Coinfection with Anaplasma phagocytophilum Alters Borrelia burgdorferi Population Distribution in C3H/HeN Mice
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感染与免疫杂志 2005年第6期
Center for Comparative Medicine, Schools of Medicine and Veterinary Medicine, University of California at Davis, Davis, California 95616
Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California at Davis, Davis, California 95616
ABSTRACT
Borrelia burgdorferi, the agent of Lyme disease, and Anaplasma phagocytophilum, the agent of human anaplasmosis, are both transmitted by Ixodes sp. ticks and may occasionally coinfect a host. The population distributions of tick-transmitted B. burgdorferi infection were assessed using quantitative PCR targeting the flaB gene of B. burgdorferi in the ear, heart base, quadriceps muscle, skin, and tibiotarsal joint tissue of C3H mice previously infected with A. phagocytophilum. Population distributions of Anaplasma infection were assessed by targeting the p44 gene. A. phagocytophilum in blood and serologic response to both agents were evaluated. Spirochete numbers were increased in the ears, heart base, and skin of coinfected mice, but Anaplasma numbers remained constant. Antibody response to A. phagocytophilum, but not B. burgdorferi, was decreased in coinfected mice. These results suggest that coinfection with A. phagocytophilum and B. burgdorferi modulates pathogen burden and host antibody responses. This may be explained by the ability of A. phagocytophilum to functionally impair neutrophils, important cells in the early defense against B. burgdorferi infection.
INTRODUCTION
Borrelia burgdorferi, the causative agent of Lyme disease, is the most common tick-borne pathogen in the United States and is a significant cause of morbidity throughout the world (30). Lyme disease is a multiphasic, multisystem syndrome that may be characterized by dermatological, musculoskeletal, and neurological manifestations. The spirochete bacterium is transmitted to humans via the bite of infected ticks belonging to the genus Ixodes. In recent years, another zoonotic pathogen, Anaplasma phagocytophilum, the agent of human anaplasmosis (formerly designated as human granulocytic ehrlichiosis, or HGE) has been detected in Ixodes ticks in both the United States and Europe (5, 26). Anaplasma phagocytophilum is an obligate, intracellular bacterium that infects the granulocytes, primarily neutrophils, of mammals. Clinical manifestations of human anaplasmosis may include a wide array of symptoms involving the hematopoietic, immune, and nervous systems; involvement can range from a mild, self-limiting disease to a severe, life-threatening condition (2). Since A. phagocytophilum shares a common vector with B. burgdorferi, cases of anaplasmosis often occur in areas where Lyme disease is endemic (2, 23). Inevitably, questing ticks coinfected with B. burgdorferi and A. phagocytophilum have been identified in these regions (17, 29). Furthermore, the simultaneous acquisition, coinfection, and transmission of both of these agents in the tick vector to the laboratory mouse have recently been established (15, 20).
Dual infections with B. burgdorferi and A. phagocytophilum have been documented in both human patients, wild rodents, and laboratory mice (20, 21, 33, 34). In humans, several distinctive clinical presentations aid in the differential diagnosis of Lyme disease from anaplasmosis. However, in coinfection scenarios, patients may present with a confusing mixture of manifestations, making diagnosis problematic (21, 24 25). Undoubtedly, anaplasmosis may complicate the disease severity and prognosis of Lyme disease (7, 31). The frequency of coinfection and the resulting clinical outcome in humans is largely unknown and has been the focus of several studies (2, 31). Ultimately, the immunosuppressive nature of anaplasmosis may invariably affect the outcome and duration of B. burgdorferi infection.
The pathogenesis of Lyme disease and anaplasmosis has been well documented in murine model systems (10, 13, 14, 16, 28, 35). However, only two studies to date have focused on investigating the coinfection phenomenon (33, 34). Zeidner et al. reported that when cotransmitted by ticks, B. burgdorferi and A. phagocytophilum act synergistically to modulate host immune responses, possibly providing a greater opportunity for either pathogen to escape initial immune surveillance (34). Moreover, Thomas et al. showed that in addition to modulation of host immune responses, coinfected mice suffered from higher pathogen burdens and more severe arthritis when B. burgdorferi and A. phagocytophilum were cotransmitted via syringe inoculation (33). Thus, simultaneous coinfection with B. burgdorferi and A. phagocytophilum appears to enhance the pathogenesis of Lyme disease in laboratory mice. However, a number of alternative coinfection scenarios may exist in nature. Perhaps a more frequent occurrence is that hosts may acquire one infection before the other. Given the evidence that Anaplasma infection can be immunosuppressive, it is important to consider this effect on subsequent tick-borne infection with B. burgdorferi, particularly in light of the fact that B. burgdorferi has been shown to significantly influence the immune status of the host (9, 18, 22, 28, 33).
The purpose of this study was to determine the effect that an established A. phagocytophilum infection has upon a subsequent infection with B. burgdorferi. Previously, our laboratory had developed accurate, sensitive, molecular techniques for assessing population dynamics and distribution of B. burgdorferi and A. phagocytophilum in mice by the use of real-time quantitative PCR (qPCR) (12, 13, 14). Herein we have utilized these tools to demonstrate that prior tick-borne infection with A. phagocytophilum alters the population distribution and antibody response in mice subsequently infected with tick-borne B. burgdorferi.
MATERIALS AND METHODS
Mice. Specific-pathogen-free, 3- to 5-week-old C3H/HeN (Frederick Cancer Research Center, Frederick, MD) and C3H/Smn.CIcr-scid (SCID) mice (Harlan, Indianapolis, IN) were used in this study based upon their susceptibility to infection and disease with both B. burgdorferi and A. phagocytophilum (16, 28, 33). Mice were maintained in individual isolator cages within an infectious disease containment room and fed commercial mouse diet and water ad libitum. Mice were euthanized by carbon dioxide asphyxiation.
