Robustness in population-structure and demographic-inference results derived from the Aedes aegypti SNP chip and whole-genome sequencing data.

The mosquito Aedes aegypti is the primary vector of many human arboviruses such as dengue, yellow fever, chikungunya and Zika, which affect millions of people world-wide. Population genetics studies on this mosquito have been important in understanding its invasion pathways and success as a vector of human disease. The Axiom aegypti1 SNP chip was developed from a sample of geographically diverse Ae. aegypti populations to facilitate genomic studies on this species. We evaluate the utility of the Axiom aegypti1 SNP chip for population genetics and compare it with a low-depth shot-gun sequencing approach using mosquitoes from the native (Africa) and invasive range (outside Africa). These analyses indicate that results from the SNP chip are highly reproducible and have a higher sensitivity to capture alternative alleles than a low-coverage whole-genome sequencing approach. Although the SNP chip suffers from ascertainment bias, results from population structure, ancestry, demographic and phylogenetic analyses using the SNP chip were congruent with those derived from low coverage whole genome sequencing, and consistent with previous reports on Africa and outside Africa populations using microsatellites. More importantly, we identified a subset of SNPs that can be reliably used to generate merged databases, opening the door to combined analyses. We conclude that the Axiom aegypti1 SNP chip is a convenient, more accurate, low-cost alternative to low-depth whole genome sequencing for population genetic studies of Ae. aegypti that do not rely on full allelic frequency spectra. Whole genome sequencing and SNP chip data can be easily merged, extending the usefulness of both approaches.

Exploring genetic diversity and variation of Ovar-DRB1 gene in Sudan Desert Sheep using targeted next-generation sequencing.

INTRODUCTION: The Ovar-DRB1 gene, a crucial element of the Major Histocompatibility Complex (MHC) Class II region, initiates adaptive immunity by presenting antigens to T-cells. Genetic diversity in sheep, particularly in MHC Class II genes like Ovar-DRB1, directly influences the specturm of presented antigens impacting immune responses and disease susceptability. Understanding the allelic diversity of Ovar-DRB1 gene in Sudan Desert Sheep (SDS) is essential for uncovering the genetic basis of immune responses and disease resistance, given the the breeds significance in Sudan's unique environment. METHODS: Utilizing Targeted Next-Generation Sequencing (NGS) we explore allelic diversity in Ovar-DRB1 gene within SDS. Successfully ampliying and and sequencing the second exon of this gene in 288 SDS samples representing six breeds provided a comprehensive allelic profile, enabling a detalied examination of the gene's genetic makeup. RESULTS: We identifed forty-six alleles, including four previously unreported, enrichness the genetic diversity of SDS breeds. These alleles exhibiting non-uniform distribution, varying frequencies across breeds, indicating a breed-specific genetic landscape. Certain alleles, known and novel, show higher frequencies in specific populations, suggesting potential associations with adaptive immune responses. Identifying these alleles sets the stage for investigating their functional roles and implications for disease resistance. Genetic differentiation among SDS breeds, as indicated by FST values and clustering analyses, highlights a unique genetic makeup shaped by geographic and historical factors. These differentiation patterns among SDS breeds have broader implications for breed conservation and targeted breeding to enhance disease resistance in specific populations. CONCLUSION: This study unveils Ovar-DRB1 gene allelic diversity in SDS breeds through targeted NGS and genetic analyses, revealing new alleles that underscore the breeds' unique genetic profile. Insights into the genetic factors governing immune responses and disease resistance emerge, promising for optimization of breeding strategies for enhanced livestock health in Sudan's unique environment.

Genetic variation and demographic history of Sudan desert sheep reveal two diversified lineages.

