The local effect of hypersensitive or inflammatory reactions to nymphal Amblyomma variegatum on simultaneous infections with Dermatophilus congolensis

Amblyomma variegatum nymphs were applied to sites infected with Dermatophilus congolensis on eight rabbits. Four rabbits were previously sensitized to the fecding of nymphal A. variegatum to produce hypersensitive reactions to the tick feeding; the remaining four rabbits had no previous exposure to nymphal A. variegatum and produced inflammatory reactions to the tick feeding.The resulting dermatophilosis infections were assessed for three weeks and there was a correlation between the position of the inflammatory tick attachment sites and the foci of infection. There was a significant increase in the lesions at sites with inflammatory reactions to the ticks, compared with sites not exposed to tick feeding; these differences appeared to be due to individual variation in the host response and were not sustained throughout the assessment.     

Experimental infection of the rabbit tick,<Emphasis Type="Italic"> Haemaphysalis leporispalustris</Emphasis>, with the bacterium<Emphasis Type="Italic"> Rickettsia rickettsii</Emphasis>, and comparative biology of infected and uninfected tick lineages

The present study consisted of two experiments that evaluated experimental infections of Haemaphysalis leporispalustris ticks by a Brazilian strain of Rickettsia rickettsii, and their effect on tick biology. In experiment I, ticks were exposed to R. rickettsii during the larval, nymphal or adult stages by feeding on rabbits (Oryctolagus cuniculus) needle-inoculated with R. rickettsii, and thereafter reared on uninfected rabbits for the entire next tick generation. Regardless of the tick stage that acquired the infection, all subsequent tick stages were shown to be infected by PCR (infection rates varying from 1.3 to 41.7%), and were able to transmit R. rickettsii to uninfected rabbits, as demonstrated by rabbit seroconversion, guinea pig inoculation with rabbit blood, and PCR on rabbit blood. In Experiment II, ticks were exposed to R. rickettsii during the larval stage by feeding on rabbits co-infested with R. rickettsii-infected adult ticks, and thereafter reared on uninfected rabbits until the next generation of larvae. Again, all subsequent tick stages were shown to be infected by PCR (infection rates varying from 3.0 to 40.0%), and were able to transmit R. rickettsii to uninfected rabbits. Thus, it was demonstrated that larvae, nymphs, and adults of H. leporispalustris were able to acquire and maintain the R. rickettsii infection by transstadial and transovarial transmissions within the tick population, with active transmission of the bacterium to susceptible rabbits by all parasitic stages. Analyses of biological parameters of uninfected and R. rickettsii-infected tick lineages were performed in order to evaluate possible deleterious effects of R. rickettsii to the infected tick lineages. Surprisingly, all but one of the four R. rickettsii-experimental groups of the present study showed overall better biological performance than their sibling uninfected control ticks. Results of the present study showed that H. leporispalustris could support infection by a high virulent strain of R. rickettsii for at least two generations, in which infected tick lineages tended to have better performance than uninfected ticks. Our results support a possible role of H. leporispalustris in the enzootic maintenance of R. rickettsii in Latin America, as previously suggested by earlier works.     

Immunization effect of recombinant P27/30 protein expressed in Escherichia coli against the hard tick Haemaphysalis longicornis (Acari: Ixodidae) in rabbits

We investigated the induction of resistance to Haemaphysalis longicornis infestation in rabbits that had been immunized with recombinant H. longicornis P27/30 protein. The success of immunological control methods is dependent upon the use of potential key antigens as tick vaccine candidates. Previously, we cloned a gene encoding 27 kDa and 30 kDa proteins (P27/30) of H. longicornis, and identified P27/30 as a troponin I-like protein. In this study, rabbits that were immunized with recombinant P27/30 expressed in Escherichia coli showed the statistically significant longer feeding duration for larval and adult ticks (P<0.05), low engorgement rates in larval ticks (64.4%), and an apparent reduction in egg weights, which suggest that H. longicornis P27/30 protein is a potential candidate antigen for a tick vaccine. These results demonstrated that the recombinant P27/30 protein might be a useful vaccine candidate antigen for biological control of H. longicornis.     

Field study of the relationship between skin-sensitizing antibody production in the cottontail rabbit, Sylvilagus floridanus, and infestation by the rabbit tick, Haemaphysalis leporispalustris (Acri: Ixodidae)

The resistance of cottontail rabbits to tick feeding appears correlated with the rabbits' development of skin-sensitizing antibodies. Resistance appeared to be greatest in adult rabbits which had been repeatedly infested with ticks. Rabbits with little exposure to ticks, usually the young cottontails, showed little or no skin-sensitizing antibody present in their blood and usually had relatively high tick loads when compared with adult rabbits. Models used to interpret the data show promise as tools for predicting tick population fluctuations and, perhaps, incidence of vector borne disease outbreaks. The existence of resistance to tick attachment has important implications for the host-parasite relationship. The research lends support to the hypothesis that the resistance may function as a homeostatic regulatory mechanism capable of maintaining the size of the tick population in equilibrium with the size of the rabbit population. In this way, host resistance may be advantageous to the parasite as well as to the host.     

