Epidemiological Analyses of the First Incursion of the Epizootic Hemorrhagic Disease Virus Serotype 8 in Tunisia, 2021-2022.

Epizootic hemorrhagic disease (EHD) is a non-contagious arthropod-transmitted viral disease and a World Organization for Animal Health (WOAH)-listed disease of domestic and wild ruminants since 2008. EHDV is transmitted among susceptible animals by a few species of midges of genus Culicoides. During the fall of 2021, a large outbreak caused by the epizootic hemorrhagic disease virus (EHDV), identified as serotype 8, was reported in Tunisian dairy and beef farms with Bluetongue virus (BTV)-like clinical signs. The disease was detected later in the south of Italy, in Spain, in Portugal and, more recently, in France, where it caused severe infections in cattle. This was the first evidence of EHDV-8 circulation outside Australia since 1982. In this study, we analyzed the epidemiological situation of the 2021-2022 EHDV outbreaks reported in Tunisia, providing a detailed description of the spatiotemporal evolution of the disease. We attempted to identify the eco-climatic factors associated with infected areas using generalized linear models (GLMs). Our results demonstrated that environmental factors mostly associated with the presence of C. imicola, such as digital elevation model (DEM), slope, normalized difference vegetation index (NDVI), and night-time land surface temperature (NLST)) were by far the most explanatory variables for EHD repartition cases in Tunisia that may have consequences in neighboring countries, both in Africa and Europe through the spread of infected vectors. The risk maps elaborated could be useful for disease control and prevention strategies.

Experimental infection of cattle, sheep, and goats with the newly emerged epizootic hemorrhagic disease virus serotype 8.

Epizootic hemorrhagic disease virus serotype 8 (EHDV-8) emerged in Europe for the first time in late 2022. In this study, we investigated the kinetics of EHDV-8 infection in cattle, sheep, and goats.  Following experimental infection with EHDV-8, four out of five calves displayed fever, while another calf exhibited ulcerative and crusty lesions of the muzzle. RNAemia peaked at day 7 post infection in all calves and remained relatively stable till the end of the study, at 78 days post infection. Infectious virus was isolated up to 21 days post infection in one calf. As far as small ruminants are concerned, one sheep experienced fever and two out of five had consistent RNAemia that lasted until the end of the study. Remarkably, infectious virus was evidenced at day 7 post infection in one sheep. In goats, no RNA was observed. All infected animals seroconverted, and a neutralizing immune response was observed in all species, with calves exhibiting a more robust response than sheep and goats. Our study provides insights into the kinetics of EHDV-8 infection and the host immune responses. We also highlight that sheep may also play a role in EHDV-8 epidemiology. Altogether, the data gathered in this study could have important implications for disease control and prevention strategies, providing crucial information to policy makers to mitigate the impact of this viral disease on livestock.

Development and validation of an RT-qPCR for detection and quantitation of emerging epizootic hemorrhagic disease virus serotype 8 RNA from field samples.

Epizootic hemorrhagic disease virus (EHDV) is a Culicoides-transmitted virus circulating in multiple serotypes. It has become a concern in the European Union as a novel strain of the serotype 8 (EHDV-8) of clear Northern African origin, has been recently discovered in symptomatic cattle in Italy (islands of Sardinia and Sicily), Spain, and Portugal. Current molecular typing methods targeting the S2 nucleotide sequences -coding for the outermost protein of the virion VP2- are not able to detect the novel emerging EHDV-8 strain as they enrolled the S2 sequence of the unique EHDV-8 reference strain isolated in Australia in 1982. Thus, in this study, we developed and validated a novel typing assay for the detection and quantitation of the novel EHDV-8 RNA from field samples, including blood of ruminants and insects. This molecular tool will certainly support EHDV-8 surveillance and control.

Characterization of Epizootic Hemorrhagic Disease Virus Serotype 8 in Naturally Infected Barbary Deer (Cervus elaphus barbarus) and Culicoides (Diptera: Ceratopogonidae) in Tunisia.

Epizootic hemorrhagic disease (EHD) is a Culicoides-borne disease of domestic and wild ruminants caused by EHD virus (EHDV). This virus circulates in multiple serotypes. In late September 2021, a novel strain belonging to EHDV-8 was reported in cattle farms in Central-Western Tunisia, and in the fall of 2022, the same virus was also detected in Italy and Spain. In the present study, we described EHDV-8 occurrence in deer and, a preliminary identification of the potential Culicoides species responsible for virus transmission in selected areas of Tunisia. EHDV-8 was identified in deer carcasses found in 2021 and 2022 in the national reserve of El Feidja, Jendouba, Northwestern Tunisia, and isolated on cell culture. Instead, insect vectors were collected in October 2021 only in the areas surrounding the city of Tozeur (Southern Tunisia) where EHDV-8 cases in cattle were confirmed. Morphological identification showed that 95% of them belonged to the Culicoides kingi and Culicoides oxystoma species and both species tested positive for EHDV-8 RNA. C. imicola was not detected in this collection and EHDV-8 RNA was not evidenced in vector pools collected in 2020, prior to official EHDV-8 emergence. EHDV whole genome sequences were also obtained directly from infected biological samples of deer and positive vectors. EHDV-8 sequences obtained from deer and vectors share a nucleotide identity ranging from 99.42 to 100% and amino acid identity from 99.18 to 100% across all genome segments with the EHDV-8/17 TUN2021 reference sequence.

