Experimental infection of lone star ticks, Amblyomma americanum (L.), with Rickettsia parkeri and exposure of guinea pigs to the agent

The maculatum agent, Rickettsia parkeri (a member of the spotted fever group rickettsiae), was inoculated into a colony of the lone star tick, Amblyomma americanum, and followed for two tick generations. In addition, guinea pigs were exposed to the agent by direct injection and by feeding infected ticks on them. Eighty (53%) of 150 nymphal A. americanum that were inoculated with suspensions of R. parkeri were positive by hemolymph test and fluorescent antibody test for rickettsial infection when examined as adults. One-month survival of R. parkeri-infected ticks was similar to that of control (noninfected) ticks. Transstadial and transovarial transmission of R. parkeri in the laboratory was demonstrated in A. americanum. When guinea pigs were exposed to the maculatum agent by either direct injection of Vero cell-grown R. parkeri, injection of homogenates of infected ticks, or by feeding of infected ticks, they developed mild fevers and occasional scrotal reactions. These data indicate that R. parkeri can remain viable in lone star ticks for two generations and suggest that guinea pigs may become infected, displaying mild clinical signs.     

Differential expression of Ixodes ricinus tick genes induced by blood feeding or Borrelia burgdorferi infection

Ixodes ricinus L. is the principal European vector of Borrelia burgdorferi sensu lato, the causative agent of Lyme borreliosis. Subtractive hybridization was used to isolate tick genes that were induced in whole ticks after blood meals on uninfected and B. burgdorferi-infected guinea pigs. Novel cDNA clones with similarity to cytochrome c oxidase, salivary secreted protein, actin, and a cysteine protease propeptide were induced after a blood meal. Novel cDNA clones with similarity to thioredoxin peroxidases, dolichyl-phosphate beta-glucosyltransferase, glutathione S-transferase, defensin, ML domain-containing protein, and von Willebrand factor were induced after B. burgdorferi infection. Virtual Northern analysis was used to verify that these genes were differentially expressed in ticks after a pathogen-infected blood meal and to detect their tissues of expression. The characterization of genes that are induced after an infected blood meal is essential for gaining an understanding of the molecular mechanisms that underlie vector-pathogen interactions.     

Isolation by a sensitive centrifugation cell culture system of 52 strains of spotted fever group rickettsiae from ticks collected in France.

Boutonneuse fever caused by Rickettsia conorii is transmitted mainly by the brown dog tick, Rhipicephalus sanguineus. We collected 540 ticks in Marseille, France, and tried to isolate as many strains of rickettsia as possible. Ticks were evaluated for the presence of rickettsia by the hemolymph test and by a new culture system, the centrifugation-shell vial technique. We avoided contamination in the culture system. Prior to ticks being submitted to the hemolymph test, they were disinfected. Only 5.6% (27 of 478) of the cultures were contaminated. A drop of hemolymph from each of 478 R. sanguineus ticks was cultured in two shell vials, and another drop was stained by the Gimenez method or indirect immunofluorescence. Since Gimenez staining in our hands was not satisfactory, comparison of the hemolymph test and culture is based on the results of indirect immunofluorescence. Thus, 50 of 369 (13.5%) examined ticks were hemolymph test positive, and 44 (11.9%) cultures were positive. After disinfection, another pool of 62 ticks were examined by the hemolymph test. The ticks were kept individually in a sterile environment. A few days later, the hemolymph of these ticks was collected again and cultured. The contamination rate was not significantly higher (6.4%) than in the above-described conditions. It allowed us to isolate eight more strains. Thus, we recommend screening ticks with the hemolymph test and culturing only the hemolymph test-positive ticks.     

Study of the seasonal dynamics, life cycle, and host specificity of Amblyomma aureolatum (Acari: Ixodidae)

In the first part of this study, monthly infestation by ticks was evaluated on dogs from December 2000 to November 2002 in the rural area of Taia?upeba, S?o Paulo. Adults of Amblyomma aureolatum (Pallas) were found on dogs in all months, with a mean prevalence per month of 46.9 +/- 15.7% (range, 25-80%). The mean tick relative abundance per month was 2.4 +/- 2.7 ticks (range, 0.5-14 ticks), and the mean tick mean intensity per month was 4.7 +/- 4.2 ticks (range, 1.5-23.3 ticks). No A. aureolatum immature ticks were found on dogs. In the second part of this study, we studied the life cycle of A. aureolatum in the laboratory. We tested the suitability of six host species for the immature stages and dogs for the adult stage. Tick developmental periods were observed at different temperatures (23, 25, or 27 degrees C), always with RH >95%, which were satisfactory for all free-living developmental stages of the tick life cycle. Chickens and guinea pigs were the most suitable hosts for larvae and nymphs (recovery rates, 18.4-52.2%). Dogs were highly suitable for adult ticks (all females exposed to them were recovered and laid eggs) but were unsuitable for the immature ticks (recovery rates, 0-10%). Based on published host records for A. aureolatum, our results indicate that dogs and birds are primary hosts for adult and immature stages, respectively, of A. aureolatum in nature. In addition, wild guinea pigs are indicated as another potential primary host for immature ticks.     

