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.     

Experimental vaccination of sheep and cattle against tick infestation using recombinant 5′‐nucleotidase

Limited prior evidence suggests that 5′‐nucleotidase, an ectoenzyme principally located in the Malpighian tubules of the tick Rhipicephalus (Boophilus) microplus, could be an effective antigen in an anti‐tick vaccine. To assess this, recombinant 5′‐nucleotidase was expressed in Escherichia coli and used in vaccination trials with both sheep and cattle. Vaccinated sheep were challenged with freshly moulted adult ticks. Those with high titres of anti‐nucleotidase antibodies showed significant protection against tick infestation, although protection was less than that found with the previously characterized antigen, Bm86. Cattle were vaccinated, in separate groups, with 5′‐nucleotidase, Bm86 and both antigens combined. Cattle, as the natural host, were challenged with larval ticks. Although Bm86 showed typical efficacy, no significant protection was seen in cattle vaccinated with 5′‐nucleotidase. Cattle receiving a dual antigen formulation were no better protected than those receiving Bm86 alone. One possible reason for the difference between host species, namely antibody titre, was examined and shown to be an unlikely explanation. This demonstrates a limitation of using a model host like sheep in vaccine studies.     

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.     

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。     

Cloning, expression and immunoprotective efficacy of rHaa86, the homologue of the Bm86 tick vaccine antigen, from Hyalomma anatolicum anatolicum

The Bm86 homologue of Hyalomma anatolicum anatolicum Izatnagar isolate was cloned and expressed in methylotropic yeast Pichia pastoris as intracellular, glycosylated and particulated form. It was named as rHaa86, the first recombinant protein of H. a. anatolicum . Seven epidermal growth factor-like domains predicted in Haa86 were structurally similar with that of its Bm86 counterpart. The identity between the corresponding EGF like domains of Bm86 and Haa86 were ranging from 51·3% to 78·3%. The molecular weight of the rHaa86 was 120–140 kDa, with possible 50–70 kDa glycosylation. The purified rHaa86 was characterized immunologically and evaluated for its immunoprotective potential against homologous challenge infestation in three groups of cross-bred calves. The immediate rejection percentage of females of H. a. anatolicum was 36 5%, 12·4% and 10·1% fed on immunized (group 1), adjuvant control (group 2) and untreated control (group 3) calves, respectively. The percent rejection of female ticks fed on immunized calves was 24·1% and 26·4% higher than for the ticks fed on control groups 2 and 3, respectively ( P <  0·05). The reduction of number of females, mean weight of eggs, adult females and efficacy of immunogen were 58·0%, 9·0%, 5·0% and 61·6%, respectively. The mean reproductive index of females fed on group 1 calves was significantly lower ( P <  0·05) than the females fed on the control groups and 44% reduction in the number of engorged larvae was recorded from the group 1 calves. The data demonstrated that rHaa86 antigen based vaccine could serve as one of the effective components in the integrated control of H. a. anatolicum.     

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 .     

Immunologic relations between cattle and ticks, specifically between cattle and Boophilus microplus]

In the present investigation, it has been demonstrated that cattle become resistant to ticks after several heavy infestations, particularly with B. microplus. During development of the infestations, antibodies against salivary glands of B. microplus were detected using 2 techniques: indirect immunofluorescence and immunoelectrophoresis. There is a positive causal relationship between antibody titer and resistance development. Two precipitating systems against B. microplus in infested cattle and 7 systems in immunized rabbits were studied. The systems 1 and 2 are similar in cattle and rabbits, but system 2 does not show any specificity, as it has been detected in cattle completely lacking tick infestations. Two one-day calves were treated with the antigen of B. microplus by injection of salivary glands and repeated infestations with a small number of larvae. They developed a pronounced resistance to the usual subsequent infestations by the ticks of the same species. Specific antibodies were found before the first usual infestation. This suggests that they might be responsible for resistance.     

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.     

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.     

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.     

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.     

Peripheral cellular and humoral responses to infestation with the cattle tick Rhipicephalus microplus in Santa Gertrudis cattle

Resistance to cattle tick infestation in single‐host ticks is primarily manifested against the larval stage and results in the immature tick failing to attach successfully and obtain a meal. This study was conducted to identify immune responses that characterize the tick‐resistant phenotype in cattle. Thirty‐five tick‐naïve Santa Gertrudis heifers were used in this study, thirty of which were artificially infested for thirteen weeks with tick larvae while five animals remained at a tick‐free quarantine property to serve as a control group. Following thirteen weeks of tick infestation, the animals in this trial exhibited highly divergent tick‐resistant phenotypes. Blood samples collected throughout the trial were used to measure peripheral immune parameters: haematology, the percentage of cellular subsets comprising the peripheral blood mononuclear cell (PBMC) population, tick‐specific IgG₁ and IgG₂ antibody titres, IgG1 avidity for tick antigens and the ability of PBMC to recognize and proliferate in response to stimulation with tick antigens in vitro. The tick‐susceptible cattle developed significantly higher tick‐specific IgG₁ antibody titres compared to the tick‐resistant animals. These results suggest that the heightened antibody response either does not play a role in resistance or might contribute to increased susceptibility to infestation.     

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.     

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.     

