Prevalence of peste des petits ruminants virus antibodies in sheep and goats sera from Central-Western Sudan.

Clinical signs suggestive of peste des petits ruminants (PPR) involved herds of small ruminants, which were described elsewhere in Sudan. Peste des petits ruminants was confirmed using an Immunocapture ELISA (IC-ELISA) assay in samples of infected and dead animals in areas of outbreaks. Therefore, to update information regarding the current situation and for assessment of the serological prevalence of PPR in small ruminants mingled at Central and Western Sudan during 2018-2019, 368 sera were collected from sheep (325 sera) and goats (43 sera) with different ages and breeds. These sera included 186 sera (173 sheep and 13 goats) from White Nile State and 182 sera (152 sheep and 30 goats) from Kordofan States. Competitive ELISA demonstrated higher prevalence of PPRV antibodies of 88.9%, 90.7% and 88.6% in both sheep and goats, goats, and sheep sera, respectively. Moreover, 100%, 94.7% and 78.5% seroprevalence values were demonstrated in South Kordofan, North Kordofan and White Nile States. The higher seroprevalence values detected in sera of unvaccinated sheep and goats indicated the wide exposure of these animals to PPRV and presence of protection following PPR viral infection. The findings of the study indicated that PPR is endemic in the surveyed areas of Sudan.Contribution: The study will contribute effectively to the global eradication programme of PPR organised by the World Organization for Animal Health (WOAH, formerly OIE) and Food and Agriculture Organization (FAO). To completely eliminate PPR from Sudan by 2030, local efforts should be directed towards effectively and wholly vaccinating small ruminants using PPRV vaccine especially in routes of seasonal animal's movement and shared grazing areas.

Seroprevalence and risk factors associated with Newcastle disease in backyard chickens in West Kordofan State, Sudan.

Background and Aim: Newcastle disease (ND), a major constraint to poultry production worldwide, is a highly contagious disease of many species of domestic, exotic, and wild birds caused by ND virus (NDV). Epidemiological studies are lacking regarding ND in village chickens, including the traditional and intensive production systems used in Sudan. However, it is necessary to develop appropriate strategies to control the disease. Therefore, this study aimed to estimate the flock- and bird-level seroprevalence of NDV in backyard chickens in West Kordofan State, Sudan, and to identify the risk factors associated with ND in the study area. Materials and Methods: The seroprevalence of the circulating NDV and bird-level risk factors associated with ND was determined in backyard chickens from March to October 2017, in six villages (Alnowara, Alleait, Geibaish, Baiad, Sougoh, and Alnuhoud) in the Geibaish and Elnuhoud localities of West Kordofan State. Results: Using the hemagglutination-inhibition test, the bird- and flock-level seroprevalences of antibodies to NDV were estimated as 20.6% (78/378) and 45% (18/40), respectively. Bird-level NDV seropositivity in backyard chickens was significantly associated with the reason for raising chickens, type of housing, contact with neighboring poultry, contact with wild birds, and chicken mortality caused by infectious diseases (p ≤ 0.05). Conclusion: This study indicated that NDV is circulating in backyard chickens and may act as a potential source of infection for other birds and thus persistence of ND among local traditionally managed chickens in the areas of West Kordofan State. Risk factors contributing to ND occurrence are important for designing appropriate prevention and control strategies.

Sero-epidemiology of foot-and-mouth disease in Darfur area, Western Sudan.

