Abstract
Ehrlichia chaffeensis, a tick-borne rickettsial organism, causes the disease human monocytic ehrlichiosis. The pathogen also causes disease in several other vertebrates, including dogs and deer. In this study, we assessed two clonally purified E. chaffeensis mutants with insertions within the genes Ech_0379 and Ech_0660 as vaccine candidates in deer and dogs. Infection with the Ech_0379 mutant and challenge with wild-type E. chaffeensis 1 month following inoculation with the mutant resulted in the reduced presence of the organism in blood compared to the presence of wild-type infection in both deer and dogs. The Ech_0660 mutant infection resulted in its rapid clearance from the bloodstream. The wild-type infection challenge following Ech_0660 mutant inoculation also caused the pathogen's clearance from blood and tissue samples as assessed at the end of the study. The Ech_0379 mutant-infected and -challenged animals also remained positive for the organism in tissue samples in deer but not in dogs. This is the first study that documents that insertion mutations in E. chaffeensis that cause attenuated growth confer protection against wild-type infection challenge. This study is important in developing vaccines to protect animals and people against Ehrlichia species infections.
INTRODUCTION
Ehrlichia chaffeensis, an Amblyomma americanum tick-transmitted rickettsial pathogen, is responsible for human monocytic ehrlichiosis (HME) and also causes persistent infections in people and several other vertebrates (1–3). The white-tailed deer is the reservoir host for E. chaffeensis, while humans, dogs, and other vertebrate hosts, such as coyotes and goats, are regarded as incidental hosts (4–7). HME disease in people may result in flu-like symptoms leading to significant morbidity, and it can be fatal if untreated, particularly in people with compromised immunity (3, 8). Patients undergoing blood transfusions and organ transplantations are also at high risk of acquiring E. chaffeensis infections (9, 10). Therapeutic options for HME disease being limited to a single antibiotic class and the nonavailability of vaccines are added challenges (11). Vaccine development is complicated due to limited understanding of the pathogen antigens involved in stimulating protective immunity (12). Deer, dog, and A. americanum tick infection studies are ideal for mapping genes essential for E. chaffeensis growth and persistence, as they are the recognized reservoir host, an incidental host, and the tick vector, respectively (13). The canine host is an ideal incidental host model that is similar to that of humans in acquiring E. chaffeensis infections from A. americanum ticks (14). We recently reported that E. chaffeensis infections in deer and dog are very similar in exhibiting clinical symptoms and pathogen persistence (13).
In an earlier study, Ohashi et al. (15) described that immunization with a recombinant p28 outer membrane protein in BALB/c mice induces transient protection against E. chaffeensis infection. A DNA vaccine targeting a p28 outer membrane protein homolog (MAP1) in another Ehrlichia species, E. ruminantium, also confers partial protection against infection challenges (16). Similarly, recent studies described that p28 outer membrane proteins of E. muris stimulate mouse memory B and T cell responses, reducing the bacterial burden after 10 to 14 days postchallenge with wild-type infection (17, 18). Several studies of Ehrlichia species also demonstrated that attenuated strains offer a high level of protection. For example, laboratory-passaged attenuated strains protect against virulent infections with E. ruminantium in sheep and goats and E. canis in dogs (19, 20). Inactivated whole-cell antigens also are capable of inducing protective host responses against virulent E. ruminantium challenge (19, 21). Although these reports suggest that the whole-cell attenuated strains are valuable in developing vaccines against Ehrlichia species, little is known about the molecular basis of attenuation. Moreover, to date there are no reports describing attenuated vaccines against E. chaffeensis.
Recently, we described several transposon insertion mutations in E. chaffeensis (22); mutations causing transcriptional inactivation of three putative membrane protein genes cause rapid clearance of the organism from deer (reservoir host). Here, we assessed the potential of attenuated mutants in conferring protection against infection challenge in both reservoir and incidental hosts.
MATERIALS AND METHODS
In vitro cultivation of E. chaffeensis.
E. chaffeensis Arkansas isolates (wild type and mutants) were cultivated in a macrophage-like cell line (DH82) (23).
Animal infections.
