RESEARCH ARTICLE

Serologic and behavioral risk survey of

workers with wildlife contact in China
Corina Monagin1¤a*, Blanca Paccha2, Ning Liang1, Sally Trufan3¤b, Huiqiong Zhou4,
De Wu4, Bradley S. Schneider1¤c, Aleksei Chmura5, Jonathan Epstein5, Peter Daszak5,
Changwen Ke4, Peter M. Rabinowitz3
1 Metabiota Inc., San Francisco, California, United States of America, 2 Yale University, New Haven,
Connecticut, United States of America, 3 University of Washington Center for One Health Research, Seattle,
Washington, United States of America, 4 Guangdong Provincial Center for Disease Control and Prevention,
Guangzhou, Guangdong, China, 5 EcoHealth Alliance, New York, United States of America
a1111111111
a1111111111 ¤a Current address: One Health Institute, School of Veterinary Medicine, University of California, Davis,
a1111111111 United States of America
a1111111111 ¤b Current address: Levine Cancer Institute, Carolinas Healthcare System, Charlotte, NC, United States of
America
a1111111111
¤c Current address: Etiologic, Oakland, California, United States of America
* cgmonagin@ucdavis.edu

OPEN ACCESS Abstract

Citation: Monagin C, Paccha B, Liang N, Trufan S, We report on a study conducted in Guangdong Province, China, to characterize behaviors
Zhou H, Wu D, et al. (2018) Serologic and
behavioral risk survey of workers with wildlife
and perceptions associated with transmission of pathogens with pandemic potential in
contact in China. PLoS ONE 13(4): e0194647. highly exposed human populations at the animal-human interface. A risk factor/exposure
https://doi.org/10.1371/journal.pone.0194647 survey was administered to individuals with high levels of exposure to wildlife. Serological
Editor: Hiroshi Nishiura, Hokkaido University testing was performed to evaluate prior infection with several wildlife viral pathogens. Follow
Graduate School of Medicine, JAPAN up serology was performed on a subset of the cohort as well as close contacts of individuals.
Received: April 5, 2017 1,312 individuals were enrolled in the study. Contact with a wide range of wildlife species
Accepted: March 7, 2018
was reported in both occupational and occasional contexts. The overall proportion of individ-
uals seropositive to any of the tested wildlife pathogens was approximately 4.0%. However,
Published: April 3, 2018
persons employed as butchers demonstrated a seropositivity of 9.0% to at least one patho-
Copyright: © 2018 Monagin et al. This is an open gen of interest. By contrast, individuals working as hunters had lower rates of seropositivity.
access article distributed under the terms of the
Creative Commons Attribution License, which
Among the study population, a number of other behaviors showed correlation with sero-
permits unrestricted use, distribution, and positivity, including contact with particular wildlife species such as field rats. These results
reproduction in any medium, provided the original demonstrate the need to further explore zoonotic risks of particular activities regarding wild-
author and source are credited.
life contact, and to better understand risks of persons working as butchers with wildlife
Data Availability Statement: All relevant data are species.
within the paper and its Supporting Information
files.

Funding: This study was made possible by Google/

Skoll and the generous support of the American
people through the United States Agency for
International Development (USAID) Emerging Introduction
Pandemic Threats PREDICT program (Cooperative
The majority of human infectious diseases have an animal origin, therefore understanding the
Agreement No. AID-OAA-A-14-00102). The
contents are the responsibility of the authors and
human-animal interface as it relates to disease emergence and risk is of upmost importance
do not necessarily reflect the views of USAID or the [1]. The increasing frequency and variety of human-wildlife interactions in China provide
United States Government. Metabiota Inc., is a opportunities for the transmission of zoonotic pathogens from animals to humans [2].

