A recurrent de novo missense mutation in COL1A1 causes osteogenesis imperfecta type II and preterm delivery in Normande cattle.

BACKGROUND: Nine male and eight female calves born to a Normande artificial insemination bull named "Ly" were referred to the French National Observatory of Bovine Abnormalities for multiple fractures, shortened gestation, and stillbirth or perinatal mortality. RESULTS: Using Illumina BovineSNP50 array genotypes from affected calves and 84 half-sib controls, the associated locus was mapped to a 6.5-Mb interval on chromosome 19, assuming autosomal inheritance with germline mosaicism. Subsequent comparison of the whole-genome sequences of one case and 5116 control genomes, followed by genotyping in the affected pedigree, identified a de novo missense substitution within the NC1 domain of the COL1A1 gene (Chr19 g.36,473,965G > A; p.D1412N) as unique candidate variant. Interestingly, the affected residue was completely conserved among 243 vertebrate orthologs, and the same substitution in humans has been reported to cause type II osteogenesis imperfecta (OI), a connective tissue disorder that is characterized primarily by bone deformity and fragility. Moreover, three COL1A1 mutations have been described to cause the same syndrome in cattle. Necropsy, computed tomography, radiology, and histology confirmed the diagnosis of type II OI, further supporting the causality of this variant. In addition, a detailed analysis of gestation length and perinatal mortality in 1387 offspring of Ly and more than 160,000 progeny of 63 control bulls allowed us to statistically confirm in a large pedigree the association between type II OI and preterm delivery, which is probably due to premature rupture of fetal membranes and has been reported in several isolated cases of type II OI in humans and cattle. Finally, analysis of perinatal mortality rates and segregation distortion supported a low level of germ cell mosaicism in Ly, with an estimate of 4.5% to 7.7% of mutant sperm and thus 63 to 107 affected calves born. These numbers contrast with the 17 cases reported and raise concerns about the underreporting of congenital defects to heredo-surveillance platforms, even for textbook genetic syndromes. CONCLUSIONS: In conclusion, we describe a large animal model for a recurrent substitution in COL1A1 that is responsible for type II OI in humans. More generally, this study highlights the utility of such datasets and large half-sib families available in livestock species to characterize sporadic genetic defects.

New insights into the genetic resistance to paratuberculosis in Holstein cattle via single-step genomic evaluation.

BACKGROUND: Bovine paratuberculosis, or Johne's disease (JD), is a contagious and incurable disease caused by Mycobacterium avium subsp. paratuberculosis (MAP). It has adverse effects on animal welfare and is very difficult to control, leading to serious economic consequences. An important line of defense to this disease is host genetic resistance to MAP, which, when it will be more fully understood, could be improved through selective breeding. Using a large dataset of Holstein cows (161,253 animals including 56,766 cows with ELISA serological phenotypes and 12,431 animals with genotypes), we applied a single-step single nucleotide polymorphism (SNP) best linear unbiased prediction approach to investigate the genetic determinism underlying resistance to this disease (heritability estimate and identification of relevant genomic regions) and estimated genetic trends, reliability, and relative risk factors associated with genomic predictions. RESULTS: Resistance to JD was moderately heritable (0.14) and 16 genomic regions were detected that accounted for at least 0.05% of the breeding values variance (GV) in resistance to JD, and were located on chromosomes 1, 3, 5, 6, 7, 19, 20, 21, 23, 25, and 27, with the highest percentage of variance explained by regions on chromosomes 23 (0.36% GV), 5 (0.22% GV), 1 (0.14% GV), and 3 (0.13% GV). When estimated for the whole chromosomes, the autosomes with the largest overall contributions were chromosomes 3 (5.3% GV), 10 (4.8%), 23 (4.7%), 1 (3.6%), 7 (3.4%), 5 (2.9%), 12 (2.5%), 11 (2.2%), and 13 (2%). We estimated a slightly favorable genetic trend in resistance to JD over the last two decades, which can be explained by a low positive genetic correlation between resistance to JD and total merit index (+ 0.06). Finally, in a validation population of 907 cows, relatively reliable genomic predictions (reliability = 0.55) were obtained, which allowed the identification of cows at high risk of infection. CONCLUSIONS: This study provides new insights into the genetic determinism of resistance to JD and shows that this trait can be predicted from SNP genotypes. It has led to the implementation of a single-step genomic evaluation that should rapidly become an effective tool for controlling paratuberculosis on French Holstein farms.