A total of 367 bovine sera positive to antibodies against non-structural proteins (NSPs) of foot-and-mouth disease (FMD) virus were screened for serotype O, A and SAT2 antibodies using the virus neutralization test (VNT). Sera had been collected in 2016 from North (228) and South (139) Darfur States in Western Sudan, where high and low circulation of FMD virus, respectively, prevailed. Tested sera represented the positive-NSPs portion in a random sample of 669 sera collected from both States. According to standard statistical methods, calculations for serial testing (NSPs ELISA and VNT) were applied to estimate prevalence rates of serotype-specific antibodies in the two States. In each State, approximately 20% of NSPs positive sera failed typing. Prevalence's detected were 49% ± 5% (O), 27% ± 5% (A) and 14% ± 4% (SAT2) in North Darfur State and 27% ± 5% (O), 17% ± 4% (A) and 8.0% ± 3% (SAT2) in South Darfur State. In both States, prevalence rates were significantly higher for serotype O, followed by A then SAT2; the same order that was known in most parts of Sudan. Consistently, estimated prevalence's were statistically significantly higher (P < 0.05) in North Darfur than in South Darfur State. Apart from serotype SAT2, detected prevalence rates were lower or similar to those inside the country in previous occasions. Frequency and pattern of distribution of serotype O prevalence were consistent with its suggested pattern of circulation from the Nile valley to other parts in Sudan and significant within the country's circulation. Alternatively, serotype SAT2 prevalence and distribution in Darfur area were suggestive of sporadic occurrence. However, slightly higher prevalence rates of SAT2 antibodies in Darfur than in neighbouring Kordofan areas in 2013 reflected the wide dissemination of SAT2 ( http://www.wrlfmd.org ) in Sudan in early 2014. Risk of FMD in Darfur seemed to be associated with the movement of animals to the North in the wet season as part of the pastoral system, and with movement related to trade into urban centers more than with pastoralism across the Western borders. Generally, the result presented little evidence to suggest presence of FMD primary endemic foci in Darfur area.

Serological detection and genetic characterization of foot-and-mouth disease virus from cattle in northern sudan, 2016­2018.

Northern Sudan is an important corridor cluster between pools of foot-and-mouth disease virus (FMDV) in East and North Africa. It involves almost the whole border area with Egypt and represents a considerable part of a projected disease-free zone in Sudan. The study monitored FMD infection between 2016 and 2018 in Northern Sudan. Clinical and serological surveillance were carried out. Results largely confirmed previous reports that have described the relatively lower circulation of FMDV in the area than in other parts of the country. Clinical FMD was confirmed, once in the three years period, as serotype O of an unnamed lineage within the topotype East Africa 3 (EA3). Using serial testing (the ID ELISA and virus neutralization test), sero-prevalence estimates of serotype-specific antibodies in the two States of Northern Sudan ranged between 15.4% (serotype A) in the River Nile State to 3.4% (serotype SAT2) in the Northern State. Striking disparities between patterns of FMD in Northern Sudan and the rest of Sudan were observed. Unlike Western, Eastern, Central and Southern Sudan, no predominance of serotype O antibodies was detected in Northern Sudan. Concurrently, a serotype O isolate from Northern Sudan in 2016 was found to be of transboundary nature circulating in East and North Africa and in the Middle East (nt. id. > 99%); like serotype O that caused the last episode of disease in Northern Sudan in 2012. Molecular findings were compatible with the inferred low circulation of FMDV in Northern Sudan. Elsewhere in Sudan, endogenous serotype O viruses seemed to be circulating more unabated. It was concluded that low animal density and limited animal movement in Northern Sudan together with the high antibody levels against serotype O in immediately neighbouring States (Khartoum and Kassala) effectively decreased infiltration of endogenous O viruses.

Detection of peste des petits ruminants virus in pneumonic lungs from clinically apparently healthy camels slaughtered at Tambul slaughterhouse, Central Sudan.

