An update of the isolation and characterization of culture-derived soluble antigens of Babesia bovis

In this paper recent progress concerning the identification of soluble antigens from cultures of Babesia bovis parasites is reviewed. Soluble antigens present in the supernatant of B. bovis cultures have been shown to be efficient immunogens for induction of protective immunity against bovine babesiosis. Immunochemical analysis of culture supernatants has demonstrated that at least 3 parasite antigens are released in vitro. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that at least 2 of these antigens have molecular weights within the range of 37 000-40 000 daltons. Crossed immunoelectrophoretic studies have further revealed an antigenic spectrum consisting of 1 major and 2 minor antigens with mobilities in the albumin and alpha 1 regions. Within the infected erythrocyte, 2 antigens have been localized on or near the erythrocytic membrane, while the third antigen appears to be directly associated with the parasite.     

Evaluation of the indirect fluorescent antibody test for Babesia major and Theileria mutans in Britain.

Use of the indirect fluorescent antibody (IFA) tests is described to detect antibodies to Theileria mutans and Babesia major in the sera of infected cattle. When antisera against T mutans and B major were tested against homologous antigens high antibody titres were recorded: when they were tested against each other or against Babesia divergens antigen insignificant titres (1/40 or less) were recorded. Thus the test was found to be species specific. Animals recovered from T mutans and B major infections retained significant levels of IFA titres for 22 and 11 months respectively. It is suggested that the IFA test could be used for field survey of the piroplasms of cattle in Britain.     

Failure of a recombinant Babesia bovis antigen to protect cattle against heterologous strain challenge

Groups of cattle were inoculated subcutaneously with (i) a recombinant DNA-derived Babesia bovis protein (KaBbl-GZ) fused to beta-galactosidase and combined with adjuvants, or (ii) native beta-galactosidase (GZ) plus adjuvant, or (iii) adjuvant only or (iv) a live, attenuated B bovis vaccine. KaBbl-GZ was produced in the lambda gt11-amp3 system as a 5-10 kD babesial polypeptide linked to GZ. KaBbl has previously been shown to be an immunodominant antigen of B bovis, localised at the apex of the parasite, and present in a range of B bovis strains. High levels of GZ antibodies were observed in KaBbl-GZ and GZ inoculated cattle, but specific KaBbl antibodies could not be detected by ELISA. Five months after primary inoculation, all cattle were blood challenged with a virulent heterologous B bovis strain. Despite four inoculations with KaBbl-GZ, significant protection against the challenge was not observed.     

Immune control of Babesia bovis infection

Babesia bovis causes an acute and often fatal infection in adult cattle, which if resolved, leads to a state of persistent infection in otherwise clinically healthy cattle. Persistently infected cattle are generally resistant to reinfection with related parasite strains, and this resistance in the face of infection is termed concomitant immunity. Young animals are generally more resistant than adults to B. bovis infection, which is dependent on the spleen. Despite the discovery of B. bovis over a century ago, there are still no safe and effective vaccines that protect cattle against this most virulent of babesial pathogens. Immunodominant antigens identified by serological reactivity and dominant T-cell antigens have failed to protect cattle against challenge. This review describes the innate and acquired immune mechanisms that define resistance in young calves and correlate with the development of concomitant immunity in older cattle following recovery from clinical disease. The first sections will discuss the innate immune responses by peripheral blood- and spleen-derived macrophages in cattle induced by B. bovis merozoites and their products that limit parasite replication, and comparison of natural killer cell responses in the spleens of young (resistant) and adult (susceptible) cattle. Later sections will describe a proteomic approach to discover novel antigens, especially those recognized by immune CD4+ T lymphocytes. Because immunodominant antigens have failed to stimulate protective immunity, identification of subdominant antigens may prove to be important for effective vaccines. Identification of CD4+ T-cell immunogenic proteins and their epitopes, together with the MHC class II restricting elements, now makes possible the development of MHC class II tetramers and application of this technology to both quantify antigen-specific lymphocytes during infection and discover novel antigenic epitopes. Finally, with the imminent completion of the B. bovis genome-sequencing project, strategies using combined genomic and proteomic approaches to identify novel vaccine candidates will be reviewed. The availability of an annotated B. bovis genome will, for the first time, enable identification of non-immunodominant proteins that may stimulate protective immunity.     