Progressive ataxia of Charolais cattle highlights a role of KIF1C in sustainable myelination.

Hereditary spastic paraplegias (HSPs) are clinically and genetically heterogeneous human neurodegenerative diseases. Amongst the identified genetic causes, mutations in genes encoding motor proteins such as kinesins have been involved in various HSP clinical isoforms. Mutations in KIF1C are responsible for autosomal recessive spastic paraplegia type 58 (SPG58) and spastic ataxia 2 (SPAX2). Bovines also develop neurodegenerative diseases, some of them having a genetic aetiology. Bovine progressive ataxia was first described in the Charolais breed in the early 1970s in England and further cases in this breed were subsequently reported worldwide. We can now report that progressive ataxia of Charolais cattle results from a homozygous single nucleotide polymorphism in the coding region of the KIF1C gene. In this study, we show that the mutation at the heterozygous state is associated with a better score for muscular development, explaining its balancing selection for several decades, and the resulting high frequency (13%) of the allele in the French Charolais breed. We demonstrate that the KIF1C bovine mutation leads to a functional knock-out, therefore mimicking mutations in humans affected by SPG58/SPAX2. The functional consequences of KIF1C loss of function in cattle were also histologically reevaluated. We showed by an immunochemistry approach that demyelinating plaques were due to altered oligodendrocyte membrane protrusion, and we highlight an abnormal accumulation of actin in the core of demyelinating plaques, which is normally concentrated at the leading edge of oligodendrocytes during axon wrapping. We also observed that the lesions were associated with abnormal extension of paranodal sections. Moreover, this model highlights the role of KIF1C protein in preserving the structural integrity and function of myelin, since the clinical signs and lesions arise in young-adult Charolais cattle. Finally, this model provides useful information for SPG58/SPAX2 disease and other demyelinating lesions.

Identification of the ABCC4, IER3, and CBFA2T2 candidate genes for resistance to paratuberculosis from sequence-based GWAS in Holstein and Normande dairy cattle.

BACKGROUND: Bovine paratuberculosis is a contagious disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), with adverse effects on animal welfare and serious economic consequences. Published results on host genetic resistance to MAP are inconsistent, mainly because of difficulties in characterizing the infection status of cows. The objectives of this study were to identify quantitative trait loci (QTL) for resistance to MAP in Holstein and Normande cows with an accurately defined status for MAP. RESULTS: From MAP-infected herds, cows without clinical signs of disease were subjected to at least four repeated serum ELISA and fecal PCR tests over time to determine both infected and non-infected statuses. Clinical cases were confirmed using PCR. Only cows that had concordant results for all tests were included in further analyses. Positive and control cows were matched within herd according to their birth date to ensure a same level of exposure to MAP. Cows with accurate phenotypes, i.e. unaffected (control) or affected (clinical or non-clinical cases), were genotyped with the Illumina BovineSNP50 BeadChip. Genotypes were imputed to whole-genome sequences using the 1000 Bull Genomes reference population (run6). A genome-wide association study (GWAS) of MAP status of 1644 Holstein and 649 Normande cows, using either two (controls versus cases) or three classes of phenotype (controls, non-clinical and clinical cases), revealed three regions, on Bos taurus (BTA) chromosomes 12, 13, and 23, presenting significant effects in Holstein cows, while only one of those was identified in Normande cows (BTA23). The most significant effect was found on BTA13, in a short 8.5-kb region. Conditional analyses revealed that only one causal variant may be responsible for the effects observed on each chromosome with the ABCC4 (BTA12), CBFA2T2 (BTA13), and IER3 (BTA23) genes as good functional candidates. CONCLUSIONS: A sequence-based GWAS on cows for which resistance to MAP was accurately defined, was able to identify candidate variants located in genes that were functionally related to resistance to MAP; these explained up to 28% of the genetic variance of the trait. These results are very encouraging for efforts towards implementation of a breeding strategy aimed at improving resistance to paratuberculosis in Holstein cows.

