Journal Pre-proof

Abundant triatomines in Texas dog kennel environments: Triatomine

collections, infection with Trypanosoma cruzi, and blood feeding
hosts

RE Busselman , R Curtis-Robles , AC Meyers , IB Zecca ,

LD Auckland , CL Hodo , D Christopher , AB Saunders ,
Hamer SA

PII: S0001-706X(23)00274-7
DOI: https://doi.org/10.1016/j.actatropica.2023.107087
Reference: ACTROP 107087

To appear in: Acta Tropica

Received date: 10 September 2023

Revised date: 26 November 2023
Accepted date: 28 November 2023

Please cite this article as: RE Busselman , R Curtis-Robles , AC Meyers , IB Zecca , LD Auckland ,
CL Hodo , D Christopher , AB Saunders , Hamer SA , Abundant triatomines in Texas dog kennel
environments: Triatomine collections, infection with Trypanosoma cruzi, and blood feeding hosts, Acta
Tropica (2023), doi: https://doi.org/10.1016/j.actatropica.2023.107087

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Highlights

• 550 triatomines collected from Texas dog kennels using manual search & trained dog
• 47% of insects infected with Trypanosoma cruzi of DTUs TcI and TcIV
• Bugs fed on dog, human, raccoon, chicken, pig, cat, vulture, and thrasher

1
Title: Abundant triatomines in Texas dog kennel environments: Triatomine collections, infection

with Trypanosoma cruzi, and blood feeding hosts

Authors: Busselman RE1, Curtis-Robles R 1, Meyers AC1, Zecca IB1, Auckland LD1, Hodo CL1,2,

Christopher D3, Saunders, AB4, Hamer SA1*

Affiliations:
1
Department of Veterinary Integrative Biosciences, Texas A&M University, College Station,

Texas, United States of America

2
The University of Texas MD Anderson Cancer Center, Michale E. Keeling Center for

Comparative Medicine and Research, Bastrop, Texas, United States of America

3
Independent Contractor, Columbus, Ohio, United States of America
4
Department of Small Animal Clinical Sciences, Texas A&M University, College Station, Texas,

United States of America

*Corresponding author: shamer@cvm.tamu.edu (Sarah A. Hamer)

Abstract:

Triatomine insects are vectors of the protozoan parasite Trypanosoma cruzi- the causative

agent of Chagas disease. Chagas disease is endemic to Latin America and the southern United

States and can cause severe cardiac damage in infected mammals, ranging from chronic

disease to sudden death. Identifying interactions among triatomines, T. cruzi discrete typing

units (DTUs), and blood feeding hosts is necessary to understand parasite transmission

dynamics and effectively protect animal and human health. Through manual insect trapping

2
efforts, kennel staff collections, and with the help of a trained scent detection dog, we collected

triatomines from 10 multi-dog kennels across central and south Texas over a one-year period

(2018-2019) and tested a subset to determine their T. cruzi infection status and identify the

primary bloodmeal hosts. We collected 550 triatomines, including Triatoma gerstaeckeri

(n=515), Triatoma lecticularia (n=15), Triatoma sanguisuga (n=6), and Triatoma indictiva (n=2),

with an additional 10 nymphs and 2 adults unable to be identified to species. The trained dog

collected 42 triatomines, including nymphs, from areas not previously considered vector habitat

by the kennel owners. Using qPCR, we found a T. cruzi infection prevalence of 47% (74/157),

with T. lecticularia individuals more likely to be infected with T. cruzi than other species. Infected

insects harbored two T. cruzi discrete typing units: TcI (64%), TcIV (23%), and mixed TcI/TcIV

infections (13%). Bloodmeal host identification was successful in 50/149 triatomines, revealing

the majority (74%) fed on a dog (Canis lupus), with other host species including humans (Homo

sapiens), raccoons (Procyon lotor), chickens (Gallus gallus), wild pig (Sus scrofa), black vulture

(Coragyps atratus), cat (Felis catus), and curve-billed thrasher (Toxostoma curviostre). Given

the frequency of interactions between dogs and infected triatomines in these kennel

environments, dogs may be an apt target for future vector control and T. cruzi intervention

efforts.

Key Words: bloodmeal, Chagas disease, triatomine vector, canine, Trypanosoma cruzi

Introduction:

Chagas disease is a neglected tropical disease caused by the parasite Trypanosoma cruzi, and

triatomine insects (Reduviidae; Triatominae; ‘kissing bugs’) are the recognized arthropod

vectors. Currently, antiparasitic treatment for infected humans is variably effective depending on

several factors, and treatment of canine infections remains challenging with limited options

