Infectiologie Animale et Santé Publique

Abstract African Swine Fever Virus (ASFV) represents a looming threat to animal health, food safety and to the livestock industry. Virulent strains of ASFV cause a severe and often fatal illness, while attenuated strains are usually associated with mild symptoms. Naturally-occurring attenuated strains are typically deleted of more than 20 genes located at the viral genome’s extremities. Whether other key differences between virulent and attenuated ASFV strains may contribute to the virulence phenotype remains however largely unexplored. In this work, we sought to determine how the dynamics of viral gene expression may shape the host’s innate immune response to ASFV infection and contribute to ASFV virulence. We conducted a medium-throughput transcriptomic study to characterize the viral transcriptome of a panel of virulent and attenuated strains (171 viral genes), as well as the host response of ASFV-infected macrophages (92 host genes). Confocal imaging allowed further characterization of cellular response to infection, by assessing the dynamics of IFN and NF-κB pathway activation in ASFV-infected cells. Our results indicate that the two types of viral pathotypes exhibit global differences in the dynamics of genome replication and viral transcription. Virulent ASFV strains displayed a burst of viral transcription early on, while attenuated strains tended to replicate to higher levels at late time points. The host response was much more pronounced in cells infected with attenuated strains compared to virulent ones, with higher expression levels of interferon-stimulated genes, some innate immunity sensors, and the inducible chaperone HSP70.2. Unexpectedly, genotype I and genotype II virulent strains exhibited some notable differences in their kinetics of viral genome replication and in the host response they provoked, with higher levels of pro-inflammatory cytokines being induced by genotype II strains. Confocal imaging analysis of ASFV-infected primary macrophages revealed that attenuated strains, but not virulent ones, caused the translocation of both p65 and STAT2 to the nucleus. Strikingly, we identified a group of 26 viral genes that were either expressed at higher levels or at an earlier stage of infection by virulent strains. Several of these genes, such as R298L , H233R , DP71L and MGF505-7R encode for proteins that inhibited the type I Interferon response in a reporter cell line system. This work sheds new light on the mechanistical drivers of ASFV virulence and will in the long run help to better understand the protection offered by ASFV Live-Attenuated Vaccine candidates. Author summary African Swine Fever (ASF), a severe infectious disease affecting domestic pigs and wild boars, presents a global threat to the livestock industry. It is caused by African Swine Fever Virus (ASFV), a large DNA virus encoding between 150 and 200 genes. While virulent ASFV strains cause a fatal illness in infected animals, attenuated strains induce only minor symptoms and some can confer subsequent protection against a pathogenic infection. While Live-Attenuated Vaccines for ASFV are under development and represent a promising tool in the fight against ASF, the mechanisms of ASFV virulence (and conversely, attenuation) are not fully understood. In particular, it is unclear whether key differences may exist between attenuated and virulent ASFV strains, beyond the extensive genomic deletions harbored by the former. In this work, we explored for the first time how the dynamics of viral gene expression may influence the innate immune response to different ASFV strains. We found that attenuated ASFV strains trigger a stronger host response compared to virulent ASFV strains, with higher expression levels of innate immune genes and a stronger activation of key signaling hubs. Finally, we identified a group of 26 ASFV genes that may drive this phenomenon and represent novel virulence factors.

Resistance to anthelmintics in gastrointestinal nematodes (GINs) is highly prevalent, as these parasites have been treated with anthelmintics for decades in ruminants and horses. Anthelmintics belong to different classes, each with a different mode of action. The most used are benzimidazoles and macrocyclic lactones and, to a lesser extent, levamisole and pyrantel in herbivores, as estimated from the literature. Combining these classes should be effective in controlling GIN. However, several farmers’ practices tend to promote GIN resistance. Therefore, it is unclear whether the use of anthelmintic associations is a sustainable solution for controlling resistance in natural conditions. It is not easy to establish the association of anthelmintic resistances on farms since rarely several anthelmintics and their combinations are used on a single farm. Composed probability calculations were employed when literature data indicated the presence of resistance (to benzimidazoles, levamisole, or macrocyclic lactones) in several ruminant GIN cases. The efficacy of different anthelmintics (benzimidazoles, pyrantel, or macrocyclic lactones) was evaluated in terms of the correlation between faecal nematode egg counts in horses in the available literature. No associations of anthelmintic resistance were found between the different classes of anthelmintics in either ruminants or horses. However, the association between anthelmintic resistance in GIN may appear in the long term. It is presumed that combining drugs may reduce the development of resistance and allow better control of infection on farms where resistance is already established to a low level.

