Identification and expression of macrophage migration inhibitory factor in Sarcoptes scabiei.

Macrophage migration inhibitory factor (MIF) is a pleiotropic proinflammatory cytokine produced by many mammalian tissues including skin. It is also found in many invertebrate parasites of mammals including ticks and may function to aid the parasite to evade the innate and adaptive immune responses in the host. In this study, the cDNA for a MIF gene was sequenced from Sarcoptes scabiei, the scabies mite, using RT-PCR and RACE molecular techniques. The resulting nucleotide sequence had a length of 405 base pairs and the putative amino acid sequences for the mite and tick (Dermacentor variabilis) proteins were identical. The initial steps for the project resulted in the production of expressed scabies mite cDNAs. A real time (qPCR) assay was performed with MIF from scabies mites and various tick species. Results show that mRNA encoding MIF homologues was three times more abundant in the mite samples when compared to RNA prepared from D. variabilis salivary glands and 1.3 times more abundant when compared with RNA prepared from D. variabilis midgut.

Pyrosequencing and characterization of immune response genes from the American dog tick, Dermacentor variabilis (L.).

Ticks continue to be a threat to animal and human health, and new and novel control strategies are needed for ticks and tick-borne pathogens. The characterization of the tick-pathogen interface and the tick immune response to microbial infections is fundamental toward the formulation of new control strategies for ticks and the pathogens they transmit. Our overall hypothesis for this research is that the tick immune system manages the maintenance of pathogens. Therefore, discovery of tick immune response genes may provide targets for novel control strategies directed toward reducing vector competency and pathogen transmission. In these studies, 454 pyrosequencing, a high-throughput genomic sequencing method was used to discover tick genes expressed in response to bacterial and fungal infections. Expressed sequence tags (ESTs) were analysed from Dermacentor variabilis ticks that had been injected with bacteria (Escherichia coli, Bacillus subtilis, Micrococcus luteus) or fungi (Saccharomyces cerevisiae and Candida albicans) and ticks that were naturally infected with the intracellular bacterium, Anaplasma marginale. By this approach, ESTs were assembled into 5995 contigs. Contigs fell into the five main functional categories of metabolism, genetic information processing, environmental information processing, cellular processes and human diseases. We identified more than 30 genes that are likely to encode for proteins involved in tick immune function. We further analysed by reverse transcriptase PCR (RT-PCR) the expression of 22 of these genes in each of our bacterial or fungal treatment groups and found that seven were up-regulated. Up-regulation of these seven genes was confirmed for bacterial, but not fungal treatment by quantitative PCR (qPCR). One of these products was novel, encoding a new tick defensin. Our results clearly demonstrate the complexities of the tick immune system and mark new directions for further study and characterization of proteins that modulate microbial infections in the American dog tick.

Macrophage migration inhibitory factor expression and protein localization in Amblyomma americanum (Ixodidae).

Amblyomma americanum (L.) ticks continue to emerge as disease vectors in many areas of the United States. Tick macrophage migration inhibitory factor (MIF) was first identified in A. americanum females and has been demonstrated to inhibit macrophage movement to the same extent as human MIF. This study was conducted to further characterize and elucidate the physiological role for MIF in tick feeding. A relative quantitative PCR assay was developed to determine the level of MIF gene expression during tick feeding. In addition, RNAi techniques were used to silence MIF prior to blood feeding. Physiological parameters of tick engorgement weight, length of feeding interval, and egg masses were observed to check for phenotypic manifestations of RNA silencing. Specific tick MIF antibody was used to localize MIF protein in frozen tick tissue sections. Tissue specific gene expression indicated that the midgut tissues were the most highly enriched for the MIF. Levels of gene expression did not parallel MIF protein pools seen in tissue sections. Of particular importance was the finding that unfed tick salivary glands appear to contain vesicles that are specific for MIF protein. This is the first demonstration of a pool of MIF that could be secreted during the first hours of tick feeding. While MIF silencing was demonstrated at the molecular level, no physiological phenotype was apparent. The MIF protein pools already available in the tissues may be sufficient to accomplish female tick feeding. Our studies show that the most prominent source of MIF during tick feeding is the midgut tissue. Future studies will address the role of MIF in blood feeding and nutrient digestion in the immature life stages of the tick.

