The animals were individually exposed to the challenge viruses (108 EID50 per animal) by connecting a SaHoMa™-II mobile ultrasonic nebulizer (NEBU-TEC International med. Produkte Eike Kern GmbH, Germany) to a head hood attached to the horse’s head; the see more aerosol was generated from 7.5 ml egg allantoic fluid. Clinical observations and

body temperature were monitored daily for 21 days post-challenge as described above. Serum samples were collected on day 28 PC to determine the accumulation of influenza virus antibodies using the HAI assay, using the native viruses A/equine/Otar/764/07 (Н3N8) and A/equine/Sydney/2888-8/07 (Н3N8) in working doses of 4 hemagglutinating units as antigens. Nasal swabs were taken from the animals on days Panobinostat nmr 1, 3, 5 and 7 post-challenge to assess the degree of viral shedding as described above. The significance of the differences between groups were determined using two-way ANOVA followed by Tukey’s

multiple comparisons test; P < 0.05 was considered significant. The vaccine was completely safe for yearlings in both single and double intranasal administration mode. After the prime and booster vaccinations, the general clinical status and body temperature of the yearlings remained within the normal limits throughout the observation period (21 days), with a rectal temperature of 37.5–38.5 °C. Lacrimation, mucopurulent discharge, 4-Aminobutyrate aminotransferase signs of conjunctivitis or discharge from the nose was not observed

in any vaccinated animal (data not shown). Low vaccine viral shedding was observed in the upper respiratory organs. After the prime vaccination, the virus was shed in 47.7% (43/90) of animals on day 1 and 26.6% (24/90) on day 3, with titers ranging from 0.75 to 1.5 log10 EID50/0.2 ml (1.02 ± 0.04 and 1.29 ± 0.05 log10 EID50/0.2 ml at 1 and 3 days PV, respectively). After the booster vaccination, the virus was only shed on day 1 by 31.1% (28/90) of yearlings at titers ranging from 0.75 to 1.25 log10 EID50/0.2 ml (0.94 ± 0.04 log10 EID50/0.2 ml). As shown in Fig. 1 or Supplementary Table 1, both prime and booster vaccination of yearlings generated a protective immune response lasting 12 months (the observation period). After challenge with the wild-type homologous virus A/equine/Otar/764/07 (H3N8), the severity and duration of the clinical signs of disease, as well as the intensity and duration of viral shedding in the upper airway were significantly lower (from P = 0.03 to P < 0.0001) throughout the observation period in the vaccinated animals than the control group.