An efficient microinjection method to generate human anaplasmosis agent Anaplasma phagocytophilum-infected ticks

Ticks are important vectors that transmit several pathogens including human anaplasmosis agent, Anaplasma phagocytophilum. This bacterium is an obligate intracellular rickettsial pathogen. An infected reservoir animal host is often required for maintenance of this bacterial colony and as a source for blood to perform needle inoculations in naïve animals for tick feeding studies. In this study, we report an efficient microinjection method to generate A. phagocytophilum-infected ticks in laboratory conditions. The dense-core (DC) form of A. phagocytophilum was isolated from in vitro cultures and injected into the anal pore of unfed uninfected Ixodes scapularis nymphal ticks. These ticks successfully transmitted A. phagocytophilum to the murine host. The bacterial loads were detected in murine blood, spleen, and liver tissues. In addition, larval ticks successfully acquired A. phagocytophilum from mice that were previously infected by feeding with DC-microinjected nymphal ticks. Transstadial transmission of A. phagocytophilum from larvae to nymphal stage was also evident in these ticks. Taken together, our study provides a timely, rapid, and an efficient method not only to generate A. phagocytophilum-infected ticks but also provides a tool to understand acquisition and transmission dynamics of this bacterium and perhaps other rickettsial pathogens from medically important vectors.


Scientific RepoRtS
| (2020) 10:15994 | https://doi.org/10.1038/s41598-020-73061-9 www.nature.com/scientificreports/ Humans get infected with A. phagocytophilum upon a bite with an infected tick 13 . The general clinical manifestations of A. phagocytophilum infection in humans include fever, thrombocytopenia, malaise, myalgia, leukopenia, headache, and increased serum aminotransferase liver enzyme activity that suggest mild injury to the liver 14 . The murine models with A. phagocytophilum infection have been noted to partially mimic the disease in humans and animals, including kinetics of infection, cytokine responses, histopathological observations similar to those observed in infected humans and other animals 15 . Different murine models such as BALB/c, C57BL/6J, C3H/HeJ, and C3H/HeN mice have been routinely used to study infection kinetics of human anaplasmosis and/or dynamics of A. phagocytophilum acquisition and transmission from mice-tick or tick-mice passages [15][16][17][18][19][20][21][22][23][24][25][26] . The A. phagocytophilum loads declines in these animals as the infection progress 27 . However, immunodeficient animals such as SCID mice can retain A. phagocytophilum infection up to 90 days 27,28 . Needle inoculation of naïve mice with A. phagocytophilum often requires use of infectious blood (from immunodeficient mice) as an inoculum 20,21,27 . Therefore, continuous maintenance of A. phagocytophilum in immunodeficient animals is expensive but required for the source of infectious inoculum to infect naïve mice.
In mammalian hosts, A. phagocytophilum primarily infects neutrophils where it resides inside an inclusion 29 . In both mammalian and tick cells, the developmental stages of A. phagocytophilum involve the formation of two morphotypes, a larger reticulate form (RC), and a smaller dense-core (DC) form 30,31 . It has been noted that only DC forms binds and enters the mammalian cells but both DC and RC forms can bind and enter tick cells 30,31 . Upon entry into mammalian cells, such as human leukemia cell line, HL-60, the DC forms transit to RC form within 12 h post infection and these forms initiate replication 31 . Inclusions with RC form can be observed by 24 h post infection of HL-60 cells 31 . By 36 h, inclusion with both DC and RC forms were evident 31 . The DC forms were ready by this time to re-infect the same host cell or infect naïve cells. Anaplasma phagocytophilum colonizes tick salivary glands and establishes persistent infection 32 . However, the presence of Anaplasma morphotypes in the tick tissues remains to be studied.
