Epidemiology of Equine Influenza in Horses in Taiwan. Hung-Chih CHOU, *Ying-Ling WU. (Received: May 3, Accepted: August 14, 2012)

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1 台灣獸醫誌 Taiwan Vet J 39 (1): Epidemiology 1-7, 2013 of Equine Influenza in Horses in Taiwan 1 Hung-Chih CHOU, *Ying-Ling WU (Received: May 3, Accepted: August 14, 2012) ABSTRACT Equine influenza (EI) is an acute, highly contagious disease worldwide. The EI virus belongs to the influenza virus A class of the family. Horses of all ages are susceptible to EI, especially foals. However, no epidemiologic evaluations of EI in Taiwan have been conducted within the past 10 years. To describe the current epidemiology of EI among horses in Taiwan, 217 samples of equine serum were collected from horses from 23 horse fields in 4 different regions of Taiwan between May and October 2010, i.e., the northern region (Taipei and Taoyuan), the central region (Hsinchu, Miaoli, and Taichung), the southern region (Kaohsiung and Pingtung), and the eastern region (Hualien and Taitung). Samples were tested with enzyme-linked immunosorbent assay (ELISA). Overall, approximately 47% (103/217) of samples were seropositive to EI. In addition, the highest prevalence of EI was seen in horse fields concentrated in northern, central, and southern regions of Taiwan. We used statistical methods to analyze the seroprevalance of EI in Taiwan and assess our ELISA results. Except for region, no significant associations between EI antibody prevalence and horse age, gender, origin, or proximity to rice or pig farms ( > 0.05). Therefore, future prevention of EI should be focused on northern, central, and southern regions of Taiwan. [Chou HC, * Wu YL.. Taiwan Vet J 39 (1): 1-7, * Corresponding author TEL: , wuyl@ntu.edu.tw] INTRODUCTION Equine influenza virus (EIV) is a single stranded, negative-sense RNA virus that belongs to the Orthomyxoviridae family and is closely related to the influenza A virus [2]. Influenza A is a zoonosis of significant concern because of its worldwide prevalence, numerous subtypes, and reassortant nature. It presents with a wide variation of pathogenic profiles ranging from asymptomatic infection to significant morbidity and death, depending on the species affected and the subtype involved [3]. EI is an acute, highly contagious viral disease that can cause rapidly spreading outbreaks of respiratory symptoms in horses and other equine species. EI is caused by 2 main strains of the virus: A/equine-1, H7N7 and A/equine-2, H3N8. H7N7 was recognized as early as 1956 in Prague, while H3N8 was first identified during a widespread EI epidemic that began in Miami, FL, in 1963 [9]. Typical symptoms include fever, cough, watery nasal discharge, and other respiratory syndromes [10]. There is no specific treatment for EI. Although vaccination is the most effective method of prophylaxis against influenza, commercially available vaccines induce only incomplete protection of a few months

2 2 Hung-Chih CHOU, Ying-Ling WU duration. Therefore, to reduce the risk of pandemic influenza and maintain the good health of animals, detection of the virus is crucial [8]. Exposure of an equine flock to influenza is determined by the measurement of antibody to influenza A in serum. Enzymelinked immunosorbent assay (ELISA) is a useful choice for efficient testing and accurate results [2]. Here, we report the epidemiology of EI among horses in Taiwan, as horses are economically very important animals in this area. This study is the first large serologic study of EI prevalence in Taiwan during the last 10 years. MATERIALS AND METHODS Sample size and selection of horses There are approximately30 registered riding schools or facilities that raise horses in Taiwan, housing approximately 1000 horses. Samples were obtained from horses from 23 chosen horse fields from May to October 2010, representing approximately 500 horses. Sample size was calculated using the following formula: N=Z 2 (P)(1 P)/E 2 where N was the required sample size, Z was the critical value for the desired confidence level (1.96), P was the expected prevalence of test results (0.5), and E was the maximum tolerable error for the confidence interval (0.05). The confidence interval was set at 95%. A sample size of 384 horses was calculated [4]. Because this large sample size was impractical to use, a second formula was used with known population to narrow the sample size. The new sample size was calculated by the following formula: New SS = SS/{1 + [(SS 1)/POP]} where New SS was the new sample size, SS was the sample size calculated before, and POP was the population. A new sample size of 217 horses was calculated. All blood samples were collected using a randomized sampling method based on number of horses per field. Horses with a recent history of untreated infectious disease or outward signs of clinical disease were excluded. Blood samples Sera were collected from the jugular vein from a total of 217 horses, each from 1 of the 4 pre-defined geographic regions: the northern region (Taipei and Taoyuan), the central region (Hsinchu, Miaoli, and Taichung), the southern region (Kaohsiung and Pingtung), and the eastern region (Hualien and Taitung).Blood samples were stored in EDTA and blank blood collection tubes. All of the samples were stored at-20 until processing [1]. Putative risk factors Putative risk factors were assessed using questionnaires for all horses, including questions on origin, breed, gender, age, and past medical or transport history. Horses were classified by age (0-5 years, 5-10 years, years, years, and >20 years), origin (place of birth or importation, such as Europe, America, Australia/New Zealand, or Taiwan), and type of activity (indoor vs. outdoor pleasure riding). Other risk factors for the horses were also recorded, including frequency of grooming and inspection, contact with other non-equine animals, management of the facilities (including frequency of environmental disinfection), and proximity of the facility to rice fields. Equine influenza ELISA The Influenza A Ab Test Kit (IDEXX Laboratories, Inc., Maine, United States) was used to detect antibodies to influenza A. The Influenza A Ab test can detect exposure in any species that generates an antibody response to the nucleoprotein, regardless of influenza subtype or antigenic variance between species. The assay is performed on 96-well plates that have been coated with influenza A viral antigen. Upon incubation of the test sample in the coated wells, influenza A specific antibody forms a complex with the coated antigen. After washing away unbound material, an anti-influenza A monoclonal antibody enzyme conjugate is added to the wells. In the absence of influenza A antibodies in the test sample, the conjugate is free to bind to the influenza A antigen on the plate. Conversely, if there are antibodies to influenza A present in the sample, the anti-influenza A conjugate is blocked from binding to the antigen. Unbound conjugate is washed away, and enzyme substrate is added. Subsequent color development is inversely proportional to the amount of anti-influenza A antibodies in the test sample.