Bacteria. A low-passage, clonal strain of B. burgdorferi N40 sensu stricto was maintained in modified BSK II medium supplemented with 6% rabbit serum (1). Cells were enumerated in a bacterial counting chamber as described previously (14). For the development of B. burgdorferi-infected ticks (see below), five C3H mice were each inoculated intradermally at the thoracic dorsal midline with 103 mid-log-phase spirochetes. The NCH-1 isolate of A. phagocytophilum was maintained via serial passage from infected SCID mice to nave SCID mice every 3 weeks by intraperitoneal inoculation of 0.1 ml EDTA-anticoagulated blood. For the development of A. phagocytophilum-infected ticks (see below), five C3H mice were inoculated intraperitoneally with blood from infected SCID mice.
Ticks. Mated adult female Ixodes scapularis ticks were kindly provided by Durland Fish of Yale University, New Haven, Connecticut. The egg mass from a single tick produced the uninfected larvae for experimental use. Three groups of five C3H mice each were infected with B. burgdorferi or A. phagocytophilum or sham inoculated with sterile BSK II medium (negative control). After 2 weeks, infection was confirmed by PCR (see below) of collected ear notches (for B. burgdorferi) or determination of morulae on peripheral blood smears and PCR (for A. phagocytophilum) using blood collected by tail bleeding. Larval ticks were allowed to attach and engorge upon three groups of five C3H mice anesthetized with a ketamine-xylazine cocktail and maintained in individual cages in order to generate B. burgdorferi-infected, A. phagocytophilum-infected, or uninfected nymphs. Engorged larvae were collected and allowed to molt and harden into nymphs. Tick rearing was conducted in an incubator at 21°C with 95% relative humidity. Ten percent of molted nymphs from each infection group were individually tested by PCR to confirm infection and to determine prevalence. None (28 individual tick samples) of the nymphs derived from larvae that fed on uninfected mice were positive for either pathogen. Infection prevalences were 93.33% (30 samples) for B. burgdorferi-infected nymphs and 31.81% (22 samples) for A. phagocytophilum-infected nymphs.
Tissue collection. At necropsy, blood was collected in EDTA-coated tubes by venipuncture. Blood was chosen for this analysis because A. phagocytophilum is found within granulocytes throughout the course of infection (7, 16). A small aliquot (100 μl) of blood from each mouse was collected for PCR. The remainder was centrifuged at 15,000 x g to pellet cells. Plasma was recovered and frozen at –20°C for use in an enzyme-linked immunosorbent assay (ELISA). Samples of urinary bladder and inoculation site (subcutaneous fascia, directly beneath the skin in the shoulder area where ticks were placed for infestation) were placed in modified BSK II medium for B. burgdorferi cultures using aseptic techniques. Ears, skin, heart base, quadriceps muscle, and left tibiotarsus joint were individually stored, snap-frozen, and kept at –20°C until they could be weighed and processed for DNA extraction. Tissues chosen for analysis were selected because they are persistently colonized by B. burgdorferi during infection (13). Right and left knees, right tibiotarsus joint, and the remainder of the heart were formalin fixed, paraffin embedded, and processed for histological analysis. Sections were mounted on slides and stained with hematoxylin and eosin. Slides were coded and examined in a blinded fashion for prevalence and severity of arthritis, as described previously (8, 13).
DNA extraction, quantitative PCR, and analysis of bacterial population distribution. DNA was extracted from B. burgdorferi cultures, ticks, and tissues using DNeasy tissue kits (QIAGEN, Valencia, CA) according to the manufacturer's suggested protocols. Prior to DNA extraction, all tissues were individually weighed and their mass was recorded. DNA extracts of tissue samples were subjected to qPCR, as described previously (12, 13, 14). Using Primer Express software (PE Biosystems, Foster City, CA), primers and probes were designed and optimized as described previously (12, 13, 14) to specifically detect B. burgdorferi in tissues or A. phagocytophilum in blood samples. Primers were specific for the target genes flaB for B. burgdorferi, which produces a 107-bp fragment, and p44 for A. phagocytophilum, which produces a 71-bp fragment. The flaB gene was selected because this gene codes for a major structural protein of the spirochete flagella and since it is required for motility and infectivity is expressed constitutively throughout infection (13, 14, 35). In addition, a single copy is known to exist within the B. burgdorferi genome, and its expression has been shown to occur at a constant level during infection, irrespective of spirochete density (27). Furthermore, this model has been previously used to elucidate spirochete populations in infections of murine hosts (13, 14, 27, 35). The p44 gene was chosen as a target to study the population distribution of A. phagocytophilum since it codes for a surface protein that is expressed constitutively throughout infection (12). Presently, at least 18 full-length copies of this gene are known to be present in the A. phagocytophilum genome (36). All reactions included positive and negative controls, to verify the validity of positive and negative findings. To reduce the risk of DNA contamination, DNA extractions and PCRs were conducted in separate, dedicated locations. Copy numbers of target genes were expressed per milligram of tissue weight. Quantitative analysis was performed as described previously (12, 13, 14).
