Comparative vaccination of cattle against Boophilus microplus with recombinant antigen Bm86 alone or in combination with recombinant Bm91

Cattle were vaccinated either with a single recombinant tick antigen, Bm86 or with a combination of two recombinant antigens, Bm86 and Bm91 from the tick Boophilus microplus . In three experiments, the responses of cattle to subsequent challenge with the tick were assessed. The addition of the Bm91 antigen enhanced the efficacy of the vaccination over that with Bm86 alone to a statistically significant degree. Moreover, co-vaccination with two antigens did not impair the response of cattle to the Bm86 antigen. Finally, responses of individual cattle to the two antigens were independent. All of these results may be relevant to the increase in efficacy expected from a dual antigen vaccine.     

Vaccination with‘concealed’antigens: myth or reality?

Cattle infested with the tick Boophilus microplus produce antibodies to intrinsic membrane glycoproteins of the tick, as well as to Bm86, a well characterized antigen from the tick gut. Several factors explain how cattle could produce antibody to such antigens, which one would expect to be 'concealed' from the host's immune system, during natural infestation. It has been shown that the carbohydrate determinants on many tick glycoproteins are cross-reactive immunologically and that the reaction of bovine antibodies with intrinsic membrane glycoprotein is at least partially blocked by low molecular weight carbohydrate. Further, antisera from cattle exposed to ticks react with a glycosylated, native Bm86 but not with a non-glycosylated, recombinant Bm86. Thus the reaction of concealed antigens with antibodies produced as a result of tick infestation appears to be due to a relatively non-specific reaction with carbohydrate determinants on tick glycoprotein. Evidence is also presented that antibodies directed against carbohydrate determinants of Bm86 are not protective. Care must therefore be exercised in interpreting the results of antibody reaction with glycoproteins in such complex organisms.     

Detection of Bm86 antigen in different strains of Boophilus microplus and effectiveness of immunization with recombinant Bm86

The control of tick populations by using conventional strategies poses several problems, including the appearance of organophosphate resistant strains, among others. The possibility of using alternative strategies such as vaccination with tick antigens has been suggested by several authors. One particular antigen (Bm86) has been described and shown to be able to induce a protective immunity against the cattle tick Boophilus microplus. In this paper we demonstrate by means of immunohistochemical staining that this antigen is conserved among several strains of this species. These results correlate with those showing that animals vaccinated with a preparation of recombinant Bm86 were protected against challenge with the four different strains tested, including one resistant to organophosphates. These results favour the immunization with recombinant Bm86 for the control of the cattle tick B. microplus.     

Larval membrane antigens protect Hereford cattle against infestation with Boophilus microplus

Hereford cattle (Bos taurus) were immunized with antigens solubilized with Triton X-100 from larval membranes of the cattle tick (Boophilus microplus). Based on tick egg production compared to control cattle, vaccinated cattle were protected (78%) against challenge with 2 x 20,000 tick larvae. The soluble Triton X-100 extract of tick larval membranes was further purified by immunoaffinity chromatography, using immunoglobulin ligands (IgG1 and IgG2) from three immune steers, previously vaccinated with membrane antigens from the midgut of partly engorged adult female ticks. Cattle vaccinated with these purified antigens were protected in two separate experiments (80 and 89% respectively), against challenge with 2 x 20,000 larval ticks compared to control cattle. Whole larval membranes used as vaccines in cattle reduced the amount of eggs produced from ticks by 47% compared to control cattle, but this difference was not significant.     

Vaccination of cattle against the Boophilus microplus using a mucin-like membrane glycoprotein

An antigen, BMA7, which induced partial immunity against tick infestation has been isolated from Boophilus microplus using two different protein fractionation protocols, accompanied by vaccination and parasite challenge trials. The antigen is a 63 kDa glycoprotein isolated from semi-engorged adult female ticks. Though significant, the induced immunity is less striking than that previously reported for antigen Bm86 from the same parasite. However, co-vaccination with Bm86 and BMA7 can enhance immunity over that seen with a commercial vaccine based on Bm86 alone. Limited peptide sequence information shows significant variation in the BMA7 protein occurs. The antigen has approximately 36 kDa of glycosylation, in both N-linked and O-linked oligosaccharides. There is evidence that both polypeptide and oligosaccharide are antigenic, but the chemical nature of the protective antigenic sites is not clear. There is little or no immunological response to the antigen during natural infestation with parasites, suggesting the antigen is 'concealed' and protective immunity dependent on artificial vaccination. The antigen has some similarities with the vertebrate mucins. It is widely distributed in tick tissues and membrane bound but its function is currently unknown .     

