Identification of virulence factors of Theileria parva

Theilaria parva, a protozoan parasite which infects bovine lymphocytes is responsible for the fatal disease East Coast Fever in Africa. The parasite, in the sporozoite stage, is injected into the host by the tick vector and invades host lymphocytes, eventually resulting in a clonal cancer-like expansion of the infected lymphocytes with pathological consequences, which may include death. Vaccination strategies are based on an infection and treatment method, involving infection of cattle with a sporozoite stabilate immediately followed with oxytetracyclin treatment. This technique could be improved through selection of a sporozoites isolates which cause a mild reaction without diminishing the protective immune responses. Such isolates will be able to give some degree of protection by inducing the immune response while keeping the infectivity down. Field observations have suggested that different parasite isolates appear to have different levels of virulence, including different degrees of morbidity and mortality in the host. To confirm that virulence differences exist and to gain more insight into possible virulence factors, a comparative analysis of isolates suspected of high and low virulence is necessary, comparing infections with an identical number of sporozoites. The use of an in vitro quantitation technique is necessary for this kind of analysis. The knowledge of the mechanisms affecting virulence is important in the development of attenuated or sub-unit vaccines.In chapter two of this thesis, we evaluated the potential differences in virulence between two T. parva isolates by comparing the infections dynamics after initiating infections in vivo and in vitro, using equivalent numbers of infective sporozoites quantified using an in vitro titration method. The parameters that were measured included prepatent period, fever, survival (based on ECF index), parasitosis, piroplasm parasitaemia and hematological parameters. The results indicated that with an equivalent dose of infective sporozoites, a T. parva Chitongo (TpC) infection resulted in a slightly later onset (1-2 days) and lower levels of parasitosis in the lymph node, while causing less and later mortality than a T. parva Muguga (TpM) infection. In addition, the pathological signs was less in the Chitongo-infected animals. In vitro comparison of sporozoite infectivity showed an even higher difference between the two isolates, with the kinetics of Chitongo transformation being almost twice as slow as for Muguga. These data confirmed that Chitongo is a less virulent parasite than Muguga, and that one of the causes of its reduced virulence may be an event that occurs during the early infection and transformation process.In chapter three, we compared the inflammatory status of animals infected with the two Theileria isolates by measuring cytokine and acute phase protein levels in serum. The results indicated significantly higher levels of two acute phase proteins, SAA and AGP in Muguga-infected calves. The levels of other parameters, likethe expression of MMP-9 involved in cell migration and metastasis, and the anti-inflammatory cytokine IL-10, did not show statistically significant differences. The higher acute phase response in animals infected with the Muguga parasite could be interpreted as a consequence of the higher parasite levels early after infection. Additional assays for inflammatory cytokines are needed to make more meaningful conclusions and identify other possible mechanisms that may contribute to the weaker virulence of Chitongo. In chapter four we demonstrated that the two Theileria isolates targeted different cell types. The study compared the susceptibility of different cell lines and purified lymphocyte populations for infection and transformation by TpC and TpM. While sporozoites from both isolates infected unfractionated peripheral blood mononuclear cells (PBMC), they differed in infection when mixed with purified subpopulations of lymphocytes. Our first observation suggested that a long term cultured cell line with the WC1+-γδ T cell phenotype was readily infected with TpM sporozoites, but not with TpC sporozoites. To rule out that this cell line had lost its capacity to be infected by Chitongo through culture, the experiment was repeated with non-cultured WC1+-γδ T cells, freshly purified from blood. The results were the same, so a systematic analysis was carried out to test the capacity of TpC and TpM sporozoites to adhere and infect different subpopulations of CD4+, CD8+ and WC1+-γδ T cells. While TpM sporozoites attached to all three subsets of T lymphocytes, TpC sporozoites only bound the CD8+ T cell population. Flow cytometry analysis of established, transformed clones confirmed that TpM clones were derived from any of the T cell subpopulations, CD4, CD8 or WC1+-γδ T, while TpC clones were exclusively of the CD8 phenotype. The two Theileria isolates infected and transformed CD8 T cells with the same kinetics, suggesting that it was exclusively the binding step to the host cell that was different between the two isolates, and not the capacity to transform the host cell. The data strongly suggest that the lower virulence of TpC is directly linked to the restricted set of T cells that the infective stage of the parasite can bind to, infect and transform. One important consequence of our observations is that the adherence process of the Chitongo isolate must be different from the Muguga isolate. However, the sequence of the p67 gene, which has always been described as the adhesion molecule that interacts and binds to the target cell, is 100% identical between TpC and TpM, suggesting the existence of at least one other adhesin. If a second binding ligand could be identified, then the possibility exists that the anti-sporozoite vaccine, which is based on one recombinant p67 fragment and has an efficacy that is too low for introduction in the field, could be thoroughly improved.Finally chapter five summarizes the data, identifies the potential mechanisms that lie atthe basis of the observed difference in virulence and discusses the potential benefit of a low virulent strain in the perspective of vaccine development. Finally, arguments are presented for further research into the adherence mechanism that may lead to a better anti-sporozoite vaccine.nrpages: 95status: publishe

