A nested PCR for detection of North American isolates of bluetongue virus based on NS1 genome sequence analysis of BTV-17.

A nested polymerase chain reaction (PCR)-based assay, for detection of bluetongue virus (BTV) ribonucleic acid in cell culture and tissue samples, was developed. Two pairs of oligonucleotide primers (BTV1 and BTV4 and BTV2 and BTV3), selected from non-structural protein 1 (NS1) gene of BTV-17, were used for the nested PCR in two amplification steps. First a 826-bp product was amplified using an outer primer pair BTV1 and BTV4. The second amplification, using nested or internal primer pair BTV2 and BTV3, produced a 517-bp PCR product. RNA from North American prototype serotypes 2, 10, 11, 13 and 17, propagated in cell cultures, were detected by this nested PCR-based assay. The nested primers BTV2 and BTV3 increased the sensitivity of the BTV PCR assay, and as little as 0.1 fg of BTV RNA (equivalent to 5 viral particles) could be detected. Amplification products were not detected when the PCR-based assay was applied to RNA from a closely related orbivirus, epizootic hemorrhagic disease virus (EHDV) prototype serotypes 1 and 2; total nucleic acid extracts from uninfected BHK-21 cells; or whole blood from calves and deer that were BTV-seronegative and virus isolation negative. Application of this nested BTV PCR-based assay to clinical samples resulted in detection of BTV RNA from a variety of tissues collected from calves and deer with natural and experimental BTV infections. The described BTV PCR-based assay provides a valuable tool to study the epidemiology of BTV infection in susceptible wild ruminants and domestic livestock.

Application of molecular biological techniques for detection of epizootic hemorrhagic disease virus (EHDV-318) recovered from a sentinel calf in central Sudan.

Epizootic hemorrhagic disease virus (EHDV), isolate 318 (EHDV-318), an untyped virus recovered from a sentinel calf herd at the Khartoum University farm in central Sudan, was characterized using molecular biological techniques. With dot blot hybridization technique, a cDNA probe derived from genome segment 6 of EHDV-2 (Alberta strain) hybridized with RNA from EHDV-318. Application of serogroup-specific EHDV polymerase chain reaction (PCR) to EHDV-318 RNA resulted in specific amplification of a 387 bp PCR product. Amplification product was visualized on ethidium bromide-stained agarose gel. Specificity of the PCR products was confirmed by chemiluminescent hybridization with a non-radiolabelled internal probe. No amplification product or hybridization signal was detected when the serotype-specific EHDV-1 or EHDV-2 PCR-based assays were applied to RNA from EHDV-318. The scientific data presented in this study indicated that cDNA probes and serogroup-specific PCR-based assay can be used to classify the virus as a member of EHDV serogroup, and as serotypically distinct from EHDV-1 and EHDV-2.

Serogrouping and topotyping of Sudanese and United States strains of epizootic hemorrhagic disease virus using PCR.

The potential use of the recently reported polymerase chain reaction (PCR) protocol for detection of United States epizootic hemorrhagic disease virus (EHDV) serotype 1 (EHDV-1) and serotype 2 (EHDV-2) ribonucleic acid in cell culture and clinical specimens was evaluated for detection of Sudanese EHDV strains. EHDV serotype 5 (EHDV-5) and EHDV, isolate 318 (untyped) designated (EHDV-318), recovered from sentinel calves at the Khartoum University farm (Sudan) were studied. RNA from EHDV-5 and EHDV-318 and a number of EHDV field isolates, propagated in cell cultures, were detected by the described PCR-based assay. The specific 387 bp PCR products were visualized on ethidium-bromide stained agarose gel. Specificity of the PCR products was confirmed by chemiluminescent hybridization with non-radiolabeled internal probe. Amplification product was not detected when the PCR-based assay was applied to RNA from blutongue virus (BTV) prototypes serotypes 2, 10, 11, 13, 16 and 17; total nucleic acid extracts from uninfected BHK-21 cells. The results of this study indicated that the previously described EHDV-PCR assay could be applied for detection of Sudanese as well as United States strains of EHDV serogroup. In addition, the described EHDV-PCR assay could be used as a supportive diagnostic assay to the current conventional virus isolation procedures used for detection of EHDV infection in susceptible ruminants.

