On an electronic microbalance (model C-31. Cahn Instruments, Cerritos, CA). Before each weighing, we. Transmission (Sunset Laboratory Thermal. Optical Transmittance Analyzer; Sunset. Laboratory, Inc., Tigard. 3 times the standard deviation of field blanks divided by the target flow rates and 24-hr sampling duration. We support the complete line of Cahn Instruments- from the old analog 4100 to the newest digital systems. From hangdown replacement to complete rebuild, we have the experience and equipments to handle all types of service.

• Cahn C 31 Microbalance Manual Transmission Reviews

Ticks secrete several anti-hemostatic factors in their saliva to suppress the host innate and acquired immune defenses against infestations. Using Ixodes scapularis ticks and age-matched mice purchased from two independent commercial vendors with two different immune backgrounds as a model, we show that ticks fed on immunodeficient animals demonstrate decreased fibrinogenolytic activity in comparison to ticks fed on immunocompetent animals. Reduced levels of D-dimer (fibrin degradation product) were evident in ticks fed on immunodeficient animals in comparison to ticks fed on immunocompetent animals.

Increased engorgement weights were noted for ticks fed on immunodeficient animals in comparison to ticks fed on immunocompetent animals. Furthermore, the LC-MS/MS and quantitative real-time-PCR analysis followed by inhibitor and antibody-blocking assays revealed that the arthropod HSP70-like molecule contributes to differential fibrinogenolysis during tick feeding. Collectively, these results not only indicate that ticks elicit variable fibrinogenolysis upon feeding on hosts with different immune backgrounds but also provide insights for the novel role of arthropod HSP70-like molecule in fibrinogenolysis during blood feeding. Ticks are obligate hematophagous ectoparasites that can transmit several pathogens to humans and animals. Understanding molecular interactions at the tick-host interface involve knowledge of the participation of host defense mechanisms against tick infestations and counter measures employed by ticks. Acquired resistance by the host to tick infestations involves both humoral and cellular immunoregulatory pathways that impair tick feeding, egg production and viability. On the other hand, ticks suppress host antibody production, complement activation and cytokine production from both antigen-presenting cells and T cell subsets.

In the United States, Ixodes scapularis ticks transmit Borrelia burgdorferi, the causative agent of Lyme disease, Anaplasma phagocytophilum, the agent of Human anaplasmosis, Babesia microti, the agent of Human babesiosis, Ehrlichia muris-like agent (EMLA), the agent of Human ehrlichioses, Powassan virus, the agent of encephalitis and B. Miyamotoi, the agent of relapsing fever. In contrast to soft ticks that feed for short periods (1–3 hours), I. Scapularis ticks feed on a host for more extended periods, up to 5 or 6 days for nymphs and even longer for adults. To establish a successful feeding niche to commence blood feeding and engorge to completion, I. Scapularis ticks secrete several pharmacologically active molecules in their saliva that include but not limited to anti-hemostatic, anti-inflammatory, immunosuppressive and immunomodulators targeting several host immune pathways,.

Tick-borne pathogens also use some of these important classes of molecules present in tick saliva to infect a vertebrate host,. Over the past few years, several studies have explored the importance of pathogen modulation of tick gene expression during tick-pathogen interactions,. However, the influence of different genetic or immune backgrounds of the vertebrate hosts on tick gene expression and blood feeding has not yet been fully evaluated. Scapularis ticks express a variety of molecules to counter host immune defense responses, including those noted previously, we used these ticks as a model to address this important question. Studies have reported significant variations in many basic hematological and coagulation parameters among many mouse strains. In addition, a recent study has shown that T-cells participate in coupling coagulation with inflammation. These studies provide strong rationale for the current study to test whether variable genetic or immune backgrounds of murine host influences tick feeding and gene expression.

The findings presented in this study report that the host’s genetic background and/or immune status does influence specific tick gene expression that subsequently impact variable fibrinogenolysis during feeding. Tick engorgement weights are increased upon feeding on immunodeficient mice We first analyzed whether the immune status of the animals influence tick feeding. Uninfected unfed larvae were fed on age and background matched immunocompetent (C57BL/6 J and BALB/c) or immunodeficient (RAG −/− and SCID) mice ordered from two different commercial vendors with independent housing conditions (Jackson Laboratoreis-C57BL/6 J, RAG −/− or Charles River Laboratories-BALB/c, SCID).

