its point-like changes in the genomic sequence of the virus and incidence of positivity in 19% of the target species in 2015 we can claim to be in a situation where the virus is endemic in the Mediterranean Sea resulting increase in the infectious pressure. Future studies will aim to understand more precisely the role of individual amino acid changes and their influence on the virulence and pathogenicity of the virus through the use of crystallography, to study the structure of the virus cellular receptor, CD150, to understand its real interaction with the virus, and to try to understand the real significance of the DMV in the ecology of the entire cetaceans population in the Mediterranean Sea

Genomic and structural investigation on dolphin morbillivirus (DMV) in Mediterranean fin whales (Balaenoptera physalus).

Dolphin morbillivirus (DMV) has been deemed as one of the most relevant threats for fin whales (Balaenoptera physalus) being responsible for a mortality outbreak in the Mediterranean Sea in the last years. Knowledge of the complete viral genome is essential to understand any structural changes that could modify virus pathogenesis and viral tissue tropism. We report the complete DMV sequence of N, P/V/C, M, F and H genes identified from a fin whale and the comparison of primary to quaternary structure of proteins between this fin whale strain and some of those isolated during the 1990-'92 and the 2006-'08 epidemics. Some relevant substitutions were detected, particularly Asn52Ser located on F protein and Ile21Thr on N protein. Comparing mutations found in the fin whale DMV with those occurring in viral strains of other cetacean species, some of them were proven to be the result of diversifying selection, thus allowing to speculate on their role in host adaptation and on the way they could affect the interaction between the viral attachment and fusion with the target host cells

Death associated to methicillin resistant staphylococcus aureus ST8 infection in two dolphins maintained under human care, Italy

The present study describes the isolation of Methicillin-resistant Staphylococcus aureus (MRSA) from respiratory tract of 2 dolphins of different origin, a stranded juvenile Risso's dolphin (Grampus griseus) and a captive born common bottlenose dolphin (Tursiops truncatus) calf, which died in the same institution at 1-month distance from the other. A complete microbiological and genetic investigation confirmed the presence of MRSA clone-complex 8, sequence type (ST) 8, spa-type t008 in both individuals. This strain differs from the one previously reported in walruses and dolphins and has never been described in dolphins before, but it is randomly isolated from Italian human patients. Vertical transmission of the infection may also occurs in other species and considering the description and location of the pathological lesions, this seems to be the most likely route of transmission implied in the young bottlenose dolphin. Staphylococcus aureus is known as an opportunistic agent, usually secondary to other pathogens, but its multiple antibiotic resistance and its zoonotic implications suggest a thorough and strict application of animal management hygiene protocols

Genomic and structural investigation on Dolphin Morbillivirus (DMV) in Mediterranean fin whales (Balaenoptera physalus)

Dolphin Morbillivirus (DMV) has been deemed as one of the most relevant threats for fin whales (Balaenoptera physalus) being responsible for a mortality outbreak in the Mediterranean Sea in the last years. Knowledge of the complete viral genome is essential to understand any structural changes that could modify virus pathogenesis and viral tissue tropism. We report the complete DMV sequence of N, P/V/C, M, F and H genes identified from a fin whale and the comparison of primary to quaternary structure of proteins between this fin whale strain and some of those isolated during the 1990-‘92 and the 2006-‘08 epidemics. Some relevant substitutions were detected, particularly Asn52Ser located on F protein and Ile21Thr on N protein. Comparing mutations found in the fin whale DMV with those occurring in viral strains of other cetacean species, some of them were proven to be the result of diversifying selection, thus allowing to speculate on their role in host adaptation and on the way they could affect the interaction between the viral attachment and fusion with the target host cells

Correlation of renal histopathology with renal echogenicity in dogs and cats: An ex-vivo quantitative study

BACKGROUND: Increased cortical or cortical and medullary echogenicity is one of the most common signs of chronic or acute kidney disease in dogs and cats. Subjective evaluation of the echogenicity is reported to be unreliable. Patient and technical-related factors affect in-vivo quantitative evaluation of the echogenicity of parenchymal organs. The aim of the present study is to investigate the relationship between histopathology and ex-vivo renal cortical echogenicity in dogs and cats devoid of any patient and technical-related biases. RESULTS: Kidney samples were collected from 68 dog and 32 cat cadavers donated by the owners to the Veterinary Teaching Hospital of the University of Padua and standardized ultrasonographic images of each sample were collected. The echogenicity of the renal cortex was quantitatively assessed by means of mean gray value (MGV), and then histopathological analysis was performed. Statistical analysis to evaluate the influence of histological lesions on MGV was performed. The differentiation efficiency of MGV to detect pathological changes in the kidneys was calculated for dogs and cats. Statistical analysis revealed that only glomerulosclerosis was an independent determinant of echogenicity in dogs whereas interstitial nephritis, interstitial necrosis and fibrosis were independent determinants of echogenicity in cats. The global influence of histological lesions on renal echogenicity was higher in cats (23%) than in dogs (12%). CONCLUSIONS: Different histopathological lesions influence the echogenicity of the kidneys in dogs and cats. Moreover, MGV is a poor test for distinguishing between normal and pathological kidneys in the dog with a sensitivity of 58.3% and specificity of 59.8%. Instead, it seems to perform globally better in the cat, resulting in a fair test, with a sensitivity of 80.6% and a specificity of 56%

Dynamics of blood circulation during diving in the bottlenose dolphin (Tursiops truncatus): the role of the retia mirabilia