To generate A. phagocytophilum infected ticks, naïve mice such as C3H/HeN have to be first infected with infectious blood obtained from SCID and RAG −/− mice or HL-60 cultures and naïve ticks (larvae or nymphs) are then allowed to feed on these infected-mice. Repleted larval or nymphal ticks can be molted to next stage to generate unfed A. phagocytophilum-infected nymphal or adult ticks, respectively. Even though this method is feasible, it requires enormous budget and more time for maintenance of these mice for longer duration. In this study, we report a timely, rapid, and an efficient method of microinjection of A. phagocytophilum into the anal pore of unfed nymphal ticks. This study not only provides a new method to generate A. phagocytophilum-infected ticks but also provides an efficient tool to study acquisition and transmission dynamics of this and perhaps other rickettsial pathogens of medical importance.

Methods
Bacterial isolate and preparation of host cell-free A. phagocytophilum dense core (DC) form. Anaplasma phagocytophilum isolate NCH-1, obtained from BEI resources, Centers for Disease Control and Prevention (CDC), was used in all studies involving in vivo work with ticks and mice; wherever necessary, NCH-1 isolate is referred as A. phagocytophilum. Maintenance of A. phagocytophilum was carried out in HL-60 (ATCC, CCL-240) cells as described 33 . Host cell-free A. phagocytophilum-DC was isolated from confluent > 70% HL-60 infected cells by sonication followed by differential centrifugation as described 33 . Briefly, infected HL-60 cells were centrifuged at 2300 g for 10 min at 4 °C and the pellet was re-suspended in 1× phosphate-buffered saline (PBS), followed by sonication (8 s bursts with 8 s resting periods) on ice and then passaged 6-8 times through 27 ½ gauge syringe needle. This sonicated lysate was processed further for differential centrifugation to separate the bacterial DC form from the host cells that included 750 g spin for 5 min to pellet host cell debris and centrifuging the supernatant again at 1000 g for 5 min. The supernatant was further centrifuged at 2300 g for 10 min. The pellet obtained is composed of host cell-free A. phagocytophilum-DC 33 . 1× PBS was added to resuspend this pellet and used for further analysis. Wherever necessary A. phagocytophilum-DC is referred as Ap-DC.
Measurement of A. phagocytophilum DC particle size and number using microfluidic resistive pulse sensing (MRPS). MRPS is based on coulter principle using a microfluidic cartridge and can be utilized to analyze particle sizes from 50 to 2000 nm in a solution 34,35 . We performed MRPS (nCS1, Spectradyne LLC, USA) on host cell-free A. phagocytophilum-DC to determine the size and concentration of DC particles. Infection in HL-60 cells was maintained as described 33 . Briefly, two cell culture flasks with 10 ml each containing approximately 1 × 10 6 /ml infected HL-60 cells were centrifuged and processed for DC form isolation as described in the previous section. Equal number of uninfected HL-60 cells was processed as control group. Final DC pellet was re-suspended in 500 μl of 1 × PBS. Control tube used for processing uninfected HL-60 cells was also treated with the same volume of 1 × PBS. Three microliters from both suspensions were processed for MRPS Spectradyne measurements. All samples were measured at − V = − 5.0 V and + V = + 6.0 V using factory calibrated TS-2000 cartridge: ~ 250-2000 nm particle size range. MRPS Spectradyne measurements are plotted to show particle size (concentration/ml) on Y-axis and particle diameter (nm) on the X-axis (Fig. 2, Supplementary Fig. S1). The ± numbers actually mean counting error. There are 2 error numbers, the first is negative error and the second is positive error. The values are calculated based on Poisson-distributed statistics, and assumption that particles arrive at the nano-constriction in a purely random fashion (i.e. no particle event is related to another, they all occur independently). The statistical counting error was calculated based upon 1/√(N) (1 divided by square root of N), where N is the number of particles counted. When using Resistive Pulse Sensing to make particle measurements there is always electrical noise in the system. All the blue dots ( Supplementary  Fig. S1) that go across bottom of the transit time scatterplot are purely noise and they were excluded from the analysis. Uninfected nymphs were generated by feeding larvae on naïve mice and were allowed to molt into nymphs. Tick rearing was conducted in an incubator at 23 ± 2 °C with 90-95% relative humidity and a 14/10 h light/dark photoperiod regiment, as described in our previous studies 22,25,26 . DC-injected (via anal pore microinjection) unfed nymphal ticks (freshly molted) were fed on naïve C3H/HeN mice to transmit A. phagocytophilum from ticks to mice (Fig. 1). Upon repletion, these fed nymphal ticks were processed for DNA extractions followed by PCR to detect A. phagocytophilum loads. After day seven-post infection in C3H/HeN mice, larvae were allowed to feed on the same mice (generated by feeding with DC-injected ticks) for pathogen acquisition into ticks (Fig. 1). Upon repletion, these fed larvae were processed for DNA extraction, followed by PCR to detect A. phagocytophilum loads. After larval repletion, mice were euthanized and blood, liver and spleen were collected and processed for DNA extractions, followed by PCR to detect A. phagocytophilum (Fig. 1). In addition, a group of repleted larval ticks that were fed on DC-injected-tick-mediated infected mice were allowed to molt to nymphs (Fig. 1). DNA was extracted from molted nymphs and processed for PCR to detect bacterial loads.