Protostrongylid nematodes (small lungworms) are very common in Mediterranean sheep and have long been recorded in North Africa. Here, the following four species are found: Muellerius capillaris, Neostrongylus linearis, Cystocaulus ocreatus, and Protostrongylus rufescens. Previous risk factors studies for protostrongylids have been conducted in a single farm and therefore have limitations. Sixty-one meat sheep farms in north-eastern Algeria were surveyed for protostrongylid infection and anthelmintic treatment in late autumn/early winter. The climates of the nine regions ranged from subhumid to arid for humidity and from mild to cool for winter temperature. The highest infection, estimated by the number of larvae per gram of faeces (LPG), was found in subhumid and semi-arid climates. The Rembi breed was more infected than the Ouled Djellal or their crosses. LPG decreased with increasing number of treatments. The latter was also associated with an increased percentage of M. capillaris and a decrease in species diversity. The anthelmintic regimen (ivermectin, levamisole, and albendazole) directly targets gastrointestinal nematodes and indirectly protostrongylids. The use of effective drugs targeting protostrongylids at key moments may provide more effective control.

BackgroundHuman cryptosporidiosis is distributed worldwide, and it is recognised as a leading cause of acute diarrhoea and death in infants in low- and middle-income countries. Besides immune status, the higher incidence and severity of this gastrointestinal disease in young children could also be attributed to the digestive environment. For instance, human gastrointestinal physiology undergoes significant changes with age, however the role this variability plays in Cryptosporidium parvum pathogenesis is not known. In this study, we analysed for the first time the impact of digestive physicochemical parameters on C. parvum infection in a human and age-dependent context using a dynamic in vitro gastrointestinal model.ResultsOur results showed that the parasite excystation, releasing sporozoites from oocysts, occurs in the duodenum compartment after one hour of digestion in both child (from 6 months to 2 years) and adult experimental conditions. In the child small intestine, slightly less sporozoites were released from excystation compared to adult, however they exhibited a higher luciferase activity, suggesting a better physiological state. Sporozoites collected from the child jejunum compartment also showed a higher ability to invade human intestinal epithelial cells compared to the adult condition. Global analysis of the parasite transcriptome through RNA-sequencing demonstrated a more pronounced modulation in ileal effluents compared to gastric ones, albeit showing less susceptibility to age-related digestive condition. Further analysis of gene expression and enriched pathways showed that oocysts are highly active in protein synthesis in the stomach compartment, whereas sporozoites released in the ileum showed downregulation of glycolysis as well as strong modulation of genes potentially related to gliding motility and secreted effectors.ConclusionsDigestion in a sophisticated in vitro gastrointestinal model revealed that invasive sporozoite stages are released in the small intestine, and are highly abundant and active in the ileum compartment, supporting reported C. parvum tissue tropism. Our comparative analysis suggests that physicochemical parameters encountered in the child digestive environment can influence the amount, physiological state and possibly invasiveness of sporozoites released in the small intestine, thus potentially contributing to the higher susceptibility of young individuals to cryptosporidiosis.

Cryptosporidiosis is a poorly controlled zoonosis caused by an intestinal parasite, Cryptosporidium parvum, with a high prevalence in livestock (cattle, sheep, and goats). Young animals are particularly susceptible to this infection due to the immaturity of their intestinal immune system. In a neonatal mouse model, we previously demonstrated the importance of the innate immunity and particularly of type 1 conventional dendritic cells (cDC1) among mononuclear phagocytes (MPs) in controlling the acute phase of C. parvum infection. These immune populations are well described in mice and humans, but their fine characterization in the intestine of young ruminants remained to be further explored. Immune cells of the small intestinal Peyer's patches and of the distal jejunum were isolated from naive lambs and calves at different ages. This was followed by their fine characterization by flow cytometry and transcriptomic analyses (q-RT-PCR and single cell RNAseq (lamb cells)). Newborn animals were infected with C. parvum, clinical signs and parasite burden were quantified, and isolated MP cells were characterized by flow cytometry in comparison with age matched control animals. Here, we identified one population of macrophages and three subsets of cDC (cDC1, cDC2, and a minor cDC subset with migratory properties) in the intestine of lamb and calf by phenotypic and targeted gene expression analyses. Unsupervised single-cell transcriptomic analysis confirmed the identification of these four intestinal MP subpopulations in lamb, while highlighting a deeper diversity of cell subsets among monocytic and dendritic cells. We demonstrated a weak proportion of cDC1 in the intestine of highly susceptible newborn lambs together with an increase of these cells within the first days of life and in response to the infection. Considering cDC1 importance for efficient parasite control in the mouse model, one may speculate that the cDC1/cDC2 ratio plays also a key role for the efficient control of C. parvum in young ruminants. In this study, we established the first fine characterization of intestinal MP subsets in young lambs and calves providing new insights for comparative immunology of the intestinal MP system across species and for future investigations on host-Cryptosporidium interactions in target species.

