Prevalence of tick-borne encephalitis virus in questing Ixodes ricinus nymphs in southern Scandinavia and the possible influence of meteorological factors.

Ixodes ricinus ticks are Scandinavia's main vector for tick-borne encephalitis virus (TBEV), which infects many people annually. The aims of the present study were (i) to obtain information on the TBEV prevalence in host-seeking I. ricinus collected within the Øresund-Kattegat-Skagerrak (ØKS) region, which lies in southern Norway, southern Sweden and Denmark; (ii) to analyse whether there are potential spatial patterns in the TBEV prevalence; and (iii) to understand the relationship between TBEV prevalence and meteorological factors in southern Scandinavia. Tick nymphs were collected in 2016, in southern Scandinavia, and screened for TBEV, using pools of 10 nymphs, with RT real-time PCR, and positive samples were confirmed with pyrosequencing. Spatial autocorrelation and cluster analysis was performed with Global Moran's I and SatScan to test for spatial patterns and potential local clusters of the TBEV pool prevalence at each of the 50 sites. A climatic analysis was made to correlate parameters such as minimum, mean and maximum temperature, relative humidity and saturation deficit with TBEV pool prevalence. The climatic data were acquired from the nearest meteorological stations for 2015 and 2016. This study confirms the presence of TBEV in 12 out of 30 locations in Denmark, where six were from Jutland, three from Zealand and two from Bornholm and Falster counties. In total, five out of nine sites were positive from southern Sweden. TBEV prevalence of 0.7%, 0.5% and 0.5%, in nymphs, was found at three sites along the Oslofjord (two sites) and northern Skåne region (one site), indicating a potential concern for public health. We report an overall estimated TBEV prevalence of 0.1% in questing I. ricinus nymphs in southern Scandinavia with a region-specific prevalence of 0.1% in Denmark, 0.2% in southern Sweden and 0.1% in southeastern Norway. No evidence of a spatial pattern or local clusters was found in the study region. We found a strong correlation between TBEV prevalence in ticks and relative humidity in Sweden and Norway, which might suggest that humidity has a role in maintaining TBEV prevalence in ticks. TBEV is an emerging tick-borne pathogen in southern Scandinavia, and we recommend further studies to understand the TBEV transmission potential with changing climate in Scandinavia.

Spatial patterns of pathogen prevalence in questing Ixodes ricinus nymphs in southern Scandinavia, 2016.

Tick-borne pathogens cause diseases in animals and humans, and tick-borne disease incidence is increasing in many parts of the world. There is a need to assess the distribution of tick-borne pathogens and identify potential risk areas. We collected 29,440 tick nymphs from 50 sites in Scandinavia from August to September, 2016. We tested ticks in a real-time PCR chip, screening for 19 vector-associated pathogens. We analysed spatial patterns, mapped the prevalence of each pathogen and used machine learning algorithms and environmental variables to develop predictive prevalence models. All 50 sites had a pool prevalence of at least 33% for one or more pathogens, the most prevalent being Borrelia afzelii, B. garinii, Rickettsia helvetica, Anaplasma phagocytophilum, and Neoehrlichia mikurensis. There were large differences in pathogen prevalence between sites, but we identified only limited geographical clustering. The prevalence models performed poorly, with only models for R. helvetica and N. mikurensis having moderate predictive power (normalized RMSE from 0.74-0.75, R2 from 0.43-0.48). The poor performance of the majority of our prevalence models suggest that the used environmental and climatic variables alone do not explain pathogen prevalence patterns in Scandinavia, although previously the same variables successfully predicted spatial patterns of ticks in the same area.

Author Correction: Spatial data of Ixodes ricinus instar abundance and nymph pathogen prevalence, Scandinavia, 2016-2017.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Spatial data of Ixodes ricinus instar abundance and nymph pathogen prevalence, Scandinavia, 2016-2017.

