Avian influenza at animal‐human interface: One‐health challenge in live poultry retail stalls of Chakwal, Pakistan

Abstract Background Live poultry retail stalls (LPRSs) are believed to be the source of human infection with avian influenza viruses (AIVs); however, little is known about epidemiology of these viruses in LPRSs of Pakistan. Objectives The current study was conducted to estimate the virological and serological prevalence of AIVs in humans and poultry and associated risk factors among seropositive butchers. Methods A field survey of LPRSs of Chakwal District was conducted between December 2015 and March 2016. In total, 322 samples (sera = 161 and throat swab = 161) from butchers and 130 pooled oropharyngeal swabs and 100 sera from birds were collected. Baseline sera (n = 100) from general population were also tested. Data were collected by structured questionnaires. Sera were tested by hemagglutination inhibition (HI) test further confirmed by micro‐neutralization test (MN). Swabs were processed by real‐time RT‐PCR. Logistic regression analyses were conducted to identify risk factors. Results In butchers, 15.5% sera were positive for antibodies against H9 virus using a cutoff of ≥40 in HI titer; 6% sera from general population were positive for H9. Seroprevalence in poultry was 89%, and only 2.30% swabs were positive for H9. Presence of another LPRS nearby and the number of cages in the stall were risk factors (OR > 1) for H9 seroprevalence in butchers. Conclusions This study provides evidence of co‐circulation of H9 virus in poultry and exposure of butchers in the LPRSs, which poses a continued threat to public health. We suggest regular surveillance of AIVs in occupationally exposed butchers and birds in LPRSs.


| INTRODUC TI ON
About hundred years ago, the 1918 influenza pandemic devastated entire communities and was the most severe pandemic in recent history, sweeping the globe quickly and killing more than 50 million people. 1 The emergence of novel influenza strains through mutation and reassortment from avian reservoir remains a constant threat to animal and public health, 2 which has been illustrated by multiple human cases due to novel H7N9 in China. 3 Phylogenetic analysis of these H7N9 viruses indicated that different gene segments were closely related to different AIV strains isolated from domestic ducks and wild birds in South-East Asia. 4 In addition, sporadic human infections with symptoms ranging from mild conjunctivitis and influenza-like illness (ILI) to pneumonia and multi-organ failure caused by multiple AIVs subtypes have been reported globally. [4][5][6][7][8] Most of these reports recognized recent exposure to poultry as the most probable cause of infection. 9,10 Multidisciplinary teams have dealt with emerging infectious diseases, for example, SARS, H5N1 AIV, and the 2009 H1N1 pandemic 11 by adopting One-Health strategy. The exponential growth of the human population has elevated the importance of human-animal-environment interface. 12 In addition, traditional smallscale poultry production systems have been transformed into industrial integrated operations in most parts of the world including Asia. 13,14 Various studies have been conducted targeting the animal-human interface and have reported different routes of human exposure to AIVs identifying potential host determinants that favored persistence of virus. [4][5][6][7][8][9][15][16][17] Early preparedness for global pandemics has become an important public health task. Preparedness includes identification of determinants of disease spread at animal-human interface and ultimately recognizing areas to implement disease prevention and outbreak control. 14 Surveillance of domestic poultry and poultry handlers has provided evidence that AIVs are evolving continuously 10,18,19 and are able to cross species barrier and infect mammals. 7,[20][21][22][23][24] Plentiful evidence is, thus, available to support the phenomenon of interspecies transmission of AIVs globally; nevertheless, very little information is available about the situation in Pakistan. 8,15,18,25,26 The main objective of the current study was to understand the role of LPRSs in poultry-to-human transmission of AIVs. We tried to eliminate bias in estimates by conducting survey and analysis with simple random sampling. We also linked human data with poultry data to identify any association among them. Very few epidemiological surveys based on probability sampling methods have been conducted in developing countries due to lack of baseline data, and this study could provide appropriate design and information for other research to plan a survey with limited resources and data. We found H9N2 virus infection in birds in the LPRSs and evidence for their spillover to occupationally exposed humans.

| Study design
The study was conducted in District Chakwal which falls at latitude and in chickens being sold in those LPRSs and to identify the potential risk factors associated with estimated prevalence. According to the Commissioner Office of Chakwal, the total number of functional LPRSs in Chakwal was 276. Live poultry sold in these markets included mostly chickens (layer, broiler, and indigenous/desi), and rarely ducks, geese, pigeons, and quail also. However, samples were collected only from broiler birds in the stalls. The sample size calculations were performed using epiR package in R software. 28 A simple random method was used to calculate the desired sample size. 29 The estimated sample size was 161 assuming 50% study design, data collection and analysis, decision to publish, or preparation of the manuscript.

