Study design and implementation
The design of this study differed from conventional epidemiological
observational studies in that it did not aim to obtain a representative
sample of the population. Rather, the focus was on active detection of
field virus and disease, elucidation of contact patterns and increasing
understanding of potential high-risk points and practices for
transmission of infection within and between different avian
populations, production systems as well as enterprise types (chicken
production Sectors and ducks). Consequently, this research consisted of
a combination of complementary research activities, rather than a single
study. Such a mixed methods research approach, which integrates
quantitative and qualitative research methods, may help to improve
understanding of potential biases of the observed data (Kristensen et
al., 2008), and allow for a deeper exploration of the topic under
investigation than can be achieved by either approach alone (Limon et
al., 2014). It also provides a framework for integrating data from
disparate sources (Vink et al., 2016).
Figure 2 provides a schematic overview of the study activities. An
initial expert consultation meeting bringing together international
experts in different fields of relevance to H5N1 HPAI was convened in
November 2015 to elicit hypotheses related to reservoirs of persistence
of the virus, risk factors for transmission of infection, production
systems and linkages between systems. Potential methods to test and
investigate these hypotheses were subsequently discussed. A subset of
eight hypotheses which could feasibly be investigated in Indonesia
(Figure 3) were utilised to inform the design of this endemicity study.
A stratification of enterprise types was applied, grouping Sector 1-3
commercial poultry farms, collector yards and live bird markets on the
one hand, and Sector 4 backyard and non-commercial producers and nomadic
duck flocks on the other. The rationale for this was that the first
group was less numerous, so that a census of all properties in
Purbalingga District could be included, whereas a sample needed to be
taken of the second group. The sampling designs for the subsequent
active sampling differed between these two strata, too.
The first part of the study aimed to characterise poultry populations
and trade and contact networks; it was conducted from April to July
2016. All Sector 1-3 commercial poultry farms (n=204), collector yards
(n=129) and LBMs (n=18) were visited and questionnaires were conducted
by 22 trained enumerators and 25 Government of Indonesia District Animal
Health Officers of Purbalingga District. If information arose about
locations that had not been included, these were also visited. In
addition, visits to all 239 villages in Purbalingga District were
performed and questionnaire surveys were conducted in a minimum of 54
households. As there was no list of households, these were randomly
selected. A total of 14,368 households were interviewed. The
questionnaires included signalment information, flock location, details
of numbers and species of poultry kept, information on biosecurity
practices and flock management (including vaccination), purchase and
sales of live poultry over the previous 12 months, information on the
incidence of disease. All data were entered into a Microsoft Access
database.
The second part of the study aimed to detect AIV in the different
enterprise types and populations by implementing a series of surveys in
two sampling rounds (October 2016 – February 2017 and August –
November 2017). For Sector 1-3 commercial farms, collector yards and
LBMs, risk-based, stratified sampling was performed. Following
geographical stratification into high, medium and low backyard poultry
population density areas, further stratification into Sector 1-3
commercial farms, collector yards and live bird markets was done.
Subsequently, random selection was carried out to identify properties,
assuming a design prevalence of 2% with a confidence level of 95% that
at least one HPAI H5 virus would be detected. All sampling was performed
by 25 District Animal Health Officers.
Thirty-two commercial farms were selected. No broiler farms were
included in the study, but all commercial duck farms (n=6) were included
in this selection, with the remainder consisting of layer farms.
Oropharyngeal swabs were taken from a fixed number of 30 birds per
property, pooling six swabs into one viral transport medium (VTM)
sample. The sampling was risk-based insofar as possible, that is, any
birds that showed suspect clinical signs as well as dead birds were
preferentially selected. Farms were sampled once during the first round
(high season) and twice during the second round (low season).
A selection of 41 collector yards was made. Twelve environmental swab
samples were pooled in two VTM samples. If any birds were observed with
suspect clinical signs, rapid testing and individual sampling were
performed. The collector yards were sampled three times during the first
round and four times during the second round.
All 18 LBMs in the District were sampled. The same sampling protocol was
based on a protocol developed for previous studies in markets in
Indonesia (Indriani et al., 2010). LBMs were also sampled three times
during the first round and four times during the second round.
An enhanced passive surveillance scheme was initiated by offering
incentive payments to District Animal Health Officers for reporting
sudden mortality events in poultry, with at least one positive AIV rapid
test result of sick and dead birds. If these birds had died or been
removed, environmental samples were taken.
In addition to the enhanced passive surveillance in Sector 4 poultry, it
was decided to expand the sampling to include nomadic duck flocks.
Twelve ducks per flock were sampled and the samples were pooled into two
VTMs. In the first round, all 20 flocks that were included in the
questionnaire survey were sampled. In the second round, 196 flocks were
sampled and the number of sampled ducks per flock was increased to 30,
pooled into 5 VTM samples.
As only four reports were received using the incentive scheme, it was
decided to actively sample backyard poultry in the second sampling
round. As the frequency of mortality events in backyard poultry during
the low season was likely to be low, in addition to which such outbreaks
were unlikely to be reported by owners of such birds, a purposive
sampling technique was used. Snowball sampling (also known as
chain-referral sampling) is a non-probability sampling technique where
initial study subjects are asked to identify future subjects from among
their acquaintances; the technique is utilised for the investigation of
‘hidden’ populations which are difficult for researchers to access, or
for rare conditions or events which are unlikely to be detected through
randomised sampling techniques (using community knowledge or ‘rumour’).
Following a sample size calculation specifying a design prevalence of
5% and a confidence level of 95%, 187 villages in Purbalingga were
visited. An initial household keeping backyard poultry was randomly
selected, and a questionnaire interview was conducted. In the interview,
the respondents were asked if they experienced any mortality in their
poultry during the previous week and if not, if they could identify any
acquaintances who may have had. If they could, this acquaintance was
requested to be interviewed. If not, the respondents’ closest neighbour
owning backyard poultry was asked to be interviewed. Using this
methodology, 54 households keeping chickens were sampled in each
village. In the interviews, numbers and type of poultry were also
recorded. If sick or dead bird were identified, individual swab samples
were taken; if the carcasses could not be identified, environmental
swabs were collected. Following this methodology, 127 households keeping
backyard chickens were sampled.