Molecular approaches to elucidating innate and acquired immune responses to Babesia bovis, a protozoan parasite that causes persistent infection.

For many vector-transmitted protozoal parasites, immunological control of acute infection leads to a state of persistent infection during which parasitemias may cycle unnoticed in infected but otherwise clinically healthy animals. Achieving persistent infection is a strategy that favors parasitism, since both host and, therefore, parasite survive, and endemically infected animal populations provide a reservoir of parasites continually available for subsequent transmission. Examples of the major economically important protozoan pathogens that cause persistent infection in mammals include the related Theileria and Babesia parasites as well as Trypanosoma species. Control of acute infection and maintenance of clinical immunity against subsequent infection are determined by the interplay of innate and acquired immune responses. This review will focus on approaches taken to gain an understanding of the molecular basis for innate and acquired immunity against the hemoprotozoan parasite of cattle, Babesia bovis. Knowledge of mechanisms used by the parasite to survive within infected cattle from acute to persistent infection combined with definition of the correlates of protective immunity in cattle should be applicable to designing effective vaccines.     

MHC class II-restricted, CD4(+) T-cell proliferative responses of peripheral blood mononuclear cells from Mycobacterium bovis-infected white-tailed deer

White-tailed deer are significant wildlife reservoirs of Mycobacterium bovis for cattle, predators, and, potentially, humans. Infection of cattle with M. bovis stimulates an antigen-specific T-cell response, with both CD4(+) and CD8(+) cells implicated in protective immunity. Few studies, however, have examined lymphocyte subset responses to experimental M. bovis infection of white-tailed deer. In this study, a flow cytometric proliferation assay was used to determine the relative contribution of individual peripheral blood mononuclear cell subsets of M. bovis-infected white-tailed deer in the recall response to M. bovis antigen. Naive deer were challenged with M. bovis by cohabitation with infected deer. These M. bovis-challenged deer developed significant in vivo (delayed-type hypersensitivity) and in vitro (proliferative) responses to M. bovis purified protein derivative (PPD). At necropsy, typical tuberculous lesions containing M. bovis were detected within lungs and lung-associated lymph nodes of infected deer. The predominant subset of lymphocytes that proliferated in response to in vitro stimulation with PPD was the CD4(+) subset. Minimal proliferative responses were detected from CD8(+), gamma delta TCR(+), and B-cells. Addition of monoclonal antibodies specific for MHC II antigens, but not MHC I or CD1 antigens, abrogated the proliferative response. Together, these findings indicate that while CD4(+) cells from infected deer proliferate in the recall response to M. bovis antigens, this response is not sufficient to clear M. bovis and immunologic intervention may require stimulation of alternate subsets of lymphocytes.     

Orientation of bovine CTL responses towards PIM, an antibody-inducing surface molecule of Theileria parva, by DNA subunit immunization

East Coast fever, an acute lymphoproliferative disease of cattle, is caused by the apicomplexan parasite Theileria parva. Protective immunity is mediated by CD8(+) cytotoxic T lymphocytes directed against schizont-infected cells. The polymorphic immunodominant molecule, although an antibody-inducing surface molecule of the schizont, has been hypothesized to play a role in protective immunity. In order to evaluate the immunogenicity of PIM for inducing CTL, cattle were immunized with PIM in isolation from other T. parva antigens, forcing the presentation of PIM-derived epitopes on the MHC class I molecules. Although parasite-specific cytotoxicity was induced in both vaccinated animals, their immune response was clearly different. One animal generated MHC-restricted parasite-specific CTL against PIM while the other calf exhibited a strong PIM-specific proliferative response but non-MHC-restricted parasite-specific cytotoxicity. Only calf 1 survived a lethal sporozoite challenge. This DNA immunization technique with an antigen in isolation of CTL-immunodominant antigens might open possibilities for directing CTL responses against predefined antigens, such as strain cross-reacting CTL antigens.     

