Effect of fatty acid-enriched black soldier fly larvae meal combined with chitinase on the metabolic processes of Nile tilapia.

The aim of this study is to determine to what extent the addition of chitinase to black soldier fly (BSF) larval meal enriched or not with long-chain PUFA (LC-PUFA) could improve growth, protein digestion processes and gut microbial composition in Nile tilapia. Two different types of BSF meal were produced, in which larvae were reared on substrates formulated with vegetable culture substrate (VGS) or marine fish offal substrate (FOS). The BSF raised on VGS was enriched in α-linolenic acid (ALA), while that raised on FOS was enriched in ALA + EPA + DHA. Six BSF-based diets, enriched or not with chitinase, were formulated and compared with a control diet based on fishmeal and fish oil (FMFO). Two doses (D) of chitinase from Aspergillus niger (2 g and 5 g/kg feed) were added to the BSF larval diets (VGD0 and FOD0) to obtain four additional diets: VGD2, VGD5, FOD2 and FOD5. After 53 d of feeding, results showed that the BSF/FOS-based diets induced feed utilisation, protein efficiency and digestibility, as well as growth comparable to the FMFO control diet, but better than the BSF/VGS-based diets. The supplementation of chitinase to BSF/FOS increased in fish intestine the relative abundance of beneficial microbiota such as those of the Bacillaceae family. The results showed that LC-PUFA-enriched BSF meal associated with chitinase could be used as an effective alternative to fishmeal in order to improve protein digestion processes, beneficial microbiota and ultimately fish growth rate.

Salinity significantly affects intestinal microbiota and gene expression in striped catfish juveniles.

In the present study, juvenile striped catfish (Pangasianodon hypophthalmus), a freshwater fish species, have been chronically exposed to a salinity gradient from freshwater to 20 psu (practical salinity unit) and were sampled at the beginning (D20) and the end (D34) of exposure. The results revealed that the intestinal microbial profile of striped catfish reared in freshwater conditions were dominated by the phyla Bacteroidetes, Firmicutes, Proteobacteria, and Verrucomicrobia. Alpha diversity measures (observed OTUs (operational taxonomic units), Shannon and Faith's PD (phylogenetic diversity)) showed a decreasing pattern as the salinities increased, except for the phylogenetic diversity at D34, which was showing an opposite trend. Furthermore, the beta diversity between groups was significantly different. Vibrio and Akkermansia genera were affected differentially with increasing salinity, the former being increased while the latter was decreased. The genus Sulfurospirillium was found predominantly in fish submitted to salinity treatments. Regarding the host response, the fish intestine likely contributed to osmoregulation by modifying the expression of osmoregulatory genes such as nka1a, nka1b, slc12a1, slc12a2, cftr, and aqp1, especially in fish exposed to 15 and 20 psu. The expression of heat shock proteins (hsp) hsp60, hsp70, and hsp90 was significantly increased in fish reared in 15 and 20 psu. On the other hand, the expression of pattern recognition receptors (PRRs) were inhibited in fish exposed to 20 psu at D20. In conclusion, the fish intestinal microbiota was significantly disrupted in salinities higher than 10 psu and these effects were proportional to the exposure time. In addition, the modifications of intestinal gene expression related to ion exchange and stressful responses may help the fish to adapt hyperosmotic environment. KEY POINTS: • It is the first study to provide detailed information on the gut microbiota of fish using the amplicon sequencing method. • Salinity environment significantly modified the intestinal microbiota of striped catfish. • Intestinal responses may help the fish adapt to hyperosmotic environment.

Analysis of the lung microbiota in dogs with Bordetella bronchiseptica infection and correlation with culture and quantitative polymerase chain reaction.

