Prevalence of ticks (Acari: Ixodidae) and Theileria annulata infection of cattle in Gezira State, Sudan.

This study was conducted in Gezira State, central Sudan during January, May and August 2014 to determine species of ticks infesting cattle and prevalence of Theileria annulata infection and antibodies. A total of 200 head of cattle were sampled for ticks, blood smears, lymph node biopsy smears and sera from five study areas. Zebu (Butana and Kenana) and cross-bred (Zebu X Friesian) cattle of both sexes were sampled. Cattle age groups were younger than one year, one year to younger than four years and four years and older. Coat colour of cattle was recorded. The results revealed that cattle were infested with Rhipicephalus evertsi evertsi (n = 562, 51.6%), Hyalomma anatolicum (n = 289, 26.5%), H. rufipes (n = 70, 6.4%), R. sanguineus sanguineus (n = 59, 5.4%), R. decoloratus (n = 39, 3.6%), Amblyomma lepidum (n = 35, 3.2%), H. impeltatum (n = 26, 2.4%) and H. dromedarii (n = 9, 0.8%). Out of 200 blood and biopsy smears, 33 (16.5%) showed piroplasms for Theileria spp., while 29 (14.5%) were positive for Theileria spp. schizonts. Seropositivity for T. annulata using indirect fluorescent antibody (IFA) test was 94 (47%). It is concluded that there are three genera of hard ticks and eight species in Gezira State and tropical theileriosis is endemic in the state. These findings are the first records in Gezira State, Central Sudan and it is highly recommended to consider the endemicity of tropical theileriosis particularly in cattle upgrading programmes.

Molecular, serological and parasitological survey of Theileria annulata in North Kordofan State, Sudan.

This survey was conducted to detect Theileria annulata infection in indigenous (Zebu) and cross (indigenous × Friesian) cattle (n 604) of different sex & age groups, and to investigate prevalence of ticks infesting cattle in North Kordofan State, Sudan. Ticks, blood smears, serum samples and blood spots on filter paper were collected from four municipalities at four monthly intervals during the period from August 2008 to July 2009. Out of 604 blood smears, 69 (11.4%, 95% CI: 16.9-5.9) were positive for Theileria piroplasms; whereas, out of 240 serum samples, 164 (68.3%, 95% CI: 89.3-47.3) were positive for antibodies against T. annulata using IFA test. Using PCR, out of 100 blood spots on filter paper, 8 (8%, 95% CI: 9.6-6.4) revealed T. annulata DNA. The highest prevalence of Theileria spp. piroplasms and T. annulata antibodies was recorded in August (15.9 ±â€¯4.6) followed by February (13.3 ±â€¯3.8) and the lowest was in May (3.9 ±â€¯1.0). Among cattle breeds, the highest prevalence of piroplasms (11.9 ±â€¯5.0) and T. annulata antibodies (68.9 ±â€¯10) was among zebu cattle. PCR assays revealed the highest prevalence rate of T. annulata DNA in February (12.5 ±â€¯1.4) followed by August (6.1 ±â€¯0.7) and the lowest was in May (5.7 ±â€¯0.7). According to cattle breeds, T. annulata DNA was detected in cross-bred cattle (3.3 ±â€¯0.4) more than zebu cattle (10 ±â€¯1.6). Seven tick species belonging to four genera and were identified, Amblyomma lepidum, Hyalomma dromedarii, H. impeltatum, H. rufipes, Rhipicephalus evertsi evertsi, Rhipicephalus decoloratus, R. annulatus. The most abundant tick species was H. rufipes followed by H. impeltatum, R. e. evertsi, H. dromedarii, R. decoloratus, A. lepidum and R. annulatus. Theileria annulata has been detected for the first time in North Kordofan State, though at a low prevalence rate. This finding is an alarming situation since tropical theileriosis is an emerging disease and it is widely believed that this economically important tick-borne parasite does not exist in this state. The possible alternative vector(s) of T. annulata in the absence of the known vector H. anatolicum is discussed.

Genotyping of Theileria lestoquardi from sheep and goats in Sudan to support control of Malignant Ovine Theileriosis.