3
(Bern et al., 2019; Hamer and Saunders, 2022; Meymandi et al., 2018). Thus, preventing

infection is the best approach to protect against T. cruzi. However, prevention options are

limited to reducing contact with infected triatomines through vector management and

environmental modification (Barr, 2009). The surveillance and control of triatomines is largely

dependent on the species of triatomines present. Timed manual searches in human dwellings,

use of indoor residual pesticide sprays, community outreach, and housing modifications are

several techniques that can be used to detect and manage triatomine species that commonly

colonize human dwellings (Gaspe et al., 2020; Grijalva et al., 2022; Gürtler et al., 2007; Gürtler

and Yadon, 2015; Pereira et al., 2022; Quinde-Calderón et al., 2016). In areas where sylvatic

species of triatomines predominate, standardized surveillance is challenged by difficulties

identifying key sylvatic habitats that are often not located around man-made structures (Bern et

al., 2011; Ibarra-Cerdeña et al., 2009; Waleckx et al., 2015). Further, sylvatic triatomines may

travel from an unknown distance to locations where humans and domestic animals are at risk of

infection (Barbu et al., 2010; Crawford and Kribs-Zaleta, 2013; Dantas et al., 2018).

Since it can be difficult to locate sylvatic triatomine populations through manual searches, other

search methods are necessary in environments where triatomines are primarily sylvatic or peri-

domestic to understand the risk posed to humans and animals by local vectors. Community

science initiatives have aided greatly in advancing our understanding of sylvatic triatomine

populations and infection prevalence (Cardinal et al., 2007; Curtis-Robles et al., 2015; Delgado-

Noguera et al., 2022; Dumonteil et al., 2009; Gürtler and Yadon, 2015; Hamer et al., 2018; Klotz

et al., 2016; Parente et al., 2017). Additionally, trained scent detection dogs have been used in

Paraguay and Texas to locate triatomines from cryptic nesting locations in areas not previously

considered vector habitat, which has yielded collection of more nymphs- the immature life

stages of triatomines- and perhaps a better representation of sylvatic populations than just

those insects encountered by humans (Christopher et al., 2023; Rolón et al., 2011).

4
In the United States, human autochthonous cases are increasingly recognized, and infestations

of houses by sylvatic triatomines occasionally occur (Beatty and Klotz, 2020; Klotz et al., 2016;

Lynn et al., 2022). In addition to human cases, domestic dogs are a population affected by T.

cruzi infections and associated disease. Infections with T. cruzi have been documented in

multiple southern states, with prevalences of 15.7% in southern Louisiana, 13.2% in Oklahoma,

and infection prevalences in Texas ranging from 3.8% along the Texas-Mexico border to 45.3%

in El Paso, TX (Allen and Lineberry, 2022; Elmayan et al., 2019; Garcia et al., 2016; Kjos et al.,

2008; Rodriguez et al., 2021). Canine Chagas disease is not only a problem in areas where

triatomines are endemic, but travel and relocation-related cases are a concern for the veterinary

community (Barr, 2009; Gavic et al., 2023; Meyers et al., 2020). In animal shelter environments

in the southern United States, infection prevalence in dogs can be high, with T. cruzi infections

in one multi-shelter study averaging 18%, the same level of infection as found for heartworms in

the same dog populations (Allen and Lineberry, 2022; Elmayan et al., 2019; Hodo et al., 2019;

Tenney et al., 2014). Multi-dog kennel environments have emerged as particulary high risk, with

our previous work in south Texas identifying a 30% annual risk of infection (Busselman et al.,

2021). Similarly, government-owned working dogs working along the US-Mexico border have

also been found to be infected, with infection prevalences up to 18.9% (Meyers et al., 2021;

Meyers et al., 2017).

Sylvatic triatomines may feed on diverse wildlife before dispersal to human dwellings or

kennels. Identifying the vertebrate hosts that support the vector populations in nature could

provide information relevant to improving vector control. Because vertebrate species vary in

their reservoir competence for T. cruzi, the local patterns of vector-host feeding may be useful

for predicting transmission risk to humans, dogs, or other target species (Hodo and Hamer,

2017). When coupled with other information on vector and host infection, bloodmeal analysis

5
can identify key hosts responsible for perpetuating the epidemiological risk (Cantillo-Barraza et

al., 2015; Gorchakov et al., 2016; Kjos et al., 2013; Ocaña-Mayorga et al., 2021; Ordóñez-

Krasnowski et al., 2020; Pessanha et al., 2023; Quiroga et al., 2022), which could later be

exploited for novel vector control initiatives. In this study, we used three collection methods-

manual searches, community science partnerships with kennel owners, and a trained scent

detection dog- to collect triatomines from large multi-dog kennels in Texas. T. cruzi infection

prevalence and bloodmeal hosts were determined in order to better understand the aspects of

vector biology that may be important for developing approaches to protect dog and human

health.

Methods:

Location of Study:

We collected triatomines from 10 multi-dog kennels in central and south Texas at three

timepoints approximately 6 months apart between May 2018 and September 2019 (Figure 1).

