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Abortion in cattle causes significant economic losses for cattle farmers worldwide. The diversity of abortifacients makes abortion diagnostics a complex and challenging discipline that additionally is restrained by time and economy. Microbial culture has traditionally been an important method for the identification of bacterial and mycotic abortifacients. However, it comes with the inherent bias of favoring the easy-to-culture species, e.g., those that do not require cell culture, pre-enrichment, a variety of selective growth media, or different oxygen levels for in vitro growth. Molecular methods such as polymerase chain reaction (PCR) and next-generation sequencing have been established as alternatives to traditional microbial culturing methods in several diagnostic fields including abortion diagnostics. Fluorescence in situ hybridization (FISH), a bridging microscopy technique that combines molecular accuracy with culture independence, and spatial resolution of the pathogen-lesion relation, is also gaining influence in several diagnostic fields. In this study, real-time quantitative PCR (qPCR), 16S rDNA amplicon sequencing, and FISH were applied separately and in combination in order to (i) identify potentially abortifacient bacteria without the bias of culturability, (ii) increase the diagnostic rate using combined molecular methods, (iii) investigate the presence of the difficult-to-culture zoonotic agents Coxiella burnetii, Chlamydia spp., and Leptospira spp. in bovine abortions in Denmark. Tissues from 162 aborted or stillborn bovine fetuses and placentas submitted for routine diagnostics were screened for pathogenic bacteria using 16S rDNA amplicon sequencing. Lesion association of fungal elements, as well as of selection of bacterial abortifacients, was assessed using specific FISH assays. The presence of Chlamydia spp. and chlamydia-like organisms was assessed using qPCR. The study focused on bacterial and fungal abortifacients, because Danish cattle is

Since the end of the ice age, spring migrating birds from Africa and Europe and autumn migrating birds from Northern Scandinavia have entered Denmark, and recently a small wave of long migrating carnivores have started arriving in Denmark from Central Europe. Theoretically, migrating birds could introduce new tick species as well as tick-associated pathogens to Denmark. These migrating animals may also carry ticks and pathogens which already exist in native tick populations in Denmark. The potential supplement of native ticks and existing pathogens to the established high density tick populations in Danish forest and nature areas can be expected to be of little practical importance. However, some of the infected ticks, introduced by migrating birds, may be deposited in private gardens and public parks that are otherwise not able to sustain a viable tick population. Migrating birds may therefore introduce a low level risk of tick borne infections to urban areas. Also the recent unexpected wave of long migrating golden jackals (Canis aureus) and grey wolves (Canis lupus), arriving at the Danish peninsula of Jutland, constitutes an emerging risk of introduction of especially Dermacentor spp ticks and their associated pathogens from Germany and Central Europe. Here, we present the results of screening migrating birds and a golden jackal for ticks as well as ticks collected by flagging in selected urban areas in Denmark. The collected ticks were screened for exotic tick species and 38 different tick borne pathogens. We show that the risk is not just theoretical and we suggest that these introductions may have a practical public health impact., Since the end of the ice age, spring migrating birds from Africa and Europe and autumn migrating birds from Northern Scandinavia have entered Denmark, and recently a small wave of long migrating carnivores have started arriving in Denmark from Central Europe. Theoretically, migrating birds could introduce new tick species as well as tick-associated pathogens to Denmark. These migrating animals may also carry ticks and pathogens which already exist in native tick populations in Denmark. The potential supplement of native ticks and existing pathogens to the established high density tick populations in Danish forest and nature areas can be expected to be of little practical importance. However, some of the infected ticks, introduced by migrating birds, may be deposited in private gardens and public parks that are otherwise not able to sustain a viable tick population. Migrating birds may therefore introduce a low level risk of tick borne infections to urban areas. Also the recent unexpected wave of long migrating golden jackals (Canis aureus) and grey wolves (Canis lupus), arriving at the Danish peninsula of Jutland, constitutes an emerging risk of introduction of especially Dermacentor spp ticks and their associated pathogens from Germany and Central Europe. Here, we present the results of screening migrating birds and a golden jackal for ticks as well as ticks collected by flagging in selected urban areas in Denmark. The collected ticks were screened for exotic tick species and 38 different tick borne pathogens. We show that the risk is not just theoretical and we suggest that these introductions may have a practical public health impact.

In recent years, focus on tick-borne diseases has increased as diseases such as Lyme disease and tick-borne encephalitis have become more common and represent a health problem in many parts of Scandinavia. More effective prevention of infections requires a better understanding of the factors affecting the vector abundance as well as human exposure to the vectors. Hence, there is a great need for analyses and models that can predict how vectors and their associated diseases are distributed now and possibly in the future. As a part of the ScandTick Innovation project, we surveyed tick nymphs at 159 sites (forests and meadows) in Denmark, southern Norway and south-eastern Sweden. At each site we measured presence/absence, and used the data obtained along with environmental data from satellite images to run Boosted Regression Tree machine learning algorithms to predict overall distribution in southern Scandinavia. Together with the predicted distribution maps, we used human density maps to identify and plot areas with high risk of exposure to ticks. The predicted distribution and the spatial variation found corresponded well with known distributions of ticks in Scandinavia (sensitivity: 91%, specificity: 60%), and we found that the model was predominantly temperature-driven. Because presence was strongly correlated with forested habitats the risk areas were much larger in Sweden and Norway compared to Denmark. When combining these distribution maps with human population density maps, we were able to quantify the proportion of people living in areas with tick presence in Scandinavia. We found that although tick nymphs were restricted to a small proportion of the modelled area, high proportions of the human populations (67-79%) lived within these same areas. The model suggests that a potential future range expansion of I. ricinus in Scandinavia is likely but may only affect a relatively small additional proportion of the human population.