Theileriosis, caused by parasitic protozoa of the genus Theileria parasites, are among the major tick-borne diseases of ruminant livestock. The largest economic losses are attributed in particular to those caused by the leukoproliferative species of Theileria: T. parva, T. annulata and T. lestoquardi. Theileria lestoquardi is transmitted by Hyalomma ticks and causes malignant ovine theileriosis (MOT), a disease that is particularly prevalent in Sudan. The disease is considered of a high economic importance in Sudan, where export of sheep is a major component of the national economy. A live vaccine based on a Sudanese isolate of T. lestoquardi (Atbara strain) was previously developed for the control of MOT in Sudan, but not yet deployed in the field. The present study aims to genetically characterize and compare samples of T. lestoquardi circulating in Sudan as well as the live vaccine isolate in order to understand vaccine breakthroughs and failure that may occur. Sheep and goats blood samples were collected from six regions in Sudan that are known to be endemic for T. lestoquardi infection or have experienced outbreaks of MOT. Blood samples infected with T. lestoquardi were identified by PCR or RLB. Genotyping was carried out by (1) sequencing the homologues of two T. parva CD8+ T cell antigen genes, Tp1 and Tp2, and (2) using a panel of seven micro- and mini-satellite markers. A total of 100 T. lestoquardi positive field samples and the T. lestoquardi (Atbara) vaccine were genotyped. The results showed that all samples had mixed genotypes, with several alleles identified at one or more loci. The gene diversity ranged from 0.7840 (TS8) to 0.2133 (TS12) with mean values of 0.5470. PCA revealed three clusters of the parasite in Sudan; interestingly one independent cluster was clearly seen, corresponding to the vaccine isolate. The T. lestoquardi Tp1 homologue showed higher homology with T. annulata than with T. parva sequences included the defined single CD8+ T cell target epitope region. The result indicates that multiple genotypes are a common feature of T. lestoquardi infection in Sudan. Both genotyping and the sequencing results clearly showed that the vaccine isolate is highly distinct from the field samples. This finding raised the question whether vaccination with the prepared lived vaccine will effectively protect animals against challenges by the field isolates of T. lestoquardi. The results of this work will inform on the best approach for controlling MOT in Sudan.

The Emergence of Theileria parva in Jonglei State, South Sudan: Confirmation Using Molecular and Serological Diagnostic Tools.

A cross-sectional survey was carried out in four counties of Jonglei State, South Sudan, between May and June 2012 to determine the distribution and northern limit of Theileria parva, the causative agent of East Coast fever in cattle, and its tick vector Rhipicephalus appendiculatus, as a prerequisite to the deployment of relevant control strategies. A total of 1636 ticks, 386 serum samples and 399 blood samples were collected from indigenous, apparently healthy, cattle of different age groups. Tick species were identified morphologically, and the identity of R. appendiculatus was confirmed by DNA barcoding. Overall, the T. parva infection rate in R. appendiculatus was 25% as shown by nested PCR. ELISA was used to assess antibodies to T. parva, and the overall seroprevalence was 22.8%. PCR of the blood samples showed 55 (13.8%) were positive for T. parva. This is the first molecular confirmation of T. parva DNA in areas north of Juba, where it was previously known and established. The northern limit of T. parva was determined as N°06.17.792, about 242 Km north from Juba. Implication of this limit on the epidemiology and control of ECF is discussed.

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.

Comparison between reverse line blot and enzyme-linked immunosorbent assay in diagnosis of major tick-borne diseases of cattle in Southern Sudan.

The performance of reverse line blot (RLB) in detecting DNA of Theileria parva, Theileria mutans and Babesia bigemina was assessed in comparison with specific antibody detection using indirect enzyme-linked immunosorbent assays (ELISA) for the same parasites. Among 90 field samples from Central Equatoria state, Southern Sudan, ELISA reported more positive samples than RLB did. The concordance of RLB showed 66.7%, 81.1% and 48.9% relative to the results of ELISA for T. parva, T. mutans and B. bigemina respectively. It has to be borne in mind that the results of ELISA might represent previous infections, while that of RLB would not only reflect an active infection, but also a carrier status. Therefore, the selection of the test would depend on the specific aims of the study.

