Paper on using detection dogs for the surveillance and mitigation of avian botulism in a national park refuge.

1. C O N T R I B U T E D P A P E R
Efficacy of detection canines for avian botulism
surveillance and mitigation
Michelle H. Reynolds1
| Kyoko N. Johnson2
| Eleni R. Schvaneveldt1
|
Daniel L. Dewey3
| Kimberly J. Uyehara3
| Steven C. Hess1,4
1
U.S. Geological Survey, Pacific Island
Ecosystems Research Center, Hawaii
National Park, Hawaii
2
Country Canine, Waialua, Hawaii
3
U.S. Fish and Wildlife Service, Kauaʻi
National Wildlife Refuge Complex,
Kīlauea, Hawaii
4
USDA-APHIS-WS National Wildlife
Research Center, Hilo, HI
Correspondence
Michelle H. Reynolds, P.O. Box 682,
Volcano, HI 96785.
Email: drdukmhr@gmail.com
Funding information
U.S. Geological Survey Pacific Islands
Ecosystem Research Center; U.S. Fish and
Wildlife Service; Science Support Program
of the U.S. Geological Survey
Abstract
Hawai'i's endangered waterbirds have experienced epizootics caused by inges-
tion of prey that accumulated a botulinum neurotoxin produced by the anaero-
bic bacterium Clostridium botulinum (avian botulism; Type C). Waterbird
carcasses, necrophagous flies, and their larvae initiate and spread avian botu-
lism, a food-borne paralytic disease lethal to waterbirds. Each new carcass has
potential to develop toxin-accumulating necrophagous vectors amplifying out-
breaks and killing hundreds of endangered waterbirds. Early carcass removal
is an effective mitigation strategy for preventing avian intoxication, toxin con-
centration in necrophagous and secondary food webs, and reducing the magni-
tude of epizootics. However, rapid detection of carcasses can be problematic
and labor intensive. Therefore, we tested a new method using scent detection
canines for avian botulism surveillance on Kaua'i Island. During operational
surveillance and a randomized double-blind field trial, trained detector canines
with experienced field handlers improved carcass detection probability, espe-
cially in dense vegetation. Detector canines could be combined with conven-
tional surveillance to optimize search strategies for carcass removal and are a
useful tool to reduce risks of the initiation and propagation of avian botulism.
K E Y W O R D S
avian botulism, avian disease, canine detection, Clostridium botulinum, Hawai'i, Kaua'i, Koloa
maoli, taro, waterbirds, wetlands
1 | INTRODUCTION
Botulinum neurotoxins (BoNT) are the most lethal poi-
sonous compounds known (Poulain & Popoff, 2019), and
avian botulism is the most common cause of death in
wild birds worldwide (Rocke & Bollinger, 2007). The
anaerobic bacteria Clostridium botulinum are Gram-
positive rods with seven serotypes producing distinct neu-
rotoxins. C. botulinum favors high temperatures for growth
(optimally 37
C; Cato, George, Finegold, 1986) and may
outcompete inhibitory species of bacteria in effluent or
eutrophic and herbicide-laden (e.g., glyphosate) wetland
environments (Krüger, Shehata, Schrödl, Rodloff, 2013;
Sandler, Rocke, Yuill, 1998; Shehata, Schrodl, Aldin,
Hafez, Kruger, 2012; Vidal et al., 2013; Woo et al., 2010).
Avian botulism outbreaks are often associated with extreme
droughts or sudden flooding that mobilize spores, and cre-
ate carcasses and environmental conditions favorable for
Received: 28 October 2019 Revised: 1 November 2020 Accepted: 14 November 2020
DOI: 10.1111/csp2.397
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
© 2021 The Authors. Conservation Science and Practice published by Wiley Periodicals LLC. on behalf of Society for Conservation Biology
Conservation Science and Practice. 2021;e397. wileyonlinelibrary.com/journal/csp2 1 of 18
https://doi.org/10.1111/csp2.397

2. toxigenesis (Reynolds, Berkowitz, Klavitter,
Courtot, 2017; Son et al., 2018). The impact of avian botu-
lism may increase globally with climate change, increasing
urbanization, and invasive aquatic species (Espelund
Klaveness, 2014; Russell, Randall, Zimmerman,
Govender, 2019).
Preventing waterbird mortality and large-scale avian
botulism epizootics often hinges on rapidly removing car-
casses that accelerate spread of the bio-toxin (Evelsizer,
Clark, Bollinger, 2010; Reed Rocke, 1992;
Wobeser, 1997). Removing carcasses is an enduring
approach to managing avian botulism, but its effective-
ness is dependent on a high rate of carcass detection and
early removal (ideally before toxigenesis and food web
contamination; Figure 1a,b). Past studies of carcass
removal efficacy in large densely vegetated prairie
marshes and lake ecosystems used pedestrian searchers,
ATVs, and airboats and reported carcass detection rates
of 7–61% and the scale and costs were decidedly
unfeasible in these ecosystems (Bollinger et al., 2011; Cli-
plef Wobeser, 1993). Our study is the first using
canines to detect experimentally manipulated carcasses
in wetland environments, and we know of no other anal-
ysis of the efficacy of canine avian botulism carcass
detection.
Initial toxigenesis (BoNT type C) may occur in any
waterbird carcass with dormant spores of C. botulinum in
the digestive tract. The spores are widespread in the envi-
ronment and are ingested inadvertently (Rocke
Bollinger, 2007; Figure 1a). Subsequent carcass decay cre-
ates anaerobic decomposition favorable for C. botulinum
growth, typically within the cecum, liver, or small intes-
tine (Reed Rocke, 1992). Outbreaks often occur during
unseasonably warm temperatures (26
C; Vidal
FIGURE 1 (a) Conceptual scenarios of carcass initiated avian botulism (type C). Spores of “Clostridium botulinum” can occur in the
sediments, algae, and in a bird's digestive tract. A waterbird carcass has the potential to undergo toxigenesis when the spores (resting stage of
the anaerobic bacteria) that are inadvertently ingested germinate as the carcass decomposes. Decomposing carcasses provide ideal substrate
for “C. botulinum” toxigenesis by providing protein, high temperatures and anoxic conditions. As the bacteria reach exponential growth they
begin producing the botulinum neurotoxin. Spores and growing bacteria may also be transferred to previously uninfected carcasses by
necrophagous vectors (e.g., blow flies, Order: Diptera, Family: Calliphoridae) that have recently fed on toxic carcasses during a botulism
outbreak (Anza, Vidal, Mateo, 2014). (b) Illustrates examples of avian botulism toxin transmission by carcass-necrophagous invertebrate-
driven amplifications. The botulinum neurotoxin accumulates in the carcass feeding invertebrates (that are unaffected by the toxin) and
then further contaminates the food web. When waterbirds feed on the toxin laden prey, they can be affected by the neurotoxin within hours,
leading to death from the toxin caused flaccid paralysis or as the onset of symptoms leads to drowning, predation or impaired
thermoregulation (Rocke Bollinger, 2007). As the number of waterbird carcasses increases, so does the potential transmission to new birds
via food poisoning from both primary and secondary sources of prey. Examples of prey items that may become toxic include diptera larvae,
snails and snail eggs, carrion beetles, and fish
2 of 18 REYNOLDS ET AL.

