Assessment of the prevalence of Theileria lestoquardi in sheep from the Sudan using serological and molecular methods.

Malignant theileriosis of sheep and goats caused by Theileria lestoquardi is considered to be among the most important tick borne diseases in the Sudan. Information on the prevalence of the disease in different parts of the Sudan is limited. The purpose of this study was to estimate the prevalence of the disease in five states of the Sudan using molecular and serological assays. A total of 393 blood and serum samples from clinically asymptomatic sheep were analysed using nested reverse line blot (nRLB) and loop mediated isothermal amplification (LAMP), as well as an enzyme-linked immunosorbent assay (ELISA). The results indicated a sero-prevalence of 33.8% while RLB and LAMP assays revealed molecular prevalences of 29.5 and 22.6% respectively. The prevalence of Theileria lestoquardi varied significantly according to the geographical origin of the infected animals, whereas age and gender did not have a significant effect. RLB data indicated that T. lestoquardi usually occurred as a co-infection with the non-pathogenic Theileria ovis. Using RLB as a gold standard, a sensitivity of 68.1% and a specificity of 96.4% were recorded for LAMP and a sensitivity of 75.9% and a specificity of 83.8% for ELISA. The Kappa coefficient between nRLB and LAMP indicated a significant level of agreement (0.692), but only moderate concordance (0.572) between nRLB and ELISA. The results of the present study confirm and extend earlier findings regarding the widespread of T. lestoquardi infections in sheep in the Sudan. The data provide evidence that should enable the veterinary authorities to deploy appropriate control measures.

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.

Isolation, identification and differentiation of Campylobacter spp. using multiplex PCR assay from goats in Khartoum State, Sudan.

The aim of this study was to identify and characterize thermophilic Campylobacter species in faecal samples from goats in Khartoum State, Sudan, by application of multiplex polymerase chain reaction. Campylobacteriosis is a zoonotic disease of global concern, and the organisms can be transmitted to human via food, water and through contact with farm animals and pets. There are five clinically related Campylobacter species: Campylobacter jejuni (C. jejuni). Campylobacter coli, Campylobacter lari, Campylobacter upsaliensis and Campylobacter fetus. Conventional cultural methods to diagnose campylobacteriosis are tedious and time consuming. Wide ranges of genes have been reported to be used for PCR-based identification of Campylobacter spp. We used a multiplex PCR assay to simultaneously detect genes from the major five clinically significant Campylobacter spp. The genes selected were hipO (hippuricase) and 23S rRNA from glyA (serine hydroxymethyl transferase) from each of C. jejuni. C. coli, C. lari, and C. upsaliensis; and sapB2 (surface layer protein) from C. fetus subsp. fetus. The assay was used to identify Campylobacter isolates recovered from 336 cultured faecal samples from goats in three localities in Khartoum State. C. coli was the most predominant isolate (234; 69.6%), followed by C. jejuni (19; 5.7%), C. upsaliensis (13; 3.9%), C. fetus subsp. fetus (7; 2.1%) and C. lari (6; 1.8%). Twenty-nine goats showed mixed infection with Campylobacter spp., 21 of which harbored two Campylobacter spp., while eight animals were infected with three species. Ten out of twelve goats that displayed diarrhea harbored C. coli only. C. coli, C. jejuni and C. upsaliensis showed significant variation with localities. The prevalence of C. coli was significantly higher (87; 25.9%) in goats from Omdurman, whereas C. jejuni and C. upsaliensis were significantly higher (11; 3.3%, 9; 2.7%) in goats from Khartoum. The multiplex PCR assay was found to be rapid and easy to perform and had a high sensitivity and specificity for characterizing the isolates, even in mixed cultures. The study demonstrated the significance of goats as reservoirs in the dissemination of Campylobacter spp. which could be considered as potential agent of caprine enteritis and abortion as well as contamination of the wider environment posing serious public health concern in Khartoum State.

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.

A serological survey of akabane virus infection in cattle in Sudan.

A cross-sectional survey was carried out in ten states in Sudan to determine seroprevalence and to assess risk factors associated with Akabane virus (AKAV) infection in dairy herds. Serum samples were collected from a total of 361 dairy cattle and tested for antibodies against AKAV using ELISA. The prevalence rates of AKAV antibodies in cattle varied between 69.6% in Khartoum state and 3.3% in Sennar State with an overall prevalence rate of 29.4%. The prevalence rates of AKAV antibodies were significantly associated with breed being high in crossbred (39.9%; P < 0.001); female sex (33%; P < 0.001), and animals in the age group of 2-3 years old (45.3%; P < 0.001). Akabane virus antibodies prevalence was also highly associated with locality (P < 0.001); season being high in winter season (58.1%; P < 0.001); and animals raised under intensive management system (37%; P < 0.001). Among 68 cases suffering from reproductive (abortion and infertility) problems the prevalence rate of AKAV antibodies in animals with infertility problem (76.2%; P < 0.03) was significantly higher than in animals with abortion (48.9%). The study revealed that AKAV infection is highly prevalent in dairy cattle in Sudan and this calls for control strategy to be implemented.

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.

Life cycle of Hyalomma anatolicum Koch, 1844 (Acari: Ixodidae) fed on rabbits, sheep and goats.

Some aspects of the biology of the tick Hyalomma anatolicum fed on rabbits, sheep and goats were studied. The non-feeding stages were maintained under laboratory conditions at 20-36°C and 75% relative humidity. The longest feeding periods of larvae and nymphs of H. anatolicum were observed when fed on rabbits (mean 4.58 ± 0.51 and 7 ± 1.15 days, respectively) while the longest feeding periods of females were observed on goats (9.61 ± 1.21). The pre-oviposition period (4.8 ± 0.42 days) and pre-eclosion periods (mean 21.3 ± 1.16 days) were shortest for females fed on rabbits. Engorged females reached heavier engorgement weights (482.92 ± 88.08 mg), and produced more eggs (4881.8 ± 842.71) when fed on rabbits. However, no significant differences were observed between the percentages hatchability of eggs laid by ticks fed on the three hosts studied. Most (94.31%) of the larvae fed on rabbits underwent a 2-host life cycle, while few (5.69%) of them behaved as a 3-host ticks. Few larvae were able to complete feeding as 3-host pattern on both sheep and goats, while the majority of the larvae failed to complete feeding or died on their way to molt on both sheep and goats.

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.

Some observations on ticks (Acari: Ixodidae) infesting sheep in River Nile Province of Northern Sudan.

Five species of ixodid ticks were found in a cross-sectional survey in which 200 sheep were examined for ticks in River Nile Province, Sudan. Hyalomma anatolicum anatolicum was the predominant species (73.6%), whereas ticks belonging to the Rhipicephalus sanguineus group (14.7%), Rhipicephalus evertsi evertsi (9.1%), Rhipicephalus simus (2%) and Hyalomma dromedarii (0.5%) were also found. The mean tick load was 11.2 per animal. In a subsequent longitudinal survey ticks were collected on a monthly basis from eight sentinel sheep that were introduced into the area. It was found that H. a. anatolicum almost disappeared during the hot period between April and August, whereas it's highest numbers were present in winter between November and February. It is concluded that there is only one generation of H. a. anatolicum per year, which may explain the year-round appearance of clinical cases of malignant ovine theileriosis indicating endemic instability of this disease in River Nile Province.

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.

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.