Peste des petits ruminants infection in domestic ruminants in Sudan.

The existence of peste des petits ruminants (PPR) in domestic ruminants and camels in Sudan during 2008-2012 was investigated. Lung tissues and serum samples were randomly collected from sheep, goats, cattle, and camels at different areas of Sudan. A total of 12,384 serum samples were collected from clinically healthy 7413 sheep, 1988 camels, 1501 cattle, 1459 goats, and 23 gazelles at different areas in the Sudan. They were examined for PPR antibodies using competitive ELISA (cELISA). The overall detected seroprevalence of PPR in tested sera was 49.4%; seroprevalence values within species were 67.1, 48.2, 25.8, 2.1, and 21.7% in sheep, goat, cattle, camels, and gazelles, respectively. The highest seroprevalence (68.1%) was observed in sera collected from Darfur states, then the central states (54.3%). A total of 1276 lung tissue samples (623 sheep, 324 cattle, 220 camels, and 109 goats) were collected. The majority of lung samples were collected from clinically healthy animals that showed lesions on PM in slaughterhouses (95%) and during PPR outbreaks; samples were tested for PPR antigen using immunocapture ELISA (IcELISA). PPR antigen was detected in 233 out of the 1276 tested samples (18.3%). Positive results were observed in samples collected from clinically healthy and diseased animals. The observed prevalence values in each species were 33.6, 21.1, 15.4, and 12.3% in camel, goat, sheep, and cattle, respectively. PPR antigen was detected in samples from different areas; however, the highest prevalence (63.9%) was found in samples collected from the eastern states, then Khartoum state (28%). Trials for virus isolation were done in different cell cultures. Out of 30 IcELISA-positive samples inoculated in primary bovine and ovine kidney cells, Vero cells, the PPR virus was successfully isolated from 15 (eight sheep, five camels, and two goats) samples in the three cell culture types. Using RT-PCR, PPRV nucleic acid was detected in all 25 IcELISA-positive tested samples.

[Morbillivirus infections of ruminants: the nearly eradicated rinderpest and the "peste des petits ruminants", an expanding disease in the South and a threat for Europe]. / Infections à Morbillivirus chez les ruminants: la peste bovine en voie d'éradication et la peste des petits ruminants en extension vers le Nord.

Rinderpest (RP) and peste des petits ruminants (PPR) are contagious viral diseases of domestic and wild ruminants producing high mortality. They are caused by viruses belonging to the Morbillivirus genus, Paramyxoviridae family. Control tools (vaccines and specific diagnostic tests) exist for these two diseases. They have been successfully used during the global rinderpest eradication programme (GREP) and the disease is expected to be eradicated by 2010. In contrast, a similar programme does not exist for PPR, which is still spreading in Africa and Asia. The persistence of PPR in Turkey and its recent introduction in Morocco, make the disease a real threat for Europe. Improvement of control measures against PPR would benefit from the development of a marker vaccine and its companion serological test, thus allowing the differentiation between infected and vaccinated animals (DIVA vaccines and tests). The recent development of reverse genetics for morbilliviruses offers this new possibility.

Mapping the Peste des Petits Ruminants virus nucleoprotein: identification of two domains involved in protein self-association.

For Mononegavirales, the template for transcription and replication is not the naked RNA but the nucleoprotein (N) encapsidated genomic and anti-genomic RNA. Because of this central role in the replication of these viruses, N has been the subject of numerous structural and functional mapping studies. Here, we report on the cloning of the Peste des Petits Ruminants virus (PPRV) N gene into the baculovirus vector and the expression of the protein in insect cells. By electron microscopy observation, we have shown that this recombinant PPRV N forms nucleocapsid-like particles in insect cells in the absence of other PPRV proteins, as reported for other paramyxoviruses. As it is known that the formation of these particles is first linked to the self-assembly of N, we have made several deletions in the PPRV N gene and expressed these mutants in insect cells. Analysis of these proteins by immunoprecipitation and electron microscopy observation enabled us to map the N-N interaction domains into two regions of PPRV N: aa 1-120 and 146-241. The fragment aa 121-145, which is not conserved within the morbillivirus group, is also required for the formation/stability of the nucleocapsid helical structure.

Long-term storage at tropical temperature of dried-blood filter papers for detection and genotyping of RNA and DNA viruses by direct PCR.