At present, very little information exists regarding what role the environmental slurry may play as an infection reservoir and/or route of transmission for bovine digital dermatitis (DD), a disease which is a global problem in dairy herds. To investigate, if DD-related bacteria belong to the indigenous microbiota of the dairy herd environment, we used deep amplicon sequencing of the 16S rRNA gene in 135 slurry samples collected from different sites in 22 dairy farms, with and without DD-infected cows. Both the general bacterial populations as well as digital dermatitis-associated Treponema were targeted in this study. The results revealed significant differences in the bacterial communities between the herds, with only 12 bacterial taxa shared across at least 80% of all the individual samples. These differences in the herd microbiota appeared to reflect mainly between-herd variation. Not surprisingly, the slurry was dominated by ubiquitous gastrointestinal bacteria, such as Ruminococcaceae and Lachnospiraceae Despite the low relative abundance of spirochetes, which ranged from 0 to 0.6%, we were able to detect small amounts of bacterial DNA from DD-associated treponemes in the slurry. However, the DD-associated Treponema spp. were only detected in samples from herds with reported problems of DD. These data indicate that treponemes involved in the pathogenesis of DD are not part of the normal environmental microflora in dairy herds without clinical DD and, consequently, that slurry is not a primary reservoir of infection.Importance Bovine digital dermatitis (DD), a dermal disease which causes lameness in dairy cattle, is a serious problem worldwide. To control this disease, the infection reservoirs and transmission routes of DD pathogens need to be clarified. The dairy herd slurry may be a possible pathogen reservoir of DD-associated bacteria. The rationale for the present study was, therefore, to examine whether DD-associated bacteria are always present in slurry or

From a migrating golden jackal (Canis aureus), we retrieved 21 live male Dermacentor reticulatus ticks, a species not previously reported from wildlife in Denmark. We identified Rickettsia raoultii from 18 (86%) of the ticks. This bacterium is associated with scalp eschar and neck lymphadenopathy after tick bite syndrome among humans.

Vector-borne diseases such as Lyme disease and tick-borne encephalitis have become more common in recent decades and present a real health problem in many parts of Europe. Risk assessment, control, and prevention of these diseases require a better understanding of vector abundance as well as risk factors determining human exposure to ticks. There is a great need for analyses and models that can predict how vectors and their associated diseases are distributed and how this relates to high risk areas for human exposure.As a part of the ScandTick Innovation project, we surveyed ticks at approximately 30 sites (forests and meadows) in each of Denmark, southern Norway and south-eastern Sweden. At each site we measured presence/absence of ticks, and used the data obtained along with environmental satellite images to run Boosted Regression Tree machine learning algorithms to predict overall spatial distribution (probability of presence) in southern Scandinavia. Together with the predicted distribution maps, we used human density maps to determine areas with high risk of exposure to ticks. For nymphs, the predicted distribution found corresponded well with known distributions of ticks in Scandinavia, with more widespread distribution in Denmark compared to Norway and Sweden. In the Norwegian region, probability of presence was markedly higher nearer the coastline and the data shows a latitudinal boundary in the Swedish region above which probability of presence was low or close to zero. Presence of larvae was much more clustered in the observed data, which was also reflected in the predicted distribution maps for the region. Whereas the predicted distribution of larvae was mostly even throughout Denmark, larvae were primarily around the coastlines in Norway and Sweden. When combining these distribution maps with human population density maps and accounting for area accessibility, we could assess the proportion of the population living in areas where ticks were present. Our

Background. While fungal infections of the bovine uterus are well-known diseases in pregnant cattle, very limited knowledge exists on the presence and significance of fungi in the uterus of non-pregnant cows. Presence of fungi in the uterine lumen of postpartum (pp) cows has been reported, but little attention has been paid to this as most studies of the bovine pp uterus have focused on bacteria. Case presentation. Microscopy of uterine lavage cytology slides of three cows from one herd revealed the presence of numerous yeast-like organisms, which were located either free in the fluid or within macrophages. Two of the cows were around 30 days pp, while the third was 7 months pp. None of the cows had been treated with antibiotics. Culturing of the flush samples was unsuccessful, but Sanger sequencing of DNA extracted from an endometrial biopsy of one of the cows revealed the presence of Candida kefyr (Kluyveromyces marxianus). Fluorescence in situ hybridization examination of endometrial tissue sections of two cows using probes targeting 18S rRNA of the K. marxianus group was performed and revealed the presence of yeast cells on the endometrium. Histology was performed and demonstrated hyphal and non-hyphal yeast-like organisms on the surface of endometrium and in the crypts. Tissue invasion was restricted to the superficial part of the epithelium and although endometrial inflammation was present, this was mild and considered as not being caused by the fungi. One of the cows became pregnant and delivered a normal calf at term, while the two others were not bred. Conclusions. Candida kefyr is commonly isolated from milk of cows with mastitis, but has not been reported in association with other diseases of cattle. The infection was present as a monoculture in all three cows, but the fungi had only colonized the uterine lumen and the endometrial surface. Only a mild non-suppurative endometrial inflammation was present, but within the uterine luminal

