Tick Hemolymph Research Articles

Effect of antibodies against β- N-acetylhexosaminidase on reproductive efficiency of the bovine tick Boophilus microplus

A polyclonal antibody (anti-HEX) was developed against a soluble N-acetylhexosaminidase (HEX) isolated from larval extracts of Boophilus microplus. Purified hexosaminidase was strongly inhibited by the IgG fraction of this antibody. The antibody inhibited the hexosaminidase activity of other sources, such as haemolymph and larval membranes. The antibody reacted with different antigens in the tick haemolymph, but did not recognize any antigen in saliva, as seen by immunoblot analysis. The anti-HEX was inoculated into fully engorged B. microplus females, resulting in a decrease in oviposition of approximately 26%, relative to the effect of pre-immune IgG. These data show the potential of the use of this tick enzyme as an antigen in vaccine development.

Control of bacterial infections in the hard tick Dermacentor variabilis (Acari: Ixodidae): evidence for the existence of antimicrobial proteins in tick hemolymph.

The ability of hard ticks to prevent infection by bacteria was investigated. During a 72-h period, virtually all Dermacentor variabilis females survived inoculation with Bacillus subtilis, Escherichia coli, and Staphyloccocus aureus but few survived infection with Pseudomonas aeruginosa. The hemocyte population increased to peak abundance at 48 h to approximately 6 times that of the uninfected controls. In contrast, the soluble hemolymph protein content decreased inversely as the hemocytes increased. D. variabilis hemolymph was found to be constitutively antimicrobial (i.e., hemolymph from bacteria-naive individuals inhibited bacterial growth). Infection with various bacterial species enhanced this innate capability. When hemolymph fractions separated by high-pressure liquid chromatography were tested for their ability to inhibit microbial growth, activity against the gram-positive bacterium, B. subtilis, was found in 2 polar fractions. Antimicrobial activity was lost when the fractions were incubated with protease. The least polar fraction contained 1 major protein, M(r) 14.5 kDa, that comigrated with human lysozyme. This protein, tentatively identified as tick lysozyme, was abundant in bacteria-naive ticks but increased greatly (43%) following challenge with B. subtilis. The identity of the other, more polar protein is unknown. Studies to characterize these antimicrobial proteins are planned.

Some quantitative aspects of natural babesial infection in the haemolymph of Boophilus microplus engorged female ticks.

Quantitative aspects of the natural babesial (Babesia bovis and Babesia bigemina) infection in Boophilus microplus engorged female ticks obtained from two herds of Holstein heifers positive by the immunofluorescent antibody test to both protozoan were evaluated. The number of kinetes/microscope field of haemolymph was determined for each tick from day 5 to 10 post-collection. A close relationship between daily and cumulative babesial infection was detected. Correlation and determination coefficients between days post-collection and the daily and cumulative infection rates, including heavily infected ticks (those ticks carrying at least 3.0 kinetes/microscope field of haemolymph), were always higher than 0.9 (P < 0.01) in ticks of both herds. The median was found to be a more representative measure than the mean to define the distribution of kinetes number amongst infected ticks since this is a negative binomial distribution. The analysis of the sequential order of days of infection more accurately showed the amplification of the babesial infection in the tick haemolymph than the evolution of kinetes number in relation to days post-collection. Sampling ticks on days 8, 9 and 10 post-collection would have detected all ticks infected with Babesia spp. from both herds. A categorization of infected or non infected ticks would be of greater epidemiological importance than the haemolymph infection level, based upon previous laboratory studies that showed a poor relationship between haemolymph infection in the female ticks and the infection rate in their eggs. However, further studies in natural infected ticks and better techniques to differentiate B. bovis and B. bigemina kinetes are needed before these laboratory results can be applied to field conditions.

Use of polymerase chain reaction in bovine babesiosis research.

