• View in gallery

    Schematic of pathogen acquisition in I. scapularis nymphs. I. scapularis nymphs micro-injected with Ponceau-S (mock) and salp14dsRNA were allowed to feed for 72 hours on C3H/HeN- and C3H/HeJ-infected mice with B. burgdorferi and A. phagocytophilum, respectively. Engorged tick nymphs were recovered from infected mice and dissected for molecular biology analysis. Three experiments were done on different dates and showed similar results.

  • View in gallery

    RNAi-mediated reduction of salp9pac expression in nymph I. scapularis does not affect the ability of ticks to feed. I. scapularis nymphs micro-injected with Ponceau-S (mock) and salp9pac-dsRNA (salp14 paralog) were allowed to feed for 4 days on C3H/HeN mice. Engorged tick nymphs (N = 13 and N = 21, respectively) were recovered and weighed.

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    Reduction of salp14 expression by RNA interference. A, Ponceau-S dye was used as a control and allowed the visualization of I. scapularis nymphs under the dissection microscope B, RNA was isolated from salivary glands of Ponceau-S (1–4) and salivary protein 14 (salp14) dsRNA (5–7)-injected nymphs and analyzed by RT-PCR for the reduction of salp14 transcription. The I. scapularis actin gene was used to determine whether all samples had similar cDNA levels. C, qRT-PCR showed ~10,000-fold decreased salp14 transcription in dsRNA-injected salivary glands compared with mock-injected nymphs. Three experiments were done on different dates using different batches of ticks and mice. One representative experiment is shown. The statistical significance was calculated using the Student t test. D, Protein was extracted from salivary glands of Ponceau-S and salp14 dsRNA-injected nymphs and immunoblotting was done. Salp14 expression was reduced in salp14dsRNA-injected (lanes KO) salivary glands compared with mock-injected salivary glands (lanes Mock). The highly immunogenic non-specific globulin binding protein of 90 kd was used as a control to determine the specificity of the salp14 dsRNA.

  • View in gallery

    A. phagocytophilum acquisition in I. scapularis nymphs. Engorged tick nymphs micro-injected with either Ponceau-S or salp14dsRNA were dissected under the microscope, and salivary glands and midguts were pooled. Total RNA was extracted and cDNA produced. A, RT-PCR followed by gel electrophoresis was done in salivary glands and midguts of engorged I. scapularis nymphs. The I. scapularis actin gene was used as a normalizing factor. Salp14 transcription showed efficiency of the RNAi. B, qRT-PCR was done to detect the transcription of 16srRNA from A. phagocytophilum in Ponceau- and salp14dsRNA–engorged I. scapularis salivary gland nymphs. Three experiments were done on different days, and the statistical significance was calculated using the Student t test.

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    Borrelia burgdorferi acquisition in I. scapularis nymphs. Engorged tick nymphs micro-injected with either Ponceau-S or salp14dsRNA were dissected under the microscope, and midguts were pooled. Total RNA was extracted and cDNA produced. A, RT-PCR followed by gel electrophoresis was done in midguts of engorged I. scapularis nymphs. B. burgdorferi flaB amplicon in mock midguts (lanes 1–4) was comparable with salp14 dsRNA-injected midguts (lanes 5–8). B, qRT-PCR was done to detect the transcription of B. burgdorferi flaB in mock- and salp14dsRNA-engorged I. scapularis midguts. The I. scapularis actin gene was used as a normalizing factor. Three independent experiments were done on different days, and the statistical significance was calculated using the Student t test.

