Genomic Dissection of an Icelandic Epidemic of Respiratory Disease in Horses and Associated Zoonotic Cases.

Iceland is free of the major infectious diseases of horses. However, in 2010 an epidemic of respiratory disease of unknown cause spread through the country's native horse population of 77,000. Microbiological investigations ruled out known viral agents but identified the opportunistic pathogen Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) in diseased animals. We sequenced the genomes of 257 isolates of S. zooepidemicus to differentiate epidemic from endemic strains. We found that although multiple endemic clones of S. zooepidemicus were present, one particular clone, sequence type 209 (ST209), was likely to have been responsible for the epidemic. Concurrent with the epidemic, ST209 was also recovered from a human case of septicemia, highlighting the pathogenic potential of this strain. Epidemiological investigation revealed that the incursion of this strain into one training yard during February 2010 provided a nidus for the infection of multiple horses that then transmitted the strain to farms throughout Iceland. This study represents the first time that whole-genome sequencing has been used to investigate an epidemic on a national scale to identify the likely causative agent and the link to an associated zoonotic infection. Our data highlight the importance of national biosecurity to protect vulnerable populations of animals and also demonstrate the potential impact of S. zooepidemicus transmission to other animals, including humans.IMPORTANCE An epidemic of respiratory disease affected almost the entire native Icelandic horse population of 77,000 animals in 2010, resulting in a self-imposed ban on the export of horses and significant economic costs to associated industries. Although the speed of transmission suggested that a viral pathogen was responsible, only the presence of the opportunistic pathogen Streptococcus zooepidemicus was consistent with the observed clinical signs. We applied genomic sequencing to differentiate epidemic from endemic strains and to shed light on the rapid transmission of the epidemic strain throughout Iceland. We further highlight the ability of epidemic and endemic strains of S. zooepidemicus to infect other animals, including humans. This study represents the first time that whole-genome sequencing has been used to elucidate an outbreak on a national scale and identify the likely causative agent.

Variability of Pasteurella multocida isolated from Icelandic sheep and detection of the toxA gene.

Pasteurella multocida can be part of the upper respiratory flora of animals, but under conditions of stress or immunocompromisation, the bacteria can cause severe respiratory symptoms. In this study, we compared 10 P. multocida isolates from Icelandic sheep with respiratory symptoms and 19 isolates from apparently healthy abattoir sheep. We examined capsule type, genetic variability and the presence of the toxA gene in the two groups. Surprisingly, we found that all ovine P. multocida isolates examined in this study carried the toxA gene, which markedly differs from what has been published from other studies. Interestingly, all isolates from abattoir animals were capsule type D, whilst bacteria isolated from animals with clinical respiratory symptoms had capsule type A, D or F. Examination of seven housekeeping genes indicated that the clinical respiratory isolates were significantly more heterogeneous than the abattoir isolates (P<0.05, two-tailed Mann-Whitney U test). The results suggest that there may be at least two groups of P. multocida in sheep - a genetically homogeneous group that resides in the respiratory tract and a genetically heterogeneous group that is the predominant cause of disease.

Morphological characteristics of Mesocestoides canislagopodis (Krabbe 1865) tetrathyridia found in rock ptarmigan (Lagopus muta) in Iceland.

Necropsies of 1010 rock ptarmigans (Lagopus muta) sampled in autumn 2006-2015 in northeast Iceland revealed Mesocestoides canislagopodis tetrathyridia infections in six birds (0.6 %), two juvenile birds (3 month old), and four adult birds (15 months or older). Four birds had tetrathyridia in the body cavity, one bird in the liver, and one bird both in the body cavity and the liver. There were more tetrathyridia in the body cavity of the two juveniles (c. 50 in each) than in three adults (10-40), possibly indicating a host-age-related tetrathyridia mortality. Approximately, half of tetrathyridia in the body cavity were free or loosely attached to the serosa, the other half were encapsulated in a thin, loose connective tissue stroma, frequently attached to the lungs and the liver. Tetrathyridia in the liver parenchyma incited variably intense inflammation. Tetrathyridia from the juvenile hosts were whitish, heart-shaped, and flattened, with unsegmented bodies with a slightly pointed posterior end. In the adult hosts, tetrathyridia were sometimes almost rectangular-shaped, slightly wider compared to those in the juveniles, but more than twice as long as the younger-aged tetrathyridia. Tetrathyridia infections are most likely acquired during the brief insectivorous feeding phase of ptarmigan chicks, and the tetrathyridia persist throughout the lifespan of the birds.

Mycoplasma ovipneumoniae--a primary cause of severe pneumonia epizootics in the Norwegian Muskox (Ovibos moschatus) population.

The Norwegian muskox (Ovibos moschatus) population lives on the high mountain plateau of Dovre and originates from animals introduced from Greenland. In the late summers of 2006 and 2012, severe outbreaks of pneumonia with mortality rates of 25-30% occurred. During the 2012 epidemic high quality samples from culled sick animals were obtained for microbiological and pathological examinations. High throughput sequencing (pyrosequencing) of pneumonic lung tissue revealed high concentrations of Mycoplasma ovipneumoniae in all six animals examined by this method and Pasteurella multocida subsp. multocida in four animals, whereas no virus sequences could be identified. Mycoplasma ovipneumoniae and P. multocida multocida were also isolated by culture. Using real time PCR on lung swabs, M. ovipneumoniae was detected in all of the 19 pneumonic lungs examined. Gross pathological examination revealed heavy consolidations primarily in the cranial parts of the lungs and it also identified one case of otitis media. Histologically, lung lesions were characterized as acute to subacute mixed exudative and moderately proliferative bronchoalveolar pneumonia. Immunohistochemical (IHC) examination revealed high load of M. ovipneumoniae antigens within lung lesions, with particularly intensive staining in the neutrophils. Similar IHC finding were observed in archived lung tissue blocks from animals examined during the 2006 epidemic. An M. ovipneumoniae specific ELISA was applied on bio-banked muskox sera from stray muskoxen killed in the period 2004-2013 and sick muskoxen culled, as well as sera from wild reindeer (Rangifer tarandus tarandus) on Dovre and muskoxen from Greenland. Serology and mycoplasma culturing was also carried out on sheep that had been on pasture in the muskox area during the outbreak in 2012. Our findings indicated separate introductions of M. ovipneumoniae infection in 2006 and 2012 from infected co-grazing sheep. Salt licks shared by the two species were a possible route of transmitting infection.

