A Tick Vector Transmission Model of Monocytotropic Ehrlichiosis

Abstract

Background. Ehrlichioses are emerging, tick-borne diseases distributed worldwide. Previously established animal models use needle inoculation as a mode of infection; however, there is limited representation of natural transmission in artificially inoculated models compared with transmission by the tick vector. The objective of this study was to develop a tick vector transmission animal model of ehrlichial infection using a human pathogen, Ehrlichia muris–like agent (EMLA).

Methods. Ixodes scapularis larvae were fed on EMLA-infected mice, and after molting, infected nymphs were used to infest naive animals.

Results. Ehrlichiae were acquired by 90%–100% of feeding larvae. The majority of animals fed upon by infected nymphs developed sublethal infection with 27% lethality. Bacteria disseminated to all tissues tested with greatest bacterial loads in lungs, but also spleen, lymph nodes, liver, kidneys, brain, and bone marrow. Numerous foci of cellular infiltration, mitoses, and hepatocellular death were observed in liver. Mice infected by tick transmission developed higher antiehrlichial antibody levels than needle-inoculated animals. Tick-feeding-site reactions were observed, but there was no observed difference between animals infested with infected or uninfected ticks.

Conclusions. For the first time we were able to develop a tick transmission model with an Ehrlichia that is pathogenic for humans.

Ehrlichioses are life-threatening emerging infectious diseases transmitted by tick vectors with worldwide distribution, affecting various animals, including humans. Different tick species are vectors of ehrlichiae; however Ehrlichia species are not maintained transovarially in the vector tick [1, 2].

The occurrence of infections with these pathogens is determined by the presence of the tick vector and vertebrate reservoirs. However, the complex effects of the vector on the success of establishment of infection are not well elucidated. The influence of tick saliva on the host response to the pathogen may determine how vector transmission affects the establishment and progression of infection. There are limited number of vector transmission models in mice that mimic natural disease using the same vector and pathogen that cause naturally occurring disease. For ehrlichioses, there is no animal model with a human pathogen that has been used to study the establishment and progression of infection. Some aspects of vector transmission of infection have been demonstrated in Ehrlichia canis and E. ruminantium infections, very important veterinary diseases [3–5]. However, the experimental use of large animals and lack of inbred and gene knockout animals and limited reagents for the study of canids and ruminants are limitations of these models. Also, most ehrlichial studies have employed artificial modes of infection with no participation of the arthropod vector, which does not mimic the naturally occurring situation. Recently, we established a new mouse model using a human pathogen, Ehrlichia muris–like agent (EMLA), which mimics several aspects of human ehrlichiosis [6]; however, as in previous models, infection of mice with EMLA by needle inoculation lacks tick vector transmission of the pathogen, limiting study of the establishment of infection and the effects of early events on the outcome of infection and protective immune response.

The critical participation of the tick vector in establishment of ehrlichial infection and pathogen evasion of the host protective response was identified in a field challenge where a potential vaccine candidate, demonstrated to be effective against challenge by experimental needle inoculation, failed when vaccinated animals were exposed to natural field tick challenge [3]. Moreover, there is vast lack of knowledge of the role of tick vector transmission on how the pathogen is presented to the immune system and its influence on the outcome of infection, either leading to pathogen evasion of host defenses or immune control of infection. Tick saliva has several identified effects on vertebrate immune responses, efficiency of pathogen transmission, establishment of infection, and disease progression [7–11]. To further investigate the mechanisms involved in tick-pathogen-host interactions, we developed a mouse model of Ixodes scapularis tick transmission (TT) of the human pathogen EMLA.

MATERIAL AND METHODS

Experimental Design

All experiments were performed in 6–8-week-old female C57BL/6 mice from Harlan Laboratories (Houston, Texas). Eight mice were infected with EMLA by intravenous (IV) inoculation for tick acquisition feeding. Two other groups of 8 mice each were infected: 1 group by IV inoculation and other by the intradermal (ID) route, for comparative studies of the routes of infection. For the TT study, 2 sets of 8 mice were infested with EMLA-infected nymphal I. scapularis. Each set was separated into 2 groups; 1 group had samples collected on day 9 post–tick detachment (p.t.d.), and other group on day 30 p.t.d. Mice were infested with uninfected I. scapularis nymphs, as negative controls. An additional group of 8 mice were infested with another batch of EMLA-infected ticks, and were observed for outcome of infection. One died of an unrelated cause. A total of 15 mice were evaluated for survival of EMLA infection transmitted by ticks until day 30 p.t.d. The animals were housed and cared for by personnel from the Animal Resource Center at the University of Texas Medical Branch at Galveston, Texas. All experiments were performed according to Institutional Animal Care and Use Committee (IACUC) guidelines and approved protocols.