Defensin from the ornate sheep tick Dermacentor marginatus and its effect on Lyme borreliosis spirochetes

Expression of the previously reported defensin of the tick Dermacentor marginatus (defDM) was analysed in different organs by RT-PCR. mRNA of the defDM gene was detected in the hemolymph, midgut and salivary glands. Moreover defDM was isolated from the tick hemolymph using RP-HPLC and its sequence was determined by mass spectrometry and Edman degradation. Synthetic peptide was used for determining biological activities. The results showed an anti-Gram-positive bacterial role for the defensin. As D. marginatus ticks appear not to be vectors of the Lyme disease agent of the complex Borrelia burgdorferi sensu lato, we tested the influence of defDM on Borrelia afzelii. There is a very clear borrelicidal activity of the defensin, which is concentration dependent and suggests a possible role in the clearing of Borrelia ingested by D. marginatus ticks.     

Comparative infections of epizootic and enzootic strains of Venezuelan equine encephalomyelitis virus in Amblyomma cajennense (Acari: Ixodidae).

To compare the potential for an enzootic or an epizootic strain of Venezuelan equine encephalomyelitis (VEE) virus to infect Amblyomma cajennense (F.), larval ticks were fed on guinea pigs (strain 13) inoculated with an enzootic viral strain of variant I-E (68U201) or on guinea pigs inoculated with an epizootic strain of variant I-A (Trinidad donkey). Peak viremias were 10(5.2) plaque-forming units (PFU)/ml and 10(7.3) PFU/ml in guinea pigs infected with enzootic and epizootic viral strains, respectively. Ticks feeding on enzootic- and epizootic-infected hosts had viral titers of 10(2.5) and 10(3.9) PFU per tick, respectively, at drop-off. Although epizootic virus was recovered from 98% (127 of 130) of larval ticks up to 16 d after drop-off, enzootic virus was recovered from 95% (19 of 20) at drop-off (mean titer, 10(2.5) PFU per tick), with recovery rates declining rapidly to 2 of 10 (mean titer, 10(1.4) PFU per tick) by 16 d after drop-off. Transstadially transmitted epizootic virus was found in 0.4% (12 of 2,950) of unfed nymphs (mean titer, 10(2.8) PFU per tick) 63 d after drop-off, 1% (5 of 521) fed nymphs 69 d after drop-off, and 1% (4 of 400) of unfed adults (mean titer, 10(3.6) PFU per tick) 106 d after drop-off. No enzootic virus was recovered from 4,600 unfed nymphs tested 63 d after drop-off.     

The effect of virus-immune hosts on Thogoto virus infection of the tick, Rhipicephalus appendiculatus

Thogoto (THO) virus infections of Rhipicephalus appendiculatus ticks were examined using tick hosts immune to the virus. In the first set of experiments, ticks were infected by feeding on viraemic hamsters. Inter-stadial infection of THO virus was not affected when ticks ingested a virus-immune bloodmeal but there was an effect on persistence of the virus. The incidence of intra-stadial infection was reduced by at least 40% when nymphs partially fed on viraemic hamsters and completed their bloodmeal on a virus-immune guinea pig. When the reverse situation was examined--feeding on a virus-immune host and then a viraemic host--no difference was observed in the number of ticks infected. In the second set of experiments, uninfected ticks acquired virus by co-feeding with infected ticks on apparently non-viraemic guinea pigs. Non-viraemic transmission of the viruses was inhibited when the guinea pigs were immune to either the Sicilian (SiAr 126) or prototype (IIA) isolates of THO virus. The laboratory data indicate that virus-immune hosts may have a significant effect on the role played by ticks in the epidemiology of tick-borne viruses.     