The study investigated the presence and prevalence of peste des petits ruminants (PPR) viral antigens among camels in Tambul area, Gezira State, Central Sudan, regardless of its sex, age and breed, and their possible contribution in the epidemiology of the disease in the Sudan. Hundred pneumonic lung tissues were aseptically collected from clinically apparently healthy camels showed no signs of illness at ante-mortem examination, from Tambul slaughterhouse, Tambul area, Gezira State, Central Sudan, between November and December 2018. Samples were collected based on presence of the pneumonic signs, at the tissue level, including congestion of the lungs, presence of abscesses, fragility, changes in colour and thickness of the tissue. In order to detect PPR viral antigen, haemagglutination (HA) test was employed on lung tissue homogenate, using chicken RBCs suspension, which gave a positive reaction in 17-19 min. PPRV antigen was detected in 98 of camel samples with an overall antigenic prevalence of 98%. Of note, the HA titres achievable ranged from 4 to 256 HA units (HAU) with mean titre of 14.4 HAU, whereas apparently most of the samples achieved HA titres of 8 HAU. The results demonstrated presence of PPR viral antigens associated with pneumonia in camels indicating exposure of these camels to PPRV and probably presence of subclinical infection. Infection of species other than small ruminants suggests the fact that camels are potential hosts for PPRV and might play a role (or not) in the epidemiology of the disease. Further studies are needed to demonstrate if camels are able to transmit PPRV for in-contact small ruminants or other animal species.

Detection of peste des petits ruminants virus in pneumonic lungs from apparently healthy sheep and goats slaughtered at Al-Hasaheisa slaughterhouse, Gezira state, central Sudan.

The study aimed to investigate the presence of peste des petits ruminants (PPR) in pneumonic lung tissues from clinically apparently healthy sheep and goats and further demonstrating its prevalence in Gezira state, central Sudan. During March 2019, 99 pneumonic lung samples were collected from apparently healthy sheep (80) and goats (19) from Al-Hasaheisa slaughterhouse located in Al-Hasaheisa locality, Gezira state. Using the haemagglutination (HA) test for the detection of peste des petits ruminants virus (PPRV) antigen, an overall antigenic prevalence of 86.9% was demonstrated in sheep and goats lung tissue homogenate. Of note, the prevalence of PPRV is higher in goats (100%) compared to sheep (83.7%). In this study, the reported increasing prevalence of PPR in central Sudan might be because of insufficient vaccination of animals. The findings of the present study indicated the widespread of PPR amongst sheep and goats in Al-Hasaheisa, Gezira state. Detection of PPRV antigen in the pneumonic lung samples is an indication of exposure of these animals to PPRV or presence of PPR viral infection and demonstrates the role of PPR as the cause of pneumonia in small ruminants. In fact, the circulation of the virus in clinically apparently healthy animals poses a threat for other in-contact susceptible animals and could play a significant role in the spread of the disease.

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.

First detection and genetic characterization of peste des petits ruminants virus from dorcas gazelles "Gazella dorcas" in the Sudan, 2016-2017.

In May 2017, many free-ranging dorcas gazelles (Gazella dorcas) with suspected signs of peste des petits ruminants (PPR) were reported in Dinder National Park, South-Eastern Sudan. Peste des petits ruminants virus (PPRV) antigen and nucleic acid were detected in specimens from these gazelles using an immunocapture ELISA and a reverse transcription polymerase chain reaction (RT-PCR) assays. PPRV was also detected in four healthy semi-captive dorcas gazelles from two areas of Khartoum State. Phylogenetic analysis showed that these PPRV strains belonged to the lineage IV genotype. The present study demonstrates that gazelles are a potential wild small ruminant host for PPRV and may influence the epidemiology of PPR in the Sudan.

Productive replication of peste des petits ruminants virus Nigeria 75/1 vaccine strain in vero cells correlates with inefficiency of maturation of the viral fusion protein.