Bovine and murine models highlight novel roles for SLC25A46 in mitochondrial dynamics and metabolism, with implications for human and animal health.

Neuropathies are neurodegenerative diseases affecting humans and other mammals. Many genetic causes have been identified so far, including mutations of genes encoding proteins involved in mitochondrial dynamics. Recently, the "Turning calves syndrome", a novel sensorimotor polyneuropathy was described in the French Rouge-des-Prés cattle breed. In the present study, we determined that this hereditary disease resulted from a single nucleotide substitution in SLC25A46, a gene encoding a protein of the mitochondrial carrier family. This mutation caused an apparent damaging amino-acid substitution. To better understand the function of this protein, we knocked out the Slc25a46 gene in a mouse model. This alteration affected not only the nervous system but also altered general metabolism, resulting in premature mortality. Based on optic microscopy examination, electron microscopy and on biochemical, metabolic and proteomic analyses, we showed that the Slc25a46 disruption caused a fusion/fission imbalance and an abnormal mitochondrial architecture that disturbed mitochondrial metabolism. These data extended the range of phenotypes associated with Slc25a46 dysfunction. Moreover, this Slc25a46 knock-out mouse model should be useful to further elucidate the role of SLC25A46 in mitochondrial dynamics.

Longitudinal study of Mycobacterium avium ssp. paratuberculosis fecal shedding patterns and concurrent serological patterns in naturally infected dairy cattle.

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiological agent of paratuberculosis, a disease that affects ruminants worldwide. Despite global interest in the control of this disease, gaps exist in our knowledge of fecal shedding patterns and concurrent serological patterns. This longitudinal study in dairy cattle herds with high MAP seroprevalence in France aimed at accurately describing fecal shedding patterns over 1 year; relating those shedding patterns to individual animal characteristics (age, breed, parity); and exploring the association between fecal shedding patterns and serological patterns. To describe temporal fecal shedding patterns and continuity of shedding, along with the standard quantitative PCR (qPCR) threshold cycle we used a cutoff value that related to low or nonculturable fecal shedding. We also defined a threshold cycle indicative of shedding in high quantities to describe infection progression patterns. Twenty-one herds completed the study, and 782 cows were tested 4 times each. We obtained 4 sets of paired fecal qPCR and serum ELISA results from 757 cows. Although we targeted highly likely infectious animals, we found a large diversity of shedding patterns, as well as high variability between herds in the proportion of animals showing a given pattern. The fecal qPCR results of almost 20% of the final study sample were positioned at least once in the range that indicated low or nonculturable fecal shedding (between the adjusted and the standard cutoff value). Although these animals would typically be classified as non-shedders, they could be important to infection dynamics on the farm. Animals that shed at least twice consecutively and animals that shed in high quantities rarely reverted to negativity. Repeated fecal qPCR can be used to detect temporal fecal shedding traits, and the decision to cull an animal could practically be based on temporal, semiquantitative results. Overall, we found a mismatch between fecal shedding and ELISA seropositivity (637 animals were ELISA-negative 4 times, but only 13% of those animals were qPCR-negative 4 times). We found that having more than 2 ELISA-positive samples was strongly related to persistent and continuous shedding. We suggest that although serological testing is much less sensitive than qPCR, it can also be used, particularly over the course of multiple testing events, to identify animals that are most likely to contribute to the contamination of the farm environment.

Which phenotypic traits of resistance should be improved in cattle to control paratuberculosis dynamics in a dairy herd: a modelling approach.