Development of a loop-mediated isothermal amplification (LAMP) assay for rapid diagnosis of Babesia canis infections.

Vector-borne diseases are rising in interest due to global warming, which is believed to impact on the distribution of vectors into new areas thus influencing the occurrence and epidemiology of vector-borne pathogens. Babesia canis belongs to the Piroplasmidae and there are three described subspecies, namely B. canis canis, B. canis rossi and B. canis vogeli. They are each transmitted by a different tick-species, Dermacentor reticulatus, Haemaphysalis leachi and Rhipicephalus sanguineus, respectively. There are also differences in the geographical distribution and pathogenicity to dogs of each subspecies. In this study, we aimed to establish a rapid and easy to perform DNA-based test using loop-mediated isothermal amplification to detect all three Babesia canis subspecies in one assay.

Seroprevalence of tick-borne diseases among cattle in the Sudan.

This study was carried out to determine the prevalence of Theileria annulata, Theileria mutans, Babesia bigemina, and Anaplasma marginale antibodies among cattle in the Sudan. A total of 600 serum samples were collected from indigenous (zebu) and crossbred cattle (zebu x Friesian) of both sex and different age groups. Indirect enzyme-linked immunosorbent assay was used to assess antibodies against tick-borne diseases in apparently healthy cattle. The overall prevalence rates of T. annulata, T. mutans, B. bigemina, and A. marginale antibodies were found to be 30.8%, 6.1%, 10.7%, and 38.9%, respectively. The highest seroprevalence of T. annulata was reported in Atbara and El Damer, Northern Sudan. There were no significant associations for the seroprevalence of all tick-borne diseases reported among different age groups. Although there were no significant differences between the two breeds of cattle examined for T. annulata, T. mutans, and B. bigemina antibodies, there was a significant difference for prevalence of antibodies against A. marginale, with highest percentages of antibodies in indigenous cattle. Six different combinations of mixed infection were detected. This is the first report in which antibodies against A. marginale among cattle in Northern Sudan is reported. The findings imply that antibodies to tick-borne infections are widely distributed in the region. The need for further investigations using more advanced techniques is recommended.

Development and evaluation of a loop-mediated isothermal amplification method for diagnosis of tropical theileriosis.

A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for diagnosis of tropical theileriosis. A set of six primers was designed based on the unique gene of Theileria annulata (Theileria annulata strain Ankara hypothetical protein (GeneDB TA04795). The protocol for the reaction was setup and the specificity and sensitivity of the assay were established. The specificity experiment showed that LAMP primers amplified T. annulata DNA successfully, while no amplification was seen for Theileria parva, Theileria mutans, Theileria sergenti, Theileria sinensis, Babesia bovis as well as bovine genomic DNA and water control. When the sensitivity of LAMP assay was compared with that of conventional PCR a 10-fold higher sensitivity was found, with a detection limit of 10 pg/microl of genomic DNA isolated from a T. annulata-infected cell line. The LAMP product was confirmed by restriction digestion and staining with SYBR Green I. Furthermore, the LAMP assay was applied for the diagnosis of T. annulata in field samples and compared with reverse line blot (RLB), demonstrating that results of the LAMP assay corresponded to those of RLB. 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 epidemiological studies on T. annulata infection of cattle.

Distribution and application of ELISA for the seroprevalence of tick-borne diseases in Central Equatoria State, Sudan.