Characterization and antiviral activity of a newly identified defensin-like peptide,HEdefensin, in the hard tick Haemaphysalis longicornis

Tick defensins are antimicrobial peptides that play a major role in the innate immunity of ticks by providing a direct antimicrobial defense. In this study, we identified and characterized a defensin-like encoding gene, HEdefensin, from the expressed sequence tags (EST) database of hemolymph from the hard tick Haemaphysalis longicornis. Expression of the gene in whole adult ticks and in different organs was upregulated during blood feeding, though not after Langat virus (LGTV) challenge. A synthetic HEdefensin peptide demonstrated significant virucidal activity against LGTV but not against an adenovirus in co-incubation virucidal assays. Moreover, the RNAi-mediated gene silencing of HEdefensin did not significantly affect the virus titer as compared to the control group. The data reported here have established the in vitro virucidal activity of the peptide against LGTV. However, its role in the innate antiviral immunity of H. longicornis remains to be explored, and further studies are needed to fully evaluate the potential biological activities of the peptide against bacteria, fungi or parasites.     

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.     

Tick Passage Results in Enhanced Attenuation of Babesia bovis

Serial blood passage of virulent Babesia bovis in splenectomized cattle results in attenuated derivatives that do not cause neurologic disease. Tick transmissibility can be lost with attenuation, but when retained, attenuated B. bovis can revert to virulence following tick passage. This study provides data showing that tick passage of the partially attenuated B. bovis T2Bo derivative strain further decreased virulence compared with intravenous inoculation of the same strain in infected animals. Ticks that acquired virulent or attenuated parasites by feeding on infected cattle were transmission fed on naive, splenectomized animals. While there was no significant difference between groups in the number of parasites in the midgut, hemolymph, or eggs of replete female ticks after acquisition feeding, animals infected with the attenuated parasites after tick transmission showed no clinical signs of babesiosis, unlike those receiving intravenous challenge with the same attenuated strain prior to tick passage. Additionally, there were significantly fewer parasites in blood and tissues of animals infected with tick-passaged attenuated parasites. Sequencing analysis of select B. bovis genes before and after tick passage showed significant differences in parasite genotypes in both peripheral blood and cerebral samples. These results provide evidence that not only is tick transmissibility retained by the attenuated T2Bo strain, but also it results in enhanced attenuation and is accompanied by expansion of parasite subpopulations during tick passage that may be associated with the change in disease phenotype.     

Quantitative description of the development of Babesia ovis in Rhipicephalus bursa (hemolymph,ovary, eggs)

The development and infection dynamics of Babesia ovis in the hemolymph, ovaries, and eggs of Rhipicephalus bursa are described quantitatively, based mainly on examination of Giemsa-stained smears. After alimentary infection of female ticks, their hemolymph became infected 5 days after repletion (p.repl.). The prevalence and mean intensity of infection increased during the course of infection studied, up to 17 days p.repl. After vertical infection of female ticks, their hemolymph was infected only during the first 3 days after the onset of infestation (p. infest.) and again after the onset of alimentary infection 5 days p.repl. There was a positive correlation between prevalence and mean intensity of infection in the hemolymph. The prevalence of infection decreased with aging of the unfed adult ticks. After alimentary infection, the ovaries became infected 6 days p.repl., and after vertical infection, 3 days p. infest; they remained infected until the death of the tick. Ticks selected for susceptibility during 18 and 19 vertically infected generations were more susceptible than ticks in their first to third vertically infected generations or alimentarily infected ticks. Eggs deposited on day 1 of oviposition were noninfected after alimentary infection of the female tick. After vertical infection of the tick, even such eggs became infected; the infection, then, was detectable in eggs produced throughout the oviposition period regardless of the infection mode. Intense hemolymph infections induced an increase of egg degeneration and a decrease of total as well as infected egg production. There was a positive correlation between the number of deposited and infected eggs as well as between prevalence and mean intensity of infection in eggs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Laboratory studies on the life cycle of Amblyomma marmoreum (Acari: Ixodidae) on two different hosts.

A comparative study was made of the life cycle of the tortoise tick, Amblyomma marmoreum Koch, on tortoises and guinea pigs under laboratory conditions. At 25 degrees C and 85% RH with natural day length, duration of off-host stages (preoviposition, oviposition, incubation, and premolt) was similar for ticks fed on both hosts. Delay in preoviposition (up to 90 d) was observed in some gravid females. Larvae, nymphs, and adults had longer feeding periods on tortoises than on guinea pigs. Adult females fed on tortoises had greater engorgement wieght and ovipositional capacity than ticks fed on guinea pigs. It is suggested that the shortened feeding period of immature stages on mammalian hosts together with the occurrence of morphogenic diapause may be more important than previously recognized in determining whether the life cycle of A. marmoreum is completed in 1 or 2 yr under natural conditions.     