Paratuberculosis is a worldwide disease causing production losses in dairy cattle herds. Variability of cattle response to exposure to Mycobacterium avium subsp. paratuberculosis (Map) has been highlighted. Such individual variability could influence Map spread at larger scale. Cattle resistance to paratuberculosis has been shown to be heritable, suggesting genetic selection could enhance disease control. Our objective was to identify which phenotypic traits characterising the individual course of infection influence Map spread in a dairy cattle herd. We used a stochastic mechanistic model. Resistance consisted in the ability to prevent infection and the ability to cope with infection. We assessed the effect of varying (alone and combined) fourteen phenotypic traits characterising the infection course. We calculated four model outputs 25 years after Map introduction in a naïve herd: cumulative incidence, infection persistence, and prevalence of infected and affected animals. A cluster analysis identified influential phenotypes of cattle resistance. An ANOVA quantified the contribution of traits to model output variance. Four phenotypic traits strongly influenced Map spread: the decay in susceptibility with age (the most effective), the quantity of Map shed in faeces by high shedders, the incubation period duration, and the required infectious dose. Interactions contributed up to 12% of output variance, highlighting the expected added-value of improving several traits simultaneously. Combinations of the four most influential traits decreased incidence to less than one newly infected animal per year in most scenarios. Future genetic selection should aim at improving simultaneously the most influential traits to reduce Map spread in cattle populations.

Effectiveness of collective treatments in the prevention and treatment of bovine digital dermatitis lesions: A systematic review.

The collective treatment (CT) of an affected herd is commonly advised to control bovine digital dermatitis (DD). Several CT are commercialized, frequently without major evidence supporting their effectiveness. The objective of this systematic review was to evaluate the published evidence that supports CT in the treatment and prevention of DD lesions in dairy herds. Across the evidence, the main limitations in the studies design were identified and the possible sources of inconsistency were investigated. An extensive literature search of publications through electronic databases and gray literature was conducted between July 2015 and January 2016. Studies that did not include an untreated or placebo control group were excluded from the review. The literature search and screening process identified 13 publications with 24 treatment trial comparisons and 18 prevention trial comparisons. The published evidence included studies mostly considered to have a low or unclear risk of bias. Descriptive analyses were performed according to the prevention and treatment outcomes, and case and success definitions were identified for each study and summarized in odds ratios (OR). Pairwise meta-analyses were conducted according to the prevention and treatment outcomes, comparing directly the intervention used in each study, and ignoring any other differences in the intervention characteristics. The results of the meta-analyses indicated a low degree of heterogeneity across the evidence for the prevention outcome [I2 = 0%, 95% CI: 0 to 37.2%, 95% prediction interval (PI): 0.72 to 1.74)] and a moderate degree for the treatment outcome (I2 = 25.3%, 95% CI: 0 to 63%, 95% PI: 0.39 to 3.73). Similarly, appraisal of the graphical L'Abbé plot suggested a considerable degree of heterogeneity across the evidence for the treatment outcome. For both outcomes, the frequent small sample sizes of the trials indicate imprecision across the included studies. Additionally, for the treatment and prevention outcomes, an asymmetric funnel plot suggested possible publication bias. The overall quality of the evidence, for both outcomes (prevention and treatment), was therefore considered to be low, indicating that the true effect of CT may be substantially different from that estimated across the included studies. Consequently, this review and meta-analysis does not support an association between the CT considered in the review and a beneficial effect in the prevention and treatment of DD lesions. The effectiveness of CT therefore remains uncertain, and the epidemiological circumstances in which it can be useful must be investigated. These findings highlight the importance of developing high quality, controlled trials to evaluate the effectiveness of CT for DD control.

A New Tool to Assess the Economic Impact of Q Fever on Dairy Cattle Farms.

