Emmanuel Couacy-Hymann

Rapid and sensitive detection of peste des petits ruminants virus by a polymerase chain reaction assay

A rapid and specific test was developed for the diagnosis of peste des petits ruminants disease. This assay is based on the rapid purification of RNA on glass beads followed by the reverse transcription-polymerase chain reaction (RT-PCR). To that effect, a set of primers (NP3/NP4) was used to amplify specifically a fragment of about 350 bp in the 3 end of the RNA messenger that encodes the nucleocapsid protein of the peste des petits ruminants virus. The PCR-product was detected by UV illumination after electrophoresis on agarose gel or by hybridisation with a digoxigenin-11-dUTP labelled oligonucleotide probe after a blot transfer. In comparison with the conventional titration technique on Vero cells, this RT-PCR assay was 1000-fold more sensitive. Compared with the popular Chomczynski and Sacchis method [Anal. Biochem. 162 (1987) 156], the purification of the RNA on the glass beads offers the advantage of being more rapid and also avoiding the use of solvents.

Genotyping field strains of African swine fever virus by partial p72 gene characterisation

Summary.u2002A PCR-based sequencing method was developed which permits detection and characterization of African swine fever virus (ASFV) variants within 5 and 48u2009h, respectively, of receipt of a clinical specimen. Amplification of a 478u2009bp fragment corresponding to the C-terminal end of the p72 gene, confirms virus presence with genetic characterization being achieved by nucleotide sequence determination and phylogenetic analysis. The method was applied to 55 viruses including those representative of the major ASF lineages identified previously by restriction fragment length polymorphism (RFLP) analysis. Results confirmed that the p72 genotyping method identifies the same major viral groupings. Characterization of additional viruses of diverse geographical, species and temporal origin using the PCR-based method indicated the presence of ten major ASF genotypes on the African continent, the largest of which comprised a group of genetically homogeneous viruses recovered from outbreaks in Europe, South America, the Caribbean and West Africa (the ESAC-WA genotype). In contrast, viruses from southern and East African countries were heterogeneous, with multiple genotypes being present within individual countries. This study provides a rapid and accurate means of determining the genotype of field and outbreak strains of ASF and is therefore useful for molecular epidemiological clarification of ASF.

Monkey CV1 cell line expressing the sheep―goat SLAM protein: A highly sensitive cell line for the isolation of peste des petits ruminants virus from pathological specimens

Peste des petits ruminants (PPR) is an important economically transboundary disease of sheep and goats caused by a virus which belongs to the genus Morbillivirus. This genus, in the family Paramyxoviridae, also includes the measles virus (MV), canine distemper virus (CDV), rinderpest virus (RPV), and marine mammal viruses. One of the main features of these viruses is the severe transient lymphopaenia and immunosuppression they induce in their respective hosts, thereby favouring secondary bacterial and parasitic infections. This lymphopaenia is probably accounted for by the fact that lymphoid cells are the main targets of the morbilliviruses. In early 2000, it was demonstrated that a transmembrane glycoprotein of the immunoglobulin superfamily which is present on the surface of lymphoid cells, the signalling lymphocyte activation molecule (SLAM), is used as cellular receptor by MV, CDV and RPV. Wild-type strains of these viruses can be isolated and propagated efficiently in non-lymphoid cells expressing this protein. The present study has demonstrated that monkey CV1 cells expressing goat SLAM are also highly efficient for isolating PPRV from pathological samples. This finding suggests that SLAM, as is in the case for MV, CDV and RPV, is also a receptor for PPRV.

Recent developments in the diagnosis of rinderpest and peste des petits ruminants