3 3 Statistical analyses Statistical analyses were performed using SAS version 9.1 (SAS Institute, NC, US). Relationships between the risk factors and ELI- SA results were analyzed using the PROC frequency procedure. Chi-square tests were used to determine influence of putative risk factors on the seroprevalence of EI. The level of significance was set at P < RESULTS According to manufacturer s instructions for the assay, the negative control mean absorbance value should be and the positive control mean value should be < 0.50, in order to assure validity. The presence or absence of antibody to EI was determined by the samples to negative control ratio (S/ N value) for each sample. S/N value = samples mean/ negative control mean. S/N values < 0.50 are considered positive for the presence of EI antibodies. Overall, approximately 47% (103/217) of samples were positive for the EI antibody. The northern, central, and southern regions of Taiwan had higher prevalence rates (P < 0.05) of EI antibody (51% [56/110], 48% [25/52], and 57% [20/35], respectively; Table 1). The positive rates of eleven northern horse fields are as following: NA: 64%, NB: 45%, NC: 100%, ND: 30%, NE: 50%, NF: 45%, NG: 73%, NH: 86%, NI: 20%, NJ: 60%, NK: 0% The positive rates of five central horse fields are as following: MA: 36%, MB: 55%, MC: 50%, MD: 67%, ME: 0% The positive rates of four southern horse fields are as following: SA: 57%, SB: 88%, SC: 44%, SD: 25% The positive rates of three eastern horse fields are as following: EA: 33%, EB: 0%, EC: 9% EI antibody seroprevalence was found to be lower in the eastern region compared to the rest of Taiwan. No horses tested positive for EI antibodies in 1 of the 3 eastern region fields; 1 horse tested positive for EI antibodies in each of the remaining 2 eastern region fields (Table 2). In the northern region, at least one horse tested positive in 10 of 11 fields (Table 3). Horses tested positive for EI antibodies in 4 of 5 central region fields and in all 4 southern region fields (Table 4 and Table 5). Accordingly, most positive EI fields are located in the western region of Taiwan. The sampled horses included geldings, mares, and stallions. Some horses were imported from European countries, America, Australia, or New Zealand, while some were born in Taiwan. Characteristics of sampled horses are summarized in Table 6, Table 7, and Table 8. No significant associations (P > 0.05) were found between EI antibody prevalence and horse age, gender, origin, or proximity to rice or pig farms. DISCUSSION Horses are important animals from an economic standpoint throughout the world, particularly in Europe and America. However, in Taiwan, horses are less important economically compared to cows, pigs, and chickens. Furthermore, there are no legal horse-related recreational activities in Taiwan; in fact, many owners regard horses only as pets. Therefore, the prevention of epidemic disease in these horses is frequently ignored. The purpose of this study was to provide information on EI antibody seroprevalence among horses in Taiwan. Our results suggest that further re- Positive rates of EI ELISA in Taiwan. Northern Central Southern Eastern Sample size Positive numbers Positive rate (%) Positive rates of EI ELISA in eastern region. EA EB EC Sample size Positive numbers Positive rate (%)