Upon repletion, engorgement weights of fed larvae were measured using an analytical balance. We found that the engorgement weights of ticks fed on immunodeficient mice (0.499 ± 0.06 mg for RAG −/−; 0.517 ± 0.05 mg for SCID) were significantly (P. Uninfected unfed larvae were fed on age- and gender-matched three immunocompetent mice (C57BL/6 J, BALB/c) or immunodeficient (RAG −/−, SCID) mice purchased from independent vendors. C57BL/6 J and RAG −/− ( A) mice are from Jackson laboratories and BALB/c and SCID NCr ( B) mice are from Charles River Laboratories. Ticks were weighed soon after repletion. Engorgement weights are shown in milligrams and were measured using a Cahn C-31 microbalance set to a range from 25 mg to 0.1 μg.

Student’s t test values are shown. Each circle represents one individual tick. Closed and open circles represent ticks fed on immunocompetent or immunodeficient mice, respectively. Levels of host fibrin degradation product (D-dimer) are reduced in ticks acquiring blood from immunodeficient mice During feeding, ticks may acquire host proteins (including fibrin/fibrinogen or its degradation products) along with the vertebrate blood.

Therefore, we tested the levels of D-dimer, a prominent product of fibrin degradation, in total lysates of whole ticks fed on immunodeficient or immunocompetent animals. Stain-free gel images showed no significant visual differences in the total protein profile between ticks fed on either group of mice. However, immunoblotting results showed dramatically low levels of D-dimer in the total lysates prepared from ticks fed on immunodeficient animals (RAG −/−, SCID) in comparison to the respective control group of ticks fed on immunocompetent (C57BL/6 J, BALB/c) animals (, respectively). Densitometry analysis of the immunoblots further supported that these observations are statistically (P. Uninfected unfed larvae were fed on three (indicated as 1, 2, 3) immunocompetent (C57BL/6 J, BALB/c) or three immunodeficient (RAG −/−, SCID) mice.

3–5 ticks fed on each mouse were pooled and total lysates were generated separately. Total protein profile ( A, B) from ticks fed on individual mouse (3 mice/group) is shown. Immunoblotting performed with 30 μg of tick total lysates and anti-D-dimer antibody showed dramatically low ( C) or reduced ( D) levels of D-dimer in ticks fed on immunodeficient animals in comparison to ticks fed on respective immunocompetent groups.

Densitometry analysis showing levels of D-dimer observed in C57BL/6 J, RAG −/− samples ( E) and BALB/c, SCID samples ( F) relative to the respective bands seen in A and B (marked with asterisk). Student’s t test values are shown. ( G) Ponceau stained membrane image showing D-dimer (2 μg) protein (as control) along with tick total lysates (30 μg). Asterisk shows the position of the bands that were considered for densitometry analysis for relative quantification of D-dimer levels. ( H) Immunoblotting assays with anti-D-dimer antibody showed presence of D-dimer in lysates prepared from ticks fed on immunocompetent animals (C57BL/6 J, BALB/c) at the same position (200 kDa) as native D-dimer protein. Detection of lower bands in the D-dimer lane could be due to degradation of the native protein.

Non-reducing and denaturation conditions were used in the SDS-PAGE analysis. 4–20% gradient SDS-PAGE gels (NuPAGE) gels were used for data in ( A–D) and 10% laboratory-made SDS-PAGE gels were used for data in G and H. Immunoblotting with Anti-D-dimer antibody was performed at least three times. The Arrow next to the immunoblots ( C, D and H) indicates position of D-dimer. Salivary gland lysates prepared from ticks fed on immunodeficient animals show reduced fibrinogenolytic activity in vitro The reduced levels of D-dimer in ticks fed on immunodeficient animals suggest reduced fibrin clot formation at bite-site during blood feeding. Therefore, we analyzed in vitro, whether salivary gland lysates prepared from ticks fed on immunodeficient animals show reduced fibrinogenolytic activity in comparison to the lysates prepared from ticks fed on immunocompetent animals.