Evaluation of the recognition of Theileria parva vaccine candidate antigens by cytotoxic T lymphocytes from Zebu cattle

East Coast fever (ECF) is a highly fatal lymphoproliferative disease of cattle caused by Theileria parva, a tick-borne intracellular apicomplexan parasite. Parasite antigens that are targets of protective cytotoxic T lymphocyte (CTL) responses are required to formulate a sub-unit vaccine against ECF. A number of CTL target antigens have recently been identified and initial evaluation has shown their vaccine potential. This study aimed to evaluate whether these antigens were recognised by CTL obtained from six genetically diverse Zebu cattle immunized with a cocktail of T. parva stocks. T. parva Muguga specific polyclonal CD8(+) CTL lines were generated and confirmed to specifically lyse autologous infected cells. CTL recognition of autologous skin fibroblasts (iSF) transduced with recombinant modified vaccinia virus Ankara strain (MVA) expressing previously identified T. parva Muguga vaccine candidate antigens was evaluated using an IFN-gamma ELISpot assay. CTL lines from one of the four calves, BY120, responded specifically to cells infected with MVA expressing the antigen Tp2 and synthetic peptides were employed to map a new CTL epitope on this antigen. Immunoscreening of the T. parva genome with these CTL lines should identify novel antigens that will constitute valuable additions to the vaccine candidates currently being evaluated.     

Vaccines designed to protect against Mycobacterium tuberculosis infection may aid the identification of novel vaccine constructs and diagnostic antigens for bovine tuberculosis

Vaccination has been identified as a promising control strategy for tuberculosis in both humans and cattle. Recent heterologous prime-boost approaches combining BCG vaccination with subunit boosts have shown considerable promise in both fields. However, the identification of further protective antigens is still a research priority. In this paper we have established the response hierarchy in Mycobacterium bovis infected or BCG vaccinated cattle of 6 Mycobacterium tuberculosis-derived proteins that were protective against M. tuberculosis in the guinea pig aerosol challenge model. Two of these proteins, Rv1806 and Rv3812, were recognised most frequently in cattle and therefore constitute potential subunit vaccine candidates that merit further evaluation in cattle. Their epitopes were mapped in infected cattle and were shown to be located primarily in the non-conserved regions of these PE/PE-PGRS protein family members. The aim of this study was to ascertain the presence of any correlation between the immunogenicity of defined antigens in different animal species. A weak association between guinea pig immunogenicity (as measured by protection) and antigenicity in M. bovis infected or BCG vaccinated cattle was found.     

A comparative study of bovine and ovine Haemophilus somnus isolates.

Bacterial isolates (including 17 Haemophilus somnus isolates and an H. somnus-like isolate) from asymptomatic or diseased cattle and sheep, were evaluated for markers associated with virulence and host predilection. The isolates were separated into 6 distinct biovariants, 3 for sheep and 3 for cattle, based on reactions in a battery of 21 test media. Three bovine isolates associated with disease caused hemolysis of bovine blood. The rest of the isolates did not hemolyze either bovine or ovine erythrocytes. Protein profiles of all H. somnus isolates were similar with the exception of the major outer membrane proteins (MOMPs). The MOMPs of isolates associated with disease in cattle had a relative molecular weight of approximately 41 kDa compared with 33 kDa for the MOMPs of isolates from asymptomatic cattle. The MOMPs from sheep isolates were either slightly higher or lower than the 41 kDa MOMPs of bovine isolates. Major antigens detected by Western blotting were similar in all isolates except the H. somnus-like isolate. An immunodominant 40 kDa antigen was conserved in all H. somnus isolates. Antibodies to this antigen have previously been found to be protective in cattle and may also be protective for sheep. Marked differences between cattle and sheep isolates were revealed by use of restriction enzyme analysis, which separated the isolates into 12 ribotypes and 15 unique DNA profiles. Thus, cattle and sheep isolates in this collection had distinctive differences in biochemical reactions, MOMP profiles, and DNA analyses. Such differences have potential value for epidemiological studies and may also be used to evaluate host specificity of H. somnus isolates.     

Cattle remain immunocompetent during the acute phase of foot-and-mouth disease virus infection

ABSTRACT: Infection of cattle with foot-and-mouth disease virus (FMDV) results in the development of long-term protective antibody responses. In contrast, inactivated antigen vaccines fail to induce long-term protective immunity. Differences between susceptible species have also been observed during infection with FMDV, with cattle often developing persistent infections whilst pigs develop more severe symptoms and excrete higher levels of virus. This study examined the early immune response to FMDV in na?ve cattle after in-contact challenge. Cattle exposed to FMDV were found to be viraemic and produced neutralising antibody, consistent with previous reports. In contrast to previous studies in pigs these cattle did not develop leucopenia, and the proliferative responses of peripheral blood mononuclear cells to either mitogen or third party antigen were not suppressed. Low levels of type 1 interferon and IL-10 were detected in the circulation. Taken together, these results suggest that there was no generalised immunosuppression during the acute phase of FMDV infection in cattle.     

