NAHDIC has been engaged in research activities over the last 20 years and has contributed more than 350 published articles to the scientific community. Research activities at NAHDIC focus on problem solving and generating information and data to support disease control and prevention measures. Research initiatives are conducted with NAHDIC technical partners found both within the country and abroad. This includes collaborations with university students. An average of 30 undergraduate and post-graduate (MSc and PhD) Ethiopian university students conduct their research at NAHDIC per year. Currently, there are about 24 active ongoing research projects. Some of the topics are done on bovine tuberculosis, PPR, CBBP, FMD, BVD, IBR, brucellosis and infectious bronchitis.
Development of Solid phase Antigen for Indirect ELISA for the Detection of Specific Antibody Response to Infection with Newcastle Disease Virus
Sahle M1, Burgess GW
A simple and inexpensive method of antigen preparation by ultrafiltration was investigated using the V4 strain of Newcastle disease virus. The antigen designated XM300 was used in an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Newcastle disease virus in chicken serum. The assay was evaluated using both experimental and field sera, as well as reference control reactor and non-reactor sera. Antigen prepared by the ultrafiltration method was compared with antigen prepared by ultracentrifugation and the ultrafiltration antigen was found to react specifically with Newcastle disease virus antiserum in this ELISA system. This antigen preparation technique is also suitable for use in developing countries. The ELISA provides an excellent method for measuring antibodies in the early stages of infection in serum samples from experimentally infected chickens. More than 14.58 % of the total serum samples which failed to be recognized as reactors by the conventional haemagglutination inhibition test were detected in the ELISA.
Evaluation of Indirect Fluorescent Antibody Test (IFAT) for the Diagnosis and Screening of Lumpy Skin Disease Using Bayesian Method
1National Animal Health Diagnostic and Investigation Center (NAHDIC), Ethiopian Ministry
of Livestock and Fisheries Development, P.O. Box 04, Sebeta, Ethiopia
The performance of indirect fluorescence antibody test (IFAT) for serological diagnosis and screening of lumpy skin disease (LSD) was evaluated using methods without gold standard. Virus neutralization test (VNT) was used as the second test and the study sites were selected from two different geographical places in Ethiopia to get different disease prevalence. The analysis of conditional dependent Bayesian model for the accuracy of IFAT showed that sensitivity, specificity, prevalence of the population Pi(1) and the population Pi(2) were 0.92 (0.89-0.95), 0.88 (0.85-0.91), 0.28 (0.25-0.32) and 0.06 (0.048-0.075), respectively. The posterior inferences obtained for VNT sensitivity, specificity and conditional correlation between the tests for sensitivity (rhoD) and specificity (rhoDc) were 0.78 (0.74-0.83), 0.97 (0.95-0.99), 0.052 (-0.03-0.15) and 0.019 (-0.01-0.06), respectively. The interval estimation of conditional correlation for both sensitivity and specificity clusters around zero and thus conditional dependence between the two tests was not significant. Although accuracy measure would not be the only basis for test selection, the result of our study demonstrated that IFAT has a reasonable high accuracy to be used for the diagnosis and sero-surveillance analysis of LSD in the target population.
Development of Capture Monoclonal Based Enzyme Linked Immunosorbent Assay for the Detection of Antibody to Newcastle Diseases Virus
National Animal Health Diagnostic and Investigation Center (NAHDIC), Ethiopian Ministry of Agriculture, P.O. Box 04, Sebeta, Ethiopia
The application of capture Mmonoclonal-based enzyme linked immunosorbent assay of antibodies against Newcastle disease viruses is described. The assay offers considerable advantage over routinely used haemagglutination inhibition test and indirect ELISA. The c-ELISA results of 289 serum samples collected from experimentally infected chickens and naturally infected flocks were compared to indirect ELISA and HI test. Overall analysis of correlation co-efficient reveals a close relationship between the two ELISAs (r=0.923, n=281) and comparatively less correlation between the two ELISA and HI test (HI and indirect ELISA r=0.631, HI and capture ELISA r=0.602, n=281). For sera collected sequentially following experimental infection, an increase in titer with time was observed in all assays. The main advantage of the new assay is its high sensitivity, allowing earlier detection of infected birds. The c-ELISA system may also be important in reducing the cross-reaction in test serum samples that contain antibodies to other avian paramyxoviruses (AMP 2-9).
Key words: ELISA, diagnostic techniques, monoclonal antibodies, Newcastle disease
Bayesian Estimation of Sensitivity and Specificity of Rose Bengal, Complement Fixation, and Indirect ELISA Tests for the Diagnosis of Bovine Brucellosis in Ethiopia
T. Getachew1, G. Getachew1, G.Sintayehu1, M. Getenet1, A. Fasil1
Test evaluation in the absence of a gold standard test was conducted for the diagnosis and screening of bovine brucellosis using three commercially available tests including RBPT, CFT, and I-ELISA in National Animal Health Diagnostic and Investigation Center (NAHDIC) Ethiopia. A total of 278 sera samples from five dairy herds were collected and tested. Each serum sample was subjected to the three tests and the results obtained were recorded and the test outcomes were cross-classified to estimate the sensitivity and specificity of the tests using Bayesian model. Prior information generated on the sensitivity and specificity of bovine brucellosis from published data was used in the model. The three test-one population Bayesian model was modified and applied using WinBug software with the assumption that the dairy herds have similar management system and unknown disease status. The Bayesian posterior estimate for sensitivity was 89.6 (95% PI: 79.9–95.8), 96.8 (95% PI: 92.3–99.1), and 94 (95% PI: 87.8–97.5) and for specificity was 84.5 (95% PI: 68–94.98), 96.3 (95% PI: 91.7–98.8), and 88.5 (95% PI: 81–93.8) for RBT, I-ELISA, and CFT, respectively. In this study I-ELISA was found with the best sensitivity and specificity estimates 96.8 (95% PI: 92.3–99.1) and 96.3 (95% PI: 91.7–98.8), compared to both CFT and RBPT.