4 4 Hung-Chih CHOU, Ying-Ling WU Positive rates of EI ELISA in northern region. NA NB NC ND NE NF Sample size Positive numbers Positive rate (%) NG NH NI NJ NK Sample size Positive numbers Positive rate (%) Positive rates of EI ELISA in central region. MA MB MC MD ME Sample size Positive numbers Positive rate (%) Positive rates of EI ELISA in southern region. The information of the horses. SA SB SC SD Sample size Positive numbers Positive rate (%) Gender numbers of horses Gelding Mare Stallion Age numbers of horses The age numbers of the horses. < > The information of rice fields and pig farms near horse fields. Rice fields nearby numbers of horse fields Pig farms nearby numbers of horse fields Yes No search should be prioritized, and preventive measures should be promptly implemented. Respiratory disease is second only to lameness as a cause of lost training in racing thoroughbreds in the UK, and viruses are indisputably an important cause of respiratory disease in the horse [5]. Among viral diseases, influenza A is a zoonosis of significant concern due to its worldwide prevalence, numerous subtypes, and reassortant nature [7]. It presents with a wide variation of pathogenic profiles ranging from asymptomatic infection to significant morbidity and death, depending on the species affected and subtype involved. In avian species, this disease is known as avian influenza. Both domestic and wild avian species are affected by the avian influenza virus. Respiratory signs and symptoms are common, along with reduced egg production, greenish diarrhea, bloodstained nasal and oral discharge, and cyanosis and edema of the head, comb, and wattle [6]. Our study objective was to describe the epidemiology of EI among horses in Taiwan using a commercially available ELISA kit to detect EI antibodies. Although this assay was designed to measure the level of antibody to avian influenza, it can also be used for hor-

5 5 ses to detect the existence of EI, as well as all other influenza A viruses. To our knowledge, this is the first report on the epidemiology of EI among horses in Taiwan. Notably, nearly half of the horses involved in this study tested positive for EI. The highest rates of EI antibody seropositivity were found in the northern, central, and southern regions of Taiwan, with EI-positive results ranging from 50% to 60%. In contrast, only 10% of horses in the eastern region tested positive for EI. However, most horses did not display clinical symptoms of infection during blood draws. Of note, this does not necessarily indicate that horses in Taiwan are not threatened by EI. Horses that are only partially immune may become subclinically infected and shed the virus. It follows that horses may contact the antigen of EI prior to the appearance of clinical symptoms. During an EI outbreak, EIV can potentially spread via droplets into the respiratory tract or via water into the digestive tract. Another mechanism for disease transmission is contagious. Rodents, flies, birds, and humans are important physical carriers during outbreaks of epidemic disease [3]. For this reason, the proximity of rice fields or pig fields to horses is important, as these fields commonly harbor large populations of rodents, flies, and birds. However, we found no significant association between rice or pig field proximity and the prevalence of EI. Furthermore, we found no difference in EI prevalence by horse age, gender, or origin. In summary, the only factor we found to be associated with EI prevalence was the region. Seropositivity to EI does not necessarily indicate an outbreak of disease. For example, horses may have previously been ill from EI but have since recovered clinically. However, our results illustrate that horses in Taiwan are still potentially at risk for EI. In particular, the western region of Taiwan should be monitored closely. Because horsemanship is not a universal activity in Taiwan, most horses are imported from Europe or America. These areas still report cases of EI; therefore, it is possible that some horses contacted the virus before arriving in Taiwan. Most horse fields in Taiwan are situated in the northern region, particularly in Taipei. Other factors contributing to the prevalence of EI include the preference of horse owners to utilize more than one horse field, as well as the tendency to change fields frequently. Furthermore, humans can physically carry the virus on their skin, hair, clothing, and shoes and can transfer the virus to horses; this can facilitate an outbreak of EI. Of note, the western region of Taiwan is the most densely populated area; this may partially explain why horses in fields with the highest prevalence of EI were found in the western region. The total samples seropositive rate is 47%, and it is a very high numeral compared with last investigation in 2000 (3.8%) [2]. During this ten year, horsemanship is more universal than before. On the other hand, the prevention of epidemic disease in horses is still not being paid attention. Besides, this investigation region is on a large scale in Taiwan and last just in local. Accordingly, we could anticipate the result. The sensitivity and specificity of ELISA kit are 99.7% and 95.4%, meaning most dates are trusty. However, we don t paired serum in this investigation because of limited funds, and it could cause a modicum inaccuracy. As mentioned above, commercially available vaccines induce only incomplete protection of a few months duration. All horses we tested stay in Taiwan over one year, and don t be vaccinated during this time. Therefore, the thing we could sure is that antibodies in seropositive horses were from natural equine influenza virus, not from vaccines. This is the first large-scale investigation in recent years of EI antibody prevalence among horses in Taiwan. In addition, we hope to further establish EI prevalence among all horses in Taiwan, as currently, these data do not exist with the World Organisation for Animal Health (OIE). Our results suggest that future studies are warranted and that design and implementation of preventive measures for EI outbreaks should be a priority. ACKNOWLEDGEMENTS This serological survey was financially supported by the Bureau of Animal and Plant Health Inspection and Quarantine, Council of Agriculture, Executive Yuan, Taiwan, R.O.C., (99AS BQ-B3 (18)). The authors thank Dr. Ching-Ho Wang for providing technical support and consultation.