Antigen presentation in vaccine development

A variety of microorganisms, nutrients or toxins are generally intrude our body through mucosal tissues or skin, where equipment for both preventing their invasions and catching their information to activate internal immune systems for adapting surroundings is arranged. Among the equipment, cells in charge of innate immunity, particularly dendritic cells (DCs), having an excellent capacity for prompt recognition of invaded pathogens via toll-like receptors (TLRs) to alert B and T cells for establishing aquired/adaptive immunity by presenting their processed antigenic fragments, have been paid great attention. These TLR-activated, antigen captured DCs are divided into two groups; one is pathogen-retaining unit and the other is pathogen-controlling unit. The latter DCs present processed antigenic molecules from the pathogens to competent β T cells together with special containers, such as class I, class II MHC and CD1 to generate specific cellular immunity. The former two MHC molecules can present processed peptide antigens, whereas the last CD1 molecule can present glycolipid/lipid antigens. In contrast, B lymphocytes that captured antigens via their specific immunoglobulin (Ig) receptors present digested peptide fragments with their class II MHC to stimulate suitable CD4⁺ helper T cells which in turn secrete various cytokines to efficiently expand and maintain antibody production from that partner B cells to establish humoral immunity. These β T cells and antibodies, recognize either processed antigenic peptide or glycolipid fragments, and thus, identification of these epitopes enables us to generate artificial pathogen-specific vaccines. Based on the recent findings about precise mechanisms of antigen processing and presentation orchestrated at the surface compartment, future development of vaccines against various pathogens are discussed.     

Characterization of the immune response to Mycoplasma bovis lung infection

To better understand the interaction between Mycoplasma bovis and its bovine host, we have characterized the immune response generated during an experimental lung infection with M. bovis. Proliferation ([3H]-thymidine blastogenesis) and Th1/Th2 cytokine production were used to monitor peripheral cellular immune responses. Flow cytometry analysis was used to determine T-cell subset activity by CD25 expression. Humoral immune response was monitored by the identification of antigen-specific IgG1 and IgG2 isotypes over time. Herein, we show that M. bovis antigen stimulates activation of CD4+ and CD8+ cells in vitro in a manner consistent with memory, and that gammadelta-T cells are activated by antigen in a manner consistent with innate immunity. In addition, the percentage of cells producing IFN-gamma during recall response is equal to that of IL-4 producing cells. Serological analysis shows M. bovis stimulates increased production of antigen-specific IgG1 while very little IgG2 is produced. We therefore submit that experimental lung infection of cattle with M. bovis results in a Th2-skewed immune response.     

牛支原体病研究进展

牛支原体是一种非常重要但容易被忽视的病原,能导致牛肺炎、乳腺炎、关节炎等多种疾病。国外已证实,该病给养牛业造成巨大的经济损失。目前,对牛支原体的毒力因子及免疫机理均不十分清楚,缺少其相应的有效疫苗和检测方法,而且,牛支原体对许多常用抗生素不敏感,从而,给临床防治带来很大困难。我国自2008年首次报道了牛支原体肺炎,但对牛支原体病危害的认识还很不全面,妨碍了对该病的有效防治。论文分析归纳了牛支原体病的国内外研究概况,从病原学、流行病学、临床症状与病理变化、诊断、毒力因子、免疫与疫苗、防治措施等方面进行了系统综述,以期为我国牛支原体病的防治提供参考。     

Babesia bovis--analysis and vaccination trial with the cryoprecipitable immune complex

Plasma samples from cattle recovering from acute Babesia bovis infection contain cryoprecipitable immune complexes (IC). Production of bovine and rabbit antisera to IC and subsequent serological assays indicated IC contained antigens of both babesial and erythrocytic origin. Vaccination of naive cattle with IC produced low titred antibody to B. bovis but the vaccinates did not survive challenge with a heterologous strain of B. bovis.     

