Health Status of the Eastern Grey Squirrel (Sciurus carolinensis) Population in Umbria: Results of the LIFE Project 'U-SAVEREDS'.

The introduction of the Eastern grey squirrel (Sciurus carolinensis) in Europe is one of the best-known cases of invasive alien species (IAS) colonisation, that poses a severe risk to the conservation of biodiversity. In 2003, it was released in a private wildlife park near the city of Perugia (Italy), where it is replacing the native Eurasian red squirrel (Sciurus vulgaris). The LIFE13 BIO/IT/000204 Project (U-SAVEREDS) was set up for the Sciurus vulgaris conservation in Umbria through an eradication campaign of grey squirrels. One hundred and fifty-four animals were analysed for bacteriological, mycological, virological, and serological investigations (C4 action). Sanitary screening showed that Sciurus carolinensis is a dermatophyte carrier, and therefore, it could cause public health issues for humans, considering its confident behaviour. Moreover, it has been marginally responsible for the spreading of Candida albicans, Coxiella burnetii, and Borrelia lusitaniae. Health status evaluation conducted on the Sciurus carolinensis population indicated that it is necessary to raise awareness of its impacts on biodiversity and human health. Moreover, the health status and behaviours of the IAS must be considered when control or eradication campaigns are planned.

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.

Genetic Diversity of Rift Valley Fever Strains Circulating in Namibia in 2010 and 2011.

Outbreaks of Rift Valley fever (RVF) occurred in Namibia in 2010 and 2011. Complete genome characterization was obtained from virus isolates collected during disease outbreaks in southern Namibia in 2010 and from wildlife in Etosha National Park in 2011, close to the area where RVF outbreaks occurred in domestic livestock. The virus strains were sequenced using Sanger sequencing (Namibia_2010) or next generation sequencing (Namibia_2011). A sequence-independent, single-primer amplification (SISPA) protocol was used in combination with the Illumina Next 500 sequencer. Phylogenetic analysis of the sequences of the small (S), medium (M), and large (L) genome segments of RVF virus (RVFV) provided evidence that two distinct RVFV strains circulated in the country. The strain collected in Namibia in 2010 is genetically similar to RVFV strains circulating in South Africa in 2009 and 2010, confirming that the outbreaks reported in the southern part of Namibia in 2010 were caused by possible dissemination of the infection from South Africa. Isolates collected in 2011 were close to RVFV isolates from 2010 collected in humans in Sudan and which belong to the large lineage containing RVFV strains that caused an outbreak in 2006-2008 in eastern Africa. This investigation showed that the RVFV strains circulating in Namibia in 2010 and 2011 were from two different introductions and that RVFV has the ability to move across regions. This supports the need for risk-based surveillance and monitoring.

Peste des Petits Ruminants outbreaks in Tunisia in 2016.

In Tunisia, 86 outbreaks of Peste des Petits Ruminants (PPR) were reported in ovine and caprine herds in 2016. Molecular characterization of PPRV strains was carried out by partial sequencing of nucleoprotein (Np) gene from diagnostic specimens. The results showed that disease outbreaks were caused by virus strains closely related to PPRV strains collected in Egypt in 2014 and 2015.

Serological Survey of Hantavirus and Flavivirus Among Wild Rodents in Central Italy.

Hantaviruses are a group of zoonotic viruses carried by rodents. Puumala virus (PUUV) and Dobrava virus (DOBV) are the causative agents of human hantavirus infections in Europe. Knowledge about hantavirus circulation in Italy is very scarce. West Nile virus (WNV) and Usutu virus (USUV) are emerging neuropathogenic flaviviruses, both endemic in most part of the Italian territories. To monitor the circulation of PUUV, DOBV, WNV, and USUV in natural environment in central Italy, we carried out serological surveillance in wild rodents. During this study, 90 animals were captured in forested areas of Abruzzo and Marche regions and tested with serological assays for the specific pathogens. Serological test provided no evidence of PUUV and DOBV circulation in the studied area. However, four rodents (Apodemus flavicollis) were found to be positive by WNV ELISA test. Two of them were confirmed as WNV by virus neutralization test.

Isolation of a Defective Prion Mutant from Natural Scrapie.