Ehrlichia Inocula

EMLA in cell culture and in splenocytes of C57BL/6 mice were used as inocula by the IV and ID routes. The inoculum dose was determined based on previous experiments in the EMLA mouse model [6]. For tick acquisition of ehrlichiae, the animals were infected with 10 median lethal dose (LD₅₀) (approximately 1 × 10⁵ bacteria) of EMLA. Comparative studies of the route of infection were performed using IV inoculation of a sublethal dose (0.1 LD₅₀) and ID inoculation of a high sublethal dose (equivalent to 100 LD₅₀ by IV infection).

Tick Vector

Ixodes scapularis were obtained from Oklahoma State University (Stillwater, Oklahoma). Uninfected I. scapularis larvae were used for acquisition experiments, and nymphs and adults were used as negative controls or for colony maintenance. All ticks were kept in desiccators at 21°C–22°C, with approximately 100% humidity and complete darkness. The ticks were fed on mice, guinea pigs, or rabbits, according to approved protocols.

Tick Infestations of Animals

Mouse infestations were performed by restriction method using a plastic capsule glued to the shaved back of the animal. Veterinary-usage skin glue (Kamar adhesive, Zionsville, Indiana) was used to affix the capsule to each mouse's back. Mice were maintained in individual cages for tick infestation according to arthropod containment level 2 regulations and IACUC-approved protocol. Preapproved numbers of larval or nymphal ticks (up to 50 larvae or 10 nymphs per mouse) were added to the capsules, which were monitored frequently to recover engorged ticks soon after detachment. During molting stage, ticks were maintained under the same conditions described above.

Acquisition Feeding by Ticks

Serial bacterial loads in IV-inoculated EMLA-infected mice were measured during the course of infection to determine the temporal quantitative bacteremia curve and the days of peak infection. Thus, uninfected larvae of I. scapularis were fed during the peak of bacteremia in mice inoculated IV with 10 LD₅₀ of EMLA. At 4–5 weeks after molting to nymphs, 10% of EMLA-acquired nymphs were tested by real-time polymerase chain reaction (PCR) to determine the percentage of ticks carrying EMLA. Ticks were maintained under the same conditions until transmission feeding.

Transmission Feeding of Ticks

Seven to 10 I. scapularis nymphs carrying EMLA were placed within capsules attached to the mouse's back. For the control group, the same number of uninfected I. scapularis nymphs was placed in the capsules, and the infestation was performed as described above. On days 4 and 5 of infestation, detached engorged nymphs were recovered, and the capsules were allowed to detach spontaneously. Blood and tissue samples were collected from infected and control mice on days 9 and 30 after detachment of nymphs for blood counts, clinical biochemistry, bacterial loads in tissues, antibody levels, histopathology, and immunohistochemistry (IHC) for EMLA.

Observations of Disease, Blood Counts, Serum Biochemistry, and Antibody Response

Samples obtained in the acute and late phases of infection from infected and control mice after tick feeding were compared to samples collected from mice inoculated by the IV or ID route. Whole blood samples were collected by cardiac puncture immediately after euthanasia. An aliquot of whole blood was placed in ethylenediaminetetraacetic acid–containing vials for blood counts. Total and differential counts of leukocytes, hematocrit, and platelet counts were obtained by Hemavet analyzer (Drew Scientific, Dallas, Texas). Serum samples were separated from aliquots of whole blood samples for determining the blood chemistry profile and electrolyte analysis, using Comprehensive Diagnostic Profile of the VetScan VS2 apparatus (Abaxis, Union City, California), which measures: albumin, alkaline phosphatase, alanine aminotransferase, amylase, blood urea nitrogen, calcium, creatinine, globulin, glucose, potassium, sodium, inorganic phosphorus, total bilirubin, and total protein.

Immunoglobulins M (IgM) and G (IgG) antibodies against EMLA antigen were measured by enzyme-linked immune assay (ELISA) in serum samples collected on days 9 and 30 p.t.d. or post infection (p.i.), using Protein Detector ELISA Kit (KPL, Gaithersburg, Maryland) [6]. The plates were coated with antigen lysate of EMLA propagated in RF6A cells following manufacturer's instructions. Uninfected RF6A cell lysate was used as antigen-negative control. Serum samples were tested at a dilution of 1:100, with secondary antibodies against mouse IgM and IgG labeled with alkaline phosphatase, provided by the manufacturer.