Avian coccidiosis. A review of acquired intestinal immunity and vaccination strategies

The gut-associated lymphoid tissues contain B and T lymphocytes responsible for acquired immunity to avian coccidiosis. Intestinal B cells begin producing parasite-specific antibodies shortly after infection although their role in protecting against coccidiosis is debated. T-cell-mediated immunity, predominantly by intestinal intraepithelial lymphocytes and lamina propria lymphocytes, confers the main component of protective immunity to Eimeria. Many of these cells display the CD8 and gammadelta T-cell receptor surface antigens, phenotypic markers of cytotoxic T cells. Although their role in eliminating Eimeria infection remains to be completely elucidated, T cells have been implicated in parasite transport, and their activity is augmented by interferon-gamma and interleukin-2. Because of the importance of cell-mediated immunity, coccidiosis vaccines must be capable of stimulating intestinal T cells. Orally delivered, live parasite vaccines, either unattenuated or attenuated, are powerful stimulators of intestinal cell-mediated immunity, but antigenic variability between Eimeria species present in the vaccine and in the field may restrict their commercial application. The newer generations of recombinant DNA and subunit protein vaccines, particularly when used in conjunction with interferon-gamma and interleukin-2, have shown preliminary promise in controlling experimental infections but have yet to be commercially developed.     

Experimentally induced infectious bovine keratoconjunctivitis: resistance of vaccinated cattle to homologous and heterologous strains of Moraxella bovis.

In studies to determine whether vaccination with one strain of Moraxella bovis would protect against challenge with virulent homologous or heterologous strains, calves were intramuscularly inoculated 3 times with formalin-killed M bovis, with 14 days between inoculations. Fourteen days after the 3rd vaccinal dose was given, all calves were exposed to homologous or heterologous virulent cultures of M bovis. The results indicated that vaccination with one strain of M bovis may induce protective immunity against homologous and heterologous challenge exposure; however, because vaccinated cattle resisted infection and disease produced by a homologous strain to a greater extent than they resisted those produced by heterologous strains, polyvalent vaccines or highly immunogenic common antigens may be needed to protect cattle against the numerous strains they might encounter under natural field conditions. There was minimal correlation between the presence of precipitating antibodies against the heterologous strains and the establishment of infection and disease.     

Productivity and health effects of anaplasmosis and babesiosis on Bos indicus cattle and their crosses, and the effects of differing intensity of tick control in Australia

Tick fever is an important disease of cattle where Rhipicephalus (Boophilus) microplus acts as a vector for the three causal organisms Babesia bovis, Babesia bigemina and Anaplasma marginale. Bos indicus cattle and their crosses are more resistant to the clinical effects of infection with B. bovis and B. bigemina than are Bos taurus cattle. Resistance is not complete, however, and herds of B. indicus-cross cattle are still at risk of babesiosis in environments where exposure to B. bovis is light in most years but occasionally high. The susceptibility of B. indicus cattle and their crosses to infection with A. marginale is similar to that of B. taurus cattle. In herds of B. indicus cattle and their crosses the infection rate of Babesia spp. and A. marginale is lowered because fewer ticks are likely to attach per day due to reduced numbers of ticks in the field (long-term effect on population, arising from high host resistance) and because a smaller proportion of ticks that do develop to feed on infected cattle will in turn be infected (due to lower parasitaemia). As a consequence, herds of B. indicus cattle are less likely than herds of B. taurus cattle to have high levels of population immunity to babesiosis or anaplasmosis. The effects of acaricide application on the probability of clinical disease due to anaplasmosis and babesiosis are unpredictable and dependent on the prevalence of infection in ticks and in cattle at the time of application. Attempting to manipulate population immunity through the toleration of specific threshold numbers of ticks with the aim of controlling tick fever is not reliable and the justification for acaricide application should be for the control of ticks rather than for tick fever. Vaccination of B. indicus cattle and their crosses is advisable in all areas where ticks exist, although vaccination against B. bigemina is probably not essential in pure B. indicus animals.     

The biological and practical significance of antigenic variability in protective T cell responses against Theileria parva