Role of eco-climatic factors in the distribution of bluetongue in endemic areas in Tunisia.

Background: Bluetongue (BT) is an important infectious, non-contagious, OIE-listed viral disease of domestic and wild ruminants. The disease is transmitted among susceptible animals by a few species of an insect vector in the genus Culicoides. Recently, during the fall of 2020 (September and October), a Bluetongue virus-4 epizootic marked the epidemiological situation in several delegations of Tunisia with clinical cases recorded in sheep and cattle. Aim: Determine the eco-climatic variables most likely associated with delegations reporting BT cases. Methods: A logistic regression model (LRM) was used to examine which eco-climatic variables were most likely associated with delegations reporting BT cases. Results: Based on the LRM, our findings demonstrated that the key factors contributing significantly to BT cases' distribution among delegations in Tunisia included day land surface temperatures (DLST), night land surface temperatures (NLST) and normalized difference vegetation index (NDVI). A positive correlation between sheep distribution and rainfall amounts was demonstrated. Statistical analysis focusing on the most affected delegations during the BT epidemic (the Sahel and the Centre of Tunisia) demonstrated that the epidemic situation seems to be a consequence of the combination of the following environmental parameters: NDVI with values ranging between 0.16 and 0.2, moderate rainfall 2-4-fold above the normal (10-50 mm) and DLST values between 32°C and 34°C in September. Conclusion: These findings suggest and develop a robust and efficient early warning surveillance program in risk areas based on eco-climatic risk factors.

Epizootic Haemorrhagic Disease Virus Serotype 8 in Tunisia, 2021.

Epizootic haemorrhagic disease (EHD) is a Culicoides-borne viral disease caused by the epizootic haemorrhagic disease virus (EHDV) associated with clinical manifestations in domestic and wild ruminants, primarily white-tailed deer (Odocoileus virginianus) and cattle (Bos taurus). In late September 2021, EHDV was reported in cattle farms in central/western Tunisia. It rapidly spread throughout the country with more than 200 confirmed outbreaks. We applied a combination of classical and molecular techniques to characterize the causative virus as a member of the serotype EHDV-8. This is the first evidence of EHDV- 8 circulation since 1982 when the prototype EHDV-8 strain was isolated in Australia. This work highlights the urgent need for vaccines for a range of EHDV serotypes.

Cationic zinc (II) phthalocyanine nanoemulsions for photodynamic inactivation of resistant bacterial strains.

BACKGROUND: The growing emergence of microbial resistance to antibiotics represents a worldwide challenge. Antimicrobial photodynamic inactivation (aPDI) has been introduced as an alternative technique, especially when combined with nanotechnology. Therefore, this study was designed to investigate the therapeutic merits of combined aPDI and nanoemulsion in infections caused by resistant bacterial strains. METHODS: Cationic zinc (II) phthalocyanine nanoemulsions (ZnPc-NE) were prepared using isopropyl myristate (IPM) as oil phase, egg phosphatidylcholine (egg PC) as emulsifier, and N-cetyl-N,N,N-trimethyl ammonium bromide (CTAB). Nanoemulsions were characterized for particle size, polydispersity, zeta potential, viscosity, and skin deposition. The in-vitro aPDI was investigated on human resistant pathogens; gram-positive methicillin-resistant Staphylococcus aureus (MRSA) and gram-negative Multidrug-resistant strain of Escherichia coli (MDR E. coli), under different experimental conditions. In addition, in-vivo model of abrasion wound infected by MDR E. coli was induced in rats to investigate the therapeutic potential of the selected formulation. RESULTS: It was evident that the selected ZnPc formulation (20 % IPM, 2 % egg PC and 0.5 % CTAB) displayed a particle size of 209.9 nm, zeta potential +73.1 mV, and 23.66 % deposition of ZnPc in skin layers. Furthermore, the selected formulation combined with light achieved almost 100 % eradication of the two bacterial strains, with superior bacterial load reduction and wound healing propertiesin-vivo, compared to either the nanoemulsion formulation or laser alone. CONCLUSION: ZnPc nanoemulsion improved antimicrobial photodynamic therapy in inactivating resistant bacterial infections and provided a promising therapeutic means of treating serious infections, and hence could be applied in diseases caused by other bacterial strains.

Transposition mechanism, molecular characterization and evolution of IS6110, the specific evolutionary marker of Mycobacterium tuberculosis complex.

The mycobacterial insertion sequence IS6110 proved crucial in deciphering tuberculosis (TB) transmission dynamics. This sequence was also shown to play an important role in the pathogenicity (transmission ability and/or virulence) of Mycobacterium tuberculosis, the main causative agent of TB in humans. In this study, we explored the usefulness of IS6110 and its potential as a phylogenetic/typing marker. We also analyzed the genetic polymorphism and evolutionary trends (selective pressure) of its transposase-encoding open reading frames (ORFs), A and B, using the maximum likelihood method. Both ORFs evolved chronologically through random single nucleotide polymorphisms. They were subjected to strict purifying selection more tight on orfA, with no evidence of significant recombination events. OrfA proved to have a crucial role in regulating the transpositional process. Several analyses showed that IS6110 acquisition antedated the emergence of the Mycobacterium tuberculosis complex. This original copy of IS6110 element was functionally optimal. In conclusion, this study not only demonstrated the usefulness of IS6110 in terms of phylogenetic and typing purposes and its transpositional mechanism, but also informed the scientific community on its evolutionary history.