Prospecting for Zoonotic Pathogens by Using Targeted DNA

any serious human pathogens result from zoo-

notic transmission, including 61% of known hu-

man pathogens and 75% of emerging human pathogens

(1). For example, rabies virus is transmitted by saliva

of infected animals (2). The plague bacteria (Yersina

pestis), the causative agent of the largest documented

pandemic in human history that reduced the popula-

tion of Europe by 30%–50%, was transmitted from rats

to humans by eas (3). Other zoonoses include Ebola

virus (4), tularemia (Francisella tularensis) (5), and tu-

berculosis (6). The SARS-CoV-2 pandemic, thought to

have a bat reservoir, has stimulated renewed emphasis

on zoonotic pathogen surveillance (7,8).

Natural history museums are repositories of bio-

logic information in the form of voucher specimens

that represent a major, underused resource for

studying zoonotic pathogens (9–13). Originally,

specimens were archived as dried skin and skel-

etal vouchers or preserved in uids (ethanol) after

xation with formalin or formaldehyde. Now, best

practices include preserving specimens and associ-

ated soft tissues in liquid nitrogen (−190°C) or me-

chanical freezers (−80°C) from the time they are col-

lected (14). Those advances in preservation make it

possible to extract high-quality DNA and RNA that

can be used for pathogen surveillance. For example,

retroactive sampling of archived tissues from the

US Southwest found that Sin Nombre virus, a New

World hantavirus, was circulating in wild rodent

populations almost 20 years before the rst human

cases were reported (15).

It is critical to develop a range of tools for extract-

ing pathogen information from museum-archived

samples. Targeted sequencing using probe enrichment

has become the tool of choice for medical genomics

(16), population genetics (17), phylogenetics (18), and

ancient DNA (19,20). Those methods are designed

to enrich small amounts of DNA target from a back-

ground of contaminating DNA. Probe-based, targeted

sequencing has been used to enrich pathogens from

complex host–pathogen DNA mixtures (21). For exam-

ple, Keller et al. used probes to capture and sequence

complete Y. pestis genomes from burial sites >1,500

years old (22). Enrichment is frequently achieved by

designing a panel of probes to specically target a

handful of pathogens of interest (23,24). Similarly,

commercial probe sets are available for many types

of viruses and human pathogens (23–25). However,

many of these probe sets are limited to specic patho-

gens that might not infect other host species.

Our goal was to develop a panel of biotinylated

baits, or probes, to identify the eukaryotic and bac-

terial pathogens responsible for 32 major zoonoses

(Table 1). We aimed to capture both known and relat-

ed pathogens, using the fact that probes can capture

sequences that are ≤10% divergent. To perform this

Prospecting for Zoonotic Pathogens

by Using Targeted DNA Enrichment

Egie E. Enabulele, Winka Le Clec’h, Emma K. Roberts, Cody W. Thompson, Molly M. McDonough,

Adam W. Ferguson, Robert D. Bradley, Timothy J. C. Anderson, Roy N. Platt II

1566 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 29, No. 8, August 2023

RESEARCH

Author aliations: Texas Biomedical Research Institute,

San Antonio, Texas, USA (E.E. Enabulele, W. Le Clec’h,

T.J.C. Anderson, R.N. Platt II); Texas Tech University, Lubbock,

Texas, USA (E.K. Roberts, R.D. Bradley); University of Michigan,

Ann Arbor, Michigan, USA (C.W. Thompson); Chicago State

University, Chicago, Illinois, USA (M.M. McDonough); Field

Museum of Natural History, Chicago (A.W. Ferguson)

DOI: https://doi.org/10.3201/eid2908.221818

More than 60 zoonoses are linked to small mammals,

including some of the most devastating pathogens in

human history. Millions of museum-archived tissues are

available to understand natural history of those patho-

gens. Our goal was to maximize the value of museum

collections for pathogen-based research by using target-

ed sequence capture. We generated a probe panel that

includes 39,916 80-bp RNA probes targeting 32 patho-

gen groups, including bacteria, helminths, fungi, and

protozoans. Laboratory-generated, mock-control sam-

ples showed that we are capable of enriching targeted

loci from pathogen DNA 2,882‒6,746-fold. We identied

bacterial species in museum-archived samples, includ-

ing Bartonella, a known human zoonosis. These results

showed that probe-based enrichment of pathogens is a

highly customizable and ecient method for identifying

pathogens from museum-archived tissues.

