The caecal microbiota promotes the acute inflammatory response and the loss of the intestinal barrier integrity during severe Eimeria tenella infection.

Introduction: Coccidiosis, a disease caused by intestinal apicomplexan parasites Eimeria, is a threat to poultry production. Eimeria tenella is one of the most pathogenic species, frequently causing a high prevalence of opportunistic infections. Objective: The objective of this study is to investigate the role of the microbiota in the pathogenesis of severe Eimeria tenella infection. Methods: We have previously shown that microbiota can promote parasite development. To study the effect of the microbiota on the pathogenesis of this infection, we used an experimental condition (inoculum of 10 000 oocysts E. tenella INRAE) in which the parasite load is similar between germ-free and conventional broilers at 7 days post-infection (pi). Thirteen conventional and 24 germ-free chickens were infected. Among this latter group, 12 remained germ-free and 12 received a microbiota from conventional healthy chickens at 4 days pi. Caeca and spleens were collected at 7 days pi. Results: Our results demonstrated caecal lesions and epithelium damage in conventional chickens at 7 days pi but not in germ-free infected chickens. Administration of conventional microbiota to germ-free chickens partially restored these deleterious effects. At day 7 pi, both infected conventional and germ-free chickens exhibited increased gene expression of inflammatory mediators, including IL15, IFNγ, TNFα and the anti-inflammatory mediator SOCS1, whereas the inflammatory mediators CXCLi2, CCL20, IL18, CSF1, NOS2, PTGS2, IL1ß, IL6, the receptor CCR2, and the anti-inflammatory mediators TGFß1 and IL10 were upregulated only in infected conventional chickens. Notably, the IL18, PTGS2 gene expression was significantly higher in the infected conventional group. Overall, the inflammatory response enhanced by the microbiota might be in part responsible for higher lesion scores. Epithelial tight junction protein gene expression analysis revealed a significant upregulation of CLDN1 with the infection and microbiota, indicating a potential loss of the intestinal barrier integrity. Conclusion: These observations imply that, during E. tenella infection, the caecal microbiota could trigger an acute inflammatory response, resulting in a loss of intestinal integrity. Increase in bacterial translocation can then lead to the likelihood of opportunistic infections. Hence, modulating the microbiota may offer a promising strategy for improving poultry gut health and limiting caecal coccidiosis.

Oral Administration of Valganciclovir Reduces Clinical Signs, Virus Shedding and Cell-Associated Viremia in Ponies Experimentally Infected with the Equid Herpesvirus-1 C2254 Variant.

Equid alphaherpesvirus-1 (EHV-1) is one of the main pathogens in horses, responsible for respiratory diseases, ocular diseases, abortions, neonatal foal death and neurological complications such as equine herpesvirus myeloencephalopathy (EHM). Current vaccines reduce the excretion and dissemination of the virus and, therefore, the extent of an epizooty. While their efficacy against EHV-1-induced abortion in pregnant mares and the decreased occurrence of an abortion storm in the field have been reported, their potential efficacy against the neurological form of disease remains undocumented. No antiviral treatment against EHV-1 is marketed and recommended to date. This study aimed to measure the protection induced by valganciclovir (VGCV), the prodrug of ganciclovir, in Welsh mountain ponies experimentally infected with an EHV-1 ORF30-C2254 strain. Four ponies were administered VGCV immediately prior to experimental EHV-1 infection, while another four ponies received a placebo. The treatment consisted in 6.5 mg/kg body weight of valganciclovir administered orally three times the first day and twice daily for 13 days. Clinical signs of disease, virus shedding and viraemia were measured for up to 3 weeks. The severity of the cumulative clinical score was significantly reduced in the treated group when compared with the control group. Shedding of infectious EHV-1 was significantly reduced in the treated group when compared with the control group between Day + 1 (D + 1) and D + 12. Viraemia was significantly reduced in the treated group when compared with the control group. Seroconversion was measured in all the ponies included in the study, irrespective of the treatment received. Oral administration of valganciclovir induced no noticeable side effect but reduced clinical signs of disease, infectious virus shedding and viraemia in ponies experimentally infected with the EHV-1 C2254 variant.

