Selective replication and vertical transmission of Ebola virus in experimentally infected Angolan free-tailed bats.

The natural reservoir of Ebola virus (EBOV), agent of a zoonosis burdening several African countries, remains unidentified, albeit evidence points towards bats. In contrast, the ecology of the related Marburg virus is much better understood; with experimental infections of bats being instrumental for understanding reservoir-pathogen interactions. Experiments have focused on elucidating reservoir competence, infection kinetics and specifically horizontal transmission, although, vertical transmission plays a key role in many viral enzootic cycles. Herein, we investigate the permissiveness of Angolan free-tailed bats (AFBs), known to harbour Bombali virus, to other filoviruses: Ebola, Marburg, Taï Forest and Reston viruses. We demonstrate that only the bats inoculated with EBOV show high and disseminated viral replication and infectious virus shedding, without clinical disease, while the other filoviruses fail to establish productive infections. Notably, we evidence placental-specific tissue tropism and a unique ability of EBOV to traverse the placenta, infect and persist in foetal tissues of AFBs, which results in distinct genetic signatures of adaptive evolution. These findings not only demonstrate plausible routes of horizontal and vertical transmission in these bats, which are expectant of reservoir hosts, but may also reveal an ancillary transmission mechanism, potentially required for the maintenance of EBOV in small reservoir populations.

Diagnostic accuracy of swine echinococcosis cytopathological tests and challenges for a differential diagnosis: slaughterhouse data.

Echinococcosis disease shows clinical signs similar to many diseases. Hence we report cases that need to be confirmed using appropriate tests. A confirmatory study has been conducted to assess the accuracy of two cytopathological tests, with the histopathology test as the reference standard. The first cytopathological test evaluates the Ziehl Neelsen staining with an epifluorescence microscope (cytopath 1). The second cytopathological test uses the same staining followed by a transmitted light microscope examination (cytopath 2). Of a total of 2524 inspected pigs, 101 suspected cases of echinococcosis were detected, of which 67 were found positive with the two cytopathological tests and the histopathological one. The specificity of cytopath 1 (100 % [95 % CI 100 - 100]) and cytopath 2 (100 % [95 % CI 100;100]) were similar, as well as their respective positive predictive values: 100 % [95 % CI 100 - 100] vs. 100 % [95 % CI 100 - 100]. The sensitivity of cytopath 1 is 79.66 % [95 % CI 69.39 - 89.93], while cytopath 2 equals 66.10 % [95 % CI 54.02 - 78.18]. The difference in sensitivity of both tests was not significant. Negative predictive values found for cytopath 1, and cytopath 2 were 40 [95 % CI 18.53 - 61.47] and 28.57 [95 % CI 11.84 - 45.3], leading to the Generalized Estimating Equations (GEE) Model estimate for an odds ratio of 1.4 [95 % CI 0.41 - 5.2], p = 0.06. Cytopath 1 and cytopath 2 are equivalent in terms of specificity (100 % [95 % CI 100 - 100] vs. 100 % [95 % CI 100;100]) and positive predictive value (100 % [95 % CI 100 - 100]. Cytopath 1 is more sensitive than cytopath 2 but not significant (79.66 % [ 95 % CI 69.39 - 89.93] vs. 66.10 % [95 % CI 54.02 - 78.18]). However, the negative predictive value of cytopath 1 is better than that of cytopath 2: 40 % [95 % CI 18.53 - 61.47] vs. 28.57 % [95 % CI 11.84 - 45.3].

Analysis of a newly discovered antigen of Bacillus cereus biovar anthracis for its suitability in specific serological antibody testing.

AIMS: The aim of this work was to identify a protein which can be used for specific detection of antibodies against Bacillus cereus biovar anthracis (Bcbva), an anthrax-causing pathogen that so far has been described in African rainforest areas. METHODS AND RESULTS: Culture supernatants of Bcbva and classic Bacillus anthracis (Ba) were analysed by gel electrophoresis, and a 35-kDa protein secreted only by Bcbva and not Ba was detected. The protein was identified as pXO2-60 by mass spectrometry. Sequence analysis showed that Ba is unable to secrete this protein due to a premature stop codon in the sequence for the signal peptide. Immunization of five outbred mice with sterile bacterial culture supernatants of Bcbva revealed an immune response in ELISA against pXO2-60 (three mice positive, one borderline) and the protective antigen (PA; four mice). When supernatants of classic Ba were injected into mice or human sera from anthrax patients were analysed, only antibodies against PA were detected. CONCLUSIONS: In combination with PA, the pXO2-60 protein can be used for the detection of antibodies specific against Bcbva and discriminating from Ba. SIGNIFICANCE AND IMPACT OF THE STUDY: After further validation, serological assays based on pXO2-60 can be used to perform seroprevalence studies to determine the epidemiology of B. cereus bv anthracis in affected countries and assess its impact on the human population.