Recently, focus on tick-borne diseases has increased as diseases such as Lyme disease and tickborne encephalitis have become more widespread and represent a real health problem in many parts of Europe. Effective control and prevention of these diseases requires a better understanding of the factors affecting the vectors. There is a great need for analyses and models that can predict how vectors and their associated diseases are distributed now and possibly in the future. As a part of the ScandTick Innovation project, we surveyed and collected ticks at approximately 30 sites in each of Denmark, southern Norway and south-eastern Sweden. At each site we measured presence/absence and relative tick abundance using north- and east- facing line transect, where number of larvae, nymphs and adult females and males were counted at eight 50 m transects. We used the data obtained along with environmental satellite images to run Boosted Regression Tree machine learning algorithms to predict overall distribution (presence/absence of ticks) and relative tick abundance of nymphs and larvae in southern Scandinavia. For nymphs, the predicted abundance had a positive correlation with observed abundance and the spatial variation found corresponded well with known abundance and distributions of ticks in Scandinavia, with higher abundance and more widespread distribution in Denmark compared to Norway and Sweden. Because abundance was strongly correlated with forested habitats the risk areas were much larger in Sweden and Norway compared to Denmark. In both the Norwegian and Swedish regions, abundance was markedly higher nearer the coastline. Presence of larvae was much more clustered in the observed data, which was also reflected in the predicted abundance and distribution maps for the region. Whereas the predicted distribution of larvae was mostly even throughout Denmark, it was primarily around the coastlines in Norway and Sweden. Abundance was fairly low overall except in some fragmen

Metritis and endometritis commonly occur in dairy cows after calving. Although numerous studies have been performed to identify the causative pathogens, a complete overview has not been done. Metagenomic studies have analyzed the bacterial populations of uterine flush samples from postpartum (pp) dairy cows, but the microbiota in the uterine luminal fluid may differ from the microbiota of the endometrium itself, and important putative pathogens may have been overlooked. In the present study, we compared the microbiota of the uterine lumen and the endometrium of healthy, metritic, and endometritic cows. Samples were collected from 68 Holstein dairy cows at 1, 4, and 7 weeks pp, and the data were analyzed by deep sequencing of the V1 and V2 hypervariable regions of the 16S ribosomal RNA gene. The results showed that Porphyromonadaceae, Fusobacteriaceae, Leptotrichiaceae, and Mycoplasmataceae may be associated with uterine disease. The microbiota of the uterine flush samples and the endometrial biopsies were correlated, but the microbiota of the biopsies was more diverse. Fusobacteriaceae and Leptotrichiaceae were not observed in the biopsies at week 7, whereas they accounted for 20% and 13%, respectively, of the bacterial populations in the flush samples. The Mycoplasmataceae family was observed in much higher quantity in the flush samples than in the biopsies of the endometritis groups at weeks 4 and 7. Our findings support the observations of previous metagenomic studies and illustrate the importance of including endometrial biopsies to obtain more detailed knowledge of the pp uterine microbiota.

Polymicrobial infections represent a great challenge for the clarification of disease etiology and the development of comprehensive diagnostic or therapeutic tools, particularly for fastidious and difficult-to-cultivate bacteria. Using bovine digital dermatitis (DD) as a disease model, we introduce a novel strategy to study the pathogenesis of complex infections. The strategy combines meta-transcriptomics with high-density peptide-microarray technology to screen for in vivo-expressed microbial genes and the host antibody response at the site of infection. Bacterial expression patterns supported the assumption that treponemes were the major DD pathogens but also indicated the active involvement of other phyla (primarily Bacteroidetes). Bacterial genes involved in chemotaxis, flagellar synthesis and protection against oxidative and acidic stress were among the major factors defining the disease. The extraordinary diversity observed in bacterial expression, antigens and host antibody responses between individual cows pointed toward microbial variability as a hallmark of DD. Persistence of infection and DD reinfection in the same individual is common; thus, high microbial diversity may undermine the host's capacity to mount an efficient immune response and maintain immunological memory towards DD. The common antigenic markers identified here using a high-density peptide microarray address this issue and may be useful for future preventive measures against DD.

Although treponemes are consistently identified in tissue from bovine digital dermatitis (DD) lesions, the definitive etiology of this debilitating polymicrobial disease is still unresolved. To study the microbiomes of 27 DD-infected and 10 healthy interdigital skin samples, we used a combination of different molecular methods. Deep sequencing of the 16S rRNA gene variable regions V1–V2 showed that Treponema, Mycoplasma, Fusobacterium and Porphyromonas were the genera best differentiating the DD samples from the controls. Additional deep sequencing analysis of the most abundant genus, Treponema, targeting another variable region of the 16S rRNA gene, V3–V4, identified 15 different phylotypes, among which Treponema phagedenis-like and Treponema refringens-like species were the most abundant. Although the presence of Treponema spp., Fusobacterium necrophorum and Porphyromonas levii was confirmed by fluorescence in situ hybridization (FISH), the results for Mycoplasma spp. were inconclusive. Extensive treponemal epidermal infiltration, constituting more than 90% of the total bacterial population, was observed in 24 of the 27 DD samples. F. necrophorum and P. levii were superficially located in the epidermal lesions and were present in only a subset of samples. RT-qPCR analysis showed that treponemes were also actively expressing a panel of virulence factors at the site of infection. Our results further support the hypothesis that species belonging to the genus Treponema are major pathogens of DD and also provide sufficient clues to motivate additional research into the role of M. fermentans, F. necrophorum and P. levii in the etiology of DD.

