The possible role of the salivary gland substrate in ixodid ticks as an adjuvant enhancing arbovirus transmission

Using a model: salivary glands of Dermacentor ticks--tick-borne encephalitis virus--guinea pig--D. marginatus ticks, it became possible to confirm the data of Jones et al. (1989) on the role of a substrate of Rhipicephalus appendiculatus glands as a strong enhancer of orthomyxovirus Togoto transmission during subcutaneous administration of a moderate virus dose to virus-resistant guinea pig. A tendency was only noticed towards better infectivity of ticks with the administration of sub- or supraoptimal virus doses together with the adjuvant (salivary gland substrate), as well as enhanced sensitivity of male individuals to a combination of virus with adjuvant. The latter fact can be explained by a transptyal way of infection typical for male individuals, which was noted earlier during joint nutrition with infected female individuals. A lower level of virus reproducibility in ticks who got it together with the adjuvant, as compared to the control, has been established. Low titer in female individuals after nutrition reduces the likelihood of transovarial transmission of virus with adjuvant.     

吸血期蓖麻硬蜱雌虫体内周身性莱姆病螺旋体生长动态的组织学研究

为查明吸血期蓖麻硬蜱雌虫体内周身性莱姆病螺旋体生长动态,用直接免疫荧光试验、银染色组织学及电子显微镜技术,对饥饿及吸血期蓖麻硬蜱雌虫脑、唾液腺和卵巢组织中莱姆病螺旋体的分布和数量进行了观察。结果显示,饥饿蟀组织中螺旋体数量极为有限,吸血期蜱组织中螺旋体的密度和数量随吸血时间的延长而快速增长,至开始吸血后4或5天,脑、唾液腺和卵巢组织中螺旋体的数量都已达到无法计数的程度;本研究首次对周身性莱姆病螺旋体的分裂增殖提出了假设。螺旋体在吸血期蜱唾液腺的大量检出,为莱姆病螺旋体经由蜱唾液分泌而传播给脊椎动物宿主的假设提供了新的证据。     

SALP16, a gene induced in Ixodes scapularis salivary glands during tick feeding

Guinea pigs infested with Ixodes scapularis acquire antibody-mediated resistance to tick bites, a phenomenon known as tick-immunity. An I. scapularis salivary gland cDNA expression library was therefore probed with sera from tick-immune guinea pigs to identify antigens that elicit humoral responses in the host. Sera from sensitized guinea pigs strongly recognized 3 of 4,500 library clones in an initial screening. The open reading frames of all 3 clones encoded a putative 16.4-kD acidic protein, designated Salp16, with an N-terminal signal sequence and signal peptidase cleavage sites specific for secretory proteins. The salp16 mRNA and Salp16 protein were detected in the salivary glands of engorged, but not unfed, nymphal and adult ticks, and Salp16 was also found in the saliva of engorged ticks. Immunization with recombinant Salp16 induced high antibody titers in guinea pigs, but did not elicit tick-immunity. Salp16 is the first feeding inducible gene that has been cloned from L. scapularis. Molecular characterization of I. scapularis salivary antigens that are induced upon tick feeding should help to facilitate our understanding of tick-host interactions.     

Experimental infection of six species of ixodid ticks with Dugbe virus (family Bunyaviridae, genus Nairovirus)

The vector potential of each of 6 species of colonized North American and African ixodid ticks was assessed by intracoelomic inoculation with Dugbe virus (IbAr 1792, 14th passage in suckling mouse brain) and viral titers were monitored after selected incubation periods. Persistence of Dugbe virus for greater than or equal to 53 days in 5 species (Dermacentor andersoni, D. variabilis, Amblyomma americanum, Rhipicephalus appendiculatus, and R. sanguineus) indicates that infection occurred. Viral titers were significantly higher in female vs. male D. variabilis, R. appendiculatus, and A. americanum after blood feeding. Blood feeding had no significant effect on the viral titers of either female or male R. sanguineus. D. andersoni males also exhibited no significant change in viral titers after blood-feeding, but 100% (20/20) of drop-off females and 96% (24/25) of post-oviposition females (36 days postinoculation) contained no detectable virus even though virus was still found in unfed specimens less than or equal to 124 days postinoculation. Virus was not recovered from greater than 30,000 1st generation progeny (eggs, larvae, nymphs, adults) collected as eggs from inoculated female D. andersoni, D. variabilis, R. sanguineus, and R. appendiculatus 27-51 days postinoculation. R. sanguineus and R. appendiculatus transmitted Dugbe virus to guinea pigs when allowed to feed 1-3 weeks postinoculation.     