Serology. Whole-cell lysates of B. burgdorferi and A. phagocytophilum were prepared for use in enzyme-linked immunosorbent assay as previously described (33), using 14-day-old cultured spirochetes and A. phagocytophilum-infected HL-60 cells maintained according to established protocols (11). Lysate protein concentrations were determined using a Bradford assay (Bio-Rad Laboratories, Hercules, CA) and stored at –20°C until use. Ninety-six-well ELISA plates (Nalge Nunc, Rochester, NY) were coated with 1 μg of either B. burgdorferi or A. phagocytophilum lysates in 100 μl of carbonate coating buffer (0.2 g of NaN3, 1.59 g of Na2CO3, and 2.93 g of NaHCO3 in 1 liter of distilled water [pH 9.6]) and kept overnight at 4°C. Plates were washed three times with wash buffer (1x phosphate-buffered saline with Triton X). Nonspecific binding sites were blocked with 200 μl of blocking buffer (10% bovine serum albumin in phosphate-buffered saline) at room temperature for 1 h. After removal of blocking buffer, duplicate samples of each mouse serum containing primary antibody (diluted 1:500 in blocking buffer), including uninfected normal mouse serum as a control, were added and incubated overnight at 4°C. Plates were then washed four times with wash buffer, and 100 μl of a secondary antibody (alkaline phosphatase-conjugated goat anti-mouse immunoglobulin G heavy and light chain [Jackson ImmunoResearch Laboratories, West Grove, PA], diluted 1:3,000 in blocking buffer) was added and incubated at room temperature for 2 h. After washing four times with wash buffer, 100 μl of an enzyme substrate, p-nitrophenyl phosphate (Sigma, St. Louis, MO), was added and the mixture was incubated at room temperature for 10 min. Optical densities at 405 nm were read on a Kinetic microplate reader (Molecular Devices, Sunnyvale, CA). The mean absorbance and standard deviation for duplicate experimental samples and control uninfected normal mouse serum were calculated. Presence of antibody was confirmed when measured absorbance exceeded three standard deviations of the mean titer of the control normal mouse serum.
Statistics. Since each treatment group consisted of five mice, a statistical average and standard deviation of the target gene copy number were derived to account for natural variances within the bacterial populations and to represent the group as a whole. To determine significant differences in these copy numbers, mean numbers of A. phagocytophilum were compared on a logarithmic scale and subjected to statistical analysis using a Student's t test.
RESULTS
This study examined the effect of ongoing tick-borne A. phagocytophilum infection in mice and subsequent susceptibility to tick-borne B. burgdorferi infection. Previous studies have shown that A. phagocytophilum infection reaches its zenith between days 5 and 10 after infected tick attachment in C3H mice (14). Thus, day 7 was chosen to examine blood smears for the presence of morulae and to extract DNA for PCR to confirm infection with A. phagocytophilum. Day 8 was chosen for exposure to tick-borne B. burgdorferi. Four experimental groups of five C3H mice each were established. Group 1 mice were infested with A. phagocytophilum-positive ticks on day 0 and then infested with B. burgdorferi-positive ticks on day 8. Group 2 mice were infested with A. phagocytophilum-positive ticks and then infested with uninfected ticks on day 8. Group 3 mice were infested with uninfected ticks and then infested with B. burgdorferi-positive ticks on day 8. Group 4 mice were infested with uninfected ticks on day 0 and day 8. At each feeding, 4 ticks were allowed to feed to engorgement, and all feedings were performed on the same time schedule, with mice maintained in individual cages. Previous studies have shown that B. burgdorferi infection and disease reach their peak in C3H mice at 14 days after syringe inoculation (13, 14). Thus, on day 24 (16 days after B. burgdorferi-positive or -negative tick exposure), mice were necropsied. Blood and tissues were collected for PCR, culture, and histology.
All mice in groups 1 and 2 (infested with A. phagocytophilum-infected ticks) had confirmed infections with A. phagocytophilum, based upon positive PCR results and the presence of morulae in peripheral blood smears on day 7. Mice in groups 3 and 4 (infested with uninfected ticks) were negative. On day 24, all mice in groups 1 and 3 were culture and PCR positive for B. burgdorferi and all mice in groups 2 and 4 were negative. Therefore, all experimental mice were determined to be suitable for analysis.
Quantitative values for A. phagocytophilum were calculated based on the number of p44 gene copy numbers present in 50 μl of blood collected at necropsy (day 24). Amplification of the A. phagocytophilum p44 gene by qPCR was successful in blood samples from all mice in groups 1 and 2 on day 24 that were previously shown to be infected with the agent on day 7 (Fig. 1). However, there was no significant difference (P = 0.063) in copy numbers, and therefore bacterial populations, between mice that were singly infected with A. phagocytophilum (group 2) and those that were coinfected with B. burgdorferi (group 1).
The populations of B. burgdorferi in various tissues were quantified by calculating the number of flaB copies present in DNA per milligram of tissue. Evaluation of two different skin sites showed differences in spirochete population distribution. Mice coinfected with A. phagocytophilum and B. burgdorferi (group 1) were found to have significantly higher levels of spirochetes present in both the ear and skin than mice singly infected with B. burgdorferi (group 3) (Fig. 2) (P = 0.026 and 0.0034, respectively). No flaB copy numbers were found in the skin of any mice that were only infected with B. burgdorferi (group 3). Analysis of heart base tissues also showed a difference in spirochete populations (Fig. 2). Coinfected mice (group 1) were consistently found to have significantly greater B. burgdorferi populations than mice singly infected with B. burgdorferi (group 3) (P = 0.017). No significant differences in spirochete population distribution were identified between coinfected (group 1) mice and B. burgdorferi-infected mice (group 3) in tibiotarsal joints and quadriceps muscle (Fig. 2) (P = 0.379 and 0.585, respectively).
Antibody titers against B. burgdorferi or A. phagocytophilum lysates were determined for each treatment group. The antibody response against A. phagocytophilum at day 24 after tick-borne infection differed between mice singly infected with the agent (group 2) and those coinfected with B. burgdorferi (group 1). With sera from five mice in each infection group, average titers for the A. phagocytophilum-infected group were 2,700, whereas average titers from the coinfected group were 900, indicating a statistically lower (P = 0.374) overall antibody response. The antibody response against B. burgdorferi at day 17 after tick-borne infection was similar for mice singly infected with B. burgdorferi (group 3) and those coinfected (group 1). With sera from five mice in each infection group, the average titer for B. burgdorferi-infected mice was 2,700 and the average titer for the coinfected mice was also 2,700.