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DISRUPTION OF THE SALIVARY PROTEIN 14 IN IXODES SCAPULARIS NYMPHS AND IMPACT ON PATHOGEN ACQUISITION

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  • 1 Section of Rheumatology and Section of Allergy and Clinical Immunology, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut

We previously examined the physiological role of the anticoagulant salivary protein 14 (salp14) in adult Ixodes scapularis and showed that Salp14 played a role in tick feeding and engorgement. We now analyze whether the disruption of the salp14 family expression by RNA interference affects tick weight in naïve nymph I. scapularis. Salp14 expression after dsRNA injection was significantly reduced, as shown by mRNA and protein analysis. However, nymph engorgement weight was not altered in salp9pac (salp14 paralog) dsRNA-injected ticks. We also determined Borrelia burgdorferi and Anaplasma phagocytophilum acquisition in I. scapularis nymphs that had reduced Salp14 expression. B. burgdorferi and A. phagocytophilum acquisition was not affected 72 hours after feeding. Our results suggest that different mechanisms govern nymph and adult feeding in I. scapularis.

INTRODUCTION

Ixodes scapularis is an important health-related tick species in the United States. I. scapularis has the potential to transmit Borrelia burgdorferi (the Lyme disease agent), Anaplasma phagocytophilum (the etiologic agent of human anaplasmosis), Babesia microti (Babesiosis agent,) and flavivirus within the tick-borne encephalitis virus complex.1 Lyme disease is the most important tick-borne disease in the United States. More than 17,000 positive cases were reported in the United States in 2000, which translates in important long-term health consequences for the population.2 A. phagocytophilum is a gram-negative, obligate intracellular bacterium that replicates within neutrophils, and it is transmitted by I. scapularis in the eastern and midwestern United States.3 Lyme disease is characterized by chronic arthritis, myocarditis, lymphocytic meningitis, and facial palsy, whereas patients with human anaplasmosis have fever, headaches, myalgias, and arthralgias. Typical laboratory findings are leukopenia, thrombocytopenia, and elevated liver enzymes.

Blood-feeding nymphal ixodid ticks remain attached to the host for 3–4 days with their mouthparts embedded in the host skin sucking blood and fluids from tissues. Components of tick saliva establish and regulate engorgement facilitating pathogen transmission.4 Tick saliva has been associated with host defense, promotion of anti-hemostatic activities that maintain the blood meal in a fluid state,5 and increase of vector borne pathogen transmission.68 Reported saliva immunosuppressive features include inhibition of the complement cascade, impairment of natural killer (NK) cell activity, reduction of circulating antibody titers, repression of cytokine production, and inhibition of T-lymphocyte proliferation.5

The establishment of the physiological relevance for ixodid salivary proteins has heavily relied on the generation of recombinant proteins that can effectively induce neutralizing antibodies against the native salivary protein. However, conventional protein immunization techniques remain a tedious task and have delayed the development of vaccines targeting the tick vector. The sequencing of the I. scapularis genome associated with the development of new investigative tools such as expressed sequence tags, microarrays, and RNA interference (RNAi) have offered new alternatives for tick and tick-borne disease control.9

RNAi provides a powerful alternative to traditional immunization and genetics techniques. Introduction of dsRNA or small interfering RNA into a cell triggers the abrogation of the target mRNA. Previous experiments10 showed the feasibility of RNAi to abrogate the expression of a histamine binding protein in the tick Amblyomma americanum. Subsequent studies with vesicular membrane proteins associated with exocytosis showed the importance of RNAi in A. americanum.11,12 The use of RNAi to address tick functional genomics was also shown in I. scapularis.7,1315

In this report, we exploit the RNAi technique to address the physiological role of the salivary gland protein 14 (Salp14) in naïve I. scapularis nymphs. Salp14 was isolated from an I. scapularis salivary gland cDNA expression library probed with tick immune rabbit serum.16,17 RNAi mediated reduction of salp14 expression and its paralog salivary gland protein 9pac in adult I. scapularis affected the ability of ticks to feed.13 Previous experiments18 showed the presence of at least 30 structural salp14 paralogs in the salivary gland transcriptome of adult I. scapularis. We observed a similar number of salp14 paralogs in the salivary glands of I. scapularis nymphs (E. Fikrig and F. S. Kantor, unpublished data). However, the physiological function of these proteins remains to be determined in nymphs.