Bacterial invasion of the uterus during the postpartum period has been well described. Recent papers using 16S rRNA gene sequencing techniques suggest that the nonpregnant uterus contains a diverse flora of bacteria that are not necessarily pathogenic. In contrast, the pregnant uterus has until now been considered a sterile environment. The aim of the present study was to investigate whether bacteria were present in the uteri of pregnant cows. Uteri from pregnant, slaughtered animals (n = 47) were sampled. The surface of the uterus was wiped with alcohol, flame sterilized, and cut open with sterile scissors. Samples were taken from the endometrium and from the placentomes. The samples were embedded in paraffin, sectioned at 3 microns, and prepared for fluorescence in situ hybridization using a probe targeting the 16S rRNA of the domain bacteria, so that all bacteria regardless of species were visualised. Using fluorescence microscopy, the presence of bacteria within or on the surface of the endometrium and within the placentomes was noted. The stage of pregnancy was estimated to range from 26 to 263 days by measuring the size of the embryo or fetus. The endometrial samples from 85.1% (40/47) of pregnant cows contained bacteria. In 22 cows, the bacteria were localised within the endometrial tissue, whereas in the remaining 18 cows, the bacteria were on the epithelial surface. Placental samples were obtained from 43 cows, and 76.7% (33/43) of these contained bacteria. The presence of bacteria in the pregnant uterus may suggest that a cow can carry a pregnancy despite the presence of few potentially pathogenic bacteria or that normal flora exist in the uterus as in, for example, the vagina. In conclusion, bacteria were present in the endometrium and placentomes of pregnant cows. Further analyses using rRNA gene sequencing techniques will aim to confirm the presence of bacteria in the bovine pregnant uterus and to investigate which species of bacteria are present in the

The objective of the study was to identify the location of the present bacteria in the uterus and oviducts of cows with pyometra. Pyometra is one of the postpartum infectious diseases in cattle that can result in infertility and thereby affect reproduction performance. Reproductive tracts (n = 21) were collected at a slaughterhouse in Denmark and send to The University of Copenhagen for examination and sampling. The uteri were included in the study when the following criteria were met: the cow was more than 21 days postpartum, the uterus was distended with pus, the cervix was closed, and a corpus luteum was present in one or both ovaries. A full thickness uterine tissue sample from the previous pregnant horn and both oviducts were sampled and then fixed in formalin. The tissues were trimmed, processed by routine methods, embedded in paraffin, sectioned at 3 microns, and prepared for fluorescence in situ hybridization using a probe targeting the 16S ribosomal RNA of the domain bacteria (i.e. targeting all bacteria regardless of species). Using fluorescence microscopy, the presence of bacteria within or on the surface of the endometrium and in the oviducts were noted. The endometrial biopsies from all cows (n = 21) contained bacteria, while 75% (16/21) of the cows had bacteria in one or both oviducts. The bacteria were located on the luminal surface and in the lamina propria in 38.1% (8/21) of the uterine biopsies. In the remaining 62% of the uterine biopsies, the bacteria were only located above the basal membrane. Regarding the oviduct biopsies, the bacteria were located on the luminal surface and in lamina propria in 9.5% (2/21) of the biopsies, whereas the bacteria were located only above the basal membrane in 90.5% of the biopsies. In conclusion, 1) bacteria are present in the uteri and oviducts of cows with pyometra and 2) the bacteria are primarily located on the luminal epithelia surface above the basal membrane. Further analyses will investigate which specifi

The bacteria present in the uterus during pyometra have previously been studied using bacteriological culturing. These studies identified Fusobacterium necrophorum and Trueperella pyogenes as the major contributors to the pathogenesis of pyometra. However, an increasing number of culture-independent studies have demonstrated that the bacterial diversity in most environments is underestimated in culture-based studies. Consequently, fastidious pyometra-associated pathogens may have been overlooked. Therefore, the primary purpose of this study was to investigate the diversity of bacteria in the uterus of cows with pyometra by using culture-independent 16S rRNA PCR combined with next generation sequencing. We investigated the microbial composition in the uterus of 21 cows with pyometra, which were obtained from a Danish slaughterhouse. Similar to the observations from the culture studies, Fusobacteriaceae, the family that F. necrophorum belongs to, was the operational taxonomic unit (OTU) observed in the largest quantities. By contrast, the Actinomycetaceae family, which includes T. pyogenes, constituted only 1% of the total number of reads. Thus we cannot confirm the previously reported role of species from this family in the pathogenesis of pyometra. Finally, we identified a large number of sequences representing three families of Gram-negative bacteria in the pyometra samples: Porphyromonadaceae, Mycoplasmataceae, and Pasteurellaceae. It is likely that these families comprise potential pathogenic species of a fastidious nature, which have been overlooked in previous studies. Our results increase the knowledge of the complexity of the pyometra microbiota and suggest that pathogens in addition to F. necrophorum may be involved in the pathogenesis of pyometra. (C) 2014 Elsevier B.V. All rights reserved.