Kennels were visited first between May 2018 and July 2018, second during December 2018 –

March 2019, and third between May 2019 and September 2019. The sampling frequency was

intended to allow collection of triatomines from the kennel environment across a full calendar

year with focus on the summer and winter seasons. Kennels enrolled in this study were part of a

larger study focusing on canine Chagas disease, which identified an annual incidence rate of T.

cruzi infection of 30% in the resident dogs (Busselman et al., 2021). These kennels each

housed between 30 and 100 resident dogs, and the dogs were primarily trained for hunting or

showing. Many of the kennels had partial or complete exposure to the outdoors, with variation in

the size of holes in the mesh or fencing (Figure 2). Environmental insecticides were used at

some kennels, although systematic data on owner-reported use were not collected.

6
Figure 1: Number of triatomines collected at 10 multi-dog kennels across central and south

Texas, 2018-2019. The location of each circle is the approximate location of each kennel.

The size of each circle indicates the relative number of triatomines collected from each kennel,

with the number of triatomines collected written near each circle. Map was created in QGIS,

version 3.28, using US Census data (QGIS.org, 2023; U.S. Census Bureau, 2018).

7
Figure 2: Examples of large multi-dog kennels around which triatomines were collected, with

various screens visible. The photo on the left shows a typical kennel with concrete floor that is

open to the outside enviroment, which often included both sylvatic habitat and a human dewlling

around the perimeter. The photo on the right shows a triatomine just outside a kennel next to

wire mesh netting that was used around the areas where dogs were housed. Additional photos

of kennels related to their environment are available as a Supplemental Figure.

Triatomine Collections:

At the initial visit, opportunistic manual searches of each property around the dog kennels were

conducted by the research team by walking around the kennel areas and nearby buildings while

visually searching for insects, with moving of debris to locate triatomines. Additionally, in some

cases, kennel owners pointed out habitats where triatomines were previously encountered, and

these historical hotspots were also searched. Occasional repeated searches at some kennels

were conducted at the second and third visits. Kennel staff were instructed to collect

triatomines- dead or alive- using a plastic bag to avoid touching the insects with their bare

hands. Triatomines were then stored in the freezer to kill any living triatomines (Muscio et al.,

1997; Pedrini et al., 2009; Salt, 1936) and to preserve DNA for laboratory diagnostics.

8
Involvement of kennel owners in collection of triatomines as part of the Kissing Bug Community

Science program was determined to not constitute human subjects research by the Institutional

Review Board at Texas A&M University.

In addition to searches by our research team and kennel staff, we used a trained scent

detection dog to augment collections. ‘Ziza,’ a 4-year old, T. cruzi-infected German shorthaired

pointer and her handler visited eight kennels throughout the course of the study: 6 between July

21 and July 28, 2018, and 4 (2 new kennels and 2 repeat visits) between August 29 and

September 3, 2019. Search methods were as previously reported (Christopher et al., 2023).

Prior to each sampling trip, Ziza participated in a reinforcement training period of one day with

her handler, which consisted of detecting live triatomines which had been contained and hidden,

as previously described (Christopher et al., 2023). Follow-up reinforcement training of one to

two practice problems was completed daily prior to any search efforts at kennels. At the

kennels, the handler walked the leashed dog to search habitats including woodpiles, storage

sheds, brushy areas, and the dog kennels (concrete and brick structures). More frequent breaks

were required when working at higher temperatures. Once Ziza signaled that she had identified

triatomines, that location was marked and followed up by human search. Triatomines caught

alive during this process were added to a laboratory triatomine colony, and a subset were tested

for T. cruzi infection using fecal spot extractions (described below). All dog handling was

conducted with support of the privately owned scent detection dog’s handler under an animal

use protocol with approval from Texas A&M University Institutional Animal Care and Use

Committee under protocol number 2018–0460 CA.

Laboratory Processing:

T. cruzi identification

9
In the laboratory, the sex and species of adult triatomines were identified based on

morphological features (Lent and Wygodzinsky, 1979). Most triatomines that were found alive

were placed into a triatomine colony on Texas A&M’s campus to contribute to future studies. For

those placed in the colony, each triatomine was housed individually until it produced a fecal

spot, which could be tested for T. cruzi DNA. Triatomines found dead or alive but not placed in

the triatomine colony were measured, and a representative subset stratified by kennel, species,

and time point was selected for further processing. The external surface of each triatomine was

washed in a 50% bleach solution for 15 seconds followed by distilled water for 15 seconds to

remove exogenous DNA. The hindgut was dissected to collect material, and DNA was extracted

from the material or fecal spots (KingFisher Cell and Tissue DNA kit, Thermo Fisher Scientific,

Waltham, MA). During dissections, the amount of blood in each triatomine hindgut was recorded

(1 = desiccated guts with no blood; 2 = guts present but no blood; 3 = traces of blood inside

guts; 4 = dried blood present; 5 = fresh blood present) (Curtis-Robles et al., 2018c). Extracted

samples were subjected to quantitative PCR using Cruzi 1/2/3 primers for detection of T. cruzi

DNA (Duffy et al., 2013). A positive control of DNA extracted from T. cruzi Sylvio-X10 CL4

(ATCC 50800, American Type Culture Collection [ATCC], Manassas, VA) was included in each

PCR. Samples that produced a Ct value <35 were considered positive for T. cruzi and were

subjected to quantitative PCR using primers and probes for the spliced leader intergenic region

(SL-IR) to differentiate between T. cruzi DTUs (Cura et al., 2015; Curtis-Robles et al., 2018d).