To support farmers in their decisions related to Q fever, a dedicated economic assessment tool is developed. The present work describes the calculator, its economic rationale, and the supporting assumptions. The calculator integrates a yearly compartmental model to represent population dynamism and the main interactions between disorders linked to Q fever, especially reproductive disorders (abortion, retained foetal membranes, purulent vaginal discharge and endometritis, extra services, and calving-conception delays). The effects of the nontangible cost of the disease on human health, the welfare of the animals, and the workload of farmers were not integrated into the model. The model shows high-level sensitivity to the prevalence of Q fever in the herd prevaccination and to the costs of abortion and extra days of calving-conception intervals. Breakeven points, i.e., cost values that allow us to achieve positive vaccination benefits, are also reported. For herds with moderate or high prevalence rates of Q fever prevaccination (>30%), a vaccination benefit is observed. The vaccine should be considered a type of insurance in herds with low prevalence rates of Q fever prevaccination (≤20%). The calculator was developed to aid decision-making at the farm level, and no conclusion can be extrapolated as a generic trend based on the present work.

Efficacy and Safety of an Inactivated Phase I Coxiella burnetii Vaccine to Control Q Fever in Ruminants: A Systematic Review.

Q fever is a disease caused by Coxiella burnetii that affects many animal species and humans. In ruminants, the disease is responsible for several reproductive disorders (such as abortions, stillbirths, premature births, weak offspring, retained foetal membranes and infertility). An inactivated vaccine based on a phase I antigen of C. burnetii is available for cattle, goats and sheep. This review aims to summarise the scientific literature regarding the efficacy and safety of this vaccine to control the infection in these three domestic ruminant species. Forty-five publications and one experimental veterinary thesis reporting on experimental studies, case reports, mathematical modelling and intervention studies were selected according to the PRISMA guidelines. Although some studies lack control groups or statistical analyses, for all three species, published data show that vaccination often results in a reduction in abortions and an improvement in reproductive performance in comparison with absence of vaccination. There is also evidence, including in infected herds and animals, that vaccination is associated with a reduction in bacterial shedding, both in intensity and duration in comparison with absence of vaccination. For these reasons, in case of human outbreaks, vaccination is one of the pillars of control measures. Vaccination is generally well tolerated, despite the rare occurrence of mild, transient side-effects, such as hyperthermia and reduction in milk yield.

Coxiella burnetii and Reproductive Disorders in Cattle: A Systematic Review.

Coxiellosis or Q fever is an infectious zoonotic disease caused by the bacterium Coxiella burnetii. A systematic review using bibliographic research was carried out, and the focus was the relationship between C. burnetii infection and reproductive disorders in cattle [abortion/stillbirth/perinatal morality/weak calves (ASPW complex); retained foetal membranes (RFMs); metritis/endometritis; and infertility/sub-fertility]. The bibliographical search yielded 443 results from databases, but only 61 were deemed eligible. For each disorder, summary tables were prepared, and a scientific evidence score was calculated for each study based on four criteria to help assess the level of evidence for the impact of C. burnetii on the reproductive disorders assessed: type of publication (peer-reviewed or other); type of study (case-control/cohort or other); type of C. burnetii test (direct or indirect); and comparative statistical analysis (yes or no). In addition, summary tables also included information on the study population, country, authors and year of publication, key findings and an assessment of the evidence for an association. For the ASPW complex, RFMs, metritis/endometritis and infertility/sub-fertility, 43, 9, 8 and 19 studies provided data, respectively. On a scale of four, nearly 50% of all study citations had evidence scores of three or four. For ASPW, RFMs and infertility/sub-fertility, there is a significant body of evidence to support a deleterious role for Q fever. In contrast, for metritis/endometritis, the evidence is unclear. It is concluded that there is a substantial need for further research, particularly involving larger animal populations in more controlled settings. To provide more consistency, it is recommended that authors follow more precise definitions of reproductive parameters and more robust diagnostic methodologies.

Molecular Investigation of Small Ruminant Abortions Using a 10-Plex HRM-qPCR Technique: A Novel Approach in Routine Diagnostics.

