First Record of an Established Population of Triatoma sanguisuga (Hemiptera: Reduviidae) in Richardson County, Nebraska
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Journal of Medical Entomology, 58(6), 2021, 2519–2523
doi: 10.1093/jme/tjab122
Advance Access Publication Date: 20 July 2021
Short Communication
First Record of an Established Population of Triatoma
sanguisuga (Hemiptera: Reduviidae) in Richardson
County, Nebraska
David H. Nielsen,1,2,4, Kyle Koch,3 Walter Roachell,1 Bernardo Delgado,1 and
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Joshua Bast1
1
Entomological Sciences Division, Public Health Command-Central, Ft. Sam Houston, TX 78234, USA, 2Entomolgy Program,
Nebraska Department of Agriculture, 301 Centennial Mall South, Lincoln, NE 68509-4756, USA, 3Entomology Department, University
of Nebraska-Lincoln, Lincoln, NE 68583-0816, USA, and 4Corresponding author, e-mail: david.h.nielsen.mil@mail.mil
Subject Editor: Gabriel Hamer
Received 12 April 2021; Editorial decision 9 June 2021
Abstract
In 2017, a single adult Triatoma sanguisuga (LeConte) (Hemiptera: Reduviidae) was identified from Indian Cave
State Park in Nemaha County, NE. The following summer, a single adult specimen was found in a 4-H insect col-
lection at the Nebraska State Fair from Richardson County. A subsequent investigation found that these were
collected from a residence in Richardson County and three more adults were collected in 2019. In 2020, the latter
three kissing bugs were submitted to Public Health Command-Central’s DoD Food Analysis and Diagnostic
Laboratory (DOD FADL) for confirmatory species identification and diagnostic testing for Trypanosoma cruzi,
the causative agent of Chagas disease. One specimen tested positive for T. cruzi using a real-time dual-target
PCR screen followed by confirmatory dual-target traditional PCR. Based on these findings, a survey plan was
developed for Richardson and surrounding counties in southeast Nebraska. In July of 2020, two adult and
seven nymphs (multiple instars) of T. sanguisuga were collected in Richardson County, one of which tested
positive for T. cruzi. This is the first record of an established population of T. sanguisuga and T. cruzi-infected
kissing bugs in Nebraska.
Key words: Nebraska, Triatoma sanguisuga, Chagas disease, Trypanosoma cruzi, kissing bug
Triatoma sanguisuga (LeConte) (Hemiptera: Reduviidae), the collected in Alabama, Florida, Georgia, Indiana, Kansas, Louisiana,
eastern conenose bug, is a member of the Triatominae subfamily, Mississippi, Missouri, Oklahoma, Tennessee, Texas, and Virginia
commonly known as kissing bugs. Triatoma sanguisuga are widely (Bern et al. 2011, 2020).
distributed in Central America and the southern United States. Survey work since 2012 conducted by the authors at Public
Surveys conducted in the 1940s recorded T. sanguisuga as far Health Command-Central (PHC-C) as well as other researchers
north as Kansas (Grundemann 1947) as well as many other states (Sarkar et al. 2010) has determined that Triatomine sanguisuga
in the southeast of the United States (Usinger 1944). Triatoma and T. cruzi are widespread across Texas and many surrounding
sanguisuga is a vector of Trypanosoma cruzi, the causative agent states. A PHC-C survey of Fort Riley, KS in 2018 found numerous
of Chagas disease. Trypanosoma cruzi is thought to have been in T. sanguisuga, some of which were positive for T. cruzi (unpublished
South America for millions of years, first appearing as a variant in data). To our knowledge, this was the first Triatomine survey con-
bats (Steverding 2014). DNA testing of mummified remains shows ducted in Kansas since the 1940s, although a single T. sanguisuga
human infection of T. cruzi as far back as 4,500 years ago in the was submitted to Texas A&M for a study conducted from 2012 to
Andean region of South America (Steverding 2014). Traditionally, 2016 (Curtis-Robles et al. 2018). More recently, new state records
Chagas disease has been considered a public health issue in Central for T. sanguisuga have also been reported in Wyoming (Reeves
and South America only, though studies to determine if Chagas and Miller 2020) and Delaware (Eggers et al. 2019). Interestingly,
is present in the United States have been ongoing since the mid- the T. sanguisuga specimen from Wyoming was collected in 2000,
20th century. Triatoma sanguisuga infected with T. cruzi have been but remained unreported in a collection until examined by the
Published by Oxford University Press on behalf of Entomological Society of America 2021. This work is written by (a) US 2519
Government employee(s) and is in the public domain in the US.