We now analyze whether the disruption of the salp14 family expression affects nymph engorgement weight and B. burgdorferi and A. phagocytophilum acquisition in naïve I. scapularis. Ponceau-S– and salp14-dsRNA–injected I. scapularis nymphs were allowed to feed on pathogen-infected mice for 72 hours, and B. burgdorferi and A. phagocytophilum acquisition was determined using quantitative real time (qRT)-polymerase chain reaction (PCR), Western blot, and reverse transcriptase (RT)-PCR. Salp14 expression was drastically reduced, as shown by mRNA and protein analysis. Contrary to adult I. scapularis, nymph engorgement weight was not altered in salp9pac (salp14 paralog) dsRNA-injected ticks, and B. burgdorferi and A. phagocytophilum acquisition was not affected.

MATERIALS AND METHODS

Ticks.

Ixodes scapularis nymphs were obtained from a tick colony at the Connecticut Agricultural Experimental Station (New Haven, CT). Ticks were reared in a biosafety level 2 laboratory at 26°C with 85% relative humidity and a daily photoperiod regimen of 14 hours.

Infection of C3H/HeN and C3H/HeJ mice with B. burgdorferi and A. phagocytophilum.

Ixodes scapularis nymphs micro-injected with Ponceau-S (mock) and salp14dsRNA were allowed to feed for 72 hours on C3H/HeN- and C3H/HeJ-infected mice with B. burgdorferi and A. phagocytophilum, respectively (Figure 1). B. burgdorferi N40 strain was recovered after plating on Barbour-Stoenner-Kelly (BSK) agar medium, and 104 bacteria was needle inoculated (intradermal injection) into three female C3H/HeN mice (3–5 weeks old).

DNA was extracted from ear punches (Stratagene, La Jolla, CA), and the presence of B. burgdorferi was confirmed by RT-PCR. Primers and RT-PCR conditions used to detect B. burgdorferi in the skin were previously described.14 Gene-specific primers were flaB-F 5′-TTGCTGATCAAGCTCAA-TATAACCA-3′ and flaB-R 5′-TTGAGACCCTGAAAGTG-ATGC3′. RT-PCR conditions were as follows: 95°C for 10 minutes followed by 40 cycles of 95°C for 60 seconds for denaturation, 55°C for 1 minute for annealing, and 72°C for 1 minute for extension. The mouse with the highest B. burgdorferi load was used for the B. burgdorferi acquisition. Three replicate experiments were done on different days.

We used the C3H/HeJ mouse strain for the A. phagocytophilum acquisition study. One hundred microliters of blood from Rag-deficient mice (immunocompromised mice) chronically infected with A. phagocytophilum (20–30% neutrophil infection rate) was used to inoculate the intraperitoneal cavity of three female C3H/HeJ mice (Figure 1). The mouse with the highest A. phagocytophilum load on Day 7 was used for A. phagocytophilum acquisition. Primers and RT-PCR conditions used to detect A. phagocytophilum in the blood were previously described.19 Gene sequences were 16srRNA-F 5′-CCATTTCTAGTGGCTATCCCATACTAC-3′ and 16srRNA-R 5′-TCGAACGGATTATTCTTTATAGCTTG-3′. RT-PCR conditions were as follows: 95°C for 10 minutes followed by 40 cycles of 95°C for 60 seconds for denaturation, 60°C for 1 minute for annealing, and 72°C for 1 minute for extension. Three replicate experiments were done on different days. All experiments were done in accordance with the Yale University Institutional Animal Care and University Committee and the National Institutes of Health Guidelines for Laboratory Animals.

Generation of dsRNA and I. scapularis pathogen acquisition.