Changes in the concentration of globulins in naive guinea pigs during feeding by the immature stages of Rhipicephalus evertsi evertsi (Acari: Ixodidae)

The weight of Rhipicephalus evertsi evertsi Neumann nymphs, which as larvae and nymphs completed the entire blood meal on the same individual guinea pig, was significantly lower than the weight of those that as larvae and nymphs fed on two separate naive hosts. Nymphs of the latter category spent 1 wk (from unfed larvae to unfed nymphs) on one animal before their transfer to the second host to complete the blood meal. The albumin concentration of the host blood decreased and was related to the time that the immature ticks spent on the hosts. The albumin/globulin ratio also decreased. The alpha 1 globulin fraction increased soon after the guinea pigs were infested with ticks. No further changes in the levels of the alpha 1 globulin fraction were observed with time. The concentration of serum beta globulins increased only in guinea pigs infested with immature ticks for the entire larval and nymphal feeding period. A second infestation of those guinea pigs with larvae of R. e. evertsi resulted in further increases in the levels of serum beta globulins. The relationships among serum beta globulin levels, the weight of engorged nymphs, and host immunity are discussed.     

Characterization of Borrelia burgdorferi isolated from different organs of Ixodes ricinus Ticks collected in nature

Borrelia burgdorferi was isolated from 22 out of 133 adult Ixodes ricinus ticks collected from vegetation at two sites in Switzerland. From 17 ticks, spirochetes could be isolated from more than one organ. When the different isolates obtained from one tick were compared by SDS-PAGE analysis, differences in the protein profiles were observed in 8 cases. The isolates were further compared by immunological methods using mono- and polyclonal antibodies. Differences were observed in the proteins of 31–35 kDA and 18–25 kDa. Genetic divergence among isolates was evaluated by use of a B. burgdorferi specific gene probe for ospA. Correlation could be observed between immunological differences in OspA defined by monoclonal antibody LA31 and genetic variation of ospA as judged by restriction fragment length polymorphism (RFLP). Our findings indicate that systemic infection in unfed I. ricinus adults, as reflected by isolation of B. burgdorferi from multiple organs of one tick, is more frequent (8/22, 36%) than previously described (5%). Moreover, the presence of different B. burgdorferi phenotypes/genotypes in one tick is described for the first time. The findings may have bearings (i) on the time of tick attachment required for spirochete transmission since borreliae are already present in the salivary glands of systemically infected ticks at the beginning of the blood meal and (ii) perhaps also on the diversity of B. burgdorferi phenotypes inoculated by these ticks.     

Granulocytic Ehrlichiosis in Tick-Immune Guinea Pigs

We investigated whether Ixodes scapularis-mediated host immunity interrupts transmission of the agent of human granulocytic ehrlichiosis (aoHGE) to guinea pigs. Ticks infected with aoHGE readily transmitted aoHGE to tick-immune guinea pigs, despite incomplete tick engorgement and host attachment. Although tick immunity can prevent Lyme borreliosis, protection is not afforded against granulocytic ehrlichiosis.     

Contrasts in tick innate immune responses to Borrelia burgdorferi challenge: immunotolerance in Ixodes scapularis versus immunocompetence in Dermacentor variabilis (Acari: Ixodidae)

The blacklegged tick, Ixodes scapularis Say, transmits the Lyme disease spirochete Borrelia burgdorferi, whereas the American dog tick, Dermacentor variabilis (Say), is unable to transmit the bacterium. We compared the innate immune response of these ticks against spirochetes directly inoculated into the hemocoel cavity of ticks. In I. scapularis, some Borrelia were found associated with hemocytes, while numerous other spiral-shaped, intact bacteria remained free in the hemolymph. In contrast, in D. variabilis only remnants of the bacteria were evident in the hemolymph, indicating lysis; intact spirochetes were rare. Spirochetes were observed bound to or within the organs of both tick species, although many more spirochetes were found associated with the I. scapularis organs. The few spirochetes observed with the D. variabilis organs appeared to be dead because D. variabilis tissues rarely contained culturable bacteria, unlike I. scapularis tissues. When spirochetes were incubated with I. scapularis hemolymph plasma in vitro, bacterial survival and motility were not reduced. In contrast, incubation of spirochetes with D. variabilis hemolymph plasma resulted in > 50% of the spirochetes becoming nonmotile by 45 min. The differences in the responses of the two different tick species indicate that I. scapularis is immunotolerant when challenged with B. burgdorferi and dependent on a slow phagocytic response to clear Borrelia from the hemolymph. In contrast, D. variabilis is highly immunocompetent (i.e., innate immunity), using plasma borreliacidal factors and a rapid increase in phagocytic cells to clear the infection and limit tissue invasion.     