3. et al., 2013), but carcasses may reach optimal tempera-
tures for toxigenesis independently of ambient air and
water temperatures (Wobeser Galmut, 1984). Fly larvae
aggregations contribute to thermal generation in car-
casses, causing temperatures to climb 11–32
C above
ambient temperatures (Ahmad Baharudin, 2018). Inva-
sive predators such as feral and domestic cats (Felis catus)
in Hawai'i (Hess, Hansen, Banko, 2007) can easily ini-
tiate avian botulism, as subtropical environmental condi-
tions are frequently ideal for spore germination and
bacterial growth within depredated waterbird remains
(Figure 1a).
Multiple pathways exist for proliferation of avian bot-
ulism (BoNT type C) that complicate its dynamics, con-
trol, and prevention (Figure 1b). One principal and rapid
transmission mode is by way of the “carcass–maggot
cycle” (Wobeser, 1997). Necrophagous flies deposit eggs
and feed on waterbird carcasses. Gravid blowflies (Order
Diptera: Family Calliphoridae) are typically first to detect
waterbird carcasses (often 10 min post-mortem) for ovi-
position and are mutualistic catalysts for C. botulinum
reproduction because both the bacteria and the blowflies
rely on carcasses for reproduction (Anza et al., 2014). The
toxin does not affect the fly larvae and other invertebrates
(Rocke Bollinger, 2007). Typically, fly eggs hatch
within 24 hr in subtropical and temperate summer condi-
tions, fly larvae feed on the carcass within 48 hr, and
may accumulate the toxin within 3–4 days (Espelund
Klaveness, 2014; Reed Rocke, 1992). As early as the
fourth day postmortem, tens of thousands of emerging
toxic maggots have infested the carcass. Thus, in wet-
lands where feasible, removing carcasses before larval
infestation, has the potential to prevent large scale mor-
tality of waterbirds as one toxic carcass with thousands of
fly larvae with BoNT could potentially kill about 100 birds
(Wobeser, 1997).
Waterbirds that consume toxin-laden fly larvae may
become intoxicated within hours and die directly from
toxin induced flaccid paralysis or indirectly as symptoms
lead to drowning, predation, or impaired thermoregula-
tion after eating as few as four toxic fly larvae (Rocke
Bollinger, 2007). Other invertebrates that fed on toxic car-
casses, or secondarily consumed toxic fly larvae also
cause intoxication of waterbirds. Invasive fish, bivalves,
and snails and their gelatinous egg masses also accumu-
late the toxin, thereby expanding sources of BoNT within
the wetland food chain (Anza, Vidal, Feliu, Crespo,
Mateo, 2016; Chi, Chen, Cheng, Ho, Lien, 2010; Dun-
can Jensen, 1976; Yin et al., 2016). Algal mats and
invasive fish such as tilapia (Oreochromis mossambicus)
serve as biotic reservoirs for spores and may facilitate
C. botulinum spore germination and bacteria growth
(Espelund Klaveness, 2014; Nol, Rocke, Gross,
Yuill, 2004). Although avian botulism toxin transfer is
typically associated with wetland food webs, carcasses
also initiate outbreaks in uplands. Terrestrial dipterans,
adult, and larval coleopterans are common terrestrial vec-
tors (e.g., Dermestidae [skin], Carabidae [predacious],
Silphidae [carrion] beetles; Duncan Jensen, 1976). Fur-
thermore, blowflies excrete viable C. botulinum to non-
toxic carcasses after feeding on intoxicated carcasses, for
up to 24 hr, amplifying toxigenesis (Anza et al., 2014).
Healthy birds (radio tagged and sentinel mallards, Anas
platyrhynchos) experimentally exposed to higher densities
of carcasses were more likely to die than birds in carcass-
free areas (Reed Rocke, 1992) and survival rates
decreased with increasing carcass densities (Bollinger
et al., 2011; Evelsizer et al., 2010). Early (preventative)
surveillance and rapid carcass removal is more effective
in reducing waterbird mortality than attempting to
remove carcasses after an outbreak has already initiated
and has spread through the food web (Evelsizer
et al., 2010). Not unlike a wildfire scenario, labor costs
and waterbird losses can spiral if avian botulism is
unchecked.
In the past decade, thousands of Hawai'i's non-
migratory endangered endemic waterbirds have died
from avian botulism outbreaks in protected habitats that
are intended to safeguard vulnerable species (State of
Hawai'i Division of Forestry and Wildlife, U.S. Fish and
Wildlife Service [USFWS] data). Endangered koloa maoli
(Hawaiian duck, hereafter “koloa”) Anas wyvilliana and
Laysan duck Anas laysanensis have particularly high
exposure to avian botulism because of their feeding
behavior and limited habitat within small geographic
ranges. Hawai'i's endemic ducks are at high risk of
extinction with small populations, thus are further jeop-
ardized by high mortality due to chronic avian botulism
epizootics (U.S. Fish and Wildlife Service, 2009; Reyn-
olds, Hatfield, Courtot, Cynthia Vanderlip, 2020; Work,
Klavitter, Reynolds, Blehert, 2010).
Given the importance of removing carcasses quickly
before toxigenesis and the carcass–necrophagous
invertebrate-driven amplifications, we tested the efficacy
of carcass surveillance methods and the utility of training
detector canines for this task in small wetlands in
Hawai'i. The domestic dog (Canis lupus familiaris) is an
excellent biosensor detecting volatile chemical com-
pounds (odors) at much lower concentration than
humans (Padodara, 2014). Carcass decomposition odor is
dynamic and dominated by polysulfide compounds
(Verheggen et al., 2017). In addition to sensitive scent dis-
crimination, dogs sample the air for environmental cues
that allow directionality and olfactory tracking (via an
“odor plume”; Prada Furton, 2018). Dogs are trained
increasingly for ecological purposes (Beebe, Howell,
REYNOLDS ET AL. 3 of 18

4. Bennett, 2016; Mendel, Furton, Mills, 2018; Mosconi
et al., 2017). Canine-aided surveillance may have utility
for mitigation of avian botulism in wetland and adjoining
habitats. Trained to detect, locate, and indicate or “alert”
the presence of specific odors, detector canines poten-
tially provide an efficient, economical method for effec-
tively inspecting some habitats susceptible to avian
botulism mortality events, and for detecting waterbird
carcasses obscured by dense vegetation.
We evaluated the feasibility of training canines to find
and alert on avian botulism carcasses. Our objectives
were to train dogs and test the efficacy of using detector
canines relative to other available surveillance methods.
We determined what factors affect detection probability
FIGURE 2 (a) Hanalei
National Wildlife Refuge on the
island of Kaua'i (371 ha), in the
Hawaiian Islands. The Hanalei
River crosses the refuge where
water is diverted for taro
production (or kalo, “Colocasia
esculenta”) in irrigated terraced
fields known as lo'i, as well as
managed wetland units. (b) A
subset of taro (33 ha) and
managed wetland units (19 ha)
within the Hanalei National
Wildlife Refuge were included in
this pilot study. Operational
surveillance for waterbird
carcasses on more than 50 ha of
taro and managed wetlands are
shown in orange and pink. The
areas used for a controlled field
experiment (double blind
detection trial) are shown
in blue
4 of 18 REYNOLDS ET AL.