Ticks carry pathogens that can cause disease in both animals and humans, and there is a need to monitor the distribution and abundance of ticks and the pathogens they carry to pinpoint potential high risk areas for tick-borne disease transmission. In a joint Scandinavian study, we measured Ixodes ricinus instar abundance at 159 sites in southern Scandinavia in August-September, 2016, and collected 29,440 tick nymphs at 50 of these sites. We additionally measured abundance at 30 sites in August-September, 2017. We tested the 29,440 tick nymphs in pools of 10 in a Fluidigm real-time PCR chip to screen for 17 different tick-associated pathogens, 2 pathogen groups and 3 tick species. We present data on the geolocation, habitat type and instar abundance of the surveyed sites, as well as presence/absence of each pathogen in all analysed pools from the 50 collection sites and individual prevalence for each site. These data can be used alone or in combination with other data for predictive modelling and mapping of high-risk areas.

Predicting the spatial abundance of Ixodes ricinus ticks in southern Scandinavia using environmental and climatic data.

Recently, focus on tick-borne diseases has increased as ticks and their pathogens have become widespread and represent a health problem in Europe. Understanding the epidemiology of tick-borne infections requires the ability to predict and map tick abundance. We measured Ixodes ricinus abundance at 159 sites in southern Scandinavia from August-September, 2016. We used field data and environmental variables to develop predictive abundance models using machine learning algorithms, and also tested these models on 2017 data. Larva and nymph abundance models had relatively high predictive power (normalized RMSE from 0.65-0.69, R2 from 0.52-0.58) whereas adult tick models performed poorly (normalized RMSE from 0.94-0.96, R2 from 0.04-0.10). Testing the models on 2017 data produced good results with normalized RMSE values from 0.59-1.13 and R2 from 0.18-0.69. The resulting 2016 maps corresponded well with known tick abundance and distribution in Scandinavia. The models were highly influenced by temperature and vegetation, indicating that climate may be an important driver of I. ricinus distribution and abundance in Scandinavia. Despite varying results, the models predicted abundance in 2017 with high accuracy. The models are a first step towards environmentally driven tick abundance models that can assist in determining risk areas and interpreting human incidence data.

A large-scale screening for the taiga tick, Ixodes persulcatus, and the meadow tick, Dermacentor reticulatus, in southern Scandinavia, 2016.

The taiga tick, Ixodes persulcatus, has previously been limited to eastern Europe and northern Asia, but recently its range has expanded to Finland and northern Sweden. The species is of medical importance, as it, along with a string of other pathogens, may carry the Siberian and Far Eastern subtypes of tick-borne encephalitis virus. These subtypes appear to cause more severe disease, with higher fatality rates than the central European subtype. Until recently, the meadow tick, Dermacentor reticulatus, has been absent from Scandinavia, but has now been detected in Denmark, Norway and Sweden. Dermacentor reticulatus carries, along with other pathogens, Babesia canis and Rickettsia raoultii. Babesia canis causes severe and often fatal canine babesiosis, and R. raoultii may cause disease in humans. We collected 600 tick nymphs from each of 50 randomly selected sites in Denmark, southern Norway and south-eastern Sweden in August-September 2016. We tested pools of 10 nymphs in a Fluidigm real time PCR chip to screen for I. persulcatus and D. reticulatus, as well as tick-borne pathogens. Of all the 30,000 nymphs tested, none were I. persulcatus or D. reticulatus. Our results suggest that I. persulcatus is still limited to the northern parts of Sweden, and have not expanded into southern parts of Scandinavia. According to literature reports and supported by our screening results, D. reticulatus may yet only be an occasional guest in Scandinavia without established populations.

Multiple infections in questing nymphs and adult female Ixodes ricinus ticks collected in a recreational forest in Denmark.