2520 Journal of Medical Entomology, 2021, Vol. 58, No. 6
authors (Reeves and Miller 2020). Furthermore, while few cases crops, ornamentals, structural and aquatic environments, as well
of locally transmitted T. cruzi infection have been documented in as humans, livestock, and pets. All citizen-collected specimens sub-
the United States (Montgomery et al. 2016), a likely case of lo- mitted prior to the 2020 field surveys were identified within the reg-
cally acquired Chagas disease was reported in Missouri in 2018 ular activities of the PPDC.
(Turabelidze et al. 2020).
Prior to 2017, T. sanguisuga had not been detected in
Nebraska. The first report was identified from an image of an Field Surveys
adult taken at Indian Cave State Park and sent to the University Surveys for T. sanguisuga were conducted the last week of July
of Nebraska-Lincoln (UNL) Entomology Department for identi- of 2020 and covered eight counties. Locations selected were state
fication. In 2018, another adult T. sanguisuga was identified by a parks, state recreation areas, and wildlife management areas.
UNL entomologist from an entry at the Nebraska State Fair. A 4-H Sites selected for survey included; Verdun State Recreation Area
entomology collection submitted by Willa Fritz from Richardson (SRA), Richardson County; Indian Cave State Park, Nemaha
County contained the specimen. This specimen was later sub- County; Riverside SRA, Otoe County, Table Rock Wildlife
mitted for diagnostic testing at Oklahoma State University for Management Area (WMA), Pawnee County; Gifford Point
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T. cruzi and tested negative. In 2019, Willa Fritz collected three WMA, Sarpy County; Randall W. Schilling WMA, Cass County;
more adults from their home in Richardson County. These Peru Bottoms WMA, Nemaha County; Rock Creek Station
were submitted to UNL for identification and retained with the SRA, Jefferson County; Bluestem WMA, Lancaster County; and
Entomology Department. In 2020, these specimens were sent to Wilderness Park, Lancaster County. Survey counties are high-
the PHC-C Food Analysis and Diagnostic Laboratory (FADL) for lighted on the map in Fig. 1. Sites were selected based on sim-
confirmatory identification and pathogen testing. One specimen ilar habitat found at Indian Cave State Park since this location
was positive for T. cruzi by PCR using a real-time dual-target had a previous record of T. sanguisuga. Indian Cave State Park
PCR (Qvarnstrom et al. 2012) screen followed by dual-target is located along the floodplain of the Missouri River along the
confirmatory traditional PCR (Reisenman et al. 2010). Based eastern border of Nebraska. Indian Cave State Park is comprised
on these findings, a survey plan was developed in the summer of of mixed tall grass prairie and contain large tracts of mixed
2020 to determine if T. sanguisuga populations were established hardwood species comprised primarily of cottonwood (Populus
in southeast Nebraska and confirm enzootic circulation of T. cruzi deltoids), oak (Quercus spp.), and eastern red cedar (Juniperus
parasites. virginiana).
Triatoma sanguisuga surveys involved systematic deconstruction
Materials and Methods of rotting logs and rodent nests found at survey sites (McPhatter
et al. 2012). Survey time per location averaged 3 h and the exami-
Diagnostic Services nation of up to 15 decomposing logs. Logs were deconstructed with
The UNL Plant and Pest Diagnostic Clinic (PPDC) is operated by the use of a small pickaxe and hand removal of separated sections
Nebraska Extension and provides diagnostic support for numerous of logs. Each piece was examined for kissing bugs and further de-
groups and individuals, including: the general public, area extension constructed if a bug was present. Collected specimens were placed
personnel, private companies, agricultural producers, pest control in plastic zipper lock bags labeled with location and collection date.
professionals, and medical professionals. Entomological samples are Specimens were placed in a cooler for transport and stored in a
submitted from a variety of sources, including field and horticulture freezer at 0°F prior to shipment to Texas.
Fig. 1. Map of surveyed counties in Nebraska. Richardson county is the the most southwest county.
Journal of Medical Entomology, 2021, Vol. 58, No. 6 2521
Triatomine Identification survey. Factors to include rain and lack of suitable decomposing logs
Triatoma sanguisuga were identified using morphological keys or rodent nests may have contributed to not finding T. sanguisuga at
(Lent and Wygodzinsky 1979). All specimens were identified to other locations during this survey.
species and life stage at 100× magnification under a stereo zoom One of the three bugs from 2019 and eight of nine of the
microscope (Leica S9i, Leica Microsystems, Wetzlar, Germany). kissing bugs from 2020 exhibited positive Ct values in the ini-
Voucher specimens of T. sanguisuga are kept with the FADL at Ft. tial screen; however, only one of three (C-E20-005-001) and one
