Sero-surveillance of emerging viral diseases in camels and cattle in Nouakchott, Mauritania: an abattoir study.

This study reports the monitoring of several emerging viral pathogens in Mauritania, which was carried out by the analysis of bovine and camel samples taken at the slaughterhouse of Nouakchott. Blood and serum were collected by random sampling from 159 camels and 118 cattle in March 2013 at the large animals abattoir in Nouakchott. Serological tests for Rift Valley Fever (RVF), Peste des Petits Ruminants (PPR), West Nile disease (WND), epizootic haemorrhagic disease (EHD) and African horse sickness (AHS) were carried out using commercial ELISA kits. The samples, which resulted positives for PPR, WND and AHS, were tested with the confirmatory virus neutralization test (VNT). According to ELISA results, serological prevalence of RVF was 45% (95% CI 52.3-37.7) in camels and 16% (95% CI 22.6-9.4) in cattle. The difference between the observed prevalences in camels and in cattle was significant (p value ≤ 0.01). PPR was absent in camels and had 12% prevalence (95% CI, 17.86-6.14) in cattle. Furthermore, camels showed 92% (95% CI, 96.1-87.9) prevalence of WNV, 73% (95% CI, 82.3-63.64) of EHD and 3% (95% CI, 5.6-0.4) of AHS. This data are of relevance since provided useful feedbacks on the circulation of the pathogens in field. Moreover, this survey provided new information on the susceptibility of camels to several emerging pathogens and on the possible use of this species as sentinel animal.

Evaluation of Humoral Response and Protective Efficacy of an Inactivated Vaccine Against Peste des Petits Ruminants Virus in Goats.

Four goats were inoculated with an inactivated peste des petits ruminants virus (PPRV) vaccine. Three unvaccinated goats were kept as controls. After 36 days, the four goats were revaccinated. The immune response was monitored by virus neutralization test showing that two doses of the vaccine were able to stimulate strong immune response in all the vaccinated animals. The vaccinated goat and the controls were challenged with virulent PPRV intranasally. After PPRV challenge, the three control goats showed fever, viremia and virus excretion through mucosal surfaces, whereas the vaccinated goats were fully protected against PPRV infection and replication.

Development and Preliminary Evaluation of a New Real-Time RT-PCR Assay For Detection of Peste des petits Ruminants Virus Genome.

A duplex real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed for a simple and rapid diagnosis of Peste des petits ruminants (PPR). qRT-PCR primers and TaqMan probe were designed on a conserved region of nucleocapsid protein (Np) of PPR virus (PPRV) genome. An in vitro transcript of the target region was constructed and tested to determine analytical sensitivity. Commercial heterologous Armored RNA(®) was used as an internal positive control (IPC) for either RNA isolation or RT-PCR steps. The detection limit of the newly designed duplex real-time RT-PCR (qRT-PCR PPR_Np) was approximately 20 copies/µl with a 95% probability. No amplification signals were recorded when the qRT-PCR PPR_Np was applied to viruses closely related or clinically similar to PPRV- or to PPR-negative blood samples. A preliminary evaluation of the diagnostic performance was carried out by testing a group of 43 clinical specimens collected from distinct geographic areas of Africa and Middle East. qRT-PCR PPR_Np showed higher sensitivity than the conventional gel-based RT-PCR assays, which have been used as reference standards. Internal positive control made it possible to identify the occurrence of 5 false-negative results caused by the amplification failure, thus improving the accuracy of PPRV detection.

Schmallenberg virus in Italy: a retrospective survey in Culicoides stored during the bluetongue Italian surveillance program.

Following the first case of Schmallenberg (SBV) in northern Italy in February 2012, virus detection was conducted on midges collected during the national entomological surveillance program for bluetongue (BT). Six cattle farms, within a radius of 50 km from the SBV case, were selected for a 12 month study, aiming to determine when the virus entered the area, if it was capable of overwintering, and the possible role played by each species of the Obsoletus complex in disseminating the infection. A total of 33,724 Culicoides were collected at the six sites between June 2011 and June 2012. Species belonging to the Obsoletus Complex were the most abundant (94.44%) and, within the complex, Culicoides obsoletus was the most prevalent species in the studying area (65.4%). Nearly 7000 Culicoides midges were screened, either in pools or individually, for SBV by real-time RT-PCR. Viral genome was detected in six pools of the Obsoletus complex, collected at three sites between September and November 2011, and in a single parous female of C. obsoletus, collected in May 2012. As a result of the BT surveillance program in Italy it was possible to demonstrate, retrospectively, that SBV has circulated in at least three Italian provinces since early September 2011, nearly 5 months prior and as far as 40 km away from the first detected case. Similarly, the survey confirmed the presence of SBV in the vector population 3 months after the outbreak, following a cold winter during which the blacklight traps failed to catch active adult midges.

