Bovine resistance to Amblyomma americanum ticks: an acquired immune response characterized by cutaneous basophil infiltrates

Purebred Holstein calves acquired resistance to Amblyomma americanum adult ticks after 1 infestation and expressed resistance during a challenge infestation 26 days later. Tick yields from resistant animals were normal, but mean tick weight and egg-mass weight were reduced significantly (31 and 32%, respectively). Cutaneous cellular responses at tick feeding sites in primary (naive) hosts consisted almost exclusively of mononuclear cells, until 24-h post-infestation when granulocytes appeared. At 48 h, basophils were the most abundant granulocyte and comprised 19% of the total cellular response. Secondary and tertiary hosts expressed significant cutaneous basophil responses as early as 6 h post-tick infestation, indicative of an anamnestic response. Cutaneous basophil levels in tertiary hosts (16-34% of the infiltrate) were increased by 2-fold over secondary hosts (12-16% of the infiltrate). Neutrophils and eosinophils were fairly abundant in secondary hosts (22-33%, and 3-13%, respectively), but decreased significantly in tertiary hosts corresponding to increased basophil levels. Mast cells comprised less than 10% of the cells found in primary hosts, and less than 5% in secondary and tertiary hosts representing a consistent, but insignificant component of the total cells. Basophils were not observed in the blood of hosts during any tick infestation, but a peripheral blood basophilia (0.1-1.1%) marked the end of each tick feeding period. A neutropenia was observed in the blood of tertiary hosts corresponding to a decreased tissue neutrophil response. Blood eosinophil, lymphocyte and monocyte levels remained unchanged during each tick infestation compared to controls. In summary, calves acquired immunity to A. americanum ticks, resulting in significant decreases in feeding and ovipositional success associated with a local cutaneous basophil response and peripheral blood basophilia. These findings suggest that basophils are involved in bovine immunity to ticks as has been established in the guinea pig model.     

Immunological control of hematophagous arthropod vectors: utilization of novel antigens

Immunological control of ixodid ticks requires development of a vaccine that stimulates an effective anti-tick response. However, it is important to avoid induction of intense host skin reactivity to tick feeding. Salivary-gland-derived molecules are introduced into the host during ixodid engorgement; therefore, use of these moieties might not be an optimal immunization strategy. Antigens not normally involved in acquired resistance can be used to induce anti-tick immunity. These "novel" antigens, obtained from tick gut absorptive surface, are not introduced into the host during tick feeding, but are exposed to host-immune effector elements in the blood meal, resulting in ixodid rejection, prevention of ova production and death. Anti-tick immunity is induced with microgram quantities of this ixodid gut antigen preparation. Sera obtained from immunized animals were used to identify antibody-reactive components of the resistance-inducing extract. Tick gut absorptive surface antigen glycoconjugates were identified by lectin blotting, using a series of probes with different carbohydrate specificities. The lectins, peanut and wheat germ agglutinin, and immunized host antibodies bound extract components with similar molecular weights. Solubilization and fractionization of tick gut proteins with the non-ionic detergent Triton X-114 is described.     

镰形扇头蜱免疫球蛋白结合蛋白的表达及免疫保护分析

为在大肠杆菌中高效表达镰形扇头蜱免疫球蛋白结合蛋白(immunoglobulin binding protein,IGBP),并对表达产物进行免疫保护效果测定,将IGBP基因克隆到pGEX-4T-1载体,转化到感受态细胞BL21,在IPTG诱导下进行表达;表达产物3次免疫家兔,剂量为500μg/只。3次免疫后进行攻蜱试验,观察免疫保护效果。结果发现在IPTG的诱导下,重组质粒pGEX-4T-1-IGBP在大肠杆菌中获得高效表达,产生GST-IGBP融合蛋白,用融和蛋白免疫家兔后,试验组与对照组成蜱饱血重量和死亡率差异显著(P<0.05)。表明重组质粒pGEX-4T-1-IGBP能在大肠杆菌中高效表达,且表达蛋白能够对家兔产生一定程度的抗蜱保护性免疫力。     

Control of Boophilus microplus ticks in cattle calves by immunization with a recombinant Bm86 glucoprotein antigen preparation.

