Comparative pathogenicity of Theileria annulata strains

Twenty three susceptible crossbred calves were inoculated with five Indian strains of Theileria annulata, collected from natural cases of tropical theileriosis at Ludhiana, Hissar, Jaipur, Uruli-Kanchan and Bangalore. All the strains produced clinical disease with typical symptoms and lesions of acute theileriosis. The strains were considered highly virulent and equally pathogenic.     

Comparative pathogenicity of Theileria annulata strains

Summary

Twenty three susceptible crossbred calves were inoculated with five Indian strains of Theileria annulata, collected from natural cases of tropical theileriosis at Ludhiana, Hissar, Jaipur, Uruli‐Kanchan and Bangalore. All the strains produced clinical disease with typical symptoms and lesions of acute theileriosis. The strains were considered highly virulent and equally pathogenic.     

Immunological relationship between strains of Theileria annulata Dschunkowsky and Luhs 1904

The immunological relationship between the Ludhiana and Hissar strains, and between the Uruli-Kanchan, Bangalore and Jaipur strains of Theileria annulata was studied. Calves were immunised against the Ludhiana strain by the infection-treatment method, against the Uruli-Kanchan strain by the infection-treatment method or by untreated low-grade infection and against the Jaipur strain by untreated low-grade infection. After 45 days, groups of immune calves and susceptible controls were challenged with 10-tick equivalent stabilates. The ensuing host responses, body temperature, swelling of the regional lymph node, appearance of schizonts in the regional lymph node and piroplasms in blood and mortality, were studied. All five strains proved very virulent and produced severe disease in susceptible calves. Immunisation with the Ludhiana, Uruli-Kanchan and Jaipur strains conferred absolute protection against severe homologous challenges. Immunisation with the Ludhiana and Uruli-Kanchan strains conferred substantial protection against heterologous challenges with the Hissar strain, and the Bangalore and Jaipur strains, respectively. Immunisation with the Uruli-Kanchan strain gave less protection against heterologous challenge with the Jaipur strain and vice versa as some of the challenged calves showed slight swelling of the regional lymph node, two had fever, all exhibited a low-grade parasitaemia and four developed anaemia.     

Cell-mediated immune responses to sporozoites and macroschizonts of Theileria annulata.

The nature of cell-mediated immune (CMI) responses was studied in cross-bred bovine calves, immunised by attenuated and allogeneic macroschizonts of Theileria annulata. The CMI responses were also investigated in calves, destined to survive or die of tropical theileriosis (Theileria annulata) induced by a virulent dose of sporozoites or macroschizont-infected lymphoblasts. Calves suffering fatal theileriosis showed poor CMI response. Microcytotoxicity assay revealed an enhanced population of specific cytotoxic cells amongst the peripheral blood lymphocytes (PBL) of calves resolving the infection successfully. The E rosette assay showed proliferation of T cells and the assay for macrophage migration inhibition factor (MIF) demonstrated antigen sensitised cells in the PBL. Calves, immunised by allogeneic and attenuated macroschizont-infected lymphoblasts or those recovering from virulent macroschizont-induced infection, showed protective CMI responses with patterns similar to those appearing after non-fatal sporozoite infection.     

A comparison of susceptibilities to infection of four species of Hyalomma ticks with Theileria annulata

In this comparative study unfed nymphs of four Hyalomma tick species (Hyalomma anatolicum anatolicum, Hyalomma anatolicum excavatum, Hyalomma detritum and Hyalomma marginatum marginatum) were allowed to engorge on calves experimentally infected with Theileria annulata. The infection prevalence in the salivary glands of the adult female and male ticks of each Hyalomma species used in the study were assessed. The infection prevalence with T. annulata was high and did not vary markedly in the four Hyalomma tick species. The mean number of infected acini per tick in female and male ticks was different with female ticks having higher numbers of infected acini than the male ticks. The sex difference was more significant between H.a. anatolicum and H.a. excavatum than between H. detritum and H.m. marginatum. This study clarifies the roles of four Hyalomma tick species, and their sex, in the development of T. annulata.     