More than 400 million sheep are raised on the African continent, the majority of which are indigenous and are primarily reared for sustenance. They have effectively adapted to various climatic and production environments, surviving and flourishing. The genetic relationships among these sheep populations remain understudied. Herein, we sequenced the entire mitochondrial DNA control region of 120 animals from Hamary and Kabashi and their crossbreed (Hamary x Kabashi) of Sudan desert sheep (SDS) to understand their maternal-inherited genetic variation and demographic history profiles and relate those to the history of sheep pastoralism on the African continent. The results show a diversified and predominant D- loop haplogroup B (n = 102, 85%), with all other sequences belonging to haplogroup A. Most of the maternal genetic variation was partitioned between haplogroup (76.3%) while within haplogroup accounted for 23.7% of the variation. However, little genetic differentiation was observed among the two breeds and their crosses, with our results supporting a Hamari maternal origin for the crossbreed. Bayesian coalescent-based analysis reveals distinct demographic history between the two haplogroups, two breeds and their crosses. Comparison of the two haplogroup showed that haplogroup B experienced an earlier expansion than haplogroup A. Unlike the breed-based comparison, the expansion of the two breeds started roughly at the same time, around 6500 years ago, with Kabashi having a slightly greater effective population size. The maternal ancestors of SDS may have diverged before their introduction to the African continent. This study provides novel insights into the early history of these two main breeds of Sudan desert sheep and their crosses.

Genomic signatures for drylands adaptation at gene-rich regions in African zebu cattle.

BACKGROUND: Indigenous Sudanese cattle are mainly indicine/zebu (humped) type. They thrive in the harshest dryland environments characterised by high temperatures, long seasonal dry periods, nutritional shortages, and vector disease challenges. Here, we sequenced 60 indigenous Sudanese cattle from six indigenous breeds and analysed the data using three genomic scan approaches to unravel cattle adaptation to the African dryland region. RESULTS: We identified a set of gene-rich selective sweep regions, detected mostly on chromosomes 5, 7 and 19, shared across African and Gir zebu. These include genes involved in immune response, body size and conformation, and heat stress response. We also identified selective sweep regions unique to Sudanese zebu. Of these, a 250 kb selective sweep on chromosome 16 spans seven genes, including PLCH2, PEX10, PRKCZ, and SKI, which are involved in alternative adaptive metabolic strategies of insulin signalling, glucose homeostasis, and fat metabolism. CONCLUSIONS: Our results suggest that environmental adaptation may involve recent and ancient selection at gene-rich regions, which might be under a common regulatory genetic control, in zebu cattle.

Epidemics of Crimean-Congo Hemorrhagic Fever (CCHF) in Sudan between 2010 and 2020.

Crimean-Congo hemorrhagic fever (CCHF) is a zoonotic arboviral disease that poses a great threat to global health in the Old World, and it is endemic in Europe, Asia, and Africa, including Sudan. In this retrospective study, we reviewed previous epidemiological reports about the major epidemics of CCHF throughout Sudan between 2010 and 2020. During these epidemics, the infection of humans with Crimean-Congo hemorrhagic fever virus (CCHFV), the causative agent of CCHF, was diagnosed using qRT-PCR. We have identified 88 cases of CCHF, including 13 fatalities reported during five epidemics that occurred in 2010, 2011, 2015, 2019, and 2020. The two epidemics in 2010 and 2011 were by far the largest, with 51 and 27 cases reported, respectively. The majority of cases (78%) were reported in the endemic region of Kordofan. Here, we document that the first emergence of CCHFV in the Darfur region, West Sudan, occurred in 2010. We were not able to investigate outbreak dynamics through phylogenetic analysis due to the limited diagnostic capacity and the lack of sequencing services in the country. These findings call for establishing a genomic-based integrated One Health surveillance and response system for the early preparedness, prevention, and control of CCHF in the country.

Population Genetic Analysis of Aedes aegypti Mosquitoes From Sudan Revealed Recent Independent Colonization Events by the Two Subspecies.

Increases in arbovirus outbreaks in Sudan are vectored by Aedes aegypti, raising the medical importance of this mosquito. We genotyped 12 microsatellite loci in four populations of Ae. aegypti from Sudan, two from the East and two from the West, and analyzed them together with a previously published database of 31 worldwide populations to infer population structure and investigate the demographic history of this species in Sudan. Our results revealed the presence of two genetically distinct subspecies of Ae. aegypti in Sudan. These are Ae. aegypti aegypti in Eastern Sudan and Ae. aegypti formosus in Western Sudan. Clustering analysis showed that mosquitoes from East Sudan are genetically homogeneous, while we found population substructure in West Sudan. In the global context our results indicate that Eastern Sudan populations are genetically closer to Asian and American populations, while Western Sudan populations are related to East and West African populations. Approximate Bayesian Computation Analysis supports a scenario in which Ae. aegypti entered Sudan in at least two independent occasions nearly 70-80 years ago. This study provides a baseline database that can be used to determine the likely origin of new introductions for this invasive species into Sudan. The presence of the two subspecies in the country should be consider when designing interventions, since they display different behaviors regarding epidemiologically relevant parameters, such as blood feeding preferences and ability to transmit disease.

