• Open Access

The Seroprevalence of Canine Influenza Virus H3N8 in Dogs Participating in a Flyball Tournament in Pennsylvania in 2010: A Follow-Up Study


Corresponding author: Dr. C.M. Otto, Department of Clinical Studies-Philadelphia, University of Pennsylvania, School of Veterinary Medicine, 3900 Delancey Street, Philadelphia, PA 19104; e-mail: cmotto@vet.upenn.edu



Canine influenza virus (CIV) H3N8 infection is thought to be highly contagious and potentially deadly. Low seroprevalence in dogs participating in the sport of flyball in 2009 was reported. It remains unknown if prevalence is changing in dogs at risk.


To determine seroprevalence of antibodies to CIV H3N8 in a population of pet dogs that participated in an indoor flyball tournament in Pennsylvania in November 2010.


Dogs participating in a flyball tournament.


Medical, travel, and activity histories for previous 10.5 months were obtained from consenting owners and serum samples were collected from dogs. Antibodies to CIV H3N8 were measured using hemagglutination inhibition.


Of 251 competing dogs, 103 dogs were tested. Overall, seroprevalence of CIV H3N8 in dogs participating in flyball was 1.9% (95% CI, 0.05–6.8%). Thirty-five dogs were tested in both 2009 (all titers <8) and 2010 (all titers <8). Ten of 103 dogs had been vaccinated for CIV H3N8. Two dogs had positive titers (>8) and both had been vaccinated twice in the previous month. Titers in remaining 8 vaccinated dogs were undetectable. Seroprevalence in vaccinated dogs was 20% (95% CI, 5.7–50.1%), whereas in unvaccinated dogs it was 0% (95% CI, 0–4.0%). Although 7 dogs had history of respiratory signs, only 1, which was vaccinated, was serologically positive for CIV H3N8.


Consistent with low prevalence of CIV H3N8 exposure previously reported in flyball dogs, unvaccinated dogs had 0% prevalence, suggesting a stable exposure rate. However, low seroprevalence may result in unprotected flyball dogs at risk for developing CIV H3N8.


canine influenza virus


equine influenza virus


interquartile range





Canine influenza virus (CIV) H3N8, an orthomyxovirus of the genus influenza A, causes acute respiratory disease with high morbidity and low mortality in dogs.[1] Clinical signs include coughing, sneezing, fever, inappetence, and nasal or ocular discharge.[1] Influenza A viruses are divided into subtypes based on their surface glycoproteins, hemagglutinin (H1–H17), and neuraminidase (N1–N9).[2] CIV originated from an H3N8 equine influenza virus (EIV) of Florida lineage.[1] The close proximity of racehorses to racing greyhounds in Florida enabled the horse virus to infect a spill-over host, dogs.[1]

Transmission of CIV H3N8 occurs by direct contact of dogs with respiratory droplets and fomites. Dogs begin shedding the CIV H3N8 virus before developing clinical signs. As a result, outbreaks could occur when clinically normal carriers come into contact with a naïve population, such as at dog sporting competitions, such as flyball, which frequently take place in indoor arenas. In addition, the H3N8 virus can remain viable for as long as 2 days on surfaces, 4 days in water at room temperature, and up to 12 hours on hands or 24 hours on clothes.[3] Therefore, inadequate handler hand-washing, contact with many dogs, close kenneling, and shared balls or toys may facilitate virus spread in flyball dogs. In an experimental study, comingling experimentally infected dogs with seronegative dogs, CIV H3N8 spread readily by direct contact and 100% of naïve dogs became infected.[4] It remains unknown if similar high levels of infectivity occur in natural settings. In June 2009, Intervet/Schering-Plough Animal Health introduced an inactivated CIV H3N8 vaccine to shorten the duration and severity of canine influenza.[5] The vaccine has been recommended for at-risk dogs in high density populations.[5] Although it has been suggested that CIV H3N8 outbreaks are not associated with any particular season, recent data suggest that infections may be more common during the winter months (January to March).1

Although EIV antibodies were identified retrospectively in stored, frozen blood samples from dogs as early as 1999,[6] sustained dog-to-dog transmission was not detected until 2004 among racing greyhounds in Florida. Over the course of 2004, outbreaks of CIV H3N8 occurred at 14 greyhound race tracks in 6 states.[1] By 2005, the virus spread beyond the greyhound population to shelters, veterinary clinics, and eventually the pet dog community.[1] CIV H3N8 now circulates as an enzootic virus in the United States; however, it remains unknown if it has reached a stable level of transmission or is still increasing in prevalence.