Uninfected unfed nymphs were fed on immunocompetent (C57BL/6 J, BALB/c) or immunodeficient (RAG −/−, SCID) mice and upon repletion they were processed for salivary gland isolation as described. Incubation of fibrinogen (purified protein from Sigma) with salivary gland lysates prepared from ticks fed on immunodeficient animals showed reduced degradation of Aα chain of fibrinogen in the presence of CaCl 2 (when incubated for 2 or 4 h) in comparison to the lysates prepared from ticks fed on immunocompetent animals. Densitometry analysis further supported this observation. The salivary gland lysates prepared from ticks fed on immunocompetent (C57BL/6 J) mice showed increased degradation of Aα chain of fibrinogen in the presence of CaCl 2 even at shorter incubation times (15, 30 min) in comparison to the lysates prepared from ticks fed on immunodeficient (RAG −/−) animals.

Cahn C 31 Microbalance Manual Transmission Reviews

The degradation of fibrinogen Aα chain (in all cases) was sensitive to EDTA treatment in comparison to the CaCl 2 treatment. Increased fibrinogenolytic activity was observed with the salivary gland lysates prepared from ticks fed on BALB/c mice even upon treatment with EDTA in comparison to the lysates prepared from ticks fed on SCID mice. Incubation of fibrinogen with CaCl 2 or EDTA alone did not show any fibrinogen degradation. The amount of salivary gland lysates (5 μg) used in the fibrinogenolysis assay did not show any prominent band with the same intensity or size of the fibrinogen, ruling out the possibility of any tick molecules being detected at the degradation product size. These results indicate that variable fibrinogenolytic activities elicited by ticks are calcium dependent and sensitive to chelating agents such as EDTA. Uninfected unfed nymphs were fed on immunocompetent mice (C57BL/6 J, BALB/c) or immunodeficient (RAG −/−, SCID) mice. Salivary glands were dissected from ticks fed on each group of mice, pooled and total lysates were generated separately.

Fibrinogen (37.5 μg) was incubated with tick salivary gland lysates (5 μg) for the indicated times (in hours) in the presence of 1 mM CaCl 2 ( A, B, C, D) or 1 mM EDTA ( E, F, G, H) in a total of 21 μl reaction volume. From the total volume, 3 μl was taken out at different time points, heated at 70 degrees with sample buffer to terminate the reactions and loaded on to a stain-free gel. 12% SDS-PAGE (BioRAD) gels were used for data in ( A, B, E, F). Fibrinogenolysis assays were performed two times for samples prepared from ticks fed on C57BL/6 J and RAG −/− mice and one time for samples generated from ticks fed on BALB/c and SCID mice. Solid arrow indicates Aα chain of fibrinogen and dotted line indicates degradation product. Densitometry analysis showing levels of degradation of Aα chain of fibrinogen in C57BL/6 J and RAG −/− samples ( C, G) or BALB/c and SCID samples ( D, H) in the presence of CaCl 2 ( C, D) or EDTA ( G, H) at the indicated time points for the images shown in ( A, B, C and D). The levels of degradation product for each sample was measured relative to the respective levels of Aα chain at 0 min time point.

The gene expression for four metalloproteases analyzed in this study is unaltered in ticks fed on immunocompetent or immunodeficient animals A tick metalloprotease belonging to reprolysin family containing pre- and pro-enzyme domains, zinc-binding motif and cysteine-rich region from salivary glands of I. Scapularis has been suggested to participate in fibrinogenolytic activities. Therefore, we analyzed expression of several of the tick metalloproteases (GenBank acc.

XM002416249, XM002416250 and XM002412196) along with the salivary gland metalloprotease (GenBank acc. AY264367), that was previously suggested to participate in fibrinogenolysis.