Structure prediction against Bm 17DIII gene product of Brugia malayi

IntroductionBrugia malayi is one of the causative agents of lymphatic filariasis. Lymphatic filariasis has threatened around 1.3 billion people in 81 countries and infected approximately 120 million people worldwide in developing and under-developed countries. This disease has been identified as the second leading cause of permanent and long term disability in year 1995. Thus WHO has targeted filariasis for elimination by year 2020. A recombinant protein BmR1 expressed from Bm17DIII gene, a highly specific and sensitive antigen for the detection of B. malayi was used as filariasis diagnosis in BRUGIArapid^TM. However, the structure of BmR1 protein is yet to be elucidated.ObjectiveTo predict the structure of Bm17DIII gene product of B. malayi.MethodsThe protein structure was predicted via in silico approaches namely comparative modeling, ab initio and threading methods. The predicted structures were evaluated for its backbone conformation, compatibility and overall quality factor.Results & DiscussionWe compared the structures modeled by two comparative modeling methods, four ab initio methods and one threading/ab initio method. Structure obtained from ab initio approach (Rosetta) showed the best scoring for the backbone conformation of 98.5% in Ramachandran plot, 97.0% for the compatibility of the model (3D) with the sequence (1D) and overall quality of 71.0%.ConclusionThe structure obtained from this study could lead to the development of antigen-based filariasis diagnostic to complement BRUGIArapid^TM when a binder specific against BmR1 is designed.     

Immunogenetic influences on tick resistance in African cattle with particular reference to trypanotolerant N'Dama (Bos taurus) and trypanosusceptible Gobra zebu (Bos indicus) cattle

In sub-Saharan Africa, tick infestation and tick-borne infections together with tsetse-transmitted trypanosomosis arguably constitute the main parasitological disease complex constraining livestock production. Resistance to tick attack and tick-borne micro-organisms (TBMs) varies among different breeds of cattle. The magnitude of losses due to these parasites is related to an extent to the degree of breed resistance. Generally, zebu (Bos indicus) cattle possess a higher resistance to ticks and TBMs than European (Bos taurus) cattle. The host's immune system would appear to be the single most important factor that regulates this resistance. This paper reports on the main effector immune mechanisms governing resistance against ticks and TBMs. The cellular immune response appears more effective and stable than humoral immunity in modulating resistance to ticks and TBMs. Similarities between the immune mechanisms employed by trypanotolerant N'Dama (B. taurus) cattle, when infected with trypanosomes, and those elicited by tick bites and TBMs seem to exist, particularly at the skin level in the early phases of parasitic invasion. Moreover, there is evidence that in the N'Dama breed, resistance against ticks per se also has a genetic basis. Therefore, the N'Dama appears to be a unique breed in that it exhibits resistance to several parasitic diseases and/or infections, including helminths, when compared to other cattle breeds in West Africa. It is concluded that the multi-parasite resistant traits of the N'Dama breed should be exploited in those areas where trypanosomosis, ticks and tick-borne diseases constrain animal production. This should be of benefit for low-input farming systems where the use of chemicals for prophylaxis and therapy is limited by their relatively high cost. Additionally, the potential contribution of multiple disease resistant N'Dama cattle should be considered in crossbreeding programmes with exotic dairy breeds for increasing milk production in West Africa.     

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。     

云南临沧地区蜱种分子生物学鉴定及埃立克体基因序列分析

目的 了解云南临沧地区蜱虫种类及其自然感染埃立克体的情况。方法 采集耕牛体表寄生的蜱虫,经形态学鉴定和分组后,从蜱虫中提取DNA,应用PCR扩增蜱虫COI基因以及埃立克体16S rRNA和groEL基因,并测序分析。结果 共采集到蜱虫42只,其中微小牛蜱38只占90.48%、血蜱4只占9.52%。将每种蜱的2只为1组,在21组样本中,检出COI片断21份(检出率100%);在相同的4组样本中均检出埃立克体16S rRNA片断和groEL片断4份(检出率19.05%)。基因序列分析,检出的COI片断序列中有2份与澳大利亚pava血蜱(JX573136)的同源性最高(89.3%),其余19份序列与马来西亚微小牛蜱B3的同源性最高(99.5%);检出的4份16S rRNA片断序列完全一致,与美国查菲埃立克体Arkansas和埃文埃立克体Aa2FT349及中国北京查菲埃立克体BY-YQ-HME-O18株的同源性均为100%;检出的4份groEL片断序列也完全一致,与日本埃立克体Yonaguni206 株的同源性最高均为93.9%。结论 经分子检测证实云南临沧地区耕牛体表微小牛蜱中存在一种类似日本Yonaguni206株的埃立克体感染,耕牛体表还可能寄生一种新的血蜱。     

Local immune response to larvae of Rhipicephalus microplus in Santa Gertrudis cattle

This study investigated the local immune response at larval attachment sites in Santa Gertrudis cattle with low and high levels of tick resistance. Skin samples with tick larvae attached were collected from Santa Gertrudis cattle at the end of a period of 25 weekly infestations, when the animals manifested highly divergent tick‐resistant phenotypes. There was a tendency for more CD3⁺, CD4⁺, CD8⁺, CD25⁺, γδ T cells and neutrophils to concentrate at larval tick attachment site in susceptible cattle than in resistant cattle but the differences were significant only for γδ T cells and CD4⁺ cells. Most of the cattle developed intra‐epidermal vesicles at the larval attachment site but the predominant cell within or around the vesicles was the neutrophil in susceptible animals and eosinophil in the resistant animals. The monoclonal antibodies (mAbs) specific for CD45 and CD45 RO antigens reacted with skin leucocytes from a higher number of susceptible cattle than resistant cattle. Our data suggest that some of the cellular responses mounted at larval attachment site are not involved in tick protection. The mAbs specific for CD45 and CD45 RO directly, or a test for CD45 genotype might be developed as markers of tick susceptibility or resistance.