West Nile virus lineage 2 in Sardinian wild birds in 2012: a further threat to public health.

West Nile virus (WNV) strains belonging to lineage 2 were detected and isolated from the tissues of a goshawk and two carrion crows in Sardinia in August 2012. According to NS3 sequence analysis, the Sardinian isolates shared a high level of similarity with those of Italian lineage 2 strains which circulated in 2011 and with the homologous sequence of the 2004 Hungarian isolate. Following the human fatality reported in 2011 in Olbia, this study is the first to report the spread and enzootic circulation of WNV lineage 2 in Sardinia.

Evidence of West Nile virus lineage 2 circulation in Northern Italy.

A West Nile virus (WNV) strain belonging to lineage 2 was for the first time detected in two pools of Culex pipiens collected in the province of Udine and in tissues of a wild collared dove (Streptopelia decaocto) found dead in the province of Treviso, in North East of Italy. It was molecularly identified by group and WNV lineage specific RT-PCRs and characterized by partial sequencing of the NS3 and NS5 genes. When compared with the sequences of same fragments of NS3 and NS5 of the WNV lineage 2 strain isolated from birds of prey in Hungary (2004), the phylogenetic analysis of these sequences revealed 100% and 99% similarity, respectively. As the Hungarian strain, the NS3 selected sequence differed from the 2010 Greek isolate by one amino-acid located at 249 site which is the site involved in genetic modulation of WNV pathogenicity. The Italian and Hungarian strains have histidine rather than proline at this site. The presence of a lineage 2 strain in regions where the lineage 1 strain is still circulating, creates a new scenario with unpredictable consequences. In this situation comprehensive investigations on the occurrence, ecology, and epidemiology of these different WNV strains circulating in Italy become the highest priority.

2009 West Nile disease epidemic in Italy: first evidence of overwintering in Western Europe?

For the second consecutive year a West Nile disease (WND) epidemic has affected Italy causing disease in horses and humans. The infection re-occurred in the same places of the 2008 and moved westerly and southerly involving new areas and regions. The whole genome sequence of the Italian 2009 West Nile disease isolate (WNDV) was compared with those responsible for the 2008 WND outbreaks. The epidemiological findings of the two years of epidemic were compared as well. The high identity between 2008 and 2009 WNV strains (>99%), the earlier virus circulation in 2009 and the re-occurrence of the disease starting from the bordering infected areas reached by the infection in the previous year, strongly support the hypothesis of the overwintering of the virus and the endemisation to local host populations.

A new duplex real-time RT-PCR assay for sensitive and specific detection of African horse sickness virus.

A new real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for a simple and rapid diagnosis of African Horse Sickness (AHS) was developed. Primers and FAM-labeled TaqMan-MGB probes specific for African horse sickness virus (AHSV) were selected from the consensus sequence of the segment 8 of all 9 serotypes of AHSV reference strains. For the determination of the analytical sensitivity, an in vitro transcript (AHS_ns2T7) of the target region was constructed and tested. Furthermore, the AHS_ns2T7 transcript was used either as positive control or as a standard for quantifying target copies. A commercial heterologous Armored RNA was used as an internal positive control (IPC) for both RNA isolation and RT-PCR steps. The qRT-PCR AHS_ns2 was able to amplify the target sequence up to 0.71 copies/reaction. Its flexibility allowed to amplify a wide dynamic range of RNA copies from 1.5 to 0.001fg. Within this range, the Ct values varied from 18 to 38 cycles with SD values always lower than 0.5 confirming their strong and constant linear correlation with the RNA target. Furthermore the newly designed duplex real-time RT-PCR proved to be strictly AHSV-specific as it did not amplify close related viruses.