5. and compared carcass detection among existing (human)
search methods and canine-assisted approaches, with
particular focus on detecting koloa. We used metrics of
detection efficacy and efficiency but lacked data to
directly assess cost effectiveness. Understanding factors
affecting surveillance efficiency (time to find carcasses
per area) and the relative efficacy (proportion of carcasses
found) of various search methods can be applied to opti-
mize carcass surveillance and collection to help mitigate
future outbreaks and prevent escalating waterbird mor-
tality, thereby reducing population impacts of avian botu-
lism on endangered waterbirds.
2 | METHODS
2.1 | Study site
Hanalei National Wildlife Refuge (NWR) on the Kaua'i
Island (371 ha, Figure 2a), was established to aid the
recovery of Hawaiian waterbirds under the Endangered
Species Preservation Act (ESA, 1973, as amended).
Hanalei NWR is believed to be the most important habi-
tat for koloa in the Hawaiian Islands (Banko, 1987).
Hanalei NWR also provides habitat for endangered ae'o
(Hawaiian stilt) Himantopus mexicanus knudseni, 'alae
ke'oke'o (Hawaiian coot) Fulica alai, 'alae 'ula
(Hawaiian gallinule) Gallinula galeata sandvicensis, and
n
en
e (Hawaiian goose) Branta sandvicensis. Chronic
avian botulism has been documented at Hanalei NWR
since 2011. Water is diverted from the Hanalei River for
taro (or kalo, Colocasia esculenta) agriculture, in irri-
gated terraced fields known as lo'i, and palustrine emer-
gent managed wetlands (Malachowski Dugger, 2018;
Figure 2a). A subset of taro (33 ha) and managed wet-
land units (19 ha) were included in this study
(Figure 2b). Average densities of endangered waterbirds
within the study site range from 15 to 24/ha in taro and
8 to 24/ha in managed wetlands (USFWS data, 2016–
April 2019). We summarized the past search effort and
waterbird carcass abundance recorded at Hanalei NWR
during surveys between October 2015 and March 2018
(USFWS data).
2.2 | Canine training
We followed guidelines and best practices of the Scien-
tific Working Group on Dog and Orthogonal Detector
Guidelines (SWGDOG 2019) and care of dogs in the
National Detector Dog Manual (U.S. Department of
Agriculture, 2012). Initial training was based on classical
conditioning, which paired target odor with reward and
transitioned to operant conditioning whereby dogs learn
to search for targets on their own initiative to earn
rewards (Premack, 1959; Pryor, 1999) to alert on specific
odors. To prevent false positives, “proofing” for nontarget
odors or training aids (e.g., carcass containers and protec-
tive gloves) was conducted to teach dogs to ignore odors
other than target odors. Distraction training also included
desensitizing dogs to live birds, initially chickens or
domestic ducks and finally waterbirds at Hanalei NWR.
Training and surveillance focused specifically on detec-
tion of koloa carcasses. Training aids (target odor
sources) consisted of previously frozen specimens col-
lected within approximately 24 hr of death without fly
larvae to reduce handling risks. Carcasses were thawed
and protected from flies and other invertebrates using
containers with screen vents or cloth bags to allow odors
to escape (Figure 3a). During an experimental double-
blind detection trial, koloa carcasses were contained
inside plastic duck decoys to improve visual search
images (Figure 3b).
After odor imprinting training 2–3 times per week for
2 months, dogs were formally tested to evaluate their
accuracy to find and alert on koloa carcasses at a neutral
field site. During evaluation trials, dog handlers were
uninformed about locations of carcasses to prevent giving
cues to dogs. We used four privately owned dogs with
prior training on wildlife carcass detection in this study:
a 5-year-old male yellow Labrador retriever, a 6-year-old
female mixed Labrador retriever, a 3-year-old male pit
bull-Labrador retriever mix, and a 1.5-year-old male Cat-
ahoula leopard dog (Figure 4). Two dogs had more than
4 years of experience, and two novice dogs had less than
1-year experience in scent detection.
Two professional dog trainers, a lead trainer (KNJ,
Country Canine) and an assistant trainer (Tarheel
Canine) participated as dog handlers. After an initial
study site visit by the lead trainer, canines were prepared
in four phases: (a) odor recognition training on training
aids, (b) demonstration of odor recognition and hunting
for odor in blind trials at neutral sites, (c) advanced field
acclimation to distractions, terrain, and climate at the
study site, and (d) training of distance or “proximity”
alerts. Dogs were taught to offer distant proximity alerts
when they detected carcasses within taro units because
they were restricted from freely entering taro units dur-
ing searches. Two novice detection dogs began training to
participate in a detection trial and began acclimating to
distractions but did not reach the final stage of proximity
alerts. Field acclimation entailed concealing training aids
and adaptation to live waterbird distractions for
10–30 min per dog, 5–6 times per week. Exposure to and
reward for alerting on carcasses in advanced stages of
decay (5 days) was also included because
REYNOLDS ET AL. 5 of 18

6. decomposition and entomofaunal succession changes
characteristics of volatile compounds (Verheggen
et al., 2017).
2.3 | Operational surveillance for avian
botulism
Surveillance protocols at Hanalei NWR involved perime-
ter surveys of assigned wetland units typically by a single
observer, driving on an all-terrain vehicle (ATV) or
walking (pedestrian) on dikes around wetland units or
taro fields. Observers searched for impaired and dead
birds using binoculars but avoided walking in taro
fields unless a carcass or sick waterbird was detected.
Intoxicated waterbirds and carcasses were identified to
species and removed. Observers recorded date, survey
start and end time, detection time, location, habitat,
and stage of intoxication or decomposition (Verheggen
et al., 2017).
FIGURE 3 (a) Training aids and search targets used during operational surveillance were previously frozen koloa maoli (Hawaiian
duck) “Anas wyvilliana” carcasses (collected approximately within 24 hr of death) enclosed so that odor could escape but invertebrates could
not make contact with the carcass (Photo credit T. Johnson, USFWS Volunteer). (b) Targets used in double blind detection trials March 1–3,
2019 were previously frozen koloa maoli (Hawaiian duck) “Anas wyvilliana” carcasses encased within plastic duck decoys (Avian-X
TopFlight Mallard Duck and Blue wing teal female Decoy Outfitter Pack Decoy; Photo credit USFWS)
FIGURE 4 Detection canines trained at Hanalei National Wildlife Refuge for avian botulism pilot study 2017 and 2018. Left to right
experienced scent detection canines (a 5-year-old male yellow Labrador retriever, a 6-year-old female mixed Labrador retriever) and novice
scent detection canines (3-year-old male pit bull-Labrador retriever mix, and a 1.5-year-old male catahoula leopard dog; Photo credits T.
Johnson (left) and T. Luxner (right), USFWS volunteers
6 of 18 REYNOLDS ET AL.

