Population Genetic Analysis and Sub-Structuring of Theileria annulata in Sudan.

Theileria annulata, which causes tropical theileriosis, is a major impediment to improving cattle production in Sudan. Tropical theileriosis disease is prevalent in the north and central regions of Sudan. Outbreaks of the disease have been observed outside the known endemic areas, in east and west regions of the country, due to changes in tick vector distribution and animal movement. A live schizont attenuated vaccination based on tissue culture technology has been developed to control the disease. The parasite in the field as well as the vaccine strain need to be genotyped before the vaccinations are practiced, in order to be able to monitor any breakthrough or breakdown, if any, after the deployment of the vaccine in the field. Nine microsatellite markers were used to genotype 246 field samples positive for T. annulata DNA and the vaccine strain. North and central populations have a higher multiplicity of infection than east and west populations. The examination of principal components showed two sub-structures with a mix of all four populations in both clusters and the vaccine strain used being aligned with left-lower cluster. Only the north population was in linkage equilibrium, while the other populations were in linkage disequilibrium, and linkage equilibrium was found when all samples were regarded as single population. The genetic identity of the vaccine and field samples was 0.62 with the north population and 0.39 with west population. Overall, genetic investigations of four T. annulata populations in Sudan revealed substantial intermixing, with only two groups exhibiting regional origin independence. In the four geographically distant regions analyzed, there was a high level of genetic variation within each population. The findings show that the live schizont attenuated vaccine, Atbara strain may be acceptable for use in all Sudanese regions where tropical theileriosis occurs.

Microsatellite and minisatellite genotyping of Theileria parva population from southern Africa reveals possible discriminatory allele profiles with parasites from eastern Africa.

The control of Theileria parva, a protozoan parasite that threatens almost 50% of the cattle population in Africa, is still a challenge in many affected countries. Theileria parva field parasites from eastern Africa, and parasites comprising the current live T. parva vaccine widely deployed in the same region have been reported to be genotypically diverse. However, similar reports on T. parva parasites from southern Africa are limited, especially in Corridor disease designated areas. Establishing the extent of genetic exchange in T. parva populations is necessary for effective control of the parasite infection. Twelve polymorphic microsatellite and minisatellite loci were targeted for genotypic and population genetics analysis of T. parva parasites from South Africa, Mozambique, Kenya and Uganda using genomic DNA prepared from cattle and buffalo blood samples. The results revealed genotypic similarities among parasites from the two regions of Africa, with possible distinguishing allelic profiles on three loci (MS8, MS19 and MS33) for parasites associated with Corridor disease in South Africa, and East Coast fever in eastern Africa. Individual populations were in linkage equilibrium (VDL) was observed. Genetic divergence was observed to be more within (AMOVA = 74%) than between (AMOVA = 26%) populations. Principal coordinate analysis showed clustering that separated buffalo-derived from cattle-derived T. parva parasites, although parasites from cattle showed a close genetic relationship. The results also demonstrated geographic sub-structuring of T. parva parasites based on the disease syndromes caused in cattle in the two regions of Africa. These findings provide additional information on the genotypic diversity of T. parva parasites from South Africa, and reveal possible differences based on three loci (MS8, MS19 and MS33) and similarities between buffalo-derived T. parva parasites from southern and eastern Africa.

Current status of tropical theileriosis in Northern Africa: A review of recent epidemiological investigations and implications for control.

Tropical theileriosis caused by the apicomplexan hemoparasite Theileria annulata is a tick-borne disease that constraints livestock production in parts of Europe, Asia and Africa. Four Hyalomma tick species transmit T. annulata in at least eight Africa countries (Mauritania, Morocco, Algeria, Tunisia, Egypt, Sudan, South Sudan and Ethiopia). The two dominant T. annulata vector ticks present in Africa, H. scupense and H. anatolicum, underlie two different patterns of transmission, which in turn greatly influence the epidemiology of tropical theileriosis. H. dromedarii and H. lusitanicum are also capable of transmitting T. annulata in North Africa, but their roles are associated with specific production systems and agro-ecological contexts. The emergence of resistance to the most widely used theilericidal compound, buparvaquone, continues to limit the effectiveness of chemotherapy. In addition, acaricide use is increasingly becoming unsustainable. Deployable T. annulata attenuated live vaccines established from local strains in Tunisia, Sudan and Egypt are available, and recent work has indicated that these vaccines can be protective under conditions of natural transmission. However, vaccination programmes may vary over space and time due to differences in the prevalence of disease amongst cattle populations, as well seasonal variation in vector activity. We review recent descriptive and analytical surveys on the epidemiology of T. annulata infection with reference to (a) demographic aspects such as breeds and ages of cattle herds previously exposed to distinct T. annulata infection pressures and (b) seasonal dynamics of tick activity and disease transmission. We then discuss how the wider endemic patterns that we delineate can underpin the development and execution of future vaccination programmes. We also outline options for integrated control measures targeting tick vectors and husbandry practices.