Non-invasive markers of affective states can help understanding animals' perception of situations and improving their welfare. These markers are scarce in avian species. In this study, we investigate the potential relation between alterations in facial skin redness in hens and their corresponding affective states. Six hens were filmed in both naturally unfolding scenarios and controlled tests designed to elicit various affective states. The facial skin redness was measured from images extracted from the videos. Our observations revealed that hens exhibited the highest degree of facial skin redness in negative situations of high arousal, a high redness in positive situations of high arousal, and the lowest in positive situations of low arousal. In a second study, we further examined whether facial skin redness and secretory immunoglobulin A (S-IgA) can serve as markers for the quality of the human-animal relationship. Two groups of hens, one habituated to humans (n=13) and one non-habituated (n=12), were compared for general fearfulness in an open field test and for fear of humans in a reactivity to human test. In the open-field test, there were no statistical differences in general fearfulness, facial skin redness or S-IgA concentrations between both groups. However, habituated hens exhibited significantly lower fearfulness and facial skin redness in the presence of humans compared to non-habituated hens in the reactivity to human test. Additionally, habituated hens showed significant lower S-IgA concentration in lachrymal fluid in the presence of humans, with no significant differences in saliva or cloacal samples. We propose that changes in facial skin redness reflect variations in affective states and can be used as a marker for assessing the quality of the human-hen relationship. The relationship between S-IgA concentrations and affective states requires further investigation.

Bile represses Salmonella enterica serovar Typhimurium (S. Typhimurium) intestinal cell invasion, but it remains unclear which bile components and mechanisms are implicated. Previous studies reported that bile inhibits the RamR binding to the ramA promoter, resulting in ramA increased transcription, and that ramA overexpression is associated to decreased expression of type III secretion system 1 (TTSS-1) invasion genes and to impaired intestinal cell invasiveness in S. Typhimurium. In this study, we assessed the possible involvement of the ramRA multidrug efflux regulatory locus and individual bile salts in the bile-mediated repression of S. Typhimurium invasion, using Caco-2 intestinal epithelial cells and S. Typhimurium strain ATCC 14028s. Our results indicate that (i) major primary bile salts, chenodeoxycholate and its conjugated-derivative salts, cholate, and deoxycholate, activate ramA transcription in a RamR-dependent manner, and (ii) it results in repression of hilA, encoding the master activator of TTSS-1 genes, and as a consequence in the repression of cellular invasiveness. On the other hand, crude ox bile extract and cholate were also shown to repress the transcription of hilA independently of RamR, and to inhibit cell invasion independently of ramRA. Altogether, these data suggest that bile-mediated repression of S. Typhimurium invasion occurs through pleiotropic effects involving partly ramRA, as well as other unknown regulatory pathways. Bile components other than the bile salts used in this study might also participate in this phenomenon.

particular concern in East Asia. Among zoonotic pathogens, Streptococcus suis causes invasive infections in humans and pigs. Kerdsin et al. reported the genomic characterization of Streptococcus suis serotype 24 clonal complex 221/234 from human patients. The strains were analyzed and classified according to their antimicrobial resistance genes, pathotyping, virulence-associated gene profiles, and minimum core genome typing. Regarding pathogen transmission, and in particular transmission of tick-borne pathogens, in a hypothesis and theory article, Chang et al. discussed the role of ranged horses in the eco-epidemiology of Rickettsia raoultii infection in China. R. raoultii gene sequences were indeed detected in both horses and their ticks. In the field of epidemiology, a study of Lang et al., performed in hospitals from Seesen/Germany, reported risk factors of patients with diarrhea for having Clostridioides (Clostridium) difficile infection. Among significant factors identified were the use of diuretics and a diet poor in vegetables. The COVID-19 disease, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged at the end of 2019 and spread rapidly in human population all over the world becoming the first major pandemic of the 21th century, affecting hundreds of millions people and causing millions of deaths. Several variants have emerged in multiple successive waves during 3 years, leading health and governmental authorities to impose drastic prevention measures, such as lockdown for several months, a situation that was not known previously to the global population, and which resulted in critical economical situations in many countries.On the other hand, this pandemic and associated research has greatly contributed to increase our knowledge regarding all aspects of a viral infectious disease, and to develop rapidly new diagnostic, therapeutic, prevention and vaccine strategies.