Fatal infection in two Icelandic stallions caused by Halicephalobus gingivalis (Nematoda: Rhabditida).

Opportunistic infections with the free living nematode Halicephalobus gingivalis are infrequently reported in horses but the cases are widespread geographically. The nematodes are believed to penetrate wounds and subsequently reproduce within the host tissues. This paper reports two cases of a fatal disease in stallions of the Icelandic breed in Iceland. Case 1: a stallion, which sustained injuries to the mouth after an accident, developed severe neurological signs and had to be euthanatized. Histological examination revealed mild inflammation and malacia in the cerebellum associated with the presence of numerous H. gingivalis nematodes. Case 2: a stallion that started swerving to one side and lost balance was euthanatized due to lack of response to therapy and rapid deterioration. Histological examination revealed numerous H. gingivalis nematodes in the cerebellum, brain stem, cervical spinal cord and in the meninges, with minimal reactive changes. In case 1 the infection presumably was acquired by nematodes from soil penetrating through wounds in the mouth. The mode of the H. gingivalis infection in case 2 is uncertain. These are the first cases of H. gingivalis infection reported from Iceland and the second report from the Nordic countries.

Skin-infiltrating T cells and cytokine expression in Icelandic horses affected with insect bite hypersensitivity: a possible role for regulatory T cells.

Equine insect bite hypersensitivity (IBH) is a seasonally recurrent, pruritic skin disorder caused by an IgE-mediated reaction to salivary proteins of biting flies, predominantly of the genus Culicoides. The aim of this study was to define T cell subsets and cytokine profile in the skin of IBH-affected Icelandic horses with particular focus on the balance between T helper (Th) 1, Th2 and T regulatory (Treg) cells. Distribution and number of CD4+, CD8+ and Forkhead box P3 (FoxP3)+ T cells were characterized by immunohistochemical staining in lesional and non-lesional skin of moderately and severely IBH-affected horses (n=14) and in the skin of healthy control horses (n=10). Using real-time quantitative reverse transcription-polymerase chain reaction, mRNA expression levels of Th2 cytokines (Interleukin (IL)-4, IL-5, IL-13), Th1 cytokines (Interferon-γ), regulatory cytokines (Transforming Growth Factor ß1, IL-10) and the Treg transcription factor FoxP3 were measured in skin and blood samples. Furthermore, Culicoides nubeculosus specific serum IgE levels were assessed. Lesions of IBH-affected horses contained significantly higher numbers of CD4+ cells than skin of healthy control horses. Furthermore, the total number of T cells (CD4+ and CD8+) was significantly increased in lesional compared to non-lesional skin and there was a tendency (p=0.07) for higher numbers of CD4+ cells in lesional compared to non-lesional skin. While the number of FoxP3+ T cells did not differ significantly between the groups, the ratio of Foxp3 to CD4+ cells was significantly lower in lesions of severely IBH-affected horses than in moderately affected or control horses. Interestingly, differences in FoxP3 expression were more striking at the mRNA level. FoxP3 mRNA levels were significantly reduced in lesional skin, compared both to non-lesional and to healthy skin and were also significantly lower in non-lesional compared to healthy skin. Expression levels of IL-13, but not IL-4 or IL-5, were significantly elevated in lesional and non-lesional skin of IBH-affected horses. IL-10 levels were lower in lesional compared to non-lesional skin (p=0.06) and also lower (p=0.06) in the blood of IBH-affected than of healthy horses. No significant changes were observed regarding blood expression levels of Th1 and Th2 cytokines or FoxP3. Finally, IBH-affected horses had significantly higher Culicoides nubeculosus specific serum IgE levels than control horses. The presented data suggest that an imbalance between Th2 and Treg cells is a characteristic feature in IBH. Treatment strategies for IBH should thus aim at restoring the balance between Th2 and Treg cells.

Mycobacterium avium subsp. paratuberculosis enters the small intestinal mucosa of goat kids in areas with and without Peyer's patches as demonstrated with the everted sleeve method.

The main lesions of paratuberculosis in ruminants are in the small intestine. Previous studies have shown that the bacterium enters the small intestine through M cells found in the follicle-associated epithelium lining the domes of the Peyer's patches. The everted sleeve method, devised for the in vitro study of intestinal absorption, was used in this study to investigate the uptake of Mycobacterium avium subsp. paratuberculosis in goat intestine. Everted small intestinal sleeves of goat kids, prepared from areas with and without Peyer's patches, were incubated for 60 min in 3H-labeled bacterial solution. The results of this study imply that the bacteria can enter the intestinal mucosa of the jejunum, both in areas with and without Peyer's patches. These findings indicate, therefore, that M. avium subsp. paratuberculosis bacteria not only enter through M cells but also through enterocytes.