PLOS ONE | https://doi.org/10.1371/journal.pone.0194647 April 3, 2018 1 / 14

 Surveillance of wildlife workers, China

commercial company that received funding from Handling, transporting, and butchering of hunted or farmed wildlife poses a risk of pathogen
Google/Skoll. The funder provided support in the spillover into humans [3]. In southern China provinces, including Guangdong, a significant
form of salaries for authors CM, BS, NL, but did
percentage of the population obtains fresh meat for consumption from wet markets, commu-
not have any additional role in the study design,
data collection and analysis, decision to publish, or nity markets that specialize in selling and butchering live animals, including animals that are
preparation of the manuscript. The specific roles of rare and endangered. Research has demonstrated that human-animal interfaces, such as within
these authors are articulated in the ‘author these wet markets, provide an ideal environment for infectious disease emergence, transmis-
contributions’ section. sion, and amplification [4–7].
Competing interests: Metabiota Inc. is a Wet markets and restaurants that butcher and sell wild animals are common in China and
commercial company that received funding from South East Asia, creating a high-risk interface where humans come into regular contact with
Google/Skoll. This does not alter our adherence to the blood and bodily fluids of wild and domesticated animals. Intermingling of wildlife in wet
PLOS ONE policies on sharing data and materials.
markets can lead to inter-species transmission of pathogens, amplifying and maintaining these
pathogens, and may result in spillover into a species that can more efficiently transmit the
pathogen to humans [8]. Furthermore, increasing numbers of individuals are traveling to
urban-rural interface areas to eat at “wildlife” restaurants. Hunted or farmed wildlife are trans-
ported and kept alive at these restaurants until consumption, increasing the risk of pathogen
spillover.
The wildlife trade has played a role in the emergence of severe acute respiratory syndrome
(SARS), avian influenza, Ebola, and other diseases of wildlife origin [2, 9]. Guangdong Prov-
ince in China was the site of the first cases of SARS in 2002. Now known to have originated in
bats [10], SARS emerged in humans and other mammals in wet markets, such as Himalayan
palm civets (Paguma larvata) and raccoon dogs (Nyctereutes procyonoides) [11]. The disease
spread to 26 countries and infected over 8,000 people; killing nearly 800 [12]. SARS shut down
trade of domestic animals and resulted in nearly $40 billion in losses to the global economy
[13]. H5N1 outbreaks during 1999–2008 and H7N9 in 2013 have demonstrated the realities of
the risk of disease transmission and spread in China and the detriments to animal and human
health, as well as human livelihoods [9, 14]. These epidemics shed new light on how quickly
and how far newly emerged zoonotic diseases can spread in today’s world. These outbreaks
continue to challenge scientists, policymakers, and public health systems, exemplifying the
need for a better understanding of the factors leading to emergence, and clarity regarding
emerging infectious disease policy, particularly as it concerns high-risk human-animal
interfaces.
Although mounting evidence implicates illegal trade and behavioral and cultural factors as
influencing disease emergence, it is clear that more research is needed to identify these drivers
[2, 4, 6, 7, 15]. A key aspect of understanding human-wildlife interfaces is the characterization
of risk behaviors and perceptions of individuals who have wildlife contact. Specifically, there is
a need to evaluate whether particular behaviors are associated with increased risk of transmis-
sion. As well, serological studies may be helpful to evaluate exposure, either current or prior,
to zoonotic agents. For example, when Guan et al. (2003) surveyed animals in markets for evi-
dence of SARS-like coronavirus [11], they also performed serological analysis on market
employees. Seroprevalence in their study ranged widely between traders (wild-animal to vege-
table), butchers and community controls.
The present study focuses on the potential for zoonotic viral transfer through contact with
wildlife in Guangdong prefectures in China, and seeks to augment our understanding and
identification of risky populations, occupations, and behaviors, as well as the perceptions of
risk at these interfaces. We performed a serological survey and concurrent behavioral ques-
tionnaire of individuals with wildlife contact in Guangdong Province, China, in order to better
characterize occupations and community-level behavioral risks that contribute to zoonotic
transmission of various wildlife pathogens with pandemic potential.

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 Surveillance of wildlife workers, China