REKAYASA SISTEM PEMBELAJARAN MANDIRI BERBANTUAN KOMPUTER MATA KULIAH TEKNIK PELABUHAN

Teknik Pelabuhan merupakan mata kuliah yang tidak sepopuler Konstruksi ]3eton, Konstruksi Baja, maupun mata kuliah keahlian lainnya di Jurusan/Program Studi Teknik Sipil. Padahal pengetahuan tentang Teknik Pelabuhan sangat penting untuk dikuasai mahasiswa Teknik Sipil di Indonesia sebagai negara yang memililci banyak pelabuhan. Terlebih lagi Pola Ilmiah Pokok Universitas Diponegoro adalah pengembangan wilayah pantai. Dengan demikian mata kuliah Teknik Pelabuhan perlu dipopulerkan dan dibuat semenarik munglcin sehingga dapat merangsang mahasiswa untuk melakukan penjelajahan informasi sesuai kemampuannya dan melakukan proses 'discovery learning' sebagai cerminan kemandirian belajar. Kemajemukan potensi mahasiswa tidak terakomodasikan dalam sistem pendidikan konvensional sehingga bagi mereka yang tergolong "di bawah normal" tidak disediakan pengajaran remidial, sebaliknya bagi yang berbakat/unggul tidak disediakan pengayaan. Dengan detnildan diperlukan suatu sistem pembelajaran yang mampu melayani kemajemukan mahasiswa. Salah satu sistem pembelajaran yang mampu melayani kemajemukan tersebut adalah sistem pembelajaran mandiri bprbasis komputer. Sistem ini dapat melayani mahasiswa yang lamban dalam belajar maupun yang cepat dalam menyerap materi perkuliahan. Penelitian ini ditujukan untuk : 1. membangun suatu Sistem Pembelajaran Mandiri Berbantuan Komputer Mats Kuliah Teknik Pelabuhan di Jurusan Teknik Sipil guna mendorong kemandirian mahasiswa dalam belajar dan menumbuhkan kebiasaan berpikir analitik sintetik di Politeknik Negeri Semarang; 2. mempopulerkan mata kuliah Teknik Pelabuhan sebagai materi yang sangat penting untuk dikuasai mahasiswa Teknik Sipil di Indonesia yang terkait dengaq Pola Ilmiah Pokok Universitas Diponegoro dalam pengembangan wilayah pantai; 3. menciptakan suatu sistem pembelajaran yang interaktif dan menarik guna merangsang mahasiswa untuk melakukan penjelajahan informasi sesuai kemampuannya dan melakukan proses 'discovery learning' sebagai cerminan dan kemandirian belajar; 4. mengoptimalkan pemakaian fasilitas komputer yang sekarang sudah banyak dimiliki oleh lembaga pendidikan maupun mahasiswa dalam proses pembelajaran interaktif dalam modus teks. Modus grafis dan audio belum akan diimplementasikan; 5. menghargai dan mengakomodasikan kemajemukan potensi mahasiswa sehingga bagi mereka yang tergolong "di bawah normal" tersedia pengajaran remidial, sebaliknya bagi yang berbakat/unggul tersedia pengayaan Sistem Pembelajaran Mandiri ini dikembangkan dengan Metode Rekayasa Sistem dalam tahapan pengembangan berdasarkan daur hidup (System Development Life Cycle = SDLC). SDLC terdiri dart Analysis, Design, Implementation, Testing, dan Evaluation. Tahapan - tahapan ini dapat dilakukan secara overlap dan bersildus. Dan kegiatan rekayasa sistem pembelajaran mandiri berbantuan komputer mats kuliah Teknik Pelabuhan ini dapat disimpulkan beberapa hal. 1. Sistem Pembelajaran Mandiri Berbantuan Komputer dapat dibangun dengan memanfaatkan paket pengolah kata yang kemudian ditransfer ke dalam berkas-berkas HTML (Hypertext Mark-up Lan-guage). Berkas ini kemudian dimuatkan ke dalam server sehingga dapat diakses oleh komputer-komputer yang terhubung ke server tersebut. 2. Akses terhadap mated tersebut dapat dilakukan melalui fasilitas Internet Explorer sehingga sekaligus memberi bake kepada mahasiswa untuk mengakses informasi dengan teknologi Iterbasis intemet. 3. Sistem ini merupakan pendukung bagi mata kuliah Teknik Pelabuhan, yang merupakan mata kuliah barn di Politeknik Undip, sebagai mated yang sangat penting untuk dikuasai karena keterkaitannya dengan Pola Ilmiah Pokok Universitas Diponegoro dalam pengembangan wilayah pantai. 4. Sistem ini merupakan suatu sistem pembelajaran yang lebih interaktif den menarik dibandingkan sistem konvensional yang menggunakan diktat. Hal ini guna merangsang mahasiswa untuk melakukan penjelajahan informasi sesuai kemampuannya dan pnelakulcan proses 'discovery learning' sebagai cerminan dad kemanditian belajar. 5. Sistem yang berbasis komputer ini menyumbang nilai tambah bagi lembaga pendidikan tinggi yang diperoleh terutama dad pemanfaatan teknologi komputer dalam proses pembelajaran, penciptaan metode pembelajaran berbasis komputer, pemberdayaan fasilitas laboratorium komputer, pengayaan mated Teknik Pelabuhan, dan pembentukan infrastruktur pendidikan berbasis komputer bagi masa kini dan yang akan datang. Dad kegiatan ini dapat disarankan agar sistem pembelajaran ini dikembangkan lebih lanjut agar menjadi sistem yang terbuka dengan mengkaitfrannya ke jaringan Internet. Server yang ada dapat ditingkatkan kemampuannya menjadi web server bagi jaringan intemet. Untuk itu diperlukan sponsor yang mendanai operasi server ini secara rutin. Keterbukaan sistem ini memerlukan masukan-masukan dad berbagai pihak untuk pienyempurnakan mated Teknik Pelabuhan agar tidak hanya menjadi konsumsi mahasiswa Politeknik saja melainkan juga untuk level pendidikan yang lebih tinggi