This study was carried out to estimate the prevalence of Theileria parva, Theileria mutans, Babesia bigemina and Anaplasma marginale antibodies among naturally infected cattle in Central Equatoria State, Southern Sudan using indirect enzyme-linked immunosorbent assay (ELISA). Three locations (Khor Rumla, Nyaing and Gumbo) were selected and surveyed every 3 months during the period from January to December 2005. Six cattle herds in the three locations were visited four times during the study period. During each visit, 150 serum samples were collected from indigenous cattle of both sex and of different age groups. Assessment of antibodies against tick-borne diseases was carried out using indirect ELISA. The overall seroprevalence rates of T. parva, T. mutans, B. bigemina and A. marginale were found to be 58.2%, 88.9%, 51.1% and 37.8% respectively. The highest seroprevalence of T. parva was observed in Gumbo (69%). An overall seroprevalence of 43.3% for T. parva was reported in July which was significantly lower than other seasons. Older animals showed higher seroprevalence than younger ones. The implications of these results on the epidemiology of tick-borne diseases are discussed with emphasis on East Coast fever.

Reliminary survey of ticks (Acari: Ixodidae) on cattle in Central Equatoria State, Southern Sudan.

In a preliminary survey conducted in 2005, the species composition and seasonality of ticks infesting cattle in Central Equatoria State, Southern Sudan was determined. Three locations were selected (Gumbo, Khor Rumla and Nyaing) and surveyed every 3 months. Two cattle herds in each of the three locations were visited four times during the study period. Total body collections of ticks were made from each of five cattle (Nilotic Zebu breed) kept in six different herds. Four tick genera and ten species were identified. The tick species identified were Amblyomma lepidum, Amblyomma variegatum, Boophilus annulatus, Boophilus decoloratus, Hyalomma marginatum rufipes, Hyalomma truncatum, Rhipicephalus appendiculatus, Rhipicephalus evertsi evertsi, Rhipicephalus praetextatus and Rhipicephalus sanguineus group. The highest number of ticks was collected in October during the rainy season. A finding of great significance was that R. appendiculatus, vector of East Coast fever, has now firmly established itself throughout the year with possible implications for cattle production in Central Equatoria State.

Determination of potential risk factors associated with Theileria annulata and Theileria parva infections of cattle in the Sudan.

A multi-variate logistic regression analysis was performed on two sets of data on the prevalence of Theileria annulata in Northern Sudan and Theileria parva in Southern Sudan, to determine the potential risk factors that might affect the distribution of the infections in those regions. The logistic regression model was fit with the tested risk factors for each disease, separately. The results indicated that locations, management systems and age could be held as risk factors for T. annulata infection in Northern Sudan, while for T. parva locations and seasons could be held as risk factors in Southern Sudan. The results of this study will assist in the development of more effective control strategies for smallholder dairy farms in the country.

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 2,661 naturally infected cattle from northern Sudan (samples collected between June 2001 and July 2002). In the ME, we detected piroplasms in 364/2,661 cattle (14%), and the kappas between the ME and the serological tests were poor (TaSP ELISA 10%; IFA 8%). The TaSP ELISA detected 885/2,661 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.

Epidemiological studies on tick-borne diseases of cattle in Central Equatoria State, Southern Sudan.

A herd-based study was carried out in Central Equatoria State, Southern Sudan, to study epidemiological aspects of tick-borne diseases. Six herds of cattle situated in three different locations were selected and investigated every 3 months during the year 2005. Blood smears for Giemsa staining and blood spots on filter paper for deoxyribonucleic acid extraction were collected from 600 apparently healthy indigenous cattle. A total of 69 (11.5%) samples showed the presence of piroplasms in Giemsa-stained blood smears, and polymerase chain reaction increased the detection limit to 297 (49.5%). Using reverse line blot, it was possible to detect and differentiate eight different piroplasms namely, Theileria parva (71.2%), Theileria mutans (73%), Theileria velifera (45.3%), Theileria taurotragi (2.7%), Theileria buffeli (0.5%), Theileria annulata (0.2%), Babesia bovis (1.7%), and Babesia bigemina (0.3%). Mixed infections were detected in 406 samples (67.7%) accounting for 17 different combinations. High infection of Theileria parva was reported among young calves compared to older cattle. The highest prevalence of Theileria parva was reported in the rainy season (October). The implications of these results on the epidemiology of tick-borne diseases are discussed with emphasis on East Coast fever.

Natural infection rates and transmission of Theileria annulata by Hyalomma anatolicum anatolicum ticks in the Sudan.