Forty Egyptian native cattle calves of 4-6 months old randomly allocated into two groups of twenty animals each were used to assess the effect of immunization of animals with a recombinant Bm86 antigen derived from Boophilus microplus ticks on induction of immunity that could protect calves during tick season. The immunization protocol involved two injections administered intramuscularly, the first was applied with complete Freund's adjuvant and the second was given with incomplete Freund's adjuvant two months later. Control calves were given saline plus adjuvant. Immunization reduced the number of adult ticks developing from a subsequent challenge infestation by 78% in immunized calves. Vaccination also, significantly reduced the weight of adult ticks in immunized calves (30.51%). The results of skin delayed hypersensitivity reaction revealed that the diameter of sites injected with the recombinant Bm86 antigen was significantly larger in immunized calves than those in controls. Analysis of the immune response indicated that there was a significant increase in the level of IgG and IgA antibodies in serum of immunized calves and protection from reinfestation was correlated with the levels of circulating antibodies.     

The interaction between the immune response of rabbits to heterologous antigens and a primary infestation with Rhipicephalus evertsi evertsi

Rhipicephalus evertsi evertsi feeding on hosts inoculated with sheep red blood cells (SRBC) and bovine serum albumin (BSA) suppressed the primary antibody response to the two antigens. In addition, while the ticks paralysed most hosts in the studies, fatality associated with this toxicosis occurred only in rabbits which had received SRBC, either alone or with BSA. Only those hosts inoculated with BSA developed any resistance against the ticks, manifested by a slight reduction of engorged weights and development of anti-tick antibodies. These results suggest that R. e. evertsi infestation induces a degree of reduced host immune responsiveness to heterologous antigens.     

Tick-Encoded serine proteinase inhibitors (serpins); potential target antigens for tick vaccine development.

Immunological protection of hosts against tick infestation is at present the most practically sustainable alternative tick control method to the current use of acaricides that is riddled with serious limitations. The current focus of tick vaccine research is the identification, cloning and in vitro production of recombinant tick vaccine candidate antigens. We have examined a selected number of reports on the roles of parasite-encoded members of the serine proteinase inhibitor (serpin) superfamily in modulation of mammalian anti-parasite defense and developed some food for thought commentaries on the possibility of targeting this class of proteins for anti-tick vaccine development.     

Cross-reactivity between antigens from Amblyomma cajennense and A. hebraeum (Acari: Ixodidae)

Laboratory animals exposed to feeding ticks develop resistance which is reflected by a decline in tick engorgement weight, egg-laying by adults and reduced egg viability. Serum antibodies from these hosts and their reaction with tick antigens have been detected by different methods, including precipitation techniques, immunofluorescent techniques, ELISA and Western blots. However, little is known about the effects of antibodies on ticks that engorge on resistant hosts, or which tissues of the tick body are possibly immunogenic. Some researchers, using immunohistochemistry, have detected host antibodies in the gut, salivary glands and haemolymph of ticks engorged on resistant animals. The same technique has helped considerably in determining antigenic sites or antibody targets in other arthropods. Consequently, immunohistochemistry techniques were used in this study to detect cross-reactivity between sera raised against Amblyomma cajennense (Fabricius, 1787) with Amblyomma hebraeum (Koch, 1844), and vice versa. The results show the existence of shared antigens between the 2 tick species. In general, our results point more to a 1-way cross-reactivity of A. hebraeum with A. cajennense than a reciprocal cross-reactivity, suggesting that A. hebraeum is more immunogenic than A. cajennense.     

Cross-reaction of tick salivary antigens in the Boophilus microplus-cattle system

Calves were immunized with Boophilus microplus saliva, filtered through Millipore membranes, in Freund's complete adjuvant. Serum samples were tested by passive hemagglutination against Babesia bigemina, Anaplasma marginale, B. microplus larvae extract, Stomoxys calcitrans extract and B. microplus saliva. After immunization, titers to saliva, larval tick-extract and to S. calcitrans were increased. The challenge with live tick larvae enhanced the formation of antibodies against larva extract, fly extract and tick saliva, which supports the idea that under natural controlled conditions this cross-reactivity could occur.     