In tropical countries the diagnosis of viral infections of humans or animals is often hampered by the lack of suitable clinical material and the necessity to maintain a cold chain for sample preservation up to the laboratory. This study describes the use of filter papers for rapid sample collection, and the molecular detection and genotyping of viruses when stored over long periods at elevated temperatures. Infected blood was collected on filter papers, dried and stored at different temperatures (22, 32 and 37 degrees C) for various periods (up to 9 months). Two animal viruses, African swine fever, a large double-stranded DNA virus and Peste des Petits Ruminants, a negative single-stranded RNA virus, were used to validate the method. Filter papers with dried blood containing virus or control plasmid DNA were cut in small 5mm(2) pieces and added directly to the PCR tube for conventional PCR. Nucleic acid from both viruses could still be detected after 3 months at 32 degrees C. Moreover, the DNA virus could be detected at least 9 months after conservation at 37 degrees C. PCR products obtained from the filter papers were sequenced and phylogenetic analysis carried out. The results were consistent with published sequences, demonstrating that this method can be used for virus genotyping.

Early detection of viral excretion from experimentally infected goats with peste-des-petits ruminants virus.

We observed 15 goats for 9 days after subcutaneous infection with 10(3) TCID(50) with isolates of peste-des-petits ruminants virus from Africa and India and five concurrent, uninfected control goats. Typical clinical signs of the infection were present in all 15 infected goats by day 8 and in most by day 6 and some signs were present by day 4. However, 6 out of 15 goats already have detectable virus shedding by day 3 and four more were shedding by day 4 and every goat had virus shedding for at least 1 day before the recognition of clinical signs. This experiment indicates that incubatory carriers therefore might play a role in the transmission of PPRV among small ruminants.

Peste des petits ruminants (PPR) outbreak in Tajikistan.

The occurrence of outbreaks of peste des petits ruminants (PPR) in three districts of Tajikistan is described. The causal strain (PPR Tajikistan) was characterized and the sequence of its N gene was compared with that of 43 other strains isolated since 1968 in Africa, the Middle East and Asia. The study demonstrated (1) the value of the N gene as a target in comparing isolates obtained over an extended period of evolution, and (2) that clustering was related to the geographical origin of strains.

Evaluation of the virulence of some strains of peste-des-petits-ruminants virus (PPRV) in experimentally infected West African dwarf goats.

Different isolates of peste-des-petits-ruminants virus (PPRV) from outbreaks in Africa and India were investigated for virulence in West African dwarf goats in the Ivory Coast. Six groups of five animals received a virulent suspension of various strains of virus at a concentration of 10(3) TCID(50)/mL and the goats were observed for 15 days after infection. The Côte-d'Ivoire 89 (CI89), Guinea Conakry and Bissau Guinea PPRV strains caused a peracute disease; the India-Calcutta strain caused acute disease; the Sudan-Sennar strain produced an acute to mild disease, while the Nigeria 75/1 wild-type strain caused a mild disease and the animals recovered. The viruses studied contained examples of PPRV from specific lineage groups based on their nucleoprotein PPRV gene. This experiment indicated that virulence characteristics might be a useful marker to help classify PPRV isolates.

Re-infection of wildlife populations with rinderpest virus on the periphery of the Somali ecosystem in East Africa.

We report surveillance for rinderpest virus in wildlife populations in three major ecosystems of East Africa: Great Rift Valley, Somali and Tsavo from 1994 to 2003. Three hundred and eighty wild animals were sampled for detection of rinderpest virus, antigen or genome and 1133 sampled for antibody in sera from Kenya, Uganda, Ethiopia and Tanzania from 20 species. This was done modifying for wildlife the internationally recommended standards for rinderpest investigation and diagnosis in livestock. The animals were selected according to susceptibility and preference given to gregarious species, and populations were selected according to abundance, availability and association with livestock. Rinderpest virus, antigen and/or genome were detected in Kenya; within Tsavo, Nairobi and Meru National Parks. Serological results from 864 animals (of which 65% were buffalo) from the region were selected as unequivocal; showing the temporal and spatial aspects of past epidemics. Recent infection has been only in or peripheral to the Somali ecosystem (in Kenya). Our evidence supports the hypothesis that wildlife is not important in the long-term maintenance of rinderpest and that wildlife are infected sporadically most likely from a cattle source, although this needs to be proven in the Somali ecosystem. Wildlife will continue to be a key to monitoring the remaining virus circulation in Africa.

Peste des Petits Ruminants Virus.

Peste des petits ruminants virus (PPRV) causes a severe contagious disease of sheep and goats and has spread extensively through the developing world. Because of its disproportionately large impact on the livelihoods of low-income livestock keepers, and the availability of effective vaccines and good diagnostics, the virus is being targeted for global control and eventual eradication. In this review we examine the origin of the virus and its current distribution, and the factors that have led international organizations to conclude that it is eradicable. We also review recent progress in the molecular and cellular biology of the virus and consider areas where further research is required to support the efforts being made by national, regional, and international bodies to tackle this growing threat.

Emergence of Lineage IV Peste des Petits Ruminants Virus in Ethiopia: Complete Genome Sequence of an Ethiopian Isolate 2010.