It is widely known that prion strains can mutate in response to modification of the replication environment and we have recently reported that prion mutations can occur in vitro during amplification of vole-adapted prions by Protein Misfolding Cyclic Amplification on bank vole substrate (bvPMCA). Here we exploited the high efficiency of prion replication by bvPMCA to study the in vitro propagation of natural scrapie isolates. Although in vitro vole-adapted PrPSc conformers were usually similar to the sheep counterpart, we repeatedly isolated a PrPSc mutant exclusively when starting from extremely diluted seeds of a single sheep isolate. The mutant and faithful PrPSc conformers showed to be efficiently autocatalytic in vitro and were characterized by different PrP protease resistant cores, spanning aa ∼155-231 and ∼80-231 respectively, and by different conformational stabilities. The two conformers could thus be seen as different bona fide PrPSc types, putatively accounting for prion populations with different biological properties. Indeed, once inoculated in bank vole the faithful conformer was competent for in vivo replication while the mutant was unable to infect voles, de facto behaving like a defective prion mutant. Overall, our findings confirm that prions can adapt and evolve in the new replication environments and that the starting population size can affect their evolutionary landscape, at least in vitro. Furthermore, we report the first example of "authentic" defective prion mutant, composed of brain-derived PrPC and originating from a natural scrapie isolate. Our results clearly indicate that the defective mutant lacks of some structural characteristics, that presumably involve the central region ∼90-155, critical for infectivity but not for in vitro replication. Finally, we propose a molecular mechanism able to account for the discordant in vitro and in vivo behavior, suggesting possible new paths for investigating the molecular bases of prion infectivity.

First evidence of West Nile virus lineage 2 circulation in Turkey.

In August 2014, a West Nile virus (WNV) strain belonging to lineage 2 was detected in the brain tissues of a 9 year old mare euthanised after showing severe clinical signs in Bursa region, Turkey. Phylogenetic analyses of 290 bp of NS3 coding region clustered the Turkish strain together with the 2010-2012 Greek isolates. Either IgG and IgM or IgG only WNV antibodies were detected in 2 and 11 horses, respectively, which were in the outbreak surrounding. No WNV RNA was detected in pools of 50 individuals of Culex pipiens (n = 2), Ochlerotatus caspius (n = 2), and Culex theileri (n = 1) collected in the infected premises.

Genetic characterization of Italian field strains of Schmallenberg virus based on N and NSs genes.

Following its first identification in Germany in 2011, the Schmallenberg virus (SBV) has rapidly spread to many other European countries. Despite the wide dissemination, the molecular characterization of the circulating strains is limited to German, Belgian, Dutch, and Swiss viruses. To fill this gap, partial genetic characterization of 15 Italian field strains was performed, based on S segment genes. Samples were collected in 2012 in two different regions where outbreaks occurred during distinct epidemic seasons. The comparative sequence analysis demonstrated a high molecular stability of the circulating viruses; nevertheless, we identified several variants of the N and NSs proteins not described in other SBV isolates circulating in Europe.

First External Quality Assessment of Molecular and Serological Detection of Rift Valley Fever in the Western Mediterranean Region.

Rift Valley fever (RVF) is a mosquito-borne viral zoonosis which affects humans and a wide range of domestic and wild ruminants. The large spread of RVF in Africa and its potential to emerge beyond its geographic range requires the development of surveillance strategies to promptly detect the disease outbreaks in order to implement efficient control measures, which could prevent the widespread of the virus to humans. The Animal Health Mediterranean Network (REMESA) linking some Northern African countries as Algeria, Egypt, Libya, Mauritania, Morocco, Tunisia with Southern European ones as France, Italy, Portugal and Spain aims at improving the animal health in the Western Mediterranean Region since 2009. In this context, a first assessment of the diagnostic capacities of the laboratories involved in the RVF surveillance was performed. The first proficiency testing (external quality assessment--EQA) for the detection of the viral genome and antibodies of RVF virus (RVFV) was carried out from October 2013 to February 2014. Ten laboratories participated from 6 different countries (4 from North Africa and 2 from Europe). Six laboratories participated in the ring trial for both viral RNA and antibodies detection methods, while four laboratories participated exclusively in the antibodies detection ring trial. For the EQA targeting the viral RNA detection methods 5 out of 6 laboratories reported 100% of correct results. One laboratory misidentified 2 positive samples as negative and 3 positive samples as doubtful indicating a need for corrective actions. For the EQA targeting IgG and IgM antibodies methods 9 out of the 10 laboratories reported 100% of correct results, whilst one laboratory reported all correct results except one false-positive. These two ring trials provide evidence that most of the participating laboratories are capable to detect RVF antibodies and viral RNA thus recognizing RVF infection in affected ruminants with the diagnostic methods currently available.