Comparative IgG recognition of tick extracts by sera of experimentally infested bovines

Enzyme-linked immunosorbent assay (ELISA) and Western blot were used to investigate the pattern of antibody responses of six bovines infested twelve times with Rhipicephalus (Boophilus) microplus (Canestrini, 1887) (Acari: Ixodidae) (six heavy infestations followed by six light infestations) against salivary gland, gut and larvae extracts. During heavy infestations, bovine IgG levels were shown to be higher, and a decrease in the number and weight of ticks that completed the parasitic cycle was observed. The pattern changed starting from the seventh infestation, showing a decrease in IgG levels. An initial increase followed by a significant decrease in the proportion of ticks that completed the parasitic cycle was also observed from the seventh infestation. The number of molecules recognized by Western blot was higher from sera collected following heavy infestations than after light infestations, although a great variation in the profiles detected could be seen when the bovines were compared. These results indicate that IgG responses to different tick antigens may not be generally associated with bovine resistance, and that infestation levels modulate the magnitude of humoral responses and possibly the immune mechanisms in the natural acquisition of tick resistance.     

American canine hepatozoonosis

American canine hepatozoonosis is an emerging, tick-transmitted infection of domestic dogs caused by a recently recognized species of apicomplexan parasite, Hepatozoon americanum. The known definitive host of the protozoan is the Gulf Coast tick, Amblyomma maculatum. Presently recognized intermediate hosts include the domestic dog and the coyote, Canis latrans. Laboratory-reared larval or nymphal A. maculatum can be infected readily by feeding to repletion on a parasitemic intermediate host; sporogony requires 35-40 days. Transmission of infection to the dog has been produced experimentally by oral administration of mature oocysts or oocyst-containing ticks. Canine disease follows experimental exposure in 4-6 weeks and is characterized by systemic illness, extreme neutrophilic leukocytosis, muscle and bone pain, and proliferation of periosteal bone. Histopathological findings include multifocal skeletal and cardiac myositis associated with escape of mature merozoites from within the host-cell environment. There is also rapid onset of periosteal activation and osteogenesis and, less frequently, glomerulopathy and amyloidosis. Sequential stages of development of H. americanum in both the dog and the tick have been elucidated. Gamonts potentially infectious to ticks have been observed in peripheral blood leukocytes of the dog in as few as 28 days after exposure to oocysts. Young coyotes experimentally exposed to a canine strain of H. americanum acquired disease indistinguishable from that of similarly exposed young dogs.     

Molecular characterization of a myosin alkali light chain-like protein, a "concealed" antigen from the hard tick Haemaphysalis qinghaiensis

There should be some differences between antibodies generated by feeding ticks on animals and those derived by immunizing animals with tick extracts. Here, we found serum collected from sheep immunized with Haemaphysalis qinghaiensis salivary gland extracts could detect two more protein bands with molecular weights of 22 and 37 kDa (P22 and P37) on Western blots of extracts of tick salivary glands than serum from tick infected animals. Rabbit anti-H. qinghaiensis differential protein immune serum was then generated from P22 and P37 and was used to immunoscreen a cDNA library constructed from salivary glands, Malpighian tubules and ovaries of partially engorged H. qinghaiensis. A cDNA contains an open reading frame of 483 bp that codes for 160 amino acid residues with a coding capacity of 18 kDa was cloned and designated Hq02. Expression analysis by RT-PCR showed that this gene is expressed in salivary glands, midguts, other organs and different developmental stages of H. qinghaiensis. The predicted amino acid sequence of the Hq02 gene had high homology to some known myosin alkali light chain (MLC) proteins. A fusion protein consisting of 130 amino acids of Hq02 protein and 335 amino acids of T7 gene 10 protein was expressed in Escherichia coli and used to immunize sheep. Western blot showed that only rabbit anti-H. qinghaiensis differential protein immune serum could recognize the expressed Hq02 protein, while rabbit anti-H. qinghaiensis saliva immune could not. This proved Hq02 protein was a "concealed" antigen. Immunization with the recombinant Hq02 conferred a 21.8% reduction of engorgement weight for adult female ticks that fed on the immunized sheep. This is the first report of tick myosin alkali light chain and the function of this protein is discussed.     