The objective of this study was to apply and preliminarily evaluate a High-Resolution Melting (HRM) analysis technique coupled with qPCR, that allows the simultaneous detection of 10 different ruminant abortogenic pathogens, for investigating abortions in sheep and goats throughout Greece. A total of 264 ovine and caprine vaginal swabs were obtained the week following the abortion from aborted females and analyzed using a commercially available kit (ID Gene™ Ruminant Abortion Multiplex HRM, Innovative Diagnostics). Results indicated a high prevalence of Coxiella burnetii and Chlamydophila spp., which were detected in 48.9% and 42.4% of the vaginal swabs, respectively. Results for these most commonly detected pathogens were compared with those of a well-established commercial qPCR kit, with near-perfect agreement. Toxoplasma gondii, Salmonella spp., Brucella spp., Anaplasma phagocytophilum, Campylobacter fetus, and Neospora caninum were also identified, the two latter reported for the first time in the country in small ruminants. Mixed infections occurred in 35.6% of the animals examined. This technique allows for the simultaneous detection of many abortogenic pathogens in an accurate and cost-effective assay. Detection of uncommon or not previously reported pathogens in various cases indicates that their role in ovine and caprine abortions may be underestimated.

Massive detection of cryptic recessive genetic defects in dairy cattle mining millions of life histories.

BACKGROUND: Dairy cattle breeds are populations of limited effective size, subject to recurrent outbreaks of recessive defects that are commonly studied using positional cloning. However, this strategy, based on the observation of animals with characteristic features, may overlook a number of conditions, such as immune or metabolic genetic disorders, which may be confused with pathologies of environmental etiology. RESULTS: We present a data mining framework specifically designed to detect recessive defects in livestock that have been previously missed due to a lack of specific signs, incomplete penetrance, or incomplete linkage disequilibrium. This approach leverages the massive data generated by genomic selection. Its basic principle is to compare the observed and expected numbers of homozygotes for sliding haplotypes in animals with different life histories. Within three cattle breeds, we report 33 new loci responsible for increased risk of juvenile mortality and present a series of validations based on large-scale genotyping, clinical examination, and functional studies for candidate variants affecting the NOA1, RFC5, and ITGB7 genes. In particular, we describe disorders associated with NOA1 and RFC5 mutations for the first time in vertebrates. CONCLUSIONS: The discovery of these many new defects will help to characterize the genetic basis of inbreeding depression, while their management will improve animal welfare and reduce losses to the industry.

Precision Livestock Farming: What Does It Contain and What Are the Perspectives?

Precision Livestock Farming (PLF) describes the combined use of sensor technology, the related algorithms, interfaces, and applications in animal husbandry. PLF technology is used in all animal production systems and most extensively described in dairy farming. PLF is developing rapidly and is moving beyond health alarms towards an integrated decision-making system. It includes animal sensor and production data but also external data. Various applications have been proposed or are available commercially, only a part of which has been evaluated scientifically; the actual impact on animal health, production and welfare therefore remains largely unknown. Although some technology has been widely implemented (e.g., estrus detection and calving detection), other systems are adopted more slowly. PLF offers opportunities for the dairy sector through early disease detection, capturing animal-related information more objectively and consistently, predicting risks for animal health and welfare, increasing the efficiency of animal production and objectively determining animal affective states. Risks of increasing PLF usage include the dependency on the technology, changes in the human-animal relationship and changes in the public perception of dairy farming. Veterinarians will be highly affected by PLF in their professional life; they nevertheless must adapt to this and play an active role in further development of technology.

Impact of Meloxicam Administration in Cows Prior to Caesarean Section on the Efficacy of Passive Immunity Transfer in Calves.

The objective of this study was to assess in the Charolais cow−calf system, the benefit of meloxicam administered to cows prior to c-section to the efficacy of passive immune transfer to the newborn under the assumption that providing analgesia to the dam could lead to an earlier or longer colostrum intake. This study was performed in Burgundy, France in two veterinary private practices. Colostrum quality, delay between the end of the c-section and the first spontaneous colostrum suckling, and the 24 h after birth calf serum IgG content from cows treated 15 min prior to c-section with meloxicam subcutaneously (0.5 mg/kg) (n = 22) or without analgesia (n = 26) were compared. No significant differences were observed in the quality of the colostrum nor the delay between the end of the surgery and the first spontaneous colostrum suckling between treatment groups. However, the number of calves showing a better transfer of passive immunity (IgG content >15 g/L) was significantly higher (p = 0.023) among those originating from dams receiving meloxicam prior to c-section. This effect was notably observed in multiparous cows (p = 0.041). This study confirms that pre-emptive analgesia in cows prior to c-section benefits the calf through an improved colostrum intake that is of paramount importance for calf short- and long-term survival.

