Am, J, Trop, Med. Hyg., 79(5). 2008. pp.

799-802
Copyright 2008 by The American Society of Tropical Medicine and Hygiene

Serologie Survey of the Sentinel Animals for Plague Surveillance and Screening for
Complementary Diagnostic Markers to Fl Antigen by Protein Microarray
Bei Li,t Ying Guo,t Zhaobiao Guo, Yun Liang, Ziwen Zhu, Qing Zhou, Yanfeng Yan, Zhizhong Song,* and
Ruifu Yang*
Yunnan Institute for Epidemic Diseases Controi and Research, Dali, Yunnan Province, China; Laboratory of Analytical
Microbiology, State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Beijing, China;
YunYang Medical College, Shiyan, Hubei Province, China

Abstract. Plague is a deadly infectious disease caused by the gram-negative bacterium, Yersinia pestis. In 2005, five
plague patients were confirmed in the Yulong County of the Yunnan Province, China. In this study, the srologie survey
of > 2,900 serum samples from domestic dogs and cats in and around the county, where human plague occurred,
confirmed that domestic dogs and cats could serve as sentinel animals for plague surveillance. Meanwhile, the antibody
responses in the infected dogs and cats were profiled by microarray containing 218 proteins of Y. pestis. In addition to
Fl, LcrV, YPCD1.28C, and YPO2118 induced humoral responses in all or most of the individuals, providing complementary candidates to Fl antigen for diagnostic markers of plague.
INTRODUCTION
Plague, a category A infectious disease caused by Yersina
pestis, is endemic in China, Mongolia, Burma, Vietnam, Indonesia, India, the large parts of South Africa, the United
States, and South America.'
In China, there remain 12 natural plague foci, covering
> 291 counties in 19 provinces.^''' The Yunnan Province includes two plague foci. Focus F and Focus E,"* the Rattus
flavipectus plague focus and the Apodemus chevrieri and Eothenomys miletus plague focus, respectively. Y. pestis isolated
from these foci belongs to biovar Orientalis and Antiqua, respectively.^ In October 2005, five people in the Yulong county
of Yunnan province, where no plague had been reported in
the last 100 years, experienced fever, cough, and hard breath.
Two of them died in 3 days after onset of the symptoms.
Detection of a specific antibody against Fl antigen in the sera
of the three survivors by the passive hemagglutination assay
(PHA) showed a 4-fold rise between the convalescent and
post-convalescent phase.^ A field investigation for finding the
cause and the ranges of this epidemic was initiated.
The domestic dog, one of the animal hosts of Y. pestis,
seems to be highly resistant to plague and has an inapparent
or mild disease if the plague bacterium does infect it.' The
srologie study of dogs can give us a clue of infectious origin
and areas. In contrast to the domestic dog, orally infected
domestic cats become acutely ill and generally develop buboes and pneumonic lesions similar to those seen in humans
with plague. Over one third die within 10 days, whereas 44%
become ill but recover.^* In the United States, there are some
plague cases that acquired Y. pestis through contact with infected domestic cats.^* In China, the roles of the domestic cat
in the spread and maintenance of Y. pestis are still unknown.
In this study, > 2,900 serum samples of domestic dogs and
cats in and around the epidemic county were collected, and a
srologie survey on antibodies against the Fl antigen was
conducted by PHA to determine the infected range of ani-

mais. Meanwhile, to overcome the false-negative results

caused by Y. pestis mutants lacking the Fl antigen in the
srologie survey to plague, the antibody responses of these
two animals to 218 proteins of Y. pestis were profiled by
protein microarray to understand the immunoresponses for
finding complementary diagnostic markers to the Fl antigen.
MATERIALS AND METHODS
Collection of serum samples. Serum samples from domestic
cats (Felis catus). One hundred fifty-one serum samples of
domestic cats in and around the village (blue dot and red dots
in Supplementary Figure SI; found online at www.ajtmh.org)
with plague epidemics in 2005 were collected. Eleven serum
samples were collected from nearby counties (yellow dots in
Supplementary Figure 5>1) where there is no reported human
plague cases in the past 10 years.
Serum samples from domestic dogs (Cards familiaris). Six
hundred eighty-nine serum samples of domestic dogs were
collected from the villages where the serum samples of cats
were collected. Two thousand one hundred thirty-six serum
samples were collected from nearby counties.
All of the above serum samples were collected in May 2006,
~7 months after human infection. The serum samples were
first collected from the village with human plague epidemics
and then extended to the surrounding villages until no positive serum of domestic dogs or cats was detected according to
the Yunnan Animal Care and Use Protocol. To collect the
sera, the captured animals were anesthetized by barbital, and
1.5 mL blood was collected from the vein on the back leg of
animals. The sera were separated and stored at -20C until
use.
Detection of antibodies against Fl by PHA. The anti-Fl
antibody titers in the sera were detected by the PHA.' The
serum samples whose antibody titers against Fl antigen were
s 1:40 were considered positive. All the PHA measurements
were validated by an accompanying inhibition assay.'" Meanwhile, all of the positive samples by PHA were confirmed by
the Fl antigen-based u|)-converting phosphor technology.' '
Diagnostic markers screening by protein microarray. The