Isolates of peste des petits ruminants virus (PPRV) can be segregated genetically into four lineages. For decades, lineages I-III have been reported across Africa whilst lineage IV has predominantly circulated across Asia. However, the lineage distribution is currently changing in Africa. Importantly, full genome sequence data for African field isolates have been lacking. Here, we announce the first complete genome sequence of a field isolate of peste des petits ruminants virus (PPRV) from East Africa. This isolate was derived from the intestine of a goat suffering from severe clinical disease during the 2010 outbreak in Ethiopia. The full genome sequence of this isolate, PPRV Ethiopia/2010, clusters genetically with other lineage IV isolates of PPRV, sharing high levels of sequence identity across the genome. Further, we have carried out a phylogenetic analysis of all of the available African partial N gene and F gene PPRV sequences to investigate the epidemiology of PPRV with a focus on the emergence of different lineages of PPRV in Africa.

Impact and Epidemiological Investigations into the Incursion and Spread of Peste des Petits Ruminants in the Comoros Archipelago: An Increased Threat to Surrounding Islands.

Late October 2012, a great number of deaths of unknown origin occurred in goat herds in the suburbs of Ngazidja, located in the Comoros archipelago. Few weeks later, laboratory testing requested by the animal health authorities resulted in the identification of peste des petits ruminants (PPR) infection. Notably, the Index case could be attributed to a sick goat imported from Tanzania. Viral isolation was successful from the lungs leading to the whole N nucleoprotein gene sequencing. Phylogenetic analysis revealed that the strain belongs to the lineage III which includes strains of eastern African origin. In addition, to evaluate the impact of PPR on the Comorian indigenous domesticated ruminant population, a cross-sectional PPR serological survey was conducted between April and July 2013. A low overall PPRV antibody prevalence 2.24% (95% CI [1.38; 3.08]) was detected with a Grande Comore prevalence of 3.34% (IC = [2.09; 4.63]) with a limited spread of the disease mainly due to farm practices such as limited contacts between farm animals and rapid slaughtering of sick animals.

Antibody seroprevalences against peste des petits ruminants (PPR) virus in camels, cattle, goats and sheep in Ethiopia.

A questionnaire-survey data indicated that 26% of 276 farmers reported the presence of respiratory disease in their herds in 2001. The incidence was perceived as "high" in small ruminants and camels, but as "low" in cattle. Simultaneously, 2815 serum samples from camels (n=628), cattle (n=910), goats (n=442) and sheep (n=835) were tested. The peste des petits ruminants (PPR) antibody seroprevalence was 3% in camels, 9% in cattle, 9% in goats and 13% in sheep. The highest locality-specific seroprevalences were: camels 10%, cattle 16%, goats 22% and sheep 23%. The animals had not been vaccinated against rinderpest or PPR. Antibody seroprevalences detected in camels, cattle, goats and sheep confirmed natural transmission of PPR virus under field conditions.

Surveillance of wildlife as a tool for monitoring rinderpest and peste des petits ruminants in West Africa.

The authors provide a report on the surveillance of rinderpest virus (RPV) and peste des petits ruminants virus (PPRV) in the wildlife population in Côte d'Ivoire. For this purpose, 266 animals from nine different species, selected according to susceptibility and abundance, were captured and sampled from Comoé, Marahoué and Lamto Parks. Two hundred and forty seven sera and 214 nasal swabs were collected and analysed by competitive enzyme-linked immunosorbent assay (cELISA) and reverse-transcriptase polymerase chain reaction (RT-PCR) techniques, respectively. Serological data demonstrated that RPV was not circulating within the national Parks and estimated the PPR seroprevalence to be less than 1%. The analysis of the nasal swabs revealed no cases of RPV infection, but PPRV infection was detected in four species, including buffalo. To minimise the cost of the study without affecting the sensitivity of the test, samples were pooled into different groups and submitted to RT-PCR using nucleoprotein gene specific primers. The RT-PCR used in this study, which was derived from the method developed by Couacy-Hymann et al. in 2002, was followed by a hybridisation step using internal specific probes to confirm the identity of the deoxyribonucleic acid product. When used in conjunction with a cELISA this method accurately demonstrated the absence of rinderpest viral persistence in Côte-d'Ivoire.

The matrix protein gene sequence analysis reveals close relationship between peste des petits ruminants virus (PPRV) and dolphin morbillivirus.

The gene encoding the matrix protein of peste des petits ruminants virus (PPRV) has been cloned and its nucleotide sequence determined. This gene is 1466 nucleotides long and contains an open reading frame (ORF) capable of encoding a basic protein of 335 amino acid residues with a predicted molecular weight of 38,057 Da. This ORF starts at position 33-35 and ends with the codon TAA at position 1038-1040 thus leaving a long untranslated region (426 nucleotides) at the 3' end of the messenger RNA. This fragment is very G/C rich (68.5%) and in contrast to the ORF region appears to be least conserved in the M gene sequence of the morbilliviruses. A comparison of the PPRV M protein with those of other viruses in the group confirms the previously noted high degree of conservation for this protein sequence. The percent of identity within the group ranges from 76.7 to 86.9%, the highest being with the dolphin morbillivirus matrix protein.