Ethics statement. All animal work in this study was carried out in strict accordance with the recommen-  www.nature.com/scientificreports/ injection, USP (2 μl from 10 mg/ml stock/30 g mouse) tranquilizer was administered to the animals prior to handling to minimize anxiety and/or discomfort, and all efforts were made to minimize suffering.
Tick microinjections. The Eppendorf FemtoJet microinjection system was used in this study. Microinjections into ticks were performed as previously described 36 . The DC preparation (as described in an earlier methods section) from A. phagocytophilum-infected HL-60 was tenfold serially diluted twice and the diluted solution was used for microinjections into ticks. Micoinjections were performed into the anal pore of uninfected unfed nymphs (~ 4.2 nl/tick). During microinjection ticks were temporarily immobilized on a double-sided tape. Microinjected ticks were incubated at room temperature for seven days for acclimatization in an incubator at 23 ± 2 °C with 90-95% relative humidity and a 14/10 h light/dark photoperiod. After 7 days of incubation, DC-microinjected ticks were fed on naïve C3H/HeN mice to transmit the pathogen. Approximately, out of 20 ticks that were placed on mice, 8-10 DC-microinjected ticks successfully fed and repleted from each mouse. Repleted ticks were processed for DNA extraction followed by quantification of A. phagocytophilum p44 loads by QRT-PCR/PCR using oligonucleotides as mentioned in Table S1. The PCR conditions for QRT-PCR/PCR are mentioned in the QRT-PCR and PCR analysis section.
Isolation of DNA, total RNA and cDNA synthesis. In non-quantitative polymerase chain reaction (PCR) and Quantitative Real-Time-PCR (QRT-PCR) experiments, DNA was used to detect presence or absence of bacteria. For reverse-transcription polymerase chain reaction (RT-PCR), RNA was used to detect presence or absence of bacterial transcripts. To detect bacteria loads in ticks, genomic DNA from A. phagocytophiluminfected (DC-injected) unfed or fed nymphs or fed larvae or molted unfed nymphs and mice samples were extracted using DNeasy Blood and tissue kit (QIAGEN) and processed for PCR with primers specific for A. phagocytophilum p44 gene as mentioned in Table S1. As an internal control, I. scapularis ribosomal 5.8s rRNA was analyzed in the same DNA samples using oligonucleotides as mentioned in Supplementary Table S1. Total RNA from A. phagocytophilum DC was generated using the Aurum Total RNA mini kit (Bio-Rad, USA) and following the manufacturer instructions. During RNA extraction, on-column DNase I digestion was performed as per manufacturer's recommendation. The eluted RNA was then converted to cDNA using BioRAD cDNA synthesis kit (BioRAD, USA). The generated cDNA was used as a template for the detection of A. phagocytophilum transcripts (dnaA and dnaK) using the oligonucleotides shown in Supplementary Table S1. In DNA samples extracted from murine blood and tissue samples, bacterial burden was quantified by normalizing the level of A. phagocytophilum p44 to mice beta-actin levels using oligonucleotides mentioned in Table S1.
