Little Evidence of Avian or Equine Influenza Virus Infection among a Cohort of Mongolian Adults with Animal Exposures, 2010–2011

2010-2011. PLoS ONE 9(1): e85616. doi:10.1371/journal.pone.0085616 Little Evidence of Avian or Equine Influenza Virus Infection among a Cohort of Mongolian Adults with Animal Exposures, 2010-2011 Nyamdavaa Khurelbaatar 0 1 Whitney S. Krueger 0 1 Gary L. Heil 0 1 Badarchiin Darmaa 0 1 Daramragchaa Ulziimaa 0 1 Damdindorj Tserennorov 0 1 Ariungerel Baterdene 0 1 Benjamin D. Anderson 0 1 Gregory C. Gray 0 1 Matthias Johannes Schnell, Thomas Jefferson University, United States of America 0 Ethics Statement This study was approved by IRBs at the University of Florida, the National Center of Communicable Diseases, Mongolia, and the US Army Medical Department. Written informed consent was obtained from each participant 1 1 Mongolian Association for Infectious Diseases Researchers, Ulaanbaatar, Mongolia, 2 College of Public Health and Health Professions, and Emerging Pathogens Institute, University of Florida, Gainesville, Florida, United States of America, 3 National Influenza Center, National Center of Communicable Diseases, Ministry of Health , Ulaanbaatar , Mongolia , 4 National Center for Zoonotic Diseases, Ministry of Health , Ulaanbaatar , Mongolia Avian (AIV) and equine influenza virus (EIV) have been repeatedly shown to circulate among Mongolia's migrating birds or domestic horses. In 2009, 439 Mongolian adults, many with occupational exposure to animals, were enrolled in a prospective cohort study of zoonotic influenza transmission. Sera were drawn upon enrollment and again at 12 and 24 months. Participants were contacted monthly for 24 months and queried regarding episodes of acute influenza-like illnesses (ILI). Cohort members confirmed to have acute influenza A infections, permitted respiratory swab collections which were studied with rRT-PCR for influenza A. Serologic assays were performed against equine, avian, and human influenza viruses. Over the 2 yrs of follow-up, 100 ILI investigations in the cohort were conducted. Thirty-six ILI cases (36%) were identified as influenza A infections by rRT-PCR; none yielded evidence for AIV or EIV. Serological examination of 12 mo and 24 mo annual sera revealed 37 participants had detectable antibody titers ($1:10) against studied viruses during the course of study follow-up: 21 against A/Equine/Mongolia/01/2008(H3N8); 4 against an avian A/Teal/Hong Kong/w3129(H6N1), 11 against an avian-like A/Hong Kong/1073/1999(H9N2), and 1 against an avian A/Migrating duck/Hong Kong/MPD268/2007(H10N4) virus. However, all such titers were ,1:80 and none were statistically associated with avian or horse exposures. A number of subjects had evidence of seroconversion to zoonotic viruses, but the 4-fold titer changes were again not associated with avian or horse exposures. As elevated antibodies against seasonal influenza viruses were high during the study period, it seems likely that cross-reacting antibodies against seasonal human influenza viruses were a cause of the low-level seroreactivity against AIV or EIV. Despite the presence of AIV and EIV circulating among wild birds and horses in Mongolia, there was little evidence of AIV or EIV infection in this prospective study of Mongolians with animal exposures. - Competing Interests: The authors have declared that no competing interests exist. Introduction Among the worlds last pastoral people groups, Mongolians often live in close proximity with flocks of migrating birds or freeranging herds of horses. Mongolias large migrating bird populations have been shown to harbor both highly-pathogenic and low-pathogenic avian influenza viruses (AIV) [1,2,3,4]. In addition, having some of the highest horse-to-man population ratios in the world, Mongolia has suffered some of the worlds largest equine influenza A virus (EIV) epizootics [5]. In recent years H3N8 EIV epizootics occurred in 20072008 (96,390 cases; 24,600 deaths) and again in 2011 (75,208 cases; 40 deaths) (Mongolias Department of Veterinary and Animal Breeding). In discussions with rural Mongolians, we learned that when their horses became sick, their children sometimes suffered upper respiratory tract infections with similar symptoms. Knowing humans can experience AIV infections and H3N8 EIV has been experimentally shown to infect volunteers who were intranasally inoculated [6], we sought to prospectively study Mongolians for evidence of AIV and EIV infections. Materials and Methods Study Design Details about the study location, study subjects, enrollment methods, database generation, and serology laboratory methods have previously been published [7]. Briefly, Mongolians greater than 18 yrs of age were recruited from 3 regions in Mongolia and followed with monthly encounters over a 24-month period for evidence of influenza-like-illness. Sera and questionnaire data were collected at enrollment, 12 months, and 24 months. Annual follow-up questionnaires collected demographic, health, and animal exposures data during the past year. Poultry or horse exposure was defined as contact $5 cumulative hour/week for at least one week. Monthly Follow-up During enrollment, cohort participants were given oral and written instructions and a digital thermometer. They were asked to contact study field staff upon developing signs and symptoms of an influenza-like illness (ILI) via a telephone call. Study staff also conducted monthly home visits to remind participants of the importance of reporting ILI and to assess whether an illness was present or had occurred during the preceding week. ILI was defined as acute onset of a respiratory illness with an oral (or equivalent from other body region) measured temperature $38uC and a sore throat, cough, shortness of breath, or respiratory distress for 4 or more hours. Investigating Influenza-like Illness When a possible ILI was reported to study staff, a home visit was performed within 24 hrs of notification. If the subject met the ILI case definition, a study nurse completed an ILI questionnaire and collected 2 respiratory swab specimens (nasal and pharyngeal). The swab specimens were stored in viral transport media and transported using cold-chain within 24 hrs after collection to local field laboratories in Khovd and Dornogovi provinces and to the National Influenza Center in Ulaanbaatar. Laboratory Methods Sera and ILI respiratory swab aliquots were preserved at 280uC and transported on dry ice to the University of Florida for testing. Sera were tested for evidence of human, equine, and avian influenza infections over time (Table 1). ILI swabs were studied for molecular evidence of influenza A virus RNA. Real-time RT-PCR influenza assay. Viral RNA was isolated from 140 ml of each swab specimen and processed using the Qiagen: QIAamp Viral RNA Mini Kit (Qiagen Inc., Valencia, California) following a mini-spin protocol. Contaminants were washed away by two wash buffers and the RNA eluted in 50 ml of elution buffer. Specimens were screened for the presence of influenza A viral RNA using the CDCs Hu (...truncated)