Serological, virological and molecular diagnosis of an outbreak of lumpy skin disease among cattle in Butana area, Eastern Sudan.

BACKGROUND: Lumpy skin disease (LSD) is a highly infectious disease of cattle caused by a virus of the Poxviridae family, genus Capripoxvirus. The disease has great economic effects on the global cattle industry. In this study, an outbreak of LSD among cattle which occurred in Nahr Atbara and Halfa El Jadida locations in Butana area, Kassala State in November, 2020 is described. METHODS: Lumpy skin disease virus (LSDV) antibody and nucleic acid were detected in specimens (n = 50) using enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). RESULTS: The virus was isolated on the chorioallantoic membrane (CAM) of embryonated chicken eggs and identified by PCR. The number of animals at risk was 295 with a morbidity rate of 4.1% and a mortality rate of 2.4%. CONCLUSIONS: LSDV was diagnosed for the first time in the Butana region, Kassala State, Eastern Sudan in 2020.

Epidemiology and molecular characterization of re-emerged virulent strains of Peste des Petits Ruminants virus among sheep in Kassala State, Eastern Sudan.

BACKGROUND: Peste des Petits Ruminants (PPR) is a severe contagious viral disease, which mainly affects small ruminants. PPR is caused by a Morbillivirus that belongs to the family Paramyxoviridae. In this study 12 suspected PPR outbreaks among sheep and goats were investigated in four localities in Kassala State, Eastern Sudan, during 2015-2017. The causative agent was confirmed by a Sandwich Enzyme-Linked Immunosorbent Assay (sELISA), and a Reverse Transcription Polymerase Chain Reaction (RT-PCR) targeting a partial sequence of nucleocapsid protein gene (N- gene) and a partial sequence of fusion protein gene (F- gene). Sequencing and phylogenetic analysis were carried out on six N- gene based RT-PCR products selected from two outbreaks occurred on border and inner localities of Kassala State to determine the circulating lineages of PPRV strains. Identity percentages were determined between isolates in this study and previous Sudanese, and other (African and Asian) isolates which clustered along with them. RESULTS: Out of 30 samples, 22 (73.3%) were positive using sandwich ELISA. From 22 s ELISA positive samples, 17 (77.3%) were positive by Ngene based RT-PCR and only 7(43.8%) out of 16 positive samples by N gene based RT-PCR were positive using Fgene based RT-PCR. The sequencing and phylogenetic analysis confirmed involvement of the lineage IV of PPRV in outbreaks among small ruminants in Kassala State and high identity percentage between our isolates and previous Sudanese and other (African and Asian) isolates. CONCLUSIONS: The present study demonstrates that genetic relationship between PPRV strains circulating in sheep in Kassala State, Eastern Sudan, and PPRV strains characterized as lineage IV in neighboring African countries such as Eretria,Ethiopia, Egypt, and other Asian countries.

Isolation, Extraction, Purification, and Molecular Characterization for Thermostable α-Amylase from Locally Isolated Bacillus Species in Sudan.

The aim of this study was to isolate some soil bacteria strain that produced α-amylase and subsequent extraction and purification. One hundred soil samples were collected from different geographical areas in Khartoum State such as north Omdurman, Toti Island, and Soba. Samples were analyzed for starch hydrolyzing bacteria. Among several bacteria isolated, Bacillus cereus and Bacillus licheniformis were identified as active α-amylase producers. Both bacteria showed a large zone of clearance of 20 mm when grown on starch-agar plates. The identity was conducted using biochemical characterization and confirmed by sequencing their 16S-rDNA. The constitutive nature of amylase was proved by amplification of the amylase gene from the genome of B. licheniformis. The α-amylase activity from the spent medium of B. cereus and B. licheniformis was optimized at pH 8.0 and temperature of 45°C and 65°C, respectively. The α-amylase produced by both bacteria is alkalophilic and thermophilic. The experiments confirmed that B. licheniformis can be a good source of amylase for industrial applications in Sudan.

A Survey of Bluetongue Infection and Associated Risk Factors among the One-Humped Camel (Camelus dromedaries) in Gadarif State, Eastern Sudan.