Histopathologic examination of mouse hearts failed to elucidate significant differences in carditis prevalence or severity between coinfected (group 1) and singly infected B. burgdorferi (group 3) mice. Analysis of joints revealed that coinfected mice showed a low prevalence of mild arthritis (data not shown), whereas singly infected mice showed no arthritis in any of the joints examined.
DISCUSSION
Previously, B. burgdorferi and A. phagocytophilum coinfection studies in mice have focused on the cotransmission of these agents simultaneously and the resulting coevolution of disease (33, 34). However, a wide range of coinfection scenarios exist in nature, and the frequency of their occurrences is unknown. Although ticks may harbor both pathogens, prevalence studies in endemic areas throughout the United States have shown that coinfection rates may be as little as 1 to 6% (6, 17) or as high as 26% (3), whereas the rate of ticks singly infected with B. burgdorferi can be up to 40% (6) and the rate of ticks singly infected with A. phagocytophilum can be up to 20% (19). In addition, transmission time, from tick to host, may differ for each pathogen (20). When one considers that natural hosts, such as mice and deer, and incidental hosts such as humans, may be infested with more than one tick at a given time, it makes it difficult to assign the order or timing in which coinfections might take place. Simultaneous infection is the simplest situation to create in a controlled experimental setting. Yet perhaps a more frequent occurrence is that hosts acquire one infection prior to the other. Due to the documented immunosuppressive nature of Anaplasma infection (2, 4) and its nature of causing either symptomatic or asymptomatic infections (7), an important avenue of exploration is the effect that this infection may have upon subsequently acquired tick-borne B. burgdorferi. It is well established that infection population dynamics and disease susceptibility in mice infected with B. burgdorferi are significantly affected by immune competence of the host (13). Thus, preinfection with an agent such as A. phagocytophilum, which may cause immunosuppression, may therefore have a significant effect upon subsequent B. burgdorferi infection. Thus, in this study, we elected to initially infect mice with tick-borne A. phagocytophilum and then at the approximate peak of infection (14) we challenged them with tick-borne B. burgdorferi. This design offered the opportunity to test the hypothesis that prior infection with A. phagocytophilum, at an interval at which effects on the host are likely to be maximal, has an effect upon subsequent infection with B. burgdorferi.
The current study found increases in B. burgdorferi populations in the subcutis, skin, ear, and heart base of mice coinfected with A. phagocytophilum. An interesting observation was that none of the mice that were singly infected with B. burgdorferi were PCR positive for skin, yet they were culture positive at the inoculation site. In contrast, skin and subcutis from all of the mice that were coinfected were PCR positive and culture positive, respectively. Although this may appear contrary, it is important to note that subcutaneous fascia directly beneath the skin (subcutis) was cultured, and PCR was performed on the overlying skin. Thus, spirochetes were present in both the skin and subcutis in coinfected mice and in significantly greater amounts in the ear samples of coinfected mice (which consist of skin, subcutis, and cartilage). This phenomenon of persistence in the skin may lead support to observations made by Thomas et al., who noted that acquisition of both B. burgdorferi and A. phagocytophilum by feeding ticks is enhanced in coinfection scenarios (33).
The immunosuppressive effects of Anaplasma infection appear to be varied. Human patients enduring this disease can readily become susceptible to opportunistic infections (4, 7). Featured defects in host immunity during this infection include T- or B-cell suppression, with notable decreases in CD4 and CD8 cell counts, and impaired lymphoproliferation of isolated lymphocytes (7). Also of importance, recent studies have shown that neutrophil function, specifically phagocytic ability, can be significantly impaired in anaplasmosis (4). This is of particular interest because studies have shown that B. burgdorferi spirochetes are susceptible to phagocytosis and a novel oxidative burst mechanism exhibited by these cells (32). Although B. burgdorferi employs a variety of immune evasion mechanisms to survive and disseminate in the host, neutrophils are thought to play a key role in the early defense against infection. Impairment of this innate ability, in concert with the overall picture of immunosuppression, may explain the increase in B. burgdorferi population distribution in coinfected mice.
Despite the increased numbers of spirochetes in several tissues of coinfected mice, including heart and joint, histopathologic observations did not reveal more severe disease. Spirochetes were increased in heart tissue of coinfected mice, yet there were no appreciable difference in carditis severity, and were found in approximately equal numbers in quadriceps muscle and tibiotarsal joint of singly and coinfected mice, and histopathology did not suggest a significant increase in arthritis severity in the coinfected mice. This may have been due to immune suppression from A. phagocytophilum and lack of an appropriate inflammatory response. Alternatively, it may be a reflection of the mouse model, as disease is relatively mild or absent in older mice, and by necessity of the experimental design, disease could not be evaluated until the mice were 8 to 9 weeks of age.
Coinfection did not significantly impair antibody response to either pathogen. In spite of the fact that coinfected mice harbored larger populations of spirochetes in several tissues, ELISA results indicated that the antibody responses to B. burgdorferi were not significantly different between coinfected and singly infected mice. This is similar to findings by Thomas et al. (33) in which mice were simultaneously coinfected. Although our results show that coinfection with B. burgdorferi does not alter levels of A. phagocytophilum in the blood, coinfection does appear to alter the antibody response to A. phagocytophilum. The observation of a diminished antibody response in coinfected mice in this study is consistent with previous reports by Zeidner et al., who further noted that differences in cytokine profiles indicated that potential immune modulation mechanisms were active in a coinfection scenario (33, 34).
We have shown that coinfection with A. phagocytophilum can influence the population distribution of spirochetes during infection of a murine host. By some means of immunomodulation, this allows B. burgdorferi to persist longer in particular tissues and to exist in overall higher numbers within the host. Although A. phagocytophilum has its own immune evasion capabilities, it is likely that such a coinfection scenario aids this bacterium in continued persistence. Additional studies are needed in order to prove that anaplasmosis invariably affects the clinical presentation and duration of Lyme disease.