Influenza A virus infections have great impact on human health and welfare and significant resources are linked to influenza epidemics due to excess hospitalizations and lost productivity. Up to 15% of the human population is affected when Influenza spreads around the world in seasonal epidemics (WHO). Animal models are essential in understanding the mechanisms involved in human infectious disease and for the development of effective prevention and treatment strategies. It is increasingly realized that large animal models like the pig are exceptionally human like and serve as an excellent model for disease and inflammation. Pigs are fully susceptible to human influenza, and have been demonstrated to be involved in influenza evolution and ecology. Pigs share many similarities with humans regarding lung physiology and innate immune cell infiltration of the respiratory system and thus seem to be an obvious large animal model for respiratory infections. This study aimed at providing a better understanding of the involvement of circulating non-coding RNA and innate immune factors in porcine blood leukocytes during influenza virus infection. By employing the pig as a model we were able to perform highly controlled experimental infections and to study changes of symptoms, viral titer, and expression of microRNAs/mRNAs as the influenza infection progresses in time, generating information that would be difficult to obtain from human patients. The Gram-negative bacterium Actinobacillus pleuropneumoniae causes pneumonia in pigs, a disease which is associated with high morbidity and mortality, as well as impaired animal welfare. The rapidly evolving pneumonia is characterized by large areas of lung necrosis resulting from the combined effect of tissue damage caused by the bacteria, and a strong proinflammatory immune response. To obtain in-depth understanding of this infection, concurrent gene expression of host and pathogen in lung samples collected from pi

Background: Actinobacillus pleuropneumoniae causes pleuropneumonia in pigs, a disease which is associated with high morbidity and mortality, as well as impaired animal welfare. To obtain in-depth understanding of this infection, the interplay between virulence factors of the pathogen and defense mechanisms of the porcine host needs to be elucidated. However, research has traditionally focused on either bacteriology or immunology; an unbiased picture of the transcriptional responses can be obtained by investigating both organisms in the same biological sample. Results: Host and pathogen responses in pigs experimentally infected with A. pleuropneumoniae were analyzed by high-throughput RT-qPCR. This approach allowed concurrent analysis of selected genes encoding proteins known or hypothesized to be important in the acute phase of this infection. The expression of 17 bacterial and 31 porcine genes was quantified in lung samples obtained within the first 48 hours of infection. This provided novel insight into the early time course of bacterial genes involved in synthesis of pathogen-associated molecular patterns (lipopolysaccharide, peptidoglycan, lipoprotein) and genes involved in pattern recognition (TLR4, CD14, MD2, LBP, MYD88) in response to A. pleuropneumoniae. Significant up-regulation of proinflammatory cytokines such as IL1B, IL6, and IL8 was observed, correlating with protein levels, infection status and histopathological findings. Host genes encoding proteins involved in iron metabolism, as well as bacterial genes encoding exotoxins, proteins involved in adhesion, and iron acquisition were found to be differentially expressed according to disease progression. By applying laser capture microdissection, porcine expression of selected genes could be confirmed in the immediate surroundings of the invading pathogen. Conclusions: Microbial pathogenesis is the product of interactions between host and pathogen. Our results demonstrate the applicability of high-through

Objective: To develop a fast and efficient method for measuring prevalence and gene expression of representatives from the three major groups of Treponema, most frequently identified in DD biopsies from cattle.

Animal models are essential in understanding the mechanisms involved in human infectious disease and for the development of effective prevention and treatment strategies. It is increasingly realized that large animal models like the pig are exceptionally human like and serve as an excellent model for disease and inflammation. Pigs are fully susceptible to human influenza, and have been demonstrated to be involved in influenza evolution and ecology. Pigs share many similarities with humans regarding lung physiology and innate immune cell infiltration of the respiratory system and thus seem to be an obvious large animal model for respiratory infections. This study aimed at providing a better understanding of the involvement of circulating non-coding RNA and innate immune factors in porcine blood leukocytes during influenza virus infection. By employing the pig as a model we were able to perform highly controlled experimental infections and to study changes of symptoms, viral titer, and expression of microRNAs/mRNAs as the influenza infection progresses in time, generating information that would be difficult to obtain from human patients.

Due to increased travel, climatic, and environmental changes, the incidence of tick-borne disease in both humans and animals is increasing throughout Europe. Therefore, extended surveillance tools are desirable. To accurately screen tick-borne pathogens, a large scale epidemiological study was conducted on 7050 Ixodes ricinus nymphs collected from France, Denmark, and the Netherlands using a powerful new high-throughput approach. This advanced methodology permitted the simultaneous detection of 25 bacterial, and 12 parasitic species (including; Borrelia, Anaplasma, Ehrlichia, Rickettsia, Bartonella, Candidatus Neoehrlichia, Coxiella, Francisella, Babesia, and Theileria genus) across 94 samples. We successfully determined the prevalence of expected (Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Rickettsia helvetica, Candidatus Neoehrlichia mikurensis, Babesia divergens, Babesia venatorum), unexpected (Borrelia miyamotoi) and rare (Bartonella henselae) pathogens in the three European countries. Moreover we detected Borrelia spielmanii, Borrelia miyamotoi, Babesia divergens, and Babesia venatorum for the first time in Danish ticks. This surveillance method represents a major improvement in epidemiological studies, able to facilitate comprehensive testing of tick-borne pathogens, and which can also be customized to monitor emerging diseases.