Demographic expansion and high level of matrilineal diversity in two populations of East African Baggara cattle.

Western Baggara cattle breed (WBCB) is an East African zebu inhabiting Sudan, well-known as beef-producing cattle. We investigated herein two phenotypically and geographically distinct populations of this breed, namely Nyalawi and Daeinawi, which are renowned for their unique meat production capabilities and adaptation attributes, with the aim to contribute to our understanding of their maternal genetic diversity and demography dynamics. Genetic polymorphism analysis of the full-length D-loop mtDNA region revealed 44 and 35 polymorphic sites defining 28 and 24 distinct haplotypes in the Nyalawi and the Daeinawi, respectively. Observed genetic diversity is high within the population with a low level of genetic differentiation between populations. Approximate Bayesian computation via the calculation of Bayesian skyline plots and neutrality tests support past expansion with a higher maternal effective population size (Ne ) in Nyalawi compared with the Daeinawi population and a population expansion beginning around 4,500 YBP and 3,500 YBP, respectively, before the arrival of zebu into the continent.

Cattle anaplasmosis and babesiosis: Major tick-borne diseases affecting the cattle industry in Khartoum State, Sudan.

Tick-borne hemoparasitic (TBH) infections are a major problem affecting livestock industries worldwide, particularly in tropical and subtropical regions. This study was carried out in response to repeated reports from local veterinarians in Khartoum State, Sudan, where TBH infections are prevalent in dairy farms. This cross-sectional study was undertaken from October 2017 to April 2018 with the objective of assessing the prevalence and potential risk factors associated with cattle anaplasmosis and babesiosis in the localities of Omdurman, Khartoum, and Khartoum North, Khartoum State. A total of 292 cattle blood samples collected from apparently healthy animals were examined for the presence of A. marginale, Babesia bigemina, and B. bovis using PCR. The overall prevalence of A. marginale and B. bigemina was found to be 40.41% and 3.42%, respectively, while B. bovis was not detected. Mixed infections with A. marginale and B. bigemina were detected in four (1.37%) cattle. The prevalence of the two pathogens was found to be significantly higher in Khartoum and Omdurman than in Khartoum North. However, no significant difference was observed for the prevalence based on sex, age, breed, and mean packed cell volume values. Our findings indicated that A. marginale is a highly prevalent parasite in Khartoum State, which may be a primary constraint to the cattle industry. Inclusion of this pathogen in the diagnostic protocols, and consequent treatment and tick control are necessary. Moreover, the role of B. bigemina infection may exacerbate the situation to some extent in this region.

Emergence of the invasive malaria vector Anopheles stephensi in Khartoum State, Central Sudan.

The emergence of the Asian invasive malaria vector, Anopheles stephensi, has been identified in Khartoum, the capital city of Sudan. This is the first report that confirms the geographical expansion of this urban mosquito into Central Sudan. We urgently recommend the launch of a national entomological survey to determine the distribution of this invasive disease vector and to generate essential information about its bionomics and susceptibility to available malaria control measures.

BoLA-DRB3 gene haplotypes show divergence in native Sudanese cattle from taurine and indicine breeds.

Autochthonous Sudanese cattle breeds, namely Baggara for beef and Butana and Kenana for dairy, are characterized by their adaptive characteristics and high performance in hot and dry agro-ecosystems. They are thus used largely by nomadic and semi-nomadic pastoralists. We analyzed the diversity and genetic structure of the BoLA-DRB3 gene, a genetic locus linked to the immune response, for the indigenous cattle of Sudan and in the context of the global cattle repository. Blood samples (n = 225) were taken from three indigenous breeds (Baggara; n = 113, Butana; n = 60 and Kenana; n = 52) distributed across six regions of Sudan. Nucleotide sequences were genotyped using the sequence-based typing method. We describe 53 alleles, including seven novel alleles. Principal component analysis (PCA) of the protein pockets implicated in the antigen-binding function of the MHC complex revealed that pockets 4 and 9 (respectively) differentiate Kenana-Baggara and Kenana-Butana breeds from other breeds. Venn analysis of Sudanese, Southeast Asian, European and American cattle breeds with 115 alleles showed 14 were unique to Sudanese breeds. Gene frequency distributions of Baggara cattle showed an even distribution suggesting balancing selection, while the selection index (ω) revealed the presence of diversifying selection in several amino acid sites along the BoLA-DRB3 exon 2 of these native breeds. The results of several PCA were in agreement with clustering patterns observed on the neighbor joining (NJ) trees. These results provide insight into their high survival rate for different tropical diseases and their reproductive capacity in Sudan's harsh environment.