Each DNA extraction and PCR reaction included no-template or sterile nuclease-free water

(VWR International LLC, Radnor, PA) negative controls, respectively. Positive controls were

included in all SL-IR PCRs, including DNA from T. cruzi Sylvio (ATCC 50800, ATCC; DTU TcI),

T. cruzi Y strain (ATCC 50832, ATCC; DTU TcII/III), and a confirmed T. cruzi positive T.

sanguisuga (DTU TcIV).

10
Bloodmeal host determination

The subsample of insects that were tested for T. cruzi infection were also processed to

determine bloodmeal hosts. Samples were subjected to an iterative series of PCRs amplifying

the vertebrate cytochrome b gene using either the ’BM’ primers (Hamer et al., 2009) or ‘herp’

primers (Cupp et al., 2004; Hamer et al., 2009), followed by Sanger sequencing of amplicons

(Eton Biosciences, San Diego, California). If no amplicon was produced or if the sequencing

reaction failed, then the other primer set (either ‘BM’ or ‘herp’) was used in an independent

attempt to identify a host. All PCRs included negative controls (sterile nuclease-free water) and

positive controls of DNA extracted from white-tailed deer (Odocoileus virginianus), lion

(Panthera leo), or the American black bear (Ursus americanus). Sequence chromatographs

were reviewed for quality in Geneious Basic (Kearse et al., 2012), and sequences were

compared to those in GenBank using BLAST (Sayers et al., 2023). A bloodmeal identification

was considered successful when the BLAST identity match in GenBank met or exceeded 98%.

Sequences with a <98% match were manually scrutinized for quality to determine if a host

identity could be determined. To mitigate potential laboratory contamination with human DNA, in

the case either PCR primer revealed a human (Homo sapiens) host match, the secondary PCR

was conducted to attempt to confirm the human result; only samples with human matches on

both the ‘BM’ and ‘herp’ primer sets were considered to have human as a bloodmeal host.

When different bloodmeal hosts were identified from one sample on multiple PCRs, sequences

were manually scrutinized for double nucleotide peaks, and confirmation of the multiple hosts

was attempted on the same PCR.

Statistical Analyses:

Chi-squared and Fisher’s exact tests were used to determine the relationships between T. cruzi

infection status and vector characteristics, including the collection method (kennel staff, manual

11
collections, or scent detection dog), sex, species, and sampling time. Variables were considered

possible risk factors when the p-value < 0.25 in bivariable analysis. These variables were added

to a logistic regression model to investigate the effects on the infection status of a triatomine.

Factors with values of p < 0.05 in the model output were considered significant. Odds ratios and

95% confidence intervals were calculated for each predictor. Infection prevalence in adults and

nymphs was compared using a Fisher’s exact test. A Fisher’s exact test was conducted to test

the influence of bloodmeal size on successful identification of a bloodmeal. All analyses were

conducted using R studio in Program R, version 4.3.0 (R Core Team, 2023; RStudio Team,

2019).

Results:

Triatomine demographics:

A total of 550 triatomines were collected from 10 kennels from May 2018 to September 2019,

ranging from 3 to 240 triatomines per kennel (mean=55; median=13). In total, 365 triatomines

were collected from May-July 2018, 25 were collected in December 2018, and 160 were

collected from May-September 2019 (Figure 3).

12
 250
Kennel staff
Number of triatomines collected

200 Research team

Scent detection dog
150

100

50

Dates of triatomine collections

Figure 3: The number of triatomines collected by each collector over time, which displays the

temporal and collector variation in triatomine collection over the course of this study. Eleven

triatomines collected by kennel staff during the summer/fall of 2018 are not included in this chart

because of the unknown collection date.

During manual searches by the research team in and around kennel environments, a total of

193 triatomines were collected across the first (n=167, collected May 2018 – July 2018), second

(n=24, collected December 2018), and third (n=2, collected May 2019 – September 2019) visits.

These triatomines were found in several locations including along kennel perimeters, in gutters

along the ground, nearby kennel screens, as well as in storage sheds, under wood piles and

railroad ties, or around nearby homes.

13
Kennel staff also submitted 315 triatomines throughout the year..Most triatominees were

collected during the summers and submitted to researchers at the time of the summer manual

searches (n = 159 at first visit and n = 155 at third visit, with n = 1 collected during the winter

months).