Upregulation of gut cathepsin L during Eimeria tenella infection.

Coccidiosis is a disease caused by Eimeria, which represents the first parasitic disease in poultry farming. Among them, E. tenella is a virulent species which specifically colonizes the caecum. The inflammatory response to infection is associated to numerous host proteases including cysteine cathepsins that can be deleterious for tissue and innate immunity integrity. Here, germ-free and conventional chickens were used as models to find out whether the microbiota could modify the intestinal expression of host cysteine cathepsins during coccidiosis. The basal caecal peptidase activity primarily relies on host proteases rather than proteases from the commensal flora. While mRNA levels of E. tenella cathepsins B and L remained unchanged in germ-free and conventional broilers, an overall increase in endopeptidase activity of cysteine cathepsins was found in E. tenella-infected caeca in both experimental models (P < 0.005). A significant decrease in avian cystatin C transcription was also observed in infected conventional, but not in infected germ-free broilers. Despite an unchanged mRNA level of avian cathepsin L (CatL), its protein expression raised following infection, in parallel with an increased transcription of antimicrobial ß-defensins (AvBD1, AvBD2, AvBD4, AvBD6, and AvBD7). Taken together, data support that host CatL is post-translationally upregulated during E. tenella infection, and thus may be involved in the alteration of the gut proteolytic balance. Furthermore, CatL may participate to inflammation occurring during coccidiosis through its known ability to proteolytically inactivates up-regulated avian ß-defensins that are key molecules of innate immunity.

Identification of a New Equid Herpesvirus 1 DNA Polymerase (ORF30) Genotype with the Isolation of a C2254/H752 Strain in French Horses Showing no Major Impact on the Strain Behaviour.

Equid herpesvirus 1 is one of the most common viral pathogens in the horse population and is associated with respiratory disease, abortion and still-birth, neonatal death and neurological disease. A single point mutation in the DNA polymerase gene (ORF30: A2254G, N752D) has been widely associated with neuropathogenicity of strains, although this association has not been exclusive. This study describes the fortuitous isolation of a strain carrying a new genotype C2254 (H752) from an outbreak in France that lasted several weeks in 2018 and involved 82 horses, two of which showed neurological signs of disease. The strain was characterised as UL clade 10 using the equid herpesvirus 1 (EHV-1) multi-locus sequence typing (MLST) classification but has not been identified or isolated since 2018. The retrospective screening of EHV-1 strains collected between 2016 and 2018 did not reveal the presence of the C2254 mutation. When cultured in vitro, the C2254 EHV-1 strain induced a typical EHV-1 syncytium and cytopathic effect but no significant difference was observed when compared with A2254 and G2254 EHV-1 strains. An experimental infection was carried out on four Welsh mountain ponies to confirm the infectious nature of the C2254 strain. A rapid onset of marked respiratory disease lasting at least 2 weeks, with significant virus shedding and cell-associated viraemia, was observed. Finally, an in vitro antiviral assay using impedance measurement and viral load quantification was performed with three antiviral molecules (ganciclovir (GCV), aciclovir (ACV) and aphidicolin (APD)) on the newly isolated C2254 strain and two other A/G2254 field strains. The three strains showed similar sensitivity to ganciclovir and aphidicolin but both C2254 and A2254 strains were more sensitive to aciclovir than the G2254 strain, based on viral load measurement.

Production of Germ-Free Fast-Growing Broilers from a Commercial Line for Microbiota Studies.

Studies of the gut microbiota contribution to the host physiology and immunocompetence are facilitated by the availability of germ-free animal models, which are considered the gold standard. Nesting birds are ideal models for the production of germ-free animals since there is no need to raise their relatives under sterile conditions. Germ-free chickens are mainly generated from specific-pathogen-free (SPF) experimental lines, which are poorly representative of commercial chicken lines. The method proposed here allowed the production of germ-free chickens from the fast growing broiler line Ross PM3, commonly used by the poultry industry. Eggs were quickly collected after laying at a broiler breeder farm. They underwent a strict decontamination process from the collection to the introduction in a sterile egg hatching isolator. The chicks have been hatched and kept in these sterile isolators during the period necessary to control their sterility. Originally developed for an experimental SPF white leghorn line, the present protocol has been adapted not only to the Ross PM3 broiler line but also to quails. It therefore represents a robust and readily adaptable procedure to other poultry species and nesting birds of economic, biological or ecological relevance.