Molecular Characterization of Ticks and Tick-Borne Pathogens in Cattle from Khartoum State and East Darfur State, Sudan.

Ticks transmit many pathogens with public health and veterinary importance. Despite the wide distribution of tick-borne pathogens in Sudan, the information on the tick-pathogen relationship needs to be updated, particularly using modern molecular techniques. This cross-sectional study, conducted between September and November 2019, used morphology, PCR, and sequencing to confirm the identity of adult cattle ticks (male and female; n = 536) from Khartoum State (n = 417) and East Darfur State (n = 119). Moreover, the presence of Theileria annulata, Babesia bigemina, B. bovis, Anaplasma marginale, and Ehrlichia ruminantium was detected and confirmed in each tick using species-specific PCR or nested PCR and sequencing. The most economically important tick genera, Rhipicephalus, Hyalomma, and Amblyomma, were prevalent in the study area, and 13 different tick species were identified. The most prevalent tick species were Rhipicephalusevertsi evertsi (34.3%) and Hyalomma anatolicum (57.3%) in Khartoum State, and Rhipicephalus annulatus (27%), Rhipicephalus decoloratus (25%), and Hyalomma rufipes (29%) in East Darfur State. We detected all five pathogens in both states. To the best of our knowledge, this is the first study to report the presence of E. ruminantium, its vector Amblyomma variegatum, and B. bovis in Khartoum State. Further, this is the first report on most tick and pathogen species identified in East Darfur State. Our findings indicate the migration of some tick and pathogen species beyond their distribution areas in the country, and this consideration is necessary to develop future control strategies.

Exploring Prokaryotic and Eukaryotic Microbiomes Helps in Detecting Tick-Borne Infectious Agents in the Blood of Camels.

Dromedary camels (Camelus dromedarius) are widely distributed in Africa, the Middle East and northern India. In this study, we aimed to detect tick-borne pathogens through investigating prokaryotic and eukaryotic microorganisms in camel blood based on a metagenomic approach and then to characterize potentially pathogenic organisms using traditional molecular techniques. We showed that the bacteria circulating in the blood of camels is dominated by Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria. At the genus level, Sediminibacterium, Hydrotalea, Bradyrhizobium and Anaplasma were the most abundant taxa. Eukaryotic profile was dominated by Fungi, Charophyta and Apicomplexa. At the genus level, Theileria was detected in 10 out of 18 samples, while Sarcocystis, Hoplorhynchus and Stylocephalus were detected in one sample each. Our metagenomic approach was successful in the detection of several pathogens or potential pathogens including Anaplasma sp., Theileria ovis, Th. separata, Th. annulate, Th. mutans-like and uncharacterized Theileria sp. For further characterization, we provided the partial sequences of citrate synthase (gltA) and heat-shock protein (groEL) genes of Candidatus Anaplasma camelii. We also detected Trypanosoma evansi type A using polymerase chain reaction (PCR) targeting the internal transcribed spacer 1 (ITS1) region. This combined metagenomic and traditional approach will contribute to a better understanding of the epidemiology of pathogens including tick-borne bacteria and protozoa in animals.

The mosaic genome of indigenous African cattle as a unique genetic resource for African pastoralism.

Cattle pastoralism plays a central role in human livelihood in Africa. However, the genetic history of its success remains unknown. Here, through whole-genome sequence analysis of 172 indigenous African cattle from 16 breeds representative of the main cattle groups, we identify a major taurine × indicine cattle admixture event dated to circa 750-1,050 yr ago, which has shaped the genome of today's cattle in the Horn of Africa. We identify 16 loci linked to African environmental adaptations across crossbred animals showing an excess of taurine or indicine ancestry. These include immune-, heat-tolerance- and reproduction-related genes. Moreover, we identify one highly divergent locus in African taurine cattle, which is putatively linked to trypanotolerance and present in crossbred cattle living in trypanosomosis-infested areas. Our findings indicate that a combination of past taurine and recent indicine admixture-derived genetic resources is at the root of the present success of African pastoralism.