Two feeding-induced proteins from the male gonad trigger engorgement of the female tick Amblyomma hebraeum

Most female ixodid ticks, once mated, feed to repletion within 6-10 days. Previous studies indicate that an engorgement factor (EF), passed to the female during copulation, may be the stimulus for engorgement. Here, we show that extracts of the testis/vas deferens of fed (but not unfed) male Amblyomma hebraeum contain EF bioactivity when injected into the hemocoel of feeding virgins. We have produced recombinant proteins (recproteins) from 28 feeding-induced genes in the male gonad and have identified a recombinant A. hebraeum engorgement factor (recAhEF) among these recproteins. recAhEF is a combination of two peptides, recAhEFalpha (16.1 kDa) and recAhEFbeta (11.6 kDa), neither of which has bioactivity on its own. recAhEF also stimulates salivary gland degeneration and partial development of the ovary, suggesting that it may be the same material as another male gonadal protein from this tick, male factor. We propose the name "voraxin" for the natural EF of ticks. When normal mated females were put on a rabbit immunized against recAhEF, 74% failed to feed beyond one-tenth the normal engorged weight within 14 days whereas all mated ticks put on a control rabbit engorged normally (mean duration of 8.8 +/- 0.8 days). This result constitutes preliminary evidence that an anti-tick vaccine might be developed from voraxin.     

Ultrastructural study of the infection process of Rickettsia conorii in the salivary glands of the vector tick Rhipicephalus sanguineus

This work was designed to study the infection process of Rickettsia conorii in the salivary glands of experimentally infected Rhipicephalus sanguineus ticks. One hundred six uninfected engorged nymphs were intracelomically inoculated with approximately 2 x 10(3) plaque-forming units of a rickettsial suspension. After the molt, unfed and fed adults were dissected, and the salivary glands were extracted and processed for transmission electron microscopy observation. Three different uninfected control groups were used for (1) evaluating the impact of the inoculation procedure, (2) establishing the feeding period of infected ticks, and (3) ultrastructural characterization of the salivary glands. Overall, 75.5% (80 of 106) of the nymphs inoculated with rickettsiae died during the molt or soon after hatching into adult instars; 50% (12 of 24) of the remaining infected adults showed severe malformations compromising their viability. In apparently healthy specimens, time of engorgement was longer. The contrast with the negative control groups was statistically significant, suggesting that R. conorii exerts a strong negative effect on the vector ticks. The ultrastructural study showed that in the salivary glands of infected ticks, rickettsial growth occurs preferentially in central, peripheral, and interstitial acini cells.     

Organ culture of Rhipicephalus appendiculatus with maturation of Theileria parva in tick salivary glands in vitro

A technique is described for the organ culture of Rhipicephalus appendiculatus ticks. Whole, unfed adult ticks with the dorsal integument removed, known as backless tick explants, were cultured in enriched Leibovitz' L-15 medium in which they remained active for at least 32 days at 28 degrees C and 9 days at 36 degrees C. Development of Theileria parva, as demonstrated by methyl green-pyronin staining, occurred in the salivary glands of infected backless tick explants held for 8 days at 28 degrees C or 3 days at 36 degrees C. Maturation in vitro of T. parva in backless tick explants was compared with that in cultured excised salivary glands. After 3-7 days at 36 degrees C glands from backless tick explants and excised salivary glands showed similar numbers of infected acini per infected tick. However, after 12 days at 28 degrees C backless tick explants showed 20-30 times as many infected acini per infected tick as excised salivary glands, in two experiments. No assessment was made of degree of parasite maturity or infectivity. It was concluded that both organ culture techniques supported development in vitro of the salivary gland stages of T. parva, but the backless tick explant technique was simpler and gave generally better results than culture of excised salivary glands.     

Isolation and characterization of salivary antigens from Hyalomma anatolicum anatolicum

This study demonstrates the involvement of a large number of salivary proteins in the acquisition of resistance to Hyalomma anatolicum anatolicum. Using immunoblotting, sera from hypersensitized rabbits were shown to react with nine proteins in the saliva and 17 in salivary gland extracts (SGE) from 96 h fed female ticks. The salivary antigens had molecular weights in the range of 14 400 to 130 000. All the antigens identified in the saliva and 12 of the SGE antigens were glycoprotein in nature and a majority of them appeared to be common to different stages of feeding. In addition antigen I (molecular weight 130 000) showed acid phosphatase and antigen III (molecular weight 96 000) showed both non-specific esterase and aminopeptidase activity. Three high molecular weight proteins isolated from saliva (antigen I, antigen II--molecular weight 103 000 and antigen III), gave immediate hypersensitivity reactions in intradermal inoculation into rabbits which had previously been exposed to ticks. Antigens II and III also elicited a strong delayed hypersensitivity reaction. These results may help to explain the nature of the immune mechanisms which effect resistance against H. a. anatolicum.     