中华硬蜱叮咬蜱抗原免疫接种宿主后中肠组织化学的动态观察

选用中华硬蜱叮咬中肠抗原免疫接种组及初次叮咬组和佐剂对照组宿主并分别在不同时间(叮咬24、48、72h以及5、8d)对蜱中肠蛋白质、糖原及多糖进行了动态观察,并对消化细胞核DNA含量进行测定。结果显示:蜱叮咬中肠抗原免疫接种宿主后可使中肠上皮发生破坏,消化细胞破损,基膜可出现松散和断裂,消化细胞蛋白质含量较初次叮咬组和佐剂对照组宿主明显降低,而糖原及多糖含量基本不变。消化细胞DNA在叮咬无免疫力宿主(初次叮咬组和佐剂对照组)早期24h主要为2倍体,随着吸血量增加、细胞核倍体数加大,第5天达7.06倍体,而叮咬中肠抗原免疫接种组宿主后,消化细胞细胞核倍体数增加不明显,与叮咬无免疫力宿主有显著性差异(P>0.01),提示中华硬蜱叮咬中肠抗原免疫接种宿主后,可干扰中肠消化细胞蛋白质和核酸(DNA)的合成代谢,为进一步揭示抗蜱免疫机理提供了理论依据。     

Evaluation of glycoproteins purified from adult and larval camel ticks (Hyalomma dromedarii) as a candidate vaccine

In order to identify antigens that can help prevent camel tick infestations, three major glycoproteins (GLPs) about 97, 66 and 40 kDa in size were purified from adult and larval Egyptian ticks, Hyalomma (H.) dromedarii, using a single-step purification method with Con-A sepharose. The purified GLPs were evaluated as vaccines against camel tick infestation in rabbits. The rabbits received three intramuscular inoculations of GLPs (20 µg/animal) on days 0, 14, and 28. In the immunoblot analysis, Sera from the immunized rabbits recognized the native GLPs and other proteins from larval and adult H. dromedarii ticks along with those from other tick species such as Rhipicephalus sanguineus but not Ornithodoros moubata. The effects of immunity induced by these GLPs were determined by exposing rabbits to adult H. dromedarii ticks. These results demonstrated that GLP immunization led to a slightly decreased reproductive index and significantly reduced rates of egg hatchability. These results demonstrated that immunization with the purified GLPs can provide protection against infestation by H. dromedarii and some other tick species. Further studies are needed to confirm the effectiveness of immunization with GLPs against other tick species.     

Rhipicephalus (Boophilus) microplus embryo proteins as target for tick vaccine

Rhipicephalus (Boophilus) microplus is one of the most widely distributed tick in the world. The control of the parasite is based mainly on the use of chemical acaricides, which are produced from a limited set of molecules. These drugs induce selection of acaricide-resistant ticks, and are an important source of environmental pollution. An approach based on anti-tick vaccines may circumvent these obstacles. Characterization of the physiological function of tick molecules may be useful to develop new vaccines. Previously, we reported the ability of some tick proteins as inducers of protective immune response. Vaccination studies using tick proteins like native (nBYC), recombinant (rBYC) egg-yolk aspartic endopeptidase and cysteine endopeptidase (VTDCE) from R. microplus and glutathione S-transferase (Hl-GST) from Haemaphysalis longicornis demonstrated the immunogenicity and antigenicity of these proteins in bovines. Eventually, immunization with these proteins triggered a partial immune response against R. microplus infestation in cattle, manifested mainly as a reduction in egg fertility (7.7% and 13.9% for nBYC, 5.9% for rBYC; 4.7% for VTDCE, 7.9% for Hl-GST), and in the number of fully engorged ticks (18.2% for rBYC, 14.6% for VTDCE, 53% for Hl-GST). The data so far obtained suggest that these proteins have potential to be used as antigens in an anti-tick vaccine. Other proteins involved in tick embryogenesis also have this potential, like THAP and BmCl1, which are enzymes with key roles in vitellin and hemoglobin hydrolysis. Moreover, the identification of analogous proteins present in other tick species may bring information about the way to develop a vaccine against multiple tick species which can help to solve the problem faced by numerous countries where animals are parasitized by more than one tick species. The aim of the present review is to comprehensibly summarize the data obtained in the last few years by our collaborative research, discussing the efforts we have made to find antigens efficient enough for a cattle tick-controlling vaccine. This review discusses tick physiology studies aimed at the selection of possible targets, characterization of the selected proteins with emphasis on their biochemical and immunological aspects and results of vaccine trials on bovines.     

