Theileria parva seroprevalence in traditionally kept cattle in southern Zambia and El Nino

Sero-epidemiological surveys involving 27,526 cattle over a period of 8 years show that Theileria parva, the parasite causing East Coast fever (ECF) is found throughout southern Zambia. Higher values of T. parva sero-prevalence were observed in the plateau districts of Monze, Choma and Mazabuka than in the valley districts of Siavonga and Sinazongwe. Our results reveal a strong association between high T. parva sero-prevalence and the presence of the periodic climatic phenomenon known as the El Nino Southern Oscillation. More T. parva sero-positive samples were recorded during El Nino years (1997/98) (P<0.001) than other years in the study period. From this association, we conclude that Multiple El Nino Southern Oscillation Indices can be used to predict years with high or low ECF infection prevalence thereby contributing to the improved control of ECF in the area.     

Immunity in theileriosis

The theilerioses can be separated on the basis of their principal pathogenic features, into a lymphoproliferative group caused by Theileria parva and T. annulata in cattle, and T. hirci in goats and sheep, and a haemoproliferative group caused by T. sergenti and T. mutans both in cattle. In the former group, proliferation of parasites within lymphoid cells followed by lymphodestruction are the main pathogenic features; whereas in the latter group, invasion and destruction of erythrocytes, causing anaemia, are more important. In addition, a number of other theilerial parasites which cause mild or inapparent infections, are found in domestic livestock. This review focuses on T. parva, the causative agent of East Coast fever (ECF) in cattle in East and Central Africa, because it is the most pathogenic species and the immunology of ECF has been more intensively studied than that of the other theilerioses.     

Effect of different East Coast Fever control strategies on fertility, milk production and weight gain of Sanga cattle in the Central Province of Zambia

Five different East Coast Fever (ECF) (Theileria parva infection) control strategies, based on tick control and/or ECF immunization, were tested in groups of traditionally managed Sanga cattle in the Central Province of Zambia over a period of 2.5 years. Two groups were kept under intensive tick control (sprayed weekly), one group immunized and one non-immunized. Two further groups were under no tick control, one group immunized and one non-immunized, while a fifth group was immunized against ECF and maintained under strategic tick control (18 sprays per year). Tick control increased milk production and weight gain but not fertility. Immunization had neither marked detrimental nor beneficial effects on the cattle productivity. The combination of strategic tick control and immunization resulted in the highest level of production and at the same time reduced the potential risk from other tick-borne diseases.     

Construction of a genetic map for Theileria parva: identification of hotspots of recombination

The tick-borne protozoan parasite Theileria parva is the causal agent of East Coast Fever (ECF), a severe lymphoproliferative disease of cattle in eastern, central and southern Africa. The life cycle of T. parva is predominantly haploid, with a brief diploid stage occurring in the tick vector that involves meiotic recombination. Resolved genetic studies of T. parva are currently constrained by the lack of a genome-wide high-definition genetic map of the parasite. We undertook a genetic cross of two cloned isolates of T. parva to construct such a map from 35 recombinant progeny, using a genome-wide panel of 79 variable number of tandem repeat markers. Progeny were established by in vitro cloning of cattle lymphocytes after infection with sporozoites prepared from Rhipicephalus appendiculatus ticks fed on a calf undergoing a dual infection with the two clonal parental stocks. The genetic map was determined by assigning individual markers to the four chromosome genome, whose physical length is approximately 8309 kilobasepairs (Kb). Segregation analysis of the markers among the progeny revealed a total genetic size of 1683.8 centiMorgans (cM), covering a physical distance of 7737.62 Kb (∼93% of the genome). The average genome-wide recombination rate observed for T. parva was relatively high, at 0.22 cM Kb⁻¹ per meiotic generation. Recombination hot-spots and cold-spots were identified for each of the chromosomes. A panel of 27 loci encoding determinants previously identified as immunorelevant or likely to be under selection were positioned on the linkage map. We believe this to be the first genetic linkage map for T. parva. This resource, with the availability of the genome sequence of T. parva, will promote improved understanding of the pathogen by facilitating the use of genetic analysis for identification of loci responsible for variable phenotypic traits exhibited by individual parasite stocks.     