Differential anti-chemokine activity of Amblyomma variegatum adult ticks during blood-feeding

Ticks secrete a cocktail of immunomodulatory molecules in their saliva during blood-feeding, including chemokine-binding factors that help control the activity of host immunocompetent cells. Here we demonstrate differential dynamics of anti IL-8 (CXCL8), MCP-1 (CCL2), MIP-1 (CCL3), RANTES (CCL5) and eotaxin (CCL11) activities in salivary gland extracts of adult Amblyomma variegatum. Unfed male and female ticks showed activity against all the chemokines except CCL5; anti-CCL11 activity was particularly high. However, during feeding the dynamics of anti-chemokine activity differed significantly between males and females, and varied between chemokines. In males, anti-chemokine activities increased, whereas in females they declined or increased slightly as feeding progressed. The exception was anti-CCL11 activity, which declined and then increased in both males and females. Comparison of salivary gland equivalents of individual ticks prepared at various feeding intervals revealed some differences that were most pronounced between individual females fed for 8 days. These observations reflect the feeding behaviour of male and female A. variegatum. They support the concept of 'mate guarding', in which males help their mates to engorge by controlling their host's immune response, and the possibility that ticks benefit from feeding together by exploiting molecular individuality.     

Dissemination, replication, and trans-stadial persistence of Dugbe virus (Nairovirus, Bunyaviridae) in the tick vector Amblyomma variegatum

The dissemination and replication of Dugbe (DUG) virus and its tissue tropisms in the tick vector Amblyomma variegatum were examined by immunohistochemical analysis using specific antibody, in situ hybridization with a viral-complementary riboprobe, and infectivity assays of dissected tissues. Dugbe virus was localized in both unfed and feeding adults inoculated as nymphs or orally infected by capillary feeding, and in nymphs infected by capillary feeding. In non-feeding ticks, the main sites of DUG virus replication were the epidermis, hemocytes associated with loose connective tissue, and a small number of phagocytic digestive cells in the gut lumen. Virus infectivity in the hemolymph was associated entirely with hemocytes. Dugbe viral antigen or infectivity was not detected in the salivary glands until after the start of feeding. Viral titers in the salivary glands of feeding ticks were about ten-fold higher than in gut, ovary, or loose connective tissue. The level of infection decreased during molting and increased during feeding. Viral particles and pathologic effects were not detected in infected ticks. The primary site of trans-stadial persistence of DUG virus is the hemocytes. Tick hemocytes and other motile cells may be important in the transmission of persistent virus infection from one cell or organ to another by diapedesis.     

Heterogeneity in the effect of different ixodid tick species on human natural killer cell activity

Tick saliva plays a vital role in blood-feeding, including manipulation of the host response to tick infestation. Furthermore, a diverse number of tick-borne pathogens are transmitted to vertebrate hosts via tick saliva, some of which exploit the immunomodulatory activities of their vector's saliva. We report that salivary gland extracts (SGE) derived from Dermacentor reticulatus adult ticks induce a decrease in the natural killer (NK) activity of effector cells obtained from healthy human blood donors. The decrease was observed with SGE from both female and male D. reticulatus fed for either 3 or 5 days on mice, but no significant effect was observed with SGE from unfed ticks or ticks that had fed for 1 day. These results indicate that the tick anti-NK factor(s) is only active after blood-feeding has commenced. Microscopic examination revealed that the first step of NK activity, namely effector/target cell conjugate formation, was affected by SGE. The observed reduction in conjugate formation occurred when effector (but not target) cells were treated with SGE for 30 min, and the effect persisted after 12 h of treatment. Similar but less potent anti-NK activity was detected for SGE from Amblyomma variegatum and Haemaphysalis inermis. By contrast, SGE derived from Ixodes ricinus and Rhipicephalus appendiculatus female ticks did not decrease NK activity. The apparent absence of such activity in these two important vectors of tick-borne viruses suggests that control of NK cells does not play an important role in promoting virus transmission, at least for these particular species.     