7. We compiled data from Hanalei NWR to estimate sea-
rch effort and carcass detection rates (birds found per
hour and per survey) from late 2015 to 2018. Carcass
abundance and search effort from previous surveillance
efforts were summarized to describe inter-annual and
seasonal outbreak variability prior to and during canine-
aided surveillance (USFWS data available from U.S. Geo-
logical Survey's ScienceBase website: https://doi.org/10.
5066/P9C4N47X; Reynolds et al., 2021).
During operational surveillance, canine-aided teams
were composed of a trained canine, a trainer/handler,
and a biological technician responsible for recording
data, carcass collection, and spotting birds to prevent
interactions between canines and birds. Technicians also
searched for intoxicated birds and carcasses. Two dogs,
two handlers, and seven biologists and technicians partic-
ipated in surveillance data collection. Daily surveillance
to find naturally occurring koloa carcasses took place in
December 2017, and February–March of 2018 to find
both naturally occurring and concealed koloa carcasses.
Technicians recorded temperature (
C), wind speed
(kph), and relative humidity using an electronic hygrom-
eter, anemometer, thermometer, wind direction using a
compass, rain index (0 no rain-3 heavy rain), carcass
location, vegetation characteristics, and categorical habi-
tat variables (growth stages, habitat description, and hab-
itat type). Vegetative cover (cover) and percent of unit
flooded (flooded) were estimated visually and calibrated
within 10% by observer consensus. Canine teams
searched systematically or proceeded directly to source
odors within fallow taro and managed wetland units.
However, searches were restricted to the perimeter of
taro lo'i in active production. Canines indicated detection
with a proximity alert by sitting before being allowed to
go to odor sources. Search areas were assigned and
rotated by the Refuge Biologist (KJU) and ranged in size
from 1.3 to 5.6 ha. Rotations generally resulted in many
areas searched by canine-aided teams every week and
human surveyors every 2 weeks. Some areas with a his-
tory of avian botulism were searched more frequently,
but typically not more than twice per week. Canines
wore working dog harnesses with a 5-m leash, and wire
basket muzzles to reduce risk to waterbirds while all-
owing food rewards. Carcasses of other species inciden-
tally encountered were also collected during operational
surveillance.
2.4 | Area, track, and detection distance
calculation
We collected tracks from several handheld and canine
collar Garmin GPS (Global Positioning System) units.
The area of each search unit was computed based on GIS
boundaries using ArcGIS 10.6 software (ESRI, 2018). We
calculated the area surveyed as the sum of individual
units. Human survey distances were estimated by conver-
ting GPS tracks into GIS lines in chronological order
using ArcGIS Pro 2.3.3 (ESRI, 2018). We computed
canine survey distances by converting Garmin Astro
320 track point files into GIS point files and smoothing a
line through survey points in chronological order
(Bodansky, Gribov, Pilouk, 2002). We quantified dis-
tances from the closest points of a canine change of
behavior (COB) to carcasses using the proximity tool in
ArcGIS. COB was identified from GPS tracks as a change
in direction toward a target odor from the down-wind
side as described by Cablk, Sagebiel, Heaton, and
Valentin (2008). Handlers also marked COB with a GPS
point where dogs detected target odors.
2.5 | Concealed surveillance carcasses
We experimentally concealed 52 koloa carcasses during
operational surveillance in 2018 to assess detection rates.
Concealment locations for carcasses were randomly gen-
erated within search areas in ArcMap 10.5 using the
“Create Random Points” sampling function. Coordinates
were unknown except to the person generating them and
the person concealing carcasses. A random sample was
taken to determine which days and whether to conceal
1 or 2 carcasses per survey. To prevent searchers from
knowing if any carcasses had been concealed, carcass
hiders went out every day a survey was scheduled even if
no carcasses were concealed.
2.6 | Double-blind timed detection trial
On March 1–3, 2018, nine canine-handler teams com-
posed of four detector dogs and three handlers were ran-
domly paired in nine timed (15-min) trials against seven
human searchers to find a total of 31 previously frozen
koloa carcasses placed inside plastic duck decoys. Decoys
were concealed randomly using GIS generated locations
in fallow taro and wetland management units totaling
about 4 ha. We used a double-blind experimental design
where both the study designer and searchers were
uninformed about carcass locations. Specific locations
and number of concealed carcasses were unknown to all
participants except carcass hiders. Carcasses were
concealed 1–2 hr before the first search each day and col-
lected after the last search of the day. The participant sea-
rch order and number of concealed carcasses (between
2 and 4 target koloa per search area) were randomly
REYNOLDS ET AL. 7 of 18