The polymerase chain reaction (PCR) in babesiosis research was originally developed to detect Babesia bovis or Babesia bigemina in blood samples containing infected erythrocytes. These preliminary studies led to development of a sensitive PCR/DNA probe assay to detect the following hemoparasites: B. bigemina and B. bovis in a single sample. This modified procedure, referred to as a duplex PCR/ nonradioactive probe assay, has an analytic sensitivity of 0.00001% for B. bigemina, and 0.00001% infected erythrocytes for B. bovis. This procedure has been modified to detect Babesia DNA in tick tissue and hemolymph. The above procedures can be performed in central laboratories that have access to a thermocycler and quality reagents. Precautions must be observed to prevent cross-contamination of samples. At the present time the procedure has application in epidemiology studies to detect carriers and the species of Babesia in the bovine population. Preliminary studies are in progress by various research groups to utilize this technique in studying the biology of the Babesia protozoans in tick vectors. The applications, advantages, and disadvantages of the technique are presented.

Functional bovine immunoglobulins in Boophilus microplus hemolymph

The aim of the present work was to quantify the passage of bovine immunoglobulins into the hemolymph of the tick Boophilus microplus during the feeding process and to determine their antibody activity. This knowledge is of paramount importance when vector control or blocking of disease transmission is attempted by vaccination of cattle. Approximately 2% of bovine immunoglobulin present in the serum as determined by competitive ELISA was demonstrated in hemolymph of B. microplus and antibody activity against an antigen of B. microplus in the hemolymph of ticks fed on bovine immunized with the antigen purified from tick eggs was detected by Western blot assay. The antibody reactivity detected against the B. microplus antigen showed that functional antibodies are present in the hemolymph of fully engorged ticks for at least 48 h after completing the parasitic life cycle.

Light microscopy diagnosis of Babesia bovis and Babesia bigemina kinetes in the haemolymph of artificially infected Boophilus microplus engorged female ticks

The length, width and position of the nucleus of Babesia bovis and Babesia bigemina kinetes from the haemolymph of Boophilus microplus engorged female ticks were recorded. Additionally, the shape of Babesia bovis kinetes were registered as curved, semi-curved or straight. To this aim Boophilus microplus tick larvae from a colony free of Babesia were fed on splenectomised calves artificially infected with either Babesia bovis or Babesia bigemina pathogenic strains. Six engorged female ticks showing an infection of at least ten mature kinetes of Babesia bovis in a sample of haemolymph 5 days after detachment were also monitored 7, 9 and 10 days after collection. The same procedure was followed with six engorged female ticks infected with Babesia bigemina. One hundred and twenty kinetes of each species of Babesia were evaluated. The mean length ± standard deviation and ranges for Babesia bovis kinetes were 14.30 ± 0.922 μm and 11.9−16.3 μm, while the corresponding measures for the kinetes of Babesia bigemina were 11.27 ± 0.900 μm and 9.0−13.1 μm ( P < 0.001, t-test). The width was 3.33 ± 0.315 μm, 2.6−4.0 μm for Babesia bovis and 2.24 ± 0.287 μm, 1.5−2.8 μm for Babesia bigemina kinetes ( P < 0.001). The most common position of the nucleus was central for both species of Babesia. A total of 58% of Babesia bovis kinetes showed the typical curved tail. No effect of time post-collection and individual host ticks in the kinete of Babesia bigemina was found while an unexpected influence of individual host tick in the width of Babesia bovis kinetes was detected ( P < 0.01, analysis of variance). The overlap in the sizes of kinetes from both species of Babesia makes it difficult to apply the results to ticks of unknown babesial infection status. This finding is further complicated by the intra-specific size variations of Babesia kinetes from different geographical origins.

Excretion of host immunoglobulin in tick saliva and detection of IgG-binding proteins in tick haemolymph and salivary glands.