Plasmodium infection-induced changes in salivary gland proteins of malaria vector Anopheles stephensi (Diptera:Culicidae)

Parasitism by Plasmodium yoelii yoelii induced 18 polypeptides in the salivary glands of aging malaria vector Anopheles stephensi. A polypeptide of low molecular size (30 kDa) could generally be induced at all infected stages. On day 5 post blood feeding (PBF), no new polypeptide could be found in the salivary glands. Seven polypeptides of low molecular size and 3 of high molecular size could be induced on day 11 PBF, which inducibility coincided with the invasion of the salivary glands by the sporozoites. Quantitatively, soluble proteins decreased in the salivary glands by about one-third in females that had consumed infected or uninfected blood meal on day 9 (oocysts stage) as compared to nonfeeding females. However, on day 15, in the salivary glands invaded by sporozoites, the amount of proteins obtained from infected females was approximately 26% lower than that obtained from uninfected females. A similar reduction was also observed in aged (20 days PBF) salivary glands of infected mosquitoes. These proteins could confer parasite tolerance to the females and enhance parasite transmission potential.     

Isolation of a Theileria species from Eland (Taurotragus oryx) infective for cattle.

Theileria infections were induced in cattle by feeding ticks on them from 3 sources: (a) adult rhipicephalid ticks obtained from the vegetation in a paddock containing an eland EAO at the Animal Orphanage, Nairobi National Park, Kenya, (b) Rhipicephalus appendiculatus adults fed as nymphs on the same eland, (c) R. pulchellus adults fed as nymphs on an eland W 68 captured in the Machakos district of Kenya. Both eland were harbouring Theileria parasites at the time nymphal ticks were fed. Mild infections were produced when adult ticks from these 3 batches were applied to cattle associated with low numbers of schizonts and piroplasms. The indirect fluorescent antibody test demonstrated that cattle recovered from infections resulting from the above 3 tick batches from eland W 68 and EAO produced antibodies which reacted with schizont antigen of the Theileria species (eland) and Theileria species (Githunguri) which had been isolated from cattle and not to antigens of other Theileria species used. The cattle recovered from the Theileria species (eland) were fully susceptible to a lethal challenge of a T. parva (Muguga) stabilate. It was concluded that the Theileria species (eland) and Theileria species (Githunguri) may be closely related and could represent a new species of Theileria infective to cattle.     

Adult Ixodes dammini on rabbits: development of acute inflammation in the skin and immune responses to salivary gland, midgut, and spirochetal components

Rabbits exposed to female Ixodes dammini (both uninfected and infected with Borrelia burgdorferi) or injected with B. burgdorferi showed an acute inflammatory response in the skin. Granulocytes and monocyte-histiocytes were the predominant infiltrating cells. Spirochetes were detected in the tick feeding cavities in the deep dermis. The inflammatory process was accompanied by polyclonal antibody responses to tick salivary gland components. Western blots showed that immune rabbit serum reacted with proteins of molecular masses of 8, 24, and 36-41 kilodaltons in both unengorged and engorged tick salivary glands. Additional reacting bands in the immunoblot of the engorged salivary gland indicated that new antigenic components of the salivary gland are synthesized during engorgement. Rabbits did not produce antibodies to tick midgut components. Murine monoclonal antibody 11G1 detected outer surface protein A of B. burgdorferi in immunoblots of midguts from unengorged ticks, faintly in engorged salivary gland, and seldomly in unengorged salivary gland, findings suggesting that the spirochete is transmitted to the host via tick saliva during the later stages of feeding.     

Acquisition and transmission of Theileria parva by vector tick, Rhipicephalus appendiculatus

In order to investigate the transmission dynamics of Theileria parva (T. parva) by the brown ear tick, Rhipicephalus appendiculatus (R. appendiculatus), under experimental conditions, detection of T. parva in ticks and cattle was performed by a quantitative real-time PCR assay. A calf inoculated with a T. parva mixture became PCR-positive for T. parva infection on day 8 post-inoculation, and subsequently, nymphal ticks were introduced and maintained to feed on the infected calf for 6 days. Engorged nymphs were collected daily and allowed to molt into adults, and overall, 70.8% (121/171) of the adult ticks acquired the T. parva infection. Furthermore, the T. parva infection rate in ticks under field conditions was monitored by real-time PCR in R. appendiculatus ticks collected from a traditionally managed pastoral land of Zambia, on which Sanga breed cattle are traditionally reared and the area has endemic East Coast fever (ECF). A total of 70 cattle were randomly selected in the same area and 67 (95.7%) were found to be serologically positive for R. appendiculatus tick antigen (RIM36). Twenty-nine (43.3%) of the 67 serologically positive cattle were real-time PCR-positive for T. parva, although no piroplasms could be detected in the blood smears. Unexpectedly, out of 614 R. appendiculatus nymphal and adult ticks collected by flagging vegetation, 4.1% were positive for T. parva DNA. However, since the rate of transmission of T. parva from infected cattle to ticks and vice versa and the serological evidence of exposure to R. appendiculatus ticks in naturally exposed cattle were relatively high, it would be wise in such a case to consider vector control as well as vaccination against ECF as control measures.     

