Antigens of Amblyomma americanum ticks recognized by repeatedly infested sheep

Sera were taken from 3 sheep that had been infested 5 times with Amblyomma americanum and that exhibited manifestations of humoral depression to homologous antigens and anti-tick resistance. Proteins extracted from the intestine or salivary glands of unfed ticks or salivary glands from partially (3-day) fed ticks were analyzed by polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE. Antigens recognized by the sheep in the same materials before and after each infestation were analyzed by western blots. The sheep responded to 44 antigens. Nine to 23 antigens were recognized by the preinfestation sera and the sera of 2 gnotobiotic sheep. Four antigens (34,000, 36,500, 38,000, and 115,000 MW) were revealed conspicuously by the serum of the first infestation but very weakly or not at all by the sera of the third infestation onward. Two antigens (35,500 and 29,000 MW) from fed salivary glands were revealed only by sera taken after manifestations of resistance had appeared. These antigens may be responsible for anti-tick protection. The 29,900 MW antigen was present also in salivary extracts of Boophilus microplus.     

Investigations into lymphocyte transformation and histamine release by basophils in sheep repeatedly infested with Rhipicephalus evertsi evertsi ticks

The immune response of a natural host of Rhipicephalus evertsi evertsi to feeding by this tick species was investigated with respect to the effects of tick salivary gland extracts on the transformation of peripheral blood lymphocytes and the release of histamine by basophils obtained from repeatedly infested sheep.The results indicated that there was no stimulation of lymphocyte transformation but that histamine release was elevated 10 fold after four infestations.Although this suggests a hypersensitivity reaction, believed to be a major factor in resistance to tick feeding, it was observed that ticks fed normally even after four infestations with 28 day intervals in between. These results emphasize the adaptation of ticks to feeding on their natural hosts.     

Amblyomma americanum: requirement for host Fc receptors in antibody-mediated acquired immune resistance to ticks

Guinea pig recipients of anti-tick immune serum or immune peritoneal exudate cells expressed 25 and 30% tick rejection, respectively, when challenged with Amblyomma americanum larval ticks. Previous studies have shown that IgG1 antibodies are responsible for the ability of immune serum to transfer resistance to ticks and to mediate the accompanying, and required, cutaneous basophil response. Since IgG1 antibodies induce mast cell-mediated passive cutaneous anaphylaxis and cutaneous basophil responses by interaction with cell surface Fc receptors, we investigated whether host Fc receptors were involved in the mechanism of antibody-mediated immune resistance to ticks. Recipients of immune serum pretreated intravenously with rabbit IgG failed to express resistance when challenged. In contrast, recipients of immune peritoneal exudate cells similarly pretreated expressed normal resistance. Sheep IgG had no inhibitory effect on the transfer of resistance by either immune serum or peritoneal exudate cells. Furthermore, recipients of immune serum pretreated with the Fc fragment from papain digestion of rabbit IgG failed to express resistance when challenged with ticks. Rabbit Fab and sheep Fc and Fab had no effect on the transfer of resistance by immune serum. Purity of rabbit Fc preparations was verified by the ability to inhibit mast cell-mediated passive cutaneous anaphylaxis due to high-titered IgG1 antiovalbumin antibodies. Rabbit Fab and sheep Fc fractions did not inhibit passive cutaneous anaphylaxis reactions. These findings suggest that immunoglobulin Fc receptors on host cells, such as mast cells and basophils, are required for antibody-mediated immune rejection of ticks from guinea pigs.     

Comparison of differentially expressed genes in the salivary glands of male ticks,Amblyomma americanum and Dermacentor andersoni