Bluetongue (BT) is an infectious, noncontagious, vector-borne viral disease that affects wild and domestic ruminants transmitted by Culicoides spp. A cross-sectional study was carried out during the period 2016-2017 in Gadarif state. A total of 276 sera samples were collected from camels in six localities of Gadarif state, eastern Sudan, to investigate bluetongue virus (BTV) seroprevalence and associated risk factors of BTV infection including age, sex, breed, locality, and ecology of the region. Enzyme-linked immunosorbent assay (ELISA) was used for estimation of BTV seroprevalence rate. The overall BTV seroprevalence rate was 96.7% in the study area ranging from 93.5% to 100% in six screened localities with no significant differences. The findings revealed similar BTV seroprevalence rates in both males and females, but high rates were found in age group of less than one year and two to three years with estimated 100%. However, the lowest seroprevalence was found in the age group of five to four years with estimated BTV to be 92.3%. BTV seropositivity was not found to be statistically associated with examined different camel breeds which revealed 93%, 94.4%, 97.6%, and 97.8% seroprevalence in Bushari, Rashide, Arabi, and Anafi, breeds, respectively. Epidemiology of BTV assessment according to the ecology of the area showed high BTV seroprevalence in desert and savanna with estimated 100% and lower BTV seroprevalence in arid and rich savanna with estimated 94.8% and 95.7%, respectively. There was no significant association between BTV ELISA positivity and sex, breed, and ecology of the area.

Sero-epidemiological survey of bluetongue disease in one-humped camel (Camelus dromedarius) in Kassala State, Eastern Sudan.

BACKGROUND: Bluetongue (BT) is a vector-borne viral disease of ruminant and camelid species which is transmitted by Culicoides spp. The causative agent of BT is bluetongue virus (BTV) that belongs to genus Orbivirus of the family Reoviridae. The clinical disease is seen mainly in sheep but mostly sub-clinical infections of BT are seen in cattle, goats and camelids. The clinical reaction of camels to infection is usually not apparent. The disease is notifiable to the World Organization for Animal Health (OIE), causing great economic losses due to decreased trade and high mortality and morbidity rates associated with bluetongue outbreaks. The objective of this study was to investigate the seroprevalence of BTV in camels in Kassala State, Eastern Sudan and to identify the potential risk factors associated with the infection. A cross sectional study using a structured questionnaire survey was conducted during 2015-2016. A total of 210 serum samples were collected randomly from camels from 8 localities of Kassala State. The serum samples were screened for the presence of BTV specific immunoglobulin (IgG) antibodies using a competitive enzyme-linked immunosorbent assay (cELISA). RESULTS: Seropositivity to BTV IgG was detected in 165 of 210 camels' sera accounting for a prevalence of 78.6%. Potential risk factors to BTV infection were associated with sex (OR = 0.061, p-value = 0.001) and seasonal river as water source for drinking (OR = 32.257, p-value = 0.0108). CONCLUSIONS: Sex and seasonal river as water source for drinking were considered as potential risk factors for seropositivity to BTV in camels. The high prevalence of BTV in camels in Kassala State, Eastern Sudan, necessitates further epidemiological studies of BTV infection in camels and other ruminant species to better be able to control BT disease in this region.

Prevalence of ticks (Acari: Ixodidae) and Theileria lestoquardi in sheep at El Huda and El Nuhud animals production research stations, Sudan.

A cross-sectional study was performed 200 blood samples and 314 tick samples in El Huda and El Nuhud animals production research stations, Sudan, in May (summer) and December (winter) in 2016, to determine the prevalence of Theileria lestoquardi in sheep and the potential risk factors associated with the infection. A total of 200 blood samples and 314 tick samples were collected from El Huda (n = 103 blood, 97 tick) and El Nuhud (n = 97 blood, 217 tick) stations. Data on the risk factors, such as age, sex, ecotype of sheep, body condition score and seasons were recorded. The overall prevalence of Theileria lestoquardi was 13% (26/200) using PCR. A significant variation in the prevalence of Theileria lestoquardi was observed between the stations and the ecotype of sheep (p ≤ 0.05), whereas the highest prevalence was recorded in El-Huda station (19.4%) as well as in Shugor (22.8%). Other risk factors, like age, sex, body condition, and seasons were not found to be significantly associated with infection. However, the highest prevalence rate was recorded in old animals (21.6%) than the other, in males (17.9%) than females (12.2%), in animals with poor body condition (26.1%) than the other, and in winter (16%) than summer (10%). Four tick species i.e. Rhipicephalus evertsi evertsi (63.1%), Hyalomma anatolicum (13.8%), Hyalomma dromedarii (8.8%), and Hyalomma impeltatum (14.3%) were recorded in El Nuhud station. While in El Huda station, only Rhipicephalus evertsi evertsi (79.4%), Hyalomma anatolicum (20.6%) were recorded. This study revealed that 13% of sheep were suffering from Theileria lestoquardi which is a considerable number at the stations. Therefore, further epidemiological investigations on disease throughout the year are required in order to set a well-coordinated control program.

