Blastocystis sp. is an intestinal protozoan with recognized zoonotic potential that is increasingly investigated in animals closely associated with humans or intended for food production. However, studies on mollusks remain limited, despite their high filtration capacity and ability to accumulate biological contaminants, making them valuable indicators of environmental pollution. Within a One Health framework, this study investigated the seasonal circulation of Blastocystis sp. in a coastal ecosystem in northern France through the analysis of mussels, cattle, and river water samples using real-time PCR. A total of 892 samples were analyzed, of which 14.5% tested positive. Mussels showed a prevalence of 22.7%, with marked seasonal variations suggesting fluctuations in environmental contamination. Cattle exhibited the highest prevalence (38.3%), while 18.8% of river water samples were contaminated. Sanger and next-generation sequencing of positive samples revealed substantial genetic diversity, with 14 subtypes (STs) identified in mussels, 8 in cattle, and 3 in water samples. The lack of host-specific STs, together with the low parasite loads assessed in mussels, suggests that these organisms act as passive accumulators rather than true hosts. Moreover, the broad ST diversity observed highlights ongoing circulation of Blastocystis sp. within this aquatic environment and widespread fecal contamination consistent with human, bovine, and avian sources. Overall, mussels appear to be effective bioindicators of multisource fecal pollution across interconnected ecosystems, emphasizing the potential risk of exposure to Blastocystis sp. through the handling or consumption of raw shellfish and the importance of strengthened environmental and public health surveillance.

Blastocystis sp. is the most commonly detected enteric protozoan in humans, yet its presence in broiler chickens remains insufficiently characterized despite its clear zoonotic potential. Here, we present the first survey conducted in France—and the largest to date in Europe—aimed at assessing the frequency, subtype (ST) and genotype distribution, and circulation patterns of Blastocystis sp. in broiler chicken production. A total of 967 cecal samples were collected at a single slaughterhouse from 66 farms representing three production systems (Label Rouge, standard, and certified). Blastocystis sp. was detected by real-time PCR in 49.7% of samples and in 83.3% of farms. Statistical analyses identified production system—reflecting differences in bird age and outdoor access—together with season, as major factors associated with parasite prevalence. Among mono-colonizations characterized by Sanger sequencing, ST7 was by far the most predominant ST, followed by ST6, reinforcing their avian origin and their capacity to co-circulate within farms. Genotyping revealed substantial genetic diversity within ST7 and moderate diversity within ST6. The dominance of specific ST7 and ST6 genotypes, potentially “adapted” to broiler chickens, suggests extensive chicken-to-chicken transmission, whereas rarer genotypes could reflect sporadic contamination from transient wild bird species that frequently harbor the parasite. Mixed colonizations involving at least two STs were detected in nearly 20% of positive animals. Next-generation sequencing of selected samples confirmed either co-colonization with ST6 and ST7 or the presence of highly divergent ST7 genotypes. Although avian STs are generally considered non-pathogenic in poultry, this is not the case in humans. Consequently, the active circulation of Blastocystis sp. in French broiler chickens highlights the potential significant zoonotic reservoir represented by these flocks. Transmission could occur through direct contact between poultry workers and birds, through ingestion of water contaminated with bird feces, or potentially via handling of contaminated carcasses—an understudied route that warrants further investigation.

The performance of dairy cows is influenced by the microbial communities hosted within their digestive tract. While the rumen microbiota has long been associated with host phenotypes, the impact of the faecal microbiota remains elusive. In this study, we collected 697 faecal samples from commercial Holstein cows and analysed them with 16S rRNA gene analyses. For each animal, routinely recorded data, i.e., milk yield, fat yield, protein yield, fat content, protein content, and an aggregate production trait (pINEL) based on the French economic dairy index, were available to assess the links between the faecal microbiota and host production. Our findings revealed a strong and significant association between the structure of the bacterial and prokaryote community (β-diversity) and dairy production. In addition, differential abundance analyses identified 48 genera whose abundances were significantly associated with pINEL, milk, fat and protein yield. Among these genera, the increased abundance of Bifidobacterium, and particularly an amplicon sequence variant with a 16S rRNA V3-V4 gene region identical to B. globosum and B. pseudolongum, was found to be the most important for high-yielding animals. Bifidobacterium seemed to be a potential key member of the bovine faecal microbiota that should be further investigated. Conversely, the p-1088-a5 gut group genus was found more abundant in low-productive cows. In conclusion, this study demonstrates significant associations between the faecal microbiota and the performance of dairy cows at the whole lactation scale. A better understanding of the physiology of the gut microbiota could help to improve dairy cow production.