Salp14 and salp9pac dsRNA generation, micro-injection procedures, primers, and qRT-PCR conditions used to detect the efficiency of salp14 and salp9pac silencing were previously described.13 Fed-adult salivary gland cDNA was used as template to amplify DNA encoding full-length salp14 (GenBank accession no. AF209921) and full-length salp9pac (GenBank accession no. AF515779). Gene specific primers for salp14 and salp9pac containing BglII and KpnI restriction sites were used. Primer sequences were as follows: Salp14-5F′-GAA-GATCTTCATGGGGTTGACCGAACC-3′ and Salp14-5R′-CGGTACCGCATAAGTTTTTCTCCTG-3′; Salp9pac-5F′-GAAGATCTTCATGGGGTTGACTGAG-3′ and Salp9pac-5R′-CGGTACCGTATCTTTATTAAG-3′. The resultant amplicons were purified and cloned into the BglII and KpnI restriction sites of the L4440 double T7 Script II vector.20 dsRNA complimentary to the DNA was synthesized by in vitro transcription using the Megascript RNAi kit (Ambion, Austin, TX). The dsRNA was purified and quantified spectroscopically. Five nanoliters of salp14 dsRNA (1 × 1010 molecules/μL) or Ponceau-S dye (1 nmol/L Ponceau S; Sigma-Aldrich, St. Louis, MO) was injected into the body of 45 I. scapularis nymphs per treatment. For the salp9pac dsRNA experiment, 5 μL of salp9pac-dsRNA (1 × 1010 molecules/μL) and Ponceau-S dye (1 nmol/L Ponceau S; Sigma-Aldrich) were injected into the body of 60 I. scapularis nymphs. The injections were carried out using 10-μL micro-dispensers (Drummond Scientific, Broomall, PA) drawn to fine point needles carrying a micropipette puller (Sutter Instruments, Long Beach, CA). The needles were loaded into a micromanipulator (Narishige, Tokyo, Japan) connected to a Nanojet microinjector (Drummond Scientific). Ticks were allowed to rest for 3 hours before placement on infected mice. Fifteen mock and 15 salp14 dsRNA–injected ticks were placed on each infected mouse. The Ponceau S dye stained the injection site in pink and enabled us to distinguish mock- from salp14 dsRNA–injected ticks. We recovered roughly 60–70% of the ticks in most of the experiments. salp14 dsRNA (1 × 1010 molecules/μL) or Ponceau-S dye (1 nmol/L Ponceau S; Sigma-Aldrich) was injected into the body of 45 I. scapularis nymphs per treatment. Twenty-one and 13 engorged nymphs injected with the salp9pac dsRNA and Ponceau-S dye were used for weight measurement. Fifteen ticks were used to determine pathogen load and salp14 mRNA expression. Another group of 15–20 ticks was used to determine salp14 protein expression levels. Salivary glands and midguts were dissected under the SZ60 Olympus dissection microscope (Olympus, Center Valley, PA). Groups of three salivary glands and midguts were pooled, totaling five replicates per treatment. Three independent experiments were done totaling ~130 ticks injected per treatment (salp14 or Ponceau group).

Total RNA from fed-nymph salivary glands was prepared as described earlier.13 Salivary gland cDNA was prepared with the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) in accordance with the manufacturer’s protocol. Specificity of primers for qRT-PCR analysis was tested using conventional RT-PCR and resulted in a single product with the desired length. One microliter of cDNA samples was used for the qRT-PCR analysis (iQ SYBR Green I Supermix; Bio-Rad, Hercules, CA) in the iCycler Thermal Cycler (Bio-Rad). qRT-PCR conditions were as follows: 95°C for 10 minutes, followed by 40 cycles of 95°C for 30 seconds for denaturation, 60°C for 30 seconds for annealing, and 72°C for 1 minute for extension. Tick salivary gland and midgut cDNA levels were normalized to the I. scapularis actin gene (GenBank accession no. AF426178). The genes B. burgdorferi N40 flagellin (flaB; GenBank accession no. X75200) and A. phagocytophilum 16s rRNA (GenBank accession no. M73224) were quantified. Gene-specific primers were flaB-F 5′-TTGCTGATCAAGC-TCAATATAACCA-3′ and flaB-R 5′-TTGAGACCCT-GAAAGTGATGC-3′; actin-F 5′-GGTATCGTGCTCGAC-TC-3′ and actin-R 5′-ATCAGGTAGTCGGTCAGG-3′; 16srRNA-F 5′-CCATTTCTAGTGGCTATCCCATAC-TAC-3′ and 16srRNA-R 5′-TCGAACGGATTATTCTT-TATAGCTTG-3′. Three experiments were done on different days using different batches of infected ticks and mice. The statistical significance was calculated using the Student t test.