Prevalence of Antibodies to Simbu Serogroup Viruses in Cattle in Sudan.

The Simbu serogroup is one of the serogroups that belong to the Orthobunyavirus genus of the family Peribunyaviridae. Simbu serogroup viruses are transmitted mainly by Culicoides biting midges. Meager information is available on Simbu serogroup virus infection in ruminants in Sudan. Therefore, in this study, serological surveillance of Simbu serogroup viruses in cattle in seven states in Sudan was conducted during the period from May, 2015, to March, 2016, to shed some light on the prevalence of this group of viruses in our country. Using a cross-sectional design, 184 cattle sera were collected and tested by a commercial SBV ELISA kit which enables the detection of antibodies against various Simbu serogroup viruses. The results showed an overall 86.4% prevalence of antibodies to Simbu serogroup viruses in cattle in Sudan. Univariate analysis showed a significant association (p=0.007) between ELISA seropositivity and states where samples were collected. This study suggests that Simbu serogroup virus infection is present in cattle in Sudan. Further epizootiological investigations on Simbu serogroup viruses infection and virus species involved are warranted.

Serological Investigations of Bluetongue Virus (BTV) among Sheep and Goats in Kassala State, Eastern Sudan.

Bluetongue (BT) is an infectious, noncontagious, vector-borne viral disease of wild and domestic ruminants. BTV is a member of the Orbivirus genus of the family Reoviridae. The present study aimed to investigate the seroprevalence of BTV in sheep and goats in Kassala State, Sudan. It also aimed to determine risk factors associated with BTV infection. The study was carried out by a structured questionnaire survey, and a total of 809 serum samples were collected from sheep (n = 459) and goats (n = 350) from 9 different localities in Kassala state. These samples were analyzed using a competitive enzyme-linked immunosorbent assay (cELISA) for the detection of BTV antibodies. The overall seroprevalence of BTV was 91.2% (738/809). In goats, the prevalence of BTV antibodies was comparatively higher (100%) than in sheep (84.5%). The prevalence differed between localities and was the highest in the center section of Kassala and Western Kassala (100%). Animals aged 6-11 months were highly infected (93.9%) compared to 1-year-old (85.5%). Caprine species was more likely to be infected (100%) than ovine (84.5%), and females were highly infected (92.8%) than males (85.5%). BTV infections were higher in the winter season (91.4%). Risk factors that showed significant associations with cELISA positivity included locality and sex (p ≤ 0.003) and species and age (p ≤ 0.000). Factors significantly associated with cELISA positivity in multivariate analysis were localities, species, age, and sex. BTV infection is prevalent in sheep and goat populations in Kassala state.

First report on molecular characterization and phylogenetic analysis of Reticuloendotheliosis virus in Sudan.

The reticuloendotheliosis virus (REV) group of retroviruses infects a wide range of avian species, including chickens, turkeys, ducks, geese, quail, and prairie chickens. The infection can result in immunosuppression, runting syndrome, high mortality, acute reticular cell neoplasia, or T- and/or B-cell lymphoma. One PCR positive chicken spleen sample obtained in a previous study in addition to one Marek's disease and three fowl pox (FP) vaccine samples were investigated in this study. A PCR assay was performed to detect the presence of REV provirus DNA in these samples. The results indicated the contamination of fowl pox virus and Marek's disease vaccines with REV. In addition, detection of integration of REV inside the genome of fowl pox vaccine was confirmed using primers corresponding to the FPV DNA regions flanking the REV integration site. Alignments of two sequences, one from the spleen tissue and the other from contaminated FP vaccine with REV, with other REV (env) gene sequences obtained from GenBank indicated their high similarity. Furthermore, phylogenetic analysis indicated that the partial part of (env) gene of our two isolates was closely related to variants from India, USA, Taiwan, and China. These results confirmed the contamination of commercial fowl pox and Marek's disease vaccines used in Sudan with REV. Phylogenetic analysis indicated that the partial part of (env) gene sequences from Sudan was closely related to variants from India, USA, Taiwan, and China.

A molecular prevalence survey on Anaplasma infection among domestic ruminants in Khartoum State, Sudan.