K E Y W O R D S
avian influenza, avian influenza viruses, birds, H9N2, live bird market butchers, Pakistan, risk factors, seroprevalence, surveillance seroprevalence, 15,30 95% confidence, and absolute precision of ±5% with population size of 276 for Chakwal District. The estimated (a priori) prevalence of 50% was used to get the largest sample size to increase the desired precision of estimate. 29 A list of randomly selected butchers (n = 161) was drawn by random digit generator (stattrek.com/statistics/random-number-generator.aspx) from the LPRSs list (N = 276). Our field teams (Veterinarian and Paramedical staff) visited the 161 selected LPRSs for consent and enrollment of butchers ( Figure 1).
In addition, serum samples from the general community that were available in the repository of a private laboratory of Chakwal District were included as control subjects. The sera from control subjects having no exposure to poultry or livestock during their daily routine were collected by the registered phlebotomist at the laboratory, who was referred by medical personnel for routine screening for medical diagnosis. The laboratory labeled these samples anonymously to keep the confidentiality of patients. Only data about sex, age, education, and pre-existing history of chronic disease were available in records.
From each LPRS, one worker was enrolled after taking consent. Inclusion criteria for butchers included ≤15 years of age, having worked in LPRSs for more than six months, having exposure to poultry slaughtering as part of daily activities for more than 8 h/wk (slaughtering and sales). Exclusion criteria included having severe respiratory illness in last three months, known history (self-reporting) of human immunodeficiency virus (HIV), tuberculosis, and those who refused to participate at the time of enrollment. No participants received a human seasonal influenza vaccine. A standard questionnaire was used to collect epidemiological information, including demographic characteristics, occupational exposure (slaughtering, selling, and handling poultry), and other potential risk factors of exposure (Appendix S1). Risk factors included education of respondent, age of respondent, smoking, having any chronic disease, stall remained open, number of cages, selling birds other than broiler, adding newly arrived birds to cages already having birds, presence of wild bird, presence of rodents, access of stray dogs, access of stray cats, preparing raw poultry using knife, touching face/food, washing facility in market, washing instruments, washing hands, wearing mask, wearing gloves, wearing protective boots, wearing apron, covering nose and mouth with handkerchief, and put carcass on ground/in drum, clean cutting board, washing gizzard, wash stall daily, another stall nearby. A trained team member administered the questionnaire in a face-to-face interview with the participant.
Each enrolled participant allowed serum and throat swab collection. From the general population, sera were collected from any age group. Information about sex, age, education, and pre-existing Children's Research Hospital, Memphis, Tennessee, USA). Throat or nasopharyngeal swab specimens obtained from participants were maintained in a viral transport medium (Brain heart infusion broth with antibiotics, Oxoid no. CM1135, Oxoid, UK) and were transported on ice to the DSL, UVAS, Lahore, where they were coded and stored at −80°C until laboratory analysis. 31 Butchers were also requested to provide oropharyngeal swabs, and blood samples from the broiler birds present in the stall at the time of survey. Apparently, healthy chickens were selected from the LPRSs after taking informed consent of stall owner. Selection of birds was an arbitrary choice of the butcher. All birds included in the study were adult broiler birds commercially available for slaughter. Only 130 stall owners allowed oropharyngeal swab to be taken from their birds, and 100 stall owners allowed collection of blood from their birds during slaughtering. A trained veterinarian collected pooled oropharyngeal swabs, that is, at each LPRS individual swabs from 5 birds. Swabs were collected and pooled in one tube containing viral transport medium. Blood was collected from one bird at each stall.