Immune responses of cattle to biochemically modified antigens from the midgut of the cattle tick, Boophilus microplus

Treatment of membrane antigens of the midgut (GM) of the cattle tick, Boophilus microplus with sodium metaperiodate (periodate), pronase and lipase significantly inhibited the reactivity of the GM with antibodies in the sera of 57 cattle vaccinated with GM. Treatment of GM with periodate only removed the correlation between antibody reactivity of sera and protection against infestation with ticks. A monoclonal antibody (MoAb QU13), which recognises protective antigens solubilized from GM (Lee + Opdebeeck 1991), did not react with GM treated with periodate. Cattle vaccinated with GM extracts were significantly protected against infestation with cattle ticks (P less than 0.05), whereas cattle vaccinated with either GM extracts treated with periodate or with antigens precipitated from GM extracts with MoAb QU13 and also treated with periodate, were not protected against infestation. These studies provide preliminary evidence that protective antigens in the tick midgut membrane either are carbohydrate or are dependent on carbohydrate for their specificity.     

Humoral immune responses of Hereford cattle vaccinated with midgut antigens of the cattle tick, Boophilus microplus

Vaccination of cattle with midgut membrane (GM) antigen derived from the cattle tick, Boophilus microplus, infected with the adjuvant Quil A, resulted in significant increases in total immunoglobulins, mainly in the IgG1 and IgG2 fractions of the serum. Analysis of the anti-GM antibody levels of vaccinated cattle showed that the levels of IgG, IgG1 and complement-fixing antibodies were significantly correlated to protection against infestation with cattle ticks. Anti-GM antibodies of the IgG2 and IgM isotype were not correlated to protection against infestation with cattle ticks. Anti-GM antibodies fixed complement (C') in the presence of GM, larval membrane antigen and live, midgut cells, but not in the presence of live, larval cells. Anti-GM antibodies were able to fix C' equally well in the presence of GM antigen and live, midgut cells. None of the antigens tested activated the alternate pathway of complement under the conditions tested. Levels of anti-GM IgG1 antibodies were used to develop a regression model for predicting levels of protection against infestation with cattle ticks in vaccinated cattle.     

Arthropod vaccines

Antigens located in the midgut of the tick are hidden from the host's immune system. Egg production of ticks can be reduced when ticks are fed on animals vaccinated with midgut antigens of the tick, and a subunit vaccine formulated with the recombinant antigen Bm86 is now available that can reduce the number of ticks infesting cattle grazing on pasture. Midgut antigens used in vaccines against insects that transmit pathogenic organisms to humans have not been as effective in reducing insect fecundity and an alternative approach may be necessary. Transmission-blocking vaccines directed at interfering with the vector-pathogen interaction could result in loss of vector competence and block the spread of disease-causing organisms.     

Cloning and expression of a protective antigen from the cattle tick Boophilus microplus.

Glycoproteins located on the luminal surface of the plasma membrane of tick gut epithelial cells, when used to vaccinate cattle, are capable of stimulating an immune response that protects cattle against subsequent tick infestation. One such tick gut glycoprotein, designated Bm86, has been purified to homogeneity and the amino acid sequences of peptide fragments generated by endoproteinase Lys-C digestion have been determined. We report here the isolation and characterization of a cDNA that encodes Bm86. The nucleotide sequence of the cDNA contains a 1982-base-pair open reading frame and predicts that Bm86 contains 650 amino acids including a 19-amino acid signal sequence and a 23-amino acid hydrophobic region adjacent to the carboxyl terminus. The main feature of the deduced protein sequence is the repeated pattern of 6 cysteine residues, suggesting the presence of several epidermal growth factor-like domains. A fusion protein consisting of 599 amino acids of Bm86 and 651 amino acids of beta-galactosidase was expressed in Escherichia coli as inclusion bodies. Ticks engorging on cattle vaccinated with these inclusion bodies were significantly damaged as a result of the immune response against the cloned antigen.     