Animal experiments with deer and dogs were performed in compliance with the Public Health Service (PHS) policy on the humane care and use of laboratory animals and the U.S. Department of Agriculture's Animal Welfare Act and Animal Welfare Regulations (24), as well as with approvals of the Oklahoma State University (OSU) and Kansas State University (KSU) Institutional Animal Care and Use Committees (IACUC) and per the guidelines of the protocols. Laboratory-reared deer and purebred laboratory-reared dogs were used for conducting infection experiments. Protocols for rearing deer were described previously (22). Infections in deer with wild-type or clonally purified organisms also were performed as reported earlier (22). Briefly, E. chaffeensis organisms cultured in the canine macrophage cell line DH82, with about 80% infectivity, were recovered by centrifugation and washing the cell pellets with phosphate-buffered saline (PBS), and the final recovered cell pellets were resuspended to a concentration of 2 × 108 Ehrlichia organisms per ml of PBS and used for intravenous infections (1 ml per animal). Five- to 6-month-old beagle dogs of either sex were obtained from Covance Research Products (Denver, PA). The animals were injected with transposon mutants as clonally purified organisms or with wild-type E. chaffeensis. Inocula were prepared and inoculated as described above with an estimated concentration of ∼2 × 108 Ehrlichia organisms in 1 ml (22). Challenge infections in deer or dogs, where applicable, were performed about 1 month following the initial infections with 1 ml each of wild-type E. chaffeensis culture (prepared as described above).
Detection of E. chaffeensis by culture recovery and by molecular methods.
About 3 ml of each sample of deer or dog blood was collected in EDTA blood collection tubes on day zero (before infection) and on different days postinfection from all groups of animals. Blood samples were centrifuged at 3,000 rpm in a Clay Adams Sero-fuge (Becton Dickinson, Sparks, MD) for 5 min. Plasma was removed, and about 1 ml of buffy coat was transferred to a 15-ml sterile Falcon centrifuge tube containing 10 ml red blood cell lysis buffer (155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA) and mixed several times until complete lysis of erythrocytes occurred. The samples then were centrifuged at 5,000 × g for 5 min, and the supernatants were discarded. The buffy coat pellet from each sample was resuspended in 200 μl of sterile 1× PBS. To assess for infection with E. chaffeensis, 100 μl each cell suspension was transferred into wells of a 12-well sterile culture plate containing 0.9 ml of DH82 cell suspension having about 80% confluence. The cultures were grown by following the detailed culture protocols reported earlier (23), and infection was monitored twice a week by microscopically examining the Hema3-stained cytospin slides for up to 8 to 10 weeks to determine if a sample was positive or negative for E. chaffeensis infection.
One hundred microliters of the buffy coats from deer or dog blood also was used for isolating total genomic DNA using the Wizard SV genomic DNA purification kit per the manufacturer's instructions (Promega, Madison, WI). Purified DNA from each sample was stored in 100 μl of buffer containing 10 mM Tris-HCl and 1 mM EDTA (pH 8.0) (TE buffer). The DNAs were used to assess E. chaffeensis infection status by performing seminested PCR targeting the p28-Omp 14 gene (Ech_1136) or the insertion-specific regions of mutant clones (primers are listed in Table 1). Briefly, 2 μl of genomic DNA from deer or dog blood was used for the first-round PCRs in a 25-μl reaction volume using Platinum Taq DNA polymerase per the manufacturer's instructions (Life Technologies, Grand Island, NY). The PCRs were performed in a GenAmp9700 instrument (Applied Biosystems, Foster City, CA) with the following temperature cycles: 94°C for 4 min; 35 cycles of 94°C for 30 s, 52°C for 30 s, and 72°C for 1 min; and 1 cycle of 72°C for 3 min. The second round of PCR was performed using the same PCR conditions as those for the first-round PCR, and the templates for the second round included 2 μl of 1:100 diluted products from the first PCR with a nested PCR primer set. The PCR products were resolved on 1.5% agarose gel to identify specifically sized products (25).
TABLE 1.