Effective implementation of control measures for rinderpest and peste des petits ruminants requires that a proper and rapid diagnosis of the disease is made. Peste de petits ruminants (PPR) can be confused clinically with other infections such as pasteurellosis or contagious ecthyma. Rinderpest, in its classical form, is easy to identify clinically; however, mass vaccination in many countries and also the emergence of mild strains of the virus have made clinical diagnosis more difficult. Clinical observations for both diseases should always be confirmed by a laboratory. Diagnostic techniques used in the past were virus neutralization, agar gel immunodiffusion and virus isolation in cell culture, followed sometimes by reproducing the disease in susceptible animals. All these techniques are either time-consuming, labour intensive, insensitive, or expensive to perform. With the advent of hybridoma and molecular biological techniques, new reagents to assist diagnosis have become available and have led to the development of specific and rapid tests for the diagnosis of each disease. The present article reviews the diagnostic techniques currently available. An indirect ELISA was used successfully to evaluate the status of cattle following the Pan African Rinderpest Campaign. More recently competitive or blocking ELISAs have been developed based on monoclonal antibodies specific for the N or H proteins of the viruses, and which enable differential diagnosis between rinderpest and PPR. This is particularly important in sheep and goats, which may be infected with either virus. In future, improved standardization and reduced costs may be expected with the introduction of ELISAs based on purified antigens expressed in gene vector systems such as baculovirus. ELISA may also be adapted to antigen detection. Nucleic acid technology has also been applied to virus detection procedures. Hybridization probes showed a disappointing sensitivity for diagnostic applications, but more recently the polymerase chain reaction method has shown great promise, providing the potential of high sensitivity combined with specificity.

Protection of goats against rinderpest by vaccination with attenuated peste des petits ruminants virus

The ability of the attenuated peste des petits ruminants vaccine virus to protect small ruminants against virulent rinderpest virus was investigated. Out of four susceptible goats that were infected with the highly virulent Saudi strain of rinderpest virus by intranasal ioculation, three developed mild clinical signs of disease and infected susceptible in-contact goats and cattle with rinderpest virus. However, four goats which had been vaccinated with the attenuated peste des petits ruminants virus resisted challenge with virulent rinderpest virus and did not infect susceptible in-contact animals.

The early detection of peste-des-petits-ruminants (PPR) virus antigens and nucleic acid from experimentally infected goats using RT-PCR and immunocapture ELISA techniques

Goats were infected subcutaneously with different African and Indian isolates of peste-des-petits-ruminants virus. Typical signs of disease were recorded from day 6 post infection for all isolates. Ocular, nasal and mouth samples were tested for the presence of virus antigen or nucleic acid using the immunocapture ELISA (ICE) and the RT-PCR technique. Using ICE, virus antigen was detected at day 4 in ocular and nasal samples of goats infected with Côte-dIvoire 89 and in the ocular, nasal and mouth samples with the India, Calcutta strains. By day 5, all samples from both these groups were positive while ocular and nasal samples from groups with Sudan-Sennar and Nigeria 75/1 strains became positive. With the RT-PCR technique virus nucleic acid, presumed to be associated with infectious virus excretion, was detected at day 3 in oral and nasal samples in groups infected with Côte-dIvoire 89 and India-Calcutta strains. From day 6-9, all samples from all groups were positive with both techniques. This experiment demonstrated that PPR virus antigens and nucleic acid, presumed to be related to infectious virus, is excreted 2-3days before the appearance of clinical signs whatever the technique used which is of epidemiological importance in controlling the spread of the disease. The ICE being easier to perform in developing countries can be recommended as a useful method to investigate PPR in small ruminants flocks at an early stage to prevent the diffusion of the disease.

The first specific detection of a highly pathogenic avian influenza virus (H5N1) in Ivory Coast.

The Virology Laboratory of the Central Laboratory of Animal Diseases in Ivory Coast at Bingerville received samples of wild and domestic avian species between February and December 2006. An RT‐PCR technique was used to test for avian influenza (AI) and highly pathogenic AI subtype viruses. Among 2125 samples, 16 were type A positive; of which, 12 were later confirmed to be H5N1. Fifteen of these 16 type A positive samples were inoculated into the chorioallantoic cavity of 11‐day‐old embryonated hens’ eggs for virus isolation. Eight produced virus with hemagglutination titres from 1/64 to 1/512. The 4/16 M‐RT‐PCR positive samples, which were H5N1 negative, were shown to be H7 subtype negative. The diagnostic efficiency of the laboratory for the surveillance of H5N1 in Ivory Coast was demonstrated. The positive cases of H5N1 were from a sparrowhawk (Accipter nisus); live market poultry and in free‐range poultry, where the mortality rate was approximately 20% (2/10) and 96.7% (29/30) respectively. Currently, investigations into intensive poultry farms have proved negative for H5N1. No human cases have been reported this time.

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.