Infection with Bordetella bronchiseptica (Bb), a pathogen involved in canine infectious respiratory disease complex, can be confirmed using culture or qPCR. Studies about the canine lung microbiota (LM) are recent, sparse, and only one paper has been published in canine lung infection. In this study, we aimed to compare the LM between Bb infected and healthy dogs, and to correlate sequencing with culture and qPCR results. Twenty Bb infected dogs diagnosed either by qPCR and/or culture and 4 healthy dogs were included. qPCR for Mycoplasma cynos (Mc) were also available in 18 diseased and all healthy dogs. Sequencing results, obtained from bronchoalveolar lavage fluid after DNA extraction, PCR targeting the V1-V3 region of the 16S rDNA and sequencing, showed the presence of Bb in all diseased dogs, about half being co-infected with Mc. In diseased compared with healthy dogs, the ß-diversity changed (P = 0.0024); bacterial richness and α-diversity were lower (P = 0.012 and 0.0061), and bacterial load higher (P = 0.004). Bb qPCR classes and culture results correlated with the abundance of Bb (r = 0.71, P < 0.001 and r = 0.70, P = 0.0022). Mc qPCR classes also correlated with the abundance of Mc (r = 0.73, P < 0.001). Bb infection induced lung dysbiosis, characterized by high bacterial load, low richness and diversity and increased abundance of Bb, compared with healthy dogs. Sequencing results highly correlate with qPCR and culture results showing that sequencing can be reliable to identify microorganisms involved in lung infectious diseases.

Putative Lineage of Novel African Usutu Virus, Central Europe.

We characterized the complete genome of a putative novel Usutu virus (USUV) strain (Usutu-BONN) detected in a dead blackbird from Germany. Genomic analysis revealed several unique amino acid substitutions among the polyprotein gene. Phylogenetic analyses demonstrated that Usutu-BONN constitutes a putative novel African USUV lineage, which was probably recently introduced to central Europe.

Characterization of a novel circo-like virus in Aedes vexans mosquitoes from Germany: evidence for a new genus within the family Circoviridae.

Over recent decades, metagenomic studies have expanded the number of newly described, often unclassified, viruses within the family Circoviridae. Using broad-spectrum circovirus and cyclovirus PCRs, we characterized a novel circo-like virus in Aedes vexans mosquitoes from Germany whose main putative ORFs shared very low amino acid identity with those of previously characterized circoviruses and cycloviruses. Phylogenetic and genetic distance analysis revealed that this new virus species defined, together with previously described mosquito- and bat faeces-derived circo-like viruses, a different genus, tentatively called Krikovirus, within the family Circoviridae. We further demonstrated that viruses of the putative genus Krikovirus all shared a genomic organization that was unique among the family Circoviridae. Further investigations are needed to determine the host range, tissue tropism and transmission route(s). This report increases the current knowledge of the genetic diversity and evolution of the members of the family Circoviridae.

Natural intrauterine infection with Schmallenberg virus in malformed newborn calves.

We surveyed morphologic alterations in calves in Belgium that were naturally infected in utero by Schmallenberg virus (SBV) and born with deformities during January-March 2012. SBV-specific RNA was distributed unevenly in different tissues. Natural intrauterine SBV infection of calves might cause serious damage to the central nervous system and muscles.

Realistic microplastics harness bacterial presence and promote impairments in early zebrafish embryos: Behavioral, developmental, and transcriptomic approaches.

The plastisphere is a newly recognized ecosystem. However, its interaction with early life stages of aquatic vertebrates is a multifaceted issue that requires further research. This study investigated the involvement of bacteria in shaping realistic microplastics hazards in zebrafish Danio rerio embryos. Fish were exposed to bottle micro-fragments (FR) and textile micro-fibers (FI) of polyethylene terephthalate (5-15 µm), concomitant with Aeromonas salmonicida achromogenes challenge from 2h post-fertilization for 3 days. Egg chorion showed affinity for FR and FI, inducing earlier embryo hatching. However, this effect was masked by biofilm invasion. Fragments were more detrimental than fibers on developmental parameters, while bacterial presence compromised body length, eye, and yolk sac surface area. In a further finding, MPs alone increased locomotor activity in zebrafish larvae, without synergistic effect when combined with bacteria. Data showed that realistic MPs had no significant effects except for downregulated sod and cyp1a gene expression, whereas bacterial challenge inhibited larval potency for most of the evaluated mRNA levels (mpx (immune system), apoeb (lipid metabolism), nfkb and tfa (inflammation), cyp and sod (oxidative stress)). This study provides new insights into realistic microplastic effects under relevant conditions when combined with environmental pathogen within the first life stages of aquatic vertebrates.

Fibroblast activation protein is a cellular marker of fibrotic activity in canine idiopathic pulmonary fibrosis.