Primers used for nested PCR in this study
| Name | Target | Size (bp) | Orientation | Sequence |
|---|---|---|---|---|
| RRG29 | Ech_1136 | Forward | 5′-GCAATAGCAGATAAGAAATATG | |
| RRG71 | 313 | Reverse | 5′-GAGCTCCTTCTAATACTAC | |
| RRG844 | 252 | Reverse nested | 5′-AACTCCGTGGTAGTATCCTCC | |
| RRG1226 | Ech_0284 | Forward | 5′-TCACTAGATATCAAATAGGTAAACGTA | |
| RRG1194 | 332 | Reverse | 5′-TATCCCTTATGTTACGATAACTTA | |
| RRG1258 | 282 | Reverse nested | 5′-TGCAACAGTTATTTAATGTATGGTTG | |
| RRG1200 | Ech_0379 | 927 | Forward | 5′-GTTACGGTGACCGTAAGGCTTG |
| RRG1202 | 419 | Forward nested | 5′-CAGTTGGAATTTGTTCACTACGT | |
| RRG1276 | Reverse | 5′-CTAAGGTTGTAGGGAATGCAACC | ||
| RRG1344 | Ech_0660 | Forward | 5′-TGTACCTGTATCCTCACCTATCACC | |
| RRG1194 | 417 | Reverse | ||
| RRG1258 | 367 | Reverse nested |
Concentrations of genomic DNAs from deer or dog blood samples were estimated using NanoDrop 3300 per the manufacturer's instructions (Thermo Fisher Scientific Inc., Wilmington, DE). Two μl of DNA was used for TaqMan-based quantitative PCR targeting the E. chaffeensis 16S rRNA gene by following the detailed protocols described in reference 26. For samples that tested positive, the bacterial copy numbers per microgram of total blood-derived DNA were calculated by comparing values to the standard curve plotted with known numbers of positive-control plasmid molecules (26).
ELISA.
Host cell-free E. chaffeensis lysate was prepared and used for enzyme-linked immunosorbent assay (ELISA) (13). Plasma samples from deer or dogs collected prior to infection and several days following infections were assessed by ELISA for the presence of the E. chaffeensis-specific IgG (13).
Statistical analysis.
Statistical analysis was performed to analyze differences in positive/negative status over time between wild-type- and Ech_0660 mutant-infected dogs. A binomial generalized estimating equation model was fit using Stata 12 software (StataCorp LP, College Station, TX) to account for repeated measures on dogs over time using a logit link and exchangeable correlation. Statistical analysis also was carried out to assess differences in average copy numbers of bacteria present in each group of animals before and after challenge to determine the effect of preimmunization with mutants. The analysis was performed using a 2-tailed unpaired Student's t test (GraphPad Software, La Jolla, CA). Similarly, Student's t test was used to evaluate the ELISA data on the last day of sampling in animals receiving inocula with wild-type and mutant E. chaffeensis clones.
RESULTS
Attenuated mutants confer protection against wild-type infection challenge in deer.
We reasoned that the attenuated growth resulting from mutations within three E. chaffeensis genes, Ech_0230, Ech_0379, and Ech_0660, in deer (22) and dogs (C. Cheng, A. D. S. Nair, D. C. Jaworski, and R. R. Ganta, unpublished data) is the result of the pathogen's inability to maintain the replication cycle continuously. We hypothesized that the attenuated mutants induce sufficient host response to protect against infection challenge with wild-type E. chaffeensis. We tested this hypothesis with two clonally purified attenuated mutants with insertions within Ech_0379 and Ech_0660 genes, as these mutations caused the loss of gene activity from putative Na+/H+ antiporter protein and phage-like structure protein, respectively (22). Five groups of deer were used (three animals each in groups 1, 3, and 4 and two animals each in groups 2 and 5): group 1 received wild-type E. chaffeensis infection; group 2 received clonally purified Ech_0284 mutant, as it is similar to the wild type in causing persistent infection, and to serve as a syngeneic positive control for other mutants (22); groups 3 and 4 received infections with clonally purified Ech_0379 and Ech_0660 mutants, respectively; and group 5 received no infection to serve as noninfection controls. Infection in all five groups was monitored in blood sampled frequently for 31 to 41 days and by performing nested PCRs on DNA recovered or by the culture recovery method (Table 2). Infection was detected frequently and persisted similarly in groups 1 and 2 (59% and 61% of the samples tested positive, respectively), while it was detected less frequently (19% of the time) in Ech_0379 mutant-infected (group 3) animals, i.e., in one animal on days 4 and 28, on day 35 in the second animal, and on day 7 in the third animal. Infection was