Studies on the transmission of Theileria annulata to cattle by the tick Hyalomma lusitanicum

The role of the ixodid tick Hyalomma lusitanicum Koch 1844 as a vector of Mediterranean or tropical theileriosis (caused by the protozoan parasite Theileria annulata Dschunkowsky et Luhs 1904) in southern Spain was studied. Hyalomma lusitanicum was the most common tick, and the only species of the genus Hyalomma L., found on T. annulata-infected cattle from the theileriosis enzootic area studied (province of Cádiz, southern Spain). Likewise, we found that all sera of the cattle previously considered as suspected of theileriosis by clinical signs, tested for T. annulata antibodies, were positive and all blood samples of these suspected cattle examined had infected erythrocytes. Partially fed H. lusitanicum adults were collected in the field on T. annulata-infected cattle in this enzootic area and fed on an uninfected calf in an experimental farm free of theileriosis and ticks. At approximately 3 weeks post-tick feeding on the calf, this became positive for T. annulata antibodies and T. annulata merozoites were found in erythrocytes from blood smears. These results show the ability of H. lusitanicum to transmit the protozoan parasite T. annulata to susceptible cattle and indicate that H. lusitanicum is probably an important vector of T. annulata in the enzootic area surveyed.     

Factors affecting the capacity of Theileria annulata sporozoites to invade bovine peripheral blood lymphocytes

The number of T. annulata sporozoites invading bovine peripheral blood lymphocytes (PBL) under different conditions (in vitro) was determined. Heat-inactivation of T. annulata sporozoites for 45 min, in a thermostatically controlled, shaking water bath preset and stabilised at 60 degrees C resulted in an almost total lack of invasion of fresh, normal PBL by the sporozoites, indicating that the interiorization process is parasite-effected. The mean number of T. annulata sporozoites interiorization (per 1000 lymphocytes) in cultures set up using sporozoites and PBL, mixed and incubated at 0 degrees C for 1 h in melting ice, was highly significantly reduced (P less than 0.01), indicating the invasion of bovine lymphocytes by T. annulata sporozoites is an active process dependent on active metabolism which is markedly affected by temperature. Pre-treatment of PBL with trypsin significantly reduced the number of invading sporozoites thus incriminating proteins or glycoproteins as constituents of receptors involved in sporozoite-lymphocyte recognition.     

Naturally occurring infections of cattle with Theileria lestoquardi and sheep with Theileria annulata in the Sudan

Theileria annulata is endemic in northern Sudan, hindering all efforts at upgrading cattle for milk production. T. lestoquardi clinical cases occur throughout the year and causes annual outbreaks that result in substantial losses in sheep. In the northern Sudan both cattle and small ruminants are frequently raised together and/or share common grazing grounds at river banks. In an attempt to evaluate field cross infectivity of Theileria lestoquardi and T. annulata in cattle and sheep respectively, a PCR analysis was carried out on samples collected from closely reared sheep and cattle using both T. annulata and T. lestoquardi specific primers. A total of 19 sheep out of 51 (37.3%) were positive for T. lestoquardi while four sheep (7.8%) showed T. annulata specific amplicons. A total of 38 out of 52 (73.1%) surveyed cattle were PCR positive for T. annulata and only two (3.8%) showed T. lestoquardi specific bands. These findings indicate complex epidemiology of both infections in areas where both parasites are transmitted by the same vector and call for further investigations of this phenomenon.     

Theileria annulata as a hazard to cattle in countries on the northern Mediterranean littoral

Theileriosis, caused by the piroplasmTheileria annulata, may spread to cattle in countries of the northern Mediterranean littoral from the west, through Morocco, and from the east, through the Middle-East. The disease is endemic in southern Spain and throughout Turkey, and epidemic in Greece, Yugoslavia and Italy. Even southern France has a resident population ofHyalomma anatolicum anatolicum, a vector tick. The paper reviews the scanty knowledge about the distribution of the disease in the area and suggests methods for improving control of the disease in countries where it is endemic or epidemic.

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Kurzfassung Die durch das PiroplasmusTheileria annulata hervorgerufene Theileriosis bedroht das Vieh in den nördlichen Mittelmeerländern vom Westen her über Marokko und vom Osten her über dan Nahen Osten. Die Krankheit tritt in Südspanien und der ganzen Türkei endemisch, in Griechenland, Jogoslawien und Italien epidemisch auf. Selbst Südfrankreich hat eine ständige Population vonHyalomma anatolicum anatolicum, einer Vektorzecke. Herausgestellt werden die geringen Kenntnisse über die Verteilung der Krankheit in diesem Bereich, und es werden Vorschläge zur Verbesserung von Kontrollverfahren bei endemischem und epidemischem Auftreten gemacht.