African and classical swine fever situation in Ivory-Coast and neighboring countries, 2008-2013.

This study was conducted from 2008 to 2013 to determine the animal health status of Ivory Coast and neighboring countries (Burkina Faso, Ghana, Togo and Benin) for African swine fever (ASF) and classical swine fever (CSF), and to assess the risk factors for ASF introduction in Ivory Coast. Ivory Coast had probably been free from ASF from 1998 to 2014 when it was re-introduced in this country. However, the ASF virus was found in all neighboring countries. In contrast, no evidence of CSF infection was found so far in Ivory Coast and neighboring countries. To assess the risk of ASF reintroduction in Ivory Coast, we surveyed 59 modern pig farms, and 169 pig owners in 19 villages and in two towns. For the village livestock, the major risk factor was the high frequency of pig exchanges with Burkinabe villages. In the commercial sector, many inadequate management practices were observed with respect to ASF. Their identification should enable farmers and other stakeholders to implement a training and prevention program to reduce the introduction risk of ASF in their farms.

African Swine Fever Diagnosis Adapted to Tropical Conditions by the Use of Dried-blood Filter Papers.

The performance of Whatman 3-MM filter papers for the collection, drying, shipment and long-term storage of blood at ambient temperature, and for the detection of African swine fever virus and antibodies was assessed. Conventional and real-time PCR, viral isolation and antibody detection by ELISA were performed on paired samples (blood/tissue versus dried-blood 3-MM filter papers) collected from experimentally infected pigs and from farm pigs in Madagascar and Côte d'Ivoire. 3-MM filter papers were used directly in the conventional and real-time PCR without previous extraction of nucleic acids. Tests that performed better with 3-MM filter papers were in descending order: virus isolation, real-time UPL PCR and conventional PCR. The analytical sensitivity of real-time UPL PCR on filter papers was similar to conventional testing (virus isolation or conventional PCR) on organs or blood. In addition, blood-dried filter papers were tested in ELISA for antibody detection and the observed sensitivity was very close to conventional detection on serum samples and gave comparable results. Filter papers were stored up to 9 months at 20-25°C and for 2 months at 37°C without significant loss of sensitivity for virus genome detection. All tests on 3-MM filter papers had 100% specificity compared to the gold standards. Whatman 3-MM filter papers have the advantage of being cheap and of preserving virus viability for future virus isolation and characterization. In this study, Whatman 3-MM filter papers proved to be a suitable support for the collection, storage and use of blood in remote areas of tropical countries without the need for a cold chain and thus provide new possibilities for antibody testing and virus isolation.

Prevalence of Newcastle disease virus and infectious bronchitis virus in avian influenza negative birds from live bird markets and backyard and commercial farms in Ivory-Coast.

Newcastle disease (ND) and infectious bronchitis (IB) are two major viral diseases affecting the respiratory tracts of birds and whose impact on African poultry is still poorly known. In the present study we aimed at assessing NDV and IBV prevalences in Ivory-Coast by molecular screening of >22,000 avian swabs by nested PCR and by serology testing of close to 2000 avian sera from 2010 through 2012. The NDV and IBV seroprevalences over the study period reached 22% and 72%, respectively. We found 14.7% pooled swabs positive by PCR for NDV and 14.6% for IBV. Both pathogens are therefore endemic in Ivory-Coast. Economic losses associated with NDV and IBV infections still need to be evaluated.

Avian influenza surveillance in Central and West Africa, 2010-2014.

Avian influenza virus (AIV) is an important zoonotic pathogen, resulting in global human morbidity and mortality and substantial economic losses to the poultry industry. Poultry and wild birds have transmitted AIV to humans, most frequently subtypes H5 and H7, but also different strains and subtypes of H6, H9, and H10. Determining which birds are AIV reservoirs can help identify human populations that have a high risk of infection with these viruses due to occupational or recreational exposure to the reservoir species. To assess the prevalence of AIV in tropical birds, from 2010 to 2014, we sampled 40 099 birds at 32 sites in Central Africa (Cameroon, Central African Republic, Congo-Brazzaville, Gabon) and West Africa (Benin, Côte d'Ivoire, Togo). In Central Africa, detection rates by real-time RT-PCR were 16·6% in songbirds (eight passerine families, n = 1257), 16·4% in kingfishers (family Alcedinidae, n = 73), 8·2% in ducks (family Anatidae, n = 564), and 3·65% in chickens (family Phasianidae, n = 1042). Public health authorities should educate human cohorts that have high exposure to these bird populations about AIV and assess their adherence to biosecurity practices, including Cameroonian farmers who raise small backyard flocks.