Materials and methods

Study location and targeted population
This study took place in Guangdong Province (23˚24’N, 113˚30’E), China from 2009–2013.
Guangdong is located on the South China Sea coast, bordering Guangxi Province, Hunan
Province, Jiangxi Province and Fujian Province. It neighbors Hong Kong and Macau, and has
over 50 million residents living in 9 cities: Guangzhou, Shenzhen, Dongguan, Foshan, Zhong-
shan, Zhuhai, Jiangmen, Huizhou and Zhaoqing. Enormous consumption of animal and ani-
mal products in this region is believed to serve an economical incentive for butchers, cooks
and hunters in less developed Guangdong prefectures, e.g. Qingyuan, Yunfu and Meizhou.
Guangdong Province has consistently reported the largest population in China since 2005,
with the highest GDP rankings for the last 25 years. Its economy is highly dependent on the
Pearl River Delta Region, which contributes around 80.0% total provincial GDP while sup-
porting 29.0% of the provincial population.
The study was conducted in 12 prefectures within the Guangdong Province: Dabu, Jiaoling,
Pingyuan, Lianping, Heping, Lianshan, Lianzhou, Yunfu, Yunan, Xinyi, Deqing and Fengkai
(Fig 1). We chose field sites within the identified prefecture areas that have wet-markets, wild-
animal restaurants, and other environments with a higher risk of animal-to-human pathogen
transmission. These environments, particularly the markets, were deliberately chosen due to
their size and character. Similar species of wildlife, both live and butchered, were present, with
similar butchering methods being utilized. Populations in these environments with high-risk
animal-to-human pathogen transmission activities, such as hunting, and butchering in wet-
markets and wild-animal restaurants were selected. We targeted high-risk individuals, defined
as individuals with high levels of exposure to wildlife (wild animal blood or bodily fluids)—pri-
marily hunters, persons working in wet markets and restaurants that butcher wild game, who
could be followed over a period of time. Inclusion criteria included participant age between 18–
65, and individuals that reported previously hunting, selling, butchering, or eating wild animals.
Individuals who did not provide informed consent and/or did not agree to biological sample
collection were excluded. Individuals were residents of the site area so that they were available
for follow up visits. More than 20 potential field sites were visited to gauge level of risk at the
site (number of high-risk animal-human interfaces). Local prefecture-level Chinese Center for
Disease Control (CDC) teams were asked to pre-enroll individuals who fit the inclusion criteria
and would be available for follow up visits for up to three years. Based on pre-enrollment num-
bers and human-animal interfaces identified at each site, 12 field sites that were identified in the
previously named prefectures: Dabu, Jiaoling, Pingyuan, Lianping, Heping, Lianshan, Lianzhou,
Yunfu, Yunan, Xinyi, Deqing and Fengkai were chosen to participate in the study (Fig 2). Study
participation was voluntary and respondents were given the equivalent of 5 USD (approxi-
mately 30 CNY) as compensation for their time. Informed consent was obtained from all
enrolled participants prior to enrollment and specimen collection. In total, 1,267 participants
were enrolled in the first round of the study. The prefecture-level CDC research teams were
trained in all enrollment procedures (biological and behavioral questionnaire).

Serum collection
Ten (10) mL of peripheral venous blood was collected using pre-labeled vacuum EDTA blood
collection tubes. All blood samples were chilled immediately at 4˚C, and transferred to partici-
pating CDC laboratories at surveillance sites within 2 hours after sampling. Upon arrival of
blood samples into the laboratories, sera were isolated by 1,500 g centrifugation for 15 min.
Serum was aliquoted, flash frozen in liquid nitrogen, and then stored at -80˚C.

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 Surveillance of wildlife workers, China

Fig 1. Prefecture maps of Guangdong Province, China. A) 1,267 participants were enrolled at Dabu, Jiaoling,
Pingyuan, Lianping, Heping, Lianshan, Lianzhou, Yunfu, Yunan, Xinyi, Deqing and Fengkai (areas colored black).
Number of respondents at each prefecture is bracketed; (B) In the brackets, in total 43 (left) seropositive respondents
and 45 (right) close contacts of theirs were enrolled in the follow-up phase at Xinyi, Yunan, Fengkai, Lianshan,
Lianzhou, Pingyuan, Jiaoling and Dabu (areas colored black). Together, 88 individuals participated in the follow up
phase.
https://doi.org/10.1371/journal.pone.0194647.g001

Risk behavior questionnaire

All study participants responded to a detailed questionnaire including questions about demo-
graphics, occupation, rural vs. urban habitation, zoonotic disease risk perceptions, and contact

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 Surveillance of wildlife workers, China

Fig 2. Flow diagram of recruitment and enrollment. 12 prefectures were selected after primary investigation by
visiting over 20 prefectures. Totally 1,267 individuals were enrolled, and serological tests and behavioral interviews
were conducted. In the follow up study, 43 respondents showing seropositive results to SARS virus, hantavirus, and/or
bunyavirus in the enrollment phase and 45 of their close contacts were enrolled. Additionally, 52 respondents showing
indeterminate or positive results were excluded in the follow up study since they moved to other locations between the
two study phases. Another round of serological analysis and behavioral investigation were conducted to 88
respondents in the follow up study.
https://doi.org/10.1371/journal.pone.0194647.g002

with particular types of wildlife through hunting, butchering, or eating. The questionnaire was
based on a previous study in Cameroon and is available in the S1 File [16, 17]. It was adapted
in English for the study environment in China and then translated into simplified Chinese.
During the training of the local CDC research teams, pretesting of the questionnaire was done
to ensure the intelligibility of the questions and proper translation. Pretesting was also done
with the study population during the pre-enrollment phase for the study sites. During enroll-
ment, a trained member of the local CDC team who was fluent in the local language of the
study site administered the questionnaire. In Guangdong Province, both Mandarin and Can-
tonese are spoken in the study sites. The questionnaire was administered in either Mandarin
or Cantonese depending on the respondent.