A standard curve was determined using dilutions of a known quantity of salp14, 16srRNA, and flab. The efficiency of each reaction was calculated from the slope of the line during the linear phase of the reaction. To make accurate comparison between samples and standard curves, PCR reactions for the samples and standards were optimized so that they came close to 100% efficiency. A melting curve was generated for the qRT-PCR reaction to confirm whether additional peaks contributed to the fluorescence.

Western blot analysis to confirm reduction of salp14 expression.

Groups of three pooled salivary glands from Ponceau-S (mock)- and salp14 dsRNA–injected I. scapularis nymphs were suspended in 100 μL of sterile 1- phosphate-buffered saline (PBS) and homogenized. Total protein was quantified using the Bradford method. Two micrograms of salivary gland proteins from mock- and salp14 dsRNA–injected I. scapularis nymphs were electrophoresed on SDS 12% polyacrylamide gel and processed for immunoblotting.17 Bound antibodies were detected using horseradish peroxidase–conjugated goat anti-guinea pig secondary antibodies (Sigma Aldrich). The immunoblots were developed using a western lightening chemiluminescence kit (Perkin Elmer, Wellesley, MA).

RESULTS

Reduction of salp9pac expression, a salp14 paralog, in I. scapularis nymphs does not impair tick feeding.

Ixodes scapularis nymphs representing mixed populations of males and females were injected with the dsRNA of the salp14 paralog (86% identity to salp14 at the nucleotide level), salp9pac, and Ponceau-S dye and allowed to feed on C3H/HeN mice. Ticks attached to the host were removed 4 days later to determine engorgement weights. Salp9pac expression levels were reduced as verified by mRNA and protein analysis (data not shown). RNAi-mediated reduction of the salivary gland protein 9pac in nymph I. scapularis did not affect the ability of ticks to feed (Figure 2). Similar results were obtained with the salp14 dsRNA tick micro-injections (data not shown).

Salp14 reduction of expression in I. scapularis nymphs.

Ixodes scapularis nymphs injected with either salp14 dsRNA or Ponceau-S dye were allowed to feed for 72 hours on B. burgdorferi–infected C3H/HeN and A. phagocytophilum–infected C3H/HeJ mice. Ticks attached to the host were removed 3 days later to determine pathogen load and Salp14 expression levels. Three days was chosen for the reason that prolonged feeding dilutes the RNAi silencing effect.10 A. phagocytophilum and B. burgdorferi acquisition also takes place within 24–72 hours of tick feeding. Therefore, 3 days of feeding is sufficient to address pathogen acquisition. Groups of salivary glands and midguts were pooled, and cDNA and proteins were prepared for RNAi verification. Ponceau-S dye was used as a control and allowed the visualization of I. scapularis nymphs (Figure 3A). Ticks injected with salp14 dsRNA showed reduced salp14 expression in salivary glands compared with the Ponceau-S–injected treatment (Figure 3B–D). The I. scapularis actin gene was used to normalize the levels of cDNA in all samples. This reduction in salp14 expression was also confirmed at the protein level by a Western blot analysis of salivary gland protein extracts from Ponceau-S (mock)- and salp14dsRNA–injected ticks (Figure 3D). A 90-kd protein that reacted non-specifically with guinea pig serum served to ensure that equal amounts of protein from mock-and salp14 dsRNA–injected ticks were transferred to the immunoblot (Figure 3D). The identity of the 90-kd protein is not known. However, it is suggested that it may be a globulin-binding protein with high affinity to guinea pig serum globulins.21

Borrelia burgdorferi and A. phagocytophilum acquisition in naïve I. scapularis nymphs.