African horse sickness outbreaks in Namibia from 2006 to 2013: clinical, pathological and molecular findings.

African horse sickness (AHS) is a vector­borne viral disease of equids, endemic in Sub­Saharan Africa. This article reports the clinic­pathological and laboratory findings observed in the framework of passive surveillance during the AHS outbreaks which occurred in Namibia between 2006 and 2013. This study was conducted in the framework of the collaboration among the Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise (Teramo, Italy), the Namibian Ministry of Agriculture Water and Forestry, and the Namibian National Veterinary Association. A total of 92 horses were investigated, showing different clinical form of AHS: peracute/acute (n = 43), sub­acute (n = 21) and mild AHS fever (n = 19). Clinical data were not available for 9 horses, because they were found dead. Pathological findings have been recorded for 35 horses. At necropsy, pulmonary and subcutaneous oedema, haemorrhages and enlargement of lymph nodes were mainly observed. Diagnosis was confirmed by laboratory testing, AHS virus (AHSV) was isolated from 50 horses and the identified serotypes were: 1, 2, 4, 6, 7, 8, and 9. The phylogenetic analysis of the S10 genome sequences segregated the Namibian AHSV strains in the same clusters of those circulating in South Africa in recent years. The description of AHS clinical, pathological, and laboratory features of AHS provided in this article is of value for differential diagnosis and control of AHS, especially in areas currently free from this disease.

Correlation between infectivity and disease associated prion protein in the nervous system and selected edible tissues of naturally affected scrapie sheep.

The transmissible spongiform encephalopathies (TSEs) or prion diseases are a group of fatal neurodegenerative disorders characterised by the accumulation of a pathological form of a host protein known as prion protein (PrP). The validation of abnormal PrP detection techniques is fundamental to allow the use of high-throughput laboratory based tests, avoiding the limitations of bioassays. We used scrapie, a prototype TSE, to examine the relationship between infectivity and laboratory based diagnostic tools. The data may help to optimise strategies to prevent exposure of humans to small ruminant TSE material via the food chain. Abnormal PrP distribution/accumulation was assessed by immunohistochemistry (IHC), Western blot (WB) and ELISA in samples from four animals. In addition, infectivity was detected using a sensitive bank vole bioassay with selected samples from two of the four sheep and protein misfolding cyclic amplification using bank vole brain as substrate (vPMCA) was also carried out in selected samples from one animal. Lymph nodes, oculomotor muscles, sciatic nerve and kidney were positive by IHC, WB and ELISA, although at levels 100-1000 fold lower than the brain, and contained detectable infectivity by bioassay. Tissues not infectious by bioassay were also negative by all laboratory tests including PMCA. Although discrepancies were observed in tissues with very low levels of abnormal PrP, there was an overall good correlation between IHC, WB, ELISA and bioassay results. Most importantly, there was a good correlation between the detection of abnormal PrP in tissues using laboratory tests and the levels of infectivity even when the titre was low. These findings provide useful information for risk modellers and represent a first step toward the validation of laboratory tests used to quantify prion infectivity, which would greatly aid TSE risk assessment policies.

Rift Valley fever in Namibia, 2010.

During May-July 2010 in Namibia, outbreaks of Rift Valley fever were reported to the National Veterinary Service. Analysis of animal specimens confirmed virus circulation on 7 farms. Molecular characterization showed that all outbreaks were caused by a strain of Rift Valley fever virus closely related to virus strains responsible for outbreaks in South Africa during 2009-2010.

Ultra-efficient PrP(Sc) amplification highlights potentialities and pitfalls of PMCA technology.