Prospecting Pathogens by Targeted DNA Enrichment

capture, we used a modied version of the ultracon-

served element (UCE) targeted sequencing technique

(26,27) to specically enrich pathogen DNA. Biotinyl-

ated baits are designed to target conserved genomic

regions among diverse groups of pathogens (Figure

1). The baits are hybridized to a library potentially

containing pathogen DNA. Bait-bound DNA frag-

ments are enriched during a magnetic bead purica-

tion step before sequencing (Figure 2). The nal li-

brary contains hundreds or thousands of orthologous

loci with single-nucleotide variants or indels from the

targeted pathogen groups that can then be used for

population or phylogenetic analyses.

Methods

We have compiled a detailed description of the meth-

ods used (Appendix 1, https://wwwnc.cdc.gov/

EID/article/29/8/22-1818-App1.pdf; https://doi.

org/10.17504/ protocols.io.5jyl8jnzrg2w/v1). Code is

available on GitHub (https://www.github.com/neal-

platt/pathogen_probes; https://doi.org/10.5281/ze-

nodo.7319915). Raw sequence data are available from

the National Center for Biotechnology Information

(BioProject PRJNA901509; Appendix 2, https://

wwwnc.cdc.gov/EID/article/29/8/22-1818-App2.

xlsx). A summary of our methods follows.

Panel Development

We developed a panel of baits for targeted sequenc-

ing of 32 zoonotic pathogens. To develop this pan-

el, we used the Phyluce version 1.7.1 (26,27) proto-

col to design baits for conserved loci within each

pathogen group. First, we simulated and mapped

reads from each species within a pathogen group

to a focal genome assembly (Table 1; Figure 1,

panel A). We used the mapped reads to identify

putative orthologous loci that were >80% simi-

lar across the group and generated in silico baits

from the focal genome (Figure 1, panel B). These

baits were mapped back to each member (Figure

1, panel C) to identify single-copy orthologs within

the group. Next, we designed 2 overlapping 80-bp

baits from loci in each member of the group (Figure

1, panel D) and removed baits with >95% sequence

similarity (Figure 1, panel E). We repeated those

steps for each pathogen group (Figure 1, panel

F). We compared the remaining baits with mam-

malian genomes and replaced them to minimize

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 29, No. 8, August 2023 1567

Table 1. Zoonotic pathogens targeted for DNA enrichment in study of prospecting for zoonotic pathogens by using targeted DNA

enrichment

Pathogen group

Taxonomic level

Focal pathogen

Zoonoses

Anaplasma

Genus

Anaplasma phagocytophilum

Anaplasmosis

Apicomplexa

Phylum

Plasmodium falciparum

Malaria

Bacillus cereus group*

Species group

Bacillus anthracis

Anthrax

Bartonella

Genus

Bartonella bacilliformis

Cat-scratch fever

Borrelia

Genus

Borrelia burgdorferi

Lyme disease

Burkholderia

Genus

Burkholderia mallei

Glanders

Campylobacter

Genus

Campylobacter jejuni

Campylobacteriosis

Cestoda

Class

Taenia multiceps

Taeniasis

Chlamydia

Genus

Chlamydia trachomatis

Chlamydia

Coxiella

Genus

Coxiella burnetii

Q fever

Ehrlichia

Genus

Ehrlichia canis

Ehrlichiosis

Eurotiales

Order

Talaromyces marneffei

Talaromycosis

Francisella

Genus

Francisella tularensis

Tularemia

Hexamitidae

Family

Giardia intestinalis

Giardiasis

Kinetoplastea

Class

Leishmania major

Leishmaniasis

Leptospira

Genus

Leptospira interrogans

Leptospirosis

Listeria

Genus

Listeria monocytogenes

Listeriaosis

Mycobacterium

Genus

Mycobacterium tuberculosis

Tuberculosis

Nematodes (clade I)