Bacterial Loads in Infected Mice

To evaluate the acquisition of EMLA by ticks, after molting, 10% of the nymphs were frozen at −80°C for 24 hours, and then nucleic acids were extracted with 1 of the 2 equally efficient methods (data not shown): DNeasy kit (Qiagen, Valencia, California) or ammonium hydroxide at 90°C for 20 minutes (using the whole tick). Tissue samples, as well as the ticks, were homogenized using Tissue Lyzer (Qiagen), and processed for DNA extraction (DNeasy kit, Qiagen) following manufacturer's instructions. Extracted DNA from blood, spleen, liver, lung, lymph node, kidney, brain, and bone marrow was assayed by amplification of the dsb (disulphide-bond formation protein) ehrlichia-specific gene fragment by real-time PCR, using primers and probe described previously [12] with few adaptations. Bacterial loads in blood, tissues, and ticks were calculated based on standard curve of dsb gene–inserted plasmid and total DNA concentration of the sample.

Histopathology and Immunohistochemistry

Tissue samples (spleen, liver, lung, lymph nodes, kidney, brain, bone marrow, and skin) were fixed in 10% buffered formalin, embedded in paraffin, and sections were stained with hematoxylin and eosin for evaluation of pathological changes or immune-stained for ehrlichial antigens using canine anti–E. canis antibody diluted 1:20 000 followed by secondary anticanine IgG antibody conjugated with streptavidin-alkaline phosphatase (1:200 dilution) (Jackson Immunoresearch, West Grove, Pennsylvania). The reactions were performed as previously described [6].

Statistical Analysis

Statistical analyses of the results were performed using GraphPad Prism software, version 5.01 for Windows (GraphPad Software, San Diego, California). All data were analyzed by Kruskal–Wallis and Mann–Whitney post-hoc nonparametric tests for comparison of infected and uninfected, infested or uninfested groups. Statistical significance was determined at 95% (P < .05).

RESULTS

Tick Vector EMLA-acquisition

After feeding, approximately 95% of engorged I. scapularis larvae molted to nymphs. Of the 10% of molted nymphs tested by real-time PCR, 90% to 100% contained the dsb gene of EMLA, indicating a high rate of acquisition. Ehrlichial levels found in individual nymphs varied from 1 × 10⁴ to 2 × 10⁵ copies of EMLA gene per mg of total DNA.

Tick Vector Transmission

From 50% to 100% engorged nymphs were recovered from the experimental TT among the control and infected mice, and no correlation was observed between the number of ticks fed and the outcome of the infection (Figure 1A).

Figure 1.

Correlation of number of ticks and survival in EMLA tick transmission (TT). A, Correlation between numbers of ticks fed on each mouse and disease outcome. B, Survival curve in EMLA TT. Bars represent median with interquartile range. Abbreviations: CTRL-IS, uninfected tick-infested controls; EMLA, Ehrlichia muris–like agent; EMLA-IS, mice infested with I. scapularis carrying EMLA; p.t.d., post–tick detachment.

Differences between EMLA infection by TT, controls infested with uninfected I. scapularis, and EMLA infection by IV and ID routes are as follows:

Clinical Observations

The mice were examined daily, and until day 7 p.t.d., no signs of illness were observed. Between days 8 and 12 p.t.d., few EMLA-infected TT–exposed mice (4 of 15 animals) demonstrated decreased activity, ruffled fur, and shallow breathing with increased respiratory rate, and they died fewer than 24 hours after onset of signs of illness, with 27% lethality (Figure 1B). The majority of the other infected animals (8 of 11 mice) showed onset of illness; however, it was less severe and they returned to normal activity after 3 days.