In QRT-PCR reactions, the standard curve for each gene fragment was generated using tenfold serial dilutions starting from 1 ng to 0.00001 ng of known quantities of respective fragments. For standard preparation, initial DNA concentration was measured by taking optical density reading in TECAN plate reader (TECAN, USA). After measurements of concentrations, tenfold serial dilutions were made to prepare various standards. PCR reactions were performed with Taq DNA polymerase (New England Biolabs Inc., USA). The PCR conditions were modified from 38 and were used for both QRT-PCR and PCR. The conditions are as follows: 95 °C for 5 min initial denaturation followed by 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min for 40 cycles with final extension of 72 °C for 7 min. For RT-PCR, following conditions were used: 94 °C for 2 min 30 s initial denaturation followed by 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 30 s for 34 cycles with final extension of 72 °C for 10 min. PCR or RT-PCR products were analyzed on 1.5% agarose gels stained with ethidium bromide. Internal quality control included parallel PCR amplification of no template control (ntc) and positive control (+, previously sequenced fragment) along with the samples. PCR products obtained from A. phagocytophilum specific oligonucleotides from every experimental group including murine blood, DC-injected fed nymphs (from transmission experiment), fed larvae (from acquisition experiment) and molted nymphs (from transstadial transmission experiment), were gel purified and sequenced at Eurofins (USA).
Statistics. Statistical significance in the data sets was analyzed using GraphPad Prism6 software (https :// www.graph pad.com/) and Microsoft Excel 2010 (https ://www.micro soft.com). For data to compare two means, the non-paired Student's t-test was performed. P values of < 0.05 were considered significant in all analyses. Wherever necessary, statistical test and P values used are reported.

Analysis of particle size and concentration of A. phagocytophilum DC form. To establish if A.
phagocytophilum-DC (Ap-DC) can be used to generate infected ticks, HL-60 cells infected with A. phagocytophilum were processed for DC isolation and used for further analysis (Fig. 1). We first measured the concentration and size of host cell-free A. phagocytophilum-DC isolated from infected HL-60 cells. We utilized MRPS Spectradyne instrument with TS-2000 cartridge that has a measurement range of ~ 250-2000 nm particle size. Measurements of undiluted Ap-DC and PBS control solutions were performed as described in the methods. MRPS spectradyne measurement showed heterogeneously sized population of DC with various sizes ranging from 250 to 1000 nm. Figure 2A shows the MRPS data for Ap-DC concentration and size in our preparations. During the measurement, a total of 20,435 particles were analyzed. The total concentration of Ap-DC was noted to be 4.73 × 10 8 ± (3.34 × 10 6 , 3.32 × 10 6 ) bacteria/ml. Ap-DC that were around size 300-400 nm were more in numbers in comparison to the other sized forms of DC ( Fig. 2A). The data also indicated that as the size of Ap-DC increased the numbers for those forms also reduced ( Fig. 2A). MRPS spectradyne measurements of PBS www.nature.com/scientificreports/ control solution that was generated from uninfected cells (processed similar way during the preparation of DC from infected cells) showed some background peaks in a size range from 250 to 550 nm (Fig. 2B). A total of 7 different particle sizes were detected in PBS solution with concentration noted to be 7.08 × 10 4 ± (7.78 × 10 4 , − 2.68 × 10 4 ) particles/ml (Fig. 2B). Comparison of particle numbers in Ap-DC preparation with PBS control solution indicated that a large number of particles were present in Ap-DC preparation (N = 20,435) compared to the PBS (N = 7) control ( Fig. 2A,B). The observation of four orders of magnitude of more particles in Ap-DC (N = 20,435) compared to PBS control (N = 7) clearly indicates successful isolation and detection of DC in the preparations. Due to the detection of increased particle numbers in Ap-DC (N = 20,435), the counting error in this sample is close to symmetrical. However, on PBS control, the counting error is much larger in proportion  both (A,B), the x-axis indicates particle diameter in nanometers and y-axis indicates particle concentration/ml/nm. Concentrations of particles are indicated above the horizontal line with ± counting error. www.nature.com/scientificreports/ to the measured concentration and very asymmetrical due to less particle numbers (N = 7). The detection of only 7 particles in PBS control over a long period of time could be due to the noise floor for the cartridge used in this measurement. The distribution of excluded and included particles/events during Ap-Dc measurement is shown in Supplementary Fig. S1. These data indicated that possibly Ap-DC exists more in 300-400 nm size form compared to other sized forms.

