Dicamba growth regulator promotes genotype independent somatic embryogenesis from immature zygotic embryos of tropical maize inbred lines

Maize is one of the most important cereal crops in Sub-Saharan Africa and an important source of energy for humans. However, the difference in the dedifferentiation frequency of immature embryos among various genotypes indicates that callus induction and genetic transformation is dependent on the genotype. This phenomenon is an impediment in the fundamental process of improving tropical maize germplasm especially through genetic engineering. Here, five tropical maize (Zea mays L.) genotypes, CML 216, CML 144, A 04, E 04 and TL 21, were evaluated for callus induction on MS medium supplemented with the growth regulator dicamba. Embryogenic and non embryogenic callus induction was independent of genotype when young immature embryos, 12 days after pollination (DAP) were used for tissue culture in combination with dicamba. The optimal concentration of dicamba for induction of embryogenic callus in all the genotypes was 3 mg/L, which was also the concentration at which non embryogenic callus formation was lowest. The frequency of embryogenic callus induction ranged from 35% to 79% among the five genotypes and somatic embryos regenerated R0 shoots that produced normal R1 progenies. This regeneration method is expected to facilitate the development of a more efficient genotype independent Agrobacterium- mediated transformation system for tropical inbred lines

A novel strategy for the identification of antigens that are recognised by bovine MHC class I restricted cytotoxic T cells in a protozoan infection using reverse vaccinology

BACKGROUND: Immunity against the bovine protozoan parasite Theileria parva has previously been shown to be mediated through lysis of parasite-infected cells by MHC class I restricted CD8(+ )cytotoxic T lymphocytes. It is hypothesized that identification of CTL target schizont antigens will aid the development of a sub-unit vaccine. We exploited the availability of the complete genome sequence data and bioinformatics tools to identify genes encoding secreted or membrane anchored proteins that may be processed and presented by the MHC class I molecules of infected cells to CTL. RESULTS: Of the 986 predicted open reading frames (ORFs) encoded by chromosome 1 of the T. parva genome, 55 were selected based on the presence of a signal peptide and/or a transmembrane helix domain. Thirty six selected ORFs were successfully cloned into a eukaryotic expression vector, transiently transfected into immortalized bovine skin fibroblasts and screened in vitro using T. parva-specific CTL. Recognition of gene products by CTL was assessed using an IFN-γ ELISpot assay. A 525 base pair ORF encoding a 174 amino acid protein, designated Tp2, was identified by T. parva-specific CTL from 4 animals. These CTL recognized and lysed Tp2 transfected skin fibroblasts and recognized 4 distinct epitopes. Significantly, Tp2 specific CD8(+ )T cell responses were observed during the protective immune response against sporozoite challenge. CONCLUSION: The identification of an antigen containing multiple CTL epitopes and its apparent immunodominance during a protective anti-parasite response makes Tp2 an attractive candidate for evaluation of its vaccine potential

Approaches to vaccination against Theileria parva and Theileria annulata

Despite having different cell tropism, the pathogenesis and immunobiology of the diseases caused by Theileria parva and Theileria annulata are remarkably similar. Live vaccines have been available for both parasites for over 40 years, but although they provide strong protection, practical disadvantages have limited their widespread application. Efforts to develop alternative vaccines using defined parasite antigens have focused on the sporozoite and intracellular schizont stages of the parasites. Experimental vaccination studies using viral vectors expressing T. parva schizont antigens and T. parva and T. annulata sporozoite antigens incorporated in adjuvant have, in each case, demonstrated protection against parasite challenge in a proportion of vaccinated animals. Current work is investigating alternative antigen delivery systems in an attempt to improve the levels of protection. The genome architecture and protein-coding capacity of T. parva and T. annulata are remarkably similar. The major sporozoite surface antigen in both species and most of the schizont antigens are encoded by orthologous genes. The former have been shown to induce species cross-reactive neutralizing antibodies, and comparison of the schizont antigen orthologues has demonstrated that some of them display high levels of sequence conservation. Hence, advances in development of subunit vaccines against one parasite species are likely to be readily applicable to the other

In vitro and in vivo antileishmanial efficacy of a combination therapy of diminazene and artesunate against Leishmania donovani in BALB /c mice