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Resume La theilériose provoquée par le piroplasmeTheileria annulata menace le bétail dans les pays riverains du nord de la Méditerranée à partir de l'ouest, au Maroc, et de l'est, au Moyen-Orient. La maladie est enzootique dans le sud de L'Espagne et dans l'ensemble de la Turquie et épizootique en Grèce, en Yougoslavie et en Italie. Même dans le midi de la France, il y a une population résidente d'Hyalomma anatolicum anatolicum, qui est la tique servant de vecteur. Le rapport étudie les maigres connaissances existant sur la distribution de la maladie dans la région et fait des propositions en vue d'en améliorer le contrôle dans les pays ou elle existe à l'état enzootique ou épizootique.

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Riassunto La teileriosi, causata dal piroplasmaTheileria annulata, può costituire un periocolo per i bovini nei Paesi del litorale mediterraneo settentrionale, da ovest attraverso il Marocco, e da est attraverso il Medio Oriente. La malattia ha carattere endemico in Spagna ed in tutta la Turchia, mentre si presenta epidemica in Grecia, Jugoslavia e Italia. Anche nella Francia meridionale è presente una popolazione stabile diHyalomma anatolicum anatolicum, una zecca che funge da vettore. Il presente lavoro prende in esame le scarse conoscenze che si hanno sulla distribuzione della malattia in tale area, e suggerisce delle metodiche atte a migliorare i sistemi di lotta in quei Paesi in cui l'affezione è presente in forma endemica o epidemica.

     

Elimination of the gametocytes of Theileria annulata of cattle by primaquin phosphate

Calves were experimentally infected with Theileria annulata and five drugs were administered in an attempt to exterminate the gametocytes of the parasite. The results of the experiment proved that primaquin phosphate is an effective parasiticide. The gametocytes from 30 calves treated with primaquin phosphate were exterminated completely. A further eight calves were treated with sulfamethoxypyrazine, trimethoprim, naganin and acaprin, but these drugs were not found to be effective in eliminating the gametocytes. The phase, size and colour of the gametocytes in these calves were the same as those of the four untreated control calves.     

Nitric oxide causes the macroschizonts of Theileria annulata to disappear and host cells to become apoptotic

The proliferation of Theileria annulata macroschizont-infected cell lines in vitro was significantly inhibited by nitric oxide (NO) generated by S-nitroso-N-acetyl-DL-penicillamine (SNAP). Incubation with SNAP caused the macroschizonts to disappear and host cells to become apoptotic. SNAP-derived NO also significantly inhibited the incorporation of tritiated thymidine by cultures of cells in which the schizonts had been induced to differentiate into merozoites by maintenance at 41°C instead of 37°C, the temperature used for culturing macroschizont-infected cells. These results point to NO as the mediator of macrophage anti-T. annulata activity and provide new evidence that the protective immune mechanisms which allow cattle to recover from primary infection and resist challenge may be attributed principally to the products of activated macrophages. These findings indicate that effective inactivated vaccines against T. annulata should include antigens able to stimulate the type of CD4+ T cell response which elicits macrophage activation and NO synthesis.     

Note on the transmission of Theileria annulata by Hyalomma ticks in the Sudan

Nymphs of Hyalomma impeltatum and H. dromedarii, fed as larvae on an infected calf transmitted fatal Theileria annulata infection to two susceptible calves. The significance of these findings is discussed in relation to the epidemiology of tropical theileriosis in the Sudan.     

Note on the transmission of Theileria annulata by Hyalomma ticks in the Sudan

Nymphs of Hyalomma impeltatum and H. dromedarii, fed as larvae on an infected calf, transmitted fatal Theileria annulata infection to two susceptible calves. The significance of these findings is discussed in relation to the epidemiology of tropical theileriosis in the Sudan.     

Comparison of MTT colorimetric assay and tritiated thymidine uptake for lymphocyte proliferation assays using chicken splenocytes.