ACKNOWLEDGMENTS
We thank Edward Lorenzana for technical support.
This work was supported by Public Health Service grant AI26815 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health.
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Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California at Davis, Davis, California 95616
ABSTRACT
Borrelia burgdorferi, the agent of Lyme disease, and Anaplasma phagocytophilum, the agent of human anaplasmosis, are both transmitted by Ixodes sp. ticks and may occasionally coinfect a host. The population distributions of tick-transmitted B. burgdorferi infection were assessed using quantitative PCR targeting the flaB gene of B. burgdorferi in the ear, heart base, quadriceps muscle, skin, and tibiotarsal joint tissue of C3H mice previously infected with A. phagocytophilum. Population distributions of Anaplasma infection were assessed by targeting the p44 gene. A. phagocytophilum in blood and serologic response to both agents were evaluated. Spirochete numbers were increased in the ears, heart base, and skin of coinfected mice, but Anaplasma numbers remained constant. Antibody response to A. phagocytophilum, but not B. burgdorferi, was decreased in coinfected mice. These results suggest that coinfection with A. phagocytophilum and B. burgdorferi modulates pathogen burden and host antibody responses. This may be explained by the ability of A. phagocytophilum to functionally impair neutrophils, important cells in the early defense against B. burgdorferi infection.
INTRODUCTION
Borrelia burgdorferi, the causative agent of Lyme disease, is the most common tick-borne pathogen in the United States and is a significant cause of morbidity throughout the world (30). Lyme disease is a multiphasic, multisystem syndrome that may be characterized by dermatological, musculoskeletal, and neurological manifestations. The spirochete bacterium is transmitted to humans via the bite of infected ticks belonging to the genus Ixodes. In recent years, another zoonotic pathogen, Anaplasma phagocytophilum, the agent of human anaplasmosis (formerly designated as human granulocytic ehrlichiosis, or HGE) has been detected in Ixodes ticks in both the United States and Europe (5, 26). Anaplasma phagocytophilum is an obligate, intracellular bacterium that infects the granulocytes, primarily neutrophils, of mammals. Clinical manifestations of human anaplasmosis may include a wide array of symptoms involving the hematopoietic, immune, and nervous systems; involvement can range from a mild, self-limiting disease to a severe, life-threatening condition (2). Since A. phagocytophilum shares a common vector with B. burgdorferi, cases of anaplasmosis often occur in areas where Lyme disease is endemic (2, 23). Inevitably, questing ticks coinfected with B. burgdorferi and A. phagocytophilum have been identified in these regions (17, 29). Furthermore, the simultaneous acquisition, coinfection, and transmission of both of these agents in the tick vector to the laboratory mouse have recently been established (15, 20).
Dual infections with B. burgdorferi and A. phagocytophilum have been documented in both human patients, wild rodents, and laboratory mice (20, 21, 33, 34). In humans, several distinctive clinical presentations aid in the differential diagnosis of Lyme disease from anaplasmosis. However, in coinfection scenarios, patients may present with a confusing mixture of manifestations, making diagnosis problematic (21, 24 25). Undoubtedly, anaplasmosis may complicate the disease severity and prognosis of Lyme disease (7, 31). The frequency of coinfection and the resulting clinical outcome in humans is largely unknown and has been the focus of several studies (2, 31). Ultimately, the immunosuppressive nature of anaplasmosis may invariably affect the outcome and duration of B. burgdorferi infection.
The pathogenesis of Lyme disease and anaplasmosis has been well documented in murine model systems (10, 13, 14, 16, 28, 35). However, only two studies to date have focused on investigating the coinfection phenomenon (33, 34). Zeidner et al. reported that when cotransmitted by ticks, B. burgdorferi and A. phagocytophilum act synergistically to modulate host immune responses, possibly providing a greater opportunity for either pathogen to escape initial immune surveillance (34). Moreover, Thomas et al. showed that in addition to modulation of host immune responses, coinfected mice suffered from higher pathogen burdens and more severe arthritis when B. burgdorferi and A. phagocytophilum were cotransmitted via syringe inoculation (33). Thus, simultaneous coinfection with B. burgdorferi and A. phagocytophilum appears to enhance the pathogenesis of Lyme disease in laboratory mice. However, a number of alternative coinfection scenarios may exist in nature. Perhaps a more frequent occurrence is that hosts may acquire one infection before the other. Given the evidence that Anaplasma infection can be immunosuppressive, it is important to consider this effect on subsequent tick-borne infection with B. burgdorferi, particularly in light of the fact that B. burgdorferi has been shown to significantly influence the immune status of the host (9, 18, 22, 28, 33).
The purpose of this study was to determine the effect that an established A. phagocytophilum infection has upon a subsequent infection with B. burgdorferi. Previously, our laboratory had developed accurate, sensitive, molecular techniques for assessing population dynamics and distribution of B. burgdorferi and A. phagocytophilum in mice by the use of real-time quantitative PCR (qPCR) (12, 13, 14). Herein we have utilized these tools to demonstrate that prior tick-borne infection with A. phagocytophilum alters the population distribution and antibody response in mice subsequently infected with tick-borne B. burgdorferi.
MATERIALS AND METHODS
Mice. Specific-pathogen-free, 3- to 5-week-old C3H/HeN (Frederick Cancer Research Center, Frederick, MD) and C3H/Smn.CIcr-scid (SCID) mice (Harlan, Indianapolis, IN) were used in this study based upon their susceptibility to infection and disease with both B. burgdorferi and A. phagocytophilum (16, 28, 33). Mice were maintained in individual isolator cages within an infectious disease containment room and fed commercial mouse diet and water ad libitum. Mice were euthanized by carbon dioxide asphyxiation.