Demonstration of differences in virulence between two Theileria parva isolates

H.S. Tindih and J. Naessens are ILRI authorsIn areas with a low incidence of infection due to unimodal presence of ticks, Theileria parva has been observed to induce a disease with relatively low pathology. This is followed by a carrier state, rather than death and therefore provides a better chance of transmission of the parasite back to the tick vector since in unimodal conditions, the different tick stages occur at different times. One isolate from such an area in Zambia, T. parva Chitongo, was compared for virulence with T. parva Muguga, isolated from an area exhibiting a continuous presence of all vector stages in East Africa. To reduce any variation due to infection dose, an in vitro standardized dose was used to initiate infection of groups of three local zebu cattle with each isolate. Parameters of virulence measured were prepatent period, fever, survival (based on ECF index), parasitosis, piroplasm parasitaemia and hematological parameters. Our results suggest that T. parva Chitongo developed a slightly later onset (1–2 days) and lower levels of parasitosis in the lymph node, causing less and later mortality. Comparison of the in vitro rate of transformation confirmed that the time needed to transform an infected lymphocyte took 4 days longer for T. parva Chitongo than T. parva Muguga. Elucidating the mechanism responsible for the lower virulence of T. parva Chitongo could be useful for designing an attenuated vaccine

Demonstration of differences in virulence between two Theileria parva isolates

In areas with a low incidence of infection due to unimodal presence of ticks, Theileria parva has been observed to induce a disease with relatively low pathology. This is followed by a carrier state, rather than death and therefore provides a better chance of transmission of the parasite back to the tick vector since in unimodal conditions, the different tick stages occur at different times. One isolate from such an area in Zambia, T. parva Chitongo, was compared for virulence with T. parva Muguga, isolated from an area exhibiting a continuous presence of all vector stages in East Africa. To reduce any variation due to infection dose, an in vitro standardized dose was used to initiate infection of groups of three local zebu cattle with each isolate. Parameters of virulence measured were prepatent period, fever, survival (based on ECF index), parasitosis, piroplasm parasitaemia and hematological parameters. Our results suggest that T. parva Chitongo developed a slightly later onset (1-2 days) and lower levels of parasitosis in the lymph node, causing less and later mortality. Comparison of the in vitro rate of transformation confirmed that the time needed to transform an infected lymphocyte took 4 days longer for T. parva Chitongo than T. parva Muguga. Elucidating the mechanism responsible for the lower virulence of T. parva Chitongo could be useful for designing an attenuated vaccine.status: publishe

A thermostable presentation of the live, attenuated peste des petits ruminants vaccine in use in Africa and Asia