The protection afforded to cattle immunized with Theileria annulata infected cell line is enhanced by subunit vaccine candidate TaSP.

Tropical theileriosis constraints the development of the dairy industry in the Sudan and vaccination using live attenuated schizont vaccines is considered a promising measure for its control. The present study was carried out to investigate the ability of recombinant T. annulata surface protein (TaSP) to improve the efficacy of the attenuated Atbara cell line in protecting calves against field challenge. To this end, 23 cross-bred (Friesian × Kenana) calves were divided into four groups. Animals in group 1 (n = 5) were left unvaccinated. Group 2 (n = 6) received the Atbara cell line, animals in group 3 (n = 6) were immunized with three doses of TaSP on days 21, 49 and 77, while animals in group 4 (n = 6) received the cell line vaccine on day 0 and three doses of TaSP in Freund's incomplete adjuvant at days 21, 49 and 77. Twenty-eight days after the last TaSP boost, all groups were challenged by exposing them to natural field tick infestation in a region known to be endemic for tropical theileriosis. No thermal reactions, piroplasms or schizonts were observed in the immunized animals following immunization. Upon challenge, all animals showed a range of symptoms of clinical theileriosis with variable degrees of severity. The application of TaSP alone appeared to have no effect in terms of protection. The efficacy of the cell line alone was lower than the 100% level of protection against mortality observed in the group that received the combined cell line vaccine and TaSP, suggesting a synergistic effect of this combination.

Antigen gene and variable number tandem repeat (VNTR) diversity in Theileria parva parasites from Ankole cattle in south-western Uganda: Evidence for conservation in antigen gene sequences combined with extensive polymorphism at VNTR loci.

Theileria parva is a tick-transmitted apicomplexan protozoan parasite that infects lymphocytes of cattle and African Cape buffalo (Syncerus caffer), causing a frequently fatal disease of cattle in eastern, central and southern Africa. A live vaccination procedure, known as infection and treatment method (ITM), the most frequently used version of which comprises the Muguga, Serengeti-transformed and Kiambu 5 stocks of T. parva, delivered as a trivalent cocktail, is generally effective. However, it does not always induce 100% protection against heterologous parasite challenge. Knowledge of the genetic diversity of T. parva in target cattle populations is therefore important prior to extensive vaccine deployment. This study investigated the extent of genetic diversity within T. parva field isolates derived from Ankole (Bos taurus) cattle in south-western Uganda using 14 variable number tandem repeat (VNTR) satellite loci and the sequences of two antigen-encoding genes that are targets of CD8+T-cell responses induced by ITM, designated Tp1 and Tp2. The findings revealed a T. parva prevalence of 51% confirming endemicity of the parasite in south-western Uganda. Cattle-derived T. parva VNTR genotypes revealed a high degree of polymorphism. However, all of the T. parva Tp1 and Tp2 alleles identified in this study have been reported previously, indicating that they are widespread geographically in East Africa and highly conserved.

Population Genetic Analysis of Theileria annulata from Six Geographical Regions in China, Determined on the Basis of Micro- and Mini-satellite Markers.

Theileria annulata, a tick-borne apicomplexan protozoan, causes a lymphoproliferative disease of cattle with high prevalence in tropical and sub-tropical regions. Understanding the genetic diversity and structure of local populations will provide more fundamental knowledge for the population genetics and epidemics of protozoa. In this study, 78 samples of T. annulata collected from cattle/yaks representing 6 different geographic populations in China were genotyped using eight micro- and mini-satellite markers. High genetic variation within population, moderate genetic differentiation, and high level of diversity co-occurring with significant linkage disequilibrium were observed, which indicates there is gene flow between these populations in spite of the existence of reproductive and geographical barriers among populations. Furthermore, some degree of genetic differentiation was also found between samples from China and Oman. These findings provide a first glimpse of the genetic diversity of the T. annulata populations in China, and might contribute to the knowledge of distribution, dynamics, and epidemiology of T. annulata populations and optimize the management strategies for control.