Re-emergence of West Nile virus in Italy.

In August 2008, West Nile disease re-emerged in Italy. The infection is affecting the North Eastern regions and, as of November 2008, has caused 33 clinical cases and five fatalities in horses. Until now, no deaths have been reported in birds. Mosquitoes, blood, serum and tissue samples, from horses and birds, within and around the outbreak area, have been collected and tested by various methods both serologically and virologically. West Nile virus strains have been isolated from blood samples of one horse and one donkey and from pools of brain, kidneys, heart and spleen of a pigeon and three magpies. When compared to the strain isolated during the 1998 Tuscany outbreak, the 255 bp sequence of the genome region coding for the envelope (E) protein of the isolated WNV strains, exhibited a 98.8% and 100% similarity at nucleotide and amino-acid level respectively.

Study of the gene expression of Th1 and Th2 cytokines in the immune response of cows vaccinated with Brucella abortus RB51.

A reverse transcriptase real-time polymerase chain reaction was developed to estimate the expression of the gamma-interferon (IFN-gamma) and interleukin-4 (IL-4) in cattle vaccinated with Brucella abortus RB51. Peripheral blood mononuclear cells of heifers vaccinated with B. abortus RB51 were stimulated in vitro with the same antigen and with Concanavalin A. The data obtained (presence of IFN-gamma expression and absence of IL-4 expression) confirmed the cell-mediated immune response to strain RB51 antigen. Furthermore, the expression of these two cytokines was quantified and the results showed values of IFN-gamma expression to be significantly higher in vaccinated than non-vaccinated animals.

Lipid fatty acid and protein pattern of equine prostasome-like vesicles.

The semen of several mammals contains vesicles of different composition and origin. We have recently reported on the presence of lipoprotein vesicles in stallion semen. To a certain extent, these resemble human prostasomes, but differ from them in amount and composition. These horse-semen prostasome-like vesicles may be important, not only in horse reproductive physiology, but also in view of stallion semen cryopreservation. In this paper, we have studied horse-semen prostasome-like vesicles and found that they possess less saturated fatty acid than human prostasomes. Moreover, their protein pattern (SDS-PAGE electrophoresis) shows that the 30-50-kDa fraction is less abundant in stallion vesicles. In addition, fluidity (measured as fluorescence anisotropy of diphenylhexatriene) is higher in horse prostasome-like vesicles than in human prostasomes, albeit being much lower than that of most membranes. These findings may be connected to some species-related differences in reproductive physiology: the vaginal milieu of the mare is not acidic and the deposition of semen is intrauterine in the horse but vaginal in humans.

Fatty acid pattern of human prostasome lipid.

Prostasomes are organelles of prostatic origin found in human semen. Their average diameter is about 150 nm and they appear as a lipoprotein membrane surrounding less organized material. Their lipid composition is peculiar, having much cholesterol and sphingomyelin. On the other hand, many of their proteins possess catalytic activity and are involved in the immune response. In previous work, we have shown that prostasomes may fuse to sperm at slightly acidic pH values, thereby modifying the composition of the sperm plasma membrane. In this paper, we examine the fatty acid pattern of prostasome lipid and find that it is completely different from that of sperm membrane lipid. Polyunsaturated phosphatidylcholines, common in sperm membrane, are rare in prostasome. Therefore, the fusion between prostasomes and sperm should stabilize sperm plasma membrane by enriching it in cholesterol, sphingomyelin, and saturated glycerophospholipid. This would prevent the untimely occurrence of the acrosome reaction.

Transfer of CD26/dipeptidyl peptidase IV (E.C. 3.5.4.4) from prostasomes to sperm.

Prostasomes are vesicles present in human semen. They are secreted by the prostate and contain large amounts of cholesterol and sphingomyelin. Some of their proteins are enzymes. Prostasomes are involved in a number of biological functions. In previous papers we demonstrated that lipid can be transferred from prostasomes to sperm by a fusion process occurring at neutral or slightly acidic pH. In this paper we demonstrate that CD26/dipeptidyl peptidase IV, an enzymatic activity absent in sperm, is transferred to sperm from prostasomes. This may be of particular interest since, by this procedure, sperm may acquire new membrane-bound enzymes and modify the catalytic activity of their surface.