Genes expressed differentially in the salivary glands of unfed and fed male ticks, Amblyomma americanum (L.), were identified, cloned and sequenced, and some were compared with those expressed in the salivary glands of Dermacentor andersoni. Total protein and RNA increased sixfold in the salivary glands of fed male A. americanum, while in fed male D. andersoni salivary glands, RNA increased approximately 3.5 times. Feeding D. andersoni in the presence of females increased total RNA by 25% over those fed in the absence of females. Complementary DNAs were synthesized from RNA obtained from unfed and fed ticks and amplified using RNA arbitrarily primed polymerase chain reaction (RAP-PCR) with three different primers in separate reactions. Differential display showed clear banding differences between the fed and the unfed ticks in A. americanum and D. andersoni. Sixty-one cDNA fragments that appeared to be from differentially expressed genes in A. americanum were isolated, cloned and sequenced. Hybridization reactions with labeled cDNA probes confirmed the differential expression of many of the genes in unfed and fed ticks' salivary glands; however, many of the bands contained more than one fragment and some of the fragments isolated from apparently differential bands were not specific. Sequences for 28 of the cDNA fragments (150-600 nucleotides in length) demonstrated similarity to genes in the databases, but nine of these were similar to sequences of unknown function. Some of the gene fragments identified may be important to tick feeding or tick salivary gland physiology, including a histamine-binding protein, an organic ion transporter, an apoptosis inhibitor, a cathepsin-B-like cysteine protease, proteins involved in gene regulation and several proteins involved in protein synthesis. Cross-hybridization of identified cDNAs from A. americanum with cDNA probes synthesized from D. andersoni total RNA did not show significant similarity between the two species.     

Systemic activity of closantel for control of lone star ticks,Amblyomma americanum (L.), on cattle

Cattle were treated once at 5 mg/kg orally or subcutaneously or daily at 0.1–5 mg/kg orally or 0.1–1 mg/kg subcutaneously with closantel, N-[5-chloro-4-[(4-chlorophenyl) cyanomethyl]-2-methylphenyl]-2-hydroxy-3,5-diiodobenzamide, and numbers and weights of engorged females, weights of egg masses and hatch of eggs of lone star ticks,Amblyomma americanum, were recorded.Effectiveness of treatments on reproduction was determined by comparing total estimated larvae (EL) (EL=wt. egg mass×est. % hatch×20000) or ticks from treated cattle with that of ticks from untreated cattle. With certain treatments, we also determined the effect of manure of treated cattle on survival of larvae of the horn fly,Haematobia irritans, or effect on survival and of fecundity of adult horn flies or stable flies,Stomoxys calcitrans, fed on blood from treated animals.The single oral treatment afforded essentially complete control of total EL only of ticks placed on the animal on the day of treatment, while the single subcutaneous treatment afforded >92% control of total EL of ticks placed on animal on treatment day and for 6 weeks posttreatment. Daily treatments of 0.5 mg/kg or greater orally and 0.1 mg/kg or greater subcutaneously afforded essentially complete control of total EL of ticks throughout the treatment period (3–12 weeks) and for 1–7 weeks after treatment was discontinued. An estimated concentration of >9 g/ml of blood was calculated by probit analysis to be necessary to provide >90% control of total EL of lone star ticks; that same concentration also provided >90% control of hatch of eggs laid by treated females. A higher concentration (40 g/ml) was necessary to prevent engorging of the females. No treatments tested were effective against larvae of the horn fly or adult horn flies or stable flies.     

Comparing scapular morphology of Amblyomma sculptum and Amblyomma dubitatum nymphs allows a fast and practical differential diagnosis of ticks in highly infested areas with dominance of these two species

Rickettsia rickettsii infection causes the highest human fatality rate among all Rickettsia species of the world and is endemic in Southeast Brazil. In this part of the country most human spotted fever cases are related to unnaturally high populations of ticks, usually a mix of two species, Amblyomma dubitatum Neumann and Amblyomma sculptum Berlese and their local host, capybara (Hydrochoerus hydrochaeris). At the same time, an increase in the number of SF notifications as well as its urbanization was observed, and a better characterization of disease epidemiology is mandatory for control measures and to halt its expansion. It was recently noticed in southeast Brazil that SF endemic areas were characterized by overgrowth populations of A. sculptum and decreased populations of A. dubitatum. Hence, characterization of areas with potential to endemicity, eco-epidemiological studies and control measures may rely on the evaluation of A. sculptum/A. dubitatum rate. However, in potentially endemic scenarios, discriminating the two tick species, particularly the nymphs considered the main vector stage, is a challenge in face of several hundreds to thousands of ticks that should be examined for quantitative studies. We herein present additional morphological features to an existing identification key for Amblyomma nymphs that considerably diminishes the labor to distinguish nymphs of these two tick species. Specifically, the oval-shaped scutum of A. dubitatum and a conspicuous scapula of A. sculptum are prominent discriminating features.