Genetic diversity and population structure of Theileria parva in South Sudan.

Theileria parva is a parasitic protozoan that causes East Coast fever (ECF), an economically important disease of cattle in eastern, central and southern Africa. In South Sudan, ECF is considered a major constraint for livestock development in regions where the disease is endemic. To obtain insights into the dynamics of T. parva in South Sudan, population genetic analysis was performed. Out of the 751 samples included in this study, 178 blood samples were positive for T. parva by species-specific PCR, were collected from cattle from four regions in South Sudan (Bor = 62; Juba = 45; Kajo keji = 41 and Yei = 30) were genotyped using 14 microsatellite markers spanning the four chromosomes. The T. parva Muguga strain was included in the study as a reference. Linkage disequilibrium was evident when populations from the four regions were treated as a single entity, but, when populations were analyzed separately, linkage disequilibrium was observed in Bor, Juba and Kajo keji. Juba region had a higher multiplicity of infection than the other three regions. Principal components analysis revealed a degree of sub-structure between isolates from each region, suggesting that populations are partially distinct, with genetic exchange and gene flow being limited between parasites in the four geographically separated populations studied. Panmixia was observed within individual populations. Overall T. parva population genetic analyses of four populations in South Sudan exhibited a low level of genetic exchange between the populations, but a high level of genetic diversity within each population.

Genes encoding two Theileria parva antigens recognized by CD8+ T-cells exhibit sequence diversity in South Sudanese cattle populations but the majority of alleles are similar to the Muguga component of the live vaccine cocktail.

East Coast fever (ECF), caused by Theileria parva infection, is a frequently fatal disease of cattle in eastern, central and southern Africa, and an emerging disease in South Sudan. Immunization using the infection and treatment method (ITM) is increasingly being used for control in countries affected by ECF, but not yet in South Sudan. It has been reported that CD8+ T-cell lymphocytes specific for parasitized cells play a central role in the immunity induced by ITM and a number of T. parva antigens recognized by parasite-specific CD8+ T-cells have been identified. In this study we determined the sequence diversity among two of these antigens, Tp1 and Tp2, which are under evaluation as candidates for inclusion in a sub-unit vaccine. T. parva samples (n = 81) obtained from cattle in four geographical regions of South Sudan were studied for sequence polymorphism in partial sequences of the Tp1 and Tp2 genes. Eight positions (1.97%) in Tp1 and 78 positions (15.48%) in Tp2 were shown to be polymorphic, giving rise to four and 14 antigen variants in Tp1 and Tp2, respectively. The overall nucleotide diversity in the Tp1 and Tp2 genes was π = 1.65% and π = 4.76%, respectively. The parasites were sampled from regions approximately 300 km apart, but there was limited evidence for genetic differentiation between populations. Analyses of the sequences revealed limited numbers of amino acid polymorphisms both overall and in residues within the mapped CD8+ T-cell epitopes. Although novel epitopes were identified in the samples from South Sudan, a large number of the samples harboured several epitopes in both antigens that were similar to those in the T. parva Muguga reference stock, which is a key component in the widely used live vaccine cocktail.

Prevalence of Theileria annulata in dairy cattle in Nyala, South Darfur State, Sudan.

AIM: This study was conducted in dairy cattle in Nyala, South Darfur State, during the period from June to September 2015, to study the prevalence of bovine tropical theileriosis. MATERIALS AND METHODS: Apparently, healthy cattle of different age groups, different breeds, and from both sexes were randomly selected from seven locations. Three age groups of cattle were selected, group one <1 year old, group two 1-3 years old, and group three older than 3 years. These cattle were indigenous and cross (Friesian X zebu). A total of 150 blood samples were collected for blood smears, blood in EDTA tubes, and serum samples as well as ticks infesting cattle. Three diagnostic techniques were used such as blood smear, indirect fluorescent antibody test (IFAT), and polymerase chain reaction (PCR). RESULTS: Of 150 samples, 11 (7.3%, 95% confidence interval [CI]: 9.1-5.5) were positive for Theileria spp. piroplasms in the blood smears, 70 (46.7%, 95% CI: 35.7-57.7) were positive for Theileriaannulata antibodies in the IFAT, and of 100 samples, 39 (39%, 95% CI: 46.6-31.4) were positive for T. annulata using PCR. The prevalence of T. annulata was higher in indigenous breed than cross cattle by the three diagnostic techniques. The highest prevalence of T. annulata was recorded among cattle older than 3 years old. There were three genera and ten species of ticks found feeding on cattle. These were Rhipicephalus evertsi evertsi, Rhipicephalus decoloratus, Rhipicephalus annulatus, Hyalomma dromedrii, Hyalomma impeltatum, Hyalomma rufipes, Hyalomma anatolicum, Hyalomma truncatum, Amblyomma variegatum, and Amblyomma lepidum. CONCLUSION: The study concluded that tropical theileriosis is prevalent among dairy cattle in Nyala. H. anatolicum was found in very low numbers, suggesting other ticks may play a role in the transmission of the disease. Molecular characterization of T. annulata is recommended for accurate mapping of the disease and evaluates the magnitude problem of tropical theileriosis in South Darfur region.