In order to investigate the potential of voles to reproduce in vitro the efficiency of prion replication previously observed in vivo, we seeded protein misfolding cyclic amplification (PMCA) reactions with either rodent-adapted Transmissible Spongiform Encephalopathy (TSE) strains or natural TSE isolates. Vole brain homogenates were shown to be a powerful substrate for both homologous or heterologous PMCA, sustaining the efficient amplification of prions from all the prion sources tested. However, after a few serial automated PMCA (saPMCA) rounds, we also observed the appearance of PK-resistant PrP(Sc) in samples containing exclusively unseeded substrate (negative controls), suggesting the possible spontaneous generation of infectious prions during PMCA reactions. As we could not definitively rule out cross-contamination through a posteriori biochemical and biological analyses of de novo generated prions, we decided to replicate the experiments in a different laboratory. Under rigorous prion-free conditions, we did not observe de novo appearance of PrP(Sc) in unseeded samples of M109M and I109I vole substrates, even after many consecutive rounds of saPMCA and working in different PMCA settings. Furthermore, when positive and negative samples were processed together, the appearance of spurious PrP(Sc) in unseeded negative controls suggested that the most likely explanation for the appearance of de novo PrP(Sc) was the occurrence of cross-contamination during saPMCA. Careful analysis of the PMCA process allowed us to identify critical points which are potentially responsible for contamination events. Appropriate technical improvements made it possible to overcome PMCA pitfalls, allowing PrP(Sc) to be reliably amplified up to extremely low dilutions of infected brain homogenate without any false positive results even after many consecutive rounds. Our findings underline the potential drawback of ultrasensitive in vitro prion replication and warn on cautious interpretation when assessing the spontaneous appearance of prions in vitro.

Gene expression profiling on sheep brain reveals differential transcripts in scrapie-affected/not-affected animals.

This study aimed at identifying genes that could mark scrapie infection in the central nervous system of sheep. We used the subtractive suppressive hybridization (SSH) technique on brain samples from sheep healthy or clinically affected by scrapie. Following subtraction, several discrete differential bands appeared between the two reciprocally subtracted samples. These bands were cloned and sequenced, allowing identifying the genes COX1, CHN1, PPP2CA, LRFN5, CAMK2A and RABEPK. Two of the genes identified, CHN1 and RABEPK, appear to locate inside a QTL region known to modulate prion disease incubation time in mice, and LRFN5 maps inside a QTL region identified in sheep. Furthermore, CHN1 and RABEKP showed new unreported differential splicing.

Interspecific chromosome-wide transcription profiles reveal the existence of mammalian-specific and species-specific chromosome domains.

A long-range exploration of expression levels through wide chromosome territories was carried out in three species (pig, cattle, and chicken) by aligning EST counts against the human genome. This strategy made it possible to produce expression profiles that were very similar between pig and cattle and that were significantly correlated with chicken levels of expression. In parallel with these alignments, we developed a statistical approach enabling us to screen genomic regions for both underexpression and overexpression at the chromosome level within a given species, as well as interspecifically. The observed correlations are indicative of the existence of interspecifically conserved domains of gene expression, not only for housekeeping genes (which are highly expressed), but also for regions where genes are significantly underexpressed. Furthermore, our strategy made it possible to point out regions that are differentially regulated between species. These expression data were crossed with available comparative mapping information for pigs and cattle, suggesting that coregulated regions are syntenic in various mammals.

Sheep/human comparative map in a chromosome region involved in scrapie incubation time shows multiple breakpoints between human chromosomes 14 and 15 and sheep chromosomes 7 and 18.

A chromosome region involved in scrapie incubation time was identified on sheep chromosome 18 (OAR18). Since OAR18 (and OAR7) share conserved chromosome segments with human chromosomes HSA14 and HSA15, a dense map of type I markers was constructed by FISH mapping of bacterial artificial chromosomes containing genes located on these human chromosomes. In this study, we used the complete human sequence information (gene positions in megabases, Mb) to locate approximately one gene every 2 Mb on HSA15 (19 genes mapped between 19.51 and 66.02 Mb) and on HSA14 (11 genes between 73.24 and 102.62 Mb). Combined with previous work carried out in cattle and goats, our results made it possible to refine the comparative map between ruminants and humans for these two highly rearranged chromosomes (10 segments on HSA15 and 7 on HSA14). Furthermore, we identified relatively short intervals containing evolutionary breakpoints, which is a prerequisite to position them precisely. This work is also the first step in the cloning of the region involved in scrapie incubation period in sheep.