8. generated for each search unit. Each search area
(1 ha) was used for one paired trial per day. Each
human searcher and canine-handler team could search
in any pattern or direction that they chose. All searchers
carried a Garmin Astro 320 GPS collar to record tracks.
Searchers were followed by a technician that recorded
time, weather and habitat covariates, and carcass loca-
tion. After a target was detected, observers continued to
search for additional targets. In contrast to operational
surveillance, technicians did not assist in searches or
remove concealed carcasses during timed trials. The sec-
ond searcher was sequestered from the search area dur-
ing the first search. After the completion of a paired trial,
searches moved to new areas with different carcasses and
searcher combinations.
2.7 | Analysis
We used logistic regression (Minitab 18.1) in an
information-theoretic model selection approach
(Burnham Anderson, 2002) to determine the causative
factors for whether carcasses were discovered during
operational surveillance with all combined carcasses. We
hypothesized the primary causative factors to be search
method (ATV, pedestrian, and canine assisted), and habi-
tat type (managed wetland or taro). Continuous variables
of wind speed, percent flooded, temperature, humidity,
and percent vegetative cover were considered as poten-
tially influential or confounding factors. Surveys missing
covariate data were excluded from logistic regression ana-
lyses. We did not hypothesize or test for interactive
effects. In lieu of a null model, we created a variable with
random numbers having no possible explanatory power
and ranked all other models against a model with this
single uninformative variable.
Similarly, for the double-blind trial, we included the
continuous variables listed above, search method
(canine-handler team or pedestrian), and search team
identification. We also compared habitat differences (per-
cent vegetative cover) between detected and undetected
targets using two sample t-tests and explored the relation-
ship between canine detection distances and wind speed
using linear regression (Minitab 19.1).
3 | RESULTS
3.1 | Previous outbreaks
Using pedestrian and ATV search methods, in 2016, a
total of 404 waterbird carcasses and 72 intoxicated birds
were collected (including incidental finds) (USFWS data;
Table 1). In 2017, 269 waterbird carcasses and 34 intoxi-
cated waterbirds were collected (including incidental
finds). Half of birds found in 2016 and 2017 were in
active and advanced decomposition and entomological
succession; Table 1 and Figure 5). In both years, koloa
was the most common species collected, accounting for
44% of birds in 2016 and 30% in 2017. Eighteen survey
records were missing start and end time data; we
assumed these searches were not atypical and used the
year's mean survey time to fill missing values (2016
x
= 103 min SD = 58.2 min, N = 828 surveys, 12 missing
survey times; 2017
x = 82 min SD = 39 min, N = 822 sur-
veys, 6 missing survey times). Twenty-two transect
searches were conducted during October 2015–December
2017 with 2–4 people searching during the time searched.
When we corrected for search effort, peak carcass and
intoxication rates do not show a consistent seasonal pat-
tern. Detection rates were 0.28 and 0.20 carcasses per sur-
vey hour in 2016 and 2017, respectively (Table 1).
Detection rates for corresponding months (February–
March) varied from 0.49 in 2016 to 0.26 in 2017, and 0.14
when canine-aided teams were added to surveillance
effort in 2018.
3.2 | Operational surveillance
We completed 118 surveillance surveys of 150 unique
wetland units (total search area of 50 ha and total sea-
rch distances 460 km; Table 2). Of these surveys, 21 were
with ATV, 23 pedestrian, and 74 were canine assisted.
For canine-assisted surveys with GPS tracks (Table 2),
total distances searched exceeded 300 km with individual
survey distances ranging from 0.6 to 6.52 km in taro
(Figure 6) and wetland management units (Figure 7).
3.3 | Carcass discovery
No koloa carcasses were discovered in 2017 during 52 sur-
veillance surveys (Supporting Information S1). In 2018,
10 koloa mortalities were discovered, nine during sur-
veys: 5 by the canine-assisted teams during 34 surveys
and 4 by pedestrian searchers during 21 surveys
(Supporting Information S1). A pedestrian searcher dis-
covered an incidental koloa carcass before starting the
survey. Twenty-five nontarget carcasses were recorded
during November 2017–March 2018, including two
endangered birds 'alae 'ula (Hawaiian gallinule)
Gallinula galeata sandvicensis, and n
en
e (Hawaiian
goose) Branta sandvicensis, cattle egret (Bubulcus ibis),
turtles (Trachemys scripta elegans), frogs and toads
(Lithobates catesbeianus and Rhinella marina), fish
8 of 18 REYNOLDS ET AL.

9. (Tilapia zillii), Rattus sp., pigeon (Columba livia), Indian
myna (Acridotheres tristis), and unknown intestinal and
passerine remains. Canine-assisted teams found 80% of
the nontarget carcasses (Supporting Information S1).
Pedestrian searchers found 20% including the two non-
target endangered species.
3.4 | Concealed surveillance carcasses
In 2018, 52 koloa carcasses were concealed during
66 operational surveillance surveys (uncontrolled field
searches) utilizing three survey methods (Table 3).
Canine-aided teams found 82% of 27 concealed carcasses
TABLE 1 Summary of waterbird carcasses and intoxicated birds found at Hanalei National Wildlife Refuge from October–December
2015, 2016–2017, and January–March 2018
Year
Total birds
collected
(I)
Estimated birds found
per hr (N = timed
surveys)
Birds
per
survey
February–
march birds per
survey
Proportion
endangered
(n = carcasses)
Proportion
decomposition
stage 1
Oct–Dec
2015
70 (15) 0.24 (N = 124) 0.41 n/a 0.76 (n = 51) 0.45
2016 476 (72) 0.28 (N = 828) 0.47 0.49 (N = 129) 0.89 (n = 393) 0.49
2017 303 (34) 0.20 (N = 822) 0.28 0.26 (N = 119) 0.86 (n = 234) 0.50
Jan–Mar
2018
54 (3) 0.14 (N = 232) 0.17 0.14 (N = 173) 0.72 (n = 39) 0.56 (n = 39)
Notes: Total birds include carcasses found suspected of avian botulism (3% were tested by the U.S. Geological Survey National Wildlife Health Center using
the mouse cross protection test; as described in Cato et al., 1986), carcasses with signs of trauma from an invasive predator, and intoxicated birds (I) showing
signs of flaccid paralysis symptomatic of avian botulism. Total birds collected include waterbirds collected during surveillance for botulism as well as those
collected incidentally. Estimated birds found per hour is the subset detected during timed surveillance (excluding incidentally found carcasses). When survey
times were missing, the mean survey time for that year's surveillance was applied. In 2018 we included the canine surveillance and the subtotal of birds/survey
found during February–March. Stage of decomposition is an index of avian botulism risk (0 = sick, 1 = fresh [≤24 hr], 2 = initial decay [24–48 hr], 3 = active
decay [48–72 hr], 4 = advanced decay, and 5 = remains; Verheggen et al., 2017). Birds captured intoxicated (stage 0) and carcasses found while fresh (stage 1)
do not contribute to transmission, whereas carcasses greater than 3 days old in active and advanced stages of toxigenesis, entomological succession and
decomposition (stages 2, 3, 4, and 5) are more likely to contribute to epizootics.
FIGURE 5 Monthly search effort with waterbird carcasses and intoxicated birds collected at Hanalei National Wildlife Refuge during
avian botulism surveillance (birds per estimated search hour and per survey) for 2016 and 2017. N = the number surveys conducted
REYNOLDS ET AL. 9 of 18

10. TABLE 2 Summary of survey effort during the pilot study at Hanalei National Wildlife Refuge with three search methods: (a) an
observer on an all-terrain vehicle (ATV), (b) an observer searching on foot (Pedestrian), and (c) a team of dog and handler, and a technician
(Canine-assisted team) during operational surveillance November 24 to December 25, 2017 and February 1 to March 30, 2018
Operational surveillance ATV Pedestrian Canine assisted
Surveys 21 23 74
Total survey time (hr:min:s) 13:05:58 23:22:00 93:04:49
Mean survey time (hr:min:s) 0:37:26 (0:14:09 SD) 1:06:46 (0:12:41 SD) 1:13:29 (0:29:29 SD)
Range (min max) 0:15–1:03 0:38–2:00 0:10:09–2:32:00
Total track distance (km) n = useable GPS
tracks recorded
83.68 (n = 21) 74.93 (n = 22) 308.42 (n = 64)
Total area searched (50 ha total)a
19 ha 24 ha 40 ha
a
Search areas were approximated using GPS.
FIGURE 6 Tracks from
GPS points during operational
surveillance February 23, 2018
by canine assisted team
conducting a perimeter search in
taro lo'i with the initial detection
distance to carcass marked as
change of behavior at 15.5 m
10 of 18 REYNOLDS ET AL.