Trypanosoma vivax: adaptation of two East African stocks to laboratory rodents

Two Trypanosoma vivax stocks from East Africa have been adapted to rats and mice. Adaptation was induced by rapid passage at two- to four-day intervals in sublethally irradiated rats. After 200 such passages, the two stocks gave rise to parasitemias of 10(9)-10(10) trypanosomes/ml in peripheral blood, and the infection was fatal in 90% of the rats. By passaging the rat-adapted T. vivax into normal mice at two- to three-day intervals for over 200 passages, the two stocks also became pathogenic to mice. One of the stocks was also capable of maintenance in non-irradiated rats. The two stocks displayed a marked degree of pleomorphism in irradiated and non-irradiated rats and mice. In the early rising parasitemia, the organisms were predominantly short, with a well formed undulating membrane, a pointed posterior end, and a large terminal kinetoplast. As parasitemia approached its peak, the organisms transformed into long, slender forms with an inconspicuous undulating membrane, an elongated posterior end, and a sub-terminal kinetoplast. The short forms associated with the early, rising parasitemia were more infective for mice than the long forms encountered at peak parasitemia. Although the two rodent-adapted stocks retained their pathogenicity for goats, neither the original stocks nor their corresponding rodent-adapted stocks could be cyclically transmitted by tsetse flies. The availability of these stocks will greatly facilitate investigations on East African T. vivax which would otherwise be difficult to carry out in experimental rodents.     

Application of a reverse line blot assay to the study of haemoparasites in cattle in Uganda

Recent advances in genomic technology have focused many veterinary researchers on the possibility of producing one multivalent recombinant vaccine against all the haemoparasites that infect cattle in the tropics. Before such a vaccine is developed it is essential to define target cattle populations as well as the range of anti-pathogen vaccines required in order to control disease. To further this objective, we have evaluated a reverse line blot (RLB) assay, which simultaneously detects the principal tick transmitted protozoan and rickettsial cattle pathogens, in different epidemiological scenarios in Uganda. A critical question is the sensitivity, particularly in relation to detecting carrier animals. As Theileria parva is considered to be the most important pathogen in the region, we assessed the sensitivity of the RLB assay for T. parva and showed that 1-2 x 10(3) parasites per ml of blood could be detected-a level comparable with previously developed PCR methods and well below conventional microscopic detection. We applied the RLB assay to evaluate the differences in pathogen profiles between crossbred and indigenous cattle and show that there were different profiles, with a low prevalence of T. parva and Theileria taurotragi in the indigenous cattle compared to a high prevalence in the crossbred cattle. In contrast, we show higher prevalences of Theileria mutans and Theileria velifera in the indigenous compared to the crossbred cattle. Interestingly Anaplasma marginale, Babesia bovis and Babesia bigemina were of low prevalence but a high prevalence of Ehrlichia bovis was seen, raising the question of whether this rickettsial species could be pathogenic in cattle. Analysis of animals with clinical symptoms of East Coast Fever showed that, while T. parva is a major cause of these symptoms, T. mutans and possibly T. taurotragi and T. velifera, may also cause clinical disease. Overall, the results presented here highlight the complexity of tick-borne pathogen infections in cattle in Uganda.     

Development of recombinant antigen vaccines for the control of theileriosis

Immunization against Theileria parva involves infection with sporozoites and simultaneous treatment with a long-acting tetracycline. For T. annulata, immunization is achieved by inoculation of attenuated schizont-infected lymphocytes. The two methods are inadequate because of the use of live organisms and the methods are also bedevilled by the multiplicity of strains, particularly of T. parva. For these reasons, alternative methods of control are being sought. In this review an attempt is made to highlight advances towards subunit vaccines against T. parva and T. annulata. Several candidate antigens which are thought to induce protective responses have been identified and recombinant DNA technology is being employed to produce these antigens in bulk. Relevant antigens may be delivered as subunit vaccines by using recombinant vaccinia virus or attenuated Salmonella spp. as carriers of the genes expressing these antigens. It is likely that effective vaccines against T. parva and T. annulata will have to elaborate immune responses against both the sporozoite and schizont stages of the parasite.     