Due to their potential impact on the host's phenotype, organ-specific microbiotas are receiving increasing attention in several animal species, including cattle. Specifically, the vaginal microbiota of ruminants is attracting growing interest, due to its predicted critical role on cows' reproductive functions in livestock contexts. Notably, fertility disorders represent a leading cause for culling, and additional research would help to fill relevant knowledge gaps. In the present study, we aimed to characterize the vaginal microbiota of a large cohort of 1171 female dairy cattle from 19 commercial herds in Northern France. Vaginal samples were collected using a swab and the composition of the microbiota was determined through 16S rRNA sequencing targeting the V3-V4 hypervariable regions. Initial analyses allowed us to define the core bacterial vaginal microbiota, comprising all the taxa observed in more than 90% of the animals. Consequently, four phyla, 16 families, 14 genera and a single amplicon sequence variant (ASV) met the criteria, suggesting a high diversity of bacterial vaginal microbiota within the studied population. This variability was partially attributed to various environmental factors such as the herd, sampling season, parity, and lactation stage. Next, we identified numerous significant associations between the diversity and composition of the vaginal microbiota and several traits related to host's production and reproduction performance, as well as reproductive tract health. Specifically, 169 genera were associated with at least one trait, with 69% of them significantly associated with multiple traits. Among these, the abundances of Negativibacillus and Ruminobacter were positively correlated with the cows' performances (i.e., longevity, production performances). Other genera showed mixed relationships with the phenotypes, such as Leptotrichia being overabundant in cows with improved fertility records and reproductive tract health, but also in cows with lower production levels. Overall, the numerous associations underscored the complex interactions between the vaginal microbiota and its host. Given the large number of samples collected from commercial farms and the diversity of the phenotypes considered, this study marks an initial step towards a better understanding of the intimate relationship between the vaginal microbiota and the dairy cow's phenotypes.

Blastocystis sp. is the most common single-celled eukaryote colonizing the human gastrointestinal tract worldwide. Because of the proven zoonotic potential of this protozoan, sustained research is therefore focused on identifying various reservoirs of transmission to humans, and in particular animal sources. Numerous groups of animals are considered to be such reservoirs due to their handling or consumption. However, some of them, including mollusks, remain underexplored. Therefore, a molecular epidemiological survey conducted in wild mussels was carried out in Northern France (Hauts-de-France region) to evaluate the frequency and subtypes (STs) distribution of Blastocystis sp. in these bivalve mollusks. For this purpose, 100 mussels (Mytilus edulis) were randomly collected in two sampling sites (Wimereux and Dannes) located in the vicinity of Boulogne-sur-Mer. The gills and gastrointestinal tract of each mussel were screened for the presence of Blastocystis sp. by real-time polymerase chain reaction (qPCR) assay followed by direct sequencing of positive PCR products and subtyping through phylogenetic analysis. In parallel, sequences of potential representative Blastocystis sp. isolates that were previously obtained from temporal surveys of seawater samples at marine stations offshore of Wimereux were integrated in the present analysis. By taking into account the qPCR results from all mussels, the overall prevalence of the parasite was shown to reach 62.0%. In total, more than 55% of the positive samples presented mixed infections. In the remaining mussel samples with a single sequence, various STs including ST3, ST7, ST14, ST23, ST26 and ST44 were reported with varying frequencies. Such distribution of STs coupled with the absence of a predominant ST specific to these bivalves strongly suggested that mussels might not be natural hosts of Blastocystis sp. and might rather be carriers of parasite isolates from both human and animal (bovid and birds) waste. These data from mussels together with the molecular identification of isolates from marine stations were subsequently discussed along with the local geographical context in order to clarify the circulation of this protozoan in this area. The identification of human and animal STs of Blastocystis sp. in mussels emphasized the active circulation of this protozoan in mollusks and suggested a significant environmental contamination of fecal origin. This study has provided new insights into the host/carrier range and transmission of Blastocystis sp. and emphasized its potential as an effective sentinel species for water quality and environmental contamination.