Host immunoglobulin G (IgG) crossed the gut wall into the haemocoel of adult Rhipicephalus appendiculatus female ticks when they fed on guinea-pigs. Guinea-pig IgG was also found in saliva of the feeding ticks. The concentration and antibody activity of IgG in haemolymph, salivary gland extract (SGE) and saliva at different stages of tick feeding were detected by enzyme-linked immunoassay. Specific activity of the IgG in tick samples was determined by feeding ticks on guinea-pigs which were immunized with killed Escherichia coli: 35-42% of the antibody activity in guinea-pig immune serum remained in the tick samples. The high relative concentration of IgG in tick saliva at later stages of feeding suggests that the tick may have a mechanism for getting rid of foreign proteins via the salivary gland. Such a mechanism could involve IgG binding proteins (IGBPs) which were found in both haemolymph and SGE of female ticks at day 6 of feeding using a guinea-pig IgG-agarose affinity column. In female ticks, the M(r) of IGBPs in SGE (23 and 57 kDa) were less than those in haemolymph (78 and > 100 kDa). The existence of IGBPs in both the tick salivary gland and haemolymph indicate that haemolymph and salivary gland cooperate to remove foreign proteins, e.g. host immunoglobulin, from the body during feeding. This mechanism may be a part of the tick self-defence system.

Comparison of the proteins in salivary glands, saliva and haemolymph of Rhipicephalus appendiculatus female ticks during feeding

To compare the proteins in salivary glands, saliva and haemolymph of Rhipicephalus appendiculatus female ticks, antisera were prepared from guinea-pigs immunized with soluble denatured salivary gland extracts (SGE). The extracts were derived from R. appendiculatus female ticks that were either unfed (day 0) or partly fed (day 6). The sera were used in immunoblotting, following SDS-polyacrylamide gel electrophoresis, to examine the antigen profiles during the course of tick feeding on guinea-pigs. Day 0 and day 6 SGE antisera appeared to detect common proteins in the different tick samples. For example, haemolymph apparently shared some of the small protein bands (31.5-34 kDa) detected in SGEs. These small proteins appeared in both samples at the same stage of feeding, suggesting that haemolymph and salivary glands not only have common antigens but may also share some functions. Furthermore, a number of protein bands were detected in haemolymph before they were apparent in the salivary glands or saliva. Thus some antigens detected in the salivary glands and saliva may be derived from the haemolymph. The results indicate that the host may be exposed to tick saliva antigens that are also present in the haemolymph. We discuss the significance of these observations with regard to the induction of host immunity to ticks and the development of tick vaccines.

QUANTITATIVE VARIATION OF ECDYSTEROIDS OF IXODID TICKS

Abstract In order to explore the role of ecdysteroids in development and reproduction of ixodid ticks, we studied the quantitative variation of ecdysteroids in the hemolymph, synganglion, ovary and whole body of the female ixodid ticks, Dermacentor niveus and Haemaphysalis longicornis, before and after engorgement and oviposition by HPLC and RIA. The ecdysteroid content in eggs of these ticks was determined by HPLC. The results indicated that before engorgement the quantitative variation of ecdysteroids in the whole female body was not significant, but their levels increased rapidly after engorgement. The ecdysteroid titer in hemolymph was peaked on the 5th day after engorgement, which was one day prior to oviposition. It may be regarded as a singnal of oviposition. In the synganglion the peak of ecdysteroid level occurred also on the 5th day after engorgement. This is coincident with the secretory activity of neurosecretory cells of synganglion. From the 3rd day after engorgement until oviposition the ecdysteroid level in the ovary increased rapidly. Ecdysteroids were detected in eggs of H. longicornis too. They stem from ovary and accumulated with the process of embryonic development.

Isolation of Borrelia burgdorferi from saliva of the tick vector, Ixodes scapularis.

A method for cultivating and isolating Lyme disease spirochetes, Borrelia burgdorferi, from the saliva of vector ticks, Ixodes scapularis (formerly known as Ixodes dammini), is described. Saliva was collected from partially engorged ticks after application of pilocarpine to induce salivation. B. burgdorferi was isolated from 8 of 14 (57%) of the saliva samples derived from ticks infected with the bacteria, as determined by direct immunofluorescent-antibody assay of tick hemolymph. A comparison of the protein profiles of the salivary isolates and a highly passaged strain (B31) showed that the salivary isolates all lacked a 22-kDa protein known to increase with continuous passage, but exhibited larger amounts of the OspA and OspB proteins than did the highly passaged B31 strain.