     

Microarray analysis of gene expression changes in feeding female and male lone star ticks,Amblyomma americanum (L)

A collection of EST clones from female tick Amblyomma americanum salivary glands was hybridized to RNA from different feeding stages of female tick salivary glands and from unfed or feeding adult male ticks. In the female ticks, the expression patterns changed dramatically upon starting feeding, then changed again towards the end of feeding. On beginning feeding, genes possibly involved in survival on the host increased in expression as did many housekeeping genes. As feeding progressed, some of the survival genes were downregulated, while others were upregulated. When the tick went into the rapid feeding phase, many of the survival genes were downregulated, while a number of transport‐associated genes and genes possibly involved in organ degeneration increased. In the males, the presence of females during feeding made a small difference, but feeding made a larger difference. Males showed clear differences from females in expression, as well. Protein synthesis genes were expressed more in all male groups than in the partially fed females, while the putative secreted genes involved in avoiding host defenses were expressed less. © 2009 Wiley Periodicals, Inc.     

Transmission of Ehrlichia chaffeensis from lone star ticks (Amblyomma americanum) to white-tailed deer (Odocoileus virginianus)

Amblyomma americanum is an aggressive ixodid tick that has been implicated as a vector for several bacterial agents. Among these is Ehrlichia chaffeensis, which causes human monocytic (or monocytotropic) ehrlichiosis. In this study, experimental tick transmission of E. chaffeensis from infected lone star ticks to deer was revisited, and the question of whether it would be possible to re-isolate the organism from deer was asked, because this had not been done previously. Here, we were able to transmit a wild strain of E. chaffeensis from acquisition-fed lone star ticks to white-tailed deer. Ehrlichia chaffeensis was re-isolated from one white-tailed deer on multiple days during the infection and from another deer on one day during the infection. Peak rickettsemias for E. chaffeensis-infected deer were 17 DPI with acquisition-fed ticks and 14 DPI with needle-inoculated deer. This study supports the role of the lone star tick and white-tailed deer as vector and reservoir host for E. chaffeensis, demonstrating culture re-isolation of E. chaffeensis in deer infected by experimental tick transmission for the first time.     

Borreliacidal activity of saliva of the tick Amblyomma americanum

Amblyomma americanum (Linneaus) (Acari: Ixodidae), an important tick vector of human and animal disease, is not a competent vector of the bacterial agent of Lyme disease, Borrelia burgdorferi, although its range overlaps the geographical distribution of Lyme disease within the United States. A possible mechanism that could prevent acquisition of B. burgdorferi spirochetes from infected hosts is the toxic effect of A. americanum saliva on B. burgdorferi. The data presented here indicate that after 24 and 48 h of exposure to A. americanum saliva, significantly fewer B. burgdorferi were alive compared to treatment controls as assessed by spirochete motility under dark-field microscopy and resistance to the dead stain, propidium iodide. After 48 h, fewer than 13% of saliva-exposed B. burgdorferi were alive. In contrast, significantly more B. burgdorferi exposed to Ixodes scapularis (Acari: Ixodidae) saliva survived after 24 or 48 h compared to A. americanum saliva or treatment controls.     

Attractivity of cattle infested by Amblyomma variegatum (Acari: Ixodidae) for conspecific adult ticks from the field in Guadeloupe