Amblyomma americanum (L): tick macrophage migration inhibitory factor peptide immunization lengthens lone star tick feeding intervals in vivo.

Immunizations of New Zealand White rabbits with specific macrophage migration inhibitory factor (MIF) tick peptide (PEP) produced circulating anti-tick PEP antibodies in the hosts. Antibody titers of greater than 1:5000 to tick MIF peptide were observed for crude sera from PEP-immunized rabbits. PEP- and BSA-vaccinated rabbits were infested with Amblyomma americanum adults. Feeding intervals, female weights, egg masses and percent egg hatch were measured for ticks feeding on control and immunized hosts. Feeding intervals were significantly lengthened to 13.3 days for PEP-vaccinated hosts compared to BSA-vaccinated controls at 12.4 days, while female engorgement weights and egg masses were unchanged. By immunizing hosts using specific tick PEP, we were able to alter the length of time the ticks fed on their hosts.

Identification and characterization of a homologue of the pro-inflammatory cytokine Macrophage Migration Inhibitory Factor in the tick, Amblyomma americanum.

Studying tick feeding and digestion, we discovered in a cDNA library from partially fed Amblyomma americanum ticks the first known arthropod homologue of a human cytokine, the pro-inflammatory Macrophage Migration Inhibitory Factor (MIF). The tick origin of the MIF cDNA clone was confirmed by sequencing a genomic fragment that contained the full-length tick MIF gene with two introns. Antiserum to a tick MIF-specific peptide as well as antiserum to complete tick MIF revealed the expression of tick MIF in the salivary gland and midgut tissues of A. americanum ticks. In an in vitro functional assay, recombinant tick MIF inhibited the migration of human macrophages to the same extent that recombinant human MIF did.

Amblyomma americanum: specific uptake of immunoglobulins into tick hemolymph during feeding.

The passage of immunoglobulins in the blood meal into hemolymph prompts development of vaccines against internal antigens of ticks, but little is known about kinetics and specificity of the immunoglobulin uptake. We used capillary feeding of adult Amblyomma americanum hard ticks to introduce compounds into the midgut and then examined the hemolymph after various times for their presence and concentration. Immunoglobulins of different sources, albumin, choramphenicol acetyltransferase, inulin, and mannitol were labeled with (125)I, (14)C, or biotin. With the exception of the carbohydrate inulin, all the compounds entered the hemolymph of tick during capillary feeding. The small molecule mannitol had the highest rate of entry at 9% after 6 h. Among proteins, the entry of immunoglobulin G (IgG) of different species into the hemolymph was greater at 6% after 6 h than for the smaller proteins albumin or choramphenicol acetyltransferase at 1 and 3%, respectively. The entry of denatured IgG was equal to that of nondenatured protein. There was no evidence of degradation of the IgG or of its binding to cells once it entered the hemolymph. A monoclonal IgG antibody labeled with biotin entered the hemolymph and retained its ability to bind to its specific antigen in an immunoassay. Although different proteins entered the hemolymph after capillary feeding, there was evidence of a specific mechanism for immunoglobulin uptake.

Antibody levels to recombinant tick calreticulin increase in humans after exposure to Ixodes scapularis (Say) and are correlated with tick engorgement indices.

The antibody responses of subjects who presented with a definite Ixodes scapularis (Say) tick bite were measured to determine the utility of the antibody response against recombinant tick calreticulin (rTC) as a biologic marker of tick exposure. Subjects bitten by I. scapularis evidenced an increase in anti-rTC antibody levels between visit 1 and visit 2 from 24.3 to 27.1 ng/microl serum (n = 88, p = 0.003), and levels remained elevated at visit 3 (p = 0.005). These anti-rTC antibody levels during visits 2 and 3 were significantly higher than those in four non-exposed controls. Tick engorgement indices, measured on the biting ticks, were found to be correlated with anti-rTC antibody levels (e.g., for visit 3: Pearson's r = 0.357, p = 0.001). Tick engorgement index (TEI), ratio of body length to scutal width, was identified to be the only independent predictor of anti-rTC antibody levels in linear regression models. Logistic regression revealed that a bite from an I. scapularis tick that became engorged (TEI >3.4) was a risk factor for anti-rTC antibody seropositivity (adjusted odds ratio for age and bite location = 7.4 (95% confidence interval 2.1-26.4)). The anti-rTC antibody test had a sensitivity of 0.50 and a specificity of 0.86 for a bite from I. scapularis that became engorged. Immunoblotting revealed that subjects made a specific anti-rTC antibody response.