Blood Cell Counts

Hematocrit was greater in mice infected with EMLA transmitted by I. scapularis (EMLA-IS) at day 9 p.t.d. compared to uninfected tick-infested controls (CTRL-IS); however with no statistical difference (Figure 2A). No differences were observed among the groups at day 30 p.t.d. White blood cell (WBC) counts did not differ between infected mice and infested control mice at day 9 p.t.d.; however, infestation with ticks infected with EMLA or uninfected ticks induced a significant decrease in leukocyte concentration (Figure 2B). At day 30 p.t.d., the WBC counts were increased in EMLA-IS–infected mice compared to CTRL-IS mice; however with no statistical difference. The changes in WBC on day 9 p.t.d. were influenced mainly by lymphocyte (Figure 2C–E). Eosinophil counts were less in CTRL-IS mice than EMLA-IS mice, and eosinophils were increased in EMLA-IS mice on days 9 and 30 p.t.d.; however with no statistical difference. Platelet counts were increased on day 9 p.t.d. in infected and uninfected tick-infested animals, but were greater in CTRL-IS mice. On day 30 p.t.d., platelet counts of infected mice did not differ from control mice (Figure 2F). Lethally infected mice had thrombocytopenia and a higher hematocrit than sublethally infected mice.

Figure 2.

Hematologic analyses on days 9 and 30 p.t.d. A, Hematocrit. B, Total leukocyte counts. C–E, Differential leukocyte counts, and platelet counts (F), comparing uninfected animals with mice infested with uninfected (CTRL-IS) and EMLA carrying I. scapularis nymphs (EMLA-IS). Bars represent means ± SD. Controls and EMLA-infected groups were compared by Kruskal–Wallis and Mann–Whitney post-hoc tests. *P < .05. Abbreviations: dpi, days post infection; EMLA, Ehrlichia muris–like agent; p.t.d., post–tick detachment.

Serum Biochemistry

Mice infected by TT did not have significant changes in serum biochemical analyte concentrations at day 9 p.t.d. However, the urine specific gravity was higher than 1.035 in all EMLA-infected animals.

Bacterial Loads and Distribution in the Organs

Bacteria were present in all tested tissues from EMLA-IS–infected mice at 9 days p.t.d., including spleen, liver, lung, lymph nodes, kidney, brain, and bone marrow (Figure 3A). Lung tissues contained the highest levels of EMLA dsb gene copies, even at 30 days p.t.d., when all animals had at least 2 tissues containing EMLA at lower levels than during acute infection. Kidney, brain, and liver contained no detected ehrlichiae at 30 days p.t.d. (Figure 3B). Organs of moribund mice contained higher bacterial loads.

Figure 3.

Bacterial loads in tissues during EMLA infection transmitted by tick feeding. A, Bacterial loads at day 9 after tick detachment. B, Bacterial loads in tissues at day 30 after tick detachment. C, Comparative levels of ehrlichiae in tissues, during early infection by IV, ID, and TT. D, Comparative levels of ehrlichiae in tissues, at day 30 p.i./p.t.d. after EMLA infection IV, ID, and by TT. *No ehrlichiae detected. Abbreviations: BM, bone marrow; EMLA, Ehrlichia muris–like agent; ID, intradermal; IV, intravenous; LN, lymph node; p.i., post infection; p.t.d., post–tick detachment; TT, tick transmission.

Histopathology

Liver, skin, and lymph node from EMLA-IS–infected mice demonstrated marked histopathologic changes. At day 9 p.t.d., all animals exhibited infiltration of liver with inflammatory cells. Mice infected with tick-transmitted EMLA demonstrated hepatocellular mitotic figures, apoptosis, and multifocal lobular infiltration of inflammatory cells in the liver (Figure 4A–C), with significantly greater severity than uninfected tick-exposed mice (Figure 5A and 5B). There were more foci of cellular infiltration in infected animals than in tick-exposed controls (Figure 4A–H). Infiltration of inflammatory cells was observed in lymph nodes on day 9 p.t.d., especially in lymph nodes draining the tick bite site. Increased eosinophils were observed in tissues of EMLA-IS–infected mice and tick-infested controls. At the skin tick attachment site, variable quantities of inflammatory cells were observed at 9 days p.t.d., with increased eosinophils in both infected and uninfected tick-exposed animals (Figure 4F). The tick bite site revealed cellular infiltration in dermis, subcutaneous tissue, and muscle layer (Figure 4G and 4H). At day 30 p.t.d., few foci of inflammatory infiltration were observed in liver, lung, and skin of EMLA-IS–infected mice. Increased infiltration of inflammatory cells and edema were observed in mice before death.

Figure 4.