* Address correspondence to Ruifu Yang, Professor No. 20, Dongdajie, Fengtai, Beijing 100071, China, E-mail: ruifuyang@gmail.com and
Zhizhong Song, Professor No. 5, Wenhualu, Dali 671000, Yunnan
Province, China, E-mail: Songl208@126.com
These authors contributed equally to this work.

protein microarray incltided 218 proteins of Y. pestis and was

constructed according to our previous reports'^''^ (Supplementary Table SI; found online at www.ajtmh.org). The purified Y. pestis proteins were printed on silylated glass slides
799

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LI, GUO AND OTHERS

from CEL (Gene Company, Hong Kong) by allowing covalent binding by amino groups on the molecules that couple to
aldehyde groups on the glass slide surface. The printed slides
were stored at 4C for further use. According to the results of
PHA against Fl antigen, 14 serum samples of cats and dogs at
different antibody titers (from 1:80 to 1:20,480 and from 1:80
to 1:1,280, respectively) were selected to profile the antibody
amounts and response magnitude against the proteins on the
microarray. Eight serum samples of cats and four serum
samples of dogs that were negative for the Fl antibody were
used as negative controls.
To determine the results in domestic cats and dogs, the
cut-off value for each protein was calculated as the average
value plus 2 SD of the fluorescence intensity (FLI) from the
negative control sera. The proteins whose antibody increased
in at least three individuals were considered to be immunogens in the animals.
RESULTS AND DISCUSSION
Prevalence of srologie antibodies against Fl in domestic
cats and dogs. All the collected serum samples were initially
screened by PHA to detect the IgG antibody titer against the
Fl antigen. Using the result of PHA s: 1:40 as positive criterion, 162 of 689 (23.5%) sera from domestic dogs and 40 of
151 (26.5%) sera from domestic cats were positive (Table 1).
None of the serum samples of dogs and cats from the control
counties were positive for Fl antibody by PHA. The sropositive rates of 23.5% and 26.5% in domestic dogs and cats in
and around the village where the human plague oecurred
were significantly higher than 0% in the dogs and cats from
the regions in which plague was not identified in the past 10
years (Table 1; Supplementary Figure SI). It was concluded
that the animals could serve as sentinel animals for plague
surveillance, and seropostivity may be a clue for recent active
prevalence of plague in the area.
The domestic dog plays an important role as a plague carrier, and srologie survey of them is an index for plague surveillance in China. Domestic cats can also be infected by Y.
pestis through ingestion of plague-infected rodents or by the
bite of infected fleas, and exposure to infected cats has recently been recognized in human plague in the United
States.^'^"* Our study showed that the seropositive rates of
domestic cats in the villages around a human-infected village
were much higher than that in villages further away (Supplementary Figure SI), and high antibody titers against the Fl
antigen were detected in domestic cats (from 1:80 to 1:
20,480), suggesting that domestic cats might play an important
role in the spread of plague in the Yunnan Province and that
srologie survey of cats would help determine the infected
origin and range.
According to the results of the PHA, the positive serum
samples of domestic dogs and cats were detected in 35 villages
around the village with human plague patients, covering -210
km^ where no Y. pestis infection was reported in the past 100
TABLE 1

Analysis of sera from different domestic hosts by PHA

Dogs
Cats

Animals from the Yulong county

(positive rate %)

Animals from the nearby counties

(positive rate %)

162/689 (23.5)
40/151 (26.5)

0/2,136 (0)
0/11 (0)