The evolution of antigenically distinct pathogen strains that fail to cross-protect is well documented for pathogens controlled primarily by humoral immune responses. Unlike antibodies, which recognise native proteins, protective T cells can potentially recognise epitopes in a variety of proteins that are not necessarily displayed on the pathogen surface. Moreover, individual hosts of different MHC genotypes generally respond to different sets of epitopes. It is therefore less easy to envisage how strain restricted immunity can arise for pathogens controlled by T cell responses, particularly in antigenically complex parasites. Nevertheless, strain restricted immunity is clearly a feature of a number of parasitic infections, where immunity is known to be mediated by T cell responses. One such parasite is Theileria parva which induces potent CD8 T cell responses that play an important role in immunity. CD8 T cells specific for parasitized lymphoblasts exhibit strain specificity, which appears to correlate with the ability of parasite strains to cross-protect. Studies using recently identified T. parva antigens recognised by CD8 T cells have shown that the strain restricted nature of immunity is a consequence of the CD8 T cell response in individual animals being focused on a limited number of dominant polymorphic antigenic determinants. Responses in animals of different MHC genotypes are often directed to different parasite antigens, indicating that, at the host population level, a larger number of parasite proteins can serve as targets for the protective T cell response. Nevertheless, the finding that parasite strains show overlapping antigenic profiles, probably as a consequence of sexual recombination, suggests that induction of responses to an extended but limited set of antigens in individual animals may overcome the strain restricted nature of immunity.     

The use of binding-prediction models to identify M. bovis-specific antigenic peptides for screening assays in bovine tuberculosis

The identification of MHC class II-restricted antigenic peptides for inclusion into vaccines and/or as diagnostic test reagents for mycobacterial infections remains a high research priority. To expedite discovery of such peptides, numerous bioinformatic tools have been developed to predict whether a given peptide is likely to form a stable binding interaction with MHC class II molecules. However, no prediction tool dedicated to the identification of bovine MHC (BoLA) class II-restricted peptides is currently available. Using experimental immunogenicity data derived from the stimulation of whole blood of Mycobacterium bovis-infected cattle with 105 individual M. bovis-derived peptides, we have compared the ability of a novel BoLA DRB3 structure-based prediction method (Hepitom) with the human MHC class II binding predictor model ProPred in predicting peptides that induce bovine T-cell activation. When a stringent cut off for considering peptide antigenicity was applied, the sensitivities of Hepitom and ProPred in detecting immunogenic peptides were 62% and 77%, respectively. In contrast, the Hepitom model showed greater specificity, with values of 66% and 34% for Hepitom and ProPred, respectively. Using all peptides, seven out of eleven M. bovis proteins were identified as being highly immunogenic. All but one of these antigens were also identified when just the Hepitom predicted peptides were used, while only four of the seven were identified using the ProPred predicted peptides. In conclusion, we demonstrate that the Hepitom model is a useful pre-screening tool to select peptides for further immunogenicity studies in cattle without major impact on the identification of antigenic M. bovis proteins.     

Functional expression of a bovine major histocompatibility complex class I gene in transgenic mice

Major histocompatibility complex (MHC) class I restricted cellular immune responses play an important role in immunity to intracellular pathogens. By binding antigenic peptides and presenting them to T cells, class I molecules impose significant selection on the targets of immune responses. Candidate vaccine antigens for cellular immune responses should therefore be analysed in the context of MHC class I antigen presentation. Transgenic mice expressing human MHC (HLA) genes provide a useful model for the identification of potential cytotoxic T lymphocyte (CTL) antigens. To facilitate the analysis of candidate CTL vaccines in cattle, we have produced transgenic mice expressing a common bovine MHC (BoLA) class I allele.The functional BoLA-A11 gene, carried on a 7 kb genomic DNA fragment, was used to make transgenic mice by pronuclear microinjection. Three transgenic mouse lines carrying the BoLA-A11 gene were established. Expression of the BoLA-A11 gene was found in RNA and the A11 product could be detected on the surface of spleen and blood cells. Functional analysis of the A11 transgene product, and its ability to act as an antigen presenting molecules in the mouse host will be discussed.     

The immune response to bovine viral diarrhea virus: a constantly changing picture.

Bovine viral diarrhea virus (BVDV) is one of the major immuno-suppressive viruses of cattle. The effect on the innate and acquired immune system is unique and results in dramatic immune dysfunction. BVDV infection also has the ability to cause persistent infection (PI) in the developing fetus. This Pl syndrome creates a requirement for high levels of BVDV immunity from vaccines to prevent these infections. BVDV vaccines and their future development continue to be an enigma in the control of BVDV.     

