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J Med Microbiol 52 (2003), 1021-1026; DOI: 10.1099/jmm.0.05234-0
© 2003 Society for General Microbiology
ISSN 0022-2615

Experimental Ehrlichia chaffeensis infection in beagles

Xiao-Feng Zhang1, Jian-Zhi Zhang1, S. Wesley Long1, Randall P. Ruble2 and Xue-Jie Yu1

Departments of Pathology and Microbiology and Immunology, Center for Biodefense and Emerging Infectious Diseases1 and Animal Resource Center2, University of Texas Medical Branch, Galveston, Texas 77555-0609, USA

Correspondence Xue-Jie Yu xuyu{at}utmb.edu

Received February 25, 2003
Accepted August 8, 2003

A canine model for human monocytic ehrlichiosis was used to assess persistent infection and antigenic variation of Ehrlichia chaffeensis. Two beagle dogs were infected subcutaneously with E. chaffeensis Arkansas strain. The dogs were observed for 6 months after inoculation for clinical signs, blood chemistry changes, antibodies to E. chaffeensis and presence of E. chaffeensis in the blood. Both dogs developed thrombocytopenia, but exhibited normal body temperatures during the entire course of infection. In one dog, E. chaffeensis was cultivated for up to 74 days post-inoculation and E. chaffeensis DNA was detected in the dog's blood for up to 81 days. In the other dog, E. chaffeensis was cultured for up to 102 days and E. chaffeensis DNA was detected in the blood for up to 117 days. PCR amplification and DNA sequence analysis indicated that there was no genetic variation in the 120 kDa outer-membrane glycoprotein gene of E. chaffeensis during infection of the dogs. The dogs developed antibodies to the immunodominant proteins of E. chaffeensis, including the 175, 140, 120, 80, 50 and 28 kDa proteins, starting in the fifth week post-inoculation. The dogs maintained high antibody titres throughout the 6-month study period. These results indicate that dogs become carriers of E. chaffeensis for 2–4 months after infection without exhibiting signs of clinical disease, suggesting that dogs may serve as a natural host for E. chaffeensis.

Abbreviations: IFA, immunofluorescence assay; UTMB, University of Texas Medical Branch.


   INTRODUCTION
TOP
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Ehrlichia chaffeensis is the pathogen of the emerging infectious disease human monocytic ehrlichiosis. Ehrlichia spp. are small, obligately intracellular, Gram-negative bacteria that reside within endosomes of host cells. The genus Ehrlichia is a member of the family Anaplasmataceae, which also includes the genera Anaplasma and Neorickettsia (Dumler et al., 2001). Anaplasma and most Ehrlichia species usually cause persistent infection in their natural animal hosts (Andrew & Norval, 1989; Telford et al., 1996; Breitschwerdt et al., 1998; Harrus et al., 1998). Persistent ehrlichial infection in humans has also been documented (Dumler et al., 1993; Roland et al., 1995; Dumler & Bakken, 1996; Horowitz et al., 1998). Experimentally infected white-tailed deer (Odocoileus virginianus) carry E. chaffeensis for up to 3 months (Davidson et al., 2001). E. chaffeensis DNA has been detected in naturally infected dogs and E. chaffeensis has been cultured from experimentally infected dogs (Dawson & Ewing, 1992; Breitschwerdt et al., 1998). Although these studies show that E. chaffeensis can infect dogs, the experiments did not determine whether dogs were persistently infected, either because experimentally infected dogs were observed for a short period of 28 days or because blood was obtained only once from naturally infected dogs. In this study, we examined the persistence of E. chaffeensis infection in two experimentally infected dogs.

Another aim of this study was to investigate whether persistence of infection by E. chaffeensis was associated with antigenic variation of a major outer-membrane protein, gp120. One striking feature of gp120 is that it contains tandem repeats. The repeat regions contain both hydrophilic and hydrophobic domains, which is typical of transmembrane proteins (Yu et al., 1997). The E. chaffeensis gp120 protein is an O-linked glycoprotein, in which carbohydrates account for about 50 % of the molecular mass (McBride et al., 2000). The gp120 protein is expressed differentially on the surface of the dense-core cell form of E. chaffeensis and accumulates in intramorular fibrillar structures (Popov et al., 2000). Antigenic variability of gp120 has been observed in E. chaffeensis strains. The number of repeats in clinical isolates of E. chaffeensis varied from two to five (Chen et al., 1997; Sims et al., 2000; Standaert et al., 2000). Other than difference in the number of repeats, no other mutation has been found in the gp120 gene of E. chaffeensis isolates (Chen et al., 1997).