QRT-PCR analysis revealed that expression of all four metalloproteases, analyzed in this study, did not show significant (P  0.05) variable expression between ticks fed on immunocompetent (C57BL/6 J) or immunodeficient (RAG −/−) animals. Identification of proteins that show variable expression in salivary glands of ticks fed on immunocompetent versus immunodeficient animals We then performed 1-D gel electrophoresis with the salivary gland lysates prepared from ticks fed on immunocompetent (C57BL/6 J) or immunodeficient (RAG −/−) animals. The results revealed increased band intensities for two of the bands at 100 kDa and 73 kDa band in lysates prepared from ticks fed on immunocompetent animals in comparison to the lysates prepared from ticks fed on immunodeficient animals ( and ). Detection of 100 kDa band was also evident in the salivary gland lysates prepared from ticks fed on BALB/c mice in comparison to lysates prepared from SCID mice. Due to the detection of 100 kDa band (with high intensity) in both groups of immunocompetent mice, this band from salivary gland lysates prepared from ticks fed on C57BL/6 J mice was excised and processed for LC-MS/MS analysis.

The LC-MS/MS analysis on the 100 kDa band revealed higher peptide matches for tick HSP70-like proteins (, ). ( A) 1-D SDS-PAGE (4–20% gradient gel, NuPAGE) analysis of total protein profile in salivary glands of ticks fed on immunocompetent (C57BL/6 J) or immunodeficient animals (RAG −/−) is shown. Solid arrow indicates band (that was excised and processed for LC-MS/MS analysis) around 100 kDa and dotted arrow indicates a band around 73 kDa that was found to be upregulated in salivary gland lysates prepared from ticks fed on immunocompetent mice.

M indicates protein marker. Asterisk indicates position of control bands that were considered for densitometry analysis. SDS-PAGE analysis to study total protein profile in samples generated from ticks fed on C57BL/6 J or RAG −/− animals was performed two times with different concentrations. QRT-PCR results showing levels of XM002433611 ( B), XM002407088 ( C), XM002406516 ( D), XM002415881 ( E), XM002402518 ( F) and XM002412155 ( G) transcripts in ticks fed on immunocompetent (C57BL/6 J, closed circles) or immunodeficient (RAG −/−, open circles) is shown. Levels of the transcripts for all hsp70-like molecules were normalized to tick beta-actin. HSP70 transcript XM00241255 was undetectable or had very low threshold levels in some of the ticks that were fed on immunodeficient animals (RAG −/−). Therefore, additional tick samples were included in this assay.

The amount of transcripts for hsp70-like molecules in tick samples that contained undetectable levels or well below threshold level were considered as “zero ng” for the analysis. Each circle represents one tick. Student’s t test P values are shown.

Level of one of the HSP70-like molecules is upregulated in ticks fed on immunocompetent animals in comparison to the immunodeficient animals QRT-PCR analysis was performed with the samples generated from whole larval ticks fed on immunocompetent (C57BL/6 J) or immunodeficient (RAG −/−) mice for all six tick hsp70-like molecules that were identified in LC-MS/MS analysis. The mRNA levels for five hsp70-like molecules (GenBank Acc.

XM002433611, XM002407088, XM002406516, XM002415881, and XM002402518) were found to be unaltered in ticks fed on either group of mice. However, one of the HSP70-like molecules (GenBank Acc.

XM002412155) was noted to be significantly (P. Fibrinogenolysis assays performed with salivary gland lysates prepared from ticks fed on immunocompetent animals in the presence of 100 μM VER155008 (HSP70 inhibitor) or equal volume of mock control and assayed at indicated time points. 12% SDS-PAGE gel (Bio-Rad) was used in the assay. Fibrinogenolysis assays with VER155008 were performed at least three times. Solid arrow indicates increased Aα chain fibrinogen degradation and dotted arrow indicates increased level of fibrinogen-degraded product at 30 min time point in mock control in comparison to HSP70-inhibitor-treated samples.

( B) Densitometry analysis (for image in ( A) showing levels of degradation of Aα chain of fibrinogen in mock- or HSP70 inhibitor-treated samples at the indicated time points. ( C) Fibrinogenolysis assays performed with salivary gland lysates prepared from ticks fed on immunocompetent animals in the presence of 50 ng of anti-HSP70 antibody or isotype control antibody and assayed at indicated time points is shown. Arrow indicates increased fibrinogen degradation in the presence of isotype-matched control antibody.

4–20% gradient SDS-PAGE gel (NuPAGE) gels were used in the analysis. Assays with Anti-HSP70 antibody was performed three times.