Gut microbiota composition before infection determines the Salmonella super- and low-shedder phenotypes in chicken.

Heterogeneity of infection and extreme shedding patterns are common features of animal infectious diseases. Individual hosts that are super-shedders are key targets for control strategies. Nevertheless, the mechanisms associated with the emergence of super-shedders remain largely unknown. During chicken salmonellosis, a high heterogeneity of infection is observed when animal-to-animal cross-contaminations and reinfections are reduced. We hypothesized that unlike super-shedders, low-shedders would be able to block the first Salmonella colonization thanks to a different gut microbiota. The present study demonstrates that (i) axenic and antibiotic-treated chicks are more prone to become super-shedders; (ii) super or low-shedder phenotypes can be acquired through microbiota transfer; (iii) specific gut microbiota taxonomic features determine whether the chicks develop a low- and super-shedder phenotype after Salmonella infection in isolator; (iv) partial protection can be conferred by inoculation of four commensal bacteria prior to Salmonella infection. This study demonstrates the key role plays by gut microbiota composition in the heterogeneity of infection and pave the way for developing predictive biomarkers and protective probiotics.

Genetic parameters of resistance to pasteurellosis using novel response traits in rabbits.

BACKGROUND: Pasteurellosis (Pasteurella infection) is one of the most common bacterial infections in rabbits on commercial farms and in laboratory facilities. Curative treatments using antibiotics are only partly efficient, with frequent relapses. Breeding rabbits for improved genetic resistance to pasteurellosis is a sustainable alternative approach. In this study, we infected 964 crossbred rabbits from six sire lines experimentally with Pasteurella multocida. After post-mortem examination and bacteriological analyses, abscess, bacteria, and resistance scores were derived for each rabbit based on the extent of lesions and bacterial dissemination in the body. This is the first study to use such an experimental design and response traits to measure resistance to pasteurellosis in a rabbit population. We investigated the genetic variation of these traits in order to identify potential selection criteria. We also estimated genetic correlations of resistance to pasteurellosis in the experimental population with traits that are under selection in the breeding populations (number of kits born alive and weaning weight). RESULTS: Heritability estimates for the novel response traits, abscess, bacteria, and resistance scores, ranged from 0.08 (± 0.05) to 0.16 (± 0.06). The resistance score showed very strong negative genetic correlation estimates with abscess (- 0.99 ± 0.05) and bacteria scores (- 0.98 ± 0.07). A very high positive genetic correlation of 0.99 ± 0.16 was estimated between abscess and bacteria scores. Estimates of genetic correlations of the resistance score with average daily gain traits for the first and second week after inoculation were 0.98 (± 0.06) and 0.70 (± 0.14), respectively. Estimates of genetic correlations of the disease-related traits with average daily gain pre-inoculation were favorable but with high standard errors. Estimates of genetic and phenotypic correlations of the disease-related traits with commercial selection traits were not significantly different from zero. CONCLUSIONS: Disease response traits are heritable and are highly correlated with each other, but do not show any significant genetic correlations with commercial selection traits. Thus, the prevalence of pasteurellosis could be decreased by selecting more resistant rabbits on any one of the disease response traits with a limited impact on the selection traits, which would allow implementation of a breeding program to improve resistance to pasteurellosis in rabbits.

The antibody response induced FMDV vaccines in sheep correlates with early transcriptomic responses in blood.