Histopathology of mice infected with EMLA by tick transmission during early infection. A, Liver with numerous hepatocellular mitoses (arrows). B and C, Liver with multifocal inflammatory infiltrates and hepatocellular apoptosis. D, Lung with mild perivascular inflammatory infiltrate. E, Kidney with multifocal lymphohistiocytic inflammation. F and G, Skin with eosinophil-rich lymphohistiocytic inflammation. H, Subcutaneous tissue with extensive lymphohistiocytic inflammation. Abbreviation: EMLA, Ehrlichia muris–like agent.

Figure 5.

Concentrations of foci of inflammatory infiltration (A) and mitotic figures (B). Microscopic field counts at 10× magnification comparing mice with EMLA transmitted by I. scapularis ticks (EMLA-IS) and control mice exposed to uninfected ticks (CTRL-IS). Bars represent means ± SD. Controls and EMLA-infected groups were compared by Mann–Whitney test. *P < .05. Abbreviations: EMLA, Ehrlichia muris–like agent; p.t.d., post–tick detachment.

Immunohistochemistry

Ehrlichiae were identified by IHC in EMLA-IS–infected mice, especially in lungs and liver, but also in other tissues, including the skin tick feeding site at day 9 p.t.d. (Figure 6A–I). Alveolar macrophages, pneumocytes, and pulmonary endothelial cells contained EMLA (Figure 6E). Hepatocytes and sinusoidal lining cells in the liver, as well as skin, lymph nodes, spleen, and brain contained morulae of EMLA after TT (Figure 6A–D and 6G–I).

Figure 6.

Immunohistochemistry of EMLA in tissues of mice infected by tick transmission. A and B, Identification of ehrlichial morulae in liver cells. C, Morula in area of inflammatory infiltration in liver. D, Presence of morula in endothelial cell in hepatic tissue. E, Presence of EMLA morulae in lung. F, Identification of morula in skin. G, EMLA morula in spleen. H, Presence of morula in peripheral lymph node. I, Identification of EMLA morula in the brain. Abbreviation: EMLA, Ehrlichia muris–like agent.

Antibody Response

IgM anti-EMLA antibody levels were elevated in EMLA-IS–infected animals on day 9 p.t.d.; however with no statistical difference (Figure 7A). At day 30 p.t.d., IgM anti-EMLA antibody levels had decreased. Infected mice produced low concentration of IgG antibodies against EMLA on day 9 p.t.d.; higher levels of IgG antibodies were observed on day 30 p.t.d. in all mice, but were highest in EMLA-IS–infected mice (Figure 7D–F).

Figure 7.

Comparative antibody titers during the course of infection by TT of EMLA. A, Levels of IgM antibodies in mice infected with EMLA transmitted by ticks. B, IgM antibody levels in EMLA-IV inoculated mice. C, IgM antibody levels in mice infected with EMLA by ID inoculation. D, IgG antibody levels in mice infected with EMLA by TT. E, Levels of IgG antibodies in mice infected with EMLA by the IV route. F, IgG antibody levels in EMLA-ID–infected mice. Bars represent means ± SD. Controls and EMLA-infected groups were compared by Kruskal–Wallis and Mann–Whitney post-hoc tests. *P < .05. Abbreviations: d.p.i., days post infection; d.p.t.d., days post-tick detachment; EMLA, Ehrlichia muris–like agent; ID, intradermal; IgG, immunoglobin G; IgM, immunoglobin M; IV, intravenous; OD, optical density; TT, tick transmission.

Comparison of Tick Transmission of EMLA With IV and ID Infection

Comparison of the disease induced by TT of EMLA with infection by needle inoculation by IV and ID routes revealed significant and interesting differences. The disease course after TT of EMLA resulted in 27% lethality, compared with no death after ID infection [6]. Tissue distribution of the bacteria was similar in IV and tick-transmitted infections, involving many organs, including kidney, brain, and bone marrow. ID infection did not result in dissemination of EMLA to those tissues (Figure 3C and 3D). Pathologic lesions induced by EMLA transmitted by ticks were similar to those in ID-inoculated mice, including significantly greater inflammatory cell infiltration in liver compared to IV infection. Antibody production after TT differed especially from IV infection. IgM antibodies after TT increased on day 9 p.t.d. and were maintained at day 30 p.t.d., similar to ID infection but at much higher levels (Figure 7A and 7C). IgM antibodies in IV-infected mice slightly increased at day 9, but were not maintained at these levels through day 30 p.i. (Figure 7B). All routes of infection induced production of IgG antibodies, increasing until day 30 of infection, but in TT, the levels were significantly higher (Figure 7D–F).