Canine idiopathic pulmonary fibrosis (CIPF) is a progressive fibrotic interstitial lung disease of unknown etiology, afflicting aging West Highland white terriers (WHWTs) and leading to progressive respiratory failure. Fibroblast activation protein (FAP), a protease overexpressed in many cancers, is upregulated in idiopathic pulmonary fibrosis in humans. The aim of this study was to investigate FAP as a marker of active fibrosis in lung biopsies from WHWTs affected with CIPF, as well as the potential of plasmatic FAP as a biomarker. After establishing a scoring system to evaluate the severity and activity of fibrosis on histopathological lung sections, anti-FAP immunohistochemistry was performed on healthy and CIPF samples. FAP expression was characterized using both visual and digital quantitative pathology software analyses and then correlated to fibrosis severity and activity. Levels of plasmatic FAP in WHWTs affected with CIPF were measured by enzyme-linked immunosorbent assay and compared with healthy dogs. Lung samples from 22 WHWTs affected with CIPF were collected. According to the fibrosis scoring system, they were classified as cases of mild (5), moderate (9) and severe (8) fibrosis and were attributed scores of fibrosis activity. Fifteen healthy lung samples were classified as non-fibrotic. Healthy lung samples were FAP-negative, whereas fibroblasts were FAP-positive in 20 CIPF samples. FAP immunohistochemical expression correlated mildly with fibrosis severity (p < 0.05; R 2 = 0.22) but highly with fibrosis activity scores (p < 0.001; R 2 = 0.68). Digital image analysis detected a higher percentage of FAP-positive cells in areas of active fibrosis (p < 0.001) and FAP-positive cells were distributed outside mature fibrosis lesions, clustered in active fibrosis areas or scattered within alveolar septa. On the other hand, plasmatic FAP was significantly lower in dogs affected with CIPF compared with healthy dogs (p < 0.01). In conclusion, this study provides a valuable histological scoring system to assess the severity and activity of fibrosis in CIPF. It demonstrates that FAP is a good cellular marker of fibrotic activity in CIPF, and thus constitutes a promising target to be exploited for diagnostic and therapeutic applications. Additionally, it suggests that plasmatic FAP, although non-specific, could be altered in CIPF.

Molecular detection and associated risk factors of Anaplasma marginale, A. ovis and A. platys in sheep from Algeria with evidence of the absence of A. phagocytophilum.

Anaplasma species are obligate intracellular rickettsial pathogens that cause significant diseases in animals and humans. Despite their importance, limited information on Anaplasma infections in Algeria has been published thus far. This study aimed to assess the infection rate, characterize Anaplasma species, and identify associated risk factors in selected sheep farms across Oum El Bouaghi region in Algeria. In 2018, we collected 417 blood samples from sheep (Ovis aries) and performed molecular characterization of Anaplasma species infecting these animals. This characterization involved the use of 16S rRNA, msp2, rpoB, and msp5 genes, which were analyzed through nested PCR, qPCR, cPCR, DNA sequencing, and subsequent phylogenetic analysis. Our findings revealed infection rates of 12.7 % for Anaplasma species detected, with Anaplasma ovis at 10.8 %, Anaplasma marginale at 1.7 %, and Anaplasma platys at 0.2 %. Interestingly, all tested animals were found negative for Anaplasma phagocytophilum. Statistical analyses, including the Chi-square test and Fisher exact test, failed to establish any significant relationships (p > 0.05) between A. ovis and A. platys infections and variables such as age, sex, sampling season, and tick infestation level. However, A. marginale infection exhibited a significant association with age (p < 0.05), with a higher incidence observed in lambs (5.2 %) compared to other age groups. Remarkably, this study represents the first molecular detection of A. platys and A. marginale in Algerian sheep. These findings suggest that Algerian sheep may serve as potential reservoirs for these pathogens. This research contributes valuable insights into the prevalence and characteristics of Anaplasma infections in Algerian sheep populations, emphasizing the need for further investigation and enhanced surveillance to better understand and manage these diseases.

Recruited atypical Ly6G+ macrophages license alveolar regeneration after lung injury.

The lung is constantly exposed to airborne pathogens and particles that can cause alveolar damage. Hence, appropriate repair responses are essential for gas exchange and life. Here, we deciphered the spatiotemporal trajectory and function of an atypical population of macrophages after lung injury. Post-influenza A virus (IAV) infection, short-lived monocyte-derived Ly6G-expressing macrophages (Ly6G+ Macs) were recruited to the alveoli of lung perilesional areas. Ly6G+ Macs engulfed immune cells, exhibited a high metabolic potential, and clustered with alveolar type 2 epithelial cells (AT2s) in zones of active epithelial regeneration. Ly6G+ Macs were partially dependent on granulocyte-macrophage colony-stimulating factor and interleukin-4 receptor signaling and were essential for AT2-dependent alveolar regeneration. Similar macrophages were recruited in other models of injury and in the airspaces of lungs from patients with suspected pneumonia. This study identifies perilesional alveolar Ly6G+ Macs as a spatially restricted, short-lived macrophage subset promoting epithelial regeneration postinjury, thus representing an attractive therapeutic target for treating lung damage.

