Confirmation of Tick Bite by Detection of Antibody to Ixodes Calreticulin Salivary Protein

Abstract

Ticks introduce a variety of pharmacologically active molecules into their host during attachment and feeding in order to obtain a blood meal. People who are repeatedly exposed to ticks may develop an immune response to tick salivary proteins. Despite this response, people usually are unaware of having been bitten, especially if they are not repeatedly exposed to ticks. In order to develop a laboratory marker of tick exposure that would be useful in understanding the epidemiology of tick-borne infection and the immune response to tick bite, we developed an enzyme-linked immunosorbent assay (ELISA) to detect antibody to a recombinant form of calreticulin protein found in the salivary glands of Ixodes scapularis, a member of a complex of Ixodes ticks that serve as the vectors for Lyme disease, human babesiosis, and human granulocytic anaplasmosis. Using this assay, we tested sera obtained from C3H/HeN and BALB/c mice before and after experimental deer tick infestation. These mice developed antibody to Ixodes calreticulin antigen after infestation. We then used the same assay to test sera obtained from people before and after they experienced deer tick bite(s). People experiencing deer tick bite(s) developed Ixodes calreticulinspecific antibody responses that persisted for up to 17 months. This Ixodes recombinant calreticulin ELISA provides objective evidence of deer tick exposure in people.

Amblyomma americanum tick calreticulin binds C1q but does not inhibit activation of the classical complement cascade

“…Data in this study builds on previous reports and show that tick CRT proteins show high amino acid identity to human than to parasite CRT proteins. Consistent with reported studies (Jaworski et al, 1995, Sanders et al, 1998, Malouin et al, 2003, Alarcon-Chaidez et al, 2006, Radulovic et al, 2014), Pichia pastoris- expressed r Aam CRT used in this study specifically reacted with antibodies to A. americanum tick saliva proteins confirming that Aam CRT is injected into the host during tick feeding. Additionally in an ongoing study, we have found a calreticulin in I. scapularis tick saliva that shows 78% amino acid identity to Aam CRT ( unpublished ) and have also verified the presence of Aam CRT in A. americanum tick saliva proteomes ( unpublished ).…”

“…In previous studies tick CRT was demonstrated to be among tick saliva proteins that are injected into the host during tick feeding (Jaworski et al, 1995, Sanders et al, 1998, Malouin et al, 2003, Alarcon-Chaidez et al, 2006, Radulovic et. al., 2014).…”

“…In tick biology, functional analysis data on CRT is limited to being a validated tick saliva protein used as the biomarker for human tick bites (Sanders et al, 1998, Malouin et al, 2003, Alarcon-Chaidez et al, 2006). Sanders et al, (1998) observed that repeated Ixodes scapularis infestations in humans conferred an anamnestic antibody response further confirming that tick CRT is injected into the host during tick feeding.…”

Differential protein expression in ovaries of uninfected and Babesia-infected southern cattle ticks, Rhipicephalus (Boophilus) microplus

“…It is secreted into saliva during the feeding process, suggesting that it may play a role in blood feeding through immunosuppression and antihemostasis in its bovine host (Jaworski et al, 1995). Using a recombinant calreticulin enzyme-linked immunosorbent assay (ELISA), Alarcon-Chaidez et al (2006) were able to detect antibodies to Ixodes calreticulin salivary protein in vertebrate hosts, confirming that these hosts had previously been bitten by ticks. It would be interesting to see if the calreticulin levels in saliva differ between infected and uninfected cattle ticks, and to investigate how that may affect a potential immune response in a bovine host.…”

Deorphanization and target validation of cross-tick species conserved novel Amblyomma americanum tick saliva protein

“…The specificity of Abs to both 24 h and 48 h TSPs as well as the reactivity of mono-specific Abs to r Aam AV422 on western blots of r Aam AV422, tick saliva, haemolymph and dissected tick organs led us to conclude that this protein was ubiquitously expressed and injected into the host within 24 h of tick feeding. In the literature two other cross-tick species conserved TSPs, tick calreticulin (Sanders et al, 1998, 1999; Ferreira et al, 2002; Xu et al, 2004, 2005; Alarcon-Chaidez et al, 2006; Gao et al, 2008; Kaewhom et al, 2008; Parizi et al, 2009) and histamine release factor (Mulenga et al, 2003a Mulenga and Azad, 2005; Dai et al, 2010) have been described. A common feature between tick calreticulin and histamine release factor is that they both have homologs in vertebrates (Mulenga et al, 2003b, Mulenga and Azad, 2005; Coe and Michalak, 2009; Martins et al, 2010).…”