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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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In China, there remain 12 natural plague foci, covering > 291 counties in 19 provinces.2,3 The Yunnan Province includes two plague foci, Focus F and Focus E,4 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.2 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 F1 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.3 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.1 The serologic 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.5,6 In the United States, there are some plague cases that acquired Y. pestis through contact with infected domestic cats.7,8 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 serologic survey on antibodies against the F1 antigen was conducted by PHA to determine the infected range of animals. Meanwhile, to overcome the false-negative results caused by Y. pestis mutants lacking the F1 antigen in the serologic 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 F1 antigen.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Serum samples from domestic dogs (Canis 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 –20°C until use.
Detection of antibodies against F1 by PHA.
The anti-F1 antibody titers in the sera were detected by the PHA.9 The serum samples whose antibody titers against F1 antigen were 
1:40 were considered positive. All the PHA measurements were validated by an accompanying inhibition assay.10 Meanwhile, all of the positive samples by PHA were confirmed by the F1 antigen-based up-converting phosphor technology.11
Diagnostic markers screening by protein microarray. The protein microarray included 218 proteins of Y. pestis and was constructed according to our previous reports12,13 (Supplementary Table S1; found online at www.ajtmh.org). The purified Y. pestis proteins were printed on silylated glass slides 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 4°C for further use. According to the results of PHA against F1 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 F1 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.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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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 F1 antibody by PHA. The seropositive rates of 23.5% and 26.5% in domestic dogs and cats in and around the village where the human plague occurred 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 S1). 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.
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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 km2 where no Y. pestis infection was reported in the past 100 years. This area has been classified as a novel natural plague focus by extended epidemiologic study and bacteriologic studies (unpublished data).
Antibody profiling showed different immunologic 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 F1 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. Thirty-four 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.
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; 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 virulence-associated 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 study12,22,23; the other five were found to be immunogenic for the first time (Figure 1, $). YPO2125, along with YPO2118 and YPO2108, locate in 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.24 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-vaccinated rabbits makes it a potential target for vaccine research.12,13
Potential complementary diagnostic markers to F1 antigen. The detection of F1 antibody by PHA is a standard method for plague serologic surveillance in mammalians and serologic diagnosis of human plague. It should also be noted that serodiagnosis of plague by detecting the F1 antibody only could miss the detection of strains lacking F1, which have been isolated from a number of different host species and from human infection.25,26 Although many alternative methods such as PCR analysis and DNA hybridization for diagnosing plague have been developed,27–29 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 serologic methods such as PHA, enzyme-linked immunosorbent assay (ELISA), and biosensor-based enzyme immunosassay (EIA) are simple and can be performed in small hospital.30,31 The LcrV captured enzyme-linked immunosorbent assay has been developed to help confirm the diagnosis of plague.32 Our results also showed that LcrV could be used with F1 for surveillance purposes and epidemiologic study. In all of the animals tested, the antibody against LcrV increased dramatically. Given 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 2003,13 other antigens are needed to help serologic surveillance and diagnosis. Except F1 and LcrV, the antibodies against YPCD1.28c (YopD) and YPO2118 were detected in most of the animals, human patients, and experimentally infected rabbits.12,13 YPCD1.28c locates in the plasmid pCD1, which is conserved among all three pathogenic Yersinia spp. and is essential for the virulence of Y. pestis. The serodiagnosis with multiple proteins located both in the pCD1 plasmid (LcrV and YPCD1.28c) and chromosome (YPO2118) will complement the plague diagnosis based on the F1 antigen to avoid missing infections caused by the F1-negative strains.
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).
* Address correspondence to Ruifu Yang, Professor No. 20, Dongdajie, Fengtai, Beijing 100071, China, E-mail: ruifuyang{at}gmail.com and Zhizhong Song, Professor No. 5, Wenhualu, Dali 671000, Yunnan Province, China, E-mail: Song1208{at}126.com 
These authors contributed equally to this work.
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: 0086-872-2125437.
Note: Supplementary Table S1 (Y. pestis proteins represented on the microarray), Supplementary Table S2 (The original data of antibody fluorescent value against the protein on the Microarray), Supplementary Figure S1 (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.
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REFERENCES |
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