Bacteria. A low-passage, clonal strain of B. burgdorferi N40 sensu stricto was maintained in modified BSK II medium supplemented with 6% rabbit serum (1). Cells were enumerated in a bacterial counting chamber as described previously (14). For the development of B. burgdorferi-infected ticks (see below), five C3H mice were each inoculated intradermally at the thoracic dorsal midline with 103 mid-log-phase spirochetes. The NCH-1 isolate of A. phagocytophilum was maintained via serial passage from infected SCID mice to nave SCID mice every 3 weeks by intraperitoneal inoculation of 0.1 ml EDTA-anticoagulated blood. For the development of A. phagocytophilum-infected ticks (see below), five C3H mice were inoculated intraperitoneally with blood from infected SCID mice.
Ticks. Mated adult female Ixodes scapularis ticks were kindly provided by Durland Fish of Yale University, New Haven, Connecticut. The egg mass from a single tick produced the uninfected larvae for experimental use. Three groups of five C3H mice each were infected with B. burgdorferi or A. phagocytophilum or sham inoculated with sterile BSK II medium (negative control). After 2 weeks, infection was confirmed by PCR (see below) of collected ear notches (for B. burgdorferi) or determination of morulae on peripheral blood smears and PCR (for A. phagocytophilum) using blood collected by tail bleeding. Larval ticks were allowed to attach and engorge upon three groups of five C3H mice anesthetized with a ketamine-xylazine cocktail and maintained in individual cages in order to generate B. burgdorferi-infected, A. phagocytophilum-infected, or uninfected nymphs. Engorged larvae were collected and allowed to molt and harden into nymphs. Tick rearing was conducted in an incubator at 21°C with 95% relative humidity. Ten percent of molted nymphs from each infection group were individually tested by PCR to confirm infection and to determine prevalence. None (28 individual tick samples) of the nymphs derived from larvae that fed on uninfected mice were positive for either pathogen. Infection prevalences were 93.33% (30 samples) for B. burgdorferi-infected nymphs and 31.81% (22 samples) for A. phagocytophilum-infected nymphs.
Tissue collection. At necropsy, blood was collected in EDTA-coated tubes by venipuncture. Blood was chosen for this analysis because A. phagocytophilum is found within granulocytes throughout the course of infection (7, 16). A small aliquot (100 μl) of blood from each mouse was collected for PCR. The remainder was centrifuged at 15,000 x g to pellet cells. Plasma was recovered and frozen at –20°C for use in an enzyme-linked immunosorbent assay (ELISA). Samples of urinary bladder and inoculation site (subcutaneous fascia, directly beneath the skin in the shoulder area where ticks were placed for infestation) were placed in modified BSK II medium for B. burgdorferi cultures using aseptic techniques. Ears, skin, heart base, quadriceps muscle, and left tibiotarsus joint were individually stored, snap-frozen, and kept at –20°C until they could be weighed and processed for DNA extraction. Tissues chosen for analysis were selected because they are persistently colonized by B. burgdorferi during infection (13). Right and left knees, right tibiotarsus joint, and the remainder of the heart were formalin fixed, paraffin embedded, and processed for histological analysis. Sections were mounted on slides and stained with hematoxylin and eosin. Slides were coded and examined in a blinded fashion for prevalence and severity of arthritis, as described previously (8, 13).
DNA extraction, quantitative PCR, and analysis of bacterial population distribution. DNA was extracted from B. burgdorferi cultures, ticks, and tissues using DNeasy tissue kits (QIAGEN, Valencia, CA) according to the manufacturer's suggested protocols. Prior to DNA extraction, all tissues were individually weighed and their mass was recorded. DNA extracts of tissue samples were subjected to qPCR, as described previously (12, 13, 14). Using Primer Express software (PE Biosystems, Foster City, CA), primers and probes were designed and optimized as described previously (12, 13, 14) to specifically detect B. burgdorferi in tissues or A. phagocytophilum in blood samples. Primers were specific for the target genes flaB for B. burgdorferi, which produces a 107-bp fragment, and p44 for A. phagocytophilum, which produces a 71-bp fragment. The flaB gene was selected because this gene codes for a major structural protein of the spirochete flagella and since it is required for motility and infectivity is expressed constitutively throughout infection (13, 14, 35). In addition, a single copy is known to exist within the B. burgdorferi genome, and its expression has been shown to occur at a constant level during infection, irrespective of spirochete density (27). Furthermore, this model has been previously used to elucidate spirochete populations in infections of murine hosts (13, 14, 27, 35). The p44 gene was chosen as a target to study the population distribution of A. phagocytophilum since it codes for a surface protein that is expressed constitutively throughout infection (12). Presently, at least 18 full-length copies of this gene are known to be present in the A. phagocytophilum genome (36). All reactions included positive and negative controls, to verify the validity of positive and negative findings. To reduce the risk of DNA contamination, DNA extractions and PCRs were conducted in separate, dedicated locations. Copy numbers of target genes were expressed per milligram of tissue weight. Quantitative analysis was performed as described previously (12, 13, 14).