Four field experiments were conducted with creole cattle in Guadeloupe to study the attractivity for adult ticks of animals previously infested with males of Amblyomma variegatum producing aggregation attachment pheromones (AAPs). In each experiment, two groups of cattle were used; the first group was infested with adult A. variegatum and the second group was kept free of ticks and pheromones by acaricidal treatment and by washing the hair and skin of previously infested sites. Both groups were then exposed to natural infestation by ticks present in the environment or by ticks bred in the laboratory and released on the pasture. The number of additional ticks infesting animals were compared between the treated and control groups. There were no significant differences between both groups in the numbers of new male ticks infesting the animals during the first days following the beginning of the experiment. Thereafter, comparison of the slopes of infestation indicated that infested cattle were no more attractive for male ticks than uninfested hosts. Uninfested cattle were spontaneously and rapidly infested by male ticks. It is concluded that carbon dioxide or another host component was a major attractant for A. variegatum males. Female ticks attached on predilection sites exclusively in the presence of AAP-producing males, i.e. 5–10 days after the attachment of the first males. Based on these results we postulate that slow-release devices impregnated with pheromones have no additional attractive effect on A. variegatum males under Caribbean conditions. However, they may be useful in inducing the attachment of females, but only in the first days preceding the arrival of pioneer AAP-producing males. © Rapid Science Ltd. 1998     

Changes in dopamine-sensitive adenylate cyclase activity in salivary glands of female lone star ticks,Amblyomma americanum (L.), during feeding

The activity of dopamine-sensitive adenylate cyclase in feeding female ixodid tick salivary glands was dependent on the state of tick feeding. Activity was significantly greater than the “basal” activity in salivary glands from ticks at all stages of tick feeding. Enzyme activity was not detected in the glands of unfed females. Enzyme activity reached a peak in glands of ticks weighing approx. 200 mg then declined as ticks increased in weight beyond 200 mg to repletion. Replete ticks (detached from the host for 12–24 hr) had similar levels of basal and dopamine-stimulated adenylate cyclase activity as that measured in salivary glands of high weight (>200 mg) ticks. Enzyme activity was 19–62% less in glands from ticks feeding on hosts that had been parasitized 2–4 months earlier by lone star ticks.     

Disrupting the Amblyomma americanum (L.) CD147 receptor homolog prevents ticks from feeding to repletion and blocks spontaneous detachment of ticks from their host

The CD147 receptor is a cell-surface glycoprotein in the IgG family that plays pivotal roles in intercellular interactions involved with numerous physiological and pathological processes such as extracellular matrix remodeling. We previously found an Amblyomma americanum (Aam) tick CD147 receptor homolog among genes that were up regulated in response to tick feeding stimuli. This study characterizes an AamCD147 receptor protein that is 72–83% conserved in other tick species and possess characteristic CD147 receptor sequence features: an extracellular (EC) region containing two IgG domains, a transmembrane and the cytoplasmic domains. Likewise, the AamCD147 EC domain folds into secondary structures that are consistent to the human homolog: an amino-terminus β-barrel that is linked to 2-carboxy-terminus β-sheets with consensus disulfide bonds conserved in each of the 2 domains. CD147 receptor signaling and regulatory mechanisms are putatively conserved in ticks as revealed by in silico analysis that show presence in the tick genome of CD147 receptor signaling protein homologs, cyclophilin (CyP) A and B, and chaperones that transport it to the plasma membrane, caveolin-1 and CyP60. The AamCD147 receptor has a dichotomous expression pattern of where it is up regulated in response to feeding in the salivary gland but remains constant at the midgut and ovary levels suggesting that it may regulate different functions in different tick organs. We speculate that biological functions of the AamCD147 receptor are essential to tick feeding success as revealed by RNAi-mediated silencing that caused ticks to obtain smaller blood meals, of which ～69% were below threshold to trigger spontaneous detachment of ticks from the host. These ticks showed unusual cuticle tenderness and assumed a reddish coloration, a phenomenon that has been attributed to tick midgut damage allowing red blood cells to leak into tick hemolymph. On the basis of the CD147 receptor being linked to tissue growth regulation in mammals, we speculate that silencing of the AamCD147 receptor blocked progression of the tick intermolt growth, a process that precedes tick engorgement and their spontaneous detachment of from the host to end feeding. The results are discussed in context of advances in tick molecular physiology.     

