Duration of equine influenza virus shedding and infectivity in immunised horses after experimental infection with EIV A/eq2/Richmond/1/07

Abstract

Equine influenza (EI) is a major respiratory disease of horses. Recent outbreaks of EI have demonstrated the ease with which EI virus (EIV) can be transmitted internationally. This study aimed to improve our understanding of EIV shedding after infection of vaccinated horses, which would inform possible changes to current quarantine requirements. Our objectives were to compare commonly used diagnostic tests and to evaluate the relative merits of nasal and nasopharyngeal swabs for detection of EIV in vaccinated and unvaccinated ponies following EIV infection and to use these data to inform optimal quarantine procedures for the safe international movement of horses. Five ponies vaccinated against EI were infected experimentally with A/eq/Richmond/1/07 (Florida clade 2), 11 weeks after V2. Nasal and nasopharyngeal swabs were taken daily for 14 days and every 2 days for another 2 weeks. The 5 vaccinates were introduced sequentially for 48 h to 3 groups of 2 naïve sentinel ponies each on days 2, 4 and 6 post-challenge respectively. Clinical signs of disease and EIV shedding were monitored for 14 days after co-mingling. EIV was detected by 3 different methods of detection (EIV nucleoprotein ELISA, EIV nucleoprotein qRT-PCR and isolation/titration in embryonated hens’ eggs). Directigen™ EZ Flu A + B tests were also performed on samples from the vaccinated ponies for 6 days after infection. Results show that nasopharyngeal swabs were superior to nasal swabs, with increased frequency and amount of virus detected. The average mean duration of shedding was 6–8 days in naïve animals. All 3 sentinel groups were infected successfully with EIV after commingling with vaccinates, indicating up to 6 days of transmission. EI protection induced by vaccination is a dynamic process, naturally fluctuating and dependent on the time since last immunisation, with periods of high immunity (peak of immunity shortly after boost immunisation) and periods of susceptibility to EIV infection. This result indicates that vaccinated horses may actively transmit EIV if the immunity gap (a usual period of susceptibility between V2 and V3) is not adequately closed by immunisation. In infected sentinels EIV was detectable up to 12 days after commingling. Results also suggest that tests such as qRT-PCR may be a suitable substitute for time spent in pre-export quarantine.

Introduction

Equine influenza (EI) is a major respiratory disease of horses induced by the highly contagious equine influenza virus (EIV) (Landolt et al., 2007). Recent EI outbreaks in countries previously considered EI-free (South Africa, Australia and Japan, Guthrie et al., 1999, Moloney, 2011, Yamanaka et al., 2008) highlight the importance of effective, science-based quarantine and associated testing practices to prevent the international spread of this important pathogen. EI remains a considerable impediment to ease of movement of horses internationally, although, the development of rapid and sensitive diagnostic tests, such as quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assays offer the prospect of improved prevention of international spread of EIV.

Since the 2007 Australian EI outbreak, exportation of horses to Australia requires a 14 day pre-export quarantine period (PEQ), and 14–21 days post-arrival quarantine periods (PAQ), depending on whether or not all animals originate from the same PEQ, respectively. Such procedures are necessary to prevent infectious diseases outbreaks. However, several problems are associated with the combined duration of PEQ and PAQ, especially with competition and breeding horses. Racing, other competition or breeding opportunities may be lost and horses need to be kept in race training to maintain fitness. In addition, horses are sampled and tested by EIV qRT-PCR at least 4 times during combined PEQ and PAQ before being released. Equine influenza virus detection by qRT-PCR is highly sensitive and was extensively used during the 2007 Australian EI outbreak. It has also more recently been routinely used for EIV surveillance worldwide.

This study aimed to evaluate several different diagnostic testing methodologies currently in use, in order to provide a scientific evidence base for optimising current quarantine procedures for preventing international spread of EIV. The objectives of the study were:

• To compare nasal and nasopharyngeal swabs for detection of EIV in ponies experimentally infected with A/eq/Richmond/1/07. This objective aimed to compare the efficiency of nasal swabbing, which is better tolerated by the horses, with the conventional nasopharyngeal swabbing.

• To evaluate the duration of detectable levels of virus shedding, up to 28 days after experimental EI infection with EIV. Infectious EIV shedding is usually detectable in nasopharyngeal secretions for 7–10 days following infection using new methods, such as the EIV NP qRT-PCR, which present a higher sensitivity of detection. This objective aimed to improve current knowledge of the kinetics of EIV shedding in order to inform decision making regarding the duration of quarantine periods.

• To correlate the levels of virus shedding detected by standard methods (with diverse levels of sensitivity) in vaccinated animals with actual EIV infection of naïve sentinel ponies. This objective aimed to determine infectivity thresholds.