   METHODS
TOP
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Inoculation of dogs with E. chaffeensis.

E. chaffeensis (Arkansas strain) was obtained from Jacqueline Dawson (Centers for Disease Control and Prevention, Atlanta, GA, USA). E. chaffeensis stock used in this study was isolated originally from dog blood after laboratory inoculation of E. chaffeensis in our laboratory and was passaged three times in DH82 cells after reisolation from a dog.

Sera from four 6-month-old male beagle dogs were obtained from Convance. The sera were tested for antibodies to E. chaffeensis and Ehrlichia canis by immunofluorescence assay (IFA). All dogs were seronegative to E. chaffeensis and E. canis. These four dogs were subsequently purchased and used in this study. Dogs ACC and AFJ were inoculated subcutaneously with 106 E. chaffeensis-infected DH82 cells (80 % infected) as described by Dawson & Ewing (1992). Two dogs (AZY and ATL) were sham-inoculated with PBS to act as controls.

The study was approved by the Institutional Care and Animal Use Committee of the University of Texas Medical Branch (UTMB) and the dogs were housed in accordance with the Guide of the Care and Use of Laboratory Animals in UTMB's Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited facility (http://research.utmb.edu/iacuc/).

Dog blood collection and blood chemistry analysis.

After inoculation with E. chaffeensis, 10 ml blood was obtained from each dog at 1-week intervals for 3 months and then at 2–3-week intervals for 3 months. Heparin anti-coagulated blood was used for cell culture. EDTA anti-coagulated blood was used for PCR and blood chemistry analysis. Complete blood counts and blood chemistry analysis were performed in the UTMB clinical laboratory and the veterinary laboratory at Texas A&M University.

Isolation of E. chaffeensis from dog blood.

Dog blood (1 ml) was lysed in ACK lysis buffer (0.15 M NH4Cl, 10.0 mM KHCO3, 0.1 mM Na2EDTA, pH 7.2) (Coligan et al., 1992) at room temperature for 10 min. Leukocytes were collected by centrifugation at 400 g for 10 min. Cells were washed twice by centrifugation at 400 g for 10 min in PBS. Leukocytes were suspended in 1 ml Eagle's minimum essential medium (MEM) supplemented with 5 % bovine serum and inoculated onto a DH82 cell monolayer in a T25 flask. Flasks were rocked for 1 h at room temperature and 4 ml of 5 % MEM was then added. Flasks were incubated at 37 °C with periodic media changes and monitoring of E. chaffeensis growth by Diff-quik staining and PCR detection of E. chaffeensis DNA. Samples were considered negative if E. chaffeensis morulae were not observed and if PCR detection of E. chaffeensis DNA was also negative in DH82 cells 3 months post-inoculation of dog blood.

DNA preparation.

Dog-blood DNA was extracted by using a QIAamp DNA Mini kit (Qiagen). E. chaffeensis genomic DNA was prepared by using an IsoQuick Nucleic Acid Extraction kit (ORCA Research) according to the instructions of the manufacturer.

PCR amplification.

PCR was used to detect E. chaffeensis DNA from dog blood and DH82 cells. The repeat region of the gp120 gene was amplified by using primers pxcf3b (5'-CAGCAAGAGCAAGAAGAT GAC-3') and pxar5 (5'-ATCTTTCTCTACAACAACCGG-3') as described previously (Yu et al., 2000a). The gp120 gene was amplified for 30 cycles of 94 °C for 30 s, 50 °C for 30 s and 72 °C for 30 s. A nested PCR assay was used to amplify the 16S rRNA gene of E. chaffeensis from dog-blood samples. The primary PCR was 30 cycles of 94 °C for 30 s, 50 °C for 1 min and 72 °C for 1 min, using primers ECC (5'-AGAACG AACGCTGGCGGCAAGCC-3') and ECB (5'-CGTATTACCGCG GCTGCTGGCA-3'). The nested PCR was 30 cycles of 94 °C for 30 s, 50 °C for 1 min and 72 °C for 1 min, using primers HE1 (5'-CAATTGCTTATAACCTTTTGGTTATAAAT-3') and HE3 (5'-TATAGGTACCGTCATTATCTTCCCTAT-3') (Swift & Thomas, 1983; Zaugg et al., 1986; Anderson et al., 1992).