Microplastics alter development, behavior, and innate immunity responses following bacterial infection during zebrafish embryo-larval development.

Although the hazards of microplastics (MPs) have been explored, no complete data exists on the effect of MPs on the egg chorion. This study aims to evaluate the modification of immune responses, metabolism, and behavior of zebrafish larvae (Danio rerio) depending on the moment of exposure. Larvae were exposed to 5 µm polystyrene microbeads at a concentration of 0, 100, or 1000 µg/l, according to a specified times of exposure (0-4, 4-8, 0-8 days postfertilization (dpf)), followed by a bacterial challenge at 8 dpf. After every 4 and 8 dpf, swimming activity, gene expression related to oxidative stress and immune system responses were assessed. During embryonic development, larvae exposed to a concentration of 1000 µg/l MPs already showed a significantly reduced tail coiling frequency, yolk sac resorption and heartbeat. At 8 dpf, swimming activity was altered, even without ingestion and a few days after the end of MP exposure. Our results indicated a difference in immune system (nfkb, il1ß) and apoptosis (casp3a, bcl2) related gene expression depending on the timing of MP exposure, which highlighted a contrasting sensitivity according to the exposure time in MP studies. This study brings new insight into how MPs might affect zebrafish larvae health and development even without ingestion.

Anti-Schmallenberg Virus Activities of Type I/III Interferons-Induced Mx1 GTPases from Different Mammalian Species.

Mx proteins are key factors of the innate intracellular defense mechanisms that act against viruses induced by type I/III interferons. The family Peribunyaviridae includes many viruses of veterinary importance, either because infection results in clinical disease or because animals serve as reservoirs for arthropod vectors. According to the evolutionary arms race hypothesis, evolutionary pressures should have led to the selection of the most appropriate Mx1 antiviral isoforms to resist these infections. Although human, mouse, bat, rat, and cotton rat Mx isoforms have been shown to inhibit different members of the Peribunyaviridae, the possible antiviral function of the Mx isoforms from domestic animals against bunyaviral infections has, to our knowledge, never been studied. Herein, we investigated the anti-Schmallenberg virus activity of bovine, canine, equine, and porcine Mx1 proteins. We concluded that Mx1 has a strong, dose-dependent anti-Schmallenberg activity in these four mammalian species.

Osteopontin and fibronectin in lung tissue, serum, and bronchoalveolar lavage fluid of dogs with idiopathic pulmonary fibrosis and control dogs.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) affects West Highland white terriers (WHWTs). Osteopontin (SPP1) and fibronectin (FN1) are associated with human IPF and are overexpressed by bronchoalveolar lavage fluid (BALF) macrophages in dogs with IPF. OBJECTIVE: To investigate the value of these proteins as biomarkers of IPF. ANIMALS: West Highland white terriers (WHWTs) with IPF, control WHWTs, and terriers. METHODS: Cross-sectional observational study. Immunohistochemistry was used to localize SPP1 and FN1 in lung tissue. Serum and BALF SPP1 and FN1 concentrations were measured using canine ELISA kits and compared between groups. RESULTS: Osteopontin stained ciliated epithelial cells, smooth muscular cells, and macrophages of all included dogs, and type-II pneumocytes and extracellular matrix of all 12 diseased WHWTs, 4/6 control WHWTs, and none of the 3 terriers. Osteopontin serum concentration was higher in diseased WHWTs (n = 22; 2.15 ng/mL [0.74-5.30]) compared with control WHWTs (n = 13; 0.63 ng/mL [0.41-1.63]; P = .005) and terriers (n = 15; 0.31 ng/mL [0.19-0.51]; P < .0001), and in control WHWTs compared with terriers (P = .005). Osteopontin BALF concentrations were higher in diseased (0.27 ng/mL [0.14-0.43]) and control WHWTs (0.25 ng/mL [0.14-0.40]), compared with terriers (0.02 ng/mL [0.01-0.08]; P < .0001 and P = .003, respectively). Fibronectin (FN1) serum concentrations were lower in diseased dogs (1.03 ng/mL [0.35-1.48]) and control WHWTs (0.61 ng/mL [0.24-0.65]) compared with terriers (2.72 ng/mL [0.15-5.21]; P < .0001 and P = .0001, respectively). There was no difference in FN1 immunostaining and FN1 BALF concentrations between groups. CONCLUSIONS: Results suggest that SPP1 is involved in pathogenesis of IPF and could predispose that breed to the disease. Osteopontin serum concentration could serve as a diagnostic biomarker of IPF.