Common and stage-specific antigens of Theileria annulata.

Western blot analysis of Theileria annulata antigens was carried out using sera collected from cattle which had been immunised and challenged with either T. annulata sporozoites or schizont-infected cells. Three antigens between 71 and 73 kDa proved to be common to the three stages of parasite studied: sporozoites, schizonts and piroplasms. An antigen was found at 32 kDa which was specific to T. annulata piroplasms. Results were reproducible using sera from Morocco and the UK. At least one of the proteins at 71-73 kDa, but not that at 32 kDa were also recognised by sera from animals infected with Babesia species.     

Detection of Theileria and Babesia species in ticks collected from cattle

The present study was carried out to detect tick species that infest cattle, and Theileria and Babesia species transmitted by these ticks in Kayseri province (Turkey). A total of 300 cattle were examined for tick infestations. Of the 300 cattle, 117 (39%) were infested with ticks. A total of 1160 ticks belonging to 11 Ixodid genera were collected from the infested animals and their shelters. The most prevalent tick species was Boophilus annulatus 26.37% (306/1160) followed by Hyalomma marginatum marginatum 21.12% (245/1160) and Rhipicephalus turanicus 18.7% (217/1160). The collected ticks were separated into 43 tick pools, according to their species. These pools were examined for bovine Theileria and Babesia species (Theileria sp., Babesia sp., Theileria annulata, T. buffeli/orientalis, Babesia bigemina, B. bovis and B. divergens) by using the reverse line blotting method (RLB). Of the 43 tick pools examined, 6 (14%) were infected with B. bigemina, 4 (9.3%) with T. annulata, and 1 (2.3%) with Babesia sp., whereas 1 (2.3%) displayed mixed infection with T. annulata + B. bigemina. The sequence and phylogenetic analyses of Babesia sp., which could not be identified to the species level by RLB, were performed. In the phylogenetic tree, Babesia sp. (Kayseri 1) grouped with Babesia sp. (Kashi 2), Babesia sp. (Kashi 1), Babesia sp. (Xinjiang) and B. orientalis with 96.8-100% identity.     

Immunity against Babesia rossi infection in dogs vaccinated with antigens from culture supernatants

Soluble parasite antigens (SPA) from different Babesia species have been shown earlier to induce protective immunity when used as vaccine. However, initial attempts to produce such vaccine against Babesia rossi infection using SPA from B. rossi culture supernatants were not or only partially successful. Here we show that when dogs were vaccinated with a vaccine comprising SPA from B. rossi combined with SPA from Babesia canis protective immunity against experimental challenge infection was induced. Immunity was reflected in reduced clinical signs that resolved spontaneously, and reduction of parasitaemia and SPA in the blood. Not a single infected erythrocyte could be found in blood smears of dogs that had been repeatedly boosted (three vaccinations in total). In contrast, three out of four control dogs required chemotherapeutic treatment to prevent death. The fourth control dog showed a transient parasitaemia that resolved spontaneously. Vaccination did not prevent the development of a transient anaemia. It is concluded that a vaccine containing a mixture of SPA obtained from in vitro culture supernatants of B. rossi and B. canis induces protection in dogs against heterologous challenge infection with B. canis (as shown before) or B. rossi.     

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.     

Piroplasms of ruminants in Switzerland and zoonotic significance of Babesia

Piroplasms are tick-transmitted blood parasites belonging to the genera Babesia and Theileria. In western and southern Switzerland, B. divergens, a small Babesia species, has been known for a long time as a parasite of cattle. Recent investigations have revealed the autochthonous occurrence of this parasite also in central and eastern Switzerland. On the occasion of an outbreak of anaplasmosis in the canton of Grisons, however, B. bigemina, a large Babesia species, and Theileria of the buffeli/sergenti/orientalis species complex were for the first time identified; the epidemiology of these two piroplasms in Switzerland remains unknown until now. The recent identification by genetic analyses of B. divergens in wild ruminants contradicts the hitherto postulated strict host specificity of this Babesia species for cattle. B. divergens as well as the closely related Babesia spp. genotype EU1 have in single cases also been identified in splenectomized humans.The rodent babesia B. microti which causes a human infection that is considered an "emerging tick-borne disease" in the U.S.A., is widespread in rodent populations in Switzerland, but seems to be of minor relevance as zoonotic pathogen here. Reasons for this could be differences in virulence of the parasites or in the transmission by the respective tick-vectors on the two continents.     