During its lifecycle, the generalist Ixodes ricinus takes up three blood meals from a wide selection of vertebrate hosts, some of which are reservoirs for multiple vector-associated pathogens. Since I. ricinus also readily bites humans, pets, and livestock, these hosts are at risk of becoming infected with more than one tick-borne pathogen. Multiple tick-borne infections are a public health concern, since they may increase diversity and duration of symptoms and complicate differential diagnosis and therapy. We used an existing Fluidigm real-time PCR chip to identify the minimum risk of exposure to infected/co-infected ticks in Denmark. We screened 509 nymphs and 504 adult female I. ricinus ticks for 17 different vector-associated pathogenic agents. The questing ticks were collected by flagging during the same season in two consecutive years in Grib forest in the capital region of Copenhagen. Overall, 19.1% of the nymphs and 52.2% of the adult female ticks harbored at least one zoonotic pathogen. The main agents were Borrelia spp., Anaplasma phagocytophilum and Rickettsia helvetica, while Candidatus Neoehrlichia mikurensis and Babesia venatorum both were present in less than 1% of the ticks. In 3.5% of the nymphs and 12.3% of adults we found more than one tick-borne pathogen. Of these, 15% were potentially triple or quadruple infections. Whereas mixed infections with Borrelia were equally distributed among both life stages, the adult ticks hosted 84.5% of the co-infections with different species of tick-borne pathogens, chiefly involving Borrelia species in combination with either R. helvetica or A. phagocytophilum. Statistical analyses indicated non-random co-occurrence of Borrelia spielmanii/Borrelia garinii in both life stages and B. garinii/Borrelia afzelii and B. garinii/Borrelia valaisiana in the nymphs. Although the overall prevalence of ticks hosting more than one infection only constituted 7.9% at the particular site investigated in this study, our results still underline that co-infections should be considered in diagnosis and treatment of tick-borne diseases in northern Europe.

Predicting and mapping human risk of exposure to Ixodes ricinus nymphs using climatic and environmental data, Denmark, Norway and Sweden, 2016.

BackgroundTick-borne diseases have become increasingly common in recent decades and present a health problem in many parts of Europe. Control and prevention of these diseases require a better understanding of vector distribution.AimOur aim was to create a model able to predict the distribution of Ixodes ricinus nymphs in southern Scandinavia and to assess how this relates to risk of human exposure.MethodsWe measured the presence of I. ricinus tick nymphs at 159 stratified random lowland forest and meadow sites in Denmark, Norway and Sweden by dragging 400 m transects from August to September 2016, representing a total distance of 63.6 km. Using climate and remote sensing environmental data and boosted regression tree modelling, we predicted the overall spatial distribution of I. ricinus nymphs in Scandinavia. To assess the potential public health impact, we combined the predicted tick distribution with human density maps to determine the proportion of people at risk.ResultsOur model predicted the spatial distribution of I. ricinus nymphs with a sensitivity of 91% and a specificity of 60%. Temperature was one of the main drivers in the model followed by vegetation cover. Nymphs were restricted to only 17.5% of the modelled area but, respectively, 73.5%, 67.1% and 78.8% of the human populations lived within 5 km of these areas in Denmark, Norway and Sweden.ConclusionThe model suggests that increasing temperatures in the future may expand tick distribution geographically in northern Europe, but this may only affect a small additional proportion of the human population.

Screening for multiple tick-borne pathogens in Ixodes ricinus ticks from birds in Denmark during spring and autumn migration seasons.

Presently, it is uncertain to what extent seasonal migrating birds contribute to the introduction of ticks and tick-associated pathogens in Denmark. To quantify this phenomenon, we captured birds during the spring and autumn migration at three field sites in Denmark and screened them for ticks. Bird-derived ticks were identified to tick species and screened for 37 tick-borne pathogens using real-time PCR. Overall, 807 birds, representing 44 bird species, were captured and examined for ticks during the spring (292 birds) and autumn migrations (515 birds). 10.7% of the birds harboured a total of 179 Ixodes ricinus ticks (38 ticks in spring and 141 in the autumn) with a mean infestation intensity of 2.1 ticks per bird. The European robin (Erithacus rubecula), the common blackbird (Turdus merula), and the common redstart (Phoenicurus phoenicurus) had the highest infestation intensities. 60.9% of the ticks were PCR-positive for at least one tick-borne pathogen. Borrelia DNA was found in 36.9% of the ticks. The Borrelia species detected were B. spielmanii (15.1%), B. valaisiana (13.4%), B. garinii (12.3%), B. burgdorferi s.s. (2.2%), B. miyamotoi (1.1%), and B. afzelii (0.6%). In addition, 10.6% and 1.7% of the samples were PCR-positive for spotted fever group rickettsiae and Candidatus Neoehrlichia mikurensis. All of the tick-borne pathogens that we found in the present study are known to occur in Danish forest populations of I. ricinus. Our study indicates that migrating birds can transport ticks and their pathogens from neighboring countries to Denmark including sites in Denmark without a sustainable tick population. Thus, a tick-borne pathogen affecting human or animal health emerging at one location in Europe can rapidly be introduced to other countries by migrating birds. These movements are beyond national veterinary control. The current globalization, climatic and environmental changes affect the potential for introduction and establishment of ticks and tick-borne pathogens in Northern Europe. It is therefore important to quantify the risk for rapid spread and long distance exchange of tick-borne pathogens in Europe.