Detection of Anaplasma marginale (Rickettsiales: Anaplasmataceae) in Hemolymph of Dermacentor andersoni (Acari: Ixodidae) with the Polymerase Chain Reaction

The polymerase chain reaction (PCR) was used to detect Anaplasma marginale in hemolymph collected from live Dermacentor andersoni Stiles ticks. Hemolymph was collected from severed legs of male and female ticks exposed to A. marginale as either nymphs or adults. Heat treatment was found to be the optimum method of hemolymph preparation for PCR. Hemolymph samples were collected and pooled from adult ticks exposed as nymphs on days 0-10 of feeding on a susceptible calf. For male and female ticks exposed as adults, samples were collected as ticks fed 7 d on an infected calf, while being held 9 d between feedings, and during a second feeding of 10 d (or to repletion) when they transmitted the parasite. Hemolymph samples were collected from uninfected ticks at the same times to serve as controls. Anaplasma marginale DNA was amplified with primers BAP-2 (5'-GTATGGCACGTAGTCTTGGGATCA-3') and AL34S (5'-CAGCAGCAGCAAGACCTTCA-3'), which flank a 409-bp fragment of the A. marginale Florida isolate msp1 beta gene. Infected tick hemolymph was PCR-positive for A. marginale at all collection times, including unfed adults infected as nymphs and previously unexposed adults that fed on infected calves for only 1 d. The PCR-based assay of tick hemolymph proved to be a sensitive method for identification of infected ticks, potentially without killing them; it would be well suited for identification of laboratory- or field-infected ticks that could then be used for further studies. The primers used in this assay were also found specific when tested with species of 18 different genera, and universal for 7 A. marginale isolates from diverse geographical areas of the United States.

Spotted fever rickettsiae in ticks from the northern Sinai Governate, Egypt.

A field study was initiated in 1988 to investigate whether spotted fever group rickettsiae occur in geographic areas in Egypt that are adjacent to an area in the southern Israeli Negev that has a defined focus of spotted fever disease. Ticks were collected from dogs, sheep, and camels at four study sites in the northern Sinai. Tick hemolymph was processed for rickettsial detection by staining with fluorescein isothiocyanate-conjugated antibody to Rickettsia rickettsii. Of the 442 hemolymphs examined, 15 contained immunofluorescent rickettsiae. Eight hemolymph test-positive (HT+) ticks were Rhipicephalus sanguineus removed from dogs; the other HT+ ticks comprised three Hyalomma species, H. anatolicum, H. impeltatum, and H. dromedarii. Both HT+ and HT- ticks were tested for rickettsial DNA using the polymerase chain reaction (PCR). Eight of 10 HT+ field-collected ticks were PCR positive (PCR+). All laboratory colony R. rickettsii-infected ticks were PCR+. No HT- ticks from field or laboratory isolates were PCR+.

Detection of an arbovirus in an invertebrate and a vertebrate host using the polymerase chain reaction

The ability of the polymerase chain reaction (PCR) to diagnose an arboviral infection in an arthropod vector or a mammalian host was examined. Dugbe (DUG) viral RNA was detected in RNA extracts from infected tissue samples by reverse transcription and enzymatic amplification of the resulting cDNA using Taq DNA polymerase, followed by characterisation of the amplified product by agarose gel electrophoresis or dot-blot hybridisation. Viral RNA was detected in the organs and haemolymph of infected Amblyomma variegatum ticks, and in the brain and blood of infected mice. The PCR technique was found to be as sensitive as a plaque assay for detecting DUG virus, but not as sensitive as intracerebral inoculation of mice. The sensitivity of the technique was greatest using crude RNA extracts combined with dot-blot analysis of the resulting PCR products using a DUG specific cDNA probe. A result was obtained within 48 h using PCR whereas biological assays took at least 8 days to diagnose the virus infection.