Foot and mouth disease (FMD) is a highly contagious viral disease with high economic impact, representing a major threat for cloven-hooved mammals worldwide. Vaccines based on adjuvanted inactivated virus (iFMDV) induce effective protective immunity implicating antibody (Ab) responses. To reduce the biosafety constraints of the manufacturing process, a non-replicative human adenovirus type 5 vector encoding FMDV antigens (Ad5-FMDV) has been developed. Here we compared the immunogenicity of iFMDV and Ad5-FMDV with and without the ISA206VG emulsion-type adjuvant in sheep. Contrasted Ab responses were obtained: iFMDV induced the highest Ab levels, Ad5-FMDV the lowest ones, and ISA206VG increased the Ad5-FMDV-induced Ab responses to protective levels. Each vaccine generated heterogeneous Ab responses, with high and low responders, the latter being considered as obstacles to vaccine effectiveness. A transcriptomic study on total blood responses at 24 h post-vaccination revealed several blood gene module activities correlating with long-term Ab responses. Downmodulation of T cell modules' activities correlated with high responses to iFMDV and to Ad5-FMDV+ISA206VG vaccines as also found in other systems vaccinology studies in humans and sheep. The impact of cell cycle activity depended on the vaccine types, as it positively correlated with higher responses to iFMDV but negatively to non-adjuvanted Ad5-FMDV. Finally an elevated B cell activity at 24 h correlated with high Ab responses to the Ad5-FMDV+ISA206VG vaccine. This study provides insights into the early mechanisms driving the Ab response induced by different vaccine regimens including Ad5 vectors and points to T cell modules as early biomarker candidates of different vaccine-type efficacy across species.

The Absence of Gut Microbiota Alters the Development of the Apicomplexan Parasite Eimeria tenella.

Coccidiosis is a widespread intestinal disease of poultry caused by a parasite of the genus Eimeria. Eimeria tenella, is one of the most virulent species that specifically colonizes the caeca, an organ which harbors a rich and complex microbiota. Our objective was to study the impact of the intestinal microbiota on parasite infection and development using an original model of germ-free broilers. We observed that germ-free chickens presented significantly much lower load of oocysts in caecal contents than conventional chickens. This decrease in parasite load was measurable in caecal tissue by RT-qPCR at early time points. Histological analysis revealed the presence of much less first (day 2pi) and second generation schizonts (day 3.5pi) in germ-free chickens than conventional chickens. Indeed, at day 3.5pi, second generation schizonts were respectively immature only in germ-free chickens suggesting a lengthening of the asexual phase of the parasite in the absence of microbiota. Accordingly to the consequence of this lengthening, a delay in specific gamete gene expressions, and a reduction of gamete detection by histological analysis in caeca of germ-free chickens were observed. These differences in parasite load might result from an initial reduction of the excystation efficiency of the parasite in the gut of germ-free chickens. However, as bile salts involved in the excystation step led to an even higher excystation efficiency in germ-free compared to conventional chickens, this result could not explain the difference in parasite load. Interestingly, when we shunted the excystation step in vivo by infecting chickens with sporozoites using the cloacal route of inoculation, parasite invasion was similar in germ-free and in conventional chickens but still resulted in significantly lower parasite load in germ-free chickens at day 7pi. Overall, these data highlighted that the absence of intestinal microbiota alters E. tenella replication. Strategies to modulate the microbiota and/or its metabolites could therefore be an alternative approach to limit the negative impact of coccidiosis in poultry.

Development and optimization of a hybridization technique to type the classical class I and class II B genes of the chicken MHC.

The classical class I and class II molecules of the major histocompatibility complex (MHC) play crucial roles in immune responses to infectious pathogens and vaccines as well as being important for autoimmunity, allergy, cancer and reproduction. These classical MHC genes are the most polymorphic known, with roughly 10,000 alleles in humans. In chickens, the MHC (also known as the BF-BL region) determines decisive resistance and susceptibility to infectious pathogens, but relatively few MHC alleles and haplotypes have been described in any detail. We describe a typing protocol for classical chicken class I (BF) and class II B (BLB) genes based on a hybridization method called reference strand-mediated conformational analysis (RSCA). We optimize the various steps, validate the analysis using well-characterized chicken MHC haplotypes, apply the system to type some experimental lines and discover a new chicken class I allele. This work establishes a basis for typing the MHC genes of chickens worldwide and provides an opportunity to correlate with microsatellite and with single nucleotide polymorphism (SNP) typing for approaches involving imputation.

Effects of gut microbiota transfer on emotional reactivity in Japanese quails (Coturnix japonica).