Bacterial species associated with interdigital phlegmon outbreaks in Finnish dairy herds.

BACKGROUND: Severe outbreaks of bovine interdigital phlegmon (IP) have occurred recently in several free stall dairy herds in Finland. We studied the aetiology of IP in such herds, and the association of bacterial species with the various stages of IP and herds of various morbidity of IP. Nineteen free stall dairy herds with IP outbreaks and three control herds were visited and bacteriological samples collected from cows suffering from IP (n = 106), other hoof diseases (n = 58), and control cows (n = 64). The herds were divided into high morbidity (morbidity ≥50%) and moderate morbidity groups (9-33%) based on morbidity during the first two months of the outbreak. RESULTS: F. necrophorum subspecies necrophorum was clearly associated with IP in general, and T. pyogenes was associated with the healing stage of IP. Six other major hoof pathogens were detected; Dichelobacter nodosus, Porphyromonas levii, Prevotella melaninogenica, Treponema spp. and Trueperella pyogenes. Most of the samples of acute IP (66.7%) harboured both F. necrophorum and D. nodosus. We found differences between moderate morbidity and high morbidity herds. D. nodosus was more common in IP lesion in high than in moderate morbidity herds. CONCLUSIONS: Our result confirms that F. necrophorum subspecies necrophorum is the main pathogen in IP, but also T. pyogenes is associated with the healing stage of IP. Our results suggest that D. nodosus may play a role in the severity of the outbreak of IP, but further research is needed to establish other bacteriological factors behind these severe outbreaks.

Prevalence of tick-borne viruses in Ixodes ricinus assessed by high-throughput real-time PCR.

Ticks are one of the principal arthropod vectors of human and animal infectious diseases. Whereas the prevalence of tick-borne encephalitis virus in ticks in Europe is well studied, there is less information available on the prevalence of the other tick-borne viruses (TBVs) existing worldwide. The aim of this study was to improve the epidemiological survey tools of TBVs by the development of an efficient high-throughput test to screen a wide range of viruses in ticks.In this study, we developed a new high-throughput virus-detection assay based on parallel real-time PCRs on a microfluidic system, and used it to perform a large scale epidemiological survey screening for the presence of 21 TBVs in 18 135 nymphs of Ixodes ricinus collected from five European countries. This extensive investigation has (i) evaluated the prevalence of four viruses present in the collected ticks, (ii) allowed the identification of viruses in regions where they were previously undetected.In conclusion, we have demonstrated the capabilities of this new screening method that allows the detection of numerous TBVs in a large number of ticks. This tool represents a powerful and rapid system for TBVs surveillance in Europe and could be easily customized to assess viral emergence.

Pathology and bacteria related to digital dermatitis in dairy cattle in all year round grazing system in Brazil.

Digital dermatitis (DD) is one of the main causes of lameness in dairy cattle worldwide, and it is frequently reported in high-yielding, free stall dairy herds from regions with a temperate climate. However, DD is also observed with high prevalence in grazing cattle with a low milk yield in tropical regions. To clarify whether these differences have an impact on the etiology of the disease, we studied DD lesions from all year round grazing cattle of mixed breed in Brazil using high-throughput 16S rRNA gene sequencing and fluorescent in situ hybridization. The study included samples from 66 skin lesions and 5 healthy skins collected from five farms. Both techniques showed Treponema spp. to be the most abundant bacteria, present in all but one of the samples with minimal epidermal alterations. We identified eleven different Treponema strains belonging to the six major phylotypes of Treponema which have all previously been identified in DD lesions. Furthermore, we identify Dichelobacter nodosus in DD lesions by gene sequencing and also by fluorescent in situ hybridization in almost half of biopsy specimens in areas with mild epithelial damage and together with Treponema. The present data support the hypothesis that Treponema constitutes the main pathogen responsible for DD, independent of the environment and region where cows are kept, and it further suggests D. nodosus as another potentially important pathogen.