11. during 34 surveys. Pedestrians found 70% of 20 concealed
carcasses during 21 surveys, and ATV searches found
40% of 5 concealed during 11 surveys. Canines detected
the target odor at distances ranging from 1 to 85 m (
x
= 25.3 SD = 24.4, n = 18) under a range of environmental
conditions (Supporting Information S2, Figure 7). Wind
speed was not a significant predictor of canine detection
distance (F1,17 = 2.80, R2
= 15.74, p = .12). There were no
FIGURE 7 Tracks from GPS points during operational surveillance February 2, 2018 using two canine teams to locate a koloa carcass in
densely vegetated wetland management unit with maximum detection distance to carcass marked as change of behavior of more than 85 m
TABLE 3 Summary of concealed koloa carcass experiment to test detection efficacy (proportion of carcasses found) and mean time to
find randomly concealed carcasses during operational surveillance (66 surveys) at Hanalei National Wildlife Refuge (February–March 2018)
Operational surveillance 2018 ATV Pedestrian Canine assisteda
Surveys 11 21 34
Concealed koloa 5 20 27
Proportion detected 0.40 0.70 0.82
Total and mean survey time (hr:min:s) 6:16:05
x = 0:34:11 23:22:00
x = 1:06:46 52:58:44
x = 1:28:18
Mean time to find concealed carcass (hr:min
range)
0:15:30 (0:11–0:20) 0:49:47 (0:08–0:50) 0:34:07 (0:1–0:56)
Mean survey area (ha) (SD) 4.02 (1.1) 4.46 (0.8) 4.43 (0.8)
Total and mean (SD) track length (km) 104.9 (n = 11)
x
= 3.5 (1.1)
41.2 (n = 19)
x
= 3.75 (1.16)
187.17 (n = 33)
x
= 3.34 (0.65)
a
Two survey areas were searched by two dogs.
REYNOLDS ET AL. 11 of 18

12. significant differences (α = 0.05) in the mean vegetative
cover for concealed carcasses detected and undetected
by canine-aided teams (t-value = −1.56, df = 11, p = .15);
however, for the carcasses not detected by humans (ATV
and pedestrians combined) mean vegetative cover
was greater than vegetative cover for carcasses found (not
detected
x = 0.75, SD = 0.15, n = 7 vs. detected
x = 0.54,
SD = 0.27, n = 15, t-value = −2.33, df = 19, p = .03).
There was no difference in mean vegetative cover
between wetland management and taro units
(t-value = −0.21, df = 89, p = .83).
The seven highest-ranked logistic regression models
with ΔAIC values less than 2 included variables of search
method, habitat type, percent flooded, temperature, and
percent vegetative cover (Supporting Information S3
[AIC ranks]). Search method occurred in all seven highly
ranked models. Habitat type occurred in six, thus we
chose these as the best explanatory variables. The model
with the single uninformative variable had a ΔAIC value
of 6.74. Odds ratios confirmed that percent flooded, tem-
perature, and vegetative cover showed less influential
effects in highly ranked models than the variables search
method and habitat type, with odds ratios near 1 (Table 4).
Carcasses were 5 times more likely to be discovered by
canine-assisted teams or pedestrian searchers than by
ATVs. Carcasses were 3 times more likely to be discov-
ered in managed wetlands than in taro.
3.5 | Timed (15-min) double-blind trials
In detection field trials that controlled for search time
and search area, canine-handler teams found 77% and
pedestrian humans found 39% of the koloa carcasses
(Table 5). One exceptional human observer had 100%
detection of four concealed carcasses (Table 5). When we
combined total detections (areas searched using a human
surveyor and a canine-handler team), 100% of koloa
carcasses were detected in seven of the nine paired
trials—because dogs and humans often found different
carcasses (Figure 8).
The seven highest-ranked logistic regression models
with ΔAIC values 2 include the variables of search type,
wind speed, percent vegetative cover, temperature, and
search team. The model with the single uninformative
variable had a ΔAIC value of 8.86 (Supporting Informa-
tion S4 [AIC ranks]). Carcasses were 5 times more
likely to be discovered by the dog-handler team than by
the pedestrian humans in the timed trials. More experi-
enced dogs had more detections than novice dogs, but
novice dogs had higher detections than humans during
timed searches (Table 6). Canines had 35 concealed tar-
gets to find during the time trial, and of those undetected
targets (8), one novice canine in training did not detect
50%. Carcasses were 10 times more likely to be detected
by the experienced dogs than pedestrian humans in the
timed trial.
Smoothed GPS tracks (N = 6 paired trials) averaged
1.0 km (SD = 0.16) for canine teams and 0.87 km for the
humans but did not differ between paired trials (t-
value = 1.25, df = 5, p = .27). Trial GPS tracks showed
canine COB ranging from 4 to 31 m (
x = 14.3, SD = 7.6,
n = 17) (Supporting Information S2). Wind speed was
not found to be a significant predictor of canine detection
distance (F = 1.51, R2
= 8.6, p = .24).
4 | DISCUSSION
4.1 | Efficiency and efficacy of carcass
detection
We found that using detection canines for avian botulism
surveillance is a potentially highly effective technique
that can be applied to many other systems. Given the
urgency to find as many carcasses as possible before
avian botulism initiation and propagation, the addition of
canine surveillance is a promising approach to detect and
pinpoint waterbird carcasses in small wetlands with
dense and flooded vegetation. We found the canine-
handler teams were 5 times more likely to find koloa
carcasses than single pedestrian searchers during timed
trials. During operational surveillance over about 50 ha,
canine-aided teams using experienced detector dogs
found 82% of concealed carcasses searching on average
4–5 ha per survey. Studies using airboats and pedestrians
over much larger search areas (420–8,200 ha) found only
7–45% of marked carcasses, 61% in smaller well-defined
areas (Bollinger et al., 2011).
Bennett, Hauser, and Moore (2020) described effi-
ciency as a function of performance, relating proportion
TABLE 4 Odds ratios of predictor levels from the highest
ranked logistic regression model of avian botulism surveillance
methods at Hanalei National Wildlife Refuge on Kaua'i Island, 2018
Predictor Level A Level B Odds ratio A:B
Search method Canine ATV 5.3:1
Search method Pedestrian ATV 5.6:1
Search method Pedestrian Canine 1:1
Habitat type Wetland Taro 3.3:1
Notes: We hypothesized the primary causative factors to be search method
(ATV, pedestrian, canine assisted), and habitat type (managed wetland or
taro). See Supporting Information S3 for logistic regression model ranks of
avian botulism surveillance.
12 of 18 REYNOLDS ET AL.

