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Short Communication
Short Communication Detection of modifiedlive equine intranasal vaccine pathogens in adult horses using quantitative PCR C. Harms, S. Mapes, N. Akana, D. Coatti Rocha, N. Pusterla DURING an equine respiratory disease outbreak, the modified-live intranasal vaccines available against strangles (Streptococcus equi subspecies equi) and equine influenza virus (EIV) are commonly used due to their ability to induce a rapid onset of mucosal immunity. However, horses displaying clinical signs of respiratory disease are routinely tested using quantitative PCR (qPCR) analysis of nasal secretions to help determine the causative pathogen(s), and these qPCR assays do not distinguish between a modified-live vaccine pathogen and the wildtype pathogen. Thus, if a horse that had recently received a modified-live intranasal vaccine subsequently develops signs of respiratory disease it would be unclear whether the qPCR detection of that pathogen in nasal secretions is from the vaccine or represents true infection. Therefore, the objective of this study was to provide a timeline for how long after vaccination will a horse continue to shed modified-live vaccine S. equi subspecies equi and EIV. A total of 23 adult horses with no intranasal vaccine history were selected from the Center for Equine Health, School of Veterinary Medicine, University of California at Davis and randomly assigned to one of two groups (12 horses in S. equi vaccine group; 11 horses in EIV vaccine group). Groups were housed according to gender in large paddocks of 3–5 horses each, with central water and food areas. Study horses ranged from 4 to 15 years of age (median=10 years), with a total of 13 geldings and 10 mares. Most horses belonged either to the Quarter Horse or Thoroughbred breed. Horses were vaccinated annually against eastern/western equine encephalitis virus, tetanus, West Nile virus, rabies virus, equine herpesvirus type 1/herpesvirus type 4 and EIV. Horses had not been vaccinated for over 12 months at the time of study commencement. Prior to study commencement, each study horse was deemed to be healthy based on a physical evaluation, and nasal secretions from both nostrils of each horse were analysed by qPCR to rule out current S. equi subspecies equi or EIV pathogen shedding. The horses to Veterinary Record (2014) C. Harms, S. Mapes, MS, N. Akana, D. Coatti Rocha, N. Pusterla, DVM, PhD, Department of Veterinary Medicine and Epidemiology, School of Veterinary Medicine, University of California,
doi: 10.1136/vr.102592 Davis, CA 95616, USA E-mail for correspondence: [email protected] Provenance: not commissioned; externally peer reviewed Accepted September 16, 2014
be vaccinated then received either a modified-live S. equi subspecies equi vaccine (Pinnacle, Zoetis) or a modified-live EIV vaccine (FluAvert, Merck Animal Health) in the left nostril. Two horses in each group remained unvaccinated to determine if the vaccine pathogens could be transmitted via direct contact. All procedures were approved by the Institutional Animal Care and Use Committee of the University of California. On each subsequent day (10 days for S. equi group; 5 days for EIV group), brief physical evaluations were performed and nasal swabs from the right and left nostril were obtained from each horse using six-inch rayon-tipped swabs (Puritan Products Company LLC). Disposable gloves were worn when handling horses, and gloves were changed between horses. Nasal secretions were processed for DNA purification using an automated extraction system (CAS-1820 X-tractor Gene, Corbett Life Science). The samples were analysed using previously validated and established qPCR assays targeting either the SeM protein gene of S. equi subspecies equi or the haemagglutinin 1 (HA1) gene of EIV (Pusterla and others 2011). To determine the sample quality and efficiency of nucleic acid extraction, all samples were assessed for the presence of the housekeeping gene eGAPDH as previously described (Pusterla and others 2008). At three weeks (S. equi group only) and six months (S. equi group and EIV group) after initial vaccination, horses were revaccinated according to the vaccine manufacturers’ recommendations. The same experimental procedure was used for vaccine administration, sample collection and processing, except that samples from both groups were only collected for five days following vaccine administration in anticipation of decreased shedding times. The first and second S. equi subspecies equi vaccines and the first EIV vaccines were given during the months of June and July, while boost vaccinations were given in late November. The average ambient temperatures for the vaccine periods during the months of June and July ranged from 20.0°C to 28.9°C (minimum temperature range 10.0–16.7°C, maximum temperature range 30.6–40.6°C), while the average ambient temperature for the vaccine periods in late November was 10.6°C (minimum 2.8°C, maximum 20.0°C). Statistical data analysis for determining differences in pathogen shedding (duration and amount) across periods and between nostrils was performed by use of an Exact Wilcoxon Signed-Rank test for paired data. A value of P