The interaction between the gut microbiota (GM) and the brain has led to the concept of the microbiota-gut-brain axis but data for birds remain scarce. We tested the hypothesis that colonization of germ-free chicks from a quail line selected for a high emotional reactivity (E+) with GM from a line with low emotional reactivity (E-) would reduce their emotional behaviour in comparison with germ-free chicks from an E+ line colonized with GM from the same E+ line. The GM composition analysis of both groups revealed a shift in terms of microbial diversity and richness between day 21 and day 35 and the GM of the two groups of quails were closer to each other at day 35 than at day 21 at a phylum level. Quails that received GM from the E- line expressed a lower emotional reactivity than quails colonized by GM from the E+ line in tonic immobility and novel environment tests carried out during the second week of age. This result was reversed in a second tonic immobility test and an open-field run 2 weeks later. These behavioural and GM modifications over time could be the consequence of the resilience of the GM to recover the equilibrium present in the E+ host, which is in part driven by the host genotype. This study shows for the first time that a GM transfer can influence emotional reactivity in Japanese quails, supporting the existence of a microbiota-gut-brain axis in this species of bird.

Melarsomine hydrochloride (Cymelarsan®) fails to cure horses with Trypanosoma equiperdum OVI parasites in their cerebrospinal fluid.

The aim of this study was to evaluate the ability of melarsomine hydrochloride (Cymelarsan®) to cure horses suffering from a nervous form of dourine, a sexually-transmitted disease caused by Trypanosoma equiperdum. The recently described experimental model for assessing drug efficacy against horse trypanosomosis allowed us to obtain eight horses (Welsh pony mares) infected by T. equiperdum with parasites in their cerebrospinal fluid. The Cymelarsan® treatment evaluated consisted of the daily administration of 0.5 mg/kg of Cymelarsan® over 7 days. Two control horses remained untreated, three horses received the treatment 36 days p.i. and three horses received the treatment 16 days p.i. Following treatment, we observed parasite clearance in blood, stabilization of rectal temperature and a relative improvement in the mean packed cell volume levels for all treated horses. However, live parasites were later observed again in the CSF of all treated horses. Our results indicate the inability of Cymelarsan® to reach Trypanozoon located in the central nervous system of infected horses and thus discourage the use of Cymelarsan® to treat animals suffering from a nervous form of dourine.

Validation of a new experimental model for assessing drug efficacy against infection with Trypanosoma equiperdum in horses.

Trypanosoma equiperdum, the causative agent of dourine, may affect the central nervous system, leading to neurological signs in infected horses. This location protects the parasite from most (if not all) existing chemotherapies. In this context, the OIE terrestrial code considers dourine as a non-treatable disease and imposes a stamping-out policy for affected animals before a country may achieve its dourine-free status. The use of practices as drastic as euthanasia remains controversial, but the lack of a suitable tool for studying a treatment's efficacy against dourine hampers the development of an alternative strategy for dourine infection management. The present study reports on the development of an experimental infection model for assessing drug efficacy against the nervous form of dourine. The model combines the infection of horses by Trypanosoma equiperdum and the search for trypanosomes in the cerebrospinal fluid (CSF) through an ultrasound-guided cervical sampling protocol. After a development phase involving four horses, we established an infection model that consists of inoculating 5 × 104T. equiperdum OVI parasites intravenously into adult Welsh mares (Equus caballus). To evaluate its efficacy, eight horses were infected according to this model. In all these animals, parasites were observed in the blood at 2 days post-inoculation (p.i.) and in CSF (12.5 ± 1.6 days p.i.) and seroconversion was detected (8.25 ± 0.5 days p.i.). All eight animals also developed fever (rectal temperature > 39 °C), low hematocrit (< 27%), and ventral edema (7.9 ± 2.0 days p.i.), together with other inconstant clinical signs such as edema of the vulva (six out of eight horses) or cutaneous plaques (three out of eight horses). This model provides a robust infection protocol that induces an acute trypanosome infection and that allows parasites to be detected in the CSF of infected horses within a period of time compatible with animal experimentation constraints. We conclude that this model constitutes a suitable tool for analyzing the efficacy of anti-Trypanosoma drugs and vaccines.

Absence of Gut Microbiota Reduces Emotional Reactivity in Japanese Quails (Coturnix japonica).