| Laboratory analysis
The hemagglutination inhibition (HI) test was used to screen human sera 31 and poultry sera. 32 Sera from butchers were tested in duplicate by parallel testing in US and Pakistan, and each assay included specific positive (chicken hyperimmune antiserum against specific subtype antigens) and negative control (sera from non-exposed adults) sera. Briefly, sera were treated with receptor destroying enzyme (RDE; Denka Seiken Co. Ltd to eliminate non-specific inhibitors. One volume of serum (50 μL) was added to 3 volume of reconstituted RDE (150 μL) and incubated at 37°C overnight. After that sera were heat inactivated at 56°C for 30 minutes. Finally, 6 volume of phosphate buffered saline (PBS) (300 μL) was added to make final dilution of 1:10 (500 ul total volume of prepared sera).

| Data analysis
R software was used to conduct all statistical analyses. 28

| Prevalence of AIV in butchers and birds in LPRSs
Among the 161 throat swabs examined, none were positive for ma-

| Risk factors for AIV H9 seroprevalence in butchers
Thirty-three variables were screened in a univariable analysis, and 6 variables showed association with seroprevalence of H9 in butchers (Table 4). In multivariable analysis, two variables namely "another stall nearby" and "number of cages" remained strongly associated with seroprevalence of H9 (P < .05) ( Table 5).

| D ISCUSS I ON
Our study results suggest subclinical infection of butchers with H9 as these workers self-reported no history of severe respiratory illness on enrollment. Absence of exposure to H5 and H7 AIVs was also documented as none of the sera from butchers and control subjects were tested positive for H7 and H5 and suggest low or no prevalence of these viruses in poultry. The latter was confirmed by the absence of H5 and H7 AIVs in oropharyngeal swabs of poultry in the current study. Similar has been reported in various studies previously. 17,30,41,42 Although H5N1 has persistently circulated in poultry in many other countries, especially in Asia, human infection has rarely been reported. 24,43-46 H5 viruses have shown strong host specificity for infection, which could be the reason for the low infection rate in poultry exposed people even in countries enzootic for the disease. 17,47 Overall, 15.5% H9 seroprevalence was found in occupationally exposed butchers providing serological evidence of human infection with antigenically similar viruses. 48 It was significantly higher (15.5% vs 6%) than controls subject (P < .05) and was strongly associated with seroprevalence (89%) in live birds in stall (P < .001) suggesting poultry-to-human transmission of H9 AIV in these LPRSs instead of human-to-human contact. 21 These results also provide confidence in the cutoff value (titer ≥ 40); we used to score seropositivity. Chakwal has a high density of commercial poultry, 49  genes, raising public health concern globally. 59,60 Concurrent circulation of H9N2 reassortants in LPRSs and evidence of human exposure to these circulating viruses represents a major risk for emergence of new pandemic in both occupationally exposed poultry handlers and the general population in Pakistan. Due to the presence of mammalian host-specific markers in these H9 viruses, they pose a serious threat to public health in Pakistan.
In current study, small sample size has put limitation on the generalization of estimates produced. Due to small number of H9seropositive persons (n = 25), the power of study may not be efficient to detect significant differences for some potential risk factors.
Furthermore, the current study was a cross-sectional survey; these study designs are not suited to estimate disease incidence, natural history of disease, or the rate of secondary human-to-human infection. Cross-sectional studies are also relatively weaker in establishing causality of risk factors than with an analytic design, such as with a cohort study. 61 Well-designed prospective epidemiological studies with follow-up of human and their birds will be better suited to answer such questions. 46 The authors acknowledge that including control human sera from repository of a private laboratory in Chakwal, which were not collected in the same time frame in which the butchers' sera were collected, might have introduced bias in our results.

| CON CLUS IONS
In summary, we have documented seroprevalence of H9 in butchers in LPRSs of Chakwal. We also established evidence of co-circulation of H9 virus in poultry in the same LPRS, which poses a continued threat for emergence of novel genotypes of AIVs through intra-and inter-subtypic reassortment. Governmental interventions to mitigate the prevalence of AIVs in these markets may reduce the risk for emergence of novel viruses. Continued active surveillance and genetic characterization of H9N2 are highly recommended to detect any ongoing public health risk. 33 Studies using one-health strategy, combined with clinical and virological surveillance in target population, would be needed to document any cross-species transmission of novel avian influenza viruses. 62

ACK N OWLED G EM ENTS
The authors are highly grateful to all the butchers in LPRSs, who participated in the study and provided data and samples. Without