PCR amplification of A. phagocytophilum transcripts from Ap-DC isolated from HL-60 cells.
To test if Ap-DC isolated using sonication and differential centrifugation method contain detectable bacterial transcripts, we processed the sample for RNA extraction followed by cDNA synthesis. In addition, DNA was extracted from the same sample. The generated cDNA was used in a RT-PCR to detect A. phagocytophilum dnaA and dnaK transcripts using the oligonucleotides mentioned in Table S1. Reactions performed in the absence of reverse transcriptase enzyme (− RT) reaction were used as negative controls. The results revealed presence of both dnaA and dnaK transcripts or gene fragments in RNA or DNA samples generated from same Ap-DC preparation, respectively. In the presence of reverse transcriptase enzyme (+RT) expected band at 175 bp for dnaA (Fig. 3A) and 178 bp for dnaK (Fig. 3B) was noted in RNA samples. Similar sized bands were noted in DNA samples extracted from Ap-DC preparation (Fig. 3A,B). No bands were evident in the absence of reverse transcriptase (−RT) enzyme and in no template controls (Fig. 3A,B). Previously sequenced fragments generated from DNA samples of Ap-DC were used as a positive control in these PCR reactions (Fig. 3A,B). The detection of dnaA and dnaK transcripts indicates that Ap-DC isolated from A. phagocytophilum-infected HL-60 contains amplifiable bacterial mRNA.

Microinjected unfed nymphal ticks with Ap-DC could transmit
A. phagocytophilum to naïve mammalian host. Next, we then analyzed whether the microinjected ticks (generated by microinjection of Ap-DC into anal pore of nymphal ticks) could transmit A. phagocytophilum to uninfected murine host. First, initial Ap-DC preparation (~ 4.7 × 10 8 bacteria/ml) was 1:100 diluted generating ~ 4.7 × 10 6 bacteria/ml suspension. This suspension was used for microinjection into unfed nymphal ticks. Our initial attempts with injection suspension taken from ~ 4.7 × 10 8 bacteria/ml solution to inject Ap-DC into tick body were unsuccessful due to higher death observed in ticks. Therefore, we microinjected ~ 4.2 nl of this solution per nymphal tick at its anal pore. Microinjected ticks were allowed to recover for 7 days to facilitate A. phagocytophilum colonization and establishment in these ticks. Approximately 90% of ticks survived microinjection and were viable after seven days of incubation. Approximately, 20 microinjected ticks were allowed to feed on each naïve mice. However, we noted that 8-10 microinjected ticks successfully fed on each mouse. Repleted ticks were collected and stored at − 20 °C and processed for DNA extractions as described in methods. After 12 days post tick placement, mice were euthanized, and blood and other tissues such as spleen and liver were harvested. DNA was extracted from mice blood, and tissue samples. PCR analysis revealed detection of A. phagocytophilum p44 gene fragment (band size = 334 bp) in all nine mice blood samples (Fig. 4A). Nucleotide sequencing of purified PCR products confirmed that the amplified band corresponds to A. phagocytophilum DNA. As expected, no bands in three uninfected mice blood samples were noted (Fig. 4A). PCR reactions performed with mice-beta actin oligonucleotides showed the presence of mice DNA (band size = 279 bp) fragment in all tested samples (Fig. 4A, Supplementary Fig. S2). We www.nature.com/scientificreports/ also tested the presence of A. phagocytophilum in liver tissues (Fig. 4A, Supplementary Fig. S3). In DNA samples extracted from liver tissues, A. phagocytophilum p44 gene fragment was amplified in six out of nine mice liver samples (samples [1][2][3][4][5][6]. Two samples showed a faint amplification (samples 7 and 9), and one sample showed no amplification (sample 8) for A. phagocytophilum p44 gene. Similarly, DNA samples from spleen tissues were analyzed (Fig. 4A, Supplementary Fig. S4). We noted that in eight out of nine mice spleen DNA samples (Fig. 4A, Supplementary Fig. S4, samples 1 and 9) clear amplification of A. phagocytophilum p44 gene fragment was evident. One sample showed no amplification (Fig. 4A, Supplementary Fig. S4). QRT-PCR further confirmed the presence of A. phagocytophilum in blood, liver, and spleen tissues (Fig. 4B). However, no statistically significant