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 Surveillance of wildlife workers, China

Follow up study
All follow up study respondents were contacted by a member of the trained research team
regarding their test results. Seropositive individuals or those with indeterminate results were
referred to a local clinic/hospital where they could follow up on their results. This study did
not provide additional financial or medical support for respondents who tested positive. All
results were kept confidential and only reported to the respondent themselves. 95 individuals
who tested seropositive from the first enrollment were asked to participate in an additional fol-
low up sampling visit. They were also asked to invite up to three of their close contacts to enroll
in the follow up study. We aimed to continue behavioral information and biological sample
collection towards gauging virus transmissibility. Prior to sampling, we re-consented seroposi-
tive individuals and consented their close contacts. Due to geographic reallocation of some
respondents, we were unable to locate 52 individuals with indeterminate or seropositive results
for inclusion in our follow up study. We successfully re-enrolled 43 seropositive respondents
and 45 of their close contacts in our follow-up study (Fig 2).

Institutional review board (IRB) approval

This study protocol was approved by Western Institutional Review Board (protocol number:
20090204, GVFI: 06-09-057-02), United States, and Ethical Committee of Center for Disease
Control and Prevention of Guangdong Province (GDCDC), China. The follow-up study pro-
tocol (protocol number: 215253–7) was approved by Ethical Committee of Center for Disease
Control and Prevention of Guangdong Province, China and the University of California,
Davis IRB.

Serological analysis
The serological investigation focused on detection of hantavirus, SARS CoV, and Severe Fever
with Thrombocytopenia Syndrome (SFTS) bunyavirus. This study aimed to demonstrate viral
transfer associated with exposure to wildlife and the virus targets for this study were selected
based on prevalence and infection associated with animal/wildlife contact as well as an interest
by the GDCDC [18–20]. To determine hantavirus specific antibodies in study respondents,
stored human sera were tested using Hantavirus Hantaan IgG/IgM ELISA in a single well for
each sample (IBL, Hamburg, Germany) [21]. Microtiter plates were pre-coated with recombi-
nant nucleo-capsidprotein of Hantaan virus. According to the manufacturer’s instructions,
control groups and experiment group sera were diluted at 1: 100 using 1×PBS, pH 7.4, and
incubated at 37˚C. Coated wells were washed, and then incubated with 100 μL diluted peroxi-
dase conjugated anti-IgG enzyme at 37˚C. Color development reaction was initiated by adding
a chromogenic substrate, tetramethylbenzidine, followed by a 10 minute incubation at room
temperature. Reaction was quenched by adding 100 μL 0.5 M H2SO4, and then measured at
450 nm to obtain its optical density.
Human sera were also tested for SARS CoV using the SARS Coronavirus IgG Diagnostic
Kit (BGI, Shenzhen, China) [22–24]. Microtiter plates were pre-coated with Vero E6 cell lysate.
Cells were previously infected by SARS coronavirus that was isolated during the SARS out-
break in 2002 in China. Sera preparation and indirect ELISA were conducted as described
above. The average Optical Density (OD) of negative control sera plus 0.13 was used as the cut
off value as suggested by the manufacturer.
We adopted recombinant coating antigen in the diagnosis of SFTS bunyavirus using indi-
rect ELISA. Gene encoding 6×His tagged SFTS bunyavirus HB29 nucleocapsid (N) protein
was expressed in E. coli, and purified using nickel affinity chromatography. Recombinant anti-
SFTSV IgG was immobilized in 96-well plate as coating antigen. Sera were diluted at 1: 100

