Fate of SARS-CoV-2 coronavirus in wastewater treatment sludge during storage and thermophilic anaerobic digestion.

Since the COVID-19 outbreak has started in late 2019, SARS-CoV-2 has been widely detected in human stools and in urban wastewater. No infectious SARS-CoV-2 particles have been detected in raw wastewater until now, but it has been reported occasionally in human stools. This has raised questions on the fate of SARS-CoV-2 during wastewater treatment and notably in its end-product, wastewater treatment sludge, which is classically valorized by land spreading for agricultural amendment. In the present work, we focused on SARS-CoV-2 stability in wastewater treatment sludge, either during storage (4 °C, room temperature) or thermophilic anaerobic digestion (50 °C). Anaerobic digestion is one of the possible processes for sludge valorization. Experiments were conducted in laboratory pilots; SARS-CoV-2 detection was based on RT-quantitative PCR or RT-digital droplet PCR. In addition to SARS-CoV-2, Bovine Coronavirus (BCoV) particles were used as surrogate virus. The RNA from SARS-CoV-2 particles, inactivated or not, was close to the detection limit but stable in wastewater treatment sludge, over the whole duration of the assays at 4 °C (55 days) and at ambient temperature (∼20 °C, 25 days). By contrast, the RNA levels of BCoV and inactivated SARS-CoV-2 particles decreased rapidly during the thermophilic anaerobic digestion of wastewater treatment sludge lasting for 5 days, with final levels that were close to the detection limit. Although the particles' infectivity was not assessed, these results suggest that thermophilic anaerobic digestion is a suitable process for sludge sanitation, consistent with previous knowledge on other coronaviruses.

Contribution to the knowledge of Culicoides (Diptera: Ceratopogonidae) host preferences in France.

Knowledge on host-feeding pattern of blood-sucking insects helps to understand the epidemiology of a vector-born disease. We determined blood meal origin from blood-fed Culicoides thanks to molecular techniques. A set of primers was used to selectively amplify segment of vertebrates' prepronociceptin gene from abdomen of engorged Culicoides (Diptera: Ceratopogonidae). Vertebrate DNA was successfully amplified in 91% of blood-fed Culicoides assayed. Direct sequencing and comparison of resultant sequences with sequences in GenBank, using BLAST, lead to the specific identification of the host in 100% of the cases. A total of 157 blood-fed females belonging to 13 different Culicoides' species were captured thanks to light traps in different areas of France between 2008 and 2009. Blood meal origin was determined for 143 blood-fed midges: 59 Culicoides obsoletus, 18 Culicoides dewulfi, 16 Culicoides scoticus, 11 Culicoides chiopterus, 10 Culicoides lupicaris, 1 Culicoides pulicaris, 8 Culicoides punctatus, 10 Culicoides pallidicornis, 3 Culicoides achrayi, 2 Culicoides furcillatus, 3 Culicoides brunnicans, 1 Culicoides picturatus and 1 Culicoides poperinghensis. The predominant species in our study belong to the C. obsoletus complex; they are considered as putative vectors of Bluetongue virus in the north of Europe. C. chiopterus sampled fed only on cattle, while blood meal origin of C. dewulfi, C. obsoletus and C. scoticus was diversified. In our sampling, we found that midges were fed mainly on cattle (54%), rabbits (20%), horses (17%), sheep (4%), pigs or wild boars (4%) and human (1%). Cattle DNA was found in at least 11 different species of Culicoides assayed.

Red deer (Cervus elaphus) Did Not Play the Role of Maintenance Host for Bluetongue Virus in France: The Burden of Proof by Long-Term Wildlife Monitoring and Culicoides Snapshots.