The research objective was to develop a thermostable vaccine against peste des petits ruminants (PPR), a morbilliviral disease of small ruminants targeted for eradication that is a major constraint on the livelihoods of the rural poor throughout much of Africa and Asia. Although existing PPR vaccines provide life-long immunity, they require continuous refrigeration. This limits their utility in developing countries. Methods for the lyophilization of a related morbillivirus, rinderpest (RP), resulted in vaccine that could be used in the field for up to 30 days without refrigeration which was a major contribution to the global eradication of RP completed in 2011. The present research applied the rinderpest lyophilization method to the attenuated Nigeria 75/1 PPR vaccine strain, and measured thermostability in accelerated stability tests (AST) at 37 °C. The shelf-life of the vaccine was determined as the time a vial retained the minimum dose required as a 25-dose presentation at the specified temperature. A lactalbumin hydrolysate and sucrose (LS) stabilizer was compared to stabilizers based on trehalose. PPR vaccine produced using the Xerovac drying method was compared to vaccine produced using the rinderpest lyophilization method in AST. LS vaccine was evaluated in AST at 37, 45 and 56 °C and an Arrhenius plot was constructed for estimation of stability at temperatures not tested. Vaccines produced using LS and the rinderpest method of lyophilization were the most stable. The shelf-life of the Xerovac preparation was 22.2 days at 37 °C. The three LS vaccine batches had shelf-lives at 37 °C of 177.6, 105.0 and 148.9 days, respectively, at 37 °C. At 56 °C, the shelf-life was 13.7 days. The projected half-life at 25 °C was 1.3 years. This is sufficient thermostability for use without a cold chain for up to 30 days which will greatly facilitate the delivery of vaccination in the global eradication of PPR

A Theileria parva Isolate of Low Virulence Infects a Subpopulation of Lymphocytes

Theileria parva is a tick-transmitted protozoan parasite that infects and transforms bovine lymphocytes. We have previously shown that Theileria parva Chitongo is an isolate with a lower virulence than that of T. parva Muguga. Lower virulence appeared to be correlated with a delayed onset of the logarithmic growth phase of T. parva Chitongo-transformed peripheral blood mononuclear cells after in vitro infection. In the current study, infection experiments with WC1(+) γδ T cells revealed that only T. parva Muguga could infect these cells and that no transformed cells could be obtained with T. parva Chitongo sporozoites. Subsequent analysis of the susceptibility of different cell lines and purified populations of lymphocytes to infection and transformation by both isolates showed that T. parva Muguga sporozoites could attach to and infect CD4(+), CD8(+), and WC1(+) T lymphocytes, but T. parva Chitongo sporozoites were observed to bind only to the CD8(+) T cell population. Flow cytometry analysis of established, transformed clones confirmed this bias in target cells. T. parva Muguga-transformed clones consisted of different cell surface phenotypes, suggesting that they were derived from either host CD4(+), CD8(+), or WC1(+) T cells. In contrast, all in vitro and in vivo T. parva Chitongo-transformed clones expressed CD8 but not CD4 or WC1, suggesting that the T. parva Chitongo-transformed target cells were exclusively infected CD8(+) lymphocytes. Thus, a role of cell tropism in virulence is likely. Since the adhesion molecule p67 is 100% identical between the two strains, a second, high-affinity adhesin that determines target cell specificity appears to exist.status: publishe

Theileria parva isolate of low virulence infects a subpopulation of lymphocytes

Theileria parva is a tick-transmitted protozoan parasite that infects and transforms bovine lymphocytes. We have previously shown that Theileria parvaChitongo (TpC) is an isolate of lower virulence than T.parvaMuguga (TpM). Lower virulence appeared correlated with a delayed onset of thelogarithmic growth phase of TpC-tranformedperipheral blood mononuclear cells after in vitro infection. In the current study, infection experiments of WC1(+)-gammadelta-T cellsrevealed thatonly TpMcould infect these cells andthat no transformed cells could be obtained withTpC sporozoites. Subsequent analysis of susceptibility of different cell lines and purified populations of lymphocytes for infection and transformation by both isolates showed that TpMsporozoites could attach and infectCD4(+), CD8(+) and WC1(+) T lymphocytes, but that TpCsporozoites were only observed to bind to the CD8(+) T cell population. Flow cytometry analysis of established,transformed clones confirmed this bias in target cells. TpM-transformed clones consisted of different cell surface phenotypes, suggesting that they were derived from either host CD4(+), CD8(+)or WC1(+)T cells. In contrast, all in vitro and in vivoTpC-transformed clones expressed CD8 but not CD4 or WC1, suggesting that the TpC-transformed target cells were exclusivelyinfected CD8(+) lymphocytes.So a role of cell tropism in virulence is likely. Since the adhesion molecule p67 is 100 % identical between the two strains, a second, high affinity adhesin that determines target cell specificity appears to exist