Evasin‐3‐like anti‐chemokine activity in salivary gland extracts of ixodid ticks during blood‐feeding: a new target for tick control

Ticks exploit many evasion mechanisms to circumvent the immune control of their hosts including subversion of the communication language between cells of the immune system provided by chemokines and other cytokines. One subversive molecule secreted in the saliva of Rhipicephalus sanguineus is Evasin‐3, a structurally unique 7 kDa protein that selectively binds the neutrophil chemoattractants, CXCL8 and (with lower affinity) CXCL1. We compared anti‐human CXCL8 and anti‐mouse CXCL1/KC activities in salivary gland extracts prepared from adult Amblyomma variegatum, Rhipicephalus appendiculatus and Dermacentor reticulatus ticks during blood‐feeding. Both anti‐CXCL8 activity and anti‐CXCL1 activity were detected in all species and in both adult females and males, with consistently higher activity levels against CXCL8. These results suggest that Evasin‐3‐like activity is common amongst metastriate ixodid tick species, and provide further evidence of the importance to ticks in controlling neutrophils during blood‐feeding. As such, Evasin‐3 offers a new target for anti‐tick vaccine development.     

微小牛蜱Bm86基因的原核表达与表达条件的优化

根据微小牛蜱Bm86基因序列设计引物,在重组质粒pMD18-T-Bm86中克隆,并将其定向亚克隆入原核表达载体pGEX-4T-1,转化至大肠埃希菌BL21（DE3）株,用不同浓度异丙基-β-D-硫代半乳糖苷（IPTG）在不同时间进行诱导表达。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）检测结果表明,37 ℃条件下经1 mmol/L IPTG诱导8 h后,目的重组蛋白表达量最大,表达相对分子质量（Mr）约为94 000的包涵体蛋白,与预期大小一致,目的蛋白约占蛋白总量的29%。蛋白质印迹（Western blotting）分析表明,该重组蛋白可被兔抗微小牛蜱全蜱阳性血清所识别。     

Exposed and concealed antigens as vaccine targets for controlling ticks and tick-borne diseases

Tick vaccines derived from Bm86, a midgut membrane-bound protein of the cattle tick, Boophilus microplus, are currently the only commercially available ectoparasite vaccines. Despite its introduction to the market in 1994, and the recognized need for alternatives to chemical pesticides, progress in developing effective antitick vaccines (and ectoparasite vaccines in general) is slow. The primary rate-limiting step is the identification of suitable antigenic targets for vaccine development. Two sources of candidate vaccine antigens have been identified: 'exposed' antigens that are secreted in tick saliva during attachment and feeding on a host and 'concealed' antigens that are normally hidden from the host. Recently, a third group of antigens has been distinguished that combines the properties of both exposed and concealed antigens. This latter group offers the prospect of a broad-spectrum vaccine effective against both adults and immature stages of a wide variety of tick species. It also shows transmission-blocking and protective activity against a tick-borne pathogen. With the proliferation of molecular techniques and their application to vaccine development, there are high hopes for new and effective antitick vaccines that also control tick-borne diseases.     

Down‐selecting circumsporozoite protein‐based malaria vaccine: A comparison of malaria sporozoite challenge model

