Genomic Dissection of an Icelandic Epidemic of Equine Respiratory Disease

The native horse population of Iceland has remained free of major infectious diseases. Between May and July 2010 an epidemic of respiratory disease swept through the population. Initial microbiological investigations ruled out known equine viral agents as the cause of the infections, but identified the opportunistic pathogen Streptococcus zooepidemicus as being frequently isolated from diseased animals. This diverse bacterial species has a broad host range and is usually regarded as a commensal of horses. By genome sequencing S. zooepidemicus recovered from horses during the epidemic we show that although multiple clones of S. zooepidemicus were present in the population, one particular clone, ST209, was responsible for the epidemic. Concurrent with the epidemic, ST209 caused zoonotic infections, highlighting the pathogenic potential of this clone. Phylogenetic analysis suggests that the original ST209 strain entered Iceland in late 2008 or early 2009. Epidemiological investigation revealed that the incursion of this strain into a training yard that utilized a submerged treadmill between the 5th and 19th of February 2010 was a critical trigger for the ensuing epidemic of disease, provided a nidus for the infection of multiple horses, and subsequent distribution of these animals to multiple sites in Iceland.


1 1
Icelandic isolates of ST209 predominantly shared the same complement of 41 spacer 2 1 2 sequences ( Figure 2). However, ST209 isolates from horses on farm 5 were found to 2 1 3 contain a novel spacer 35 sequence. This unique spacer was present in isolates 2 1 4 recovered from four resident horses (3, 5, 7 and 14), but not from three other 2 1 5 residents (1, 2 and 9) on the 2 nd June, or from any other affected horses from other 2 1 6 farms throughout Iceland. The ST209 isolates that were recovered from the horses,  became secondary centers of transmission. However, two horses from yard A, which 2 2 8 had returned to farm 16 on the 4 th of February, were not incubating the disease.

9
Therefore, it is likely that the transmission of ST209 strains to horses at yard A began 2 3 0 between the 5 th and 19 th of February 2010. probably due to the relatively long incubation period permitting apparently healthy 2 3 7 horses to return to their original premises post-training. Instead, the most likely 2 3 8 source of transmission of the epidemic strain at yard A was a water treadmill, which 2 3 9 horses used on a daily basis. The water used in the treadmill contained no 2 4 0 disinfectant and was changed on a once-or twice-weekly basis, providing ideal 2 4 1 conditions for the transmission of S. zooepidemicus between the visiting horses.

4 2
Transmission via the water treadmill at yard A provides one possible explanation for

7 9
Interestingly, this rate is faster than the closely-related host-restricted pathogen 2 8 0 and Waller 2015). Therefore, it is also possible that more than one variant of ST209, 2 9 4 potentially originating from the same animal, was introduced to Iceland prior to the 2 9 5 start of the epidemic. In this latter scenario, the ST209 variants may have circulated 2 9 6 for a shorter period of time within the Icelandic horse population prior to February 2 9 7 2010. However, regardless of the date at which the epidemic strain was introduced to 2 9 8 Iceland, the transfer of an infected horse to yard A, identified through our network 2 9 9 analysis, remains essential to the rapid spread of the ST209 strains that was 3 0 0 characteristic of the epidemic.  isolated from Scandinavia and it is interesting to note the close relationships between 3 0 8 many training yards in Iceland and this region of Europe. The import of horses to 3 0 9 Iceland has been prohibited since 1882 for biosecurity reasons. Although the import 3 1 0 of used riding equipment is also prohibited, it is difficult to control. Therefore, 3 1 1 contaminated tack represents a possible horse-related route of introduction of the 3 1 2 ST209 strain.

4
This study emphasizes the importance of national biosecurity as a barrier to protect   connected farms and training centers to be investigated.  respiratory tract and conjunctiva post-mortem were also used to inoculate these cells.

9 3
Inoculated cells were passaged and the conditioned cell culture media was also used 3 9 4 to inoculate new cell cultures. The sample was judged as being virus negative if no 3 9 5 cytopathic effect could be seen after the third passage. At each passage cells were 3 9 6 processed by cytospin and examined by indirect fluorescent antibody staining using 3 9 7 pooled convalescent serum samples from 6 horses as the primary antibody.

9 8
Additional virus isolation attempts were made in the same way with nasal swabs from      Iceland. The human isolates from the United Kingdom were provided by Professor

2 7
Androulla Efstratiou at Public Health England. Bovine isolates were provided by Dr.

2 8
Adrian Whatmore of the Animal and Plant Health Agency. The human isolate from 5 2 9 Finland was a kind gift from Professor Sinikka Pelkonen and Dr. Tamara