Cryptosporidium apicomplexan protozoa are ubiquitous intracellular agents affecting humans and animals. In particular, bovine cryptosporidiosis is recognized as endemic worldwide. However, epidemiological investigations remain limited in France regarding the burden of these parasites in cattle. To improve our understanding of the epidemiology of cryptosporidiosis, the main aim of this study was to determine the frequency and the genetic diversity of Cryptosporidium in adult Prim’Holstein dairy cattle farms in the north of France. Fecal specimens were collected from 1454 non-diarrheic and non-pregnant animals (nulli-, primi-, or multiparous) throughout 20 farms in an area of 110 km around Lille. For Cryptosporidium species identification, nested PCR followed by sequence and phylogenetic analyses were used. The overall frequency of Cryptosporidium spp. in-fection was 30.00% (C.I. 95%: 12.83–54.33) in farms and 0.89% (C.I. 95%: 0.498–1.57) at the individual level. In primi- or multiparous cows, only C. andersoni was found. C. ryanae, C. bovis/xiaoi and C. andersoni were detected in heifers. The phylogenetic tree confirmed that analyzed sequences were grouped with known reference sequences reported in dairy cattle. Further studies on the cumulative prevalence, risks factors and pathogenicity are needed to give a more accurate assessment of the impact of Cryptosporidium infection in dairy cattle in France.

The fecal microbiota of ruminants constitutes a diversified community that has been phenotypically associated with a variety of host phenotypes, such as production and health. To gain a better understanding of the complex and interconnected factors that drive the fecal bacterial community, we have aimed to estimate the genetic parameters of the diversity and composition of the fecal microbiota, including heritabilities, genetic correlations among taxa, and genetic correlations between fecal microbiota features and host phenotypes. To achieve this, we analyzed a large population of 1,875 Holstein cows originating from 144 French commercial herds and routinely recorded for production, somatic cell score, and fertility traits. Fecal samples were collected from the animals and subjected to 16S rRNA gene sequencing, with reads classified into Amplicon Sequence Variants (ASVs). The estimated α- and β-diversity indices (i.e., Observed Richness, Shannon index, Bray-Curtis and Jaccard dissimilarity matrices) and the abundances of ASVs, genera, families and phyla, normalized by centered-log ratio (CLR), were considered as phenotypes. Genetic parameters were calculated using either univariate or bivariate animal models. Heritabilities estimates, ranging from 0.08 to 0.31 for taxa abundances and β-diversity indices, highlight the influence of the host genetics on the composition of the fecal microbiota. Furthermore, genetic correlations estimated within the microbial community and between microbiota features and host traits reveal the complex networks linking all components of the fecal microbiota together and to their host, thus strengthening the holobiont concept. By estimating the heritabilities of microbiota-associated phenotypes, our study quantifies the impact of the host genetics on the fecal microbiota composition. In addition, genetic correlations between taxonomic groups and between taxa abundances and host performance suggest potential applications for selective breeding to improve host traits or promote a healthier microbiota.