The in vitro and in vivo activity of diminazene (Dim), artesunate (Art) and combination of Dim and Art (Dim-Art) against Leishmania donovani was compared to reference drug; amphotericin B. IC50 of Dim-Art was found to be $2.28 \pm 0.24 \mu$ g/mL while those of Dim and Art were $9.16 \pm 0.3 \mu$ g/mL and $4.64 \pm 0.48 \mu$ g/mL respectively. The IC50 for Amphot B was $0.16 \pm 0.32 \mu$ g/mL against stationary-phase promastigotes. In vivo evaluation in the L. donovani BALB/c mice model indicated that treatments with the combined drug therapy at doses of 12.5 mg/kg for 28 consecutive days significantly ($p < 0.001$) reduced parasite burden in the spleen as compared to the single drug treatments given at the same dosages. Although parasite burden was slightly lower ($p < 0.05$) in the Amphot B group than in the Dim-Art treatment group, the present study demonstrates the positive advantage and the potential use of the combined therapy of Dim-Art over the constituent drugs, Dim or Art when used alone. Further evaluation is recommended to determine the most efficacious combination ratio of the two compounds.Comment: 4 Pages, 3 Figure

A longitudinal assessment of the serological response to Theileria parva and other tick-borne parasites from birth to one year in a cohort of indigenous calves in western Kenya

Tick-borne diseases are a major impediment to improved productivity of livestock in sub-Saharan Africa. Improved control of these diseases would be assisted by detailed epidemiological data. Here we used longitudinal, serological data to determine the patterns of exposure to Theileria parva, Theileria mutans, Babesia bigemina and Anaplasma marginale from 548 indigenous calves in western Kenya. The percentage of calves seropositive for the first three parasites declined from initial high levels due to maternal antibody until week 16, after which the percentage increased until the end of the study. In contrast, the percentage of calves seropositive for T. mutans increased from week 6 and reached a maximal level at week 16. Overall 423 (77%) calves seroconverted to T. parva, 451 (82%) toT. mutans, 195 (36%) to B. bigemina and 275 (50%) to A. marginale. Theileria parva antibody levels were sustained following infection, in contrast to those of the other three haemoparasites. Three times as many calves seroconverted to T. mutans before seroconverting to T. parva. No T. parva antibody response was detected in 25 calves that died of T. parva infection, suggesting that most deaths due to T. parva are the result of acute disease from primary exposure

Fusion of the Mycobacterium tuberculosis Antigen 85A to an Oligomerization Domain Enhances Its Immunogenicity in Both Mice and Non-Human Primates

To prevent important infectious diseases such as tuberculosis, malaria and HIV, vaccines inducing greater T cell responses are required. In this study, we investigated whether fusion of the M. tuberculosis antigen 85A to recently described adjuvant IMX313, a hybrid avian C4bp oligomerization domain, could increase T cell responses in pre-clinical vaccine model species. In mice, the fused antigen 85A showed consistent increases in CD4+ and CD8+ T cell responses after DNA and MVA vaccination. In rhesus macaques, higher IFN-γ responses were observed in animals vaccinated with MVA-Ag85A IMX313 after both primary and secondary immunizations. In both animal models, fusion to IMX313 induced a quantitative enhancement in the response without altering its quality: multifunctional cytokines were uniformly increased and differentiation into effector and memory T cell subsets was augmented rather than skewed. An extensive in vivo characterization suggests that IMX313 improves the initiation of immune responses as an increase in antigen 85A specific cells was observed as early as day 3 after vaccination. This report demonstrates that antigen multimerization using IMX313 is a simple and effective cross-species method to improve vaccine immunogenicity with potentially broad applicability

Characterization of the fine specificity of bovine CD8 T-cell responses to defined antigens from the protozoan parasite Theileria parva

Immunity against the bovine intracellular protozoan parasite Theileria parva has been shown to be mediated by CD8 T cells. Six antigens targeted by CD8 T cells from T. parva-immune cattle of different major histocompatibility complex (MHC) genotypes have been identified, raising the prospect of developing a subunit vaccine. To facilitate further dissection of the specificity of protective CD8 T-cell responses and to assist in the assessment of responses to vaccination, we set out to identify the epitopes recognized in these T. parva antigens and their MHC restriction elements. Nine epitopes in six T. parva antigens, together with their respective MHC restriction elements, were successfully identified. Five of the cytotoxic-T-lymphocyte epitopes were found to be restricted by products of previously described alleles, and four were restricted by four novel restriction elements. Analyses of CD8 T-cell responses to five of the epitopes in groups of cattle carrying the defined restriction elements and immunized with live parasites demonstrated that, with one exception, the epitopes were consistently recognized by animals of the respective genotypes. The analysis of responses was extended to animals immunized with multiple antigens delivered in separate vaccine constructs. Specific CD8 T-cell responses were detected in 19 of 24 immunized cattle. All responder cattle mounted responses specific for antigens for which they carried an identified restriction element. By contrast, only 8 of 19 responder cattle displayed a response to antigens for which they did not carry an identified restriction element. These data demonstrate that the identified antigens are inherently dominant in animals with the corresponding MHC genotypes

The cytotoxic T cell: MHC restriction, strain specificity and role in immunity to Theileria parva infection. Annales de Recherches Vétérinaires, 1992, 3,311-318.

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