New advances in the diagnosis of diseases in marine mammals

Tesis de la Universidad Complutense de Madrid, Facultad de Veterinaria, Departamento de Sanidad Animal, leída el 14-06-2017Los mamíferos marinos son considerados buenos centinelas de la salud de los ecosistemas marinos y producen un gran interés social. Por ello, el estudio de las enfermedades que les afectan es de vital importancia y, además, urgente por encontrarse amenazadas numerosas especies.El objetivo principal de esta tesis doctoral es aumentar el conocimiento existente sobre la sanidad de los mamíferos marinos desarrollando y aplicando herramientas de diagnóstico precoz y de estudio de agentes infecciosos. A partir de éste, se plantearon tres objetivos concretos.En el objetivo 1 se aplicó la termografía a la medición de la temperatura corporal a distancia en cetáceos y pinnípedos. En el objetivo 2 se estudió a nivel clínico, histopatológico y bacteriológico, el primer caso de muerte por septicemia por E. rhusiopathiae en un delfín mular varado en el Mediterráneo valenciano. Además, se desarrolló la primera aplicación de la tecnología Luminex® a los mamíferos marinos, mejorando la determinación de anticuerpos frente a E. rhusiopathiae y estableciendo el primer paso para un protocolo multiplex. En el objetivo 3, centrado en el estudio de virus de interés en mamíferos marinos, se describió la primera determinación de herpesvirus y poxvirus en morsa del Pacífico y de herpesvirus en rorcual común y rorcual aliblanco. También, se realizó el análisis sistemático de la presencia de ADN y ARN con la secuencia de herpesvirus en todas las muestras de cetáceos varados en la Comunidad Valenciana durante tres años consecutivos. Los resultados de esta tesis doctoral describen el desarrollo y la aplicación de herramientas novedosas en el campo de la sanidad de los mamíferos marinos. Se estudiaron un total de 10 especies, pero resulta útil también como primer paso en su aplicación a otras especies similares y a otras enfermedades.Depto. de Sanidad AnimalFac. de VeterinariaTRUEunpu

Mecanismo de defensa del erizo europeo (Erinaceus europaeus)

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Aplicación de la termografía en la valoración de la fertilidad en huevos de Cernícalo primilla

Habitualmente la valoración de la fertilidad de los huevos se realiza mediante un ovoscopio. El principal inconveniente es la necesidad de manipulación del huevo, que impide su aplicación a la fauna salvaje. Por ello se estudió su posible sustitución por termografía, analizándose los patrones térmicos de 190 huevos de Cernícalo primilla, Falco naumanni. Los resultados obtenidos muestran que la termografía nos permite diferenciar los huevos en fértiles, infértiles y abortados gracias a sus diferentes patrones térmicos y velocidades de enfriamiento desde tres días después de la puesta. Es posible emplearlo tanto durante incubación artificial como en la naturaleza con diferentes condiciones ambientales, siendo necesario tener en cuenta el lugar de medición, los días transcurridos desde la puesta así como la temperatura de partida.Usually the evaluation of eggs fertility is done with ovoscopy. The principal inconvenient is the need to manipulate the egg, this prevents its application in wildlife. This is why it was studied to substitute this method for termography, analizing the termic patterns of 190 eggs of Falco naumanni. The obtained results show that termography allows us to differentiate the fertile eggs, from unfertile, and aborted ones thanks to its different termic patters and cooling speeds since 3 days after the lay. Its possible to use it both during the artificial incubation and in nature with different environment conditions, being necessary to have in mind the place, the number of days that have passed since the lay as well as the temperature at the beginning

Thermal reference points as an index for monitoring body temperature in marine mammals

BACKGROUND Monitoring body temperature is essential in veterinary care as minor variations may indicate dysfunction. Rectal temperature is widely used as a proxy for body temperature, but measuring it requires special equipment, training or restraining, and it potentially stresses animals. Infrared thermography is an alternative that reduces handling stress, is safer for technicians and works well for untrained animals. This study analysed thermal reference points in five marine mammal species: bottlenose dolphin (Tursiops truncatus); beluga whale (Delphinapterus leucas); Patagonian sea lion (Otaria flavescens); harbour seal (Phoca vitulina); and Pacific walrus (Odobenus rosmarus divergens). RESULTS The thermogram analysis revealed that the internal blowhole mucosa temperature is the most reliable indicator of body temperature in cetaceans. The temperatures taken during voluntary breathing with a camera held perpendicularly were practically identical to the rectal temperature in bottlenose dolphins and were only 1 °C lower than the rectal temperature in beluga whales. In pinnipeds, eye temperature appears the best parameter for temperature control. In these animals, the average times required for temperatures to stabilise after hauling out, and the average steady-state temperature values, differed according to species: Patagonian sea lions, 10 min, 31.13 °C; harbour seals, 10 min, 32.27 °C; Pacific walruses, 5 min, 29.93 °C. CONCLUSIONS The best thermographic and most stable reference points for monitoring body temperature in marine mammals are open blowhole in cetaceans and eyes in pinnipeds

Tracto digestivo de un insectívoro: erizo europeo (Erinaceus europaeus)

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First molecular determination of herpesvirus from two mysticete species stranded in the Mediterranean Sea