Plasmodium falciparum circumsporozoite protein (PfCSP) is the main target antigen in development of pre‐erythrocytic malaria vaccines. To evaluate PfCSP vaccines in animal models, challenge by intravenous sporozoite injection is preferentially used. However, in clinical trials, vaccinated human volunteers are exposed to the bites of malaria‐infected mosquitoes. In this study, we down‐selected Escherichia coli‐produced full‐length PfCSP (PfCSP‐F) and its three truncated PfCSPs based on their abilities to elicit immune response and protection in mice against two challenge models. We showed that immunization with three doses of PfCSP‐F elicited high anti‐PfCSP antibody titres and 100% protection against the bites of infected mosquitoes. Meanwhile, three‐dose truncated PfCSP induced 60%‐70% protection after immunization with each truncated PfCSP. Heterologous prime‐boost immunization regimen with adenovirus‐PfCSP‐F and R32LR greatly induced complete protection against intravenous sporozoite injection. Our results suggest that Abs to both anti‐repeat and anti‐nonrepeat regions induced by PfCSP‐F are required to confer complete protection against challenge by the bites of infected mosquitoes, whereas anti‐repeat Abs play an important role in protection against intravenous sporozoite injection. Our findings provide a potential clinical application that PfCSP‐F vaccine induces potent Abs capable of neutralizing sporozoites in the dermis inoculated by infected mosquitoes and subsequently sporozoites in the blood circulation.     

Preclinical efficacy and immunogenicity assessment to show that a chimeric Plasmodium falciparum UB05‐09 antigen could be a malaria vaccine candidate

Although it is generally agreed that an effective vaccine would greatly accelerate the control of malaria, the lone registered malaria vaccine Mosquirix? has an efficacy of 30%‐60% that wanes rapidly, indicating a need for improved second‐generation malaria vaccines. Previous studies suggested that immune responses to a chimeric Plasmodium falciparum antigen UB05‐09 are associated with immune protection against malaria. Herein, the preclinical efficacy and immunogenicity of UB05‐09 are tested. Growth inhibition assay was employed to measure the effect of anti‐UB05‐09 antibodies on P. falciparum growth in vitro. BALB/c mice were immunized with UB05‐09 and challenged with the lethal Plasmodium yoelii 17XL infection. ELISA was used to measure antigen‐specific antibody production. ELISPOT assays were employed to measure interferon‐gamma production ex vivo after stimulation with chimeric UB05‐09 and its constituent antigens. Purified immunoglobulins raised in rabbits against UB05‐09 significantly inhibited P. falciparum growth in vitro compared to that of its respective constituent antigens. A combination of antibodies to UB05‐09 and the apical membrane antigen (AMA1) completely inhibited P. falciparum growth in culture. Immunization of BALB/c mice with recombinant UB05‐09 blocked parasitaemia and protected them against lethal P. yoelii 17XL challenge infection. These data suggest that UB05‐09 is a malaria vaccine candidate that could be developed further and used in conjunction with AMA1 to create a potent malaria vaccine.     

广西地区家畜牛、羊体表硬蜱情况调查及种类鉴定

目的 掌握广西地区牛羊场硬蜱种类及其分子特征,了解该地区蜱类的分类地位,为养殖户防控硬蜱及蜱媒传染病提供参考依据。方法 本实验于2019年1月至2021年11月,采用动物体表法采集寄生的蜱类;提取蜱基因组,PCR扩增3种硬蜱的线粒体16S rDNA和COI基因片段,并进行同源性分析。基于邻接法,用MEGA6.0软件分别构建系统发生树,进行遗传进化分析。结果 牛羊体表上共采集蜱2 030只,隶属1科2属3种,其中微小扇头蜱1 968只,长角血蜱49只,具角血蜱13只。PCR扩增微小扇头蜱、长角血蜱和具角血蜱16S rDNA和COI基因片段长度分别是460 bp和710 bp左右,分别与GenBank中已登录的相应种类相似较高且在一个进化分支上。结论 广西地区牛羊场优势蜱种是微小扇头蜱且存在长角血蜱和具角血蜱。三蜱种16S rDNA和COI序列存在多样性和地域差异性。     

Comparative efficacy of rHaa86 and rBm86 against Hyalomma anatolicum anatolicum and Rhipicephalus (Boophilus) microplus

Hyalomma anatolicum anatolicum and Rhipicephalus (Boophilus) microplus are the most economically important tick species in India and other tropical and subtropical regions of the world and transmit pathogens causing animal and human diseases. We demonstrated that vaccination of animal by rHaa86 could be used for the control of both H. a. anatolicum and R. (B.) microplus infestations. By comparing the efficacy of rHaa86 and rBm86, it was observed that vaccine based on rHaa86 will be more effective in controlling homologous challenge infestations (68·7% against larvae and 45·8% against adults). The results of this trial demonstrated that species-specific antigens are the better choice for vaccine development and could serve as an effective tool for the integrated control of H. a. anatolicum.     