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 Surveillance of wildlife workers, China

using 1×PBS, pH 7.4, and incubated at 37˚C. We used horseradish peroxidase (HRP)-conju-
gated anti-human IgG antibody (Sigma, Saint Louis, MO) as the secondary antibody for color
development. OD was measured at 450 nm, with a reference wavelength of 620 nm. Cut-off
value was determined by adding 3× SD to the mean of OD values resulting from analyses of
negative control sera. Moreover, a microneutralization assay was performed to detect neutral-
izing antibodies against SFTS bunyavirus [25]. This was done to ensure the highest level of cer-
tainty given the newness of the SFTS virus assays. Fifty (50) μL serially diluted human sera
were mixed with an equal volume of 100 TCID50 /0.1ml of SFTS bunyavirus HB29, and then
incubated at 37˚C for 1.5 h. Incubated mixture was added to cultured Vero cells in a 96-well
plate in quadruplicate. Subsequently, the plates were incubated at 37˚C with 5.0% CO2 for 7 d.
Viral infection was assessed via an immunofluorescence assay using mouse polyclonal anti-
SFTS bunyavirus antibody. End-point titer was then expressed as the reciprocal of the highest
dilution of serum with viral infection prevention capability.

Risk factor questionnaire analysis

Laboratory results and behavioral data from the questionnaire were manually entered twice
(double entry system) and compared for entry error. Hand checks were also done on 10.0% of
the database to further reduce chances of human entry error. Questionnaire responses and
serological test results were then analyzed and compared using SAS (version 9.4; SAS Institute
Inc., Cary, NC, USA). We assigned primary occupation to the categories of hunter, agriculture
worker, butcher, market worker, and restaurant worker based on questionnaire responses. We
also aggregated reported behaviors to assign an “ever” status to each individual in terms of eat-
ing, butchering, or hunting wildlife. Basic descriptive univariate statistics were carried out on
the data set. We then examined bivariate associations between serological status for the three
viruses of interest and independent demographic and risk behavior variables. Variables show-
ing bivariate association with seropositivity at a p value of 0.1 or less were included in multi-
variate logistic models for seropositivity to individual viruses or any of the three viruses using
Firth’s Penalized Likelihood adjustment to address small sample size and possible collinearity
[26].
Since butchers were the only occupational group showing increased risk of seropositivity,
we also performed a subanalysis of specific behaviors performed by butchers, again including
behaviors with bivariate association of p = 0.1 or less in a multivariate logistic model.

Results
Demographics
A total of 1,267 individuals provided a baseline blood sample for serological testing and com-
pleted a behavioral risk factor survey. The demographics of these individuals are shown in
Table 1.
The study population was predominantly younger than age 60, with a mean age of 36.97
±11.03 years. Junior high school was most frequently reported as the highest level of education
attained. Most of the respondents were married or living with their partner and had between
1–2 children, with a total of 3–6 people living in the house.
Traveling to a forest at least once a month, which may provide additional exposure to wild
animals, was reported by 67.0% of respondents (data not shown). Some of the most common
reasons for traveling to the forest were fieldwork (56.0%), gathering of fruit and vegetables
(21.0%), collecting firewood (11.0%), and hunting (10.0%). More respondents reported ever
living in a city than not, with the most common urban occupations being restaurant work fol-
lowed by “other” and factory jobs.

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 Surveillance of wildlife workers, China

Table 1. Demographics of the study population.

Variable Total (%)
N = 1,267
Gender
Female 232 (18)
Male 985 (78)
No answer 50 (4)
Age (y) 36.97 ± 11.03
Education level
Primary school 219 (17)
Junior school 791 (62)
Senior school 177 (14)
University 18 (1)
Apprenticeship 49 (4)
None 13 (1)
https://doi.org/10.1371/journal.pone.0194647.t001

Table 2 describes type and location of occupational activities. Restaurant-related occupa-

tions such as cook or kitchen worker were the most commonly reported primary occupation,
followed by agriculture, butcher, and jobs working in a market.
Table 3 summarizes reported exposures to wild animals. Almost all respondents indicated
that they had ever touched any live or dead wild animal. Having butchered and eaten wild ani-
mals were the most common types of animal interactions, and injury was experienced by less
than a quarter of the respondents.
Table 4 shows the beliefs and reported protective behavior regarding contact with animals
and zoonotic disease transmission. Less than one third of respondents reported believing that
they could get infected from contact with blood or other types of contact with wild animals. A
smaller proportion reported taking at least one type of protective measure to prevent infection
in such circumstances. Among those who reported taking protective measures, use of gloves,
and avoiding touching the animal were the most commonly reported protective practices.

Table 2. Primary occupation and urban residence.