Peste des petits ruminants virus (PPRV), a member of the genus Morbillivirus, in the family Paramyxoviridae expresses two membrane glycoproteins, the fusion (F) and haemagglutinin (H) glycoproteins which mediate virus-to-cell fusion and cell-to-cell fusion leading to the induction of syncytia in PPRV infected cells. In the context of the characterization of the virulent lineage IV strain PPRV Kurdistan 2011, isolated from wild goats from the Kurdistan region in Iraq, we observed that both PPRV Kurdistan 2011 and the PPRV Nigeria 75/1 vaccine strain led to induction of large syncytia in Vero-dogSLAM cells within 48 h whereas both failed to induce detectable cell-cell fusion events in two Vero cell lines of differing passage histories. We were unable to detect syncytium formation in transiently transfected cells expressing PPRV F or H alone whereas co-expression of F and H induced large syncytia - in Vero-dogSLAM cells only. In VeroMontpellier cells expressing PPRV F and H, fused cells were rarely detectable indicating that PPRV mediated cell fusion activity is impaired in this cell line. Surprisingly, on Vero-dogSLAM cells the vaccine strain grew to titers of 105.25 TCID50/ml, whereas infectious virus yield was about 200-fold higher on VeroMontpellier and Vero-76 cells. In contrast, the virulent Kurdistan 2011 strain grew to a maximum titer of 107.0 TCID50/ml on Vero-dogSLAM cells and only 104.5 TCID50/ml on normal Vero cells. This was as expected since Vero cells lacking the SLAM receptor for PPRV are regarded as not so permissive for infection. To elucidate the divergent productive replication behaviour of PPRV Nigeria 75/1 vaccine strain on Vero vs Vero-dogSLAM cells, we examined whether intracellular transport and/or maturation of the viral envelope glycoproteins F and H might be implicated with this phenomenon. The results indicate that F in contrast to the H glycoprotein matures inefficiently during intracellular transport in VeroMontpellier cells, thus leading to an absence of detectable syncytia formation. However, in the case of the PPRV Nigeria 75/1 vaccine strain this did not impair efficient virus assembly and release.

Serological investigations of peste des petits ruminants among cattle in the Sudan.

During 2015 and 2016, from five different States of the Sudan, a total of 1000 cattle sera were purposively collected from many herds of apparently healthy cattle regardless of their age, sex, and breed. Assessment of the sero-prevalence of PPRV antibodies using competitive ELISA (C-ELISA) yielded a higher overall sero-prevalence of 42.0% (420/1000) among cattle populations in the Sudan which is higher than previously reported. Within Sudanese States under study, the highest sero-prevalence of 53.5% (107/200 sera) was demonstrated in Khartoum State while the least sero-prevalence of 31.5% (63/200 sera) was demonstrated in White Nile State. The higher PPRV sero-prevalence values detected in cattle suggested the potential exposure of cattle to PPRV via contact with infected small ruminants and thus might be an indicator of infection of small ruminants. There is a need to include serological surveillance of PPR in cattle within the sero-monitoring program of PPR to give a better indication of the national herd immunity and to assess in the ongoing eradication program.

Sero-prevalence of peste des petits ruminants virus antibodies in sheep and goats from the Sudan, 2016-2017.

Recently, severe outbreaks of PPR among small ruminants were reported regularly in different parts of the country leading to significant economic losses. Between 2016 and 2017, a total of 320 sera were collected from sheep (258) and goats (62) from PPR suspected outbreaks from four different States in the Sudan. Screening of sera for the presence of PPRV antibodies by competitive ELISA revealed an overall antibodies sero-prevalence of 80.9% (259/320, 95% CI 20.5-28) among sheep and goats. On the species basis, sheep sera yielded the higher antibodies sero-prevalence of 84.5% (218/258, 95% CI 16.7-24.1) compared to a lower sero-prevalence of 66.1% (41/62, 95% CI 28.5-51.1) obtained from goats sera. Within Sudanese States where outbreaks occurred, the highest overall sero-prevalence of PPRV antibodies in sheep and goats was demonstrated in River Nile State 90.3% (159/176 sera, 95% CI 12.8-18.2) while the lowest incidence was present in Northern State 00.0% (0/2 sera, 95% CI 69.9-72.2). Of note, higher sero-prevalence values were achieved in this study than previously documented. Results of the present study indicated that PPR is currently circulating widely in the Sudan and still is a leading cause of disease outbreaks and higher fatalities in small ruminants. Therefore, the effective PPR vaccine is recommended to cover all parts of the Sudan to prevent the occurrence of disease outbreaks.