undetectable throughout the study in group 4 animals (Ech_0660 mutant group), similar to uninfected controls (group 5). To determine if the Ech_0379 and Ech_0660 mutants confer protection, deer infected with these two mutants were intravenously challenged with wild-type E. chaffeensis after a month, and the infection was monitored in blood by nested PCR and by in vitro culture recovery methods for another month or longer (monitored for 32 days for group 3 deer and 44 days for the group 4 animals) (Table 2). To serve as a positive control, infection in deer with the wild-type E. chaffeensis (group 1, described above) was carried out with this challenge experiment using the inoculum from the same batch of culture, and infection monitoring of challenged animals and the wild-type infection controls also was carried out simultaneously. One animal in the Ech_0379 mutant-infected group tested positive on day 7 postchallenge, while all three challenged animals in the Ech_0660 mutant-infected and -challenged group tested negative for the organism for the entire 44 days of assessment (Table 3). The prior exposure of animals with an attenuated mutant, Ech_0379 or Ech_0660, reduced E. chaffeensis circulating in blood when challenged with wild-type organisms. While these data are encouraging, the conclusions are based on three animals per group. Additional experimental evidence is needed if these mutants are to be investigated further as vaccine candidates for deer. Tissue samples (liver and spleen) collected at the endpoint of the study were assessed for the presence of E. chaffeensis by nested PCR; the Ech_0660 mutant-infected and -challenged group and noninfected controls tested negative, while deer in groups 1 and 3 (wild-type and Ech_0379 mutant groups) tested positive in both tissues (Table 4).
TABLE 2.
White-tailed deer infection status with three different clonally purified mutants or with wild-type E. chaffeensisa
| Group | Infection status by day postinfectionb |
|||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 0 | 3 | 4 | 5 | 7 | 8 | 10 | 11 | 12 | 14 | 18 | 21 | 22 | 23 | 24 | 27 | 28 | 30 | 31 | 35 | 37 | 41 | |
| 1 | ||||||||||||||||||||||
| Wild-type-1 | − | − | + | + | + | + | + | + | + | + | ||||||||||||
| Wild-type-2 | − | − | − | − | + | + | + | + | + | − | ||||||||||||
| Wild-type-3 | − | + | − | − | − | + | + | − | − | − | ||||||||||||
| 2 | ||||||||||||||||||||||
| Ech_0284-1 | − | − | + | + | + | + | + | − | − | − | ||||||||||||
| Ech_0284-2 | − | − | − | + | + | + | + | + | − | + | ||||||||||||
| 3 | ||||||||||||||||||||||
| Ech_0379-1 | − | + | − | − | − | − | + | − | ||||||||||||||
| Ech_0379-2 | − | − | − | − | − | − | − | + | ||||||||||||||
| Ech_0379-3 | − | − | + | − | − | − | − | − | ||||||||||||||
| 4 | ||||||||||||||||||||||
| Ech_0660-1 | − | − | − | − | − | − | − | − | − | − | ||||||||||||
| Ech_0660-2 | − | − | − | − | − | − | − | − | − | − | ||||||||||||
| Ech_0660-3 | − | − | − | − | − | − | − | − | − | − | ||||||||||||
| 5 | ||||||||||||||||||||||
| Control-1 | − | − | − | − | − | − | − | − | − | − | ||||||||||||
| Control-2 | − | − | − | − | − | − | − | − | − | − | ||||||||||||
Samples were collected on different days postinfection from each group. Group 4 (Ech_0660-infected animals) contained zero positives, Ech_0379-infected animals (group 3) had a few positives (19%), and the wild-type-infected group had 59% positives. The Ech_0284 mutant-infected group had 61% blood positives, similar to the wild-type-infected group.
The minus and plus signs refer to samples tested negative or positive by culture recovery and/or nested PCR, respectively.
TABLE 3.
Assessing Ech_0379 and Ech_0660 mutants in conferring protection against E. chaffeensis challenge in white-tailed deer
| Group | Infection status by day postinfectiona |
|||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 4 | 7 | 11 | 12 | 18 | 19 | 22 | 23 | 24 | 27 | 30 | 32 | 34 | 37 | 41 | 44 | |
| 3 | ||||||||||||||||
| Ech_0379-1 | − | + | − | − | − | − | ||||||||||
| Ech_0379-2 | − | − | − | − | − | − | ||||||||||
| Ech_0379-3 | − | − | − | − | − | − | ||||||||||
| 4 | ||||||||||||||||
| Ech_0660-1 | − | − | − | − | − | − | − | − | − | − | − | |||||
| Ech_0660-2 | − | − | − | − | − | − | − | − | − | − | − | |||||
| Ech_0660-3 | − | − | − | − | − | − | − | − | − | − | − | |||||
The minus and plus refer to samples tested negative or positive by culture recovery and/or nested PCR, respectively.