Trypanosoma cruzi: variability of stocks from Colombia determined by molecular karyotype and minicircle Southern blot analysis

Nineteen Trypanosoma cruzi stocks, most of them of wild origin, and four Trypanosoma rangeli stocks from Colombia were analysed by molecular karyotype analysis with cloned DNA cruzipain as the probe. Another 27 cloned stocks of T. cruzi from different geographic areas of South America were used as reference for T. cruzi lineages. Phenetic analysis of chromosome size polymorphism demonstrated a great variability of Colombian T. cruzi stocks, suggesting that most belong to lineage I, although two of them belong to lineage II. The 2 lineage II T. cruzi, 17 T. cruzi lineage I, and 3 T. rangeli stocks from Colombia were studied further by Southern blot analysis with a panel of kinetoplast DNA minicircle probes. Hybridisation results indicate that the two T. cruzi II stocks are genetically distant from each other and from T. cruzi lineages IIb, IId, and IIe from Chile. Finally, T. cruzi minicircle probes do not cross-hybridise in any stringency condition tested with T. rangeli minicircles, a clear indication that these parasites can be easily distinguished by this method.     

Molecular and morphological studies of Brazilian Trypanosoma evansi stocks: the total absence of kDNA in trypanosomes from both laboratory stocks and naturally infected domestic and wild mammals

The kinetoplast DNA (kDNA) minicircle molecules of 14 Brazilian stocks of Trypanosoma evansi were studied by morphological approaches (Giemsa and 4'-6'-diamidino-2-phenylindole staining and transmission electron microscopy) and molecular approaches (probing with an oligonucleotide complementary to the minicircle origin of replication and polymerase chain reaction amplification of a minicircle sequence). All methods indicated the absence of both a typical kinetoplast and kDNA minicircles, even in a very small number of parasites of a single stock or in small numbers of copies of molecules per cell. We did not detect any altered kDNA molecules. There were no kDNA molecules in either old or new stocks of T. evansi maintained by successive passages in mice. Similarly, no kDNA minicircles were detected in trypanosomes in blood smears from naturally infected domestic and wild animals. Thus, the total absence of kDNA in Brazilian stocks of T. evansi from both domestic and wild mammals is probably the natural state of Brazilian T. evansi.     

A Sporozoite-based vaccine for Theileria parva

East Coast fever, which is caused by Theileria parva infection in cattle, is of major economic importance in eastern and central Africa. Until recently, the only available method of immunization against East Coast fever was the infection with live sporozoites and simultaneous treatment with a long-acting oxytetracycline. This method has two major disadvantages: (I) it uses live organisms; and (2) the immunity engendered is parasite strain specific. In this article, Antony Musoke, Vishvonath Nene and Subhosh Morzoria review the progress made in developing an alternative method o f immunization based on a defined sporozoite antigen.     

Methods currently used for the control of East Coast fever: their validity and proposals for future control strategies

Restriction of cattle movements, vector control, treatment and immunization are identified as the main control methods against East Coast fever (ECF). The effectiveness of these methods is very much influenced by cultural practices, economic and political pressures and development of resistance by ticks to acaricides. The proposed strategies for the future include continued restriction of cattle movements, less intensive vector control in enzootic areas so as to maintain enzootic stability, chemotherapy and immunization of the improved cattle. In addition, research efforts to evaluate strategic vector control, develop cheaper drugs, improve the quality and delivery of the stabilate vaccine, and identify and mass produce appropriate immunogenic subunit vaccines should be intensified.     