Antibody to a cDNA-derived calreticulin protein from Amblyomma americanum as a biomarker of tick exposure in humans.

The antibody responses of human and animal hosts were studied to determine the utility of antibody against recombinant tick calreticulin (rTC), a cDNA-derived protein isolated from salivary glands of Amblyomma americanum L., as a biologic marker of tick exposure. Rabbits fed upon by either A. americanum or Dermacentor variabilis Say developed significant anti-rTC antibody responses, as measured by both ELISA and immunoblot assay. In contrast, gerbils exposed to Aedes aegypti did not develop anti-rTC antibodies, as measured by ELISA or immunoblot assay. The utility of the assay was next evaluated in humans at high risk for tick exposure. During April through September 1990, 192 military personnel who originated from either Fort Chaffee, Arkansas or Fort Wainwright, Alaska were studied during maneuvers in tick infested areas at Fort Chaffee. Study subjects completed a questionnaire and had pre- and post-maneuvers serum specimens analyzed for antibodies to rTC. In adjusted analysis (controlling for age, fort of origin, attached tick during maneuvers, and bed netting use), the use of bed netting and home station were associated with post-maneuvers anti-rTC antibody seropositivity by ELISA. Subjects from Fort Wainwright were more likely to be seropositive for anti-rTC antibody (adjusted odds ratio = 5.3, 95% confidence interval [CI] = 1.1-25.6). Personnel who did not report the use of bed netting were more likely to be anti-rTC seropositive (adjusted odds ratio = 6.8, 95% CI = 1.4-32.4). Immunoblot assays showed that humans had specific anti-rTC antibody responses. The animal experiments demonstrate that hosts exposed to naturally feeding ticks develop anti-rTC antibodies. The data also indicate that hosts exposed to Ae. aegypti saliva may not develop antibodies against rTC. Observations in tick-exposed humans support the hypothesis that anti-rTC antibody seropositivity is a biologic marker of tick exposure.

Cold-hardiness of a laboratory colony of lone star ticks (Acari: Ixodidae).

The cold-hardiness of a lone star tick, Amblyomma americanum (L.), laboratory colony was characterized. Fed and unfed larvae, fed and unfed nymphs, and unfed adults did not survive exposure to -17 degrees C for 7 d. After an 8-d exposure to -10 degrees C, adults tolerated cold better than immatures and unfed specimens fared better than fed ticks. Exposing unfed 6-wk-old (postmolt) adult males and females to -15 degrees C for increasing intervals up to 2 h suggests that males were more tolerant to cold than were females. Half of all adults were alive 3 d after the 2-h low-temperature treatment. Males may have survived because of a significantly higher hemolymph osmotic pressure, although the solute concentration increased for both sexes after a 2-h exposure to 0 degree C. Acclimation to 5 degrees C for 7 d had no influence on supercooling points for unfed males and females, engorged nymphs and larvae, and eggs. None of the life stages survived supercooling, which strongly suggests that this species is freeze intolerant. Intolerance of immature stages to chilling may be a limiting factor in the northern distribution of lone star ticks in North America.

Presence of calreticulin in vector fleas (Siphonaptera).