Major outer membrane proteins of Brucella spp.: past,present and future

The major outer membrane proteins (OMPs) of Brucella spp. were initially identified in the early 1980s and characterised as potential immunogenic and protective antigens. They were classified according to their apparent molecular mass as 36–38 kDa OMPs or group 2 porin proteins and 31–34 and 25–27 kDa OMPs which belong to the group 3 proteins. The genes encoding the group 2 porin proteins were identified in the late 1980s and consist of two genes, omp2a and omp2b, which are closely linked in the Brucella genome, and which share a great degree of identity (>85%). In the 1990s, two genes were identified coding for the group 3 proteins and were named omp25 and omp31. The predicted amino acid sequences of omp25 and omp31 share 34% identity. The recent release of the genome sequence of B. melitensis 16 M has revealed the presence of five additional gene products homologous to Omp25 and Omp31. The use of recombinant protein technology and monoclonal antibodies (MAbs) has shown that the major OMPs appear to be of little relevance as antigens in smooth (S) B. abortus or B. melitensis infections i.e. low or no protective activity in the mouse model of infection and low or no immunogenicity during host infection. However, group 3 proteins, in particular Omp31, appear as immunodominant antigen in the course of rough (R) B. ovis infection in rams and as important protective antigen in the B. ovis mouse model of infection. The major OMP genes display diversity and specific markers have been identified for Brucella species, biovars, and strains, including the recent marine mammal Brucella isolates for which new species names have been proposed. Recently, Omp25 has been shown to be involved in virulence of B. melitensis, B. abortus and B. ovis. Mutants lacking Omp25 are indeed attenuated in animal models of infection, and moreover provide levels of protection similar or better than currently used attenuated vaccine strain B. melitensis Rev.1. Therefore, these mutant strains appear interesting vaccine candidates for the future. The other group 3 proteins identified in the genome merit also further investigation related to the development of new vaccines.     

Immune responses of cattle following vaccination with living and non-living Babesia bovis antigens

Twenty-four yearling Hereford (Bos taurus) cattle were vaccinated against Babesia bovis using either live parasites or non-living antigens obtained from the supernatant of in vitro cultures. A single dose of live parasites was given subcutaneously, while the non-living supernatant antigen (NLSA) was combined with saponin and 2 doses given, 2 weeks apart. Following vaccination with live parasites, serum antibodies remained at high levels for 6 months, but the lymphocyte transformation response was low and lasted only 10-18 days. In contrast, NLSA vaccination was followed, after 21-28 days, by a peak of serum antibodies which then slowly declined. The lymphocyte transformation response in these animals was much higher and persisted for 6 months. Following heterologous challenge all unvaccinated cattle had severe reactions and required treatment to prevent death. Cattle vaccinated with live parasites had mild reactions with only 1 of the 12 requiring treatment. Cattle vaccinated with NLSA were only partially protected and 6 of the 12 required treatment.     

Evaluation of an enzyme immunoassay for serodiagnosis of infections with Theileria parva and T annulata

An enzyme linked immunosorbent assay (ELISA) was used to determine antibody levels in cattle infected with Theileria parva and T annulata, using antigens prepared from the intra-erythrocytic piroplasm stage of the parasites. Antibody levels in calves infected with T parva increased from the 16th day after infection to reach peak values at days 28 to 35 and then declined rapidly, but in calves infected with T annulata antibody levels rose steadily up to day 40. Similar patterns of antibody production were shown by indirect fluorescent antibody tests. Sera from animals infected with T parva gave higher ELISA values with the antigen prepared from the homologous parasite species than with the antigen prepared from T annulata, but sera from cattle infected with T annulata gave similar high ELISA values with antigens prepared from both T parva and T annulata. Sera from animals infected with T mutans, T sergenti, T velifera, Babesia divergens, B major and B bovis gave only slight or no cross reactions with the piroplasm antigens, but serum from a calf infected with B bigemina cross reacted at a significant level with both piroplasm antigens.     