Genetic relatedness among Trypanosoma evansi stocks by random amplification of polymorphic DNA and evaluation of a synapomorphic DNA fragment for species-specific diagnosis.

In this study we employed randomly amplified polymorphic DNA patterns to assess the genetic relatedness among 14 Brazilian Trypanosoma evansi stocks from domestic and wild hosts, which are known to differ in biological characteristics. These akinetoplastic stocks were compared with one another, to three Old World (Ethiopia, China and Philippines) dyskinetoplastic stocks of T. evansi, and also with Trypanosoma equiperdum, Trypanosoma brucei brucei, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. Randomly amplified polymorphic DNA analysis showed limited heterogeneity in T. evansi stocks from different hosts and geographical regions of the world, or in other species of the subgenus Trypanozoon. However, minor variations generated random amplification of polymorphic DNA analysis disclosed a pattern consisting of a unique synapomorphic DNA fragment (termed Te664) for the T. evansi cluster that was not detected in any other trypanosome species investigated. Pulsed field gel electrophoresis analysis demonstrated that the Te664 fragment is a repetitive sequence, dispersed in intermediate and minichromosomes of T. evansi. Based on this sequence, we developed a conventional PCR assay for the detection of T. evansi using crude preparations of blood collected either on glass slides or on filter paper as template DNA. Our results showed that this assay may be useful as a diagnostic tool for field-epidemiological studies of T. evansi.     

Alteration of host cell phenotype by Theileria annulata and Theileria parva: mining for manipulators in the parasite genomes

The apicomplexan parasites Theileria annulata and Theileria parva cause severe lymphoproliferative disorders in cattle. Disease pathogenesis is linked to the ability of the parasite to transform the infected host cell (leukocyte) and induce uncontrolled proliferation. It is known that transformation involves parasite dependent perturbation of leukocyte signal transduction pathways that regulate apoptosis, division and gene expression, and there is evidence for the translocation of Theileria DNA binding proteins to the host cell nucleus. However, the parasite factors responsible for the inhibition of host cell apoptosis, or induction of host cell proliferation are unknown. The recent derivation of the complete genome sequence for both T. annulata and T. parva has provided a wealth of information that can be searched to identify molecules with the potential to subvert host cell regulatory pathways. This review summarizes current knowledge of the mechanisms used by Theileria parasites to transform the host cell, and highlights recent work that has mined the Theileria genomes to identify candidate manipulators of host cell phenotype.     

Investigations into human serum sensitivity expressed by stocks of Trypanosoma brucei evansi

Trypanosoma bruceievansi, a widely distributed species of trypanosome infecting different livestock species in many countries in Africa, Asia and South America, has recently been reported as a pathogen causing a case of human trypanosomiasis in India. To date, there is little information regarding the natural resistance of animal-infective stocks of T. b. evansi to normal human serum (NHS). In this study, we investigated the degree of sensitivity to NHS of 15 stocks of T. b. evansi from different geographical origins and found that 10 of the stocks were completely susceptible to the action of NHS; parasites disappeared from the blood of infected mice within a few hours and the mice remained free from infection for more than 1 month. The remaining five stocks were partially resistant to NHS; although parasites initially disappeared from the circulation more than 50% of the mice showed relapse infection 10-18 days later. Studies on one stock, T. b. evansi STIB 810, showed that the changes in parasitaemia in the infected mice were correlated with the amount of NHS inoculated (correlation factor −0.584 and P = 0.001). When this stock was passaged 25 times in mice in the presence of NHS it was found that the trypanosomes’ serum resistance increased compared with the parent stock from which they were derived; 40% of the passaged parasites survived after in vitro incubation with 50% NHS for 7 h, while only 1% of individual trypanosomes of the parent stock survived under the same conditions. These findings show, to our knowledge for the first time, that human serum sensitivity varies amongst stocks of T. b. evansi, that some of them naturally display resistance to NHS and that, furthermore, T. b. evansi serum resistance can be increased by sub-passage in the presence of NHS.     