Phylum (clade)

Trichinella spiralis

Trichinosis

Nematodes (clade III)

Phylum (clade)

Brugia malayi

Filariasis

Nematodes (clade IVa)

Phylum (clade)

Strongyloides stercoralis

Strongyloidiasis

Nematodes (clade IVb)

Phylum (clade)

Steinernema carpocapsae

None

Nematodes (clade V)

Phylum (clade)

Haemonchus contortus

None

Onygenales

Order

Histoplasma capsulatum

Histoplasmosis

Pasteurella

Genus

Pasteurella multocida

Pasteurellosis

Rickettsia

Genus

Rickettsia rickettsii

Typhus

Salmonella

Genus

Salmonella enterica

Salmonellosis

Streptobacillus

Genus

Streptobacillus moniliformis

Rat-bite fever

Trematoda

Class

Schistosoma mansoni

Schistosomiasis

Tremellales

Order

Cryptococcus neoformans

Cryptococcosis

Trypanosoma*

Genus

Trypanosoma cruzi

Sleeping sickness

Yersinia

Genus

Yersinia pestis

Plague

*Supplemented with additional probes/baits.

RESEARCH

cross-reactivity with the host. Finally, we combined

baits to capture 49 loci from each pathogen group

into a panel that was synthesized by Daicel Arbor

Biosciences (https://arborbiosci.com).

Museum-Archived and Control Samples

We extracted DNA from 38 museum samples by

using the DNeasy Kit (QIAGEN, https://www.qia-

gen.com) (Table 2). We generated control samples

1568 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 29, No. 8, August 2023

Figure 1. Probe panel design for

study of prospecting for zoonotic

pathogens by using targeted

DNA enrichment. A) Simulated

reads from each pathogen within

a group were mapped back to

a single focal genome. B) We

identied regions with consistent

coverage from each member of

the pathogen group to identify

putative, orthologous loci and

generated a set of in silico probes

from the focal genome. C) Those

in silico probes were then mapped

back to the genomes of each

member in the pathogen group

to nd single copy, orthologous

regions, present in most

members. D, E) We designed

2 overlapping 80-bp baits to

target the loci in each member

of the pathogen group (D) and

compared them with each another

to remove highly similar probes

(E). One probe was retained from

each group of probes with high

sequence similarity (>95%). F) We

identied the probes necessary to

capture 49 loci in that pathogen

group. This process was repeated

for the next pathogen group. Finally, all probes were combined together into a single panel. Chr, chromosome; Sp, specimen.

Figure 2. Targeted DNA

enrichment workow for study

of prospecting for zoonotic

pathogens by using targeted

DNA enrichment.

A) Genomic DNA extracted

using the DNeasy Kit

(QIAGEN, https://www.qiagen.

com). B) Next-generation

sequencing libraries prepared

using KAPA Hyperplus Kit

(https://www.biocompare.

com) and barcoding each

library with IDT xGen Stubby

Adaptor-UDI Primers (https://

www.idtdna.com). C) RNA

probes hybridization using

the high sensitivity protocol

of myBaits version 5. (https://

arborbiosci.com). D) Probes

bound to streptavidin-

coated magnetic beads and

sequestered with a magnet (E)

15 cycles PCR amplication of

enriched libraries. F) Libraries sequenced on an Illumina Hi-Seq 2500 platform (https://www.illumina.com).

Prospecting Pathogens by Targeted DNA Enrichment

by spiking naive mouse DNA with 1% microorga-

mism DNA from Mycobacterium bovis, M. tuberculo-

sis, Plasmodium vivax, P. falciparum, and Schistosoma

mansoni. We then further diluted an aliquot of this

1% pathogen mixture into mouse DNA to create

a 0.001% host–pathogen mixture. This range was

designed to test the lower limits of detection but also

represent a reasonable host–pathogen proportion.