Genetic Diversity and Sequence Polymorphism of Two Genes Encoding Theileria parva Antigens Recognized by CD8+ T Cells among Vaccinated and Unvaccinated Cattle in Malawi.

East Coast fever (ECF) is an acute fatal tick-borne disease of cattle caused by Theileria parva. It causes major losses in exotic and crossbreed cattle, but this could be prevented by a vaccine of T. parva if the vaccine is selected properly based on information from molecular epidemiology studies. The Muguga cocktail (MC) vaccine (Muguga, Kiambu 5 and Serengeti-transformed strains) has been used on exotic and crossbreed cattle. A total of 254 T. parva samples from vaccinated and unvaccinated cattle were used to understand the genetic diversity of T. parva in Malawi using partial sequences of the Tp1 and Tp2 genes encoding T. parva CD8+ antigens, known to be immunodominant and current candidate antigens for a subunit vaccine. Single nucleotide polymorphisms were observed at 14 positions (3.65%) in Tp1 and 156 positions (33.12%) in Tp2, plus short deletions in Tp1, resulting in 6 and 10 amino acid variants in the Tp1 and Tp2 genes, respectively. Most sequences were either identical or similar to T. parva Muguga and Kiambu 5 strains. This may suggest the possible expansion of vaccine components into unvaccinated cattle, or that a very similar genotype already existed in Malawi. This study provides information that support the use of MC to control ECF in Malawi.

Mutations in the TaPIN1 peptidyl prolyl isomerase gene in Theileria annulata parasites isolated in Sudan.

The tick-borne parasite Theileria annulata is the causative agent of tropical theileriosis or Mediterranean theileriosis. Infection of bovine leukocytes by the obligate intracellular parasites induces proliferative and invasive phenotypes associated with activated signaling pathways. The transformed phenotypes of infected cells are reversible by treatment with the theilericidal drug buparvaquone. Recent reports of resistance to buparvaquone in Africa and Asia highlight the need to investigate the mechanisms and prevalence of drug resistance. We screened 67 T. annulata isolates from Sudan to investigate mutations in the T. annulata prolyl isomerase I gene (TaPIN1). The secreted TaPin1 interacts with host proteins to induce pathways driving oncogenic transformation and metabolic reprogramming. We found an Alanine-to-Proline mutation at position 53 (A53P) in the catalytic loop that was previously found in Tunisian drug-resistant samples. This is the first study reporting independent confirmation of the A53P mutation in geographically isolated samples. We found several additional mutations in the predicted N-terminal signal peptide that might affect TaPin1 processing or targeting. We found that many parasites also share mutations in both the TaPIN1 and the cytochrome b genes, suggesting that these two genes represent important biomarkers to follow the spread of resistance in Africa, the Middle East and Asia.

Species Diversity and Seasonal Distribution of Hard Ticks (Acari: Ixodidae) Infesting Mammalian Hosts in Various Districts of Riyadh Province, Saudi Arabia.

Hard ticks are among the most important blood sucking arthropods that transmit pathogens to humans and animals. This study was designed to determine prevalence, mapping, geographical distribution, and seasonal activity of hard tick species infesting the most common domestic and wild mammals in various districts of Riyadh Province, Saudi Arabia, during the period January to December 2017. In total, 10,832 adult hard ticks were collected from the bodies of 8,435 animals belonging to 18 different mammalian species. The ticks were preserved in 70% alcohol and microscopy was used to identify species. Two genera, Hyalomma and Rhipicephalus, were identified, comprising 10 species of hard ticks, with Hyalomma comprising 68.3% and Rhipicephalus comprising 31.7% of species. The most common species on domestic mammalian hosts was Hyalomma dromedarii (Koch 1844) (39.9%) followed by Rhipicephalus turanicus (Pomerantsev, Matikashvili & Lotosky 1936) (34.9%), whereas on wild mammalian hosts Rhipicephalus sanguineus (Latreille 1806) was by far the most prevalent species (83.0%). However, ticks were most abundant during May through July (36.0%) in the studied areas, and tick intensity and abundance differed among seasons. Our results provide information for human and animal health service managers, as well as governmental authorities, to gain a better understanding of hard ticks infesting mammalian hosts in Riyadh Province, Saudi Arabia, which can help improve prevention and control of tick-borne diseases, especially during outbreaks.