The retia mirabilia are vascular nets composed of small vessels dispersed among numerous veins, allowing blood storage, regulation of flow and pressure damping effects. Here, we investigated their potential role during the diving phase of the bottlenose dolphin (Tursiops truncatus). To this effect, the whole vertebral retia mirabilia of a series of dolphins were removed during post-mortem analysis and examined to assess vessel diameters, and estimate vascular volume and flow rate. We formulated a new hemodynamic model to help clarify vascular dynamics throughout the diving phase, based on the total blood volume of a bottlenose dolphin, and using data available about the perfusion of the main organs and body systems. We computed the minimum blood perfusion necessary to the internal organs, and the stroke volume and cardiac output during the surface state. We then simulated breath-holding conditions and perfusion of the internal organs under the diving-induced bradycardia and reduction of stroke volume and cardiac output, using 10 beats min 121 as the limit for the heart rate for an extended dive of over 3 min.Within these simulated conditions, the retia mirabilia play a vital role as reservoirs of oxygenated blood that permit functional performances and survival of the heart and brain. Our theoretical model, based on the actual blood capacity of the retia mirabilia and available data on organ perfusion, considers the dynamic trend of vasoconstriction during the diving phase and may represent a baseline for future studies on the diving physiology of dolphins and especially for the blood supply to their brain

Environmental and pathological factors affecting the hatching success of the two northernmost loggerhead sea turtle (Caretta caretta) nests

In recent years, the report of loggerhead sea turtle (Caretta caretta) Mediterranean nesting range has expanded together with new records of nests becoming northward on the Italian coastline of the Tyrrhenian and Adriatic seas. These areas are characterized by intensive human activities, such as tourism, fishery, and marine traffic, all possibly involved in the influence of the use of coastal habitat by marine species. These anthropic threats, in addition to the natural ones and the changing environmental characteristics of the beach, may influence the growth of microorganisms causing hatching failures. Among microorganisms, fungal infection by the genus Fusarium (Link, 1809) is considered one of the main causes of globally declining sea turtle populations. In summer 2021, the two northernmost worldwide loggerhead sea turtle nests were monitored along the Northern Adriatic coastline (Veneto, Italy). These first records may potentially candidate this area as suitable for a large part of the loggerhead turtle's life cycle and it could represent a minor sea turtle nesting area that, according to Prato and colleagues, remained unnoticed due to the lack of specific monitoring. Sea Turtle Egg Fusariosis (STEF) was deemed to have deeply compromised the hatching success of the northmost one. Climate change and anthropogenic impacts have been scored as one of the highest hazards to sea turtle health and could have played a role in the STEF development. Environmental changes, human activities, and emerging pathogens deserve the highest attention in terms of health research, and conservation management

Assessment of Antimicrobial Effects on Broiler Gut Barrier Through Histopathology and Immunohistochemistry of Tight-Junction Proteins

In recent years, antimicrobial (AM) use in poultry farming has been attracting attention worldwide mainly due to AM resistance spreading. The role of AM prophylaxis in the modulation of gut microbiota, as well as of gut health, is still not clearly understood. Therefore, this study aimed to investigate the role of different prophylaxis protocols in the modulation of the gut barrier in broilers by applying a histopathological approach. Intestinal tissue samples were collected from a total of 240 male broilers (Ross 306), reared and treated with different AM protocols. Haematoxylin and Eosin (HE) staining and a multiple scoring system were used to evaluate the presence of lesions in ileum, cecum and colon of treated broilers. Moreover, immunohistochemistry (IHC) was performed to assess the expression of claudin-3 and ZO-1 proteins in intestinal tissues. The application of a semi-quantitative scoring system was used in IHC stained samples. HE results revealed that intestinal tissues were mainly characterized by epithelial detachment and fusion of the intestinal villi, but also by the presence of lymphocytic infiltrate in the mucosa and submucosa of AM-treated broilers. However, the IHC approach for the evaluation of claudin-3 and ZO-1 proteins showed that their expression was not affected by the different AM treatments. Nevertheless, the presence of intestinal lesions highlighted by histopathology suggests that AM treatments could harm the gut health of broilers, inducing an inflammatory response and consequent epithelial lesions. In order to clarify the role of AM treatments in the modulation of gut barrier in broilers, further studies are needed

Morphological evidence for the sensitivity of the ear canal of odontocetes as shown by immunohistochemistry and transmission electron microscopy

The function of the external ear canal in cetaceans is still under debate and its morphology is largely unknown. Immunohistochemical (IHC) analyses using antibodies specific for nervous tissue (anti-S100, anti-NSE, anti-NF, and anti-PGP 9.5), together with transmission electron microscopy (TEM) and various histological techniques, were carried out to investigate the peripheral nervous system of the ear canals of several species of toothed whales and terrestrial Cetartiodactyla. This study highlights the innervation of the ear canal with the presence of lamellar corpuscles over its entire course, and their absence in all studied terrestrial mammals. Each corpuscle consisted of a central axon, surrounded by lamellae of Schwann receptor cells, surrounded by a thin cellular layer, as shown by IHC and TEM. These findings indicate that the corpuscles are mechanoreceptors that resemble the inner core of Pacinian corpuscles without capsule or outer core, and were labelled as simple lamellar corpuscles. They form part of a sensory system that may represent a unique phylogenetic feature of cetaceans, and an evolutionary adaptation to life in the marine environment. Although the exact function of the ear canal is not fully clear, we provide essential knowledge and a preliminary hypothetical deviation on its function as a unique sensory organ.Peer ReviewedPostprint (published version