Genetic fusion of peste des petits ruminants virus haemagglutinin and fusion protein domains to the amino terminal subunit of glycoprotein B of bovine herpesvirus 1 interferes with transport and function of gB for BHV-1 infectious replication.

Peste des petits ruminants is an emerging, often fatal viral disease of domestic and wild small ruminants caused by peste des petits ruminants virus. The haemagglutinin and the fusion protein are viral envelope glycoproteins and essential for the infection process and both induce a protective immune response in infected or vaccinated animals. Attempts to generate pseudotyped bovine herpesvirus 1 recombinants firstly by integration of expression cassettes for PPRV-H and PPRV-F into the herpesviral genome or secondly to generate pseudotyped BHV-1 by genetically fusing relevant parts of both PPRV glycoproteins to the amino-terminal subunit of glycoprotein B, approaches that had been successful for heterologous viral membrane glycoproteins in the past, failed repeatedly. We therefore analyzed at which intracellular stage generation of viable BHV-1 hybrid-gB recombinants might be inhibited. Results obtained from transient protein expression experiments revealed that, dependent on the fusion protein, transport of the hybrid glycoproteins beyond the endoplasmic reticulum is impeded. Thus, expression of heterologous glycoproteins using BHV-1 interferes more than expected from published experience with BHV-1 gB transport and consequently with virus replication.

Antibody seroprevalences against Peste des Petits Ruminants (PPR) virus in sheep and goats in Sudan.

Counter immnuo-electrophoresis (CIEP) and Competitive ELISA (C-ELISA) tests were employed for seroprevalence of Peste des Petits Ruminants (PPR) infection in Sudan. The result of both tests showed high prevalence of PPRV antibodies in sheep and goats sera collected from six different regions of Sudan. Of the 519 serum samples examined for the presence of PPRV antibodies 307(59.15%) were positive by CIEP while 263(50.67%) were positive by C-ELISA. CIEP technique was shown to be more sensitive than C-ELISA technique for detection of PPRV antibodies (Kappa statistics 0.259). C-ELISA allowed rapid, simple, specific, sensitive and differential sero-diagnosis of PPRV and RPV in sheep, goats and cattle. CIEP is, unlike competitive ELISA, is group-specific test and can not differentiate between PPR and RP infections. Despite its low specificity CIEP can be a useful indicative screening test for PPRV antibodies in flocks that neither been vaccinated nor otherwise exposed to PPR or RP virus. Results obtained suggest that CIEP, like the HI test, could be a useful screening test where it is not possible to use C-ELISA.

Rapid detection of Peste des Petits Ruminants (PPR) virus antigen in Sudan by agar gel precipitation (AGPT) and haemagglutination (HA) tests.

AGPT and HA tests were employed for rapid diagnosis of PPRV infection in sheep and goats in Sudan. Forty lymph nodes and spleen samples from suspected cases of PPR in both sheep and goats were examined by AGPT and HA tests for detection of PPRV antigen. Viral antigen was detected from (77.5%) of the samples tested by AGPT and (92.5%) tested by HA test. The results of both tests revealed that HA test was more sensitive than AGPT for detection of PPRV antigen (Kappa statistics 0.4366). Another advantage of the HA test over AGPT was that it can differentiate PPRV from RPV. Thus the HA test represents a quick, easy, simple, cheap and reliable confirmatory test for the diagnosis of PPR and differential diagnosis of PPRV and RPV. The HA test was carried out using chicken, goat and pig RBCs. Chicken RBCs were found to be the most sensitive for detection of PPRV antigen, followed by goat then pig RBCs. The HA time when using chicken RBCs was 20-25 minutes, using goat RBCs was 25-30 minutes and using pig RBCs was 40-45 minutes. The distribution of PPR infection in four different regions of Sudan was investigated.