( D) Densitometry analysis (for gel image in ( C) showing levels of degradation of Aα chain of fibrinogen in isotype- or HSP70 antibody-treated samples at the indicated time points. In ( B and D), the levels of Aα chain degradation for each sample was measured relative to the respective levels of Aα chain at 0 min time point.

To acquire a blood meal, ticks mechanically attach to their vertebrate host, insert their mouthparts in to the host skin, secrete saliva to encounter host defense mechanisms and cement themselves to the attachment site, engulf blood and then fall off upon completion of blood feeding,. Several tick molecules play essential roles in the blood feeding,. In this study, we report a novel role for the participation of tick HSP70-like molecule in fibrinogenolysis during blood feeding. HSP70s are usually 70 kDa proteins. Tian et al., (2011) have shown that antibody generated against recombinant Haemaphysalis longicornis HSP70 recognized bands of approximately 100, 72 and 28 kDa in the egg lysates prepared from these ticks.

The variation in sizes suggests that tick HSP70-like molecules could undergo several posttranslational modifications to an active form. A recent study has shown that HSP70 forms antiparallel dimers that are stabilized by post-translational modifications to position clients for transfer to HSP90. Therefore, the observation of an HSP70-like peptide sequence (with highest peptide match and scores) in the LC-MS/MS analysis (for a 100 kDa band) in our study is not surprising. Fibrinogen is an important factor in normal vertebrate host blood clotting.

Our hypothesis is that increased engorgement weights in ticks fed on immunodeficient mice is due to the reduced fibrin/fibrinogen clots in these mice. Our finding that ticks fed on immunodeficient animals express low level of HSP70-like protein is consistent with the reduced fibrinogen degradation around the tick bite site on these animals in comparison to the immunocompetent animals. The low levels of murine D-dimer in ticks fed on immunodeficient mice in comparison to ticks fed on immunocompetent mice strongly support this hypothesis.

Low levels of fibrin/fibrinogen clots could account for less clotting that may subsequently affect increased blood in-take by ticks fed on the immunodeficient animals and possibly increased transmission of pathogens. The later argument is in agreement with a study that has reported an increased rate of transmission of pathogen from ticks to immunodeficient animals in comparison to transmission of pathogens from ticks to the immunocompetent animals. HSP70 was initially thought to be an intracellular protein. However, work from Tytell and Hightower laboratories provide evidence that HSP70 could transit through the extracellular spaces. HSP70 is also found in the human serum in substantial quantities suggesting its role as an extracellular or secreted protein.

In addition, a proteasome component Sec61p and HSP70 are reported to be involved in the degradation of fibrinogen in the mammalian cells. Our data provides the evidence that suggests contribution of arthropod HSP70-like molecule in fibrinogenolysis during tick blood feeding. The observation of higher mRNA levels for only one of the HSP70-like molecule out of six molecules analyzed in this study does not rule out the possibility for the participation of other five HSP70-like molecules at different stages of tick blood feeding. In addition, role for other HSP70-like molecules in fibrinogenolysis cannot be ruled out in ticks during feeding on diverse vertebrate hosts such as deer or other animals. Future comparative studies on all tick HSP70-like molecules would reveal whether they have similar or redundant function during arthropod blood feeding.

We hypothesize that HSP70-like molecules are secreted in tick saliva and may aid in proper folding of proteins that are involved in the degradation of fibrinogen at the arthropod bite site to facilitate blood feeding. However, the contribution of arthropod HSP70 molecules to fibrinogenolysis either directly or indirectly would not rule out other salivary activities by these molecules important during feeding. HSP70s are molecular chaperones that consist of three domains: N-terminal ATPase domain, substrate binding domain and C-terminal domain. The N-terminal ATPase domain hydrolyses ATP to ADP.

The HSP70-substrate interactions are determined by the nucleotide status of the N-terminal domain. In the ATP-bound state, HSP70s have a high association and dissociation rates for substrate exchange.

In contrast, in the ADP-bound state, HSP70s have low substrate exchange rates. VER155008, an ATP-competitive inhibitor, arrests the N-terminal domain of HSP70 that subsequently affect substrate binding. The observation of reduced fibrinogenolysis upon treatment of salivary glands lysates generated from ticks fed on immunocompetent animals with VER155008 suggests that N-terminal domain of arthropod HSP70 is critical for the recognition of substrate proteins that could be involved in fibrinogen degradation. Future studies on these aspects will reveal interesting findings on the role for arthropod HSP70-like molecules in fibrinogenolysis.