Transstadial and intrastadial experimental transmission of Ehrlichia canis by male Rhipicephalus sanguineus

The acquisition and transmission of rickettsial pathogens by different tick developmental stages has important epidemiological implications. The purpose of this study was to determine if male Rhipicephalus sanguineus can experimentally acquire and transmit Ehrlichia canis in the absence of female ticks. Two trials were performed where nymphal and male R. sanguineus were simultaneously acquisition fed on the same infected donor hosts, and transstadially or intrastadially exposed male ticks were fed on separate pathogen-free dogs as a test for transmission. A single-step p30-based PCR assay was used to test canine and tick hosts for E. canis infections before and after tick feeding. E. canis was detected after either intrastadial or transstadial passage in male ticks, the organism remained detectable in both tick groups after transmission feeding, and both tick groups transmitted the rickettsia to susceptible dogs. Infection of dogs via tick feeding resulted in milder clinical signs and lower antibody titers than intravenous inoculation of carrier blood, but further investigation is needed to understand the mechanisms responsible for this observation. These results demonstrate that male R. sanguineus can take multiple feedings, and that they can both acquire and transmit E. canis in the absence of female ticks. This tick development stage could be important in transmission of E. canis, and perhaps related pathogens, between vertebrate hosts under natural and experimental conditions.     

Detection of immune response in rabbits infested with Rhipicephalus appendiculatus and Rhipicephalus evertsi evertsi

Two groups of rabbits infested repeatedly with Rhipicephalus evertsi evertsi and R appendiculatus acquired resistance as evidenced by reduced ectoparasite engorged weights and development of anti-tick antibodies in the hosts. The enzyme-linked immunosorbent assay (ELISA) using salivary gland antigens of R appendiculatus was able to detect antibody responses to both the homologous and heterologous tick challenges. It was concluded that the immune response in hosts infested by these two tick species stimulates the production of antibodies which show cross reactions.     

Evidence for regurgitation by Amblyomma americanum

Western blot analysis of Amblyomma americanum-derived midgut extracts using tick-resistant rabbit and guinea pig antisera demonstrated several unique polypeptides ranging from 7 to 355 kDa. These polypeptides were not observed in similar blots of salivary gland extracts or serum from the respective host animals. The data from this study suggest that female A. americanum ticks regurgitate gut material during feeding. Such a finding explains the ability of immunization with gut-derived preparations to induce immunity and may help to understand the mechanism of transmission of several tick-borne pathogens that have been only clearly demonstrated to infect the gut of ticks.     

Antibodies to Anaplasma marginale major surface proteins 1a and 1b inhibit infectivity for cultured tick cells

Major surface protein 1 (MSP1) of the cattle pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) is a complex of two proteins, MSP1a and MSP1b. Previous studies demonstrated that MSP1a and MSP1b are adhesins for bovine erythrocytes, while only MSP1a proved to be an adhesin for tick cells. In this study, a tick cell culture system for propagation of A. marginale was used to develop an infection inhibition assay for testing the ability of antisera to block infection of A. marginale for cultured tick cells. A. marginale derived from cell culture was incubated with various antisera prior to inoculation onto cell monolayers. The monolayers were harvested 7 days post-inoculation and A. marginale in the cultures was quantified using an antigen detection ELISA. Antisera tested in the infection inhibition assay were derived from persistently infected cattle, from cattle immunized with A. marginale purified from bovine erythrocytes, and from rabbits and cattle that were immunized with the recombinant MSP1a, MSP1b and MSP1 complex. Antibodies from cattle persistently infected with A. marginale, cattle immunized with A. marginale from bovine erythrocytes or cattle immunized with the recombinant MSP1 complex did not inhibit the infectivity of A. marginale for tick cells. Antiserum from rabbits immunized with MSP1a and MSP1b (individually or combined) reduced infection of both the Virginia and Oklahoma isolates of A. marginale for tick cells by 25-70%. Likewise, antisera from cattle immunized with recombinant MSP1a or MSP1b inhibited infection of tick cells by 26-37%. These results further confirm the role of MSP1 complex proteins in infection of tick cells. Lack of inhibition of infection by antisera from naturally infected cattle or cattle immunized with whole organisms suggests that the bovine immune response is not directed toward blocking infection of A. marginale for tick cells and may contribute to the continued infectivity of the pathogen for ticks.