Current situation of tropical theileriosis in the Sudan.

Tropical theileriosis has long been recognized as a hindrance to the development of sound dairy industry in the Sudan and is a cause of major economic losses. Serological surveys indicated that Theileria annulata infection is widespread in the country but the disease mostly affects exotic dairy breeds and their crosses with indigenous breeds. The disease has recently been identified in Darfur and southern parts of Blue Nile State in dairy farms around large urban areas where it has never been detected before. These new introductions were accompanied by the establishment of Hyalomma anatolicum tick which is the main vector of the parasite in the Sudan. The disease is routinely diagnosed using microscopic examination of stained blood and lymph node biopsy smears. More advanced techniques are mainly used for research purposes. Tropical theileriosis in the Sudan is mainly controlled by using anti-theilerial drugs and acaricide application. It is recommended that live attenuated schizont vaccines developed from locally isolated T. annulata strains be used to control the disease. In addition, every care should be taken to prevent introduction of the disease into new areas.

Development of a loop-mediated isothermal amplification method for detection of Theileria lestoquardi.

A loop-mediated isothermal amplification (LAMP) assay was developed for the diagnosis of Theileria lestoquardi infection. The primers were designed based on the clone-5 sequence of T. lestoquardi. The specificity and sensitivity of the assay were established. Analysis of the specificity showed that the selected LAMP primers amplified the target sequence from T. lestoquardi DNA successfully, while no amplification was seen with DNA from Theileria annulata, Theileria ovis, Babesia ovis, Anaplasma ovis, or ovine genomic DNA. The specificity of the LAMP product was further confirmed by restriction digestion and sequencing. The sensitivity of the LAMP assay was analyzed in comparison to PCR resulting in a detection limit of 10 fg/µl of plasmid DNA containing the clone-5 sequence. The suitability for utilizing the LAMP assay in the field for the diagnosis of T. lestoquardi infection was tested on 100 field samples collected in Sudan and compared with results obtained by PCR. The relative specificity and sensitivity of the established LAMP assay was determined to be 92.1% and 87.5%, respectively, indicating that it may be regarded as an alternative molecular diagnostic tool to PCR which could be used for epidemiological surveys on T. lestoquardi infection.

Seroprevalence of East Coast fever in Central Equatoria State, South Sudan.

A cross-sectional survey was conducted in 2005 in different cattle camps in Juba, Mangalla and Terekeka localities of Central Equatoria State, South Sudan. Serum samples were collected from 514 cattle of different age groups. Samples were analysed using an indirect enzyme-linked immunosorbent assay (ELISA) with commercially available polymorphic immunodominant molecule (PIM) ELISA kits. The overall serological prevalence of Theileria parva was 70.8% (364/514). The highest rate of prevalence was observed in Mangalla (91.2%) and the lowest in Juba (61.8%), with Terekeka recording 71.8%. Regarding the age groups, older cattle (over four years of age) showed a significantly higher rate of prevalence (p>0.001) than calves (below one year of age). The implications of these results in the overall epidemiology of East Coast fever in South Sudan are discussed and possible recommendations for future implementation of disease control measures are outlined.

First confirmed report of outbreak of malignant ovine theileriosis among goats in Sudan.

An outbreak of malignant ovine theileriosis among goats was confirmed and documented. In this outbreak, 16 out of 22 (72.7%) goats died within 4 days showing clinical signs of malignant ovine theileriosis as well as in the postmortem findings. The goats were reared in a mixed flock with sheep in Atbara Town, Northern Sudan. The infection was detected microscopically and confirmed serologically by IFA test and molecularly by PCR technique using specific primer for Theileria lestoquardi. Hyalomma anatolicum was the most prevalent (dominant) tick species found in the farm. It is recommended to undertake future research on the role of goats on the epidemiology of malignant ovine theileriosis.