Transstadial transmission of A. phagocytophilum from larval to nymphal stage in ticks fed on mice infected with Ap-DC-microinjected ticks. We then analyzed if larvae fed on mice infected with
Ap-DC-microinjected ticks could retain A. phagocytophilum after molting into next development stage. A group of larvae were therefore allowed to molt to nymphal stage. Freshly molted nymphs were individually processed for DNA extractions and analyzed by PCR for the presence of A. phagocytophilum. PCR analysis revealed the presence of A. phagocytophilum in three out nine freshly molted nymphs (Fig. 7) indicating successful transstadial transmission of this bacterium from larval to nymphal stage. In addition, nucleotide sequencing of the purified PCR products confirmed the presence of A. phagocytophilum DNA in molted nymphs. Amplification of tick 5.8s rRNA fragment was evident in all samples (Fig. 7, Supplementary Fig. S9). DNA from uninfected nymphs that were molted from larvae fed on uninfected mice was used as a control in this analysis. As expected, no A. phagocytophilum specific PCR amplified product was observed in larvae fed on uninfected mice (Fig. 7). These results elucidate that larvae fed on mice infected with Ap-DC-microinjected ticks could transstadially transmit A. phagocytophilum into the next developmental stage.  www.nature.com/scientificreports/

Discussion
Hard ticks such as I. scapularis requires several days to feed on an animal to acquire or transmit pathogens 7 . Therefore, development of an alternative way of infection in this species would rapidly advance research in studying tick-pathogen interactions. Several artificial membrane-feeding methods have been developed for both hard and soft ticks for the generation of infected ticks 39,40 . Even though these methods are simpler and cost effective, generation of tick cohorts with equal number of pathogens in each tick is limited. The success of anal pore microinjection system to generate Lyme disease pathogen B. burgdorferi-infected ticks 41 or tick-borne encephalitis virus family Langat virus-infected ticks 42 suggests that this technique can be easily adopted for generating both extracellular or intracellular tick-borne pathogen-infected ticks, respectively. In this study, we report the use of microinjection system to generate obligate intracellular bacterium A. phagocytophilum-infected unfed nymphs in the laboratory condition and showed that these ticks can efficiently transmit this bacterium to the murine host. Quantification of A. phagocytophilum by competitive PCR is reported 20 . In addition, electron microscopy methods have been used to count DC isolated from mammalian cells 31 . The competitive PCR method is based upon amount of DNA and electron microscopy method requires multiple microscopic sections to count several and hundreds of DC forms. This study reports the use of Spectradyne system as a novel method to evaluate Ap-DC numbers from HL-60 cells. Even though competitive PCR and electron microscopy methods can be used to evaluate A. phagocytophilum concentration in different cells, our study shows efficiency on the use of Spectradyne system to count and also analyze sizes of DC forms in a rapid way. A previous study has reported findings from in vitro cell culture experiments and has provided microscopic evidence that within one to four hour post Anaplasma infection of tick cells, bacteria attaches to tick plasma membrane and encloses inside an endosome 30 . By eight hours post infection, evidence of possible first bacterial division was observed 30 . At 12 h p.i., appearance of morulae-like shaped inclusions were observed and by 48 h p.i. endosomes harboring large numbers of Anaplasma were noted 30 . At 72 h p.i. highly pleomorphic sizes of Anaplasma was observed 30 . The observation of heterogeneous population of DC in the range from 300 to 1200 nm noted from Spectradyne measurements in this study corroborates with the reported microscopic observations from the previous study 30 . This method not only provides quantitative information on Ap-DC numbers but also provides quantitative information on the size of Ap-DC forms. Compared to the other existing methods, we believe that by using Spectradyne measurement one could rapidly quantitate both numbers and size of Ap-DC in samples.