years. This area has been classified as a novel natural plague

focus by extended epidemiologic study and bactriologie
studies (unpubhshed data).
Antibody profiling stiowed different immunotogic responses of dogs and cats against Y. pestis. To better understand the different susceptibility to Y. pestis between domestic
dogs and cats, a protein microarray containing 218 known or
putative virulence-associated proteins of Y. pestis was used in
this study to profile humoral immune responses of domestic
dogs and cats whose antibodies against the Fl capsule antigen
were > 1:40. Figure 1 gives an overall picture of these antibody profiles for different hosts: 1) for domestic cats, antibody responses to 45 proteins were found in at least three
individuals (Figure 1, @); 2) for domestic dogs, 26 proteins
were identified as immunogens (Figure 1, &). In total, 59
proteins were detected to induce humoral immune responses
by using the above-mentioned protein microarray. Thirtyfour of them are hypothetical or putative proteins. The detection of antibodies suggests that the corresponding proteins
can be expressed by the bacterium in vitro and/or during the
course of infection. Overall, the numbers and response magnitude of antibodies in domestic cats were above those in
domestic dogs (Supplementary Figure S2 and Table S2; found
online at www.ajtmh.org). The immunogenicity of 33 proteins
was found in domestic cats but not in dogs, indicating that
these proteins might be expressed specifically when the bacterium invaded cats or is processed specifically by cat immune
cells.
Antibody responses to some known virtilence proteins were
different in domestic dogs and cats. Fl capsular antigen
(YPMT1.84), PsaA (YPO1303), and the proteins encoded by
type III secretion system (T3SS) are important virulence proteins, which are involved in inhibiting phagocytosis of V. pestis by macrophages and neutrophils.'^"^' The antibody response intensities to these proteins were obviously different
between dogs and cats. The antibodies to YPCD1.39c
(YopN), YPCD1.19 (YopK), and YPCD1.67c (YopH) were
only detected in cats and those to YPCD1.26c (YopM),
YPCD1.28C (YopD), and YPO1303 (PsaA) were detected in
more cats than dogs. Although the antibodies against
YPMT1.84 (Fl) and YPCD1.31c (LcrV) were detected in all
tested sera of both hosts, their increasing magnitude in dogs
was obviously lower than that in cats (Figure 1; Supplementary Table S2). The above results implied that the fewer bacteria proliferate in dogs for inducing the immune responses,
which might partially explain the dog's high resistance to
y. pestis. In contrast to the domestic dog, the bacteria should
proliferate rapidly in cats, and the expression of virulenceassociated proteins helps Y. pestis counteract the defense
mechanisms of cats, resulting in the apparent symptoms. The
detection of antibodies against these known virulence proteins and 37 other proteins in the sera of cats showed that they
might be synthesized during disease development and played
roles in plague pathogenesis.
Proteins whose antibodies were detected in both hosts. A
total of 12 proteins induced humoral responses in both hosts
(Figure 1, *). Because the infection in domestic dogs is transitory, the antibodies detected in them indicated the dominant immunogenicity of these proteins. Seven of them were
found to be immunogenic in a previous study'^'^^'^^; the other
five were found to be immunogenic for the first time (Figure
1, $). YPO2125, along with YPO2118 and YPO2108, locate in

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SEROLOGIC SURVEY FOR PLAGUE SURVEILLANCE

Domestic Cats
12 3 4

5 6 7 8 9 10 111213 141 2 3 4

Domestic Dogs
5 6 7 8 9 10111213 14 GenaiDinco92
YPMT1.84
YPCD1.31C
YPCD1.28C
YPO2118
YPCD1,26c
YPO1303
YPO1023 $
YPO2108
YPO1387 $
YPO2319 $
YPO0816 $
YPO212G $
YPO0397
YPMT1,22
YPCD1.39C
YPO2958
YPCD1.40
YPMT1.46BC
YPMT1.34
YPO2321
YPO2130
YPO1045
YPO1071
YPO1089
YPO1435
YPMT1.12C
YPO2094
YPO3382
YPCD1.67C
YPCD1.19
YPO2127
YPCD1.42
YPMT1.43
YPMT1.86C
YPO0687
YPO23d4
YPO3516
YPO3247
YPO2132
YPO3278
YPO0448
YPO10S2
YPO1053
YPO3827
YPPCP1.05
YPO37S4
YPMT1.87
YPO2313
YPO0686
YPO2120
YPMT1.39
YPCD1.68C
YPMT1.76A
YPMT1.35C
YPO3879
YPO2124
YPO2190
YPO2664
YPO3118

F1 capule antigen
V ontiQen,IcrV
putative Yop negative regiiiation/targeting component, yopO
hypothettcai phage protein
targeted effector protein.yopM
pH 6 antigen precursor (atitigen 4) (adhcsin)
N-acetylmuramoyi-L-aianiiie amidase AmiC precursor
hypothetlcai phage protein
putative exported protein
putative pepetidase
general secretion pathway protein D
putative phage regulatory protein
hypotheticai protein
hypotheticai protoin
putative membrane-bound Yop targeting protein, yopN, icrE
lron(lli)-binding perlplasmic protein
putative Yops secretion A t P synthase, yscN
hypothetical protoin
hypotheticai protoin
putative phage protein
hypothetical phage protein
elongation factor Ta
putative lipoproteln
putative regulatory prophnge protein
putative outer membrane porin A protoin
hypotheticai protoin
hypotheticai phage protein
giobal stress requirement protein GsrA
putative protein-tyrosine phosphata3e Yop effector, yopH
putative viruienco determinant protein, yopK, yopQ
putative phage-related membrane protein
putative type iil secretion protein, yscP
hypothetical protein
hypothetical protein
conserved hypothetical piotein
major outer membnine pisprotein
maiato dehydrogonase
putative adhesin
hypothetical phage protein
putative llpoprotein
putative iipoprotein
putative surface antigen
catlonfc 19 kDa outor membrane protein precursor
giycerophosphoryl dlestei' phosphodlosterase
pesticin
eiongation factor Tu
putative porphyrin blosynthetic protein
hypothetical protein
putative membrane protein
putative phage protein
hypotheticai protein
'
conserved hypotheticai protein
hypotheticai protein
hypotheticai protein
putative outer membrane usher protein
hypotheticai phage protein
attachment invasion iocus protein precursor
putative membrane protein
adenylate itinase