Background: Recent studies have demonstrated an effect of the gut microbiota on brain development and behavior leading to the concept of the microbiota-gut-brain axis. However, its effect on behavior in birds is unknown. The aim of the present study was to determine the effect of the absence of gut microbiota on emotional reactivity in birds by comparing germ-free (GF) quails to those colonized (COL) with gut microbiota. Material and Methods: From hatching, the quails of both groups GF (n = 36) and COL (n = 36) were reared in sterile isolators. The COL quails were colonized at day 2 by introducing a pool of droppings from conventional adult females into the drinking water and feed. The quails were weighed individually on day 2, 6, and 14. From day 8, emotional reactivity was assessed in each group in the isolators through several behavioral tests. Results: GF quails showed a considerable decrease in emotional reactivity demonstrated by spending less time in tonic immobility during the tonic immobility test (242 s ± 31 vs. 331 s ± 32, p ≤ 0.05), traveling a shorter distance (3,897 cm ± 242 vs. 4,827 cm ± 278, p ≤ 0.05) at a lower velocity (6.55 cm/s ± 0.4 vs. 8.1 cm/s ± 0.5, p ≤ 0.05) during the social separation test and spending more time near an object at the beginning of the novel object test (33.7 s ± 6.4 vs. 18.5 s ± 4.1, p ≤ 0.05). No difference in growth was found between the 2 groups. Conclusion: For the first time, this study demonstrates that the absence of gut microbiota reduces emotional reactivity in Japanese quails in situations of fear and social perturbation without influence on growth.

Local immunization impacts the response of dairy cows to Escherichia coli mastitis.

Current vaccines to Escherichia coli mastitis have shown some albeit limited efficacy. Their mode of action has not been documented, and immune responses protecting the mammary gland against E. coli are not completely understood. To improve our knowledge of mammary gland immune protection, cows immunized either intramuscularly or intramammarily with the E. coli P4 were submitted to a homologous mastitis challenge. A third group of mock-immunized cows serve as challenge controls. Local immunization modified favorably the course of infection, by improving bacterial clearance while limiting inflammation. Systemic clinical signs and reduction in milk secretion were also contained. This occurred with a modification of the cytokine profile, such as an increase in IFN-γ and a reduction in TNF-α concentrations in milk. Concentrations of IL-17A and IL-22 increased in milk at the onset of the inflammatory response and remained high up to the elimination of bacteria, but concentrations did not differ between groups. Accelerated bacteriological cure was not linked to an increase in the initial efficiency of phagocytosis in milk. Results support the idea that antibodies did not play a major role in the improvement, and that cell-mediated immunity is the key to understanding E. coli vaccine-induced protection of the mammary gland.

A High-Performance Multiplex Immunoassay for Serodiagnosis of Flavivirus-Associated Neurological Diseases in Horses.

West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV) are flaviviruses responsible for severe neuroinvasive infections in humans and horses. The confirmation of flavivirus infections is mostly based on rapid serological tests such as enzyme-linked immunosorbent assays (ELISAs). These tests suffer from poor specificity, mainly due to antigenic cross-reactivity among flavivirus members. Robust diagnosis therefore needs to be validated through virus neutralisation tests (VNTs) which are time-consuming and require BSL3 facilities. The flavivirus envelope (E) glycoprotein ectodomain is composed of three domains (D) named DI, DII, and DIII, with EDIII containing virus-specific epitopes. In order to improve the serological differentiation of flavivirus infections, the recombinant soluble ectodomain of WNV E (WNV.sE) and EDIIIs (rEDIIIs) of WNV, JEV, and TBEV were synthesised using the Drosophila S2 expression system. Purified antigens were covalently bonded to fluorescent beads. The microspheres coupled to WNV.sE or rEDIIIs were assayed with about 300 equine immune sera from natural and experimental flavivirus infections and 172 nonimmune equine sera as negative controls. rEDIII-coupled microspheres captured specific antibodies against WNV, TBEV, or JEV in positive horse sera. This innovative multiplex immunoassay is a powerful alternative to ELISAs and VNTs for veterinary diagnosis of flavivirus-related diseases.