This study was conducted in Khartoum State, Sudan to determine the prevalence and the risk factors associated with Anaplasma and Ehrlichia species infections in domestic ruminants. Blood samples were collected from a total of 594 animals from 32 different farms distributed in the three provinces of Khartoum State. Among the 196 cattle, 200 sheep, and 198 goats examined using PCR, 13.27%, 32.50%, and 35.86% were infected with Anaplasma spp., respectively, with an overall prevalence of 27.27%. Cattle were infected with A. marginale (10.71%), A. centrale (2.04%), and A. ovis (0.51%), while sheep and goats were infected with A. ovis being significantly higher compared with cattle. No Ehrlichia spp. was detected in domestic ruminant in Khartoum State. Prevalence rates of Anaplasma infections were highly associated with breed, location, season, and sex. The prevalence rates of Anaplasma infection were significantly higher in exotic goat breeds compared with indigenous, and the infection in sheep and cattle was significantly higher in summer and in autumn in goats. The Anaplasma spp. infection rate in goats was significantly higher in females. The infection rate was also significantly higher in Khartoum North in both sheep and goats. It could be concluded that Anaplasma infection is prevalent in small and large ruminants in Khartoum State. Therefore, further studies on the epidemiology of anaplasmosis, possible tick, lice, and flea vectors and reservoirs in Sudan are important.

Serological and molecular identification of Reticuloendotheliosis virus (REV) in chickens in Sudan.

BACKGROUND: Reticuloendotheliosis virus (REV) is a gammaretrovirus that belongs to the family of Retroviridae. The infection can result in immunosuppression, runting syndrome, high mortality, acute reticular cell neoplasia or T- and/ or B-cell lymphoma, in a variety of domestic and wild birds. The disease is widespread around the world. No related data have been reported in Sudan about the disease. The present study was conducted to determine the prevalence of REV antibodies and DNA in local and commercial breeds of chickens older than 20 weeks from June 2014 to February, 2017. METHODS: A total of 460 sera samples and 150 (50 liver and 100 spleen) tissue samples were collected from local and commercial breeds of chickens older than 20 weeks and screened for anti-REV antibodies in four states of Sudan using a commercial REV antibody ELISA test kit (IDEXX). Polymerase chain reaction (PCR) was performed to detect REV DNA in tissue samples in Khartoum State. RESULTS: The results revealed that the overall seroprevalence of REV was 74.6% among local and commercial chicken breeds, but in commercial it was 79.5% (190/239) and 69.2% in local breeds (153/221). One hundred and fifty tissue samples of chickens (50 liver, 100 spleen) were tested using PCR for detection of REV using primer sets of the conserved region in envelope glycoprotein (env) gene with a band length of 850 bp. Five out of 50 (10%) liver samples were RE provirus DNA positive detected by PCR, whereas 15 out of 100 (15%) spleen samples were PCR positive. Univariate analysis revealed there was a difference (p ≤ 0.05) between locality and breed of chickens and seropositivity to REV. CONCLUSIONS: The prevalence of the disease was high in Sudan and more studies are needed to evaluate the epidemiology and pathogenesis of the virus.

Seroprevalence of spotted fever group (SFG) rickettsiae infection in domestic ruminants in Khartoum State, Sudan.

Spotted fever group (SFG) rickettsiosis is caused by obligatory intracellular Gram-negative bacteria that belong to the genus Rickettsia. Ticks belonging to the family Ixodidae can act as vectors, reservoirs or amplifiers of SFG rickettsiae. This study was conducted to estimate the seroprevalence of SFG rickettsioses in cattle, sheep and goats from Khartoum State, Sudan. Blood samples were collected from a total of 600 animals (sheep, goats and cattle) from 32 different farms distributed in three locations in Khartoum State during the period January to December 2012. Sera were tested for antibodies against SFG rickettsiae using IFAT. The prevalence of seropositivity was 59.3% in sheep, 60.1% in goats and 64.4% in cattle. Season was significantly (P < 0.05) associated with seroprevalence of SFG rickettsiae in cattle during winter. The SFG rickettsiae antibodies prevalence was significantly higher in female compared with male in sheep, but there were no significant differences between male and female in either cattle or goats. The prevalence was significantly higher in adult animals compared with young in both sheep and goats. With regard to management system, there was a significant difference in the prevalence in cattle raised in closed system compared with those raised in semi-intensive system. In contrast, there was significant difference in the seroprevalence of SFG in sheep where the prevalence was higher in the sheep raised in semi-intensive system compared with those raised in close system. There was no significant difference in the seroprevalence in goats with regard to management systems. The unexpected high prevalence of SFG rickettsia antibodies in domestic ruminants sera suggest that the veterinary and public health impact of these agents in Sudan need further evaluation especially in humans.

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

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