Changes in Total Hemolymph and Ovarian Proteins During Oogenesis in Argas (Argas) hermanni (Acari: Argasidae)

Changes in the total protein concentrations in the hemolymph and ovaries of fed, mated female Argas (Argas) hermanni Audouin during oogenesis and in the hemolymph of males were studied. There was a decline in the total protein level in hemolymph of mated ticks for 2 d after feeding. This decrease was followed by an increase, reaching a maximum on day 6 in males and days 5 and 6 in females. Protein levels in the hemolymph of female ticks were higher than that of the male during the period of vitellogenesis and onset of oviposition (3–6 d after feeding). At the end of the oviposition period (day 20), the protein level in the hemolymph decreased, approaching that of the male. These data suggest an increase in the demand and use of proteins in the hemolymph of females by the developing oocytes. The increase of the hemolymph proteins in the females coincided with an increase of the ovarian protein level, ovarian weight, and the number of mature oocytes inside.

Quantitative studies of host immunoglobulin G in the hemolymph of ticks (Acari).

A radioimmunoassay was used to measure the concentration of host immunoglobulin G (IgG) in the hemolymph of female hard and soft ticks. Hyalomma excavatum Koch, Rhipicephalus sanguineus Latreille, Ornithodoros tholozani (Laboulbene and Megnin), and O. moubata (Murry) were fed on rabbits immunized with ovalbumin; Argas persicus (Oken) was fed on chickens immunized with cytochrome 'C.' At 24 h after feeding, the concentration of antiovalbumin IgG in the hemolymph was 7 micrograms/ml for H. excavatum, 5 micrograms/ml for R. sanguineus, and 0.15 micrograms/ml for O. moubata; the percentage of intact IgG molecules was 30, 44, and 100%, respectively. Host IgG was not detected in the hemolymph of O. tholozani and A. persicus. There was no increase in the concentrations of host IgG in the hemolymph of the soft ticks during the first week following the blood meal. The potential contribution to the resistance of hosts against ticks by host antibodies that cross into the tick hemocoel is discussed.

Relationship between feeding, mating, vitellogenin production and vitellogenesis in the tick Dermacentor variabilis.

Anti-vitellin IgG directed against Dermacentor variabilis egg vitellin was used in sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gradient gel immunoblots to detect the presence of vitellin and its precursor, vitellogenin, in the organs of feeding adults and in the immature stages of this tick. Vitellin polypeptides were found in the egg, larvae, nymph, and in the unfed adult stages of both sexes. Vitellin polypeptides were first detected in the ovary of mated females during the rapid-engorgement feeding period. These polypeptides were also present in the ovaries of ovipositing females, unmated females fed for extended periods, and fed unmated females that were detached from the host and held for 12 h before dissection. The same anti-vitellin antibody was used in immunoblots to monitor the appearance of vitellogenin in the organs and hemolymph of female ticks. Immunoreactive peptides of vitellogenin were found in the fat body, midgut, and hemolymph of pre-rapid-engorging mated and unmated females. These polypeptides were not found in fed males nor in Malpighian tubes of feeding or ovipositing females. Our data supported the following conclusions: 1) presence of immunoreactive vitellogenin in the adult female fat body, hemolymph, and midgut was dependent upon feeding; 2) in mated feeding females, we could not detect the uptake of vitellogenin by the ovary until rapid engorgement; 3) in unmated females, vitellogenesis did not begin unless prolonged feeding occurred; and 4) during the early developmental stages of this tick, vitellin served as an embryonic nutrient reserve and as a reserve against starvation between feedings.

A new method for determining vitellogenin in hemolymph of female Ornithodoros moubata (Acari: Argasidae).

A simple technique is described that allows evaluation of vitellogenin (Vg) concentration in hemolymph of ticks by scanning hemolymph spotted on filter paper with a spectrophotometer. This “spotting–scanning” method was applied for the determination of Vg titers during vitellogenesis in engorged female Ornithodoros moubata sensu Walton (1962) and was compared with the analysis of rocket immunoelectrophoresis.