TABLE 4.
Infection status of tissue samples in deer
| Tissue | Infection status by group and animal numbera |
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 (wild type) |
3 (Ech_0379) |
4 (Ech_0660) |
5 (Control) |
||||||||
| 1 | 2 | 3 | 1 | 2 | 3 | 1 | 2 | 3 | 1 | 2 | |
| Spleen | + | + | − | + | + | − | − | − | − | − | − |
| Liver | + | + | + | + | − | − | − | − | − | − | − |
The deer group numbers are the same as those used in Table 2. The minus and plus signs refer to samples tested negative or positive by nested PCR, respectively. Groups 1, 3, and 4 each had three animals, while the control group had two animals.
DNA recovered from blood samples of wild-type- and Ech_0379 and Ech_0660 mutant-infected groups before and after challenge with wild-type infection (groups 1, 3, and 4, respectively) from all days postinfection also were assessed by TaqMan-based real-time PCR to compare E. chaffeensis copy numbers among these groups when samples tested positive. Average values of positives of each animal from each group were used to determine the quantitative differences between the wild-type- and the two mutant-infected groups before and after wild-type infection challenge (Fig. 1). E. chaffeensis copy numbers in deer infected with the wild type and Ech_0379 mutant were not significantly different before or after the wild-type infection challenge. E. chaffeensis DNA copy numbers in the Ech_0660 mutant-infected group were undetectable by the quantitative PCR assay in animals both before and after wild-type infection challenge (P = 0.001).
FIG 1.
White-tailed deer infection status following infections with mutants or the wild type, assessed in blood by TaqMan assay to measure DNA copy numbers. Ehrlichia DNA copy numbers were assessed by TaqMan real-time PCR assay to measure the DNA copy numbers in deer blood following infections with wild-type (Wt) E. chaffeensis and mutant Ech_0379 or Ech_0660 (Ech_0379-wt and Ech_0660-wt) before and after challenge with the wild-type bacteria. The average values for the positive samples from each mutant group of animals were compared to those of the wild-type-infected group, and the data are presented on a semilog graph. Significant differences (P ≤ 0.05) observed between groups are identified with asterisks.
Plasma samples from all deer in all five groups were evaluated for the total IgG antibody response against E. chaffeensis whole-cell antigens (Fig. 2). Deer in groups 1 and 4 (wild-type- and Ech_0660 mutant-infected groups, respectively) had detectable IgG responses, whereas the deer receiving Ech_0379 mutant infection (group 3) and controls (group 5) had undetectable IgG responses. The Ech_0284 mutant-infected animals (group 2) had a weak but detectable response. The IgG levels were higher in the wild-type-infected group, which steadily increased with time postinfection. The IgG response for Ech_0660 mutant-infected animals was similar for the wild type; the IgG levels for these two groups for the last day of sample analysis were not significantly different (Fig. 2, bottom right). In contrast, the IgG responses in Ech_0284 and Ech_0379 mutant-infected animals were significantly lower than those of the wild type (P = 0.05 and P = 0.002, respectively). Similarly, Ech_0379 and Ech_0660 mutant-infected animals differed significantly (P = 0.02). The group 3 (Ech_0379 group) animals receiving E. chaffeensis infection challenge had very little change in antibody response, while two of the three challenged animals in the Ech_0660 mutant group (group 4) had a steady rise in antibody response (Fig. 3).
FIG 2.
E. chaffeensis-specific IgG response in deer infected with wild-type and mutant E. chaffeensis. Groups 1 to 5 represent the ELISA performed on animals infected with the wild type, Ech_0284, Ech_0379, Ech_0660, and noninfected controls, respectively. Line graphs within each group represent the IgG responses for individual animals (data are presented as mean values ± standard deviations [SD] from triplicate samples). For simplicity, we presented the line graphs in each panel, with different colors to represent different animals within each group. The bottom right panel has the comparison of IgG data for each group for the last day of sample analysis. Significant IgG differences (P ≤ 0.05) observed between groups are identified with asterisks. Error bars for this panel represent mean values ± SD for animals within each group.