FiGURi: 1. Antibody responses in domestic cats and dogs. The hosts and the individuals are indicated on the top, whereas Y. pestis proteins
are listed on the right column. Protein IDs are derived from the genome annotation of Y. pestis CO92. Black represents that antibody was detected
in the corresponding individual; white represents antibody not detected. * Proteins whose antibody were detected in both hosts. @ Domestic cats.
& Domestic dogs. $ The immunogenicity of these proteins was first described in this study.
a 33-kb chromosomal fragment YPO2095-2135 that was absent from the biovar Microtus strains, which are supposed to
be avirulent to humans, although they are highly lethal to
mice; therefore, this fragment likely contributes to the ability
to infect humans with fully virulent strains.^"* The detection of
antibodies against the proteins encoded by this fragment
showed that this fragment could play a role in plague pathogenesis. Additionally, the obvious increase of antibody to
YPO2118 in plague patients and EV76-vaccitiated rabbits
makes it a potential target for vaccine research.'^'''
Potential complementary diagnostic markers to Fl anti-

gen. The detection of Fl antibody by PHA is a standard

method for plague srologie surveillance in mammalians and
srologie diagnosis of human plague. It should also be noted
that serodiagnosis of plague by detecting the Fl antibody only
could miss the detection of strains lacking Fl, which have
been isolated from a number of different host species and
from human infection.^^-^'' Although many alternative methods such as PCR analysis and DNA hybridization for diagnosing plague have been developed,^^"^' these methods are

not widely available for on-site applications. The handheld

diagnostic assay should be developed for field applications in
the daily surveillance of plague.
The srologie methods such as PHA, enzyme-linked immunosorbent assay (ELISA), and biosensor-based enzyme immunosassay (EIA) are simple and can be perfornied in small
hospital.'"'"" The LcrV captured enzyme-linked imtnunosorbent assay has been developed to help confirm the diagnosis
of plague."''^ Our restilts also showed that LcrV cotild be used
with Fl for surveillatice purposes and epidetniologic study. In
all of the animals tested, the antibody against LcrV increased
dramatically. Giveti the variability of the LcrV antigen and
the fact that no antibodies were detected in five Yunnan patients who were infected with Y. pestis in 2003,'"' other antigens are needed to help srologie sut veillance and diagnosis.
Except Fl and LcrV, the antibodies against YPCD1.28c
(YopD) and YPO2] 18 were detected in most of the animals,
human patients, and experimentally infected rabbits.'^"
YPCD1.28C locates in the plasmid pCDl, which is conserved
amotig all three pathogenic Yersinia spp. and is essential for

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, GUO AND OTHERS

the virulence of Y. pestis. The serodiagnosis with multiple

proteins located both in the pCDl plasmid (LcrV and
YPCD1.28c) and chromosome (YPO2118) will complement
the plague diagnosis based on the Fl antigen to avoid missing
infections caused by the Fl-negative strains.
Received April 17, 2008. Accepted for publication August 4, 2008.

14.
15.
16.

Note: Supplementary Table SI (K pestis proteins represented on the

microarray). Supplementary Taljte S2 (The original data of antibody
fluorescent value against the protein on the Mieroarray), Supplementary Figure SI (The geographic distribution of the seropositive rate in
the satellite map of Yulong and around County) and Supplementary
Figure S2 (Antibody profile by the protein microarray) appear online
at www.ajtmh.org.

17.

Financial support: This work was supported by the National 863

Project (Contract 2006AA02Z438), the National Natural Science
Foundation of China (Grants 30430620 and 30771920), and the Social
Development Foundation of Yunnan Province (Grant 2008CA010).

19.

Authors' addresses: Bei Li, Zhaobiao Guo, Ziwen Zhu, Yanfeng

Yan, and Ruifu Yang, No. 20, Dongdajie, Fengtai, Beijing 100071,
China, Tel: 0086-10-66948595, Fax: 0086-10-63815689. Ying Guo, Yun
Liang, Qing Zhou, and Zhizhong Song, No. 5, Wenhualu, Dali
671000, Yunnan Province, China, Tel: 0086-872-2192058, Fax: 0086872-2125437.

18.

20.
21.
22.

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