Molecular diagnosis of African Swine Fever by a new real-time PCR using universal probe library.

A highly sensitive and specific real-time PCR method was developed for the reliable and rapid detection of African swine fever virus (ASFV). The method uses a commercial Universal Probe Library (UPL) probe combined with a specifically designed primer set to amplify an ASFV DNA fragment within the VP72 coding genome region. The detection range of the optimized UPL PCR technique was confirmed by analysis of a large panel (n = 46) of ASFV isolates, belonging to 19 of the 22 viral p72 genotypes described. No amplification signal was observed when closely clinically related viruses, such as classical swine fever, or other porcine pathogens were tested by this assay. The detection limit of the UPL PCR method was established below 18 DNA copies. Validation experiments using an extensive collection of field porcine and tick samples (n = 260), coming from Eastern and Western African regions affected by ASF, demonstrated that the UPL PCR technique was able to detect over 10% more positive samples than the real-time TaqMan PCR test recommended in the OIE manual, confirming its superior diagnostic sensitivity. Clinical material collected during experimental infections with different ASFV p72 genotypes was useful for assuring both the capacity of the UPL PCR for an early viral DNA detection and the competence of the technique to be applied in any ASF diagnostic target sample. The reliability and robustness of the UPL PCR was finally verified with a panel of ASFV-infected clinical samples which was repeatedly tested at different times. Additionally, an internal control PCR assay was also developed and standardized using UPL probes within the endogenous ß-actin gene. Finally, the complete study offers a new validated real-time PCR technique, by means of a standardized commercial probe, providing a simple, rapid and affordable test, which is ready for application in the routine diagnosis of ASF.

Surveillance for avian influenza and Newcastle disease in backyard poultry flocks in Côte d'Ivoire, 2007-2009.

Between 2007 and 2009, active surveys were conducted on backyard poultry (chickens, guinea fowls and ducks) in four areas of Côte d'Ivoire, including two areas where avian influenza H5N1 outbreaks occurred in 2006. Each bird underwent clinical examination. In total, 5,578 sera, 4,580 tracheal swabs and 5,120 cloacal swabs were collected, plus tissues from 35 sick chickens. Using the haemagglutination inhibition (HI) test, 277 and 36 serum samples were positive for H5 and H7, respectively; all were negative for H9. All samples were negative by reverse transcription polymerase chain reaction. These results confirm the circulation of H5 and H7 influenza subtypes in backyard poultry in Côte d'Ivoire. Given that the seropositive birds were healthy, the circulating subtypes may be low pathogenicity avian influenza strains. Half (2,680) of the sera collected from chickens were tested by HI for Newcastle disease virus (NDV) antibody: 531 were positive. The seroprevalence of 19.8% confirms the endemic status of NDV, but may underestimate its true prevalence in Côte d'Ivoire.

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-d'Ivoire 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-d'Ivoire 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.

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.

Observations on rinderpest and rinderpest-like diseases throughout West and Central African countries during rinderpest eradication projects.

Between 1998 and 2005, the Regional Reference Laboratory at Bingerville (Ivory-Coast) received samples for analysis from Western and Central African countries. From a total of 606 sera; 65 tissue samples and 75 swabs received, no rinderpest virus or specific gene products or antibodies against rinderpest were detected. Use of the PCR on the tissue and swabs (total of 140 samples) identified the genomic presence of BVD (4/140), MCF (2/140), IBR (1/140) and FMD (6/140) viruses. These cause diseases that produce similar clinical signs to rinderpest. The quality of many samples sent to the reference laboratory did not meet the laboratory requirements and this compromised analysis of some specimens.

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.

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.

Interference in the vaccination of cattle against rinderpest virus by antibodies against peste des petits ruminants (PPR) virus.