Infected greyhounds carried CIV from Florida and transmitted the virus to shelters in other states.[7] Dense populations and high numbers of seronegative, susceptible hosts entering shelters provide an ideal environment for high rates of virus transmission.[7] Management practices of shelter facilities also can contribute to CIV spread.[7] Molecular evolution analysis of CIV in New York, Pennsylvania, and Colorado shelters suggests that each shelter sustains the virus independently with limited mixing.[7] CIV is now considered enzootic in Florida, Colorado, New Jersey, New York, and Pennsylvania. It remains unknown if the virus can be sustained or spread in the absence of a reservoir, such as a shelter. Theoretically, any high density population environment with direct contact among dogs, such as dog shows, agility events, and flyball tournaments, presents a higher risk for transmission of this acute respiratory disease. Seroprevalence among pet dogs has been reported to be low in enzootic states,[8] but the change in seroprevalence over time has not been reported for the pet population or from any shelters.

Although dogs that participate in the competitive team sport of flyball have many risk factors for exposure to CIV H3N8, a previous study of seroprevalence in a population of dogs at a tournament in Downingtown, Pennsylvania in 2009 showed a prevalence of only 3%.[9]

The low seroprevalence in 2009 indicates low exposure in this potentially susceptible population, which emphasizes the importance of continued surveillance to understand this relatively new virus. This study determined the seroprevalence of CIV antibodies in a population of pet dogs that participated in the same flyball tournament in Downingtown, Pennsylvania, 1 year after the original study of CIV H3N8 in flyball dogs.

This prospective serologic survey was reviewed and approved by the University of Pennsylvania Institution Animal Care and Use Committee. Using the same protocol as employed for the 2009 study,[9] owner consent was obtained for all samples collected. Any dog attending the flyball tournament on the weekend of November 13–14, 2010 in Downingtown, Pennsylvania was eligible for testing. Owners were asked to complete a questionnaire2 regarding their dog's lifestyle, travel history, and health history over the previous year (from January 2010 through November 2010), and their dog's vaccination status.

All blood samples (1.5–3.0 mL) were collected at the tournament site on November 13 and 14, 2010 by a veterinarian or trained veterinary student from the jugular, cephalic or saphenous vein using a 1 inch 22G needle on a 3 mL Luer lock syringe. Sample site was determined based on patient size and personal preference. Samples were placed in serum separator tubes3 and centrifuged4 for 5 minutes at 1,048 × g within 15 minutes of collection. Serum was refrigerated and stored at 4°C immediately after centrifugation until shipping. Samples were submitted to the Animal Health Diagnostic Laboratory at Cornell University for hemagglutination inhibition analysis using a 2004 Florida isolate, the same strain used for the CIV vaccine. 5[5] All samples were shipped overnight in an insulated, sealed container with cold packs. A titer >8 was considered positive. Positive titers could have resulted from exposure, vaccination, or maternal transfer of antibodies. Titers of 8 or 16 were considered to be nonspecific or acute phase. Dogs with titers <8 were considered unexposed.

Data normality was rejected using Tukey's ladder test. Summary statistics determined the quartiles for the variables of interest. Confidence intervals were calculated using the method of Newcombe (www.vassarstats.net/prop1.html).[10] Summary statistical analysis was performed using Stata 10.1 statistical software.6 Values of P ≤ .05 were considered significant.

Out of 251 competing dogs, 103 dogs (56 females and 48 males) were tested. Median age was 5 years (IQR = 2.5, 8.2) and median weight was 32.0 lb (14.5 kg; IQR = 18.4, 40.0 lb or 8.2, 18.2 kg). Twenty flyball teams and 30 breeds were represented. Border Collies (n = 22), mixed breed (n = 14), and Jack Russell Terriers (n = 12) were the most common breeds. The prevalence of titers >8 in the group of 103 dogs was 1.9% (95% CI, 0.05–6.8%). Thirty-five of the dogs tested also were tested in 2009.[9] None of the 35 dogs previously tested had titers >8 in either 2009 or 2010, including 1 dog that had been vaccinated in the interim. The prevalence in this group was 0% (95% CI, 0–10%) in both years. Ten of the 103 dogs had been vaccinated for CIV. The 2 dogs with positive titers of 32 completed their series of vaccines less than 2 weeks before samples were obtained. The remaining 8 vaccinated dogs had titers <8. One of these dogs also was vaccinated within 2 weeks of the flyball tournament. Of the remaining vaccinated dogs, 2 completed the series 1 year previously, 2 were last vaccinated approximately 2 years before the tournament, and the time of vaccination remain unknown for 2 dogs. The seroprevalence in vaccinated dogs was 20% (95% CI, 5.7–50.1%), whereas in unvaccinated dogs, it was 0% (95% CI, 0–4.0%).

Although 7 dogs had a history of respiratory signs, only 1, which was vaccinated, was serologically positive. The seroprevalence of CIV in the total population was approximately 2%, but there were no detectable antibodies in unvaccinated dogs and only 20% of vaccinated dogs had measurable titers. None of the 11 dogs the owners of which also fostered dogs from shelters had positive titers.