Utilization of crude and recombinant ELISAs for serodiagnosis of camel trypanosomosis in Sudan.

This study was carried out to evaluate the application of CATT/T. evansi, crude and recombinant (TeGM6-4r) antigen ELISAs in the diagnosis of camel trypanosomosis caused by two trypanosome species, T. evansi and T. vivax, in Sudan. Concurrently, the current situation of camel trypanosomosis was investigated based on the results of a serological analysis. The recombinant tandem repeat antigen TeGM6-4r is conserved among salivarian trypanosome species and was highly sensitive in the detection Trypanozoon, and T. vivax. It has been validated in the diagnosis of surra in cattle and water buffalo but not in camels. A comparative evaluation of a crude antigen ELISA and a recombinant antigen GM6 (rTeGM6-4r) ELISA was performed using 189 blood samples, which included 148 samples obtained from different camel herds in Eastern Sudan and 41 samples from camels that had been brought from Western Sudan to local markets. The results showed that the rTeGM6-4r ELISA detected the greatest number of positive samples (n = 118, 62%), while CATT/T. evansi and the crude antigen ELISA detected the lowest number of positive samples (n = 73, 39%). The kappa value of rTeGM6-4r as compared to TeCA ELISA was 0.5515, which indicated moderate agreement. We concluded that the rTeGM6-4r ELISA is the test of choice for use in screening camel for trypanosomosis caused by T. evansi and T. vivax in Sudan.

Potential role of dogs as sentinels and reservoirs for piroplasms infecting equine and cattle in Riyadh City, Saudi Arabia.

Canine tick-borne diseases have been considered emerging and re-emerging threats, given their increasing global prevalence. In this molecular survey, we aimed to detect and identify common tick-borne pathogens in dogs from Riyadh city in Saudi Arabia. Initially, the study included 36 dogs visiting private veterinary clinics. PCRs targeting the 18S ribosomal RNA gene (rDNA) of haemoparasites (Babesia, Theileria and Hepatozoon) and the 16S rDNA of Anaplasmataceae were performed. The results showed that 26 (72.2%) dogs were infected by some of the haemoparasites under investigation. The sequencing analysis of the amplicons confirmed the infections due to two parasite species Theileria equi and Theileria velifera. Further examination of guard dogs kept in the horse stables of the Riyadh Municipality revealed that the majority of the tested dogs (65.2%: 30 out of 46) were infected with either of the parasites. In addition, the genotypes of all the parasites in these dogs were identical to those of the parasites in the dogs from the veterinary clinics. Thus, it can be concluded that dogs are infected with these haemoparasites and serve as a reservoir for both T. equi and T. velifera in the study area; however, the clinical implication of this finding is to be studied.

Evidence of multiple point mutations in Theileria annulata cytochrome b gene incriminated in buparvaquone treatment failure.

Drug resistance is one of the emerging and re-emerging epidemics affecting both veterinary and public health sectors. Buparvaquone provides the most satisfactory means in the treatment of bovine tropical theileriosis. However, recently there has been widespread reports of development of resistance of Theileria annulata to buparvaquone. To investigate the situation in Sudan where bovine tropical theileriosis is endemic, fifty blood samples from T. annulata-positive cattle. were used for DNA extraction, PCR and cytochrome b gene nucleotide sequencing. Analysis of the two buparvaquone binding site regions Q01 (130-148) and Q02 (244-266), revealed three non- synonymous mutations at codon 146; alanine (GCT) to threonine (ACT) within the Q01 region across all 50 isolates and the other mutation at codon 129; serine (AGC) to glycine (GGC) in 18 isolates which is very close to the Q01 binding site. However, we documented another mutation at position 227; valine (GTG) to methionine (ATG) close to the close to the Q02 binding site, in three isolates with mutation at codon 129. We concluded that this study has provided evidence of point mutations in the cytochrome b gene of T. annulata that might be associated with buparvaquone treatment failure in Sudan.