Hyalomma anatolicum anatolicum nymphs were collected from two localities in the Sudan: Eddamer in Northern Sudan and Wad-Medani in Central Sudan. They were allowed to moult to adult ticks, which were assessed for Theileria infection in their salivary glands using Feulgen stain. At Eddamer, 49.6% of 123 ticks examined were infected with Theileria and the mean intensity of infection was 1.3 (i.e. the number of infected acini/number of infected ticks). At Wad-Medani, 8.6% of 162 ticks were infected and the mean intensity of infection was 7.9. The prevalence of infection was higher in female than in male ticks at both localities. When adult H. a. anatolicum were applied onto two susceptible calves, both animals developed the severe form of theileriosis.

Application of the recombinant Theileria annulata surface protein in an indirect ELISA for the diagnosis of tropical theileriosis.

The recombinant surface protein of Theileria annulata (TaSP) was used in the standardization and validation of an enzyme linked immunosorbent assay (ELISA) for the detection of circulating antibodies against tropical theileriosis. ELISA data were expressed as the percentage positivity (PP) of the reactivity of an internal positive control. A total of 50 sera samples from a disease-free area were used for the calculation of the cut-off value which served as a threshold between the positive and the negative sera samples. This was determined as the mean PP plus two standard deviations or the twice the mean PP of the results obtained with these negative samples. The obtained thresholds were 17.8% and 18.3%, respectively. Accordingly, the reactivity of 140 field sera samples collected at random from an area known to be endemic for tropical theileriosis in Sudan was determined as PP values which were then compared to the results obtained using the indirect fluorescence antibody test (IFAT) from the same samples. Both tests showed a high degree of correlation. The TaSP-ELISA had a sensitivity of 99.1% and specificity of 90.47% when taking the IFAT as a reference test. Our test has proved its suitability for the diagnosis of tropical theileriosis and could be used in serological surveys to map out the prevalence of the disease or to monitor vaccination efficiencies in disease-free populations.

Preliminary survey of ticks (Acari: Ixodidae) on cattle in northern Sudan.

In a cross sectional survey conducted during the period June 2001 to July 2002, the geographical distribution of ticks on cattle in the Sudan was determined. Seventeen locations were surveyed from Northern, Central, Eastern, Western, Blue Nile and White Nile Provinces. Total body collections of ticks were made from 20 cattle at each location. Four tick genera and 11 species were identified. The tick species collected included Amblyomma lepidum, Amblyomma variegatum, Boophilus decoloratus, Hyalomma anatolicum anatolicum, Hyalomma dromedarii, Hyalomma impeltatum, Hyalomma marginatum rufipes, Hyalomma truncatum, Rhipicephalus evertsi evertsi, Rhipicephalus sanguineus group and Rhipicephalus simus simus. Major ecological changes have occurred due to extensive animal movement, deforestation, desertification and establishment of large mechanized agricultural schemes. These factors have certainly affected the distribution of ticks and tick-borne diseases in the Sudan. The absence of A. variegatum and A. lepidum in northern Sudan was not surprising, since these tick species are known to survive in humid areas and not in the desert and semi-desert areas of northern Sudan. The absence of B. annulatus in northern and central Sudan is in accordance with the finding that this tick species is restricted to the southern parts of the central Sudan. The presence of H. anatolicum anatolicum in Um Benin in relatively high abundance is an interesting finding. The present finding may indicate that the southern limit of this species has changed and moved southwards to latitude 13 degrees N. It is concluded that major changes in tick distribution have taken place in the Sudan.

Survey of Theileria lestoquardi antibodies among Sudanese sheep.

The prevalence of Theileria lestoquardi antibodies in Sudanese sheep from nine geographical areas in Sudan was determined using indirect fluorescent antibody "IFA" test. Out of 315 samples examined, 51 (16.2%) were found positive and ranged between 23.4% in River Nile State and 10% in Kasala and Darfour Provinces with an overall prevalence of 16.2% indicating widespread distribution of the infection. We also report on presence of antibodies reactive to Theileria annulata in sheep sera.