Variable Total (%)
N = 1,267
Primary occupation
Cook /Other restaurant worker 463 (37)
Agriculture 220 (17)
Butcher 111 (9)
Market worker 92 (7)
Housework 62 (5)
Hunting 38 (3)
Other 145 (11)
No Answer 135 (11)
Ever lived in the city
No 442 (35)
Yes 825 (65)

Numbers add up to more than 100% because the respondent had the option to select multiple occupations

https://doi.org/10.1371/journal.pone.0194647.t002

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 Surveillance of wildlife workers, China

Table 3. Reported exposures to wild animals.

Variable Total (%) N = 1,267
Hunted any wild animal
No 1,043 (82)
Yes 224 (18)
Butchered any wild animal
No 301 (24)
Yes 966 (76)
Eaten any wild animal
No 419 (33)
Yes 848 (72)
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Serological results
Risk factors for seropositivity. Table 5 summarizes the results of baseline testing of blood
for evidence of antibodies against zoonotic wildlife viruses. The overall rate of seropositivity to
any of the other three tested pathogens (Hantavirus, SARS CoV, or SFTS bunyavirus) was low
among the study population (approximately 4%). Seropositivity in respondents was only
found in the first phase of enrollment. Among contacts of these seropositive respondents
(n = 45), all individuals were seronegative for the pathogens of interest. The most common
seropositivity was for Hantavirus (2.3% of the study population). However, among the 112 per-
sons reporting that their occupation was a butcher, the rate of seropositivity to at least one
pathogen was higher (9.0%), and this elevated risk was statistically significant (Fisher’s Exact
Test, p = 0.005). Butchers had elevated seropositivity rates to all three of the viruses compared
to the overall study population. No other occupational group showed an increased risk of sero-
positivity. In a multivariate model of the butchers, those who reported butchering porcupines
showed an elevated risk of seropositivity (data not shown). There was no increased risk among
the butchers associated with not wearing gloves or taking fewer protective measures.
By contrast, the 38 persons reporting hunting as a profession showed a decreased risk for
seropositivity as did those reporting their profession as housework. No other occupational
groups showed significantly increased or decreased risk. Persons who reported ever hunting
also had a lower risk of seropositivity compared to those who never hunted. Age and gender

Table 4. Beliefs and reported behaviors.

Variable Total (%) N = 1,267
Believe one can get infection from animals/blood
No 594 (47)
Yes 402 (32)
Don’t Know/Refused 271 (21)
Take Any type of protective measure
None 810 (64)
At least one type 218 (17)
Don’t Know 239 (19)
If yes, type of protective measure (n = 218)
Gloves 128 (59)
Avoidance of exposure 42 (19)
Other 48 (22)
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 Surveillance of wildlife workers, China

Table 5. Serological results, by primary occupation and reported activities.

Risk Factor SARS Hanta Bunya Any
Total Number (%) Total Number (%) Total Number (%) Total Number (%)
Primary Occupation
Butcher (n = 112) 3 (2.7%) 5 (4.5%) 2 (1.8) 10 (9.0%)
p = 0.18 p = 0.17 p = 0.18 p = 0.005
Hunter (n = 38) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%)
p = 0.34 p = 0.9 p = 0.19
Cook or Restaurant (n = 463) 5 (1.1%) 11 (2.4%) 0 (0%) 16 (3.5%)
p = 0.62 p = 0.88 p = 0.62 p = 0.37
Agriculture (n = 220) 4 (1.8%) 3 (1.4%) 2 (0.9%) 9 (4.1%)
p = 0.52 p = 0.31 p = 0.52 p = 0.98
Housework (n = 62) 0 (0%) 1 (1.6%) 0 (0%) 1 (1.6%)
p = 0.9 p = 0.71 p = 0.9 p = 0.31
Market (n = 92) 0 (0%) 3 (3.3%) 1 (1.1%) 3 (3.3)
p = 0.6 p = 0.51 p = 0.63 p = 0.67
All (n = 1267) 17 (1.3%) 29 (2.3) 7 (0.6%) 53 (4.2%)
Specific Behavior
Ever hunting (n = 224) 1 (0.4) 0 2 (0.9) 3 (1.3%)
p = 0.33 p = 0.01 p = 0.33 p = 0.02
Ever butchering (n = 966) 14 (1.4) 26 (2.7) 6 (0.6) 46 (4.8%)
p = 0.77 p = 0.09 p = 0.77 p = 0.07
Ever eaten wild animals (n = 848) 12 (1.4) p = 0.74 18 (2.1) 7 (0.8) 36 (4.3%)
p = 0.57 p = 0.9 p = 0.72
https://doi.org/10.1371/journal.pone.0194647.t005

did not show a significant association with seropositivity, nor did reporting living in a city or
going frequently into the forest. While 111 persons reported butchering as a profession, a
larger number (966) reported ever butchering a wild animal; this was associated with an ele-
vated risk of seropositivity, however when the professional butchers were removed from this
group, the risk was no longer significant. As shown in the Table S1 Table, reported contact
with certain wildlife species such as civets (for Hanta and Bunyavirus) and field rats (for
SARS) showed elevated risk for certain viruses in bivariate analyses.
In multivariate models of specific wildlife contact, (Table 6), the principal association
remaining significant was eating civets (associated with Hantavirus seropositivity). The risk
estimate for Bunyavirus was elevated but did not reach statistical significance.