A 38-kDa protein from Babesia gibsoni and its antibody response in an experimentally infected dog

A cDNA encoding the Babesia bovis 12D3 antigen homologue was obtained by immunoscreening the expression library prepared from Babesia gibsoni merozoite mRNA. The complete nucleotide sequence of the gene was 1406 bp. Computer analysis suggested that the sequence contains an open reading frame of 1052 bp encoding an expected protein with a molecular weight of 36kDa. Based on homology analysis, this putative protein was designated as the B. gibsoni 12D3 antigen (Bg12D3). The Bg12D3 gene was expressed in the Escherichia coli BL21 strain, and the chronically infected dog serum reacted with the recombinant protein. The antiserum against the recombinant Bg12D3 protein can recognize a 38-kDa native protein, which is consistent with its expected size. Moreover, the purified recombinant proteins were used as the antigen to detect the antibody response in an experimentally infected dog by the enzyme-linked immunosorbent assay (ELISA). Our results indicated that the Bg12D3 protein was recognized by the host immune system and that it induced an antibody response in chronic B. gibsoni infection. These results allowed us to identify a new member of the 12D3 antigens and its characteristic immune response in canine B. gibsoni infection.     

Radioimmunoassay for Anaplasma marginale antibodies in cattle

A radioimmunoassay is described for use in the detection of Anaplasma marginale antibodies in cattle sera. Optimal sensitivity and specificity were obtained by using 2 antigens, an A marginale antigen and a RBC antigen (obtained before infection was established) from the same calf. In addition, sera were preabsorbed with RBC from healthy cattle and with sonicated Babesia bovis. Of 86 sera obtained from cattle with A marginale infection (as determined by blood smear examination or by results of subinoculation of blood from such infected cattle into splenectomized calves), 85 had positive results by use of this test. Of 100 sera obtained from cattle raised in an anaplasmosis-free area, 98 yielded negative results, and sera obtained from 35 cattle (97 sera) infected with B bigemina and from 18 cattle infected with Theileria orientalis yielded negative results. By use of this test, 99 of 100 sera obtained from cattle with B bovis infection were negative for A marginale. Anaplasma marginale antibodies were detected in 18 cattle that had been pastured in a Boophilus microplus-free area for 2 years after natural infection. After 3 years, 16 of these cattle were still seropositive for A marginale. Sixteen cattle pastured in a Bo microplus-infested area had detectable antibody against A marginale 27 months after initial infection with A marginale. Sensitivity and specificity of the test were assessed as 98.8% for each.     

Hematological findings and antibody responses in Syrian hamster (Mesocricetus auratus) infected with Babesia microti

Hematological findings during the course of infection and the antibody response in Syrian hamsters infected with Babesia microti were examined. A macrocytic hypochromic anemia with an increase of the reticulocyte count was detected as a rise in the parasitized erythrocyte rate. White blood cell counts also remarkedly increased with the increases of both neutrophils and active-shaped monocytes, and thus they particularly play an important role in eliminating the parasite. In Western blotting with the sera from the hamsters infected with B. microti, a 38 kDa protozoan antigen reacted to the early-term sera, and additionally 28, 32, and 34 kDa antigens also reacted to the medium- and latter-term, and convalescent sera. These antigens were immunodominant and the antibodies against these antigens had also important roles for inhibition of this parasite.     

Identification of immunoreactive polypeptides of Babesia equi parasite during immunization

This study was carried out to identify immunoreactive polypeptides in Babesia equi merozoite antigen. Three fractions of killed B. equi merozoite antigen viz.; whole merozoite (WM), cell membrane (CM) and high speed supernatant (HSS) antigens were prepared from the parasite infected erythrocytes. These antigenic preparations along with ghost antigen from non-infected erythrocytes were fractionated on 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with sera showing high antibody titres. On SDS-PAGE, 16 polypeptides with molecular weight (Mr) in the range of 112-17kDa were obtained from the WM and CM antigens. But only six polypeptides were detected (96.5-28kDa) in the HSS antigen. On immunoblotting with high titred serum collected from donkeys following two immunizations with a killed B. equi merozoite immunogen, 11 polypeptides were observed in the WM and CM antigens (Mr 112-18kDa). Of these, four polypeptides (Mr 112, 45, 33 and 18kDa) were identified as most immunoreactive. Besides these, a 28kDa was observed as strong immunoreactive protein in WM and CM antigens. The HSS antigen showed only six polypeptides and one peptide (28kDa) was identified as immunoreactive. When high titred serum collected from immunized donkeys following challenge with B. equi infected blood and was used for immunoblotting, the protein profile of WM and CM antigens remained the same. However, three additional polypeptides (Mr 81, 54.5 and 39kDa) were detected in HSS antigen.