Genetic Features of Mycobacterium avium subsp. paratuberculosis Strains Circulating in the West of France Deciphered by Whole-Genome Sequencing.

Paratuberculosis is a chronic infection of the intestine, mainly the ileum, caused by Mycobacterium avium subsp. paratuberculosis in cattle and other ruminants. This enzootic disease is present worldwide and has a negative impact on the dairy cattle industry. For this subspecies, the current genotyping tools do not provide the needed resolution to investigate the genetic diversity of closely related strains. These limitations can be overcome by the application of whole-genome sequencing (WGS), particularly for clonal populations such as M. avium subsp. paratuberculosis. The purpose of the present study was to undertake a WGS analysis with a panel of 200 animal field M. avium subsp. paratuberculosis strains selected based on a previous large-scale longitudinal study of Prim'Holstein and Normande dairy breeds naturally infected with M. avium subsp. paratuberculosis in the West of France. The pangenome analysis revealed that M. avium subsp. paratuberculosis has a closed pangenome. The phylogeny, based on alignment of 2,786 nonhomoplasic single nucleotide polymorphisms (SNPs), showed that the strain population is structured into three clades independently of the cattle breed or geographic distribution. The increased resolution of phylogeny obtained by WGS confirmed the homoplasic nature of the markers variable-number tandem repeat (VNTR) and short sequence repeat (SSR) used for M. avium subsp. paratuberculosis genotyping. These phylogenetic data also revealed independent introductions of the different genotypes in two main waves since at least 2003. WGS applied to this sampling demonstrated the presence of mixed infections in herds and at the individual animal level. Collectively, the phylogeny results inferred with French isolates compared to M. avium subsp. paratuberculosis isolates from around the world suggest introductions of M. avium subsp. paratuberculosis genotypes through the animal trade. Relationships between genetic traits and epidemiological data can now be investigated to better understand transmission dynamics of the disease. IMPORTANCE Mycobacterium avium subsp. paratuberculosis causes Johne's disease in ruminants, which is present worldwide and has significant negative impacts on the dairy cattle industry and animal welfare. Prevention and control of M. avium subsp. paratuberculosis infection are hampered by knowledge gaps in strain virulence, genotype distribution, and transmission dynamics. This work has revealed new insights into M. avium subsp. paratuberculosis strains currently circulating in western France and how they are related to strains circulating globally. We applied whole-genome sequencing (WGS) to obtain comprehensive information on genome evolution and discrimination of closely related strains. This approach revealed the history of M. avium subsp. paratuberculosis infection in France, refined the pangenomic characteristics of M. avium subsp. paratuberculosis, and demonstrated the existence of mixed infection in animals. Finally, this study identified predominant genotypes, which allow a better understanding of disease transmission dynamics. This information will facilitate tracking of this pathogen on farms and across agricultural regions, thus informing transmission pathways and disease control points.

Risk of seropositivity to Coxiella burnetii in humans living in areas with endemically infected cattle: No way for specific prevention.

Q fever, a zoonotic disease caused by Coxiella burnetii, is endemic among cattle in Western France. However, studies assessing the risk of human infection in such areas are lacking to date, while they may provide information about key specific preventive actions which could be advised to the human populations living with or close to cattle. Therefore, we conducted a cross-sectional study in two departments of Western France during the 2017/18 winter in order to explore possible risk factors for seropositivity among two distinct populations, i) an occupational risk group, that is, the cattle farmers, and ii) the general adult population (approached by blood donors). Sera were collected in 176 cattle farmers and 347 blood donors respectively, and tested for phase I and II antibodies using an indirect immunofluorescence antibody assay. Each participant was asked to fill in a questionnaire containing socio-demographic characteristics, occupational and non-occupational risk exposure. Identified risk factors were: in the general population, working in contact with ruminants, comparatively to any other activity (OR = 4.41; 95% CI: [1.59-6.55]); among farmers, managing an itself infected cattle herd (OR = 3.20; 95% CI: [1.59-6.55]). No other controllable risk factor (lifestyle, outdoor activities, proximity to pets and livestock animals, occupational practices) was here evidenced. In areas with endemically infected cattle, human exposure to Coxiella burnetii is to some extent unavoidable. This strengthens the need for physicians' awareness of the symptoms of Q fever and the appropriateness of general biosecurity measures, especially among at-risk groups living there.