Toxicoses of ticks (Acari: Ixodida)

Toxins have been shown to present in the salivary glands, whole body extracts, and eggs of ticks. They cause histological lesions in the skin, and in various organs of tick hosts. Among toxicoses, tick paralysis is of the greatest medical and veterinary importance. Toxins are secreted by cells "b" of acinus II in salivary glands during tick feeding.     

Salivary gland proteins of the human malaria vector, Anopheles dirus B (Diptera: Culicidae)

Salivary gland proteins of the human malaria vector, Anopheles dirus B were determined and analyzed. The amount of salivary gland proteins in mosquitoes aged between 3--10 days was approximately 1.08 +/- 0.04 microg/female and 0.1 +/- 0.05 microg/male. The salivary glands of both sexes displayed the same morphological organization as that of other anopheline mosquitoes. In females, apyrase accumulated in the distal regions, whereas alpha-glucosidase was found in the proximal region of the lateral lobes. This differential distribution of the analyzed enzymes reflects specialization of different regions for sugar and blood feeding. SDS-PAGE analysis revealed that at least seven major proteins were found in the female salivary glands, of which each morphological region contained different major proteins. Similar electrophoretic protein profiles were detected comparing unfed and blood-fed mosquitoes, suggesting that there is no specific protein induced by blood. Two-dimensional polyacrylamide gel analysis showed the most abundant salivary gland protein, with a molecular mass of approximately 35 kilodaltons and an isoelectric point of approximately 4.0. These results provide basic information that would lead to further study on the role of salivary proteins of An. dirus B in disease transmission and hematophagy.     

Plasmid requirements for infection of ticks by Borrelia burgdorferi

Borrelia burgdorferi strain B31 MI commonly loses one or more of its complement of 21 extrachromosomal plasmids during normal handling procedures and during genetic manipulations. Certain plasmid losses cause an inability or reduction in the ability of spirochetes to infect mice. In the current study, nine strains of spirochetes with varying plasmid profiles were used to identify plasmids necessary for nymphal tick infection. Nymphal ticks were artificially fed the nine spirochete strains as well as the parental strain containing a full complement of plasmids. The capillary fed nymphs were allowed to feed on mice for at least 63 h and then examined for the presence of spirochetes in their guts and salivary glands. All spirochete strains tested were able to infect ticks guts, but to different degrees. We determined that the plasmids lp5, lp28-1, and cp9 were not required for infecting tick guts, whereas loss of lp25 and lp28-4 was associated with reduced gut infectivity. A reduction in the ability of spirochetes to invade salivary glands was seen in bacteria that did not have lp28-1, whereas cp9 was not required for salivary gland infection. This study has pinpointed specific plasmids whose absence is deleterious to infecting nymphal tick guts and salivary glands.     

斑须按蚊唾液腺中疟原虫配子体激活因子的活性动态

目的　探讨斑须按蚊生长、发育过程唾液腺中疟原虫配子体激活因子的活性动态。方法　应用体外雄配子体出丝分析方法 ,检测雌性斑须按蚊羽化后、吸血及产卵前后唾液腺中疟原虫配子体激活因子对柏氏疟原虫雄配子体出丝诱导活性的变化。结果　羽化后未吸血组斑须按蚊唾液腺配子体激活因子活性与按蚊生长、发育呈同步变化 ;吸血后未产卵组按蚊唾液腺抽提物的配子体激活因子活性在吸血后显著下降 ,吸血后第 8天恢复到吸血前水平 ;而吸血后产卵组按蚊唾液腺抽提物的配子体激活因子活性在吸血后第 4天恢复到吸血前水平。结论　吸血后斑须按蚊唾液腺配子体激活因子的消长与按蚊产卵相关。