An HRM Assay to Differentiate Sheeppox Virus Vaccine Strains from Sheeppox Virus Field Isolates and other Capripoxvirus Species.

Sheep poxvirus (SPPV), goat poxvirus (GTPV) and lumpy skin disease virus (LSDV) affect small ruminants and cattle causing sheeppox (SPP), goatpox (GTP) and lumpy skin disease (LSD) respectively. In endemic areas, vaccination with live attenuated vaccines derived from SPPV, GTPV or LSDV provides protection from SPP and GTP. As live poxviruses may cause adverse reactions in vaccinated animals, it is imperative to develop new diagnostic tools for the differentiation of SPPV field strains from attenuated vaccine strains. Within the capripoxvirus (CaPV) homolog of the variola virus B22R gene, we identified a unique region in SPPV vaccines with two deletions of 21 and 27 nucleotides and developed a High-Resolution Melting (HRM)-based assay. The HRM assay produces four distinct melting peaks, enabling the differentiation between SPPV vaccines, SPPV field isolates, GTPV and LSDV. This HRM assay is sensitive, specific, and provides a cost-effective means for the detection and classification of CaPVs and the differentiation of SPPV vaccines from SPPV field isolates.

A gel-based PCR method to differentiate sheeppox virus field isolates from vaccine strains.

BACKGROUND: Sheeppox (SPP) and goatpox (GTP) caused by sheeppox virus (SPPV) and goatpox virus (GTPV), respectively of the genus Capripoxvirus in the family Poxviridae, are severely afflicting small ruminants' production systems in Africa and Asia. In endemic areas, SPP and GTP are controlled using vaccination with live attenuated vaccines derived from SPPV, GTPV or Lumpy skin disease virus (LSDV). Sometimes outbreaks occur following vaccination. In order to successfully control the spread of the virus, it is essential to identify whether the animals were infected by the field strain and the vaccine did not provide sufficient protection. Alternatively, in some cases the vaccine strain may cause adverse reactions in vaccinated animals or in rare occasions, re-gain virulence. Thus, diagnostic tools for differentiation of virulent strains from attenuated vaccine strains of the virus are needed. The aim of this study was to identify an appropriate diagnostic target region in the capripoxvirus genome by comparing the genomic sequences of SPPV field isolates with those of the most widely used SPP vaccine strains. RESULTS: A unique 84 base pair nucleotide deletion located between the DNA ligase gene and the VARV B22R homologue gene was found only in SPPV vaccines derived from the Romanian and Yugoslavian RM/65 strains and absent in SPPV field isolates originated from various geographical locations of Asia and Africa. In addition, we developed and evaluated a conventional PCR assay, exploiting the targeted intergenic region to differentiate SPPV vaccine virus from field isolates. The assay produced an amplicon size of 218 bp for the vaccine strains, while the SPPV field isolates resulted in a 302 bp PCR fragment. The assay showed good sensitivity and specificity, and the results were in full agreement with the sequencing data of the PCR amplicons. CONCLUSION: The developed assay is an improvement of currently existing diagnostic tools and, when combined with a capripox virus species-specific assay, will enhance SPP and GTP diagnosis and surveillance and facilitate epidemiological investigations in countries using live attenuated SPP vaccines. In addition, for laboratories with limited resources, the assay provides a simple and cost-effective alternative for sequencing.

Development and validation of an epitope-blocking ELISA using an anti-haemagglutinin monoclonal antibody for specific detection of antibodies in sheep and goat sera directed against peste des petits ruminants virus.