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) colorimetric assay was compared with the conventional tritiated thymidine deoxyriboside (3H-TdR) incorporation for assay of lymphocyte blastogenesis using mononuclear cells isolated from the spleens of specific-pathogen-free chickens. The study was undertaken in an effort to simplify methods for assessing avian lymphocyte proliferation, specifically for evaluating response to mitogens or for indirect measurement of T-cell growth factors. The results from stimulated cells in both assay methods were significantly different from results from the control cells, and the MTT assay results regressed in a significant linear manner on counts from 3H-TdR incorporation. On this basis, the MTT assay is a valid test for evaluation of lymphocyte proliferation of chicken splenocytes.     

Comparisons among two serological tests and microscopic examination for the detection of Theileria annulata in cattle in northern Sudan

We tested the agreement between microscopic examination (ME), a surface protein-detecting enzyme-linked immunosorbent assay (TaSP ELISA) and an indirect fluorescent assay (IFA) for detection of Theileria annulata in 2661 naturally infected cattle from northern Sudan (samples collected between June 2001 and July 2002). In the ME, we detected piroplasms in 364/2661 cattle (14%), and the kappas between the ME and the serological tests were poor (TaSP ELISA 10%; IFA 8%). The TaSP ELISA detected 885/2661 cattle as positive, and the Rogan-and-Gladen corrected true prevalence of this sample was estimated to be 30%. The relative sensitivity and specificity of the IFA (compared to the previously validated TaSP ELISA) were 70.7% and 81.8%, respectively.     

基于转录组数据分析环形泰勒虫对诱导宿主细胞转化相关基因的影响

本试验主要通过研究布帕伐醌(BW720)处理前后,环形泰勒虫转化牛单核细胞(TaNM Ⅰ)转录组数据的变化情况,为进一步研究环形泰勒虫诱导宿主细胞转化的分子机制奠定基础。以环形泰勒虫感染的牛单核细胞为试验材料,设置对照组(二甲基亚砜,DMSO)和试验组(BW720),利用Illumina HiSeq4000测序平台进行高通量测序,将测序得到的原始数据(Raw reads)与GenBank和Rfam数据库进行比对过滤,通过质控获得最终数据(Clean read)。利用Venn分析软件筛选出DMSO组和BW720组中差异表达明显的基因,进行功能注释(GO)以及信号通路(KEGG)分析。随机选择10个基因,利用荧光定量PCR (qPCR)检测基因在不同处理条件下细胞中的表达量。在验证测序结果的准确性后,选出差异表达明显的基因,分析其在TaNM Ⅰ细胞的凋亡、增殖等信号通路方面的作用。结果显示：BW720组和DMSO组中分别得到6 854 019 704和6 627 265 854条Raw reads,过滤、质控后分别得到22 925 606和22 171 427条Clean reads。BW720组和DMSO组中筛选出差异表达明显的基因共计4 054个(P＜0.05),其中,2 146个基因显著上调,1 908个基因显著下调;进一步筛选出共同差异表达的基因367个,其中,显著上调的196个,显著下调的171个。同时,qPCR验证随机选择10个基因的表达趋势与转录组测序结果一致,表明测序结果可靠。然后对差异表达基因进行GO功能注释,筛选到与细胞增殖相关的20个生物学过程、15个细胞组分以及11个分子功能条目。KEGG富集分析结果显示,在Top20的信号通路中筛选出与环形泰勒虫转化细胞有关的一些信号通路,如：癌症信号通路、PI3K-Akt信号通路、MAPK信号通路等。对这些信号通路进一步分析发现：PI3KR3、FOXO1、IL23A、FZD3、AKT、MMP9等基因在环形泰勒虫转化细胞的研究中也有相关文献的报道。本研究表明,在TaNM Ⅰ细胞中DAPK1、FZD3、FOXO3、PI3KR3等可能在感染细胞无限增殖的过程中发挥作用,为后续研究TaNM Ⅰ细胞无限增殖机制奠定了基础。     

Evaluation of purified Theileria annulata sporozoite antigen from the tick Hyalomma anatolicum anatolicum

Rabbits immunized with purified sporozoites from Hyalomma anatolicum anatolicum produced antibody to Theileria annulata specific antigens as measured by the indirect fluorescent antibody test. Nonspecific fluorescence which compounded the interpretation of the test was observed. Incorporation of Eriochrome black and Evans blue as a counterstain in the diluent buffer for the conjugate significantly reduced the background fluorescence.