Large animal models are essential in understanding the mechanisms involved in human infectious disease. To study the expression of host and bacterial genes involved in defense and survival mechanisms, we analyzed lung tissue from pigs experimentally infected with the Gram-negative bacterium A. pleuropneumoniae. All steps including RNA extraction and high-throughput real-time qPCR were carried out simultaneously for the two organisms. By applying this dual-organism approach, we obtained unique insights into the host-pathogen interaction at the site of infection. Differential expression of host genes involved in innate immune responses towards Gram-negative infections, including pattern recognition receptors and cytokines concurrent with expression of bacterial genes involved in lipopolysaccharide biosynthesis and adhesion was demonstrated. We also studied the gene expression in blood leukocytes after experimental H1N2 virus infection of pigs, and found the regulation of several swine encoded miRNAs and cytokines to mimic key findings from influenza studies in human patients. By employing the pig as a model we were able to perform highly controlled experimental infections and to study changes of symptoms, viral titer, and expression of microRNAs/mRNAs as the influenza infection progressed in time, generating information that would be difficult to obtain from human patients.

The bacteria associated with the infectious claw disease bovine digital dermatitis (DD) are spirochetes of the genus Treponema; however, their environmental reservoir remains unknown. To our knowledge, the current study is the first report of the discovery and phylogenetic characterization of rRNA gene sequences from DD-associated treponemes in the dairy herd environment. Although the spread of DD appears to be facilitated by wet floors covered with slurry, no DD-associated treponemes have been isolated from this environment previously. Consequently, there is a lack of knowledge about the spread of this disease among cows within a herd as well as between herds. To address the issue of DD infection reservoirs, we searched for evidence of DD-associated treponemes in fresh feces, in slurry, and in hoof lesions by deep sequencing of the V3 and V4 hypervariable regions of the 16S rRNA gene coupled with identification at the operational-taxonomic-unit level. Using treponeme-specific primers in this high-throughput approach, we identified small amounts of DNA (on average 0.6% of the total amount of sequence reads) from DD-associated treponemes in 43 of 64 samples from slurry and cow feces collected from six geographically dispersed dairy herds. Species belonging to the Treponema denticola/Treponema pedis-like and Treponema phagedenis-like phylogenetic clusters were among the most prevalent treponemes in both the dairy herd environment and the DD lesions. By the high-throughput approach presented here, we have demonstrated that cow feces and environmental slurry are possible reservoirs of DD-associated treponemes. This method should enable further clarification of the etiopathogenesis of DD.

Skin lesions often seen in pig production are of great animal welfare concern. To study the potential role of Treponema bacteria in porcine skin ulcers, we investigated the presence and distribution of these organisms in decubital shoulder ulcers (n=51) and ear necroses (n=54) by fluorescence in situ hybridization (FISH) and high-throughput sequencing. In addition, two cases of facial ulcers and five cases of other skin ulcers were included in the study. Samples from all 112 skin lesions and intact skin from pigs without skin ulcers (n=14) were screened by FISH. Three different oligonucleotide probes targeting 16S rRNA were used, specific for domain bacterium, Treponema spp. and species T. pedis. Screening showed that two cases each of facial and other ulcers, 35 (69%) of shoulder ulcers and 32 (59%) of ear necroses were positive for Treponema spp. T. pedis was the unequivocally, predominant species typically constituting more than 90% of the treponemes in a lesion, assessed visually by microscopy. Altogether, T. pedis was demonstrated in 69 of the 71 Treponema spp. positive lesions. We conclude that Treponema spp. are frequently present and abundant in various skin ulcers of pigs. The results from this study point toward an important role of T. pedis as a secondary bacterial infection in porcine skin ulcers, especially in severe and chronic lesions.

Recent years’ advance in sequencing technology has resulted in extensive new knowledge of the microbial ecology of different environments. We used the technology to investigate the causality of endometritis, which is an inflammation in the inner lining of the uterus affecting up to 20% of dairy cows in Denmark post partum. Endometritis is linked to reduced reproductive performance, which is costly for the farmer and often leads to culling of the affected cows. With incomplete knowledge of the bacteria involved, treatment is performed without an option for choosing the best suited antimicrobial agent, which may lead to unnecessary antibiotic resistance development. Slaughterhouse samples were analysed in order to obtain information on the uterine microbiota from both cows with endometritis and seemingly healthy cows from a variety of herds. We sampled uteri from cows (n=50) from a slaughterhouse in Holstebro, Denmark. An incision was made into the right uterine horn and an endometrial biopsy was taken with a pair of sterilised scissors. The endometrial surface was sampled with a cotton-swab through the same incision. All samples were immediately put in RNAlater. The DNA was extracted with the Maxwell 16 LEV Blood kit (Promega), the 16S rRNA PCR was performed with primers targeting the V2 region, and the 454 next generation sequencing was performed by GATC. Previous results have shown that Proteobacteria and Tenericutes are the most important bacteria phyla in the uterus of healthy cows (Machado et al. 2012 and Galvão et al. 2012) while Escherichia coli, Trueperella Pyogenes, Prevotella species and Fusobacterium necrophorum have commonly been associated with endometritis (Sheldon 2006). Preliminary results indicate that we often find bacteria from phylum Actinobacteria in the healthy cows and often bacteria from phyla Bacteroidetes and Fusobacteria in cows with endometritis.

