Bashir Salim

Emergence of new types of Theileria orientalis in Australian cattle and possible cause of theileriosis outbreaks

Theileria parasites cause a benign infection of cattle in parts of Australia where they are endemic, but have, in recent years, been suspected of being responsible for a number of outbreaks of disease in cattle near the coast of New South Wales. The objective of this study was to identify and characterize the species of Theileria in cattle on six farms in New South Wales where disease outbreaks have occurred, and compare with Theileria from three disease-free farms in Queensland that is endemic for Theileria. Special reference was made to sub-typing of T. orientalis by type-specific PCR and sequencing of the small subunit (SSU) rRNA gene, and sequence analysis of the gene encoding a polymorphic merozoite/piroplasm surface protein (MPSP) that may be under immune selection. Nucleotide sequencing of SSU rRNA and MPSP genes revealed the presence of four Theileria genotypes: T. orientalis (buffeli), T. orientalis (ikeda), T. orientalis (chitose) and T. orientalis type 4 (MPSP) or type C (SSU rRNA). The majority of animals showed mixed infections while a few showed single infection. When MPSP nucleotide sequences were translated into amino acids, base transition did not change amino acid composition of the protein product, suggesting possible silent polymorphism. The occurrence of ikeda and type 4 (type C) previously not reported to occur and silent mutation is thought to have enhanced parasite evasion of the host immune response causing the outbreak.

Diagnosis of Babesia caballi and Theileria equi infections in horses in Sudan using ELISA and PCR

The purpose of this study was to estimate the prevalence of equine piroplasmosis in Sudan. The presence of antibodies against Babesia caballi and Theileria equi was determined in serum samples obtained from 158 horses raised in different locations in Sudan by enzyme-linked immunosorbent assay (ELISA). The B. caballi 48-kDa and the T. equi EMA-2 purified recombinant proteins were used as antigens in the ELISA test. Results showed that seven (4.4%) were positive for B. caballi and 80 (63.5%) were positive for T. equi. Polymerase chain reaction (PCR) assays have been applied using primers targeting the B. caballi 48-kDa merozoite antigen, the T. equi SSUrRNA and the T. equi EMA-1 genes. PCR performed on 131 blood spots in filter paper revealed that 33 (25.2%) samples were positive for T. equi but no positives were found for B. caballi. It is concluded that equine piroplasmosis is endemic in the country. This is the first study on serological and molecular epidemiological diagnosis on equine piroplasmosis in Sudan.

Molecular epidemiology of camel trypanosomiasis based on ITS1 rDNA and RoTat 1.2 VSG gene in the Sudan

BackgroundInternal transcribed spacer one (ITS1) of the ribosomal DNA is known to be a suitable target for PCR-based detection of trypanosomes. The analysis of this region provides a multi-species-specific diagnosis by a single PCR. Using ITS1 primer-based PCR, a cross sectional study was carried out in the period from September to November 2009 on samples collected from 687 camels from geographically distinct zones in the Sudan to detect all possible African trypanosomes, which can infect camels.ResultsThe results showed that all PCR-positive camels were infected with a single parasite species; Trypanosoma evansi. The highest prevalence, 57.1% (117/205), was observed in the Butana plains of mid-Eastern Sudan and the lowest, 6.0% (4/67), was in the Umshadeeda eastern part of White Nile State. In another experiment, the RoTat 1.2 gene encoding the variable surface glycoprotein (VSG) of T. evansi was analyzed for its presence or absence by a polymerase chain reaction (PCR) using T. evansi species-specific primers. The study showed that the RoTat 1.2 VSG gene was absent in thirteen out of thirty T. evansi-positive samples.ConclusionsIt is concluded that camel trypanosomiasis in Sudan is apparently caused by a single parasite species T. evansi and there were no other typanosomes species detected. In addition, the disease is highly prevalent in the country, which strengthens the need to change control policies and institute measures that help prevent the spread of the parasite. To our knowledge, this is the first molecular diagnosis report, which gives a picture of camel trypanosomiasis covering large geographical areas in Sudan.

