An Outbreak of Pestivirus Infection in Sheep in West Kordofan, Sudan

During AprilMay 2010 an outbreak of a disease with obvious nervous and respiratory signs in sheep was reported in west Kordofan State in western Sudan. Four flocks at four different locations in the State were investigated. The deaths by the disease versus the total number of animals in the different areas were 18/150, 1/200, 3/280 and 17/300. The main clinical symptoms observed in adults and young lambs were central nervous signs and respiratory signs; most lambs were weak and emaciated with rough coats. Sera were collected from 11, 18, 19 and 4 of animals in the four Original Research Article Ali et al.; BMRJ, 8(4): 540-545, 2015; Article no.BMRJ.2015.146 541 flocks, respectively. Bacteriological examination on tissue samples did not reveal any positive results. Using competitive ELISA, antibodies against pestivirus were detected in 10/11, 7/18, 7/19 and 1/4, respectively of examined sheep sera in the different locations. RT/PCR using pestvirus specific primers to examine the lung and sera samples showed positive results for one lung and 17 sera samples, and sequence data indicated that the causative agent was Bovine viral diarrhea virus-1 (BVDV-1). This is the first report of BVDV-1 outbreak in sheep in Sudan.


INTRODUCTION
Pestiviruses includes bovine viral diarrhea virus (BVDV) of cattle, border disease virus (BDV) of sheep, and classical swine fever virus (CSFV) of pigs. These viruses are typically isolated from primary host species, but are capable of infecting other species [1]. Under natural farming conditions there is no evidence so far of border disease viruses causing disease in cattle, but approximately 15% of outbreaks of BDV in the UK are caused by BVDV-1 viruses which probably originated from cattle [2].
BD was first reported in 1959 at the English/Welsh border. It is also called 'Hairyshaker' or 'fuzzy-lamb' disease and has been recognized in most sheep-rearing areas of the world [3].
The disease is caused by BDV, which is closely related to CSFV and BVDV. It is a member of the genus Pestivirus in the family Flaviviridae [4].
Serological evidence of BDV in sheep sera (antigen and antibody) has been apparently studied worldwide, in Spain [5], Tunisia [6], Turkey [7] and Iran [8]. The first outbreak of Border disease in a sheep flock in Austria was described [9].
In Sudan, the sheep population is estimated to be 51.6 million [10]. Outbreaks of clinical disease with symptoms of border disease are continuously reported, however, no study had been conducted to investigate the existence of this disease in the country so far. In a recent study, we have detected pestivirus antigen using ELISA in 7 out of 98 samples from sheep lung collected from slaughter houses and in 5 out of 20 lungs of sheep collected from outbreaks of respiratory infections, abortion, diarrhea and nervous signs in Sudan (unpublished data). In a serological survey, we reported the first detection of antibodies to pestivirus in sheep (39.1%) and goats (14.8%) in different areas of Sudan [11].
Here we describe the first outbreak of pestivirus in sheep in Sudan.

Area of the Outbreak
The outbreak was reported in four areas in West Kordofan State in western Sudan (Al Mafrya, Abu Hasheem, Huzeiran and Jabrat Elsheikh).

History of the Outbreak
During April-May 2010, which is the dry season where shortage in pasture and water is dominated in western Sudan, an outbreak of a disease with prominent nervous and respiratory signs had been reported to the veterinary authority from the fore mentioned areas. The total number of animals and the deaths of the disease in the different areas were 150 with 18 deaths, 200 with 1, 280 with 3 and 300 with 17, respectively. The main clinical signs observed in adults, especially in pregnant sheep and young lambs, were central nervous signs and respiratory signs; most of lambs were weak and looked emaciated with rough coats.

Collected Samples
The clinical signs (nervous and respiratory signs) were observed mainly in pregnant animals and young lambs. Most of samples were collected from clinically healthy animals with few sick ones. Samples were collected randomly from adult and young animals at 8 -18 months of age.
Lung and brain tissues samples were collected from a recently dead young animal at Jabrat Elsheikh.

Screening for PPR
PPR is the main causative agent of respiratory infections in sheep. peste des petits ruminants (PPR) antigen and antibodies were screened in collected samples using PPR specific ELISA kits (CIRAD, Montpellier, France).

Bacteriological examination
Collected tissue samples were screened for bacterial growth in different media.

Investigation of pestivirus antibodies
Collected sera were examined for pestivirus antibodies using competitive ELISA kits (BIO X Diagnostics, Jemelle, Belgium). The test was performed according to the instructions of the manufacturer.

RNA extraction
RNA was extracted from serum samples using QIAamp and from brain and lung samples using RNeasy. Both kits were purchased from Qiagen, Germany.

RT/PCR
The extracted RNA samples were subjected to RT/PCR using One step RT/PCR Kits (Qiagen), and a pair of primers; 5'-CATGCCCWYAGTAGGACTAGC-3' and 5'-AACTCCATGTGCCATGTACAG-3' [12]. synthesized by Bioneer Corporation. The reverse transcription and thermocycling were carried out using a TC-512 (Techne) thermocycler. The conditions were, reverse transcription at 50°C for 30 minutes followed by 94°C for 15 minutes; thermocycling for 40 cycles at 94°C for 30 seconds, 55°C for 30 sec and 72°C for 30 sec; one cycle at 72°C for 10 minutes.

Agarose gel electrophoresis
The amplified fragments were separated on a 1.5% agarose (Vivantis) gel using a horizontal mini-gel electrophoresis system (MSMINI, Cleaver Scientific). DNA bands were visualized by a gel documentation system (Ingenius, Syngene Bio Imaging).

Sequencing and sequence analysis
The amplicon fragments were isolated from the agarose gel, purified by gel purification kit (QIAGEN) and sent to Macrogene Incorporation for unidirectional sequencing. The sequences were analyzed by the BioEdit software package and the Basic Local Alignment Search Tool (BLAST) (http://blast.ncbi.nlm.nih.gov).

Screening for PPR
Neither antigen nor antibodies of PPR were detected in any tested samples.

Bacteriological Examination
Screening of collected tissue samples for bacterial growth in different media did not reveal any positive result.

Electrophoresis
One lung sample and 17 sera samples gave amplicon fragments of about 300 bp. The positive results for each area were, 7/19, 5/11, 4/18 and 1/4; bands are shown for the lung sample and four selected sera samples (Fig. 1).

Sequence analysis
All samples were identical in nucleotide sequence and length (286 nucleotide) after analysis with the BioEdit software package as shown for one sample (Fig. 2). Sequence analysis with the BLAST, indicated that the sequence is identical to the BVDV-1 (sequence accession number AF220247.1.), except for a single nucleotide substitution from A to T at position 9.
The detection of the pestivirus genome during outbreaks using RT/PCR is indicative for the existence of the virus and its role in the outbreak [18][19][20][21][22][23].
In this study, pestivirus genome could be detected using RT/PCR in 32.7% of 52 sera tested, as well as in lung tissue of newly died sheep; this confirms the role of pestivirus in causing the described outbreak. Sequence analysis of the PCR products indicated that the infection is due to BVDV-1. This is the first report of an outbreak of BVDV-1 infection in sheep in Sudan. This viral infection should be considered in investigations of epidemics as well as infertility diseases of sheep in Sudan.

CONCLUSION
This study investigated an outbreak of nervous and respiratory signs in sheep in West Kordofan State of Sudan. The causative agent of the outbreak was found to be BVDV-1. This is the first report of pestivirus outbreak in sheep and its sequence analysis in Sudan