The aim of this study was to assess infectious foot diseases, including identification and characterization of Dichelobacter nodosus and Treponema spp., in herds having problems with interdigital dermatitis (ID) and heel horn erosion (E) and in control herds expected to have few problems. We also wanted to compare diseased and healthy cows in all herds. The study included 14 dairy herds with a total of 633 cows. Eight herds had a history of ID and E, and 6 were control herds. All cows were scored for lameness, and infectious foot diseases on the hind feet were recorded after trimming. Swabs and biopsies were taken from the skin of 10 cows in each herd for bacterial analyses. In total, samples were taken from 34 cows with ID, 11 with E, 40 with both ID and E, and 8 with digital dermatitis (DD), and from 47 cows with healthy feet. Swabs were analyzed for identification and characterization of D. nodosus by PCR, culture, virulence testing, and serotyping. Biopsies were analyzed by fluorescent in situ hybridization regarding histopathology, identification, and characterization of Treponema spp., and identification of D. nodosus. Interdigital dermatitis was the most frequent foot disease, with a prevalence of 50.4% in problem herds compared with 26.8% in control herds. Heel horn erosion was recorded in 34.8% of the cows in problem herds compared with 22.1% in control herds. Dichelobacter nodosus was detected in 97.1% of the cows with ID, in 36.4% with E, in all cows with both ID and E, in all cows with DD, and in 66.0% of cows with healthy feet. All serogroups of D. nodosus except F and M were detected, and all isolates were defined as benign by the gelatin gel test. Treponema spp. were detected in 50.0% of the cows with ID, in 9.1% with E, in 67.5% with ID and E, in all cows with DD, and in 6.4% of those with healthy feet. In total, 6 previously described phylotypes (PT) of Treponema were detected: PT1, PT3, PT6, PT13, and PT15 in cows with ID, PT1 in a cow with E, and

Recent years’ advance in sequencing technology has resulted in extensive new knowledge of the microbial ecology of different environments. We used the technology to investigate the causality of endometritis, which is an inflammation in the inner lining of the uterus affecting up to 20% of dairy cows in Denmark post partum. Endometritis is linked to reduced reproductive performance, which is costly for the farmer and often leads to culling of the affected cows. With incomplete knowledge of the bacteria involved, treatment is performed without an option for choosing the best suited antimicrobial agent, which may lead to unnecessary antibiotic resistance development. Slaughterhouse samples were analysed in order to obtain information on the uterine microbiota from both cows with endometritis and seemingly healthy cows from a variety of herds. We sampled uteri from cows (n=50) from a slaughterhouse in Holstebro, Denmark. An incision was made into the right uterine horn and an endometrial biopsy was taken with a pair of sterilised scissors. The endometrial surface was sampled with a cotton-swab through the same incision. All samples were immediately put in RNAlater. The DNA was extracted with the Maxwell 16 LEV Blood kit (Promega), the 16S rRNA PCR was performed with primers targeting the V2 region, and the 454 next generation sequencing was performed by GATC. Previous results have shown that Proteobacteria and Tenericutes are the most important bacteria phyla in the uterus of healthy cows (Machado et al. 2012 and Galvão et al. 2012) while Escherichia coli, Trueperella Pyogenes, Prevotella species and Fusobacterium necrophorum have commonly been associated with endometritis (Sheldon 2006). Preliminary results indicate that we often find bacteria from phylum Actinobacteria in the healthy cows and often bacteria from phyla Bacteroidetes and Fusobacteria in cows with endometritis.

Endometritis is inflammation of the inner lining of the uterus, affecting up to 20% of the dairy cows after calving in Denmark. The disease causes reduced pregnancy rates, which often leads to culling of the cows and is costly for the farmer. Until now, investigations of which pathogens may cause the disease have been based on microbiological culturing, and no conclusive evidence has been found. Only a fraction of the bacterial flora is cultivable, and therefore more than 90% of the uterine microbiome has not been characterised. With incomplete knowledge of the pathogens, treatment is performed without an option for choosing the best suited antimicrobial agent, which may lead to unnecessary antibiotic resistance development. The present study is based on 16S rRNA PCR, which in combination with 454 next generation sequencing allows phylogenetic identification of the bacteria present in the sample. Not being limited to bacteria that are suited to growth under laboratory conditions, this study promises a more comprehensive insight into the microbiome of the dairy cow uterus than has previously been offered. Cows (n=40) on a Danish dairy herd were randomly selected on the basis of a uterine score indicating that the cows had uterine pathology. Uterine fluid was aspirated and if necessary the uterus was flushed with 30 ml sterile saline solution in order to retrieve uterine material. The fluid was placed in RNAlater. An endometrial biopsy was retrieved and the tissue placed in RNAlater. The cows were sampled on days 5-11 (week 1), days 26-32 (week 4), and on days 47-53 (week 7). This sampling schedule provided an opportunity to follow the development of any infection, and the combination of biopsy and uterine flush samples offered insights into whether tissue-invasive bacteria were present. The DNA was extracted with the Maxwell 16 LEV Blood kit (Promega), the 16S rRNA PCR was performed with primers targeting the V2 region, and the 454 next generation sequencing was