FIG 3.
E. chaffeensis-specific IgG changes in deer receiving Ech_0379 or Ech_0660 mutant followed by challenge with wild-type infection. IgG levels were assessed in animals receiving mutant that had been inoculated and challenged with the wild type for several days postinfection. The data were plotted as described for Fig. 2. The day of challenge is identified with down arrows. As described for Fig. 2, the line graphs in each panel are presented with different colors to represent different animals within each group.
Attenuated mutants confer protection against wild-type infection challenge in dogs.
To further investigate whether the attenuated mutants, Ech_0379 and Ech_0660, generate a protective response in an incidental host, the infection and challenge experiments were repeated in dogs. Four dogs each were infected with Ech_0660 mutant or with wild-type E. chaffeensis, two dogs were infected with Ech_0379, and two dogs were kept as uninfected controls. Infection in blood was monitored by nested PCR and culture recovery methods. The wild-type E. chaffeensis-infected dogs tested positive 78% of the time, while the uninfected controls tested negative for the same time period. Ech_0379 mutant-infected dogs also tested positive 45% of the time postinfection; one dog tested positive on days 3, 7, and 10, and the second dog tested positive on days 3, 10, 15, 30, 32, and 35 (Table 5). The four dogs receiving Ech_0660 mutant infection tested positive less frequently (10.5% of the time) than wild-type-infected animals. In this group, the dogs tested positive only for the first week after receiving the inoculum (positives were detected on days 5 and 7 for one dog and day 5 or 7 for another dog each) (Table 5). Dogs infected with Ech_0379 and Ech_0660 mutants were challenged after a month with wild-type E. chaffeensis. The infection was monitored in blood sampled for up to 38 days (Ech_0379 mutant group) or 35 days (Ech_0660 mutant group) (Table 5). The Ech_0379 mutant-challenged dogs tested positive for several days of the first 17 days postchallenge, while the Ech_0660 mutant group tested negative on all days after infection challenge, with the exception of one dog on day 5 and two dogs on day 8 postchallenge. The Ech_0660 mutant-infected dogs were positive on significantly fewer days than wild-type-infected dogs (P = 0.017). DNA recovered from spleen and liver samples from the challenged dogs at the endpoint of the study for both the Ech_0379 and Ech_0660 mutant groups tested negative for the organism, while dogs that received the wild-type infection tested positive in both tissue samples (not shown).
TABLE 5.
Assessing Ech_0379 and Ech_0660 mutants in conferring protection against wild-type E. chaffeensis challenge in dogsa
| Group | Infection status by days after: |
||||||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Infection |
Challenge |
||||||||||||||||||||||||||||||||||
| 0 | 2 | 3 | 5 | 7 | 9 | 10 | 12 | 14 | 15 | 16 | 18 | 19 | 21 | 22 | 24 | 26 | 29 | 30 | 32 | 35 | 42 | 2 | 5 | 8 | 10 | 14 | 17 | 20 | 22 | 24 | 28 | 31 | 35 | 38 | |
| Wild-type-1 | − | + | + | + | + | + | + | + | + | − | + | + | + | ||||||||||||||||||||||
| Wild-type-2 | − | + | + | + | + | + | + | − | − | − | + | + | + | ||||||||||||||||||||||
| Wild-type-3 | − | + | − | + | + | − | − | − | + | + | − | + | + | ||||||||||||||||||||||
| Wild-type-4 | − | + | − | + | + | + | + | + | + | + | |||||||||||||||||||||||||
| Ech_0379-1 | − | + | + | + | − | − | − | − | − | − | − | + | + | + | − | − | − | − | |||||||||||||||||
| Ech_0379-2 | − | + | − | + | + | − | − | − | + | + | + | − | + | + | + | − | − | − | |||||||||||||||||
| Ech_0660-1b | − | + | + | − | − | − | − | − | − | − | − | + | − | − | − | − | − | − | − | ||||||||||||||||
| Ech_0660-2b | − | − | + | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | ||||||||||||||||
| Ech_0660-3b | − | − | − | − | − | − | − | − | − | − | − | + | − | − | − | − | − | − | |||||||||||||||||
| Ech_0660-4b | − | + | − | − | − | − | − | − | − | − | − | + | − | − | − | − | − | − | |||||||||||||||||
The minus and plus signs refer to samples tested negative or positive by culture recovery and/or nested PCR, respectively. The day of challenge for Ech_0379-infected dogs was day 35 postinfection, and for Ech_0660-infected dogs it was day 29 postinfection. Total average positives in samples collected from wild-type-infected and Ech_0379- and Ech_0660-infected animals before challenge are 78%, 45%, and 10.5%, respectively (day 0 data were not included in this calculation).