Table 6. Multivariate models for seropositivity–common activities, animal specific.

Any Seropositive Hanta SARS Bunya
OR 95% CI OR 95% CI OR 95% CI OR 95% CI
Boar-Eat - - - - - - 1.5 0.3–8.7
Boar-Hunt 0.3 0.01–5.2 - - - - - -
Civet-Eat 1.8 0.8–4.0 3.5 1.4–8.5 - - 4.2 0.9–19.4
Field rat-Eat 2.3 0.99–5.4 - - 1.1 0.2–8.2 - -
Field rat-Butcher - - - - 2.3 0.3–16.9 - -
Rat-Eat - - - - 2.8 0.7–10.7 - -
Rat-Butcher - - - - 0.99 0.2–4.4 - -
Wildbird-Eat - - - - - - 2.6 0.5–13.5
Wildbird-Butcher 1.7 0.96–2.9 - - - - - -
Wildbird-Hunt 0.3 0.06–1.6 0.1 0.007–1.9 - - - -
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 Surveillance of wildlife workers, China

Discussion
This study, examining the association between reported wildlife contact and seropositivity for
wildlife zoonotic viruses, found detectable levels of antibodies for several pathogens in the pop-
ulation surveyed. Overall, the population had contact with multiple species of animals. Among
reported primary occupations, working as a butcher was associated with an increased risk of
infection with wildlife pathogens, while professional hunters were at decreased risk, and no
increased risk was seen for market or restaurant workers. Contact with certain animals, such
as civets and field rats, showed evidence of increased risk of seropositivity. Few people in the
study reported using protective measures such as gloves when working with wild animals, and
less than a third of individuals reported believing that they could become infected through
wildlife contact. When seropositive individuals were followed over time, no evidence of infec-
tion of close contacts was seen, although the number of contacts enrolled was small and there
are limited conclusions that can be draw about alternative transmissions risks. Further investi-
gation is warranted to gauge transmissibility of the viruses studied.
These findings have a number of implications for prevention of zoonotic disease transmis-
sion and outbreaks. Those individuals at the animal-human interface (such as persons butch-
ering wild animals) that are highly exposed to wild animals are influential in disease
amplification and dissemination and reduction of risk factors can play a large role in mediat-
ing potential outbreaks.
While we identified butchers as an occupational group at increased risk, better understand-
ing of the specific exposures driving this increased risk would require more in-depth study.
Among butchers, we did find an elevated risk of virus seropositivity associated with butchering
porcupines, but due to small numbers, such a finding must be considered preliminary. This
study also identified an increased risk of seropositivity with contact with civets and field rats
which could be due to multiple factors. Civets and field rats are among the most popular types
of wild animals found in markets and restaurants and prolific contact may result in higher
risk. There is also a bias related to the viruses that the study targeted with two of the three
(SARS CoV and hantavirus) associated with both civets and rats.
This study had a number of limitations, including selection bias. We deliberately targeted
high risk individuals, making our sample less representative of the general population. Con-
versely, it is possible that there could be differential participation rates between high risk and
lower risk individuals, further limiting any extrapolation to community levels. We focused on
wildlife contact, and therefore were unable to assess the risk impact of contact with other ani-
mal species including livestock and companion animals. Further research should aim to
include larger sample sizes from more geographically diverse regions, and assess seropreva-
lence rates in random population samples. Underreporting of risk activities on the behavioral
survey may have occurred, since many activities related to wild animals are illegal in China. In
addition, since this was a hypothesis generating study, the analysis involved multiple compari-
sons. Therefore, any reported association should be viewed with caution. While this study
involved serological testing for more than one potential wildlife pathogen, there are numerous
other wildlife pathogens, including bacterial and viral agents, which the study did not investi-
gate. It is therefore possible that this study failed to detect other significant transmission events
of human health relevance. Future studies should consider such possibilities.
The finding that less than one third of the surveyed population, most of whom reported wild
animal contact, believed that they could become infected through such contact, indicates the need
for targeted educational programs for prevention, especially among those involved in butchering
wildlife for consumption. Targeted education programs should aim to increase knowledge of dis-
ease risk, perception of risk, and risky behaviors in the identified high-risk sub groups.