For example, Theileria parva, a tick-transmitted

apicomplexan, is present in samples from 0.9%

through 3% (28), and 1.5% of DNA sequence reads

in clinical blood samples is from P. vivax (29).

Library Preparation

We generated standard DNA sequencing librar-

ies from 500 ng of DNA per sample. We combined

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 29, No. 8, August 2023 1569

Table 2. Specimens examined using targeted sequencing in study of prospecting for zoonotic pathogens by using targeted DNA

enrichment*

Museum

accession no.

Source species (common name)

Locality, country: state, county

Date

SRA ID

TK48533

Myotis volans (long-legged myotis)

Mexico: Durango, Arroyo El Triguero

1995 May 18

SAMN31718202

TK49668

Didelphis virginiana (Virginia opossum)

United States: Texas, Kerr

1996 May 14

SAMN31718203

TK49674

Peromyscus attwateri (Texas mouse)

United States: Texas, Kerr

1996 May 14

SAMN31718204

TK49686

Peromyscus laceianus (deer mouse)

United States: Texas, Kerr

1996 May 14

SAMN31718205

TK49712

Dasypus novemcinctus (nine-banded

armadillo)

United States: Texas, Kerr

1996 May 16

SAMN31718206

TK49732

Lasiurus borealis (eastern red bat)

United States: Texas, Kerr

1996 May 17

SAMN31718207

TK49733

Myotis velifer (vesper bat)

United States: Texas, Kerr

1996 May 16

SAMN31718208

TK57832

P. attwateri

United States: Texas, Kerr

1997 May 14

SAMN31718209

TK70836

Desmodus rotundus (common vampire

bat)

Mexico: Durango, San Juan de

Camarones

1997 Jun 27

SAMN31718210

TK90542

Sigmodon hirsutus (southern cotton rat)

Mexico: Chiapas, Comitán

1999 Jul 9

SAMN31718211

TK93223

Peromyscus melanophrys (plateau

mouse)

Mexico: Oaxaca, Las Minas

2000 Jul 13

SAMN31718212

TK93289

Carollia subrufa (gray short-tailed bat)

Mexico: Chiapas, Ocozocoautla

2000 Jul 16

SAMN31718213

TK93402

Chaetodipus eremicus (Chihuahan

pocket mouse)

Mexico: Coahuila

2000 Jul 22

SAMN31718214

TK101275

Glossophaga commissarisi

(Commissaris’ long-tongued bat)

Honduras: Comayagua, Playitas

2001 Jul 10

SAMN31718215

TK136205

Heteromys desmarestianus

(Desrmarest’s spiny pocket mouse)

Honduras: Atlantida, Jardin Botanico

Lancetilla

2004 Jul 16

SAMN31718216

TK136222

Peromyscus mexicanus (Mexican deer

mouse)

Honduras: Colon, Trujillo

2004 Jul 17

SAMN31718217

TK136228

H. desmarestianus

Honduras: Colon, Trujillo

2004 Jul 17

SAMN31718218

TK136240

Glossophaga soricine (Pallas’s long-

tongued bat)

Honduras: Colon, Trujillo

2004 Jul 16

SAMN31718219

TK136756

Eptesicus furinalis (Argentine brown

bat)

Honduras: Colon, Trujillo

2004 Jul 17

SAMN31718220

TK136783

Glossophaga leachii (gray long-tongued

bat)

Honduras: Colon, Trujillo

2004 Jul 17

SAMN31718221

TK148935

Rhogeessa tumida (back-winged little

yellow bat)

Mexico: Tamaulipas, Soto la Marina

2008 Jul 27

SAMN31718222

TK148943

M. velifer

Mexico: Tamaulipas, Soto la Marina

2008 Jul 27

SAMN31718223

TK150290

Balantiopteryx plicata (gray sac-winged

bat)

Mexico: Michoacan, El Marqués

2006 Jul 22

SAMN31718224

TK154677

Gerbilliscus leucogaster (bushveld

gerbil)