Our study indicates that using this method, we can rapidly and efficiently generate A. phagocytophiluminfected unfed nymphs in the laboratory conditions. In nature, both I. scapularis nymphs and adults could transmit tick-borne pathogens to humans 7 . Several studies, including our own, have characterized several tick molecules in A. phagocytophilum-nymphal I. scapularis interactions [22][23][24][25][26] . However, studies in understanding A. phagocytophilum-adult tick interactions are limiting. We believe that the method reported in this study could be easily adapted to directly inject DC into adult I. scapularis to generate A. phagocytophilum-infected unfed adult ticks. Characterization of bacterial and/or tick molecules in adult ticks would provide new information in understanding A. phagocytophilum-adult tick interactions. The observation of bacterial loads in only 3 out of 9 freshly molted nymphs suggests that bacterial loads could be very low and are at undetectable levels in some of the nymphs. In addition, the observation of increased death in nymphs upon initial injection with undiluted stock (injection suspension taken from ~ 4.7 × 10 8 bacteria/ml solution) suggests that ticks cannot tolerate high dose of bacteria. Future experiments can be performed with different doses of DC to determine lethal dose of 50% (LD 50 ) for ticks. Tick molecules have been considered as candidates for the development of transmission-blocking vaccines 43 . Therefore, availability of methods, such as this one, to generate unfed A. phagocytophilum-infected unfed nymphal or adult ticks would facilitate rapid testing on the effect of active or passive immunization strategies (developed against tick molecules) to block transmission of A. phagocytophilum from vector to the vertebrate host.
The success in the transmission of A. phagocytophilum from I. scapularis that were microinjected with DC into anal pore of unfed ticks is not surprising. In the natural acquisition route, A. phagocytophilum migrates from an infected animal to the gut of an engorging tick through a blood meal 2,20 . The bacterium then enters tick gut cells and undergoes first round of replication 2,20 . Later, A. phagocytophilum migrate and colonize tick salivary gland cells 2,20 . In the natural transmission route, A. phagocytophilum that are colonized in tick salivary glands cells, are transmitted to the vertebrate host along with saliva during blood feeding 2,20 . As anal pore connects to the gut, we believe that injection of Ap-DC into this provides a partial/artificial acquisition route for this bacterium similar to its natural route to enter tick gut first, undergo initial replication and then colonize in tick salivary glands. Incubation of Ap-DC-microinjected ticks for seven days may additionally facilitate replication and establishment of A. phagocytophilum in ticks. Even though we observed increased trend in bacterial loads in samples generated from murine blood compared to levels noted in samples generated from other tissues, we did not noted statistical difference between them. This could be due to high variability in bacterial numbers between murine tissue samples. The successful transmission of A. phagocytophilum from Ap-DC-injected ticks to murine host suggests that this method could also serve as an alternative method to generate a natural transmission route environment for this bacterium.
Collectively, we describe a timely, rapid and an efficient microinjection method to generate A. phagocytophilum-infected ticks in the laboratory. Studies like this would substantially enhance research not only in understanding this and perhaps other rickettsial pathogen-tick interactions but could also accelerate anti-vector/ transmission-blocking vaccine research in the field of tick-borne diseases.