We determined whether the reduction of salp14 expression in I. scapularis had an effect on B. burgdorferi and A. phagocytophilum acquisition. B. burgdorferi–infected C3H/HeN and A. phagocytophilum–infected C3H/HeJ mice were generated, and naive tick nymphs were placed on the mice as described in the Materials and Methods section.14,19 The acquisition of A. phagocytophilum and B. burgdorferi in I. scapularis after a 72-hour feeding was not statistically significant when mock- and salp14 dsRNA–injected treatments were compared as described by RT-PCR (Figures 4A and 5A) and qRT-PCR (Figures 4B and 5B). The genes 16srRNA (A. phagocytophilum) and flaB (B. burgdorferi) were used as surrogate markers.

DISCUSSION

We previously showed that Salp14 functions as an anticoagulant protein in the salivary glands of adult I. scapularis, facilitating the interruption of host hemostatic mechanisms during tick feeding. We also showed that RNA interference–mediated reduction of salp14 expression in adult ticks resulted in diminished engorgement weights,17 which is an indicative of impaired tick feeding. When ticks feed on an infected host, ticks acquire the pathogen, and when an infected tick feeds on a naïve host, ticks transmit pathogens to the host. The process of feeding is therefore quintessential both for pathogen acquisition and for pathogen transmission. In this report, we examine the physiologic role of the salivary gland protein 14 (Salp14) in naïve nymph I. scapularis.

Recent examination of the I. scapularis transcriptome has shown that gene expression in this arthropod is very complex.22 Ticks feed for several days and need to have well-equipped machinery to cope with the host innate and adaptive immune response. Gene duplication, antigenic variation, and perhaps redundancy in biochemical pathways may have evolved to support feeding habits of I. scapularis. For example, the number of blood anti-clotting and pharmacologically active proteins has been shown to be high in I. scapularis salivary glands.22 On the other hand, the adaptation of I. scapularis to long-term blood feeding in mammals may have allowed pathogens to explore new pathways to be efficiently acquired from the host. Pathogens may interact with tick proteins and avoid host inflammation, immunity, and homeostasis. We now describe that, contrary to adult I. scapularis, reduction of the salp14 paralog (salp9pac) expression in I. scapularis nymphs does not affect engorgement and also does not impair B. burgdorferi and A. phagocytophilum acquisition.

However, the assessment of nymph feeding success based on nymph engorgement weights may not be conclusive; a mixed population of male and female ticks was used in these experiments because nymphs are sexually immature, and female and male nymphs are indistinguishable. Female ticks take bigger blood meals than males, and unequal numbers of males and females in the two groups may have a confounding effect. It is, however, likely that evolutionary events shaped by host and tick natural selection such as gene duplication may have created alternative networks to be exploited by pathogens and ticks during different developmental stages (nymphs and adults) of the tick vector.

Figure 1.
Figure 1.

Schematic of pathogen acquisition in I. scapularis nymphs. I. scapularis nymphs micro-injected with Ponceau-S (mock) and salp14dsRNA were allowed to feed for 72 hours on C3H/HeN- and C3H/HeJ-infected mice with B. burgdorferi and A. phagocytophilum, respectively. Engorged tick nymphs were recovered from infected mice and dissected for molecular biology analysis. Three experiments were done on different dates and showed similar results.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 75, 4; 10.4269/ajtmh.2006.75.677

Figure 2.
Figure 2.

RNAi-mediated reduction of salp9pac expression in nymph I. scapularis does not affect the ability of ticks to feed. I. scapularis nymphs micro-injected with Ponceau-S (mock) and salp9pac-dsRNA (salp14 paralog) were allowed to feed for 4 days on C3H/HeN mice. Engorged tick nymphs (N = 13 and N = 21, respectively) were recovered and weighed.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 75, 4; 10.4269/ajtmh.2006.75.677

Figure 3.
Figure 3.