QTest: A new way to easily sample, store, and ship samples to perform Q fever PCR analysis on bulk tank milk.

Bulk tank milk (BTM) is an easy, inexpensive, and representative sample for detection of Coxiella burnetii infections (Q fever) in dairy herds using real-time PCR. Bulk tank milk PCR can be performed either for initial herd screening or for monitoring the effectiveness of preventive measures. However, one major limitation under field conditions is the need to deliver BTM samples in adequate condition (quickly, safely, and under refrigeration) to a qualified laboratory. In addition, sending non-inactivated biological samples via normal mail may be prohibited. We developed an innovative, easy, and accurate diagnostic tool (QTest) for Q fever to support veterinarians and farmers in overcoming these constraints. The farmer or veterinarian simply places some drops of BTM on a Whatman FTA Elute Micro Card (FTA card) and lets the card dry before mailing it to the laboratory. In a 2-step study, we tested the hypotheses that (1) BTM samples stored on FTA cards are stable over time and at different temperatures, and (2) PCR results obtained via FTA cards are consistent with those obtained from raw BTM samples. The stability of C. burnetii DNA in milk preserved on an FTA card was maintained for at least 29 d at room temperature or 37°C to mimic field conditions. In our field study, of the original 70 positive BTM samples (when tested on raw BTM just after sampling), 58 samples were positive (on either raw BTM or FTA card) by the time of the direct comparison study (10 to 14 d later). Of these 58 samples, 45 raw BTM samples still tested positive after aging, and 53 FTA card BTM samples tested positive, indicating that detection was higher using FTA cards (91.4%) than raw milk (77.6%). Therefore, with inactivation and shipping advantages, this technique facilitates an easier and more practical approach to diagnosis of Q fever at the herd level and would support Q fever control strategies, especially in countries lacking adequate and close laboratory facilities.

Draft Genome Sequences of 142 Mycobacterium avium subsp. paratuberculosis Strains Isolated from Naturally Infected Dairy Cattle.

Mycobacterium avium subsp. paratuberculosis is the etiological agent of Johne's disease in ruminants. Here, we report the annotated draft genome sequences of 142 M. avium subsp. paratuberculosis strains that were isolated from dairy cattle in France between 2014 and 2018. The genomes of these strains were sequenced using Illumina technology.

Atypical actinobacillosis affecting hind limbs and lungs in a single beef cattle herd.

Actinobacillosis usually is a sporadic infection that affects the tongue in cattle ("wooden tongue") with possible spread to the digestive tract. Two 4-year-old Rouge-des-Prés cows from a single French beef herd were referred for chronic (2-6 months) swelling and cutaneous nodules in the distal hind limbs. In addition to cutaneous signs, physical examination disclosed cachexia, lameness, lymphadenitis of the hind limbs, and pneumonia in both cows. Cytologic examination of direct skin smears was inconclusive, and no parasites were observed in examination of multiple skin scrapings. Histopathological examination of skin and lung biopsy specimens identified chronic, diffuse, severe pyogranulomatous dermatitis, associated with Splendore-Hoeppli phenomenon and intralesional Gram-negative bacteria. Cultures from skin, lymph nodes, and lungs (both cows were euthanized for welfare reasons) identified a Pasteurellaceae organism, confirmed as Actinobacillus lignieresii by partial sequencing of the rpoB gene. This report emphasizes that actinobacillosis can appear as a small outbreak in cattle with cutaneous and respiratory signs.