Comparison of Preservation Methods of Rhipicephalus appendiculatus (Acari: Ixodidae) for Reliable DNA Amplification by PCR

Five differently preserved groups of adult Rhipicephalus appendiculatus specimens were compared for quality of DNA extracted. Three methods were used to extract DNA from specimens i.e. two simple mosquito validated DNA extraction methods and a tick validated method. Extraction of DNA from tick legs was attempted. The quality of DNA extracted was evaluated by the success of PCR amplification of the ITS2 gene and the mitochondrial COI gene fragment. Fresh specimens (i.e. killed just before extraction) had the highest success of DNA amplification followed by specimens killed in ethanol and subsequently stored in the refrigerator (4 °C). There was no significant difference in amplification success between cryopreserved and 70% ethanol preserved specimens. It was possible to amplify DNA from legs of ticks. Sequenced ITS2 amplicon of template obtained from legs of ticks was as legible as those from whole tick extract. The two mosquito validated DNA extraction methods showed a significantly lower amplification success than the tick validated protocol.     

Histological study of the attachment sites of adult Rhipicephalus appendiculatus on rabbits and cattle

Biopsies of tick attachment sites on ears of five rabbits and four calves (Bos taurus) were taken at first and third infestations and were examined by histochemistry, light microscopy and electron microscopy. Resistance was expressed by all hosts by reduction of tick engorgement weights. Attachment sites at first infestations on rabbits and cattle were similar and were characterized by an acute inflammatory abscess with a preponderance of neutrophils and macrophages infiltrating the site. Attachment sites at third infestations on rabbits and cattle were similar and were characterized by infiltration of neutrophils and macrophages and a 2- to 5-fold increase in the proportions of eosinophils and basophils. Lymphoblasts and plasmacytes were found in third infestation sites. In rabbits there was much necrosis and in cattle there were large intraepidermal pustules in third infestations. The cement attachment cone of the ticks was of lipoprotein and contained aminopeptidase. Salivary glycoproteins and esterases were detected in the attachment sites.     

Augmentation of host's naturally acquired immunity by solubilized membrane-bound midgut proteins of the tick Rhipicephalus appendiculatus.

Immune resistance of rabbits to the hard tick Rhipicephalus appendiculatus induced by combined tick infestations and immunization with solubilized midgut membrane proteins were compared with resistance due to 1-3 infestations or immunization alone. Results demonstrate that the level of exposure of rabbits to ticks used in the study does not significantly affect the immune expression resulting from immunization and that the latter augments the resistance due to infestation.     

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.     

Resistance and cross-resistance in guinea-pigs and rabbits to immature stages of ixodid ticks

Infestation of guinea-pigs and rabbits with larvae of any one of five species of ticks, Rhipicephalus appendiculatus Neumann, Rhipicephalus evertsi evertsi Neumann, Amblyomma hebrauem Koch, Amblyomma variegatum Fabricius and Ixodes ricinus L., conferred resistance in the animals when exposed to subsequent infestations with the same tick species. Resistance to infestations by other tick species was not observed.     

Immunisation of rabbits against the brown ear tick,Rhipicephalus appendiculatus using tick haemolymph

Tick naive rabbits were immunised with haemolymph components from partially fed Rhipicephalus appendiculatus adult ticks and subsequently challenged with all the developmental instars of the tick. The results obtained showed that the rabbits were rendered resistant to all the instars of the tick. However, the resistance was more pronounced in adult ticks than in the immature stages. The resistance was manifested as a reduction in the number of ticks that fed successfully to engorgement, reduced engorgement weights and reduced fecundities. Re challenging the resistant experimental rabbits with all the developmental instar stages of Rhipicephalus appendiculatus showed that resistance was maintained in subsequent infestations.     

Biological aspects of <Emphasis Type="Italic">Amblyomma brasiliense</Emphasis> (Acari: Ixodidae) under laboratory conditions