Serology. Whole-cell lysates of B. burgdorferi and A. phagocytophilum were prepared for use in enzyme-linked immunosorbent assay as previously described (33), using 14-day-old cultured spirochetes and A. phagocytophilum-infected HL-60 cells maintained according to established protocols (11). Lysate protein concentrations were determined using a Bradford assay (Bio-Rad Laboratories, Hercules, CA) and stored at –20°C until use. Ninety-six-well ELISA plates (Nalge Nunc, Rochester, NY) were coated with 1 μg of either B. burgdorferi or A. phagocytophilum lysates in 100 μl of carbonate coating buffer (0.2 g of NaN3, 1.59 g of Na2CO3, and 2.93 g of NaHCO3 in 1 liter of distilled water [pH 9.6]) and kept overnight at 4°C. Plates were washed three times with wash buffer (1x phosphate-buffered saline with Triton X). Nonspecific binding sites were blocked with 200 μl of blocking buffer (10% bovine serum albumin in phosphate-buffered saline) at room temperature for 1 h. After removal of blocking buffer, duplicate samples of each mouse serum containing primary antibody (diluted 1:500 in blocking buffer), including uninfected normal mouse serum as a control, were added and incubated overnight at 4°C. Plates were then washed four times with wash buffer, and 100 μl of a secondary antibody (alkaline phosphatase-conjugated goat anti-mouse immunoglobulin G heavy and light chain [Jackson ImmunoResearch Laboratories, West Grove, PA], diluted 1:3,000 in blocking buffer) was added and incubated at room temperature for 2 h. After washing four times with wash buffer, 100 μl of an enzyme substrate, p-nitrophenyl phosphate (Sigma, St. Louis, MO), was added and the mixture was incubated at room temperature for 10 min. Optical densities at 405 nm were read on a Kinetic microplate reader (Molecular Devices, Sunnyvale, CA). The mean absorbance and standard deviation for duplicate experimental samples and control uninfected normal mouse serum were calculated. Presence of antibody was confirmed when measured absorbance exceeded three standard deviations of the mean titer of the control normal mouse serum.
Statistics. Since each treatment group consisted of five mice, a statistical average and standard deviation of the target gene copy number were derived to account for natural variances within the bacterial populations and to represent the group as a whole. To determine significant differences in these copy numbers, mean numbers of A. phagocytophilum were compared on a logarithmic scale and subjected to statistical analysis using a Student's t test.
RESULTS
This study examined the effect of ongoing tick-borne A. phagocytophilum infection in mice and subsequent susceptibility to tick-borne B. burgdorferi infection. Previous studies have shown that A. phagocytophilum infection reaches its zenith between days 5 and 10 after infected tick attachment in C3H mice (14). Thus, day 7 was chosen to examine blood smears for the presence of morulae and to extract DNA for PCR to confirm infection with A. phagocytophilum. Day 8 was chosen for exposure to tick-borne B. burgdorferi. Four experimental groups of five C3H mice each were established. Group 1 mice were infested with A. phagocytophilum-positive ticks on day 0 and then infested with B. burgdorferi-positive ticks on day 8. Group 2 mice were infested with A. phagocytophilum-positive ticks and then infested with uninfected ticks on day 8. Group 3 mice were infested with uninfected ticks and then infested with B. burgdorferi-positive ticks on day 8. Group 4 mice were infested with uninfected ticks on day 0 and day 8. At each feeding, 4 ticks were allowed to feed to engorgement, and all feedings were performed on the same time schedule, with mice maintained in individual cages. Previous studies have shown that B. burgdorferi infection and disease reach their peak in C3H mice at 14 days after syringe inoculation (13, 14). Thus, on day 24 (16 days after B. burgdorferi-positive or -negative tick exposure), mice were necropsied. Blood and tissues were collected for PCR, culture, and histology.
All mice in groups 1 and 2 (infested with A. phagocytophilum-infected ticks) had confirmed infections with A. phagocytophilum, based upon positive PCR results and the presence of morulae in peripheral blood smears on day 7. Mice in groups 3 and 4 (infested with uninfected ticks) were negative. On day 24, all mice in groups 1 and 3 were culture and PCR positive for B. burgdorferi and all mice in groups 2 and 4 were negative. Therefore, all experimental mice were determined to be suitable for analysis.
Quantitative values for A. phagocytophilum were calculated based on the number of p44 gene copy numbers present in 50 μl of blood collected at necropsy (day 24). Amplification of the A. phagocytophilum p44 gene by qPCR was successful in blood samples from all mice in groups 1 and 2 on day 24 that were previously shown to be infected with the agent on day 7 (Fig. 1). However, there was no significant difference (P = 0.063) in copy numbers, and therefore bacterial populations, between mice that were singly infected with A. phagocytophilum (group 2) and those that were coinfected with B. burgdorferi (group 1).
The populations of B. burgdorferi in various tissues were quantified by calculating the number of flaB copies present in DNA per milligram of tissue. Evaluation of two different skin sites showed differences in spirochete population distribution. Mice coinfected with A. phagocytophilum and B. burgdorferi (group 1) were found to have significantly higher levels of spirochetes present in both the ear and skin than mice singly infected with B. burgdorferi (group 3) (Fig. 2) (P = 0.026 and 0.0034, respectively). No flaB copy numbers were found in the skin of any mice that were only infected with B. burgdorferi (group 3). Analysis of heart base tissues also showed a difference in spirochete populations (Fig. 2). Coinfected mice (group 1) were consistently found to have significantly greater B. burgdorferi populations than mice singly infected with B. burgdorferi (group 3) (P = 0.017). No significant differences in spirochete population distribution were identified between coinfected (group 1) mice and B. burgdorferi-infected mice (group 3) in tibiotarsal joints and quadriceps muscle (Fig. 2) (P = 0.379 and 0.585, respectively).
Antibody titers against B. burgdorferi or A. phagocytophilum lysates were determined for each treatment group. The antibody response against A. phagocytophilum at day 24 after tick-borne infection differed between mice singly infected with the agent (group 2) and those coinfected with B. burgdorferi (group 1). With sera from five mice in each infection group, average titers for the A. phagocytophilum-infected group were 2,700, whereas average titers from the coinfected group were 900, indicating a statistically lower (P = 0.374) overall antibody response. The antibody response against B. burgdorferi at day 17 after tick-borne infection was similar for mice singly infected with B. burgdorferi (group 3) and those coinfected (group 1). With sera from five mice in each infection group, the average titer for B. burgdorferi-infected mice was 2,700 and the average titer for the coinfected mice was also 2,700.
Histopathologic examination of mouse hearts failed to elucidate significant differences in carditis prevalence or severity between coinfected (group 1) and singly infected B. burgdorferi (group 3) mice. Analysis of joints revealed that coinfected mice showed a low prevalence of mild arthritis (data not shown), whereas singly infected mice showed no arthritis in any of the joints examined.