Ticks infesting cattle in Central Equatoria region of South Sudan.

Ticks infesting cattle represent a serious problem for improvement of cattle productivity in South Sudan. There has been limited information on ticks and tick-borne diseases in southern Sudan. This study was initiated to update the current distribution of ticks infesting cattle in the Central Equatoria region of South Sudan. The surveys for the present study were conducted at various cattle camps in Juba, Mangalla and Terekeka between December 2004 and June 2005. A total of 2322 ticks were collected from the bodies of 88 randomly selected cattle. Ticks were preserved in 70% ethanol for later identification. Seven ixodid tick species were found to infest cattle in Juba whilst six species were recorded in Mangalla and only four species in Terekeka. Amblyomma variegatum was the most common and widely distributed species found on cattle across all the study locations. Amblyomma lepidum was not found during this study. Based on these findings, it would be advisable to preempt the situation and institute containment procedures before possible East Coast fever outbreaks occur.

Epidemiological studies on tropical theileriosis (Theileria annulata infection of cattle) in Kurdistan Region, Iraq.

In an ad hoc survey conducted during 2006, the epidemiology of tropical theileriosis in Kurdistan Region, Iraq, was addressed. For this purpose, a total of 299 blood samples were collected from female cattle older than 1 year reared under open system management in Duhok (n = 99), Sulaimanyia (n = 100) and Erbil (n = 100) governorates. The samples were subjected to TaSP indirect ELISA as well as polymerase chain reaction (PCR) and nested PCR assays. The results indicated that the seroprevalence was 77.9%, and PCR reported an infection rate of 68.9% in the Kurdistan Region of Iraq. The implication of the results in the epidemiology of tropical theileriosis in the region is discussed with emphasis on comparisons between the two tests used and recommendations for the future work are outlined.

Development of loop-mediated isothermal amplification (LAMP) assay for rapid diagnosis of ovine theileriosis in China.

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid detection method in which the target deoxyribonucleic acid (DNA) can be efficiently amplified with high specificity and sensitivity under isothermal conditions using a set of either four or six specific primers. In this study, we have identified a conserved sequence for Theileria luwenshuni (UTRlu8) and for T. uilenbergi (UTRu6) suitable for designing a set of six primers for the simultaneous detection by LAMP of these pathogens causing theileriosis in sheep and goats in China. LAMP was performed at 63 degrees C, and the amplified DNA was detectable within 15 min. The specificity of the reaction was confirmed through EcoRI restriction enzyme digestion analysis and sequencing. The assay was proven sensitive since specific amplification was obtained from 0.1 pg DNA of T. luwenshuni or T. uilenbergi. The LAMP assay was evaluated by testing 86 field samples in comparison to the reverse line blot method, showing a sensitivity and specificity of 66.0% and 97.4%, respectively. These results indicate that the LAMP assay is rapid and simple to run, cost effective, sensitive, and specific and has potential usefulness for application in diagnostics of and epidemiological studies on T. luwenshuni and T. uilenbergi infection of small ruminants.

Innate immunity to tropical theileriosis.

The intracellular protozoan parasite Theileria annulata causes a severe, and often fatal, disease of pure and cross-bred cattle in tropical and subtropical countries. The present review refers to the importance of innate immunity as far as it is known to date in this infectious disease. Specifically, macrophages and the mediators produced by these cells are outlined. In addition, the latest findings concerning cattle breed differences in susceptibility to T. annulata infection in relation to macrophage activation are discussed.

Influence of subculturing on gene expression in a Theileria lestoquardi-infected cell line.

In this study potential molecular markers for identification of attenuation in a Theileria lestoquardi-infected cell line to be used in vaccination trials were identified. Two markers associated with attenuation in Theileria annulata vaccine strains were analyzed (metalloproteinase activity and TNF? mRNA expression). The result showed a decreased activity of MMP 9 and decreased mRNA expression of TNF? with increasing passage number. Suppression subtractive hybridization was used to identify potential new markers of attenuation. Random screening revealed nine differentially expressed genes, one from the parasite and eight from the host. Quantitative real time-PCR confirmed mRNA expression of the parasite vacuolar H+ATPase to be downregulated at higher passages.