13. of targets found to time, and area searched. Our results
indicated canine-aided teams were more efficient at
finding carcasses than pedestrian searchers and more
effective than ATVs in dense vegetation. During 15-min
blind detection trials, most paired trials had 100% detec-
tion of concealed carcasses because pedestrian
observers and canine-handler teams often found differ-
ent carcasses. Experience was an important factor
influencing carcasses not detected by canine teams,
with newer handlers and novice canines detecting
fewer carcasses. Ideally, surveillance could maximize
the abilities of both detection dogs and human
observers.
Habitat type and search methods influenced both the
efficacy and efficiency of carcass detection. Detection
probability of carcasses in dense vegetation is often
higher for canine-assisted teams, but human searchers on
foot also demonstrated high probability of detection with
more time to search. ATV searches were efficient for
large areas with less than 55% vegetative cover and for
habitats of lower botulism risk such as managed wet-
lands. Optimizing the allocation of search effort within
differing and dynamic habitats involves managers
weighing the costs of surveillance against the risk of not
detecting carcasses while considering environmental con-
ditions (Glen Veltman, 2018). Efficiency and efficacy
TABLE 5 Summary of paired double-blind detection trials March 1–3, 2018 at Hanalei National Wildlife Refuge
Trial
number
and
date 2018
Search area
and habitat
Time
start
Searcher
(order)
Raw
distance (km)
Concealed
carcasses
found/
total
First
detection
(min:s)
Wind
range
(kph)
1–1 Mar 0.97 ha Flooded Fallow 9:39 (1) Bodhi/
Kyokoa
1.17 2/3b
3:15 2.2-7.5
10:10 (2) MM 1.20 3/3 3:10 1.1–4.6
2–1 Mar 0.73 ha Dry Fallow 11:05 (2)Solo/Nini 1.41 4/4 8:18 0–5.2
10:55 (1)KR 1.13 0/4 n/a 2.8
3–1 Mar 0.82 ha Flooded Wetland 13:40 (2)Pandac
/
Kyoko
1.17 2/3 1:30 7.5–9.4
13:15 (1)TL 1.57 2/3 2:30 6.8–8.6
4–1 Mar 0.9 ha Flooded Fallow 14:21 (1)Dukec
/Nini 1.07 1/4 4:40 1.8–5.2
14:40 (2)VP 1.2 1/4 0:41 4.8–6.8
5–2 Mar 0.74 ha Flooded Fallow 9:35 (1)Pandac
/Nini –d
2/2 3:56 4.5-4.7
10:00 (2)SD 0.67 0/2 n/a 13.5
6–2 Mar 0.73 ha Dry Fallow 10:50 (2)Dukec
/
Kyoko
1.04 3/4 3:33 3.5–16.1
10:26 (1)DG 0.68 2/4 5:20 10.9
7–2 Mar 0.82 ha Flooded Wetland 13:05 (1)Bodhi/KR 1.05 3/3 1:42 7.5–12.4
13.29 (2)LB 0.77 1/3 1:18 12–15.3
8–2 Mar 0.90 ha Flooded Fallow 14:10 (1)KU 1.23 1/4 2:19 6.1–9.2
14:39 (2)Solo/Kyoko 1.17 3/4 1:08 4.1–8.6
9–3 Mar 0.90 ha Dry and Flooded
Fallow
14:05 (2)Pandac
/Nini 1.49 3/4 1:20 2.8–11.8
14:30 (3)Bodhi/
Kyoko
1.37 4/4 0:29 2.8–7
13:40 (1)TJ –d
2/4 1:38 4–11.2
Notes: Search order (order) and search pairs were randomized. Each human searcher (represented with initials) and canine handler team had 15 min to search
for koloa carcasses placed inside a female mallard plastic decoy (for visual or olfactory detection).
a
Searcher cells with two names refer to canine handler teams.
b
Search area poorly defined on first run.
c
Novice dog.
d
Track missing.
REYNOLDS ET AL. 13 of 18

14. may be weighed to consider costs of escalating epizootics
and the conservation value of waterbird survival.
Another important factor affecting efficiency and effi-
cacy is the distance of detection. In this study, canines
were exceptional, detecting carcasses up to 85 m and
averaging 25 m under typical environmental conditions,
with no significant difference related to vegetation cover.
Detection was more efficient if dogs could navigate
directly to the source of odor, thus increasing both detec-
tion probability and efficiency. For example, in the timed
double-blind trial, perimeter searches with proximity
alerts were not required and canine detection signifi-
cantly exceeded humans. However, despite the
inefficiency, proximity alerts can have important advan-
tages including allowing handlers more time to evaluate
hazards such as deep mud, flowing water, or fragile vege-
tation, and may allow waterbirds and broods more time
to move away.
In future studies, analyses of cost-effectiveness could
include detailed records on fixed and variable costs of
person-hours, equipment, and maintenance relative to
the costs of canine surveillance to determine the cost per
carcass and per area by each method. An experiment
could be designed to optimize carcass detection costs
according to some of the influential factors that we
found, such as vegetation type and cover, and observer
FIGURE 8 Tracks from the
double blind timed detection
trial March 2, 2018 with
examples from a human
searcher (top) and canine-
handler team (bottom). The
human searcher found one of
the four concealed carcasses
during the 15-min time limit.
The canine-handler team found
three of four hidden carcasses
during the 15-min time limit.
Because each searcher found
different hidden carcasses,
together carcass detection
was 100%
14 of 18 REYNOLDS ET AL.