DISCUSSION
Previously, B. burgdorferi and A. phagocytophilum coinfection studies in mice have focused on the cotransmission of these agents simultaneously and the resulting coevolution of disease (33, 34). However, a wide range of coinfection scenarios exist in nature, and the frequency of their occurrences is unknown. Although ticks may harbor both pathogens, prevalence studies in endemic areas throughout the United States have shown that coinfection rates may be as little as 1 to 6% (6, 17) or as high as 26% (3), whereas the rate of ticks singly infected with B. burgdorferi can be up to 40% (6) and the rate of ticks singly infected with A. phagocytophilum can be up to 20% (19). In addition, transmission time, from tick to host, may differ for each pathogen (20). When one considers that natural hosts, such as mice and deer, and incidental hosts such as humans, may be infested with more than one tick at a given time, it makes it difficult to assign the order or timing in which coinfections might take place. Simultaneous infection is the simplest situation to create in a controlled experimental setting. Yet perhaps a more frequent occurrence is that hosts acquire one infection prior to the other. Due to the documented immunosuppressive nature of Anaplasma infection (2, 4) and its nature of causing either symptomatic or asymptomatic infections (7), an important avenue of exploration is the effect that this infection may have upon subsequently acquired tick-borne B. burgdorferi. It is well established that infection population dynamics and disease susceptibility in mice infected with B. burgdorferi are significantly affected by immune competence of the host (13). Thus, preinfection with an agent such as A. phagocytophilum, which may cause immunosuppression, may therefore have a significant effect upon subsequent B. burgdorferi infection. Thus, in this study, we elected to initially infect mice with tick-borne A. phagocytophilum and then at the approximate peak of infection (14) we challenged them with tick-borne B. burgdorferi. This design offered the opportunity to test the hypothesis that prior infection with A. phagocytophilum, at an interval at which effects on the host are likely to be maximal, has an effect upon subsequent infection with B. burgdorferi.
The current study found increases in B. burgdorferi populations in the subcutis, skin, ear, and heart base of mice coinfected with A. phagocytophilum. An interesting observation was that none of the mice that were singly infected with B. burgdorferi were PCR positive for skin, yet they were culture positive at the inoculation site. In contrast, skin and subcutis from all of the mice that were coinfected were PCR positive and culture positive, respectively. Although this may appear contrary, it is important to note that subcutaneous fascia directly beneath the skin (subcutis) was cultured, and PCR was performed on the overlying skin. Thus, spirochetes were present in both the skin and subcutis in coinfected mice and in significantly greater amounts in the ear samples of coinfected mice (which consist of skin, subcutis, and cartilage). This phenomenon of persistence in the skin may lead support to observations made by Thomas et al., who noted that acquisition of both B. burgdorferi and A. phagocytophilum by feeding ticks is enhanced in coinfection scenarios (33).
The immunosuppressive effects of Anaplasma infection appear to be varied. Human patients enduring this disease can readily become susceptible to opportunistic infections (4, 7). Featured defects in host immunity during this infection include T- or B-cell suppression, with notable decreases in CD4 and CD8 cell counts, and impaired lymphoproliferation of isolated lymphocytes (7). Also of importance, recent studies have shown that neutrophil function, specifically phagocytic ability, can be significantly impaired in anaplasmosis (4). This is of particular interest because studies have shown that B. burgdorferi spirochetes are susceptible to phagocytosis and a novel oxidative burst mechanism exhibited by these cells (32). Although B. burgdorferi employs a variety of immune evasion mechanisms to survive and disseminate in the host, neutrophils are thought to play a key role in the early defense against infection. Impairment of this innate ability, in concert with the overall picture of immunosuppression, may explain the increase in B. burgdorferi population distribution in coinfected mice.
Despite the increased numbers of spirochetes in several tissues of coinfected mice, including heart and joint, histopathologic observations did not reveal more severe disease. Spirochetes were increased in heart tissue of coinfected mice, yet there were no appreciable difference in carditis severity, and were found in approximately equal numbers in quadriceps muscle and tibiotarsal joint of singly and coinfected mice, and histopathology did not suggest a significant increase in arthritis severity in the coinfected mice. This may have been due to immune suppression from A. phagocytophilum and lack of an appropriate inflammatory response. Alternatively, it may be a reflection of the mouse model, as disease is relatively mild or absent in older mice, and by necessity of the experimental design, disease could not be evaluated until the mice were 8 to 9 weeks of age.
Coinfection did not significantly impair antibody response to either pathogen. In spite of the fact that coinfected mice harbored larger populations of spirochetes in several tissues, ELISA results indicated that the antibody responses to B. burgdorferi were not significantly different between coinfected and singly infected mice. This is similar to findings by Thomas et al. (33) in which mice were simultaneously coinfected. Although our results show that coinfection with B. burgdorferi does not alter levels of A. phagocytophilum in the blood, coinfection does appear to alter the antibody response to A. phagocytophilum. The observation of a diminished antibody response in coinfected mice in this study is consistent with previous reports by Zeidner et al., who further noted that differences in cytokine profiles indicated that potential immune modulation mechanisms were active in a coinfection scenario (33, 34).
We have shown that coinfection with A. phagocytophilum can influence the population distribution of spirochetes during infection of a murine host. By some means of immunomodulation, this allows B. burgdorferi to persist longer in particular tissues and to exist in overall higher numbers within the host. Although A. phagocytophilum has its own immune evasion capabilities, it is likely that such a coinfection scenario aids this bacterium in continued persistence. Additional studies are needed in order to prove that anaplasmosis invariably affects the clinical presentation and duration of Lyme disease.
ACKNOWLEDGMENTS
We thank Edward Lorenzana for technical support.
This work was supported by Public Health Service grant AI26815 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health.
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