Attempts to immunize sheep against Haemonchus contortus using a cocktail of recombinant proteases derived from the protective antigen,H‐gal‐GP

The objective of this study was to determine whether an antigen cocktail containing recombinantly expressed versions of most of the protective proteases of H‐gal‐GP, a known protective antigen from Haemonchus contortus, would confer any protection to lambs in a vaccine‐challenge trial. Haemonchus contortus metalloendopeptidases, MEP1, MEP3 and MEP4, were expressed as soluble recombinant proteins in insect cells, but attempts to express the H. contortus aspartyl proteases, PEP1 and PEP2, by the same techniques were not successful. Recombinant H. contortus PEP1 was therefore expressed in Escherichia coli and refolded. Groups of sheep were immunized thrice with either native H‐gal‐GP, a cocktail of recombinantly expressed proteins (rMEP1, rMEP3, rMEP4 and rPep1), or adjuvant only (QuilA in PBS). All sheep were challenged with 5000 infective larvae 1 week after the final vaccination. High levels of serum antibodies that recognized H‐gal‐GP were detected in both the native antigen and recombinant cocktail‐immunized groups by the time of challenge, but protective immunity was only observed in the group immunized with native H‐gal‐GP.     

Antigenic differences between the EG95‐related proteins from Echinococcus granulosus G1 and G6 genotypes: implications for vaccination

Cystic echinococcosis caused by Echinococcus granulosus remains an important and neglected issue in public health. The study of the likely efficacy of the currently available EG95 vaccine against other genotypes of the parasite is important to improve the vaccine as a potential tool to be used in control programmes. The recombinant vaccine EG95‐1G1 was developed based on the G1 genotype of E. granulosus. Characterization of the eg95 gene family in the G6 genotype by genomic DNA cloning previously produced the first unequivocal information about the composition of the gene family in a different genotype. The information was used in this study to predict and express two EG95‐related proteins from the G6 genotype as recombinants, for assessment of their capacity to bind antibodies raised in sheep vaccinated with the EG95‐1G1 vaccine. The proteins (EG95‐1G6 and EG95‐5G6) from the G6 genotype of E. granulosus were unable to bind all the antibodies raised by sheep vaccinated with EG95‐1G1. Differences in the amino acid sequence of EG95‐related proteins from G6 and likely the differences in the encoded FnIII domain may be responsible for changes in the conformation of these epitopes.     

Survey of tick species parasiting domestic ruminants in Ghaemshahr county,Mazandaran province,Iran

ObjectiveTo determine the tick species parasitizing domestic ruminants in Ghaemshahr county in Mazandaran, a Caspian province in the north of Iran.MethodsAbout 361 sheep, 54 goats and 10 cattle of 18 herds in several villages in Ghaemshahr were inspected for tick infestation. Separated ticks were preserved in 70% alcohol and identified.ResultsAbout 323 ticks (207 female, 116 male) were collected, the occurrence of ticks on sheep, goats and cattle were 28.3%, 22.2% and 20.0% respectively. The mean number of ticks on each animal was low (3-5 ticks per animal). Rhipicephalus sanguineus, Rhipicephalus bursa, Ixodes ricinus, Boophilus annulatus, Haemaphysalis punctata and Haemaphysalis numidiana were the tick species we found. Rhipicephalus sanguineus were the most abundant species in the study area. The largest number of ticks were generally present from April to July, mostly in animal ears and tails. Ixodes, Boophilus and Haemaphysalis occurred in mountainous areas of Ghaemshahr, whereas Rhipicephalus were present in both mountains and plains of the study area.ConclusionsThe result of this study is a survey of tick species from domestic animals in Iran and implication of possible prevention measures for diseases transmitted by ticks.