The development ofAmblyomma americanum andDermacentor variabilis (Acari: Ixodidae) fed on rabbits immunized with tick hemolymph

The possibility of interfering with the normal function of tick hemolymph using antihemolymph antibodies taken in with the bloodmeal, was investigated. Cell free hemolymph from repleteAmblyomma americanum andDermacentor variabilis ticks was used to immunize rabbits. Immunized rabbits developed high antihemolymph antibody titers (ca. 10⁵) and had no ill side effects. Rabbits were simultaneously infested with larvae, nymphs, and adult ticks. The biological performance of ticks fed on immunized rabbits was virtually identical to that of ticks fed on nonimmunized rabbits. Usually, the mean engorgement weights of nymphs and females and the weights of the egg masses of both species were slightly higher for ticks fed on the nonimmunized rabbits but differences were not significant (P>0.05) due to a large standard deviation. The possibility of deactivating a single hemolymph component with specific antibodies is discussed.Journal article 4979 of the Agricultural Experiment Station, Oklahoma State University, Stillwater, OK, USA     

Microwave fixation: in situ tick (Acari: Ixodidae) histoanatomy, thin sectioning of tick tissues, and antigen preservation in mouse spleen

Microwave irradiation was used for the fixation of eggs, nymphs, and adult Boophilus spp. ticks. Although optimal temperatures for fixation of the different tick stages varied, heating to 58 degrees C of adult ticks submerged in either PBS or fixative was found to be sufficient. After microwave fixation, whole adult ticks, hand held, were sectioned with a sharp razor blade. The resulting sections revealed the in situ histoanatomy of the tick. Thin sections of ticks were obtained after either paraffin or polyester wax embedding. Microwave fixation combined with polyester wax embedding made serial thin sections of the different stages of Boophilus ticks possible. The technique preserved antigens as demonstrated by the immunostaining of lymphocytes and erythrocytes infected with Babesia microti in mouse tissues subjected to the same treatment as the ticks. With the microwave fixation-polyester wax technique, the specimen preparation time from fixation to the section on the glass slide was reduced to less than 8 h.     

Rectal infusion and aspiration of material through the guts of ixodid ticks (Acari: Ixodidae).

A technique for inoculating and removing substances via the anus of vector ticks was devised to define features of vector competence precisely. Calibrated inocula (greater than 5 nanoliter) containing aqueous dye and polystyrene beads as well as infectious agents were infused into the rectal sacs of ticks using glass microcapillary pipettes placed within the expanded anal orifice. The guts of preadult and adult ticks, Ixodes dammini Spielman, Clifford, Piesman & Corwin, Dermacentor variabilis (Say), Hyalomma impeltatum Schulze & Schlottke, and Amblyomma americanum (L.), were thereby infused with these inocula. Distribution of inocula was determined by examining hemolymph and sectioned ticks and confirmed that material placed in the rectal sac spread throughout the midgut diverticula. Ticks survived for greater than 6 mo after this procedure and were able to feed, molt to the next stage, or oviposit. In contrast, fewer ticks survived after intracelomic inoculation. The course of infection in ticks receiving anal infusions of Borrelia burgdorferi (the Lyme disease spirochete) was assessed. Such infections appear to differ from those established by feeding on infected hosts. Contents of the tick gut can be sampled nondestructively by anal perfusion to diagnose infection by this spirochete.     

Replication and tissue tropisms of Crimean-Congo hemorrhagic fever virus in experimentally infected adult Hyalomma truncatum (Acari: Ixodidae).

Adult Hyalomma truncatum Koch ticks were inoculated intracoelomically with Crimean-Congo hemorrhagic fever (CCHF) virus to examine tissue tropisms of this virus in ticks. Virus was recovered from all 185 ticks examined between 2 and 42 d after inoculation with CCHF virus. Titers or virus recovered from unfed male and female ticks were essentially the same (means, 10(2.4) and 10(2.5) plaque-forming units [PFU] per tick, respectively). Infection rates and titers recovered per gland for both salivary glands and reproductive tissues (ovaries and testes) were positively associated with blood feeding; average titers were 10-fold greater in organs from fed compared with those from unfed individuals. In contrast, neither the tick's sex nor feeding status (unfed or engorged) affected the titer of virus recovered from hemolymph (mean, 10(1.6) PFU/microliters). Although virus was recovered from Malpighian tubules, midgut, muscle, and nervous tissues from nearly all of the ticks tested, viral titers were consistently low. The increase in viral titer associated with blood feeding by ticks appeared to be due primarily to proliferation of tissue (e.g., salivary gland and reproductive tissues), rather than to increased replication in tissue already present.