The scent detection dog/handler team visited a total of eight kennels: six in July 2018 (first visit)

and four (two repeat and two new) in August/September 2019 (third visit). They found a total of

42 triatomines from nine different localities across six kennels, including in storage areas near

kennels, in and around the kennels themselves, and under woodpiles and railroad ties within 38

meters of the kennels. Thirty-nine triatomines were collected during the first summer, and three

during the second summer. Of the 42 triatomines collected by the dog/handler team, seven

adults and 10 nymphs were found alive; 25 adults were found dead. The nymphs were located

in three separate areas.

In total, four triatomine species were identified, including 515 Triatoma gerstaeckeri, 15

Triatoma lecticularia, six Triatoma sanguisuga, and two Triatoma indictiva. All 10 nymphs and

two damaged adults were not identified to species.

T. cruzi infection:

Overall, 47.1% (74/157) of the triatomines tested using qPCR were positive for T. cruzi. Based

on the logistic regression (Tables 1 and 2), Triatoma lecticularia insects had a slightly higher

infection prevalence than other species (p-value = 0.0495, OR = 4.38, 95% CI = 1.13 - 22.31).

The submission source, sex, and time of year were not significant predictors of the level of

infection in triatomines. In total, 72 (n = 147, 49%) adults and 2 (n = 10, 20%) nymphs were

positive for T. cruzi, with no significant difference in infection prevalence (Fisher’s exact test, p-

value = 0.104).

14
Of the 74 positive insects, 64 (86.5%) were successfully strain typed to their DTU, including 41

with TcI (64.1%), 15 with TcIV (23.4%), and 8 with mixed infection of TcI/TcIV (12.5%). When

looking across species and life stages, DTU assignment was successful for 56 T. gerstaeckeri

(n=61), of which 34 (60.7%) were TcI, 14 (25%) were TcIV, and 8 (14.3%) were mixed TcI/TcIV

infections. DTU assignment was successful for 5 T. lecticularia (n = 9), of which 4 (80%) were

TcI and 1 (20%) was mixed TcI/TcIV infection. The one T. cruzi-positive T. indictiva and 2

positive nymphs were determined to be TcI. No DTU was determined for the one positive T.

sanguisuga.

Table 1: Bivariable analysis using Chi-square and Fisher’s exact tests (denoted with *) to

determine predictors of T. cruzi infection in triatomines (n = 550) collected from multi-dog kennel

environments across Texas, 2018-2019.

Number Number T. cruzi

Risk Factor collected Number tested positive (%) P-value

Collection method 0.1194

Kennel staff 315 101 52 (51)

Research team 193 34 16 (47)

Scent detection dog 42 22 6 (27)

Sex 0.0895*

Male 336 67 28 (42)

Female 197 78 43 (55)

Nymph 10 10 2 (20)

Unknown 7 1 1 (100)

Species 0.045*

15
 T. gerstaeckeri 515 127 61 (48)

T. indictiva 2 2 1 (50)

T. lecticularia 15 12 9 (75)

T. sanguisuga 6 6 1 (17)

nymph 10 10 2 (20)

Sampling time 0.2907*

Summer 2018 355 126 63 (50)

Winter 35 20 8 (40)

Summer 2019 160 11 3 (27)

Table 2: Logistic regression models to identify risk factors for T. cruzi infection in triatomines

collected from multi-dog kennel environments across Texas, 2018-2019.

Infection

Variable prevalence Odds Ratio 95% CI p-value

Submission source

Research team 47 Referent

Scent detection dog 27 0.85 0.17 - 3.82 0.8349

Kennel staff 51 1.64 0.71 - 3.93 0.2573

Sex

Female 55 Referent

Male 42 0.5 0.25 - 1.00 0.0513

Nymph 20 0.33 0.04 - 2.42 0.2894

Unknown 100 0.96 0.03 - 26.94 0.9757

Species of adults

T. gerstaeckeri 48 Referent

16
 T. indictiva 50 0.98 0.04 - 25.88 0.9863

T. lecticularia 75 4.38 1.13 - 22.31 0.0459

T. sanguisuga 17 0.29 0.01 - 2.05 0.2773

nymph 20 NA NA NA

Bloodmeal analysis:

Overall, 50/149 (33.6%) triatomines had at least one identifiable bloodmeal host (Supplemental

File 1). These insects included 48 T. gerstaeckeri and 2 T. indictiva. T. cruzi infections were

detected in 32/50 (64%) individuals. DTUs identified included TcI (n=16, 50%), TcIV (n=10,

31.2%), and mixed TcI/TcIV infections (n=3, 9.4%), with 3 (9.4%) not determined.

The majority of bloodmeal hosts (39; 73.6%) were canine (Canis lupus or Canis lupus

familiaris), with additional bloodmeal sources including five raccoons (Procyon lotor, 9.4%), two

humans (Homo sapiens, 3.8%), chickens (Gallus gallus, 3.8%), and wild pigs (Sus scrofa, 3.8%)

, and a single black vulture (Coragyps atratus, 2%), curve-billed thrasher (Toxostoma curviostre,

2%), and cat (Felis catus, 2%) (Figure 4). We also recorded 19 additional human bloodmeals

that could not be replicated on the secondary PCR, and these triatomines were not considered

to have a bloodmeal host determined and are not included in the presentation of host taxa.