6 6 Hung-Chih CHOU, Ying-Ling WU REFERENCES 1. 林嘉勇 臺灣馬匹日本腦炎流行病學調查 國立臺灣大學臨床動物醫學研究所碩士論文, 台北,17-18, 張詠樂 臺灣地區馬病 性疾病之血清學檢測 國立臺灣大學獸醫學研究所碩士論文, 台北,9-15, Ainsworth DM, Cheetham J. Disorders of the respiratory system. In: Reed SM, ed. Equine internal medicine. 3 rd ed. Saunders Elsevier, USA, , Chan KY, Wang CH, Wu YL. Serological survey of equine Piroplasmosis, equine granulocytic Anaplasmosis, and equine Lyme disease in Taiwan. Taiwan Vet J 36: , Chapman PS, Green C, Main JPM, Taylor PM, Cunningham FM, Cook AJC, Marr CM. Retrospective study of the relationships between age, inflammation and the isolation of bacteria from the lower respiratory tract of thoroughbred horses. Vet Rec 146: 91-95, Guo Y, Wang M, Kawaoka Y, Gorman O, Ito T, Saito T, Webster RG. Characterization of a new avian-like influenza A virus from horses in China. Virology 188: , Kawaoka Y, Bean WJ, Webster RG. Evolution of the hemagglutinin of equine H3 influenza viruses. Virology 169: , Nelson KM, Schram BR, McGregor MW, Sheoran AS, Olsen CW, Lunn DP. Local and systemic isotype-specific antibody responses to equine influenza virus infection versus conventional vaccination. Vaccine 16: , Quinlivan M, Zamarin D, Sastre AG, Cullinane A, Chambers T, Palese P. Attenuation of equine influenza viruses through truncations of the NS1 protein. Virology 79: , Wood J, Smith KC, Daly JM, Newton RJ. Viral infections of the equine respiratory tract. In: McGorum BC, ed. Equine respiratory medicine and surgery. 1 st ed. Saunders Elsevier, China, , 2007.

7 7 臺灣馬匹馬流行性感冒之流行病學調查 周宏致 * 吳應寧 國立臺灣大學獸醫專業學院臨床動物醫學研究所 台北市 ( 收稿日期 :101 年 5 月 30 日 接受日期 :101 年 8 月 14) 摘要馬流行性感冒 (Equine Influenza, EI) 為高接觸之傳染性疾病 馬流感病 為正黏液病 科 (Orthomyxoviridae) 之 A 型流行感冒病 (Influenza virus A), 可感染所有年齡層之馬匹, 其中以年輕馬較易感染 然而近十年內臺灣地 區並無馬流行性感冒之流行病學調查 為了解臺灣馬匹馬流行性感冒之現況, 於 2010 年 5 月至 10 月分別於臺灣北 部 ( 台北 桃園 ) 中部 ( 新竹 苗栗 台中 ) 南部 ( 高雄 屏東 ) 和東部 ( 花蓮 台東 ), 採集全臺馬場 217 匹馬 之頸靜脈血液樣本, 對馬流行性感冒病 抗體進行酵素連結免疫吸收分析 (enzyme-linked immunosorbent assay, ELI- SA) 血清學檢測 結果顯示 217 個樣本的陽性率約為 47% (103/217), 其中大部分血清學檢驗呈陽性的馬場集中於北 部 中部和南部 藉由統計軟體分析後, 血清學陽性之馬場只和地域有關, 和年齡 品種 來源 以及馬場附近是否 有稻田或豬舍等因素皆無顯著差異 因此, 未來防治馬流行性感冒的重點應集中於臺灣的北部 中部和南部地區 [ 周 宏致 * 吳應寧 臺灣馬匹馬流行性感冒之流行病學調查 台灣獸醫誌 39 (1):1-7,2013 * 通訊作者 TEL: , wuyl@ntu.edu.tw]