Tendon-Derived Mesenchymal Stem Cells (TDSCs) as an In Vitro Model for Virological Studies in Wild Birds.

The use of wild animals in research is complicated due to the capture and housing conditions, as well as to legal aspects, making it difficult to develop in vivo and in vitro models for the study of pathologies that affect these species. Here we validate an in vitro model of tendon-derived mesenchymal cells (TDSC) from Eurasian blackbird (Turdus merula) cadaveric samples. Through the expression of surface markers and the ability to differentiate into multiple lineages, the nature of the cells was confirmed. We then evaluated Mesenchymal Stem Cells (MSCs) as an infection model for the Usutu Flavivirus. To this aim, blackbird TDSCs were compared to Vero E6 cells, commonly used in Flavivirus studies. Both cells showed permissiveness to USUV infection as confirmed by immunocytochemistry. Moreover, TDSCs exhibited replication kinetics similar to, although slightly lower than, Vero E6, confirming these cells as a pertinent study model for the study of the pathogenesis of USUV. In this work, we isolated and characterized tendon-derived mesenchymal stem cells, which represent an interesting and convenient in vitro model for the study of wildlife species in laboratories.

Relationship between the Cycle Threshold Value (Ct) of a Salmonella spp. qPCR Performed on Feces and Clinical Signs and Outcome in Horses.

The objective of this retrospective study was to evaluate the clinical significance of fecal quantitative real-time polymerase chain reaction (qPCR) Salmonella results when taking the cycle threshold values (Ct) into account. The study included 120 Salmonella qPCR-positive fecal samples obtained from 88 hospitalized horses over a 2-year period. The mean Ct of the qPCR test was evaluated in regard to (1) clinical outcome and (2) systemic inflammatory response syndrome (SIRS) status (no SIRS, moderate SIRS, or severe SIRS) of the sampled horses. An ROC analysis was performed to establish the optimal cut-off Ct values associated with severe SIRS. The mean ± SD Ct value was significantly lower in samples (1) from horses with a fatal issue (27.87 ± 5.15 cycles) than in surviving horses (31.75 ± 3.60 cycles), and (2) from horses with severe SIRS (27.87 ± 2.78 cycles) than from horses with no (32.51 ± 3.59 cycles) or moderate (31.54 ± 3.02 cycles) SIRS. In the ROC analysis, the optimal cut-off value of Ct associated with a severe SIRS was 30.40 cycles, with an AUC value of 0.84 [95% confidence interval 0.76-0.91] and an OR of 0.64 [0.51-0.79]. Results suggest that including the Ct value in the interpretation of fecal qPCR results could improve the diagnostic value of this test for clinical salmonellosis in horses.

Correction: Rivas et al. Tendon-Derived Mesenchymal Stem Cells (TDSCs) as an In Vitro Model for Virological Studies in Wild Birds. Viruses 2023, 15, 1455.

In the original publication [...].

Schmallenberg virus in domestic cattle, Belgium, 2012.

To determine prevalence of antibodies against Schmallenberg virus in adult cows and proportion of infection transmitted to fetuses, we tested serum samples from 519 cow/calf pairs in Belgium in spring 2012. Of cattle within 250 km of location where the virus emerged, ≈91% tested positive for IgG targeting nucleoprotein. Risk for fetal infection was ≈28%.

Epizootic spread of Schmallenberg virus among wild cervids, Belgium, Fall 2011.

Schmallenberg virus was detected in cattle and sheep in northwestern Europe in 2011. To determine whether wild ruminants are also susceptible, we measured antibody seroprevalence in cervids (roe deer and red deer) in Belgium in 2010 and 2011. Findings indicated rapid spread among these deer since virus emergence ≈250 km away.