Recognition of soluble Theileria parva antigen by bovine helper T cell clones: characterization and partial purification of the antigen

Theileria parva-specific bovine BoT4+ Th cell clones were used to characterize Ag associated with T. parva schizont-infected lymphoblastoid cells. All of the clones tested responded to cells infected with the immunizing (Muguga) as well as heterologous stocks of T. parva, indicating that the T cells are specific for an Ag shared by several geographically diverse parasites. The response was apparently MHC-restricted, and induced by Ag expressed on the infected cell surface. In the presence of autologous APC, the clones were also stimulated by a soluble high speed supernatant (HSS), but not by a schizont membrane-enriched, subcellular fraction prepared from homogenates of infected cells. The clones produced IFN-gamma and T cell growth factor in response to HSS. The soluble Ag was absent in cells from which schizonts had been eliminated by treatment with the anti-theilerial drug, parvaquone. Fractionation of HSS by hydroxylapatite chromatography revealed two antigenic peaks that separated from the majority of the protein. Fractionation of HSS by gel filtration with the use of HPLC revealed several peaks of activity ranging in Mr from 270 kDa to less than 5 kDa. Further fractionation of HSS by both hydroxylapatite chromatography and gel filtration yielded three major peaks of activity (Mr 43, 12, 4.2 kDa). We conclude that a T cell-dependent schizont-associated soluble Ag is also expressed on the surface of T. parva-infected cells.     

Trypanosoma brucei gambiense: study of population genetic structure of Central African stocks using amplified fragment length polymorphism (AFLP)

To understand the maintenance and resurgence of historical Human African Trypanosomiasis (HAT) foci, AFLP was used to genotype 100 Central African Trypanosoma brucei s.l. stocks. This technique confirmed the high genetic stability of T. b. gambiense group 1 stocks and the micro genetic variability within Central African T. b. gambiense stocks. It revealed several T. b. gambiense genotypes and allowed the identification of minor and major genotypes in HAT foci. The coexistence of these genotypes in the same focus suggests that clustering of stocks according to HAT focus does not provide the true genetic picture of trypanosome circulating within the disease focus because the minor genotypes are generally underestimated. The presence of minor and major genotypes in HAT foci may explain the persistence and the resurgence of Central African sleeping sickness foci.     

肺心病患者血清甲状腺激素的变化

本文分析30例慢性肺心病心衰并呼衰患者(心衰并呼衰组)及30例慢性肺心病心衰无呼衰患者(心衰无呼哀组)和慢性肺心病死亡组的血清甲状腺激素水平。结果表明心衰并呼衰组T_3、T_4水平均值显著低于心衰无呼衰组和健康组,心衰无呼衰组T_3水平均值显著低于健康组,肺心病死亡组T_3、T_4水平均值显著低于存活组,并发现血清T_3、T_4水平与动脉血氧分压(PaO_2)呈正相关。作者认为T_3明显降低是重症肺心病的损伤性结果,预示病情严重,预后差。而T_4明显下降,可能是死亡的信号之一。     

Mexican Trypanosoma cruzi stocks: analysis of minicircle kDNA homologies by cross-hybridization

Homologies of minicircle kDNA of 27 Mexican stocks were studied by cross-hybridization with four kDNA probes derived from three reference stocks belonging to groups Trypanosoma cruzi I (SO34 cl4 and Silvio) and T. cruzi II (MN) and one Mexican stock. High homologies were only observed with Silvio (six stocks) and Mexican probes (11 stocks). After 30 min exposure (low homology) additional stocks were recognized with SO34 cl4 (three stocks) and Silvio (six stocks) probes; with the Mexican probe only five stocks remained non-reactive. All the stocks were typed by isoenzyme (16 loci) and Mexican stocks belonged to T. cruzi I. Hybridization patterns were not strictly correlated with the observed clustering and cross-hybridization of kDNA minicircles is not available to distinct Mexican stocks.