Bovine digital dermatitis, the cause of lameness and wasting in cattle, was first reported in 1974. Today, this disease has considerable negative effects on animal welfare and production economy in many parts of the world. A bacterial etiology of digital dermatitis is now well documented, and the current view on this disease points towards a complicated etiology involving co-infection of more than one, and probably multiple species belonging to the genus Treponema. Still, the pathogenic role of each of the digital dermatitis-associated phylotypes remains unclear. The aim of this investigation was to obtain a better understanding of digital dermatitis in general, including possible predisposing skin alternations and the role of the bacteria Dichelobacter nodosus. Finally, we wanted to determine if any Treponema phylotypes could be singled out as having a particularly prominent role in the etiology of the disease. Here, a PCR-based approach targeting the 16S rRNA gene along with fluorescence in situ hybridization was used to determine the prevalence and diversity of 17 Treponema phylotypes in 85 digital dermatitis lesions from six Danish dairy herds as well as additional biopsies of healthy skin and previously examined digital dermatitis lesions. All skin samples were evaluated histopathologically for possible predisposing abnormalities. Furthermore, fluorescence in situ hybridization tests for Fusobacterium. necrophorum and D. nodosus was applied. All lesions revealed intermingled infections with multiple Treponema phylotypes (mean > 7). In six herds, the mean number of phylotypes identified varied between 12 and 15. D. nodosus was present in forty-nine (51%) of the lesions and in three of the apparently healthy skin samples. One “healthy” sample also contained Treponema spp. and D. nodosus, and were histopathologically categorized as subclinical digital dermatitis. We propose that external noxious stimuli allow D. nodosus to break down the epide

Background Gene expression profiles of bacteria in their natural hosts can provide novel insight into the host-pathogen interactions and molecular determinants of bacterial infections. In the present study, the transcriptional profile of the porcine lung pathogen Actinobacillus pleuropneumoniae was monitored during the acute phase of infection in its natural host. Methodology/Principal Findings Bacterial expression profiles of A. pleuropneumoniae isolated from lung lesions of 25 infected pigs were compared in samples taken 6, 12, 24 and 48 hours post experimental challenge. Within 6 hours, focal, fibrino hemorrhagic lesions could be observed in the pig lungs, indicating that A. pleuropneumoniae had managed to establish itself successfully in the host. We identified 237 differentially regulated genes likely to encode functions required by the bacteria for colonization and survival in the host. This group was dominated by genes involved in various aspects of energy metabolism, especially anaerobic respiration and carbohydrate metabolism. Remodeling of the bacterial envelope and modifications of posttranslational processing of proteins also appeared to be of importance during early infection. The results suggested that A. pleuropneumoniae is using various strategies to increase its fitness, such as applying Na+ pumps as an alternative way of gaining energy. Furthermore, the transcriptional data provided potential clues as to how A. pleuropneumoniae is able to circumvent host immune factors and survive within the hostile environment of host macrophages. This persistence within macrophages may be related to urease activity, mobilization of various stress responses and active evasion of the host defenses by cell surface sialylation. Conclusions/Significance The data presented here highlight the importance of metabolic adjustments to host conditions as virulence factors of infecting microorg

Background Comparative analysis of gene expression among serotypes within a species can provide valuable information on important differences between related genomes. For the pig lung pathogen Actinobacillus pleuropneumoniae, 15 serotypes with a considerable variation in virulence potential and immunogenicity have been identified. This serotypic diversity can only partly be explained by amount of capsule and differences in the RTX toxin genes in their genomes. Iron acquisition in vivo is an important bacterial function and in pathogenic bacteria, iron-limitation is often a signal for the induction of virulence genes. We used a pan-genomic microarray to study the transcriptional response to iron restriction in vitro in six serotypes of A. pleuropneumoniae (1, 2, 3, 5b, 6, and 7), representing at least two levels of virulence. Results In total, 45 genes were significantly (p <0.0001) up-regulated and 67 genes significantly down-regulated in response to iron limitation. Not previously observed in A. pleuropneumoniae was the up-regulation of a putative cirA-like siderophore in all six serotypes. Three genes, recently described in A. pleuropneumoniae as possibly coding for haemoglobin-haptoglobin binding proteins, displayed significant serotype related up-regulation to iron limitation. For all three genes, the expression appeared at its lowest in serotype 3, which is generally considered one of the least virulent serotypes of A. pleuropneumoniae. The three genes share homology with the hmbR haemoglobin receptor of Neisseria meningitidis, a possible virulence factor which contributes to bacterial survival in rats. Conclusions By comparative analysis of gene expression among 6 different serotypes of A. pleuropneumoniae we identified a common set of presumably essential core genes, involved in iron regulation. The results support and expand previous observations concerning the identification of new potential iron acquisition systems in A. pleuropneumoniae, showing that t