RAG −/− mice do not produce any mature T or B-lymphocytes and this is described as a “non-leaky” immune deficiency animal model. Whereas, some SCID mice appear to develop few clones of T and B cells.

The differences in the in vitro fibrinogenolysis observed between salivary gland lysates prepared from ticks fed on SCID in comparison to ticks fed on RAG −/− mice could be due to the two different genetic backgrounds (BALB/c versus C57BL/6 J, respectively) or due to the leakiness in the generation of some clones of T and B cells in the SCID mice. T and B-cells play important roles in the host immune responses against tick bites. The lack of T and B cells or other metabolites in the host blood could also account for increased blood in ticks fed on immunodeficient animals independent of the host blood clotting factors.

Several studies have now characterized various tick salivary proteins involved in T and B cell inhibition. It remains interesting to analyze whether tick HSP70-like proteins affect activation of host T and B cells and thereby facilitate arthropod blood feeding. The finding of a decreased level of fibrinogenolysis from lysates prepared from ticks fed on T and B-cell deficient background host provides important future avenues in understanding the role of these cells and their relevant signaling pathways in the formation of fibrin/fibrinogen clots around the feeding cavity formed by the arthropod bites. In summary, using I. Scapularis ticks as a model we provide evidence that ticks elicit variable fibrinogenolysis upon feeding on hosts with different genetic background and/or immune status.

This study is not only important in understanding the molecular basis of the interactions at the tick-host interface but may also potentially lead for the development of anti-tick vaccines to interfere with the life cycle of this and perhaps other medically important vectors. & Magnarelli, L.

Biology of ticks. 22, 195–215 (2008). & Piesman, J. Overview of tick-borne infections of humans.

In Tick-Borne Diseases of Human, D.T.D. Goodman, Daniel E. Sonenshine, Editor.

American Society for Microbiology: Washington D.C., 3–11 (2005). Sonenshine, D. The Biology of Tick Vectors of Human Disease. In Tick-Borne Diseases of Human, D.T.D.

Goodman, Daniel E. Sonenshine, Editor. American Society for Microbiology: Washington D.C., 12–35 (2005). Ticks and tick-borne pathogens at the cutaneous interface: host defenses, tick countermeasures, and a suitable environment for pathogen establishment. Frontiers in microbiology 4, 337, doi: 10.3389/fmicb.2013.00337 (2013). Tick immunobiology. Parasitology 129 Suppl, S161–176 (2004).

Francischetti, I. M., Sa-Nunes, A., Mans, B. J., Santos, I.

& Ribeiro, J. The role of saliva in tick feeding. Front Biosci (Landmark Ed). 14, 2051–2088 (2009).

Salivating for knowledge: potential pharmacological agents in tick saliva. PLoS medicine 5, e43, doi: 10.1371/journal.pmed.0050043 (2008). & Armstrong, P. Prevalence and genetic characterization of Powassan virus strains infecting Ixodes scapularis in Connecticut. The American journal of tropical medicine and hygiene 87, 754–759, doi: 10.4269/ajtmh.20 (2012). Borrelia miyamotoi sensu lato seroreactivity and seroprevalence in the northeastern United States. Emerging infectious diseases 20, 1183–1190, doi: 10.3201/eid2007.131587 (2014).

10. Emergence of a new pathogenic Ehrlichia species, Wisconsin and Minnesota, 2009. The New England journal of medicine 365, 422–429, doi: 10.1056/NEJMoa1010493 (2011). 11. Sonenshine, D. Biology of Ticks, Second Edition. Oxford University Press 2 (2014).

M., Makoul, G. T., Levine, J., Robinson, D.

& Spielman, A. Antihemostatic, antiinflammatory, and immunosuppressive properties of the saliva of a tick, Ixodes dammini. The Journal of experimental medicine 161, 332–344 (1985). 13. & Francischetti, I. Role of arthropod saliva in blood feeding: sialome and post-sialome perspectives.

Annual review of entomology 48, 73–88, doi: 10.1146/annurev.ento.48.012 (2003). 14.