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New advances in the diagnosis of diseases in marine mammals

Mar Melero Asensio defended the PhD Thesis at the Faculty of Veterinary Medicine of the Complutense University of Madrid

June 14th, 2017

Marine mammals have long been considered good indicators of marine ecosystem health because they have long lifespans; blubber stores that can serve as depots for chemicals and toxins and some species occupy a high trophic level. In addition to this, many species reside along coasts; human activities like fishing and maritime traffic affect them, and they are vulnerable to many infectious diseases. Some of these have implications for human public health and others may serve as indicators of environmental distress syndrome. Furthermore, marine mammals are very charismatic animals, and thus provide great social interest, and their conservation has repercussions for many other species that share their environment. All of these reasons make the study of diseases of marine mammals not only very important, but also urgent, as many species of marine mammals are currently endangered.
The main objective of this doctoral thesis, entitled “New advances in the diagnosis of diseases in marine mammals”, is to expand the current knowledge of marine mammals’ health through the development and application of tools for early diagnosis and the study of the infectious agents. The work performed in this thesis have been included in five research articles published in scientist journals with international impact and in a draft that is currently in preparation. The latter describes the systematic determination of herpesvirus in all the sampled tissues from cetaceans stranded in the Valencian Community over a period of three years.
In order to reach this main objective, three specific objectives were established. Objective 1 consisted of the application of thermography as a tool for the determination from distance of body temperature in marine mammals. Different anatomic points were evaluated thermographically in two cetacean species (beluga and bottlenose dolphin) and three pinnipeds (South American sea lion, harbor seal and Pacific walrus). In cetaceans, the best thermographic reference point for body temperature was the mucosa inside the blowhole, when was measured during voluntary breathing. In pinnipeds, the best thermographic reference point was the eye, when was measured after a stabilization period after the animal had left the water, which is characteristic for each species. The thermography allows rapid, remote, non-invasive measurement of body temperature, which avoids stressing the animals and is also safe for the technicians. These characteristics made thermography a very useful tool in the evaluation of wildlife.
The 2nd objective focused on studying the pathology caused by Erysipelothrix rhusiopathiae and the detection of antibodies against this bacteria in samples taken from cetaceans. The first case of septicemia for this bacteria in a bottlenose dolphin stranded in the Valencian coast of Mediterranean sea was evaluated via clinical examination, bacteriology and histopathology analysis. Moreover, a Luminex® technique was developed for antibodies against E. rhusiopathiae determination in three cetacean species: bottlenose dolphin, striped dolphin and Risso’s dolphin. This technique was validated by using serum samples of vaccinated and non-vaccinated captive bottlenose dolphins. Antibodies were determined in all of the vaccinated dolphins but in none of non-vaccinated dolphins. Then, samples from free ranging cetaceans were evaluated in order to study the interaction between cetaceans and this bacteria, including samples from the clinic case of erysipelas septicemia described previously. Antibodies were determined in none of the samples from the clinical case, probably because the disease progressed too quickly to allow the dolphin to mount a specific antibody response. However, antibodies were determined in the serum of a Risso’s dolphin which lacked any signs or lesions that might be indicative of E. rhusiopathiae infection. The fluorescence intensity value obtained from the serum sample of this Risso’s dolphin was similar to that measured in dolphins around 12 months after vaccination. This suggested that the cetacean had previously been exposed to the bacteria, though, it seemed unlikely that the animal was experiencing the disease at the time of death. This is the first application of Luminex® technology in marine mammals, being a technique of great importance because the relevance of this bacteria in cetaceans and also because is the first step in the development of a multiplex technique. This is the main advantage of Luminex®; it allows the simultaneous determination of up to 100 analytes in the same sample. This characteristic is especially advantageous in wildlife applications, when samples are difficult to obtain and therefore very valuable.
Objective 3 deals with the necessity of determining several virus in samples of captive and free-ranging marine mammals. This thesis included the first determination of herpesvirus and poxvirus in a Pacific walrus, and herpesvirus in a fin whale and a common minke whale. To increase knowledge about the relationship between herpesviruses and cetaceans, a systematic determination of herpesvirus in all tissues sampled from cetaceans stranded in the Valencian Community over three years was performed. A total of 966 samples were collected from four different species of cetaceans (striped dolphin, bottlenose dolphin, Risso’s dolphin and Cuvier`s beaked whale). Since herpesvirus could be in a period of latency, for all positive samples, the determination of RNA with herpesvirus sequence was also performed, as an indicator of viral replication. Herpesvirus was determined in more than the 80% of individuals and in almost 13% of tissues evaluated, with viral replication in half of them. These results, together with herpesvirus characteristics, such as its capacity to produce latent infections, (that could be reverse because of stress or immunosuppression) and the low number of reported cases in which herpesvirus has been considered the cause of death, suggest herpesvirus as a valuable parameter for evaluation and assessment of the health of marine mammals populations. Therefore, based on the results of this study, analysis of the variation in the prevalence of herpesvirus over time and in the percentage of tissues in which it is active in cetaceans stranded in the Valencia Community can provide very useful information for the study of the health of these populations.
In the set of objectives of this doctoral thesis, the thermography is applied to the measurement of the body temperature at a distance; the first application of Luminex® technology to marine mammals is developed, improving the determination of antibodies against E. rhusiopathiae and establishing the first step for a multiplex protocol; the first case of death by septicemia by E. rhusiopathiae in a bottlenose dolphin in the Valencian Mediterranean is described; the first determination of herpesvirus and poxvirus in Pacific walrus and of herpesvirus in fin whale and common minke whale; and the first systematic analysis of the presence of DNA and RNA with herpesvirus sequence in all samples from cetacean stranded over a long period of time.
The results of this doctoral thesis describe the development and application of novel tools in marine mammal health field and represent an important advance in the study of diseases that affect them. In this thesis a total of 10 species of marine mammal have been studied, but it is also useful as a first step in its application to other similar species and other diseases.

Mar Melero Asensio PhD Thesis: New advances in the diagnosis of diseases in marine mammals Mar Melero Asensio

TITLE: New advances in the diagnosis of diseases in marine mammals

TYPE: PhD Thesis

AUTHOR: Mar Melero Asensio

DIRECTORS: Sanchez-Vizcaino JM.

DATE: June 14th, 2017

LANGUAGE: English-spanish


Mar Melero Asensio. New advances in the diagnosis of diseases in marine mammals. Universidad Complutense de Madrid. June 14th, 2017. (PhD Thesis)