Goat immune response to capripox vaccine expressing the hemagglutinin protein of peste des petits ruminants.

Sheep-pox and capripox are contagious diseases of domestic small ruminants for which the causal agent is a poxvirus classified into the Capripoxvirus genus. Viruses of this group have a host range specific to sheep, goats, cattle, and possibly buffalo. Thus, they are clearly indicated as vectors for the development of recombinant vaccines for peste des petits ruminants (PPR). Here we report the immune response of goats inoculated with a recombinant capripox-PPR hemagglutinin.

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 Sacchi's 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.

Comparison of rinderpest and peste des petits ruminants viruses using anti-nucleoprotein monoclonal antibodies.

Monoclonal antibodies (MAbs) were obtained using a purified preparation of the RBOK strain of a rinderpest vaccine virus. The cytoplasmic immunofluorescent staining test showed that these clones had specificity for the nucleoprotein (N) of the virus. Six clones which immunoprecipitated the N protein corroborated these results. Thirteen anti-N MAbs were used to compare geographically widespread rinderpest viruses (RPV) and peste des petits ruminants viruses (PPRV) to two other morbilliviruses, measles (MV) and canine distemper (CDV). The N protein antigen profiles of the 23 isolates determined by immunofluorescent staining and enzyme linked immunosorbent assay (ELISA) on infected cells enabled us to classify the strains into groups. A differential identification of the morbilliviruses can be made using one MAb or associations of the MAbs. The potential to distinguish between RPV and PPRV and between virulent and avirulent strains of rinderpest is of primary interest.

A competitive ELISA using anti-N monoclonal antibodies for specific detection of rinderpest antibodies in cattle and small ruminants.

A competitive ELISA (C-ELISA) using monoclonal antibodies (mAbs) which bind to the nucleo-protein (NP) of rinderpest virus (RPV) for detection of RPV antibodies in cattle and small ruminant sera is described. Unlike virus neutralisation test (VNT), this test using mAb IVB2-4, can detect specific RPV antibodies without showing a cross-reaction with antibodies to peste-des-petits ruminants-virus (PPRV); by contrast, when mAb VE4-1 is used the test detects both RPV and PPRV antibodies, including low levels of antibodies that can be found in sera containing maternal antibodies. Although antibodies to the PPRV 75-1 strain are also detected with mAb 51-5-6, the test is suitable for assessing the immune status of cattle against the Rinderpest Old Kabete (RBOK) strain. The results from a panel of sera with a known status of vaccination provide evidence for a highly significant correlation between C-ELISA and VNT. This test may be a useful tool for a standardized and accurate determination of the immunity status of both cattle and small ruminants.

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.

The use of monoclonal antibodies in the diagnosis of contagious caprine pleuropneumonia (CCPP).

Contagious caprine pleuropneumonia is a severe disease affecting goats in Eastern Africa and the Middle East, caused by Mycoplasma sp. type F38. Its exact geographical distribution is however not exactly known due to the lack of specificity of the available serological tests and the difficulty in cultivating M. sp. F38. A panel of monoclonal antibodies (mAbs) was produced, using crude or membrane proteins antigens from type F38 strains to immunize mice. The reactivity of the mAbs was tested by an immunobinding assay with crude mycoplasma antigens spotted on nitrocellulose filters. One hundred and twelve antigens, standardized at 0.5 mg protein/ml, were used. Mycoplasma strains were chosen among closely related species of the "mycoides cluster", M. capricolum, Group 7 of Leach, M. mycoides mycoides LC, M. mycoides mycoides SC, M. mycoides capri, as well as among species that are isolated from goat lungs, M. arginini, M. ovipneumoniae, M. putrefaciens, M. agalactiae. Out of 60 mAbs, 4 were chosen to build an identification test for mycoplasmas of the "mycoides cluster". Controls showed that accurate identification could be hampered by antigenic heterogeneity within the M. capricolum species. One mAb was used for the direct detection of M. sp. F38 antigen in pleural fluid from goats suspected of CCPP. The sensitivity of the test can be estimated at 0.5 micrograms protein/ml. Comparison with isolation results show a 74% agreement between the two methods. The same mAb was used to build a blocking ELISA. This serological test was strictly specific for CCPP. It detects antibodies in sera of naturally infected or artificially immunized animals while it remained negative with hyperimmune sera to related strains such as PG 50. Direct antigen detection and blocking ELISA are tools that may enable a better assessment of CCPP distribution.