Identification of Dermacentor reticulatus Ticks Carrying Rickettsia raoultii on Migrating Jackal, Denmark.

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.

Colonization of the bovine uterus by Candida kefyr.

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 content, many macrophages having phagocytized yeast cells were present. Re-examination of the cows did not reveal a persistent infection, so the infection probably resolved spontaneously.

Presence of bacteria in the endometrium and placentomes of pregnant cows.

Bacterial invasion of the bovine uterus during the postpartum period occurs in most cows, but the general consensus is that these bacteria are eliminated before the next pregnancy. The pregnant uterus has therefore hitherto been considered a sterile environment, but this assumption has now been challenged by recent studies in humans, which indicate that bacteria can be present in the placenta of term pregnancies without causing abortion. The aim of the present study was therefore to investigate whether bacteria are present in the uterus of pregnant cows. Specimens were taken from the inter-caruncular endometrium and from placentomes of slaughtered pregnant cows (n = 43) and subjected to histology, fluorescence in situ hybridization and massive parallel sequencing. Bacteria were observed in the tissue from 90.7% (39/43) of the cows by fluorescence in situ hybridization. Fusobacterium necrophorum, Porphyromonas levii and Trueperella pyogenes were located within the endometrium, on the endometrial surface and in the caruncular stroma, but their presence was not associated with inflammation. Data from massive parallel sequencing of the 16S rRNA gene from a subset of 15 cows indicated that the most abundant bacteria were the families Porphyromonadaceae, followed by Ruminococcaceae and Lachnospiraceae. Our results indicate that the bovine uterus is not a sterile environment during pregnancy as previously assumed and that a cow can carry a pregnancy despite the presence of a few potentially pathogenic bacteria in the uterus.

Microbiota Analysis of an Environmental Slurry and Its Potential Role as a Reservoir of Bovine Digital Dermatitis Pathogens.

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 whether 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 and 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 detected only in samples from herds with reported DD problems. 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 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 if they are found only in DD-afflicted herds. The results strongly indicated that DD Treponema spp. are not part of the indigenous slurry and, therefore, do not comprise an infection reservoir in healthy herds. This study applied next-generation sequencing technology to decipher the microbial compositions of environmental slurry of dairy herds with and without digital dermatitis.

Transmission differentials for multiple pathogens as inferred from their prevalence in larva, nymph and adult of Ixodes ricinus (Acari: Ixodidae).

Ixodes ricinus serves as vector for a range of microorganisms capable of causing clinical illness in humans. The microorganisms occur in the same vector populations and are generally affected by the same tick-host interactions. Still, the instars have different host preferences which should manifest in different transmission patterns for various microorganisms in the tick populations, i.e., most microorganisms increase in prevalence rate from larvae to nymphs because their reservoirs are among small mammals and birds that serve as blood hosts for larvae. Other microorganisms, like Anaplasma phagocytophilum, mainly increase in prevalence rates from nymphs to adults, because their reservoirs are larger ungulates that serve as primary blood hosts for nymphs and adults. We sampled a representative sample of ticks from 12 locations on Zealand and Funen, Denmark, and investigated the differences in prevalence rate of infection in larvae, nymphs and adults for multiple pathogens. Prevalence of infection for larvae, nymphs and adults, respectively, was: 0, 1.5 and 4.5% for Borrelia burgdorferi; 0, 4.2 and 3.9% for Borrelia garinii; 0, 6.6 and 6.1% for Borrelia afzelii; 0, 0 and 0.6% for Borrelia valaisiana; 0, 3.7 and 0.6% for Borrelia spielmanii; 0, 0.7 and 1.2% for Babesia divergens; 0, 0, 0.6% for Babesia venatorum; 0, 1.5 and 6.1% for A. phagocytophilum. The results were in general compatible with the hypothesis i.e., that differences in blood host for larvae and nymphs define differences in transmission of infectious agents, but other factors than differences in blood hosts between larvae and nymphs may also be important to consider.

A novel approach to probe host-pathogen interactions of bovine digital dermatitis, a model of a complex polymicrobial infection.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

An investigation of the microbiota in uterine flush samples and endometrial biopsies from dairy cows during the first 7 weeks postpartum.

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.

Potential bacterial core species associated with digital dermatitis in cattle herds identified by molecular profiling of interdigital skin samples.

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.