Short Communication
Short Communication Detection of modifiedlive equine intranasal vaccine pathogens in adult horses using quantitative PCR C. Harms, S. Mapes, N. Akana, D. Coatti Rocha, N. Pusterla DURING an equine respiratory disease outbreak, the modified-live intranasal vaccines available against strangles (Streptococcus equi subspecies equi) and equine influenza virus (EIV) are commonly used due to their ability to induce a rapid onset of mucosal immunity. However, horses displaying clinical signs of respiratory disease are routinely tested using quantitative PCR (qPCR) analysis of nasal secretions to help determine the causative pathogen(s), and these qPCR assays do not distinguish between a modified-live vaccine pathogen and the wildtype pathogen. Thus, if a horse that had recently received a modified-live intranasal vaccine subsequently develops signs of respiratory disease it would be unclear whether the qPCR detection of that pathogen in nasal secretions is from the vaccine or represents true infection. Therefore, the objective of this study was to provide a timeline for how long after vaccination will a horse continue to shed modified-live vaccine S. equi subspecies equi and EIV. A total of 23 adult horses with no intranasal vaccine history were selected from the Center for Equine Health, School of Veterinary Medicine, University of California at Davis and randomly assigned to one of two groups (12 horses in S. equi vaccine group; 11 horses in EIV vaccine group). Groups were housed according to gender in large paddocks of 3–5 horses each, with central water and food areas. Study horses ranged from 4 to 15 years of age (median=10 years), with a total of 13 geldings and 10 mares. Most horses belonged either to the Quarter Horse or Thoroughbred breed. Horses were vaccinated annually against eastern/western equine encephalitis virus, tetanus, West Nile virus, rabies virus, equine herpesvirus type 1/herpesvirus type 4 and EIV. Horses had not been vaccinated for over 12 months at the time of study commencement. Prior to study commencement, each study horse was deemed to be healthy based on a physical evaluation, and nasal secretions from both nostrils of each horse were analysed by qPCR to rule out current S. equi subspecies equi or EIV pathogen shedding. The horses to Veterinary Record (2014) C. Harms, S. Mapes, MS, N. Akana, D. Coatti Rocha, N. Pusterla, DVM, PhD, Department of Veterinary Medicine and Epidemiology, School of Veterinary Medicine, University of California,
doi: 10.1136/vr.102592 Davis, CA 95616, USA E-mail for correspondence: [email protected] Provenance: not commissioned; externally peer reviewed Accepted September 16, 2014
be vaccinated then received either a modified-live S. equi subspecies equi vaccine (Pinnacle, Zoetis) or a modified-live EIV vaccine (FluAvert, Merck Animal Health) in the left nostril. Two horses in each group remained unvaccinated to determine if the vaccine pathogens could be transmitted via direct contact. All procedures were approved by the Institutional Animal Care and Use Committee of the University of California. On each subsequent day (10 days for S. equi group; 5 days for EIV group), brief physical evaluations were performed and nasal swabs from the right and left nostril were obtained from each horse using six-inch rayon-tipped swabs (Puritan Products Company LLC). Disposable gloves were worn when handling horses, and gloves were changed between horses. Nasal secretions were processed for DNA purification using an automated extraction system (CAS-1820 X-tractor Gene, Corbett Life Science). The samples were analysed using previously validated and established qPCR assays targeting either the SeM protein gene of S. equi subspecies equi or the haemagglutinin 1 (HA1) gene of EIV (Pusterla and others 2011). To determine the sample quality and efficiency of nucleic acid extraction, all samples were assessed for the presence of the housekeeping gene eGAPDH as previously described (Pusterla and others 2008). At three weeks (S. equi group only) and six months (S. equi group and EIV group) after initial vaccination, horses were revaccinated according to the vaccine manufacturers’ recommendations. The same experimental procedure was used for vaccine administration, sample collection and processing, except that samples from both groups were only collected for five days following vaccine administration in anticipation of decreased shedding times. The first and second S. equi subspecies equi vaccines and the first EIV vaccines were given during the months of June and July, while boost vaccinations were given in late November. The average ambient temperatures for the vaccine periods during the months of June and July ranged from 20.0°C to 28.9°C (minimum temperature range 10.0–16.7°C, maximum temperature range 30.6–40.6°C), while the average ambient temperature for the vaccine periods in late November was 10.6°C (minimum 2.8°C, maximum 20.0°C). Statistical data analysis for determining differences in pathogen shedding (duration and amount) across periods and between nostrils was performed by use of an Exact Wilcoxon Signed-Rank test for paired data. A value of P
