Sarcocysts were detected in routinely processed histological sections of skeletal muscle, but not cardiac muscle, of two adult male otters (Lutra lutra; Mustelidae) from northern Norway following their post-mortem examination in 1999 and 2000. The sarcocysts were slender, spindle-shaped, up to 970 μm long and 35-70 μm in greatest diameter. The sarcocyst wall was thin (∼ 0.5 μm) and smooth with no visible protrusions. Portions of unfixed diaphragm of both animals were collected at the autopsies and kept frozen for about 14 years pending further examination. When the study was resumed in 2013, the thawed muscle samples were examined for sarcocysts under a stereo microscope, but none could be found. Genomic DNA was therefore extracted from a total of 36 small pieces of the diaphragm from both otters, and samples found to contain Sarcocystidae DNA were used selectively for PCR amplification and sequencing of the nuclear 18S and 28S ribosomal (r) RNA genes and internal transcribed spacer 1 (ITS1) region, as well as the mitochondrial cytochrome b (cytb) and cytochrome c oxidase subunit 1 (cox1) genes. Sequence comparisons revealed that both otters were infected by the same Sarcocystis sp. and that there was no genetic variation (100 % identity) among sequenced isolates at the 18S and 28S rRNA genes (six identical isolates at both loci) or at cox1 (13 identical isolates). PCR products comprising the ITS1 region, on the other hand, had to be cloned before sequencing due to intraspecific sequence variation. A total of 33 clones were sequenced, and the identities between them were 97.9-99.9 %. These sequences were most similar (93.7-96.0 % identity) to a sequence of Sarcocystis kalvikus from the wolverine in Canada, but the phylogenetic analyses placed all of them as a monophyletic sister group to S. kalvikus. Hence, they were considered to represent a novel species, which was named Sarcocystis lutrae. Sequence comparisons and phylogenetic analyses based on sequences of the 18S and 28S rRNA genes and cox1, for which little or no sequence data were available for S. kalvikus, revealed that S. lutrae otherwise was most closely related to various Sarcocystis spp. using birds or carnivores as intermediate hosts. The cox1 sequences of S. lutrae from the otters were identical to two sequences from an arctic fox, which in a previous study had been assigned to Sarcocystis arctica due to a high identity (99.4 %) with the latter species at this gene and a complete identity with S. arctica at three other loci when using the same DNA samples as templates for PCR reactions. Additional PCR amplifications and sequencing of cox1 (ten sequences) and the ITS1 region (four sequences) using four DNA samples from this fox as templates again generated cox1 sequences exclusively of S. lutrae, but ITS1 sequences of S. arctica, and thus confirmed that this arctic fox had acted as intermediate host for both S. arctica and S. lutrae. Based on the phylogenetic placement of S. lutrae, the geographical location of infected animals (otters, arctic fox) a...

BackgroundInfectious keratoconjunctivitis (IKC) is a transmissible disease in semi-domesticated Eurasian reindeer (Rangifer tarandus tarandus). It is regarded as multifactorial and a single causative pathogen has not yet been identified. From clinical outbreaks we have previously identified Cervid herpesvirus 2 (CvHV2) and Moraxella bovoculi as candidates for experimental investigations. Eighteen reindeer were inoculated in the right eye with CvHV2 (n = 5), M. bovoculi (n = 5), CvHV2 and M. bovoculi (n = 5) or sterile saline water (n = 3; controls).ResultsAll animals inoculated with CvHv2, alone or in combination with M. bovoculi, showed raised body temperature, increased lacrimation, conjunctivitis, excretion of pus and periorbital oedema; clinical signs that increased in severity from day 2 post inoculation (p.i.) and throughout the experiment, until euthanasia 5–7 days p.i. Examination after euthanasia revealed corneal oedema, and three animals displayed a corneal ulcer. CvHV2 could be identified in swab samples from both the inoculated eye and the control eye from most animals and time points, indicating a viral spread from the inoculation site.ConclusionsThis study showed that CvHV2 alone and in combination with M. bovoculi was able to cause the characteristic clinical signs of IKC in reindeer, whereas inoculation of M. bovoculi alone, originally isolated from a reindeer with IKC, did not produce clinical signs. Previous studies have suggested that herding procedures, animal stress and subsequent reactivation of latent CvHV2 infection in older animals is a plausible mechanism for IKC outbreaks among reindeer calves and young animals in reindeer herds. However, further studies are needed to fully understand the infection biology and epidemiology associated with IKC in reindeer.

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