Calreticulin has been defined in the cat flea, Ctenophalides felis (Bouché), and oriental rat flea, Xenopsylla cheopis (Rothschild). Calreticulin, a major endoplasmic reticulum protein, was previously identified as a component of ixodid tick saliva. Using a riboprobe generated from tick calreticulin complementary DNA (cDNA), we distinguished 2 transcripts for calreticulin in cat fleas by Northern blot analysis. Increased expression of calreticulin was not evident in fed versus unfed adult fleas. We were able to amplify a calreticulin flea product from fed female messenger RNa (mRNA) using primers designed from the tick calreticulin gene. One of these products hybridized to the tick riboprobe. Localization of specific antibody to cat flea tissues showed calreticulin in the midgut with no detection in the salivary glands. We also observed specific labeling of calreticulin with antibody in the ovaries of fed females. Several cat flea polypeptides appear to crossreact with anticalreticulin antibody in Western blots. We did not detect a calreticulin using antibody to the tick-secreted protein in cat flea salivary glands. This antibody did recognize a protein in the rate flea salivary glands. Our results show that fleas have calreticulin and, possibly, several isoforms. It appears that the salivary glands of the cat and oriental rat flea differ in detectable levels of calreticulin. The specific antibody labeling of the ovaries is interesting and remains to be understood. Calreticulin's appearance in the midgut suggests a possible source of calreticulin as a flea secretion. Further studies are in progress to complete the sequencing of the flea polymerase chain reaction (PCR) product to compare to tick-secreted calreticulin. Comparisons to other blood-feeding arthropods at the protein and gene level are also being done. We hope to define further the expression of calreticulin in fleas, and in general, blood-feeding arthropods, with respect to its role in feeding and pathogen transmission.

Acquisition of the cat scratch disease agent Bartonella henselae by cat fleas (Siphonaptera:Pulicidae).

We assayed the ability of cat fleas to become infected with Bartonella henselae, using an artificial feeding device. Fleas fed a concentration of 1 x 10(5) cfu/ml in blood were examined using immunofluorescent antibody assay and polymerase chain reaction. Bacteria were present in the gut at 3 h, and persisted up to 9 d after infection. Qualitatively, the density of B. henselae was greater in the flea gut at 9 d, indicating that replication was occurring in the gut. B. henselae also was detected in the feces of infected fleas 9 d after infection, and produced viable colonies upon inoculation onto heart infusion agar/rabbit blood plates. Our results indicate that fleas can maintain infection with B. henselae, and may play a role in the transmission of this bacterium from infected cats to humans.

Isolation, cultivation, and partial characterization of the ELB agent associated with cat fleas.

ELB rickettsiae from cat flea homogenates were recovered in tissue culture cells following sequential passage through laboratory rats and the yolk sacs of embryonated chicken eggs. Seven days after inoculation of ELB from the infected yolk sacs, Vero cells and L929 cells were observed to contain intracellular bacteria as demonstrated by Diff Quik and indirect immunofluorescence assay staining. The rickettsial and ELB identity of the cultured agent was confirmed by PCR detection of the 16S rRNA and citrate synthase genes and PCR-restriction fragment length polymorphism analysis of the 17-kDa conserved rickettsial antigen gene. The ELB rickettsiae induced plaques in Vero cells on day 11 postinfection. Rat anti-ELB serum reacted at 1:4,096 to cultured ELB and had lower reactivity to Rickettsia typhi Wilmington (1:1,024), Rickettsia akari Kaplan (1:512), and Rickettsia australis JC (1:64). Spotted fever group polyclonal sera also exhibited lower reactivity to ELB than to the homologous antigen. Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of the ELB isolate and two R. typhi strains were identical.

In vitro and in vivo antibiotic susceptibilities of ELB rickettsiae.

The activities of doxycycline, rifampin, chloramphenicol, and erythromycin against ELB rickettsiae (Rickettsia azadi) were determined by dye uptake and plaque assays. Plaque formation in Vero cells was inhibited by 0.12 microgram of doxycycline per ml. The data presented demonstrate the susceptibility of ELB rickettsiae to commonly used antibiotics for the treatment of rickettsial diseases.

Partial biochemical characterization of venom from the ant, Pseudomyrmex triplarinus.