Triatomines that had a higher bloodmeal score (indicating a fuller abdomen) had higher success

on bloodmeal identification (Fisher’s exact test, p-value < 0.001, Figure 5).

Two T. gerstaeckeri had more than one host identified from their hindgut samples using Sanger

sequencing methods. One triatomine had three hosts amplify in three independent PCRs using

the ‘BM’ primers with <98% matches. These matches were manually scrutinized to confirm the

presence of the identified hosts, which included chicken (Gallus gallus), wild pig (Sus scrofa),

and raccoon (Procyon lotor). The second triatomine had a human bloodmeal (Homo sapiens)

17
confirmed on both ‘BM’ and ‘herp’ PCR; however, the ‘herp’ PCR amplicons also sequenced to

dog (Canis lupus). In both cases, all host identities were included in the total bloodmeal

matches.

Figure 4: Bloodmeal results from triatomines collected at 10 multi-dog kennels across central

and south Texas, 2018-2019. The number of hosts are identified to the left of the species name

below each image. Images are from the public domain or used with permission.

18
Figure 5: Bloodmeal scoring as it relates to success identifying bloodmeal hosts in triatomines.

The higher the bloodmeal score- i.e. the more blood/gut contents in the triatomine at the time of

dissection- the greater percent success identifying the bloodmeal host using Sanger sequencing

(p-value < 0.001).

Discussion:

We characterized triatomine populations related to their locations, infection prevalence, parasite

strains, and bloodmeal hosts in kennel environments that have high densities of dogs and a

history of canine Chagas disease (Busselman et al., 2021).

Multiple collection methods are routinely used to collect triatomines during research efforts,

especially in scenarios where triatomine species are primarily sylvatic. Timed manual searches

19
are a mainstay for studies of domestic and peridomestic infestations (Dumonteil et al., 2009),

but community participation, natural/artificial shelter units, traps and other approaches are also

useful (Barros et al., 2021; Enriquez et al., 2020; Gürtler et al., 2001). Efforts to update the

distribution of triatomines in Texas have employed community science involvement as well as

manual searches, increasing the number of triatomines the research team alone could collect

(Curtis-Robles et al., 2018c; Kjos et al., 2009). Using a multimodal approach, we collected 550

triatomines during repeated visits over the course of one year. Within this study, our triatomine

collections ranged from 3 to 240 triatomines at each kennel. This variation in samples collected

from kennels could reflect our opportunistic sampling efforts, variation in the abundance of

triatomines around each kennel, the potential use of insecticides near some kennels, and

variable search efforts of kennel staff. Our collection methods focused on finding triatomines

with a high likelihood of contact with dogs. The triatomines collected by kennel staff originated

from around the dog kennels and associated areas as they were observed during the year.

Thus, the high number of triatomines found by kennel staff in the summers (n=159 and 155) and

low number in the winter (n=1) may be more reflective of the expected triatomine phenology

(Curtis-Robles et al., 2018c) than our manual searches. Across visits to the kennels, manual

search efforts were performed only opportunistically, with 167 collected at the first visit and only

2 collected at the third, with both sampling dates in the summer when triatomines are expected

to be active in our search areas.

The use of a trained scent detection dog allowed collection of 42 triatomines from habitats close

to kennels that would have otherwise been neglected in our searches. We are aware of two

prior efforts that used a trained scent detection dog to look for triatomines, including prior work

with the dog we used in the current study and foundational work with a dog in Paraguay

(Christopher et al., 2023; Rolón et al., 2011). In Paraguay, the detection dog was critical for the

confirmation of the occurrence of sylvatic populations of T. infestans and other triatomine

20
species which were found in dry branches, hollows of trees, rodent nests in standing trees, and

cut firewood; these sylvatic populations had gone undetected for decades prior (Rolón et al.,

2011). In our current study, the dog was particularly useful at kennels where the human search

efforts were low yield, perhaps reflecting a lower density of triatomines. For example, at one

kennel the only triatomines found over the course of the study (n=3) were found by the scent

detection dog; at another kennel, half of the samples (n=3 of 6) were found by the dog; and at

another kennel, 1 of the 3 bugs found was found by the scent detection dog. Additionally, all

nymphs in our study were found by the scent detection dog from areas slightly removed from

the kennels. Importantly, the bugs the dog found were in areas not apparent to the owners as

infested, and so vector control can now be performed in recognition of these cryptic vector

habitats.