Based on a binomial generalized estimating equation model used to account for repeated measures on dogs over time, Ech_0660 mutant-infected dogs were positive on significantly fewer days than wild-type-infected dogs (P = 0.017).
Blood-derived DNAs from wild-type-infected dogs and Ech_0379 and Ech_0660 mutant-infected dogs before and after challenge also were assessed by TaqMan-based real-time PCR. Average copy numbers of samples that tested positive from each group were compared to levels detected in wild-type-infected dogs (Fig. 4). E. chaffeensis copy numbers for wild-type-infected animals were significantly higher than those observed for dogs infected with Ech_0379 or Ech_0660 mutant before challenge (P = 0.007 and P = 0.007, respectively). After challenge, the copy numbers were significantly lower for Ech_0379 (P = 0.001) and Ech_0660 (P = 0.008) mutant-infected animals.
FIG 4.
Infection status in dogs following infections with mutants or the wild type assessed in blood by TaqMan assay to measure DNA copy numbers. Ehrlichia DNA copy numbers were assessed by TaqMan real-time PCR assay to measure the DNA copy numbers in dog blood following infections with the wild type (Wt) and E. chaffeensis mutant Ech_0379 or Ech_0660 before and after challenge (Ech_0379-wt and Ech_0660-wt) with the wild-type bacteria. The average values for the positive samples from each mutant group of animals were compared to values for the wild-type-infected group, and the data are presented on a semilog graph. Significant differences (P ≤ 0.05) observed between wild-type- and Ech_0660-infected groups are identified with asterisks. Due to the small sample in the Ech_0379 mutant group, statistical analysis was not performed.
Dogs infected with both Ech_0379 and Ech_0660 mutants induced IgG responses against E. chaffeensis antigens, and the responses were very similar for both groups before and after wild-type infection challenge (Fig. 5); dogs in both groups had a rise and fall in pathogen-specific IgG responses following mutant infections, and the responses were boosted initially with wild-type infection challenges and then declined with time.
FIG 5.
E. chaffeensis-specific IgG response in dogs with Ech_0379 or Ech_0660 mutant followed by challenge with wild-type E. chaffeensis. IgG levels were assessed in animals receiving mutant that had been inoculated and challenged with the wild type for several days postinfection. The data were plotted as described for Fig. 2. The day of challenge is identified with down arrows. The line graphs in each panel have different colors to represent different animals within each group.
DISCUSSION
Recently, we reported the first experiments for E. chaffeensis mutagenesis (22). In addition, we presented the first evidence demonstrating the attenuated growth of some of the E. chaffeensis mutants in vivo (22). In the current study, we assessed the potential of two attenuated mutant clones in serving as vaccine candidates to protect against Ehrlichia species infections in deer and dogs. The data presented here demonstrate that the attenuated mutants induce host response in both the reservoir host and in an incidental host, which aided in altering the infection progression in the animals when challenged with wild-type E. chaffeensis. The data presented in the current study for the incidental host are well supported by statistical analysis; however, additional experiments would be necessary to further authenticate the results for the reservoir host.