The ability of the attenuated vaccine 75/1 of peste des petits ruminants to interfere with rinderpest vaccination in cattle was investigated experimentally. Young cattle (93) were selected and tested as being negative for antibodies against RP or PPR viruses. These were vaccinated with the peste des petits ruminants attenuated vaccine strain PPR75/1. All animals produced specific antibodies against the peste des petits ruminants vaccine after one or two doses. The cattle were then vaccinated with attenuated rinderpest vaccine. Two months later, 88 of these animals were sampled and 21/88 were positive for antibodies specific for rinderpest. The 67 negative animals received a second rinderpest vaccine dose after which 31seroconverted. The 36 animals which failed to seroconvert were re-vaccinated, of these 28 seroconverted. This study highlights the interference by peste des petits ruminants vaccination, presumably through production of antibodies that cross react with the live rinderpest virus in the vaccine used. This interference is also observed after vaccination against rinderpest followed by subsequent administration of peste des petits ruminants vaccine.

[Retrospective study of foot and mouth disease in West Africa from 1970 to 2003]. / Etude rétrospective de la fièvre aphteuse en Afrique de l'Ouest de 1970 à 2003.

A retrospective study of foot and mouth disease in seven West African countries was conducted for the period 1970 to 2003. The study included three cattle-exporting Sahel countries (Burkina-Faso, Mali and Niger) and four cattle-importing coastal countries (Benin, Côte d'lvoire, Ghana and Togo). Foot and mouth disease has been enzootic in these countries since 1990/1991. Four of the seven serotypes are regularly notified (O, A, SAT 1 and SAT 2). In the seven countries as a whole, 198 biological samples from identified foot and mouth disease outbreaks confirmed the involvement of the following serotypes: O (62 outbreaks); A (32 outbreaks); SAT 1 (18 outbreaks); SAT 2 (86 outbreaks). This result, which is largely underestimated, clearly demonstrates the seriousness of foot and mouth disease in West Africa, whose livestock production system characterised by continual uncontrolled animal movements facilitates the spread of the disease. Unlike in Southern Africa, for foot and mouth disease to be controlled in West Africa it is necessary immediately to introduce a regional strategy involving all countries which takes into account the real situation in the field: transhumance, nomadism and live-animal imports by coastal countries.

In ovo inhibition of fowlpoxvirus replication by a gall extract from Guiera senegalensis.

Several field isolates of fowlpoxvirus (FPV) from Burkina Faso, West Africa, were isolated and partly evaluated by molecular analysis. In addition, the in ovo antiviral activity against FPV of a gall extract from Guiera senegalensis was determined. Three viral isolates were obtained from suspected fowlpox cases after passage in embryonating chicken eggs and their poxviral identity confirmed by electron microscopy. All isolates were found to be pathogenic for chicks and all grew well in cell culture. Polymerase chain reaction and sequencing of amplicons revealed sequences identical with those of other FPV strains. The most studied isolate was then employed for use in an antiviral assay. An aqueous acetone extract from the galls of G. senegalensis was found to inhibit both virus-induced pock formation and to reduce viral titre in embryonating chicken eggs. The suggested mechanism of action is the activation of the alternative complement pathway and the inhibition of FPV-induced cholesterogenesis in ovo by constituents of the gall extract.

The phytochemical composition and in vitro antiviral activity of decoctions from galls of Guiera senegalensis J.F. Gmel. (Combretaceae) and their relative non-toxicity for chickens.

Aqueous decoctions obtained from the galls of Guiera senegalensis were screened to determine their phytochemical composition and in vitro antiviral activity against fowlpox virus. In addition, we wanted to investigate the toxic effects, if any, of crude extracts in chickens. Steroids as well as cardiac glycosides not previously reported, an alkaloid, polyphenols and saponins were detected in the various fractions of organic solvents used for extracting the decoctions. Antiviral activity was determined by cytopathic effect inhibition assay in primary chicken embryo skin cells. The 50% inhibitory concentration (EC50) was shown to be 15.6 microg/ml. Toxicity for cells was established by determining the 50% cytotoxic concentration (CCy50). A value of 90 microg/ml and a selectivity index (CCy50/EC50) of 5.8 were obtained. In vivo studies of toxicity were performed in chickens that were dosed orally with decoctions of several concentrations for 2 weeks and then monitored for 3 months. No significant changes in several blood chemical parameters were obtained, except for a significant decline in SGOT levels in birds dosed with 100 mg/kg. These levels were nevertheless within the accepted normal range. The findings suggest that aqueous decoctions of galls from G. senegalensis are non-toxic for chickens when administered orally, even at a daily dose of 100 mg/kg for 14 days.