Abstract

Gammaherpesviruses have recently been implicated in genital^2,3, oral³, digestive and respiratory (unpublished) mucosal pathology in several species of dolphins and whales. These cetacean viruses remain, for the most part, uncharacterized as to their genetic relatedness, disease-inducing potential, and in the role that they may play in triggering stranding in which significant mucosal lesions are observed. It also remains to be determined whether dolphins from the same species harbor genetically dissimilar gammaherpesviruses or whether there is genetic heterogeneity among gammaherpesviruses that infect different cetacean species.

The main obstacle in understanding the impact that herpesviruses, in general, may have on cetacean health is the fact that these viruses have not been isolated or characterized.¹ However, molecular approaches that use DNA extracted from lesions that contain these viruses make it possible to obtain sequence of complete genes for genetic and phylogenetic analyses. Preliminary multigenic analyses of gammaherpesviruses associated with mucosal lesions in three dolphin species, suggest that different strains of gammaherpesviruses may infect different dolphin species (unpublished). These analyses are limited, due to the lack of complete gene sequences of gammaherpesviruses from other species of dolphins and whales.

To accelerate the detection and identification of gammaherpesviruses from lesions and tissues, a real-time polymerase chain reaction (PCR) assay was developed for the rapid diagnosis of gammaherpesviruses from three dolphin species, Atlantic bottlenose dolphin (Tursiops truncatus), Risso's dolphin (Grampus griseus), and rough-toothed dolphin (Steno bredanensis), all members of the Delphinidae family. The assay targets the highly conserved herpesvirus glycoprotein B gene and is driven by a set of primers that has been shown to amplify a DNA fragment of 138-nt in length by conventional PCR (unpublished). Specificity and sensitivity of the assay are enhanced by the use of an LNA probe (Invitrogen) with a FAM dye attached to its 5' end and a quencher on its 3' end. Identification of gammaherpesvirus DNA from the three dolphin species was accurate and there was no cross-reaction with a gammaherpesvirus of a Blainville's beaked whale (Mesoplodon densirostris), a member of the Ziphidae family. This real-time PCR assay is rapid, specific and sensitive in the detection gammaherpesviruses contained in dolphin lesions and tissues and takes less than one hour to obtain the final results, in both graphic and quantitative modes. In addition to sensitivity, the real-time assay does not require the handling of post-amplification reactions, an advantage over conventional PCR assays. As a rule, cycle threshold (Ct) values for most of the gammaherpesvirus-containing lesions and tissues begin after cycle 14, while negative samples do not reach the Ct 30 value during the 40 cycles of the assay. In addition, hundreds of DNA samples from lesions and tissues taken from many different cetacean species and determined to be negative for gammaherpesviruses by conventional PCR, have also tested negative in the real-time assay. The real-time PCR assay has properly identified gammaherpesviruses in pharyngeal lesions of T. truncatus and S.bredanensis, genital lesions of T. truncatus and G. griseus, and oropharyngeal, esophageal, and first stomach lesions of T. truncatus and S.bredanensis. It is not known at this time whether the real-time PCR assay described here will also detect all gammaherpesviruses of the Delphinidae, as we have not assayed infected lesions or tissues from all the genera within the Delphinidae family.

Acknowledgements

This work was supported by a grant from the Save the Wild Dolphin Program from Harbor Branch Oceanographic Institution, and the Aquatic Animal Health Program of the University of Florida.

References

1.  Manire CA, Smolarek KA, Romero CH, Kinsel MJ, Clauss TM, Byrd L. 2006. Proliferative dermatitis associated with a novel alphaherpesvirus in an Atlantic bottlenose dolphin, Tursiops truncatus. Journal of Zoo and Wildlife Medicine 37: 174-181.

2.  Saliki JT, Cooper EJ, Rotstein DS, Caseltine SL, Pabst DA, McLellan WA, Govet P, Harms C, Smolarek KA, Romero CH. 2006. A novel gammaherpesvirus associated with genital lesions in a Blainville's beaked whale (Mesoplodon densirostris). Journal of Wildlife Diseases 42 (1): 142-148.

3.  Smolarek Benson KA, Manire CA, Ewing RY, Saliki JT, Townsend FI, Ehlers B, Romero CH. 2006. Identification of novel alpha- and gamma herpesviruses from cutaneous and mucosal lesions of dolphins and whales. Journal of Virological Methods 136: 261-266.