To date, there are no reports in the literature describing vaccine development to control E. chaffeensis infections in humans or animals. Ohashi et al. reported nearly 2 decades ago that immunization with a recombinant p28 outer membrane protein in BALB/c mice induced transient protection against E. chaffeensis infection (15). The current study is the first to investigate the vaccine potential of attenuated transposon insertion mutant clones in vertebrate hosts. Live attenuated pathogenic bacteria are ideal in developing host responses to confer protection against infection challenge, as they are likely to activate all arms of the immune system to provide long-lasting immunity. We reported in this study that the attenuated mutation in the phage-like protein-coding gene, Ech_0660, generated sufficient host response to clear infection in dogs. Another attenuated mutant with a mutation located in a putative Na+/H+ antiporter protein-coding gene (Ech_0379), while valuable in reducing bacteremia, did not completely eliminate wild-type E. chaffeensis. The E. chaffeensis genome (NC_007799) contains several genes having significant homology to predicted antiporter protein genes (Ech_0179, Ech_0466, Ech_0467, Ech_0469, Ech_0474, and Ech_0944), whereas no known homologs for Ech_0660 are present. As E. chaffeensis replicates in acidified endosomes (27), we reasoned that the Na+/H+ antiporter proteins are critical for the bacterium to maintain its intracellular pH for optimal growth. It is likely that in the absence of Ech_0379 gene product, other antiporter proteins complement its function to some extent. At this time, we do not know how antiporter proteins contribute to the continued survival of E. chaffeensis. Similarly, much remains to be understood about the importance of the Ech_0660 gene product having homology to head-tail connector protein gp6 of bacteriophage HK97 for the pathogen's growth in vivo. The E. chaffeensis genome contains several phage-related protein genes in addition to Ech_0660. Interestingly, they include genes also located at close proximity to Ech_0660, namely, Ech_0659, Ech_0661, Ech_0662, and Ech_0665, encoding gp15 family protein, small conductance ion channel protein, phage major tail protein, and phage uncharacterized protein, respectively. The genes Ech_0663 and Ech_0664 encode hypothetical proteins with unknown functions. The gene cluster spanning from Ech_0659 to Ech_0665 also differed in G+C content (25%) compared to that for the entire genome (33%) (28). At this time, we do not know the significance of this gene cluster to E. chaffeensis pathogenesis and its adaptation to the vertebrate host. The absence of a functional Ech_0660 leading to attenuated growth suggests that the genomic region plays a critical role. This hypothesis remains to be tested.
IgG responses in deer and dogs following infection and challenges with the mutants revealed both host-specific and mutant-specific differences. It is unclear why the antibody profiles differed in deer and dogs. Additional investigations are necessary to understand these differences. Bacterial numbers appeared to vary between deer and dogs when infected with the two different mutants; it is unclear if the observed differences in the IgG responses play a role in inducing protection against E. chaffeensis. Prior reports on other intracellular bacteria suggest that induction of T cell responses played a greater role in generating protection against infection than B cell responses (29, 30). Interestingly, we observed a good correlation between the E. chaffeensis copy numbers and IgG response. In particular, the Ech_0660 mutant-infected deer produced IgG levels similar to those of wild-type infection, whereas the deer infected with Ech_0379 produced very little IgG. A significant decline in E. chaffeensis copy numbers also was observed in deer infected with Ech_0660 compared to the level for Ech_0379. Dogs receiving infections with Ech_0379 and Ech_0660 mutants induced very similar IgG responses. Consistent with this observation, both groups of dogs had a significant decline in E. chaffeensis copy numbers compared to that of wild-type E. chaffeensis-infected animals. We have yet to determine how various immunological components are activated after infecting the vertebrates with wild-type or attenuated E. chaffeensis organisms. The present study is the critical first step in furthering such studies evaluating the potential of both Ech_0379 and Ech_0660 mutants as vaccine candidates.
In summary, we reported that two clonally purified insertion mutants in genes Ech_0379 and Ech_0660 of E. chaffeensis, causing attenuation of the organism, are useful in reducing rickettsemia when challenged with wild-type infections and in dropping the bacterial copy numbers in an incidental host. We also presented data demonstrating the complete clearance of the pathogen from both the reservoir and an incidental host when animals receive the attenuated clone containing a mutation in the Ech_0660 gene. This study is important in extending future investigations assessing the growth kinetics of mutants and molecular studies to define how gene expression is altered in E. chaffeensis and to further study the potential of attenuated mutants as immunogens to protect animals and people against E. chaffeensis infection.
ACKNOWLEDGMENTS
This work was supported by PHS grant number AI070908 from the National Institute of Allergy and Infectious Diseases.
We acknowledge Lisa Coburn at the OSU Tick Facility for support in rearing ticks. We also thank Mal Rooks Hoover for preparing the figures.
Footnotes
This article is a contribution from the Kansas Agricultural Experiment Station (number 15-203-J).
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