DISCUSSION

Several studies have shown the importance of the vector in pathogen transmission and establishment of infection. Vector salivary components induce pathogen gene expression changes within the salivary gland during vector feeding transmission that may represent important factors for establishment of disease [8, 11, 13–15]. No mouse model of TT of ehrlichiae has been reported. The newly described human pathogen, EMLA, enabled the development of a tick vector transmission model of ehrlichiosis in mice.

Tick acquisition of EMLA (90%–100%) by the larval stage was higher than reported in experiments using other ehrlichial species (12%–38% of E. canis by Rhipicephalus sanguineus) [4, 16]; however, the different rate of acquisition could be explained, in our study, as a result of very high inoculum dose (10–100 LD₅₀) by IV route [17], inducing higher bacteremia during the peak of feeding, which was used to determine the better feeding time.

Mice infected with EMLA transmitted by ticks developed clinical disease with moderate lethality, similar to naturally occurring ehrlichioses in animals and humans. The infection persists beyond the acute phase, with low levels of ehrlichiae in tissues.

Clinical laboratory changes in acute EMLA infection transmitted by ticks suggested hemoconcentration, based on the mild increased hematocrit and urine specific gravity. Similar pathophysiological changes were observed in lethal IV EMLA infection of mice [6]. Leukopenia due to lymphopenia was also observed in lethal IV EMLA infection; however, uninfected tick infestation induced a decrease in leukocyte concentration, probably due to an effect of tick saliva on lymphocyte proliferation as reported in other studies of host immune response to tick saliva [11, 18, 19].

Differences in tissue distribution of the pathogen had been observed in mice infected by the IV route compared to ID inoculation of EMLA [6]. TT of EMLA, similar to IV infection, induced dissemination of the bacteria throughout the organs tested, during early infection. These data suggest that TT of EMLA has some features similar to IV inoculation and others similar to ID inoculation. These events might be explained by the formation of a pool of blood and inflammatory cells around the tick mouth parts at the site of feeding, with increased vasodilation caused by tick saliva [20, 21]. The route of infection can influence how the pathogen is presented to the immune system, or even how it evades the host response, which will be focus of future studies.

The histopathology in animals infested with ticks revealed host reaction, especially in skin and draining lymph nodes, but also in liver, spleen, lungs, and kidney that comprised significantly more extensive cellular responses in mice infected with EMLA transmitted by ticks. More severe inflammation and tissue damage were observed in the liver of these mice during the acute stage. Animals infected by TT demonstrated a greater host response than IV-inoculated mice, albeit with fewer deaths. Interestingly, sublethal ID infection induced more severe histopathological changes than lethal IV infection [6]. Tissue damage, especially in the liver, did not correlate directly with a lethal outcome, and TT of EMLA induced pathological changes similar to ID infection.

More severe histopathological and hematological changes associated with higher bacterial loads also could be associated with the inoculum size in IV and TT models, as demonstrated in other experimental studies [17], which could explain the differences in the outcome of the disease.

Cellular infiltration observed in skin did not differ between infestation with infected and uninfected ticks. However, intense infiltration of inflammatory cells, including eosinophils at 9 days p.t.d., indicated that there was a prolonged host reaction to the damage, the arthropod, or the capsule, than just during tick attachment. The effect of tick saliva controlling host responses is well recognized; however, little is known about the effects at the feeding site after tick detachment. Thus, the response in the skin several days after tick detachment might be a rebound response to earlier local inhibition of host response by tick saliva. Changes in lymph nodes paralleled the skin pathology with increased inflammatory cellular infiltration, including eosinophils. Eosinophilia was observed during early infection, and was the only difference in blood cell counts between infestation with infected and uninfected ticks observed at 9 days p.t.d.

EMLA transmitted by ticks stimulated a significantly greater humoral immune response than after IV or ID needle inoculation. Challenge by those routes reveals differences in protection according to how the pathogen enters the host. However, the outcome does not directly correlate with the levels of antibody. Challenge experiments have not been performed with the TT model, but the antibody levels could indicate a different memory response with this mode of transmission. The pattern of IgG and IgM antibody response, especially at 30 days p.t.d., is similar to that in ID-inoculated mice, which is protective against a subsequent ordinarily lethal challenge, although ID mice present much lower IgG levels.

In conclusion, TT of EMLA is a promising model that includes more natural elements to study tick-host-pathogen interactions, mechanisms of protection, and vaccine efficacy.