New generation vaccines and delivery systems for control of bovine tuberculosis in cattle and wildlife

Advances in the understanding of protective immune responses to tuberculosis are providing opportunities for the rational development of improved vaccines for bovine tuberculosis. Protection requires activation of macrophages through stimulation of a Th 1 type immune response. Ideally, a vaccine for cattle should induce protection without causing animals to react in a tuberculin test when exposed to Mycobacterium bovis. A number of new tuberculosis vaccines including attenuated M. bovis strains, killed mycobacteria, protein and DNA vaccines have been developed and many of these are being assessed in cattle. The requirements for a tuberculosis vaccine for wildlife differ from those for cattle. The major goal of a wildlife vaccine is to prevent the transmission of M. bovis to cattle and other wildlife. Although there are a number of technical problems associated with the development of a vaccine delivery system for wildlife, attenuated M. bovis vaccines administered via oral baits or aerosol spray to possums have already been shown to reduce the severity of a subsequent M. bovis infection.     

Genetic conservation of potentially immunogenic proteins among Brazilian isolates of Babesia bovis

Bovine babesiosis caused by Babesia bovis remains an important constraint for the development of cattle industries worldwide. Effective control can be achieved by vaccination with live attenuated phenotypes of the parasite. However, these vaccines have a number of drawbacks, which justifies the search for better, safer vaccines. In recent years, a number of parasite proteins with immunogenic potential have been discovered. However, there is little information on the genetic conservation of these proteins among different parasite isolates, which hinders their assessment as immunogens. The aim of the present study was to evaluate the conservation of the genes ama-1, acs-1, rap-1, trap, p0 and msa2c among five Brazilian isolates of B. bovis. Through polymerase chain reaction, genetic sequencing and bioinformatics analysis of the genes, a high degree of conservation (98-100%) was found among Brazilian isolates of B. bovis and the T2Bo isolate. Thus, these genes are worth considering as viable candidates to be included in a recombinant cocktail vaccine for cattle babesiosis caused by B. bovis.     

Modulation of host immune responses by protozoal DNA

The pathology caused by acute Babesia bovis infection is similar to that seen in severe human malaria caused by Plasmodium falciparum infection, which is related to dysregulated production of inflammatory cytokines and nitric oxide (NO). We have observed induction of NO, inducible nitric oxide synthase (iNOS) and inflammatory cytokines in macrophages by B. bovis. Furthermore, proliferation of lymphocytes from individuals never exposed to certain protozoal pathogens can be induced by crude protozoal parasite extracts. We have repeatedly observed stimulation of naive PBMC from cattle to antigenic extracts of Babesia bovis. Based on recent studies demonstrating the mitogenicity of bacterial and other non-vertebrate DNAs for murine B cells and macrophages, the mitogenic properties of B. bovis DNA were examined. B. bovis and E. coli DNAs induced proliferation of PBMC and purified B cells from non-exposed cattle. Stimulatory activity was reduced by DNase treatment and methylation with CpG methylase, indicating the presence of stimulatory non-methylated CpG motifs in the B. bovis genome. B. bovis and E. coli DNAs enhanced IgG secretion by cultured B cells, stimulating IgG1 and more strongly, IgG2. Several hexameric CpG immunostimulatory sequences (ISS) active for murine B cells were identified in an 11 kb fragment of B. bovis DNA. An oligodeoxyribonucleotide containing one of these (AACGTT), located in the rhoptry associated protein-1 (rap-1) open reading frame, stimulated B cell proliferation. These studies identify a potential mechanism by which protozoal parasites may modulate host immune responses, leading to consequences such as hypergammaglobulinemia and splenomegaly. These results also support the use of ISS as vaccine adjuvants to enhance Type 1 immune responses in cattle.     