Sam Houston, TX. of nine bugs (C-E20-0352-002), respectively, were confirmed for
T. cruzi following the traditional PCR assay. Results have been
summarized in Table 1 below. All NTC, EC, and positive controls
Laboratory Analysis
were analyzed and met acceptance criteria for each RT–PCR ABI
Laboratory detection of T. cruzi-infected kissing bugs was performed
7500 DX run.
using a combination of real-time (Qvarnstrom et al. 2012) and tra-
ditional PCR (Reisenman et al. 2010). After receiving the midgut
portion of the kissing bug, 800 µl of Dulbecco's phosphate-buffered Discussion
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saline was added to the sample tube with one BB. Homogenization
Prior to 2020, only four adult T. sanguisuga have been collected
of the kissing bug was accomplished using a tissue lyser set to 8 min
in Nebraska. Based on the collection of adults over multiple
with a frequency of 20 cycles per second, followed by DNA extrac-
years (2018–2020) and a collection of both adults and nymphs
tion using the QIAGEN DNEasy Blood and Tissue Spin Protocol
at varying stages of development, we believe that there is an es-
(Qiagen DNeasy Blood and Tissue Handbook). The eluate was
tablished population of T. sanguisuga in Richardson County, NE.
screened for the presence of T. cruzi by two satellite DNA (Cruzi
Considering Richardson County borders Missouri and Kansas, a
1F/2R/3P) and kDNA (32F/148R/71P) (Qvarnstrom et al. 2012)
state that has records of T. sanguisuga and T. cruzi dating back
real-time primer sets. Presumptive positive samples underwent a con-
decades, their discovery is not surprising. The detection of T. cruzi
firmatory traditional PCR protocol, followed by gel electrophoresis.
in kissing bugs from this survey and from previous submissions
A positive sample is defined by evidence of both 330 bp (S35/S36)
is a public health concern that needs to be investigated. Published
and 188 bp (TCZ1/TCZ2) traditional PCR products (Reisenman
records of T. sanguisuga collections in other states (Grundemann
et al. 2010). Trypanosoma cruzi positive control (PC), no template
et al. 1947, Reisenman et al. 2010, Eggers et al. 2019, Reeves
control (NTC), and extraction controls (EC) were included to assess
and Miller 2020, Turabelidze et al. 2020) show that when
RT–PCR run acceptability.
T. sanquisuga is present, T. cruzi is as well. Based on this, there is
the possibility residents of Richardson County have been exposed
to T. cruzi for decades, since there is no evidence to suggest a re-
Results
cent arrival of T. sanguisuga in Nebraska.
Nine T. sanguisuga (adults (2); fourth instar (4); third instar (1); Further statewide surveillance for T. sanguisuga and T. cruzi
second instar (2)) were collected from deconstructed logs (Fig. 2) to characterize the distribution and risk of Chagas disease are
at Verdon SRA (Fig. 3) in Richardson County during the 2020 field warranted.
Fig. 2. Kissing bug nymph from deconstructed log at Verdon Lake State Recreation Area, Nebraska.2522 Journal of Medical Entomology, 2021, Vol. 58, No. 6
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Fig. 3. Map of Verdon Lake State Recreation Area, Nebraska. Location of kissing bug collection is northeast between Verdon Lake and the Big Nemaha River.
Table 1. Molecular results of Trypanosoma cruzi from Triatoma sanquisuga collected in Nebraska
Sample accession # Initial dual RT–PCR Ct S35/S36 gel band TCZ1/TCZ2 gel band
C-E20-0005-001 22.01/18.72 Present Present
C-E20-0005-002 Undet. Not present Not present
C-E20-0005-003 Undet. Not present Not present
C-E20-0352-001 27.04/22.74 Present (no match) Present (multiple)
C-E20-0352-002 15.14/16.20 Present Present
C-E20-0352-003 36.35/32.81 Present (weak) Not present
C-E20-0352-004 32.55/29.61 Present Not present
C-E20-0352-005 32.32/27.53 Present Not present
C-E20-0352-006 35.41/34.13 Not present Not present
C-E20-0352-007 36.70/31.66 Present (weak) Not present
C-E20-0352-008 Undet./Undet. Not tested Not tested
C-E20-0352-009 36.20/32.30 Present (weak) Not present
Acknowledgments Bern, C., L. A. Messenger, J. D. Whitman, and J. H. Maguire. 2020. Chagas
disease in the United States: a public health approach. Clin. Microbiol.
We would like to thank the PHC-C Commander and FADL Director for
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providing diagnostic testing and support for this field survey. We would
Curtis-Robles, R., S. A. Hamer, S. Lane, M. Z. Levy, and G. L. Hamer. 2018.
also like to thank the Fritz family for communicating with authors and
Bionomics and spatial distribution of Triatomine vectors of Trypanosoma cruzi
their efforts to collect specimen. Funding and collaboration for this pro-
in Texas and other southern states, USA. Am. J. Trop. Med. Hyg. 98: 113–121.
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Eggers, P., T. N. Offutt-Powell, K. Lopez, S. P. Montgomery, and G. G. Lawrence.
Diagnostic Laboratory, the University of Nebraska-Lincoln, and the
2019. Notes from the field: identification of a Triatoma sanguisuga “kissing
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