Owners reported a history of clinical signs (eg, coughing, sneezing, fever, inappetence, nasal discharge, ocular discharge) in 7 dogs. Of these, 1 dog, which also had a history of vaccination, had a positive titer (32).

Dogs resided in 7 states, including states in which CIV is considered enzootic: Pennsylvania (n = 50) and New Jersey (n = 5). In addition, dogs had traveled to 28 states, Canada (n = 27), and France (n = 1) in the past 10.5 months. All 5 states in which CIV is considered enzootic were visited by at least 1 dog tested (CO, n = 1; FL, n = 1; NJ, n = 41; NY, n = 25; PA, n = 103). Dogs tested attended a median of 8 flyball tournaments (IQR = 4, 11 tournaments) in the previous 10.5 months.

No unvaccinated dogs in this group of potentially high risk dogs had positive titers to CIV H3N8, suggesting that the incidence in this pet population is low and a change in seroprevalence could not be detected between 2009 and 2010. The only seropositive dogs were vaccinated for CIV, but only 20% of vaccinated dogs had detectable titers. The seroprevalence among pet dogs in Colorado also is similarly low at 3.6%,[8] suggesting that CIV H3N8 is not common in the pet populations even where the virus is considered enzootic.

The low percentage of vaccinated dogs with positive titers calls into question the duration of immunity provided by the CIV vaccine. Current vaccination recommendations include an initial series of 2 doses given 2 weeks apart and annual boosters, but duration of immunity remains unknown.[6] Three dogs completed their series of vaccines less than 2 weeks before samples were obtained, but only 2 had positive titers. Ineffective vaccination, such as failure to receive the second dose or annual booster and individual variation in response to the vaccine, also can contribute to the lack of titers.

Mutation of CIV also may be a reason for the low seroprevalence. The virus could accumulate mutations in such a way that protective antibodies in the dog do not bind to the antigen used in the hemagglutination inhibition assay. Hoelzer et al investigated evolution dynamics of CIV in vaccinated and naïve dogs, and found mutation rates in vaccinated animals to be higher than in naïve animals.[11] Furthermore, antigenic escape is possible during the course of a single infection.[11] The assay could be missing dogs exposed to an antigenically distinct CIV, but this would not explain the absence of titers in vaccinated dogs.

One of the 2 dogs with a positive titer also had a history of clinical signs, and thus the source of the titer remains unclear. This dog's positive titer could have arisen from either natural infection or vaccination. Of 7 dogs with a history of clinical signs, only 1 had a positive titer. These dogs could have been infected with an influenza virus similar to the CIV strain used for HI testing and no longer have circulating antibodies. Another possibility is that these dogs could have been infected with a new influenza virus altered by antigenic drift, but that seems unlikely.7 The clinical signs also could be attributable to another etiology, such as Bordetella complex. The duration of positive titers after natural infection has not been studied beyond 21 days,[5] and thus further investigation of antibody protection from natural infection also is warranted.

This study had several limitations, similar to those reported in the 2009 study.[9] The sample size was relatively small and represented a fraction of the dogs at risk, participation was voluntary which could have introduced bias, survey information could have been impacted by recall bias, and the geographic region was limited. Furthermore, the lack of seropositive dogs made statistical evaluation difficult, and could have been influenced by assay sensitivity or antigenic drift of the virus.

Finally, the low seroprevalence suggests that should CIV H3N8 affect a dog involved in competitive team sports, particularly those where competitions allow for close dog contact (e.g, flyball in indoor arenas) and occur during higher risk periods (eg, winter months),1 many unprotected dogs could be exposed and at risk for developing CIV H3N8. It remains unknown, however, if the extent of exposure at these venues is sufficient to result in infection of other dogs. This potential risk warrants continued surveillance of flyball dogs and the pet dog community to monitor CIV H3N8.


Grant support: Schering Plough/Intervet directly paid the cost of sample analysis.

Conflict of Interest: Authors disclose no conflict of interest.


  1. 1

    Leutenegger CM, Estrada M, Armstrong RD. Canine influenza virus H3N8: A 4 year review of seasonal patterns, geographical frequency differences and age distribution. J Vet Intern Med. 2012;26:794–795 (abstract)

  2. 2

    The full survey is available from the corresponding author.

  3. 3

    Luer-Lok syringe, Becton, Dickinson, and Co, Franklin Lakes, NJ

  4. 4

    Vacutainer, Becton, Dickinson, and Co

  5. 5

    Personal correspondence with Dr. Edward Dubovi of Animal Health Diagnostic Laboratory at Cornell University

  6. 6

    Stata Statistical Software, release 10, StataCorp LP, College Station, TX

  7. 7

    Personal correspondence with Dr. Edward Dubovi of Animal Health Diagnostic Laboratory at Cornell University