Susceptibility of Amblyomma americanum to natural and experimental infections with Theileria cervi

One hundred fifty Amblyomma americanum were examined between March and September 1986 from Cookson Hills Wildlife Refuge in eastern Oklahoma (USA). Of these ticks, 11% (17 of 150) were infected with Theileria cervi. Field-collected nymphal ticks had an 8% (3 of 37) prevalence of infection averaging 1.0 infected acini/nymph. Female ticks had a 16% prevalence of infection averaging 1.6 infected acini/female; T. cervi was not observed in salivary glands of field-collected male ticks. When laboratory reared A. americanum nymphs were allowed to feed on experimental white-tailed deer (Odocoileus virginianus) with varying T. cervi parasitemias (less than 1, 2, 6 and greater than 20%), only ticks which fed on deer with parasitemias greater than 1% became infected. Although prevalence and intensity of infection varied in the infected ticks, there was no significant difference in prevalence of infection between males and females. However, females did acquire significantly greater intensities than males. The data from these studies confirm that T. cervi overwinters in A. americanum and suggests that the prevalence, intensity and abundance of infection of T. cervi in ticks is influenced by the parasitemia of the deer host. Furthermore, fawns may play a more important role in the epidemiology of T. cervi transmission than do adult deer because of the coordination between tick activity patterns and deer fawning.     

Adenosine-3',5'-monophosphate in salivary glands of unfed and feeding female lone star ticks,Amblyomma americanum (L.)

1. Basal levels of cAMP in salivary glands of female lone star ticks were found to be about 5 pmoles/mg protein during all stages of feeding.2. Glands stimulated with 10⁻⁵M dopamine and 10⁻⁵M dopamine plus theophylline exhibited significant increases in cAMP/mg protein.3. After stimulation by 10⁻⁵ M dopamine was removed, cAMP decreased faster in glands from slowly feeding ticks (< 200 mg) than in glands of rapidly feeding ticks ( > 200 mg).     

A conceptual model of the Amblyomma americanum life cycle in northeast Missouri

The geographic distribution of Amblyomma americanum (the lone star tick) has increased as has its role as a pathogen vector. The objectives of this study were to determine seasonal activity patterns of each life stage of A. americanum in the northwestern part of the species range and the relationship of these activity patterns among life stages and degree days. Tick activity was monitored over four years since 2007 in a forest and old field habitat located in northeast Missouri. Every other week from February to December, ticks were collected using bait and drag methods. Autocorrelations demonstrated yearly seasonal patterns in each life stage and cross‐correlations between life stages depicted a relationship between activity at a life stage and the previous stage's activity. Cross‐correlations indicated that degree days were related to activity. These data indicated that A. americanum generally complete their life cycle in a minimum of two years in northeast Missouri, with overwintering occurring predominantly in the nymphal and adult stages. These data provide a baseline to compare the life cycle of A. americanum in northeast Missouri to populations in different parts of the species range or at different times in the region.     

Ultrastructure of Haller's organ in the tick Amblyomma americanum (L.)

Summary Haller's organ on the tarsus of the tick Amblyomma americanum (L.) (Acarina: Ixodidae; nymphal stage) was studied by scanning and transmission electron microscopy. It consists of a distal bristle group, (the anterior pit), and a proximal capsule which encloses several sensilla. The seven sensilla of the anterior pit (A1–A7) are all thick-walled and multi-innervated (2–9 neurons), but at least three different types can be differentiated. Sensilla A1 and A2 possess large, plugged pores (>1000 Å) and are the only sensilla with branching dendrites. A3 and A5 are characterized by a spoke-wheel arrangement of the cuticle wall and very fine pores (100–200 Å) penetrating the spokes centrally; A4, A6, and A7 do not exhibit any pore system but a single opening at the bristle tip is assumed.The capsule contains seven thin-walled, blunt-tipped sensilla, and several non-sensory cuticular projections (pleomorphs). All of these sensilla possess large plugged pores in the cuticle wall and numerous dendritic branches of several neurons (3–5) in the lumen. Glandular openings were found inside the capsule; their significance is discussed.The fine structure of Haller's organ supports the functions postulated by Lees (1948), namely olfaction for the capsule and humidity reception (among others) for the anterior pit.This research was supported in part by the Office of Naval Research, and by NIH Training grant ES 00069. Paper no. 3459 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh.