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 Association of High Prevalence of Trophozoites in Peripheral Blood with Lower Antibody Response to P. falciparum Infected Erythrocytes among Asymptomatic Children in Sudan.

Background. The most prominent variant surface antigens (VSAs) of Plasmodium falciparum are the var gene-encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family, which serves as a parasite-sequestering ligand to endothelial cells. In this study we have examined the antibody reactivity of autologous plasma from symptomatic and asymptomatic malaria infected children against the infected erythrocytes' surface antigens using flow cytometry. Methods. Ethidium-bromide-labelled erythrocytic mature forms of P. falciparum parasites obtained from symptomatic and asymptomatic children were sequentially incubated with autologous plasma and fluorescein isothiocyanate-conjugated (FITC) antihuman IgG. Plasma antibody reactivity was detected by flow cytometry. Results. Asymptomatic children had more prevalence of trophozoites in peripheral blood (66%) compared to symptomatic children (16%), p = 0.002. The mean percentage of infected RBCs reacting with autologous sera was 89.78 among symptomatic children compared to 79.62 among asymptomatic children (p = 0.09). Moreover, the mean fluorescence intensity (MFI) in the asymptomatic was significantly higher compared to symptomatic children (p value = 0.040). Conclusion. Variant surface antigens on Plasmodium falciparum infected RBCs from symptomatic malaria children tend to be better recognized by IgG antibodies. This may suggest a role of some IgG antibodies in severity of malaria.

The pulmonary involvement in Theileria lestoquardi naturally infected sheep.

Malignant Ovine Theileriosis (MOT) caused by Theileria lestoquardi is considered a major constraint for sheep production in many areas of the world including Sudan. Pulmonary oedema is thought to be the main cause of animal death, but the mechanism, the cell types involved and/or the probable cause of this pneumonia has yet to be defined. The present study was carried out to investigate the pulmonary involvement post T. lestoquardi infection and to identify the cell types involved in pneumonia. Apparently healthy sheep were exposed to ticks challenge in T. lestoquardi endemic area. Lungs impression smears and tissue sections for histopathology were processed. At necropsy, fifteen infected sheep revealed severe pneumonia associated with oedema and accumulation of creamy-grayish frothy exudates. The microscopic findings of examined lungs showed emphysema, congestion, collapse and proliferation of immense amount of different kinds of cells. The current study indicates that T. lestoquardi infections are accompanied with remarkable pulmonary involvements and may lead to respiratory failure and death.

Molecular identification of different Theileria and Babesia species infecting sheep in Sudan.

The epidemiological aspects of sheep piroplasmosis in Sudan are poorly studied, and further investigations using sensitive and precise techniques are required. In this study, the Reverse Line Blot (RLB) hybridization assay was used to detect and simultaneously differentiate between Theileria and Babesia species. DNA was extracted from blood collected on filter paper (n=219) from apparently healthy sheep from six different geographical localities in Sudan. Results indicated that Theileria ovis (88.6%), T. separata (20.1%), T. lestoquardi (16.4%) and T. annulata (16.4%) DNA could be detected in the blood samples. Single and mixed Theileria infections were detected in 74 (33.8%) and 124 (56.6%) respectively and T. ovis being the most prevalent species in the country. T. ovis and T. separata were reported for the first time in sheep in Sudan.

Seroprevalence of Toxoplasma gondii in Dairy Cattle with Reproductive Problems in Sudan.

Toxoplasmosis, caused by Toxoplasma gondii, is one of the most common parasitic infections of humans and other warm-blooded animals in most parts of the world. The disease is common among sheep and goats and it is recognized as one of the major causes of reproductive failure in these animals. Cattle, on the other hand, can be infected, but abortion or perinatal mortality has not been recorded. This survey was carried out to study the prevalence of this disease in cattle in Khartoum and Gazira States (Sudan). 181 sera samples collected from dairy cattle with reproductive problems were assayed for antibodies to T. gondii by ELISA. The prevalence rate of T. gondii antibodies in cattle at herd level was 44.8% (13/29). Herd level infection rates were 50% and 33.3% in Khartoum and Gazira States, respectively. The overall prevalence of T. gondii at individual level in both states was 13.3% (24/181). The prevalence was 12.7% (17/134), was 14.9% (7/47) in Khartoum and Gazira State, respectively. There was significantly higher (P < 0.05) prevalence of T. gondii antibodies in the age group less than one year old (36.4%) than in other age groups and in males (30.8%) than in females (11.9%) while no significant relationship was discerned regarding breed, location, season, or signs of reproductive disease.

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.

Prevalence and genetic diversity of Babesia and Anaplasma species in cattle in Sudan.

Disease prevalence studies are one of the most valuable tools to demonstrate the risk or impact of certain infections in local and global economies. The data obtained in these studies contribute to develop strategies for disease control. The present study aims to provide information about the prevalence of babesiosis and anaplasmosis in the northern regions of Sudan. Blood samples from four different states of Sudan were collected from apparently healthy cattle (n=692), DNA was extracted and the prevalence of Babesia and Anaplasma species was analyzed by PCR. The results confirmed the presence of Babesia bigemina, Babesia bovis and Anaplasma marginale in cattle in northern Sudan with overall prevalence rates of 4.0%, 1.9% and 6.1%, respectively. Statistical analysis revealed that the prevalence of B. bigemina, B. bovis and A. marginale varies significantly between Sudanese states as well as in different age groups, while gender seems not to have a significant effect on the prevalence of these pathogens among Sudanese cattle. The highest prevalence for B. bigemina was found in the Aljazirah State while the highest number of A. marginale positive samples was reported in River Nile.

Validation of the indirect TaSP enzyme-linked immunosorbent assay for diagnosis of Theileria annulata infection in cattle.

An ELISA based on a recombinant Theileria annulata surface protein (TaSP) was evaluated for detection of antibodies in sera from cattle exposed to tropical theileriosis in Sudan. The reference positive samples, used in this study, were from Theileria-infected populations and consisted of 80 cattle from an endemic area in Khartoum State, with high antibody titers in the indirect fluorescent antibody test (IFAT). The reference negative samples were taken from non-exposed populations and consisted of 120 cattle maintained under strict tick control at a commercial farm in Sudan. The cut-off value determined by Two-Graph Receiver-Operating Characteristic (TG-ROC) curves was set at 31.6%, based on the positive reference samples. Further diagnostic validation was performed, which consisted of the measurement of the area under the ROC (AUC) and by valid range proportion (VRP), which was 0.97 and 0.98 for the cut-off, respectively. There were no cross-reactions with antibodies raised against Babesia spp. It is concluded that the TaSP ELISA is a useful test for the diagnosis of T. annulata infection in cattle under field conditions.