Ancient and modern DNA reveal dynamics of domestication and cross-continental dispersal of the dromedary

Significance The dromedary is one of the largest domesticates, sustainably used in arid and hostile environments. It provides food and transport to millions of people in marginal agricultural areas. We show how important long-distance and back-and-forth movements in ancient caravan routes shaped the species’ genetic diversity. Using a global sample set and ancient mitochondrial DNA analyses, we describe the population structure in modern dromedaries and their wild extinct ancestors. Phylogenetic analyses of ancient and modern dromedaries suggest a history of restocking from wild animals from the southeast coast of the Arabian Peninsula. Dromedaries now extend the list of species for which classic models of domestication from a single center and from wild conspecific individuals in isolation are rejected. Dromedaries have been fundamental to the development of human societies in arid landscapes and for long-distance trade across hostile hot terrains for 3,000 y. Today they continue to be an important livestock resource in marginal agro-ecological zones. However, the history of dromedary domestication and the influence of ancient trading networks on their genetic structure have remained elusive. We combined ancient DNA sequences of wild and early-domesticated dromedary samples from arid regions with nuclear microsatellite and mitochondrial genotype information from 1,083 extant animals collected across the species’ range. We observe little phylogeographic signal in the modern population, indicative of extensive gene flow and virtually affecting all regions except East Africa, where dromedary populations have remained relatively isolated. In agreement with archaeological findings, we identify wild dromedaries from the southeast Arabian Peninsula among the founders of the domestic dromedary gene pool. Approximate Bayesian computations further support the “restocking from the wild” hypothesis, with an initial domestication followed by introgression from individuals from wild, now-extinct populations. Compared with other livestock, which show a long history of gene flow with their wild ancestors, we find a high initial diversity relative to the native distribution of the wild ancestor on the Arabian Peninsula and to the brief coexistence of early-domesticated and wild individuals. This study also demonstrates the potential to retrieve ancient DNA sequences from osseous remains excavated in hot and dry desert environments.

Rapid discrimination and quantification of Theileria orientalis types using ribosomal DNA internal transcribed spacers

We report the population structure analysis of Theileria orientalis types (Ikeda, Buffeli and Chitose), the causative agent of theileriosis in cattle and its cohorts, using ITS1 and ITS2 spacers by fragment genotyping. We utilized primers flanking the two ribosomal RNA internal transcribed spacers (ITS1 and ITS2). Due to varying degrees of sequence polymorphism in the ITS regions found within and between species, we exploited the insertions and or deletions in these regions which resulted in different fragment sizes. On the basis of fragment size polymorphism, we could discriminate the three commonly found types of T. orientalis. ITS1 was capable of discriminating all three types (Ikeda-251 bp, Chitose-274 bp and Buffeli-269 bp) in one single reaction by fragment genotyping. In contrast, using ITS2, Ikeda (133-bp) a more pathogenic type was distinguishable from Buffeli/Chitose (139-bp). When compared with previous PCR detection method using, ITS1 and ITS2 genotyping was found to be more sensitive method with high specificity in population analysis and can be deployed in molecular epidemiology studies.

Population Genetics of Trypanosoma evansi from Camel in the Sudan

Genetic variation of microsatellite loci is a widely used method for the analysis of population genetic structure of microorganisms. We have investigated genetic variation at 15 microsatellite loci of T. evansi isolated from camels in Sudan and Kenya to evaluate the genetic information partitioned within and between individuals and between sites. We detected a strong signal of isolation by distance across the area sampled. The results also indicate that either, and as expected, T. evansi is purely clonal and structured in small units at very local scales and that there are numerous allelic dropouts in the data, or that this species often sexually recombines without the need of the “normal” definitive host, the tsetse fly or as the recurrent immigration from sexually recombined T. brucei brucei. Though the first hypothesis is the most likely, discriminating between these two incompatible hypotheses will require further studies at much localized scales.

Molecular detection of equine trypanosomes in the Sudan

Equine trypanosomosis (ET) is a protozoan disease affecting equines in many parts of the world. We examined 509 samples collected from geographically distinct regions in eastern, central and western Sudan to estimate the endemicity of ET using the generic ITS1-PCR diagnostic methods. Results revealed that horses and donkeys were infected by Trypanosoma brucei subgroup, Trypanosoma vivax, Trypanosoma simiae and Trypanosoma congolense. The prevalence of Trypanosoma spp. was higher in horses (12.7%, n=393) than in donkeys (3.4%, n=116). The highest prevalence was observed in South Darfur State (19.3%, n=202), followed by Kassala State (15.1%, n=86), Gadaref State (3.7%, n=82), and Khartoum State (2.6%, n=76). No trypanosomes were detected in the 63 samples collected from North Kurdofan State. We report for the first time the presence of T. simiae and T. congolense in horses in the Sudan. This study should alert veterinary services, authorized bodies to take action toward ET by undertaking countrywide epidemiological studies of the disease and adopting control strategies.