DNA sequencing.

PCR products were purified by using a QIAquick PCR Purification kit (Qiagen) and were sequenced directly by using PCR primers. An ABI PRISM DNA sequencer (Perkin-Elmer Applied Biosystems) was used to sequence the DNA by the UTMB Protein Chemistry Laboratory.

IFA.

Indirect IFA was used to titrate E. chaffeensis antibodies in the dog sera. Sera were diluted, starting at 1 : 8, in twofold increments. A dilution of 1 : 64 was used as the cut-off for a positive titre. Antigen slides were prepared by using E. chaffeensis-infected L929 cells (rather than DH82 cells) to avoid cross-reaction with conjugate, as DH82 is a canine histiocyte cell line. FITC-labelled anti-dog IgG (H+L-chains) was purchased from Kirkegaard & Perry Laboratories.

Protein immunoblotting.

Antigens were prepared from E. chaffeensis-infected L929 cells. L929 cell monolayers were inoculated with E. chaffeensis. When 100 % of cells were infected with ehrlichiae, cells were centrifuged at 17 400 g for 20 min. Pellets were disrupted with a Braun-Sonic 2000 sonicator at 40 W for 30 s twice on ice. The suspension was centrifuged at 200 g for 10 min. The resulting pellet, which consisted primarily of host-cell debris, was discarded and the supernatant was centrifuged at 17 400 g for 20 min. The pellet was suspended in SPK (sucrose/potassium phosphate) buffer and was centrifuged through 30 % sucrose solution at 17 400 g for 30 min.

The repeat region of the E. chaffeensis gp120 gene was expressed in Escherichia coli as described previously (Yu et al., 1996, 2000b). The p28-15 gene, a member of the p28 multigene family, was amplified by PCR using primers 19f (5'-GAAGCGCAATATCCAACTCCTC-3') and 19r (5'-TGACCAATAAACACAGAAG-3'). The PCR product was cloned into pCRT7/CT-TOPO vector (Invitrogen) and the recombinant plasmid was used to transform E. coli BL21 to express the p28-15 protein.

Proteins were separated on NuPAGE SDS-PAGE gels (Invitrogen) and electroblotted onto nitrocellulose membranes. Alkaline phosphatase-labelled anti-dog IgG (H+L-chains) was purchased from Kirkegaard & Perry Laboratories.


   RESULTS
TOP
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Clinical manifestation and laboratory results of the dogs

During the 6-month observation period, neither dog manifested a fever or exhibited loss of body mass. However, thrombocytopenia was observed in both infected dogs, but not in sham-infected dogs. Thrombocytopenia within infected dogs appeared by the second week after subcutaneous inoculation with E. chaffeensis and was still present at the end of the study period, despite the fact that E. chaffeensis was not demonstrable within the blood at that time.

Culture of E. chaffeensis from dog blood

E. chaffeensis was cultured from blood samples of dog ACC from days 23–102 post-inoculation. E. chaffeensis was isolated from blood samples of dog AFJ from days 23–74 post-inoculation (Tables 1 and 2). E. chaffeensis was not isolated from the sham-infected dogs at any time.


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  		Table 1. Detection of Ehrlichia chaffeensis and antibodies from blood samples of dog ACC
 

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  		Table 2. Detection of Ehrlichia chaffeensis and antibodies from blood samples of dog AFJ
 

PCR amplification of E. chaffeensis DNA from dog blood

E. chaffeensis DNA was amplified from dog-blood samples by nested PCR (Tables 1 and 2). DNA sequence analysis confirmed that PCR-amplified DNA was that of the E. chaffeensis 16S rRNA gene. Dog ACC was PCR-positive for approximately 4 months (117 days) and dog AFJ was PCR-positive for 81 days.

Indirect IFA

Pre-inoculation dog sera and sera from the first week to the sixth month after inoculation of E. chaffeensis were analysed for antibodies to E. chaffeensis antigens by IFA. Both infected dogs developed antibodies to E. chaffeensis (Tables 1 and 2). Antibodies to E. chaffeensis were detected at the fourth week post-inoculation (day 23) at a titre of 1 : 64 or higher. Peak titres in both dogs were 1 : 16 384 at the seventh or eighth week post-inoculation. Titres remained high throughout the remainder of the study. Uninfected dogs did not seroconvert to E. chaffeensis.