IntroductionChronic diarrhoea is one of the most recurrent health issues in pig herds. Among Q9 the causative agents of diarrhoea, Lawsonia intracellularis is quite frequent with worldwide distribution leading to detrimental economical losses, as a result of the poor growth rate of the infected pigs. A growing body of evidence has shown that branched yeast b-glucans are excellent inducers of innate immunity memory in humans and in pigs.MethodsIn this study, yeast branched b-glucans were evaluated as a potential solution to control infection by L. intracellularis in pigs by reducing the shedding of L. intracellularis in the faeces of infected animals. To address this hypothesis, naturally infected grow finishing pigs were divided into two treatment groups (n=80) and fed either with a basal diet (Control) or the same basal diet supplemented with 250 g/ton b-glucans (BG-fed group).ResultsOur results have shown that BG-supplemented pigs during the growing and finishing period had significantly increased body weight (BW) by 4.22 kg on average on day 97 and ADG up to 5.6% over the whole experiment (p<5.0 x 10-2) with no significant increase in ADFI. Similarly, supplementation of pigs with BG during the grow-finish phase has significantly reduced (p<4.0x10-4) faecal shedding of the bacteria in infected pigs on day 97. Furthermore, our results indicate that microbial a-diversity was negatively correlated with the presence of L. intracellularis. Animals fed with yeast BG show a reduction of the two genera Sarcina and Catenibacterium and a deeper analysis indicated that the ratio of Fibrobacter to Lacnospiraceae_AC2044 group could be a potential marker for L. Intracellularis susceptibility.ConclusionThis study demonstrated the beneficial effect of adding branched yeast BG in the diet of grow-finish pigs in reducing the dissemination of L. intracellularis between pigs in a given herd.

Background Because of its potential influence on the host’s phenotype, increasing attention is paid to organ-specific microbiota in several animal species, including cattle. However, ecosystems other than those related to the digestive tract remain largely understudied. In particular, little is known about the vaginal microbiota of ruminants despite the importance of the reproductive functions of cows in a livestock context, where fertility disorders represent one of the primary reasons for culling.Results In the present study, we aimed at better characterizing the vaginal microbiota of dairy cows through 16S rRNA sequencing, using a large cohort of Holstein cows from Northern France. Our results allowed to define a core microbiota of the dairy cows’ vagina, and highlighted that 90% of the sequences belonged to the Firmicutes, the Proteobacteria, and the Bacteroidetes phyla. The core microbiota was composed of four phyla, 16 families, 14 genera and only one amplicon sequence variant (ASV), supporting the idea of the high diversity of vaginal microbiota within the studied population. This variability was partly explained by various environmental factors such as the herd, the sampling season, the lactation rank and the lactation stage. In addition, we investigated potential associations between the diversity and the composition of the vaginal microbiota and several health-, performance-, and fertility-related phenotypes. Our analyses highlighted significant associations between the α and β- diversities and several traits including the first insemination outcome, the productive longevity, and the culling. Besides, relevant phenotypes were correlated with the abundance of several genera, some of which, such as Leptotrichia , Streptobacillus , Methylobacterium-Methylorubrum , or Negativibacillus , were linked to multiple traits.Conclusion Considering the large number of samples, which were collected in commercial farms, and the diversity of the phenotypes considered, this study represents a first step towards a better understanding of the close relationship between the vaginal and the dairy cow’s phenotypes.### Competing Interest StatementLB, SD, GE, SMa, SMe and CA were employed by GD Biotech / Genes Diffusion company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.* AI : Artificial insemination ANCOM-BC : Analysis of compositions of microbiomes with bias correction ANOVA : Analysis of variance ASV : Amplicon sequence variant C-AIf : Calving to fertilizing insemination interval CI : Calving interval DADA2 : Divisive amplicon denoising algorithm 2 FIS : First insemination success OTU : Operation taxonomic variant PERMANOVA : Permutational multivariate analysis of variance

The basis of any animal experimentation begins with the housing of animals that should take into account the need for splitting animals into similar groups. Even if it is generally recommended to use the minimum number of animals necessary to obtain reliable and statistically significant results (3Rs rule), the allocation of animals is currently mostly based on randomness. Since variability in gut microbiota is an important confounding factor in animal experiments, the main objective of this study was to develop a new approach based on 16S rRNA gene sequencing analysis of the gut microbiota of animals participating in an experiment, in order to correctly assign the animals across batches. For this purpose, a pilot study was performed on 20 mouse faecal samples with the aim of establishing two groups of 10 mice as similar as possible in terms of their faecal microbiota fingerprinting assuming that this approach limits future analytical bias and ensures reproducibility. The suggested approach was challenged with previously published data from a third-party study. This new method allows to embrace the unavoidable microbiota variability between animals in order to limit artefacts and to provide an additional assurance for the reproducibility of animal experiments.