Development of an Epitope Blocking-ELISA and Evaluation of its Diagnostic Performance for Specific Detection of Antibodies Anti-haemagglutinin Protein of Peste des Petits Ruminants Virus in Sheep and Goats sera
Sanne Charles Bodjo*, Jean de Dieu Baziki, Nick Nwankpa, Ethel Chitsungo, Yao Mathurin Koffi, Emmanuel Couacy-Hymann, Mariam Diop, Daniel Gizaw , Tajelser Idris Badri Adam, Mamadou Lelenta, Adama Diallo, Karim Tounkara
Peste des Petits Ruminants (PPR) is a contagious and economically important disease affecting small ruminant (i.e. sheep and goat) production. Taking into consideration lessons learnt from the Global Rinderpest Eradication Programme (GREP), PPR is targeted as the next animal disease to be eradicated. The Enzyme-linked immunosorbent assays (ELISAs) which enables the testing of large number of samples for detection of antibodies was one the tools using during that programmed for disease surveillance and the vaccination efficacy monitoring. Here, we report on the development of a new blocking ELISA (bELISA) and its performance evaluation for the specific detection of PPR-specific antibodies in sera from sheep and goats. This new test is based on the use of a monoclonal antibody anti-haemagglutinin able to neutralize the PPR virus in- vitro. PPR negative sera from non-infected countries were used to establish the assay cut-off point at a percentage inhibition of 20% and diagnostic specificity (DSp) of 100%. Diagnostic sensitivity (DSe) was determined at 94.18% using sera from vaccinated animals. Comparison of PPR-bELISA performances with virus neutralization test (gold standard test) showed Kappa agreement at 0.947 similar to the N-based PPR-cELISA commercial test, indicating good correlation. The PPR-bELISA showed high repeatability and reproducibility in addition to good diagnostic performance which makes this test a good test for PPR antibody detection for disease surveillance and sero-monitoring of PPR vaccination campaign.
Keywords: Peste des petits ruminants (PPR); Blocking Enzyme-Linked Immunosorbent Assay (bELISA); diagnostic performances
An HRM Assay to Differentiate Sheeppox Virus Vaccine Strains from Sheeppox Virus Field Isolates and other Capripox Virus Species
Tesfaye Rufael Chibssa1,2,3, Tirumala Bharani K. Settypalli 1, Francisco J. Berguido1, Reingard Grabherr2, Angelika Loitsch4, Eeva Tuppurainen5, Nick Nwankpa6, Karim Tounkara6, Hafsa Madani7, Amel Omani7, Mariane Diop8, Giovanni Cattoli1, Adama Diallo8,9 & Charles Euloge Lamien 1
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 Vrus Field Isolates from Vaccine Strain
Tesfaye Rufael Chibssa1,2,3, Reingard Grabherr2, Angelika Loitsch4, Tirumala Bharani K. Settypalli1,Eeva Tuppurainen5, Nick Nwankpa6, Karim Tounkara6, Hafsa Madani7, Amel Omani7, Mariane Diop8,Giovanni Cattoli1, Adama Diallo8,9 and Charles Euloge Lamien1
1Animal Production and Health Laboratory, Joint FAO/IAEA Agricultural and Biotechnology laboratory, Division of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Wagramer Strasse 5, P.O. Box 100, A1400 Vienna, Austria. 2Institute of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Muthgasse 18, 1190 Vienna, Austria. 3National Animal Health Diagnostic and Investigation Center (NAHDIC), P.O Box, 04, Sebeta, Ethiopia.4Institute for Veterinary Disease Control, Austrian Agency for Health and Food Safety (AGES), Modling, Austria. 5Capripoxvirus Reference Laboratory, Pirbright Institute, Pirbright, Surrey, UK. 6Pan African Veterinary Vaccine Centre, African Union (PANVAC), P. O. Box 1746, Debre Ziet, Ethiopia. 7Institut National de la Médecine Vétérinaire, Laboratoire Central Vétérinaire d’Alger, Algiers, Algeria. 8Laboratoire National de l’Elevage et de Recherches Vétérinaires, Institut Sénégalais de Recherches Agricoles Route du Front de Terre Hann, Dakar,Sénégal. 9UMR CIRAD-INRA, Animal, Santé, Territoires, Risques et Ecosystèmes (ASTRE), cedex, 05 Montpellier, France.
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. 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. 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.