We identified four triatomine species- T. lecticularia, T. gerstaeckeri, T. sanguisuga, and T.

indictiva- all of which have previously been identified in central and south Texas (Bern et al.,

2011; Curtis-Robles et al., 2018c; Kjos et al., 2009). Overall, the triatomine infection prevalence

was 47.1%, with no differences in the submission source, sex, or time of year. Similar infection

prevalences in triatomines have been reported across Texas (54%) (Curtis-Robles et al.,

2018b), in El Paso, TX (66%) (Rodriguez et al., 2021), and as high as 88% in Louisiana

(Dumonteil et al., 2020). The odds of infection in T. lecticularia were 4.38 times higher than

those in T. gerstaeckeri (p-value = 0.46, 95% CI = 1.13 - 22.31). In contrast, a prior study found

no significant difference in infection between the two species (Curtis-Robles et al., 2018b).

Further, we found a higher percent of adults were infected (49%) compared to nymphs (20%),

like the findings of (Curtis-Robles et al., 2018b); although the difference in our current study was

not statistically significant. We identified exclusively TcI and TcIV DTUs in triatomines, including

mixed TcI/TcIV infections, consistent with prior findings from Texas, with TcI infections being

most common (Curtis-Robles et al., 2018b; Rodriguez et al., 2021). Both TcI and TcIV infections

21
have been identified in dogs with systemic T. cruzi infections and clinical Chagas disease,

highlighting the risk of infection and subsequent disease to dogs with exposure to local

triatomines (Curtis-Robles et al., 2018a; Meyers et al., 2021).

One-third (50/149) of the triatomines which we subjected to the bloodmeal analysis pipeline

resulted in an identified bloodmeal host. This percent success is consistent with other

PCR/Sanger sequencing bloodmeal studies in triatomines and is likely less than 100% due to

digestion of the bloodmeal and DNA degradation leading to low amounts of host DNA relative to

triatomine DNA in our extracted sample (Curtis-Robles et al., 2018d; Kjos et al., 2013;

Rodriguez et al., 2021). Metagenomic and deep sequencing analysis pipelines, such as those

applied to characterize hosts of triatomines in Colombia (Murillo-Solano et al., 2021), are

associated with higher success in assigning hosts to individual insects. On the other hand,

RADseq-based approaches applied to triatomines in Guatemala were less successful for

bloodmeal host identification with success in only 25% of the abdomens analyzed and

contamination from human handling uniformly present, yet this approach showed great promise

for genetic characterization of the parasite, vector, and gut microbiome (Orantes et al., 2018), In

our study, we identified 53 hosts, including nine distinct species that served as hosts for

triatomines around these dog kennels. The majority of bloodmeal hosts identified (73.6%) were

Canis lupus or Canis lupus familiaris, which may be expected given the proximity of triatomine

collection to high densities of dogs and the known triatomine-dog contacts that result in a high

prevalence of canine Chagas disease in these regions (Busselman et al., 2021; Kjos et al.,

2013). T. gerstaeckeri and T. indictiva have been known to feed on canine hosts and have been

collected around dog kennels (Gorchakov et al., 2016; Kjos et al., 2013). This result supports

the hypothesis that dogs are an important bloodmeal host for triatomines in kennel

environments. Even if the triatomines are found dead, they are getting the opportunity to feed on

dogs prior to death, potentially increasing the risk a dog would be exposed to T. cruzi during the

22
process. Similarly, dogs are recognized as critically important in maintaining T. cruzi infections

and feeding triatomines in northern Argentina (Gurtler et al., 1991) and in Mexico (Estrada-

Franco et al., 2006); in both areas dogs are sentinels for human health risk of Chagas disease.

In contrast, despite the widespread availability of dogs in two Chagas endemic regions of

Ecuador, humans, rodents, and chickens predominated as triatomine hosts whereas dogs were

blood sources for only 5 of 416 (1.2%) of triatomines for which the host was determined (Ocaña-

Mayorga et al., 2021). In a region of diverse triatomine species in Colombia, dogs were the

second most commonly used host behind humans, with domestic pigs and wildlife including

squirrels, opossums, porcupines and anteaters and high levels of host switching common

among the triatomines (Murillo-Solano et al., 2021). The degree to which dogs are used by

triatomines likely depends not only on the availability of dogs in the habitats under study, but

also the triatomine species, relative abundance of other potential hosts, and other biotic and

abiotic factors.

Chickens were identified as hosts (3.8%) and have previously been reported in studies of

bloodmeals of triatomines in the USA (Curtis-Robles et al., 2018d; Dumonteil et al., 2020;

Stevens et al., 2012). Although anecdotally some kennel owners keep chickens on their

property with the assumption that the chickens will eat triatomines, thereby lowering disease risk

to dogs, it is evident that chickens also provide bloodmeals to vectors that survive. The

presence of these additional hosts in the ecosystem may support a higher carrying capacity of

the vector population. In domestic transmission settings, chickens appear to play a complex role

in increasing or decreasing the risk of T. cruzi infection to humans (Gürtler et al., 2014;

Ordóñez-Krasnowski et al., 2020; Zahid and Kribs, 2020). Additionally, we report triatomine

bloodmeal hosts in these kennel settings include raccoon, black vulture, cat, wild pig, humans,

and the curve-billed thrasher, the latter of which has not previously been implicated as a host of

triatomines. The curve-billed thrashers range extends across the Southwestern United States,

23
from Arizona to central Texas and further south into Mexico (Rojas-Soto, 2003). They often

build their nests in dense vegetation (Fischer, 1980); it is in these locations they may be most

likely to interact with a nest-dwelling or dispersing triatomine. This further indicates the broad

hosts and environments from which triatomines feed.