Bluetongue virus (BTV) is a Culicoides-borne pathogen infecting both domestic and wild ruminants. In Europe, the Red Deer (Cervus elaphus) (RD) is considered a potential BTV reservoir, but persistent sylvatic cycle has not yet been demonstrated. In this paper, we explored the dynamics of BTV1 and BTV8 serotypes in the RD in France, and the potential role of that species in the re-emergence of BTV8 in livestock by 2015 (i.e., 5 years after the former last domestic cases). We performed 8 years of longitudinal monitoring (2008-2015) among 15 RD populations and 3065 individuals. We compared Culicoides communities and feeding habits within domestic and wild animal environments (51,380 samples). Culicoides diversity (>30 species) varied between them, but bridge-species able to feed on both wild and domestic hosts were abundant in both situations. Despite the presence of competent vectors in natural environments, BTV1 and BTV8 strains never spread in RD along the green corridors out of the domestic outbreak range. Decreasing antibody trends with no PCR results two years after the last domestic outbreak suggests that seropositive young RD were not recently infected but carried maternal antibodies. We conclude that RD did not play a role in spreading or maintaining BTV in France.

Antibiotic treatment of the hard tick Ixodes ricinus: Influence on Midichloria mitochondrii load following blood meal.

Midichloria mitochondrii is the most prevalent symbiont of the hard tick Ixodes ricinus, present in 100% of eggs and adult females of wild ticks. This bacterium is intracellular, and is the only known symbiont able to invade the mitochondria of the host cells. However, the role that M. mitochondrii plays in the host metabolism has yet to be elucidated. Multiple lines of evidence indicate the possibility of transmission of this bacterium to the vertebrate host during the tick blood meal. In order to investigate the role of M. mitochondrii in the biology of the tick host, we performed an antibiotic treatment on Ixodes ricinus individuals, with the aim of reducing/eliminating the symbiont, and to potentially observe the dynamic of bacterial infection in the tick host. We microinjected engorged adult females of I. ricinus with tetracycline, and we allowed the resulting larvae to feed on gerbils treated with the same antibiotic. The amount of M. mitochondrii was evaluated at different stages of the experiment using molecular techniques. In addition we evaluated the presence/absence of the symbiont DNA in the blood of gerbils used for the larval feeding. The performed treatments did not allow to eliminate the symbiont population from the host tick, however it allowed to reduce the multiplication that occurs after the larval blood meal. These results open the way for future experiments, using different antibiotic molecules, different administration methods and antibiotic administration on subsequent tick stages, to fulfill the goal of eliminating M. mitochondrii from the host I. ricinus, a major step in our understanding of the impact of this bacterium on ticks.

Characterization of two cryptic species, Culicoides stigma and C.parroti (Diptera: Ceratopogonidae), based on barcode regions and morphology.

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are insect vectors of economically important veterinary diseases such as African horse sickness, bluetongue, and Schmallenberg virus. The identification of Culicoides based on morphological features can be difficult. Three species of biting midges, Culicoides nubeculosus, C. stigma, and C. parroti have emerged in the laboratory from mud collected around watering troughs on a farm in northern France. Emerging Culicoides were characterized morphologically and molecularly using molecular markers. The closely related species C. stigma and C.parroti showed highly divergent sequences for both mitochondrial (cytochrome B and cytochrome oxidase I) and ribosomal DNA first internal transcribed spacer. A RFLP based on a single restriction using the same enzyme (HaeIII) for both cytochrome C oxidase I and cytochrome B is proposed to identify these species.

Emergence of Culicoides obsoletus from indoor and outdoor breeding sites.

Culicoides obsoletus (Diptera: Ceratopogondae) is considered as the probable main vector of Bluetongue virus (BTV) in northern Europe. Its breeding sites are poorly documented at the present time. We report numerous emergences of C. obsoletus s. str. from soil samples collected in two holdings between August and September 2010. Specimens were collected regularly in the laboratory during 80 days. In one holding, 1584 C. obsoletus midges emerged from used litter collected inside a dairy cow building and 211 C. obsoletus midges emerged from manure left outside the farm buildings. In the second holding, the number of emergences observed was much lower, especially for indoor samples. We discuss the impact of the farming practices on the abundance of emergence between both holdings on one side and between indoor and outdoor breeding sites on the other side. The observation of a peak in emergence and high emergence rates recorded in the laboratory for indoor samples let us assume that these breeding sites are of great importance, especially when it comes to understand the biology of this widespread species and its role in the transmission of BTV.