OprD mutant Pseudomonas aeruginosa (PA) is a strain that is resistant to carbapenems by loss of functional OprD porin and that shows increased pathogenicity towards the airway epithelium. To understand what could lead to the virulence rise and identify new potential therapeutic targets, we sought to determine essential genes (EGs) of OprD mutant PA14 by TnSeq (Transposon Sequencing). Transposon mutant libraries were constructed for PA14 WT and PA14∆oprD, inactivating in each bacterium a different gene from the entire chromosome. These libraries were then cultured in LB medium and bacterial DNA was extracted and sequenced at high throughput in single-read on Nextseq 500. EGs were determined by comparing different methods and analysis parameters with TRANSIT and FiTnEss software. The identified genes were annotated and the results for the two strains were compared. Specific genes of the OprD mutant strain were biologically verified by clean deletion and inducible transcriptional repression with CRISPRi/dCas9. A list of 510 GEs was determined for PA14∆oprD versus 609 GEs for PA14 WT, 86 genes were specific to the WT strain and 31 to the OprD mutant strain. After having annotated these 31 GEs, the focus was made on 2 involved in Krebs cycle precursors obtainment. One of these two genes showed a growth defect during CRISPRi/dCas9 induction in growth curves. The repression of this gene led to a 65% diminution of CFU compared to the control (p<0.05). Among the EGs found in the mutant strain linked to the loss of the OprD porin, one was confirmed to reduce growth in the mutant strain only, making it a new potential therapeutic target in the case of carbapenem-resistant infection.

In the fight against SARS-COV-2, the development of serological assays based on different antigenic domains represent a versatile tool to get a comprehensive picture of the immune response or differentiate infection from vaccination beyond simple diagnosis. Here we use a combination of the Nucleoprotein (NP), the Spike 1 (S1) and Spike 2 (S2) subunits, and the receptor binding domain (RBD) and N-terminal domain (NTD) of the Spike antigens from the CoViDiag® multiplex IgG assay, to follow the immune response to SARS-CoV-2 infection over a long time period and depending on disease severity. Using a panel of 209 sera collected from 61 patients up to eight months after infection, we observed that most patients develop an immune response against multiple viral epitope, but anti-S2 antibodies seemed to last longer. For all the tested IgGs, we have found higher responses for hospitalized patients than for non-hospitalized ones. Moreover the combination of the five different IgG responses increased the correlation to the neutralizing antibody titers than if considered individually. Multiplex immunoassays have the potential to improve diagnostic performances, especially for ancient infection or mild form of the disease presenting weaker antibody responses. Also the combined detection of anti-NP and anti-Spike-derived domains can be useful to differentiate vaccination from viral infection and accurately assess the antibody potential to neutralize the virus.

Despite the major impact of Blastocystis sp. in terms of prevalence in human and animal populations and the risk of zoonotic transmission, no epidemiological survey has yet been conducted in cattle herds in France. The aim of this study was thus to assess the prevalence and molecular diversity of Blastocystis sp. and associated factors in dairy cattle from the north of France. A total of 1581 fecal samples were collected from 1246 animals reared in 20 farms. Molecular detection of the protozoan was performed by real-time PCR and indicated an overall prevalence of Blastocystis sp. reaching 54.8% in the study population. Important inter-herd variation (from 22.2% to 76.5%) of Blastocystis sp. prevalence was also reported. Sequence analysis of 159 positive samples highlighted a very large predominance of ST10 (36/159) and ST14 (64/159), and ST2 was only found in 2 samples. Mixed subtype infections were common, representing 35.8% of sequenced samples (57/159). A putative correlation between Blastocystis sp. colonization and various animal and herd characteristics or feed intake was subsequently investigated. The protozoan was less prevalent in cows that have recently calved but Blastocystis sp. carriage was not significantly related to age. Blastocystis sp. colonization also decreased with high beet pulp and pasture grass consumption and increased with corn silage intake. Finally, the only significant association between Blastocystis sp. STs and animal and herd characteristics was the number of lactations of cows, with a predominance of ST14 in cows that calved once only.