We found that bloodmeal scoring was associated with the success of triatomine host

identification, which is in line with previous work (Curtis-Robles et al., 2018d; Kjos et al., 2013).

One limitation of Sanger sequencing is that it often allows detection of only one bloodmeal host.

This contrasts with other bloodmeal techniques, such as next generation sequencing, which can

routinely detect multiple hosts in individual insects, if present (Balasubramanian et al., 2022;

Dumonteil et al., 2018; San Juan et al., 2023). Even while using Sanger sequencing, we

identified multiple hosts (chicken, wild pig, and raccoon in one triatomine, and human and dog in

another) in individual triatomines, adding to our understanding of the multiple hosts used to

sustain triatomine populations and host sharing that occurs within a single triatomine’s life cycle.

Pest management efforts varied across kennels, including the use of environmental insecticides

and vegetation removal around kennels, although systematic data on owner-reported pest

management were not collected. Further, the use of common heartworm/flea/tick preventatives

on dogs may have an added consequence of killing triatomines, as has been shown for

triatomine species found in South America (Laiño et al., 2019; Loza et al., 2017; Reithinger et

al., 2005) and recently for T. gerstaeckeri (the most common species in our study) exposed to

blood from treated dogs (Busselman et al., 2023). The high proportion of dog-feeding and

findings of many dead triatomines in this study suggests that systemic insecticides given to

dogs may be effective in killing triatomines in the local environment, although migration from

sylvatic settings and the feeding on non-dog hosts may modulate the efficacy of suppressing the

vector population.

24
Understanding triatomine populations and their host associations around dog kennels can help

us improve vector control measures to reduce the risk of disease in dogs and detect patterns

that would be translatable to other ecological settings. Detecting wildlife hosts that are

sustaining populations of triatomines and possibly contributing to T. cruzi transmission around

dog kennels may allow host-targeted intervention. As dogs play such a large role in serving as a

source of bloodmeals for triatomines in these kennel environments, dog-targeted vector control

interventions may allow us to interrupt transmission cycles of disease in a meaningful way to

decrease the risk of disease to dogs (Fiatsonu et al., 2023; Gürtler et al., 2022; Laiño et al.,

2021).

Acknowledgements

We thank Ester Carbajal and summer research students Hannah Meyers, Andy Castro, and

Becky Dowd for assistance in triatomine collections and molecular processing. We are grateful

for the support and collaboration with kennel owners and staff for allowing us access to their

facilities, dogs, and for collecting triatomines throughout the year. American Kennel Club Canine

Health Foundation Grant No 02448, AgriLife Research Insect Vector grant program, and the

Harry Willett Foundation provided funding. The funders had no role in study design, data

collection and analysis, decision to publish, or preparation of the manuscript.

CREDIT Statement:

Busselman RE: Data curation, Formal analysis, Investigation, Project administration,

Visualization, Writing - original draft, Writing - review & editing; Curtis-Robles R:

Conceptualization, Funding acquisition, Investigation, Visualization, Writing - review & editing;

Meyers AC: Conceptualization, Funding acquisition, Investigation, Writing - review & editing;

Zecca IB: Conceptualization, Investigation, Writing - review & editing; Auckland LD: Data

25
curation, Investigation, Writing - review & editing; Hodo CL: Conceptualization, Funding

acquisition Investigation, Writing - review & editing; Christopher D: Conceptualization, Data

curation, Investigation, Methodology, Validation, Writing - review & editing; Saunders AB:

Funding acquisition, Investigation, Supervision, Writing - review & editing; Hamer SA:

Conceptualization, Funding acquisition, Investigation, Project administration, Resources,

Supervision, Writing - review & editing.

26
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Graphical abstract

CREDIT Statement:

Busselman RE: Data curation, Formal analysis, Investigation, Project administration,

Visualization, Writing - original draft, Writing - review & editing; Curtis-Robles R:

Conceptualization, Funding acquisition, Investigation, Visualization, Writing - review & editing;

Meyers AC: Conceptualization, Funding acquisition, Investigation, Writing - review & editing;

Zecca IB: Conceptualization, Investigation, Writing - review & editing; Auckland LD: Data

curation, Investigation, Writing - review & editing; Hodo CL: Conceptualization, Funding

acquisition Investigation, Writing - review & editing; Christopher D: Conceptualization, Data

curation, Investigation, Methodology, Validation, Writing - review & editing; Saunders AB:

Funding acquisition, Investigation, Supervision, Writing - review & editing; Hamer SA:

Conceptualization, Funding acquisition, Investigation, Project administration, Resources,

Supervision, Writing - review & editing.

40
Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:

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