Prospects for molecular vaccines in veterinary parasitology

Despite the profound developments in recombinant DNA technology there is only one marketed recombinant vaccine (for human viral hepatitis B). The development of others proceeds with great difficulty. Molecular vaccines against veterinary parasites are at the utmost pole of complexity in the spectrum of potential vaccines since these parasites are complex eukaryotic organisms, often dwelling at mucosal surfaces where anamnestic responses are problematic, where the immunogenicity of the parasite components is poorly understood and where the effector mechanisms of immunity are unresolved. Cloning a "protective" gene is only the first step, and perhaps the easiest, in a long process which will be necessary to develop vaccines against parasites. Additional steps will involve comprehensive analyses of the immunological responses to ensure that vaccine antigens contain the correct epitopes to induce appropriate immune effector mechanisms for parasite elimination and immunological memory and that these responses are not genetically restricted. The great expectations for recombinant vaccinia-based vaccines must be modified substantially in the light of recent evidence indicating immunological and other constraints on this approach. The use of anti-idiotype vaccines is an underexplored opportunity for practical parasite vaccines since they have several potentially important advantages. The need to include T cell antigenic peptides in peptide vaccines to extend the range of genetic responsiveness and to induce anamnestic responses is now clear. New algorithms for the prediction of such sites exist and these can be tested experimentally with synthetic peptides. There are no major technical obstacles to the development of vaccines for parasites which cannot be overcome. However substantial long term basic research is needed over a range of disciplines to achieve this worthwhile objective.     

The WC1(+) gammadelta T-cell population in cattle: a possible role in resistance to intracellular infection

Intracellular infections are important in veterinary medicine and detailed understanding of the associated immune responses is needed for optimal development of strategies based on diagnosis and vaccination. It is generally accepted that cell-mediated immune responses are of greatest importance in intracellular infections and recent studies from several bovine models of infection indicate that WC1(+) gammadelta T-cells have a number of possible levels of involvement, which remain incompletely defined. Investigations of experimental infection with Mycobacterium bovis in cattle have indicated that WC1(+) gammadelta T-cells are among the first cells to accumulate at initial sites of infection, an observation which has been linked with decreased numbers of these cells in the circulation within days of infection. These WC1(+) gammadelta T-cells have been shown to respond in vitro, both to protein antigens and to non-protein, phosphate containing antigens of M. bovis and to be capable of producing IFN-gamma. Studies of M. bovis infection in calves depleted of WC1(+) gammadelta T-cells by monoclonal antibody have suggested that the presence of these cells is associated with development of a Th1-biased acquired immune response. In combination, these observations allow speculation regarding a possible role for WC1(+) gammadelta T-cells as a link between the innate and acquired immune systems which is instrumental in establishing an appropriate response.     

Emerging perspectives in the research of bovine babesiosis and anaplasmosis

The Babesia bovis and B. bigemina apicomplexan protozoa in conjunction with the rickettsia Anaplasma marginale are intraerythrocytic pathogens that are responsible for the most prevalent and costly tick borne diseases (TBD's) of cattle worldwide. These organisms are historically associated as they can cause clinically related hemolytic diseases in cattle, are all transmitted by Rhiphicephallus (Boophilus) ticks, and share an uncanny ability to evade the immune systems of the vertebrate hosts, causing persistent disease. In addition, acute babesiosis and anaplasmosis can be prevented quite effectively by combining tick control and vaccination with living attenuated organisms. However these methods of control have numerous limitations and improved approaches are needed. Importantly, immunizations of cattle with inactivated experimental Babesia and Anaplasma vaccines can elicit variable degrees of protection, indicating the feasibility for the development of inactivated or subunit vaccines. A new research toolbox that includes full genome sequencing combined with the improved ability to genetically modify the organisms is enhancing our understanding of their biology. An emerging paradigm is the use of recently developed Babesia and Anaplasma transfection methods for functional gene characterizations and for vaccine development. Promising recently identified subunit vaccine candidates are also emerging, including babesial proteases, putative rhoptry, microneme, and sexual stage antigens, as well as subdominant, conserved, A. marginale outer membrane major surface proteins. However, significant knowledge gaps on the role of key parasite molecules involved in cell invasion, adhesion, asexual and sexual reproduction, tick transmission, and evasion of the immune system, remain. A better understanding of the biology of these organisms and the protective immune responses will positively contribute toward the goal of developing improved immunological and pharmacological interventions against these elusive pathogens that are responsible for the most devastating TBD's of cattle. Importantly, the currently available research toolbox provides basic research instruments for helping close current knowledge gaps which will aid the design and production of effective vaccines and alternative pharmacological interventions.