Abstract

Gastrointestinal disease is a common problem in marine mammals. The origin of this disease used to be environment, parasites, diet, toxics, and rarely bacteria and viruses. With respect to the bacteria origin, only clostridial enterotoxemia has been described as a causative agent of primary gastrointestinal disease in captive marine mammals.¹ In cetaceans, Helicobacter spp. is a well-recognized problem and has been isolated from gastric mucosal samples producing gastritis and gastric ulcers.¹ With respect to viruses, adenovirus has been associated with gastrointestinal signs including abdominal splinting and intermittent blood-tinged diarrhea in sea lions.^2,3 Adenoviruses are common pathogens in vertebrates, including humans. In marine mammals, adenovirus has been associated to fatal hepatitis in sea lions (discussed above); however, in cetaceans, adenoviruses have been isolated from wild cetaceans, including a sei whale (Balaenoptera borealis),⁴ two bowhead whales (Balaena mysticetus)⁵ and a beluga whale, (Delphinapterus leucas).⁶ These viruses were isolated from gastrointestinal samples, and the correlation between disease and these viruses in cetaceans remains unclear and they had not been characterized.

In this study, we report two outbreaks of gastrointestinal disease in captive bottlenose dolphins (Tursiops truncatus) in two different parks of Spain with a distance of one year. In both cases we identified a novel adenovirus, the first well-characterized cetacean adenovirus. The animals were sampled (fecal and orina samples). Viral detection and identification were achieved by PCR-amplification from faecal samples, and sequencing, of partial adenovirus polymerase (pol)⁷ and hexon genes;⁸ and by producing the virus in HeLa cells, with PCR-detection/sequencing of the indicated gene fragments, observing virions within the cells by EM. We obtained the same sequence for all animals and it was different to any sequence from GenBank. The causative role of this adenovirus for the gastroenteritis was supported by our failure to find other potential etiological agents; by the exclusive detection in the sick dolphins but not in healthy individuals, of this novel adenovirus in faeces and of seropositivity for canine adenoviruses 1 and 2; and by the disappearance of the virus from faeces soon after the clinical signs disappeared. Therefore, we present partial genomes and phylogenetic analyses of Tursiops adenovirus 1 and the relationship between the gastrointestinal disease and this novel adenovirus. Further, we present development of quantitative PCR assays to be used for surveillance and epidemiologic studies of these viruses.

Acknowledgments

This work was carried out under the auspices of a collaborative agreement on virology studies in sea mammals between The Oceanogràfic park of the Ciudad de las Artes y las Ciencias of Valencia and the VISAVET Center of Complutense University of Madrid. We thank Vicente Rubio (IBV-CSIC, Valencia) for critical reading of the manuscript; Carmen Martín Espada for assays to detect rotavirus, coronavirus, Cryptosporidium and parvovirus; Belén Rivera and Rocío Sánchez for technical assistance; Francisco Javier García Peña (Laboratorio Central de Sanidad Animal de Algete) for bacteriological assays; Narcisa Martinez Quiles for providing HeLa cells; and Mario Soriano (Electron Microscopy service of the Centro de Investigación Principe Felipe de Valencia) for assistance with EM sample preparation and analysis. C.R-G. is the recipient of a predoctoral fellowship from the FPU programme of the Spanish Ministry of Education. M.M. is the recipient of a predoctoral fellowship from the PhD student grant programme of Complutense University of Madrid.

* Presenting author
⁺ Student presenter

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