Venom from the ant, Pseudomyrmex triplarinus, contains 12 proteins with mol. wts of > 100,000-4200, and they constitute 41.5% of the dry weight. In comparison with published data on ant, wasp, and bee venoms, whole venom has intense phospholipase activity and intermediate hemolytic activity. Four major proteins were isolated and purified by low pressure chromatography. The most abundant protein had a mol. wt of 4200 and weak hemolytic activity. The second most common protein was 20,400 and had phospholipase A2 activity. The other two major proteins had mol. wts of 24,500 and 14,100 and both exhibited phospholipase and direct hemolytic activities. There are eight minor proteins (> 100,000-40,000), each present at about 1% or less of the total protein. Assayed as a mixture, they had hyaluronidase activity. Seventeen free amino acids were detected with aspartic acid, glutamic acid, and proline together making up 72% of the total mass of amino acids. Glycerol was present at a concentration of 3.1% of the dry weight and the venom was devoid of lipids.

Tick (Acari: Ixodidae) attachment cement and salivary gland cells contain similar immunoreactive polypeptides.

A specific antiserum (12C) raised to a 90-kDa immunogenic component of salivary glands of the tick Rhipicephalus appendiculatus recognized similar 90-kDa polypeptides from salivary glands of the American dog tick, Dermacentor variabilis, and the lone star tick, Amblyomma americanum, as well as 70-kDa polypeptides in the cement of D. variabilis, A. americanum, and R. sanguineus (brown dog tick). The reduction in size of the polypeptide for these ticks suggests that it is modified in some way during or after secretion. Immunostaining of salivary glands of unfed- and partially-fed female D. variabilis localized an immunoreactive protein in the d- and e-cells of the type III acini. The quantity of label in granules of glands from unfed ticks was visibly greater than in the granules of glands from partially fed ticks, suggesting that this component is secreted within the first 2 d of feeding. Collectively, these data support the conclusion that a 90-kDa polypeptide of saliva is conserved among ixodid tick genera and is a component of the attachment cement.

Amblyomma americanum: identification of tick salivary gland antigens from unfed and early feeding females with comparisons to Ixodes dammini and Dermacentor variabilis.

Salivary gland antigens involved in host resistance to tick feeding by Amblyomma americanum (lone star tick) have been identified. Gland extracts from unfed and partially fed 12-, 48-, 72-, 96-, and 120-hr females and their corresponding midgut tissues were analyzed by immunoblotting with sera from naturally immune and hyperimmune sheep and rabbits. Polypeptides at 90, 75, 58, 45, 33, and 23 kDa from the salivary glands of A. americanum females were consistently observed with antibodies from both sheep and rabbits. No antigens unique to tick midgut tissue were detected with immune sera. Female Dermacentor variabilis and Ixodes dammini shared 90- and 45-kDa salivary gland antigens with A. americanum, and these may represent conserved polypeptides. We speculate that some of the salivary gland antigens represent components of tick cement, while others are playing some other yet undetermined role in tick feeding.

Characterization of ixodid tick salivary-gland gene products, using recombinant DNA technology.

Ticks secrete an array of lesion-maintenance factors into the host via the salivary glands while feeding, some of which elicit an immune response by the host that adversely affects the ability of the tick to feed and reproduce. Our approach to characterizing these factors has been to make expression libraries from mRNA of salivary glands (from unfed and 3-day-feeding Amblyomma americanum females) which will serve as sources of the genes (clones) that code for them. Thus far, we have detected 10 positive clones in primary screens using polyspecific antiserum from rabbits hyperimmunized to 3-day-feeding tick salivary glands. We also report making a cDNA library from whole unfed females, and a genomic library from whole unfed ticks, which will serve as additional sources of genetic information for characterizing salivary-gland secretory products. Immunoblots of salivary glands from A. americanum females feeding for various intervals (unfed, and 12, 48, 72, and 96 h) revealed the presence of several prominent polypeptides (90 & 45 kDa) when probed with the same rabbit antiserum that was used to screen the expression library. Ixodes dammini had several immunogens in common with A. americanum at 96 h (90, 45, 43 and 23 kDa). We plan to use monospecific antiserum raised to antigens detected in our immunoblots (e.g. 90 kDa) to further screen the expression libraries, in addition to using the polyspecific antiserum already in hand. We discuss the future use of the salivary-gland genes for characterizing secretory products which facilitate attachment to the host (cement) and maintain the lesion during the lengthy feeding interval.