Peste des petits ruminants (PPR) is a contagious and economically important disease affecting production of small ruminants (i.e., sheep and goats). Taking into consideration the lessons learnt from the Global Rinderpest Eradication Programme (GREP), PPR is now targeted by the international veterinary community as the next animal disease to be eradicated. To support the African continental programme for the control of PPR, the Pan African Veterinary Vaccine Centre of the African Union (AU-PANVAC) is developing diagnostics tools. Here, we describe the development of a blocking enzyme-linked immunosorbent assay (bELISA) that allows testing of a large number of samples for specific detection of antibodies directed against PPR virus in sheep and goat sera. The PPR bELISA uses an anti-haemagglutinin (H) monoclonal antibody (MAb) as a competitor antibody, and tests results are interpreted using the percentage of inhibition (PI) of MAb binding generated by the serum sample. PI values below or equal to 18% (PI ≤ 18%) are negative, PI values greater than or equal to 25% (PI ≥ 25%) are positive, and PI values greater than 18% and below 25% are doubtful. The diagnostic specificity (DSp) and diagnostic sensitivity (DSe) were found to be 100% and 93.74%, respectively. The H-based PPR-bELISA showed good correlation with the virus neutralization test (VNT), the gold standard test, with a kappa value of 0.947. The H-based PPR-bELISA is more specific than the commercial kit ID Screen® PPR Competition (N-based PPR-cELISA) from IDvet (France), but the commercial kit is slightly more sensitive than the H-based PPR-bELISA. The validation process also indicated good repeatability and reproducibility of the H-based PPR-bELISA, making this new test a suitable tool for the surveillance and sero-monitoring of the vaccination campaign.

Molecular identification of Mycobacterium bovis from cattle and human host in Mali: expanded genetic diversity.

BACKGROUND: Bovine tuberculosis (BTB) is a contagious, debilitating human and animal disease caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex. The study objective were to estimate the frequency of BTB, examine genetic diversity of the M. bovis population in cattle from five regions in Mali and to determine whether M. bovis is involved in active tuberculosis (TB) in humans. Samples from suspected lesions on cattle at the slaughterhouses were collected. Mycobacterial smear, culture confirmation, and spoligotyping were used for diagnosis and species identification. Mycobacterium DNA from TB patients was spoligotyped to identify M. bovis. RESULTS: In total, 675 cattle have been examined for lesions in the five regions of Mali. Out of 675 cattle, 79 specimens presented lesions and then examined for the presence of M. bovis. Thus, 19 (24.1 %) were identified as M. bovis; eight (10.1 %) were non-tuberculous Mycobacterium (NTM). Nineteen spoligotype patterns were identified among 79 samples with five novel patterns. One case of M. bovis (spoligotype pattern SB0300) was identified among 67 TB patients. CONCLUSION: This study estimates a relatively true proportion of BTB in the regions of Mali and reveals new spoligotype patterns.

An international collaborative study to determine the prevalence of contagious caprine pleuropneumonia by monoclonal antibody-based cELISA.

BACKGROUND: Few serological tests are available for detecting antibodies against Mycoplasma capricolum subsp. capripneumoniae, the causal agent of contagious caprine pleuropneumonia (CCPP). The complement fixation test, the test prescribed for international trade purposes, uses a crude antigen that cross-reacts with all the other mycoplasma species of the "mycoides cluster" frequently infecting goat herds. The lack of a more specific test has been a real obstacle to the evaluation of the prevalence and economic impact of CCPP worldwide. A new competitive ELISA kit for CCPP, based on a previous blocking ELISA, was formatted at CIRAD and used to evaluate the prevalence of CCPP in some regions of Kenya, Ethiopia, Mauritius, Tajikistan and Pakistan in an international collaborative study. RESULTS: The strict specificity of the test was confirmed in CCPP-free goat herds exposed to other mycoplasma species of the "mycoides cluster". Prevalence studies were performed across the enzootic range of the disease in Africa and Asia. Seroprevalence was estimated at 14.6% in the Afar region of Ethiopia, whereas all the herds presented for CCPP vaccination in Kenya tested positive (individual seroprevalence varied from 6 to 90% within each herd). In Mauritius, where CCPP emerged in 2009, nine of 62 herds tested positive. In Central Asia, where the disease was confirmed only recently, no positive animals were detected in the Wakhan District of Afghanistan or across the border in neighboring areas of Tajikistan, whereas seroprevalence varied between 2.7% and 44.2% in the other districts investigated and in northern Pakistan. The test was also used to monitor seroconversion in vaccinated animals. CONCLUSIONS: This newly formatted CCPP cELISA kit has retained the high specificity of the original kit. It can therefore be used to evaluate the prevalence of CCPP in countries or regions without vaccination programs. It could also be used to monitor the efficacy of vaccination campaigns as high-quality vaccines induce high rates of seroconversion.