Population Genetics and Reproductive Strategies of African Trypanosomes: Revisiting Available Published Data

Trypanosomatidae are a dangerous family of Euglenobionta parasites that threaten the health and economy of millions of people around the world. More precisely describing the population biology and reproductive mode of such pests is not only a matter of pure science, but can also be useful for understanding parasite adaptation, as well as how parasitism, specialization (parasite specificity), and complex life cycles evolve over time. Studying this parasite’s reproductive strategies and population structure can also contribute key information to the understanding of the epidemiology of associated diseases; it can also provide clues for elaborating control programs and predicting the probability of success for control campaigns (such as vaccines and drug therapies), along with emergence or re-emergence risks. Population genetics tools, if appropriately used, can provide precise and useful information in these investigations. In this paper, we revisit recent data collected during population genetics surveys of different Trypanosoma species in sub-Saharan Africa. Reproductive modes and population structure depend not only on the taxon but also on the geographical location and data quality (absence or presence of DNA amplification failures). We conclude on issues regarding future directions of research, in particular vis-à-vis genotyping and sampling strategies, which are still relevant yet, too often, neglected issues.

Current status of equine piroplasmosis in the Sudan.

This is a cross-sectional molecular epidemiological study on equine piroplasmosis (EP) affecting horses and donkeys in the Sudan. The study evaluated 499 samples from geographically distinct regions in eastern, central and western parts of the country. PCR amplification of the 18S rRNA gene of both Thelieria equi and Babesia caballi was carried out. Horses from all sampled areas were found positive to T. equi DNA but no B. caballi was detected. Absence of B. caballi infection was confirmed by another PCR targeting the B. caballi 48-kDa merozoite antigen. The overall prevalence was found to be 35.95%. The highest prevalence was detected in Showak 13 (81.3%) and the lowest was in Shearia locality in South Darfur 1 (5.6%). In another experiment, capillary electrophoresis was used to detect and differentiate between T. equi and B. caballi using one set of primers designed to amplify the 18S rRNA gene in a single PCR. Capillary electrophoresis method was found to be powerful in detecting mixed infections in artificially mixed controls samples. The data obtained in this study would contribute to the development of a national control strategy of EP in the Sudan.

An outbreak of bovine trypanosomiasis in the Blue Nile State, Sudan

BackgroundIn this paper, we report an outbreak of bovine trypanosomiasis in Kurmuk District, Blue Nile State, Sudan that involved an infection with four Trypanosoma species in cattle. The outbreak occurred in June 2010 when indigenous cattle, mainly Kenana and Fulani breed types, crossed the national Sudanese border to Ethiopia and returned. A veterinarian was notified of massive deaths in the cattle populations that recently came from Ethiopia. All animals involved in the outbreak were from the nomadic Fulani group and resident local cattle were not infected and no death has been reported among them. A total of 210 blood samples were collected from the ear vein of cattle. A few samples were also collected from other domestic animals species. Parasitological examinations including hematocrit centrifugation techniques (HCT) and Giemsa-stained thin blood films were carried out. ITS1-PCR, which provides a multi-species-specific diagnosis in a single PCR, was performed.FindingsParasitological examinations revealed that 43% (91/210) of the affected cattle population was infected with two morphologically distinct trypanosomes. Seventy animals (33.3%) were infected with T. vivax and twenty one (10%) with T. congolense. In contrast, ITS1-PCR was able to identify four Trypanosoma species namely T. vivax, T. congolense, T. simiae and T. brucei in 56.7% (80/141). T. brucei showed the highest prevalence of 36.9% (52/141) and the lowest 19% (27/141) was displayed by T. congolense. Furthermore, and because ITS1-PCR could not differentiate between T. brucei subspecies, serum resistance-associated (SRA) gene based PCR was used to detect the human T. brucei rhodesiense in T. brucei positive samples. None of the samples was shown positive for T. b. rhodesiense. The identity of the 400 bp PCR product originating from T. simiae, was further confirmed by sequencing and subsequent phylogenetic analysis.ConclusionsThe outbreak of bovine trypanosomiasis occurred in the Blue Nile State was caused by mixed infection of two or more Trypanosoma species and the conventional parasitological examinations were not reliable in identifying all the species of Trypanosoma involved in the outbreak. It is difficult to determine the cause of the disease for the reason that the current enzootic situation in the resident cattle in the region is poorly understood. The study concluded that there are at least four species of trypanosomes that caused this outbreak in the Blue Nile State. The presence of mixed infections might have exacerbated the severity of the disease. It is hypothesized that variant parasite type(s) might have been introduced to Sudanese cattle when they crossed to Ethiopia, a tsetse belt region.