Detection of epizootic hemorrhagic disease virus serotypes 1 and 2 in cell culture and clinical samples using polymerase chain reaction.

The potential for the use of the polymerase chain reaction (PCR) in detecting epizootic hemorrhagic disease virus (EHDV) ribonucleic acid in cell cultures and clinical samples was studied. Using oligoribonucleotide primers selected from genome segment 6 of EHDV-2 (Alberta strain), the PCR-based assay resulted in a 387-base pair (bp) PCR product. EHDV RNA from US prototype serotypes 1 and 2 and a number of EHDV field isolates, propagated in cell cultures, were detected by this EHDV PCR-based assay. Amplification products were visualized on ethidium bromide-stained agarose gels or detected by chemiluminescent hybridization. The sensitivity of the PCR assay was 100 fg of virus RNA (equivalent to 6 x 10(3) virus particles) with ethidium bromide-stained agarose gels. Chemiluminescent hybridization increased the sensitivity of the PCR assay 1,000 times, and specific signals were detected from 0.1 fg of virus RNA (equivalent to 6 virus particles). Amplification product was not detected when the PCR-based assay was applied to RNA from the US bluetongue (BT) virus prototype serotypes 2, 10, 11, 13, and 17 or total nucleic acid extracts from uninfected baby hamster kidney-21 cells, Vero cells, and blood cells from deer that were EHDV seronegative and virus isolation negative. Application of this EHDV PCR-based assay to clinical samples resulted in detection of EHDV RNA from blood and spleen samples from a deer in California with clinical hemorrhagic disease. This EHDV PCR-based assay could provide a rapid, sensitive, and specific assay for detection of EHDV infection in susceptible ruminants.

Comparison of polymerase chain reaction and virus isolation for detection of epizootic hemorrhagic disease virus in clinical samples from naturally infected deer.

We compared our recently reported reverse transcriptase polymerase chain reaction (PCR)-based assay for detection of epizootic hemorrhagic disease virus (EHDV) in clinical samples with different virus isolation (VI) procedures. Thirty-six blood samples and 1 spleen sample from deer were assessed by the EHDV PCR assay and VI in baby hamster kidney (BHK)-21 cells and embryonated chicken eggs (ECE). The EHDV PCR assay detected EHDV RNA from 6 blood samples obtained from deer during 1988-1989 outbreaks of epizootic hemorrhagic disease and from the spleen and blood samples of a deer with clinical hemorrhagic disease in 1992. The 6 blood samples from the 1988-1989 outbreaks and the spleen sample from the 1992 case were VI positive on BHK-21 cell culture. The blood from the same deer with the PCR- and VI-positive spleen was VI negative in BHK-21 cells and ECE. All EHDV isolates were identified as EHDV serotype 2 by a plaque inhibition test. The results of this study indicate that the sensitivity of the previously described EHDV PCR assay is comparable to or greater than that of the VI method in BHK-21 cell culture or ECE. The EHDV PCR assays could provide a superior diagnostic alternative to the current cumbersome and time-consuming VI procedures.

A molecular survey of cystic echinococcosis in Sudan.