Although Amblyomma brasiliense Aragão 1908 has been reported as one of the most aggressive ticks to humans in Brazil, information about the biology of this tick species is virtually inexistent. This work reports data on the life cycle of A. brasiliense fed on rabbits and pigs and maintained in an incubator at 20°C, 90% RH and 12 h of light for off-host development. Tick yield of adult females fed on pigs and rabbits was 81.2% and 58.3%, respectively. Females fed on pigs had mean engorgement weight of 862.3 mg and egg mass of 208 mg, while females fed on rabbits had mean engorgement weight of 606.1 mg and egg mass of 160 mg; these values did not differ statistically between host species. Feeding period of female ticks fed on pigs (10 days) was significantly shorter than that on rabbits (17 days). Mean preoviposition period was slightly longer (35.9 days) for ticks fed on pigs than on rabbits (30 days). The minimum incubation period of eggs of ticks from both host species was similar and over 100 days. Egg production efficiency was low for females fed on both hosts (less than 30% and 20% for ticks from pigs and rabbits, respectively). More than 55% of larvae and 79% of nymphs fed on rabbits, set free inside the feeding chambers, engorged successfully. These ticks attained an engorgement weight of 1.3 and 18.2 mg, respectively, and fed for approximately 5 days. The minimum pre-molt period was 30 days for engorged larvae and over 44 days for nymphs. Molting success was low, less than 50% in the case of larvae and less than 20% for nymphs. Further studies are required to better determine the off-host requirements of this tick species.     

Identification and characterization of Ixodes scapularis antigens that elicit tick immunity using yeast surface display

Repeated exposure of rabbits and other animals to ticks results in acquired resistance or immunity to subsequent tick bites and is partially elicited by antibodies directed against tick antigens. In this study we demonstrate the utility of a yeast surface display approach to identify tick salivary antigens that react with tick-immune serum. We constructed an Ixodes scapularis nymphal salivary gland yeast surface display library and screened the library with nymph-immune rabbit sera and identified five salivary antigens. Four of these proteins, designated P8, P19, P23 and P32, had a predicted signal sequence. We generated recombinant (r) P8, P19 and P23 in a Drosophila expression system for functional and immunization studies. rP8 showed anti-complement activity and rP23 demonstrated anti-coagulant activity. Ixodes scapularis feeding was significantly impaired when nymphs were fed on rabbits immunized with a cocktail of rP8, rP19 and rP23, a hall mark of tick-immunity. These studies also suggest that these antigens may serve as potential vaccine candidates to thwart tick feeding.     

Testosterone depresses innate and acquired resistance to ticks in natural rodent hosts: a force for aggregated distributions of parasites

The effects of testosterone on acquired resistance to ticks, Ixodes ricinus, in their natural rodent hosts (voles, Clethrionomys glareolus, and wood-mice, Apodemus sylvaticus) were investigated by manipulating testosterone levels and exposing the hosts to repeated tick infestations. Testosterone reduced both innate and acquired resistance to tick feeding. During primary infestations, attachment rates were higher on rodents with high testosterone levels than on oil-implanted controls. Successive infestations on voles were accompanied by a decrease in tick feeding success and survival, but this decrease was significantly greater in ticks fed on control voles than in those fed on voles implanted with testosterone. When reduced feeding success had been induced, either by vaccination with tick salivary gland extract or by 4 successive infestations, implantation with testosterone partially reversed the acquired resistance. These effects of testosterone will generate heterogeneities within the rodent population with respect to tick distribution and microparasite transmission. The lowest innate and acquired resistance to tick feeding occurs in that fraction of the host population, i.e., sexually active males, most actively involved in the transmission of both Babesia microti and Borrelia burgdorferi s.l.     

Rhipicephalus appendiculatus feeding on rabbits and cattle: Salivary-gland responses to varying host resistance

AdultRhipicephalus appendiculatus ticks were fed as three sequential infestations on both rabbits and cattle. The feedings at first infestation on naive hosts were optimum for the ticks, whereas at third infestation the hosts were resistant, as expressed by reduced tick feeding performance. Ticks from naive and resistant hosts were examined for histological differences of salivary glands. In ticks fed on resistant rabbits there was a large increase in the synthesis of glycoprotein secretory granules in thec ₁ cells compared with ticks fed on naive rabbits. In ticks fed on naive and resistant cattle, the activity of thec ₁ cells was less than in ticks fed on naive and resistant rabbits. It was concluded that the salivary glands are able to respond selectively to conditions at the feeding site, and that this may be advantageous to 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.     

Effects of immunization of rabbits on establishment, survival, and reproductive biology of the tick Hyalomma anatolicum anatolicum.

Four antigenic preparations, viz. salivary gland antigen (SG Ag), whole tick extract antigen (WTE Ag) and 30,000-g supernatant fraction, and pellet of WTE Ag (TES Ag and TEP Ag, respectively), were made from partially fed adult female Hyalomma anatolicum anatolicum. Four groups of 5 rabbits each were immunized with the antigens, and a fifth group was kept as control. Following challenge with adult H. a. anatolicum, a significant decrease in engorgement weight and egg mass weight and an increase in engorgement period and preoviposition period were observed in WTE Ag-immunized rabbits. Similar results were observed with TES Ag and SG Ag, except that change in the engorgement period was insignificant. However, none of the tick parameter measurements showed significant changes with TEP Ag. None of the antigens produced significant changes in the percentage of engorgement or oviposition period of the challenged ticks. Thus, WTE Ag was the most effective in altering tick performances.     