In this article, we propose a new approach for the cow heat detection from endoscopic images. Our approach permits to identify on the fly the cow heat state through two successive stages, namely cervix detection then heat classification. For this purpose, images are analyzed by a Transformer based detection model to localize the cervix, in which case they are analyzed by a CNN-based heat classification model. The proposed approach permits to assist the farmer during the insemination operation by localizing the cervix in an accurate way. Moreover, the confidence level of the final decision of the classification model is increased by focusing its analysis only on cervix images. The effectiveness of our method is demonstrated on our generated dataset and the obtained performance outperform the state of the art.

Human gut microbial communities are mainly composed of bacteria, but also include fungi, viruses, archaea, and protozoa, whose role in the gut ecosystem has only recently begun to be recognized. For example, humans colonized by Blastocystis (a gut protozoan with controversial pathogenicity) host a more diverse bacterial microbiota than individuals not carrying it, suggesting that its presence may be beneficial for the host. In parallel, the presence of non-pathogenic Entamoeba spp. has been associated with an increased diversity and compositional shifts in the bacterial microbiota of healthy rural individuals in Cameroon. However, Entamoeba and Blastocystis, the two most prevalent human gut protozoa, have never been studied in the same individuals, preventing the study of their interaction. As Blastocystis is one of the few gut protozoa commonly found in industrialized populations, which are otherwise mostly devoid of gut eukaryotes, we need to focus on rural "traditional" populations, who harbor a higher diversity of gut eukaryotes (whether pathogenic or commensal) in order to study protozoa interactions in the gut ecosystem. To this end, we profiled the gut bacterial microbiota of 134 healthy Cameroonian adults using 16S rRNA gene amplicon sequencing data. Entamoeba and Blastocystis presence and co-occurrence pattern in the same individuals were determined using metagenomic shotgun data. We found that, when taking into account both protozoa jointly, Blastocystis was associated with both a higher richness and a higher evenness of the gut bacterial microbiota, while Entamoeba was associated only with a higher richness. We demonstrated a cumulative influence of these protozoa on bacterial microbiome diversity. Furthermore, while the abundance of several common taxa (for example, Ruminococcaceae, Coprococcus and Butyrivibrio) varied according to Blastocystis colonization, only a single Bacteroides amplicon sequence variant was found to be differentially abundant between Entamoeba-negative and Entamoeba-positive samples. Given the specific signature of each protozoan on the gut microbiota and the seemingly stronger association for Blastocystis, our results suggest that Blastocystis and Entamoeba interact with gut bacteria each in its own way, but experimental studies are needed to explore the precise mechanisms of these interactions.

In cattle farming, the artificial insemination technique is a biotechnology that brings to farmers a wide range of benefits namely health security, genetic gain and economic costs. The main condition for the success of artificial insemination within cattle is the heat (or estrus) detection. In this context, several cow heat detection systems have been recently proposed in the literature to assist the farmer in this task. Nevertheless, they are mainly based on the analysis of the physical behavior of the cow which may be affected by several factors related to its health and its environment. In this paper, we present a new vision system for cow heat detection which is based on the analysis of the genital tract of the cow. The main core of our system is a CNN model that has been designed and tailored for analyzing endoscopic images collected using an innovative insemination technology named Eye breed. The conducted experiments on two datasets namely our own dataset and a public dataset show the high accuracy of our CNN model (more than 97% for both datasets) outperforming 19 methods from the state of the art. Moreover, we propose an optimized version of our model for an Android deployment by exploiting several techniques namely quantization, GPU acceleration and video downsampling. The conducted tests on a smart-phone shows that our heat detection system has a response time of a few seconds.