BACKGROUND Herpesvirus can infect a wide range of animal species: mammals, birds, reptiles, fish, amphibians and bivalves. In marine mammals, several alpha- and gammaherpesvirus have been identified in some cetaceans and pinnipeds species. To date, however, this virus has not been detected in any member of the Balaenoptera genus. CASE PRESENTATION Herpesvirus was determined by molecular methods in tissue samples from a male fin whale juvenile (Balaenoptera physalus) and a female common minke whale calf (Balaenoptera acutorostrata) stranded on the Mediterranean coast of the Region of Valencia (Spain). Samples of skin and penile mucosa from the fin whale and samples of skin, muscle and central nervous system tissue from the common minke whale tested positive for herpesvirus based on sequences of the DNA polymerase gene. Sequences from fin whale were identical and belonged to the Alphaherpesvirinae subfamily. Only members of the Gammaherpesvirinae subfamily were amplified from the common minke whale, and sequences from the muscle and central nervous system were identical. Sequences in GenBank most closely related to these novel sequences were viruses isolated from other cetacean species, consistent with previous observations that herpesviruses show similar phylogenetic branching as their hosts. CONCLUSIONS To our knowledge, this is the first molecular determination of herpesvirus in the Balaenoptera genus. It shows that herpesvirus should be included in virological evaluation of these animals

Novel adenovirus detected in captive bottlenose dolphins (Tursiops truncatus) suffering from self-limiting gastroenteritis

BACKGROUND Adenoviruses are common pathogens in vertebrates, including humans. In marine mammals, adenovirus has been associated with fatal hepatitis in sea lions. However, only in rare cases have adenoviruses been detected in cetaceans, where no clear correlation was found between presence of the virus and disease status. CASE PRESENTATION A novel adenovirus was identified in four captive bottlenose dolphins with self-limiting gastroenteritis. Viral detection and identification were achieved by: PCR-amplification from fecal samples; sequencing of partial adenovirus polymerase (pol) and hexon genes; producing the virus in HeLa cells, with PCR and immunofluorescence detection, and with sequencing of the amplified pol and hexon gene fragments. A causative role of this adenovirus for gastroenteritis was suggested by: 1) we failed to identify other potential etiological agents; 2) the exclusive detection of this novel adenovirus and of seropositivity for canine adenoviruses 1 and 2 in the four sick dolphins, but not in 10 healthy individuals of the same captive population; and 3) the virus disappeared from feces after clinical signs receded. The partial sequences of the amplified fragments of the pol and hexon genes were closest to those of adenoviruses identified in sea lions with fatal adenoviral hepatitis, and to a Genbank-deposited sequence obtained from a harbour porpoise. CONCLUSION These data suggest that adenovirus can cause self-limiting gastroenteritis in dolphins. This adenoviral infection can be detected by serology and by PCR detection in fecal material. Lack of signs of hepatitis in sick dolphins may reflect restricted tissue tropism or virulence of this adenovirus compared to those of the adenovirus identified in sea lions. Gene sequence-based phylogenetic analysis supports a common origin of adenoviruses that affect sea mammals. Our findings suggest the need for vigilance against adenoviruses in captive and wild dolphin populations

First molecular detection and characterization of herpesvirus and poxvirus in a Pacific walrus (Odobenus rosmarus divergens)

BACKGROUND Herpesvirus and poxvirus can infect a wide range of species: herpesvirus genetic material has been detected and amplified in five species of the superfamily Pinnipedia; poxvirus genetic material, in eight species of Pinnipedia. To date, however, genetic material of these viruses has not been detected in walrus (Odobenus rosmarus), another marine mammal of the Pinnipedia clade, even though anti-herpesvirus antibodies have been detected in these animals. CASE PRESENTATION In February 2013, a 9-year-old healthy captive female Pacific walrus died unexpectedly at L'Oceanografic (Valencia, Spain). Herpesvirus was detected in pharyngeal tonsil tissue by PCR. Phylogenetic analysis revealed that the virus belongs to the subfamily Gammaherpesvirinae. Poxvirus was also detected by PCR in skin, pre-scapular and tracheobronchial lymph nodes and tonsils. Gross lesions were not detected in any tissue, but histopathological analyses of pharyngeal tonsils and lymph nodes revealed remarkable lymphoid depletion and lymphocytolysis. Similar histopathological lesions have been previously described in bovine calves infected with an alphaherpesvirus, and in northern elephant seals infected with a gammaherpesvirus that is closely related to the herpesvirus found in this case. Intracytoplasmic eosinophilic inclusion bodies, consistent with poxviral infection, were also observed in the epithelium of the tonsilar mucosa. CONCLUSION To our knowledge, this is the first molecular identification of herpesvirus and poxvirus in a walrus. Neither virus was likely to have contributed directly to the death of our animal

Unusual striped dolphin mass mortality episode related to cetacean morbillivirus in the Spanish Mediterranean sea

BACKGROUND In the last 20 years, Cetacean Morbillivirus (CeMV) has been responsible for many die-offs in marine mammals worldwide, as clearly exemplified by the two dolphin morbillivirus (DMV) epizootics of 1990-1992 and 2006-2008, which affected Mediterranean striped dolphins (Stenella coeruleoalba). Between March and April 2011, the number of strandings on the Valencian Community coast (E Spain) increased. CASE PRESENTATION Necropsy and sample collection were performed in all stranded animals, with good state of conservation. Subsequently, histopathology, immunohistochemistry, conventional reverse transcription polymerase chain reaction (RT-PCR) and Universal Probe Library (UPL) RT-PCR assays were performed to identify Morbillivirus. Gross and microscopic findings compatible with CeMV were found in the majority of analyzed animals. Immunopositivity in the brain and UPL RT-PCR positivity in seven of the nine analyzed animals in at least two tissues confirmed CeMV systemic infection. Phylogenetic analysis, based on sequencing part of the phosphoprotein gene, showed that this isolate is a closely related dolphin morbillivirus (DMV) to that responsible for the 2006-2008 epizootics. CONCLUSION The combination of gross and histopathologic findings compatible with DMV with immunopositivity and molecular detection of DMV suggests that this DMV strain could cause this die-off event