A survey of cystic echinococcosis in livestock was conducted from May 2001 to July 2003 in central, western and southern Sudan. Hydatid cysts were present in 59% (466/779) of camels, 6% (299/4893) of cattle, 11% (1180/10,422) of sheep and 2% (106/5565) of goats, with little variation among different geographical areas. 532 of these cysts were examined by PCR and could be overwhelmingly (98.7%) allocated to Echinococcus canadensis G6/7 (all of 215 cysts from camels, 112 of 114 cysts from cattle, 134 of 138 cysts from sheep, and all of 65 cysts from goats); the genotype G6 was identified by sequencing 13 of these isolates. Only 2 cysts from cattle belonged to Echinococcus ortleppi. The mean number of cysts per infected animal was much higher in camels (5.1) than in the other species (1.0-1.3), and cyst fertility was higher in camels and cattle (74% and 77%) than in goats and sheep (31% and 19%). Fertile cysts from five human patients from hospitals in Khartoum and Juba belonged to E. canadensis (G6). This study confirms the predominance of the 'camel strain' in Sudan and the infectivity of this strain for humans. This is the first genetic characterization of human CE in Sudan.

Development of polymerase chain reaction for specific identification of epizootic hemorrhagic disease virus serotype 1.

The diagnostic potential of the polymerase chain reaction (PCR) for specific identification of epizootic hemorrhagic disease virus serotype 1 (EHDV-1) in cell culture and clinical specimens was evaluated. Using oligonucleotide primers, selected from genome segment 2 of EHDV-1 (New Jersey strain), the PCR-based assay resulted in a 862 base pair (bp) PCR product. EHDV-1 RNA from United States prototype serotype 1 and a number of EHDV-1 field isolates, propagated in cell cultures, were detected by this PCR based assay. The specific 862 bp PCR products were visualized on ethidium bromide-stained agarose gel. Identity of the PCR product was confirmed by chemiluminescent hybridization with non radiolabelled internal probe. Using chemiluminescent hybridization, the sensitivity of the PCR assay was 1.0 fg of virus RNA (equivalent to 60 virus particles). Amplification product was not detected when the PCR-based assay was applied to RNA from EHDV serotype 2 (EHDV-2); the United States bluetongue virus (BLU) prototypes serotypes 2, 10, 11, 13, and 17; total nucleic acid extracts from uninfected BHK-21 cell; or blood cells from calves and deer that were EHDV-seronegative and virus isolation negative. Application of this EHDV-1 PCR-based assay to clinical samples resulted in detection of EHDV-1 RNA from blood samples, collected from a calf experimentally infected with EHDV-1. The described PCR-based assay provides a simple, rapid, sensitive, specific and inexpensive method for specific identification of EHDV-1 infection in susceptible ruminants.

PCR detection of North American and Central African isolates of epizootic hemorrhagic disease virus (EHDV) based on genome segment 10 of EHDV serotype 1.

PCR amplification technology for the detection of epizootic hemorrhagic disease virus (EHDV) ribonucleic acid in cell culture and clinical specimens was developed. With oligoribonucleotide primers selected from genome segment 10 of EHDV serotype 1 (EHDV-1), which codes for two nonstructural proteins (NS3 and NS3a), the PCR-based assay resulted in a 535-bp PCR product. RNAs from North American EHDV-1 prototype, EHDV-2 prototype, and a number of EHDV field isolates, including the Central African isolates of EHDV-5 and EHDV-318 propagated in cell cultures, were detected by this PCR-based assay. The specific 535-bp PCR products were visualized onto agarose gels, and the identity of the PCR products was confirmed by chemiluminescent hybridization with a 352-bp internal probe. The sensitivity of the EHDV PCR assay was increased by chemiluminescent hybridization; by this EHDV-NS3 PCR, 10 fg of EHDV RNA was detected (equivalent to 600 viral particles). Amplification product was not detected when the PCR-based assay was applied to RNAs from North American bluetongue virus prototype serotypes 2, 10, 11, 13, and 17; total nucleic acid extracts from uninfected BHK-21 cells; or unfractionated blood from calves and deer that were EHDV seronegative and virus isolation negative. The described EHDV PCR-based assay with primers derived from segment 10 of EHDV-1 resulted in detection of EHDV RNA from blood and tissues collected from calves and deer with natural and experimental EHDV infections and provides a valuable tool to study the epidemiology of EHDV infection in susceptible ruminants.