Reduction of salp14 expression by RNA interference. A, Ponceau-S dye was used as a control and allowed the visualization of I. scapularis nymphs under the dissection microscope B, RNA was isolated from salivary glands of Ponceau-S (1–4) and salivary protein 14 (salp14) dsRNA (5–7)-injected nymphs and analyzed by RT-PCR for the reduction of salp14 transcription. The I. scapularis actin gene was used to determine whether all samples had similar cDNA levels. C, qRT-PCR showed ~10,000-fold decreased salp14 transcription in dsRNA-injected salivary glands compared with mock-injected nymphs. Three experiments were done on different dates using different batches of ticks and mice. One representative experiment is shown. The statistical significance was calculated using the Student t test. D, Protein was extracted from salivary glands of Ponceau-S and salp14 dsRNA-injected nymphs and immunoblotting was done. Salp14 expression was reduced in salp14dsRNA-injected (lanes KO) salivary glands compared with mock-injected salivary glands (lanes Mock). The highly immunogenic non-specific globulin binding protein of 90 kd was used as a control to determine the specificity of the salp14 dsRNA.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 75, 4; 10.4269/ajtmh.2006.75.677

Figure 4.
Figure 4.

A. phagocytophilum acquisition in I. scapularis nymphs. Engorged tick nymphs micro-injected with either Ponceau-S or salp14dsRNA were dissected under the microscope, and salivary glands and midguts were pooled. Total RNA was extracted and cDNA produced. A, RT-PCR followed by gel electrophoresis was done in salivary glands and midguts of engorged I. scapularis nymphs. The I. scapularis actin gene was used as a normalizing factor. Salp14 transcription showed efficiency of the RNAi. B, qRT-PCR was done to detect the transcription of 16srRNA from A. phagocytophilum in Ponceau- and salp14dsRNA–engorged I. scapularis salivary gland nymphs. Three experiments were done on different days, and the statistical significance was calculated using the Student t test.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 75, 4; 10.4269/ajtmh.2006.75.677

Figure 5.
Figure 5.

Borrelia burgdorferi acquisition in I. scapularis nymphs. Engorged tick nymphs micro-injected with either Ponceau-S or salp14dsRNA were dissected under the microscope, and midguts were pooled. Total RNA was extracted and cDNA produced. A, RT-PCR followed by gel electrophoresis was done in midguts of engorged I. scapularis nymphs. B. burgdorferi flaB amplicon in mock midguts (lanes 1–4) was comparable with salp14 dsRNA-injected midguts (lanes 5–8). B, qRT-PCR was done to detect the transcription of B. burgdorferi flaB in mock- and salp14dsRNA-engorged I. scapularis midguts. The I. scapularis actin gene was used as a normalizing factor. Three independent experiments were done on different days, and the statistical significance was calculated using the Student t test.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 75, 4; 10.4269/ajtmh.2006.75.677

*

Address correspondence to Erol Fikrig, Section of Rheumatology, Department of Internal Medicine, Yale University School of Medicine, The Anlyan Center for Medical Research and Education, 300 Cedar Street, Room 525, APO Box 208031, New Haven, CT 06520-8031. E-mail: erol.fikrig@yale.edu

These authors contributed equally to this work.

Authors’ addresses: Joao H. F. Pedra, Sukanya Narasimhan, and Erol Fikrig, Section of Rheumatology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520. Kathleen DePonte, Nancy Marcantonio, and Fred S. Kantor, Section of Allergy and Clinical Immunology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520.

Financial support: This study was supported by National Institutes of Health (NIH) Grants 5R01AI032947-12, 5R01AI041440-08, and 5R21AI054630-02 to Erol Fikrig. Fred Kantor and Sukanya Narasimhan were supported by NIH Grants 5U01AI054971-03 and 5R21AI057940-02, respectively.

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