Protein immunoblotting of dog sera

Major proteins of E. chaffeensis that reacted with dog sera were the 175, 140, 120, 80, 50 and 28 kDa proteins (Fig. 1). Antibodies to major E. chaffeensis proteins appeared on the fifth to the seventh week post-inoculation and lasted until the end of the study period. Reactions of the sera with gp120 and p28 were confirmed by using recombinant proteins (Fig. 2).



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  		Fig. 1. Protein immunoblotting of E. chaffeensis antigens reacting with sera of dogs ACC (top) and AFJ (bottom). Day, days post-inoculation of E. chaffeensis.

 


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  		Fig. 2. Protein immunoblotting of sera of dogs ACC and AFJ reacting with recombinant E. chaffeensis gp120 and p28-15 proteins. Day, days post-inoculation.

 

Genetic variation of the gp120 gene

The repeat region of gp120 was amplified from all E. chaffeensis isolates from dogs ACC and AFJ. PCR products had identical sizes to that of the parental strain of Arkansas, which contained four repeat units of 240 bp each (Fig. 3). DNA sequencing indicated that there was no change in the repeat region of the gp120 gene among the isolates of E. chaffeensis from both dogs. This indicates that any antigenic variation of the gp120 protein did not originate from insertion or deletion of repeat units within the gp120 gene.



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  		Fig. 3. PCR amplification of gp120 repeat region of E. chaffeensis isolated from blood samples of dogs ACC (top) and AFJ (bottom). Day, days post-inoculation of E. chaffeensis; ch, parental strain of E. chaffeensis; M, molecular marker (bp).

 


   DISCUSSION
TOP
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGEMENTS
 REFERENCES
 
The life-cycle of Ehrlichia involves a tick vector and a mammalian host. Mammals are infected with Ehrlichia by the bite of infected ticks; non-infected ticks acquire Ehrlichia by taking a blood meal from an infected mammal. Ehrlichia are not transmitted transovarially from one generation of ticks to the next (Stich et al., 1989); therefore, mammalian hosts are essential for maintenance of Ehrlichia in the environment. Carrier animals serve as reservoirs for Ehrlichia organisms (Swift & Thomas, 1983; Zaugg et al., 1986). Our results indicate that dogs carry E. chaffeensis for 3–4 months with no clinical symptoms; thus, dogs can be considered a carrier for E. chaffeensis. Currently, no suitable animal model is available for human monocytic ehrlichiosis. Immunocompetent mice are not susceptible to E. chaffeensis infection and immunodeficient mice would not be suitable for studying E. chaffeensis infection. Our results indicate that canines may serve as a model for the study of E. chaffeensis infection.

The persistence of infection by Ehrlichia may be the result of antigenic variation of surface proteins, especially the two major outer-membrane proteins of E. chaffeensis, gp120 and p28. The p28 protein is encoded by a multigene family (Reddy et al. 1998; Yu et al., 2000b). The gp120 gene of E. chaffeensis contains a tandem-repeat region; each repeat is 240 bp in length. Clinical isolates of E. chaffeensis contain two to five repeats. Immune escape mediated by antigenic variation of the number of repeats in outer-membrane protein genes is a well-established feature of many bacteria. Group B streptococcal {alpha}C protein, a protective surface protein antigen, contains a maximum of 16 tandem repeats. Loss of repeats from the {alpha}C protein of bacteria used in a challenge inoculation resulted in decreased protection of the vaccinated host (Gravekamp et al., 1996). Variants of group B streptococci that contain fewer tandem repeats have been isolated from neonates; in the presence of {alpha}C protein-specific antiserum, human polymorphonuclear leukocytes killed the maternal, but not the neonatal, isolates (Madoff et al., 1996). Deletion of repeat(s) in the M6 protein of group A streptococci alters the ability of certain antibodies, produced originally in response to sequences of the parent M protein, to bind to the mutant M protein (Jones et al., 1988). Change in the repeats causes change in epitopes; presumably, such conformational changes in the protein give micro-organisms the ability to escape the host immune system. However, our results indicate that the repeat region of gp120 was stable during a 3–4-month period of infection in dogs. It is most likely that the number of repeats in the gp120 gene of E. chaffeensis is relatively stable in natural infection over time.


   ACKNOWLEDGEMENTS
TOP
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
REFERENCES
 
This study was supported by a grant from the National Institute of Allergy and Infectious Disease (AI45871).


   REFERENCES
TOP
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 

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