The establishment of infection in the salivary glands of Rhipicephalus appendiculatus ticks by transplantation of kinetes of Theileria parva and the potential use of the method for parasite cloning

Fujisaki K., Irvin A. D., Voigt W. P., Leitch B. L. and Morzaria S. P. 1988. The establishment of infection in the salivary glands of Rhipicephalus appendiculatus ticks by transplantation of kinetes of Theileria parva and the potential use of the method for parasite cloning. International Journal for Parasitology 18: 75–78. A simple method was developed to establish infection of a single salivary gland acinus of the tick Rhipicephalus appendiculatus with kinetes of Theileria parva. Kinetes were obtained from haemolymph of newly-moulted infected adult ticks and injected into the haemolymph of unfed adult ticks. The latter ticks were subsequently fed on rabbits; salivary glands were dissected out and acinar infection rates established. Transplantation of 16–138 kinetes into unfed female ticks resulted in infection of one or more salivary gland acini. Infection could not be reliably established in male ticks, nor in female ticks with fewer than 16 kinetes. The potential use of this method to obtain cloned populations of T. parva sporozoites is discussed.

Host immunoglobulin G titre and antibody activity in haemolymph of the tick, Ornithodoros moubata.

Immunoglobulin G (IgG) in tick haemolymph was analysed immunochemically and biochemically for its antigenicity, antibody activity and relative concentration in a soft tick, Ornithodoros moubata (Murray) sensu Walton 1962 (Acari: Argasidae). Ouchterlony immunodiffusion tests showed that haemolymph from a tick engorged on rabbit IgG (or human IgG) through an artificial membrane, reacted with anti-rabbit IgG (anti-human IgG) but not with anti-human IgG (anti-rabbit IgG). This indicates that haemolymph of the fed tick contains IgG with a similar antigen specificity to host blood IgG. IgG from tick haemolymph was demonstrated by enzyme immunoassay to have the same antibody activity as ingested IgG. The IgG concentration in tick haemolymph was measured by a quantitative single immunodiffusion test. Changes of IgG titre after a bloodmeal were correlated with IgG activity, which was low for 5 days after a bloodmeal and then suddenly increased. The IgG titre reached a maximum 7 days post-engorgement, and remained high for over 4 months during and after oviposition. 125I-labelled IgG was injected into the tick haemocoel to determine the persistence of IgG in the haemolymph. Recovery of labelled IgG was low at 1 and 3 days, and high at 5, 8 and 16 days after engorgement. The data suggest that IgG in haemolymph disappears quickly soon after engorgement possibly by degradation and/or absorption (adhesion to tissues).

Quantification of Host Immunoglobulin in the Hemolymph of Ticks

The inhibition of tick enzymes and neutralization of their hormones with specific antibodies have been suggested as methods for interfering with tick feeding and development (Roberts, 1968, Journal of Parasitology 54: 657-662; Galun, 1978, Control of livestock pests by interference in their development, Bogota, Colombia, UC/AID Pest Management and Relative Environmental Protection Progress Report; Allen and Humphreys, 1979, Nature 280: 491-493; Ackerman et al., 1980, Journal of Medical Entomology 17: 391-397; McGowan et al., 1980, Journal of Parasitology 66: 42-48). Antibodies of mammalian hosts, imbibed with a blood meal, have been demonstrated in the hemolymph of several soft and hard tick species. Human and rabbit immunoglobulin G (IgG) were found in the hemolymph of Dermacentor variabilis (Say) (Ackerman et al., 1981, Journal of Parasitology 67: 737-740). Hemolysin antibodies were detected in the hemolymph of Ixodes ricinus (L.) following a blood meal on rabbits immunized with sheep red blood cells (Bossard and Rais, 1984, Experientia 40: 561-563). Bovine IgG specific for Theileria sergenti was found in the hemolymph of both Ornithodoros moubata (Murray) and Haemaphysalis longicornis Neumann (Fujisaki et al., 1984, Annals of Tropical Medicine and Parasitology 78:449-450). Specific (antiovalbumin) rabbit IgG was found in the hemolymph of 0. moubata following a blood meal on rabbits immunized with ovalbumin (Minoura et al., 1985, Experimental Parasitology 60: 355363). The hemolymph samples tested in the above studies were usually pooled from several ticks, and therefore provided no information about individual variation in the presence of IgG in the hemolymph. Furthermore, when hemolymph samples from individual ticks were assayed (Brossard and Rais, 1984, loc. cit.), only a small fraction contained hemolysin antibodies. Though the concentration of host antibodies in tick he-