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 Surveillance of wildlife workers, China

Previous studies indicate the need to target the high-risk sub group of hunters [16, 17]. In
this study, however, hunters and persons reporting ever hunting or hunting as a profession
exhibited a lower rate of seropositivity compared with the overall population. It is possible that
hunting activities may be underreported, but the results of this study indicate some of the diffi-
culties of relying on self report to identify individuals at increased risk.
Due to the huge demand by the local residents and immigrants in the larger Pearl River
Delta area, which includes Guangdong, Hong Kong and Macau, increasing number of individ-
uals engaged in animal production and value chain systems are believed to continue to come
into contact with wild animals and engage in risky behaviors increasing the risk of a possible
infectious disease outbreak that could have a potentially severe impact. Wild animals are an
important part of Southern Chinese economy and culture, and pragmatic policy changes
should be implemented to control the risks of possible disease outbreaks. These policy changes
should aim to increase risk perception and uptake of precautionary behaviors without risking
the cultural and financial security of the community. Results of the analyses of these research
findings can be used to inform public health strategies such as communication and educa-
tional campaigns, targeting those behaviors that place individuals at higher risk of disease.
Our study provides evidence towards recommendations that include: an increase in the
level of collaboration between animal and human health programs targeting human-animal
interfaces to increase efforts to control and prevent outbreaks; development of new educa-
tional campaigns to increase knowledge and awareness of infectious diseases that target spe-
cific sub-groups, such as butchers, that are at increased risk; and increase monitoring of high-
risk groups to detect continued transmission of zoonotic diseases in a timely manner.

Supporting information
S1 File. Study questionnaire for persons hunting, butchering, eating and/or keeping wild
animals as pets.
(PDF)
S1 Table. Prevalence of seropositivity for tested viruses, by reported wildlife exposure.
(PDF)

Acknowledgments
We thank prefecture CDCs at Dabu, Jiaoling, Pingyuan, Lianping, Heping, Lianshan, Lianz-
hou, Yunfu, Yunan, Xinyi, Deqing and Fengkai for their efforts in participant enrollment and
specimen collection. There are no competing interests in this scientific work.

Author Contributions
Conceptualization: Corina Monagin, Huiqiong Zhou, De Wu, Changwen Ke.
Data curation: Corina Monagin, Blanca Paccha, Sally Trufan, Bradley S. Schneider, Peter M.
Rabinowitz.
Formal analysis: Corina Monagin, Blanca Paccha, Sally Trufan, Bradley S. Schneider, Chang-
wen Ke, Peter M. Rabinowitz.
Funding acquisition: Peter Daszak, Changwen Ke.
Investigation: Corina Monagin, Huiqiong Zhou, De Wu, Changwen Ke.
Methodology: Corina Monagin, Huiqiong Zhou, De Wu, Changwen Ke, Peter M. Rabinowitz.

PLOS ONE | https://doi.org/10.1371/journal.pone.0194647 April 3, 2018 12 / 14

 Surveillance of wildlife workers, China

Project administration: Corina Monagin, Ning Liang, De Wu, Aleksei Chmura, Jonathan
Epstein, Peter Daszak, Changwen Ke.
Resources: Corina Monagin, Sally Trufan, Huiqiong Zhou, De Wu, Aleksei Chmura, Jonathan
Epstein, Peter Daszak, Changwen Ke.
Software: Blanca Paccha, Sally Trufan, Peter M. Rabinowitz.
Supervision: Corina Monagin, Huiqiong Zhou, Changwen Ke.
Validation: Corina Monagin, Blanca Paccha, Ning Liang, Sally Trufan, De Wu, Bradley S.
Schneider, Changwen Ke, Peter M. Rabinowitz.
Visualization: Corina Monagin, Ning Liang, Sally Trufan, De Wu, Peter M. Rabinowitz.
Writing – original draft: Corina Monagin, Ning Liang, De Wu, Bradley S. Schneider, Chang-
wen Ke, Peter M. Rabinowitz.
Writing – review & editing: Corina Monagin, Ning Liang, Sally Trufan, Bradley S. Schneider,
Peter M. Rabinowitz.

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