15. experience. Cost-effectiveness of human-only versus
canine-aided teams is not something that we could
directly evaluate in this study due to the lack of cost data.
However, the efficiency and efficacy data presented were
proxies for cost effectiveness. Volunteers do much of the
pedestrian surveillance at the refuge; here they also par-
ticipated with staff in double-blind trials. In contrast,
only trained staff used ATVs. Furthermore, waterbirds at
the refuge are primarily endangered species; thus, the
value of finding carcasses will be greater for endangered
species with small populations. Working under the chal-
lenging conditions of this study with endangered species
provided a model that can be instructive for other studies
that may have different species, staffing costs, or
environments.
4.2 | Ecological complexities and
management of avian botulism in wetlands
Interactions between depredated carcasses, habitat, and
epizootic risk are difficult to disentangle but are interre-
lated because introduced predators create carcasses,
which could serve as substrate for avian botulism. Like-
wise, intoxicated birds may be easy prey that attract inva-
sive predators. Effective control of feral cats at Hanalei
NWR and beyond may benefit waterbirds by reducing
mortality, thus resulting in fewer carcass substrates. Half
(n = 398) of all the carcasses found at Hanalei NWR (Oct
2015–Dec 2017, n = 798) were suspected to be due to
avian botulism, the majority of which were discovered in
taro units (USFWS data). Likewise, more than 96% of the
depredated carcasses collected were also found in the taro
habitats (USFWS data).
We did not observe strong seasonal patterns in car-
cass occurrence at Hanalei NWR during 2015–2018 after
correcting for search effort; however, the magnitude of
outbreaks varied between years. In all years, nearly half
of the carcasses found were in risk-prone stages of
decomposition (≥stage 2), indicating that surveillance
optimization may reduce avian botulism risks and costs
by detecting carcasses earlier.
The high avian botulism risk at Hanalei NWR may be
related to the floodplain landscape that includes man-
aged wetlands and agriculture. This landscape configura-
tion includes increased predator access (taro dikes used
as hunting corridors), eutrophic agricultural inputs, and
many invasive aquatic species and potential vectors
(e.g., tilapia, clams). Invasive predator control, water
management, carcass surveillance, and rehabilitation of
intoxicated birds are ongoing at Hanalei NWR. In 2019,
243 waterbird carcasses and 33 intoxicated birds were col-
lected during avian botulism surveillance at Hanalei
NWR (USFWS data).
Other practices to potentially reduce the magnitude
of an avian botulism outbreak include capture and reha-
bilitation of sick birds to reduce bird mortality, vaccina-
tion of endangered species (Martinez Wobeser, 1999),
and in some cases managing wetlands to be less favorable
to bacterial growth of Clostridia (Espelund
Klaveness, 2014). Vegetative growth and toxin production
of C. botulinum is complex and affected by many factors
including temperature (optimally between 25 and 40
C),
high pH, oxygen tension/redox potential, nutrient and
pesticide inputs, as well as complexity of the microbial
community and ecosystem health (Riley, Munkittrick,
Evans, Krueger, 2008; Rocke Bollinger, 2007;
Rocke Samuel, 1999). Other biotic factors that have
potential for management may include invasive bivalves
(William Chi, Asia University, DMV oral communica-
tion, January 15, 2020), necrophagous vectors
(e.g., Calliphoridae blowflies, Sarcophagidae flesh flies,
and Dermestidae beetles; Wobeser, 1997; Anza
et al., 2014), and filamentous algae and invasive fish that
are described as “biotic incubators” for C. botulinum in
Espelund and Klaveness (2014). Applied field experi-
ments could be used to learn more about the role of inva-
sive aquatic species in avian botulism proliferation.
4.3 | Conservation value
This is the first study exploring the efficacy of canines for
avian botulism carcass detection and removal. We found
TABLE 6 Odds ratios of predictor levels from the highest
ranked logistic regression model of search team detection
probability during double-blind detection trial at Hanalei National
Wildlife Refuge on Kaua'i Island, 2018
Level A Level B Odds ratio A:B
B Any human 14.3:1
D Any human 11.1:1
F Any human 5.5:1
G Any human 1.6:1
D B 0.8:1
F B 0.4:1
G B 0.1:1
F D 0.5:1
G D 0.1:1
G F 0.3:1
Notes: See Supporting Information S4 for logistic regression model ranks.
Experienced detector canines are indicated by B, D and novice canines F
and G.
REYNOLDS ET AL. 15 of 18

16. this approach feasible at Hanalei NWR. Because canine-
assisted surveillance can improve detection probability
and efficiency, especially within heavily vegetated areas
that often obscured or slowed detection for human
observers, this approach is a valuable conservation tool.
Finding and removing carcasses before toxigenesis could
prevent a larger avian botulism outbreak and has been
demonstrated to reduce waterbird mortality (Evelsizer
et al., 2010; Reed Rocke, 1992). Combined with con-
ventional (human) searches, trained detector canines can
contribute to the optimization of search strategies (for
maximum probability of success), maximizing detection
efficacy and minimizing time to detection, thus curtailing
toxigenesis and toxin accumulation in food webs, and
ultimately reducing waterbird mortality.
Avian botulism has been compared with wildfires
and infectious epidemics, emphasizing the importance of
preemptive surveillance for early detection, early inter-
vention, and rapid response (Chowell Nishiura, 2014;
Soos Wobeser, 2006). Time to discovery or delay in car-
cass removal can also influence epizootic magnitude and
duration because of complex disease dynamics. Although
environmental conditions greatly influence the progres-
sion of avian botulism, heuristics drawn from the litera-
ture suggest that detection and removal of any waterbird
carcasses by or before day three or four postmortem may
help prevent initiation of toxigenesis (Anza et al., 2016).
In addition, detection and removal of sick birds and
foodborne toxic carcasses by day three (Evelsizer
et al., 2010; Wobeser, 1997) may reduce the intoxication
pathways in food webs, decreasing the magnitude of out-
breaks. In the subtropical Hawaiian Islands, in particular,
a paradigm shift from botulism emergency response to
botulism prevention and risk reduction is important
because most wetlands habitats are small and affected
birds are endangered and nonmigratory. If outbreaks are
typically seasonal, intensive surveillance, beginning prior
to the anticipated onset of botulism and concentrating
efforts on areas with past high carcass densities may help
prevent or reduce outbreak severity (Soos
Wobeser, 2006) and may ultimately reduce the cost of
managing epizootics.
ACKNOWLEDGMENTS
We appreciate the support and cooperation provided by
Kaua'i NWR Complex, particularly Heather Tonneson,
Michael Mitchell, Jennifer Waipa, and Megan Nagel. We
thank numerous volunteers for dedicated field assistance,
and Hanalei taro farmers for access during training and
surveys. We thank James Jacobi, Jeff Hatfield, and
Rebecca Ostertag for reviewing this manuscript. We
thank canine handler and assistant trainer Nini Kuo, and
canines Solo, Bodhi, Duke, and Panda for their intensive
efforts. We thank Ted Young for help with odor recogni-
tion training, Meaghan Lyon for data entry, trial organiz-
ing, concealing carcasses, and field support. We thank
artist Ilana Nims for creating the conceptual illustrations
for Figure 1a,b. Thanks to Tor Johnson for photo and
video documentation. Thanks to Jerry Bradshaw of
Tarheel Canine for helping to envision this canine effi-
cacy project and for providing important connections
with ecological detection experts (William Chi) and
canine handler (Nini Kuo). Paul Berkowitz processed
data and created track maps. Any use of trade, firm, or
product names is for descriptive purposes and does not
imply endorsement by the U.S. Government. This
research was funded by the Science Support Program of
the U.S. Geological Survey and the U.S. Fish and Wildlife
Service and supported by the U.S. Geological Survey
Pacific Islands Ecosystem Research Center.
CONFLICTS OF INTEREST
The authors declare no potential conflicts of interest.
AUTHOR CONTRIBUTIONS
Michelle H. Reynolds and Kyoko N. Johnson: Designed
and coordinated the study. Steven C. Hess and Michelle
H. Reynolds: conducted the statistical analyses. Michelle
H. Reynolds and Steven C. Hess: Led writing of the man-
uscript. Kim J. Uyehara, Kyoko N. Johnson, Eleni
R. Schvaneveldt, and Daniel L. Dewey: Conducted the
research, reviewed, edited, and commented on the manu-
script. All authors conceived of ideas in the study imple-
mentation and have given final approval of the version to
be published.
DATA AVAILABILITY STATEMENT
Data available from U.S. Geological Survey's ScienceBase
website: https://doi.org/10.5066/P9C4N47X; Reynolds
et al. (2021).
ORCID
Eleni R. Schvaneveldt https://orcid.org/0000-0002-7416-
3836
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SUPPORTING INFORMATION
Additional supporting information may be found online
in the Supporting Information section at the end of this
article.
How to cite this article: Reynolds MH,
Johnson KN, Schvaneveldt ER, Dewey DL,
Uyehara KJ, Hess SC. Efficacy of detection canines
for avian botulism surveillance and mitigation.
Conservation Science and Practice. 2021;e397.
https://doi.org/10.1111/csp2.397
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