The effect of various host species on the feeding performance of immature stages of the tick Hyalomma truncatum (Acari: Ixodidae)

In order to determine the effect of various hosts on the feeding performance of the tick Hyalomma truncatum, we used three mammalian species as hosts. Larvae and nymphs of H. truncatum were fed, under controlled laboratory conditions, on gerbils, guinea-pigs and rabbits. The larvae fed for 4.3±1.4 days on gerbils, 5.6±1.3 days on guinea-pigs and 4.7±1.2 days on rabbits. The mean weights of the larvae which fed on the rabbits, guinea-pigs and gerbils were 0.58± 0.09, 0.46±0.04 and 0.45±0.04 mg. respectively. The feeding periods of the nymphs on gerbils, guinea-pigs and rabbits were 7.9±1.3, 8.6±1.3 and 9.6±2.2 days respectively. The mean weights of the nymphs which fed on the gerbils, guinea-pigs and rabbits were 22.5±2.8, 19.7±1.3 and 15.8±1.4 mg, respectively. Hyalomma truncatum demonstrated a life cycle of a three-host tick on gerbils and guinea-pigs and of a two-host tick on rabbits. The evolutionary advantage of a two-host cycle over a three-host cycle in metastriate ticks is discussed.     

Ornithodoros moubata: host immunoglobulin G in tick hemolymph

Hemolymph proteins of a soft tick, Ornithodoros moubata, were analyzed immunochemically and biochemically. The components of tick hemolymph proteins were shown to be totally different from the host (rabbit) serum proteins by polyacrylamide gel electrophoresis with sodium dodecyl sulfate and Coomassie blue or silver stain. However, in the hemolymph of ticks engorged from rabbits immunoglobulin G was detected by immunoblotting analysis with goat anti-rabbit immunoglobulin G. The concentration of rabbit Immunoglobulin G in tick hemolymph changed with the physiological stages after a blood meal. Immunoglobulin G was isolated from tick hemolymph by affinity chromatography on a Protein A-Sepharose 4B column. Analysis of the isolated immunoglobulin G from tick hemolymph with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Ouchterlony double diffusion test showed it to be composed of the same subunits as heavy and light chains of host (rabbit) immunoglobulin G. Tracer experiments showed that 125I-labeled heavy and light chains of immunoglobulin G were detected in an intact form in hemolymph from ticks that sucked 125I-labeled rabbit immunoglobulin G through an artificial membrane. These facts suggested that the host rabbit immunoglobulin G ingested in the tick midgut passed through the gut wall without digestion. By solid-phase enzyme immunoassay, immunoglobulin in the hemolymph was shown to retain its antibody activity.     

Immunosuppression and feeding success of Ixodes ricinus nymphs on BALB/c mice

Abstract. The effect of repeated infestations of Ixodes ricinus (L.) nymphs on BALB/c mice was studied. Four successive infectations resulted in an increase of tick feeding success. Tick yield and mean engorged weight increased and the length of the feeding period was reduced significantly (P <0.05-0.01). The increase of specific anti-tick antibodies was not significant (P > 0.05). The blastogenic response of spleen lymphocytes to T-cell mitogens (Con A and PHA-P) was unimpaired or slightly enhanced, whereas the response to B-cell activators (LPS and PWM) was suppressed, as was the total antibody generation in vitro. The numbers of mast cells in murine skin at the tick attachment sites slightly decreased during the third infestation. The suppression of B-cell competence and of antibody generation, together with decrease of skin mast cell numbers in tick attachment sites, are considered to be responsible for enhancement of tick feeding success.     

Ticks Ixodes uriae and the breeding performance of a colonial seabird, king penguin Aptenodytes patagonicus

High densities of penguins in their colonies and the continuous use of these sites over consecutive reproductive periods increase the risk of development of tick populations. We have studied the effects of tick parasitism by Ixodes uriae in a colony of king penguins Aptenodytes patagonicus at Possession Island during three breeding seasons. We investigated the prevalence and periods of tick infestation during the one-year breeding cycle of penguins. The effects of tick parasitism on penguin breeding performance were assessed from photographs of the colony and with an automatic penguin identification system. We compared two groups of penguins carrying individual subcutaneous electronic tags, one group breeding in an infested area and the other in a non-infested area. Tick feeding activity was coincident with the periods when adult penguins stayed ashore for six days or more, i.e. during the incubating period. This duration corresponds to the duration of a tick meal on the host. The level of infestation varied between years. Penguins showed a lower incubating success in infested areas during a year of high infestation. In an infested area, individuals seen with ticks had a lower breeding success in rearing a one-year old chick than those seen without ticks.