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.

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.

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.

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.

Concurrent host-pathogen gene expression in the lungs of pigs challenged with Actinobacillus pleuropneumoniae.

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-throughput RT-qPCR for the elucidation of dual-organism gene expression analysis during infection. We showed differential expression of 12 bacterial and 24 porcine genes during infection and significant correlation of porcine and bacterial gene expression. This is the first study investigating the concurrent transcriptional response of both bacteria and host at the site of infection during porcine respiratory infection.

Discovery of bovine digital dermatitis-associated Treponema spp. in the dairy herd environment by a targeted deep-sequencing approach.

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.

Targeting the treponemal microbiome of digital dermatitis infections by high-resolution phylogenetic analyses and comparison with fluorescent in situ hybridization.

Modern pyrosequencing technology allows for a more comprehensive approach than traditional Sanger sequencing for elucidating the etiology of bovine digital dermatitis. We sought to describe the composition and diversity of treponemes in digital dermatitis lesions by using deep sequencing of the V3 and V4 hypervariable regions of the 16S rRNA gene coupled with species-level taxonomic identification. Treponema-specific 16S rRNA gene PCRs and pyrosequencing were performed on biopsy specimens originating from 10 different Catalan dairy herds (n = 36) with digital dermatitis, and this analysis yielded 75,297 sequences. We identified 20 different taxa, including a potentially novel phylotype that displayed 95% sequence identity to members of the Treponema denticola/Treponema pedis-like cluster. Species frequencies and abundances that were determined by pyrosequencing analysis were highly correlated with the results of fluorescent in situ hybridization using phylotype-specific oligonucleotide probes. In a limited number of animals from a single geographic region, we detected most of the Treponema phylotypes that were described in previous investigations of digital dermatitis. Additionally, we identified a number of phylotypes that mapped to oral treponemes of humans and dogs that had not been reported for digital dermatitis lesions. The results presented here support previous observations of a polytreponemal etiology of infections, with Treponema phagedenis-like, Treponema medium/Treponema vincentii-like, and T. denticola/T. pedis-like phylotypes being highly associated with disease. Using this new approach, it has become feasible to study large herds and their surrounding environments, which might provide a basis for a better understanding of the pathogenesis of this disease.

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.

Comparative profiling of the transcriptional response to iron restriction in six serotypes of Actinobacillus pleuropneumoniae with different virulence potential.

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 this bacterium has evolved several strategies for scavenging the limited iron resources of the host. The combined effect of iron-depletion and serotype proved to be modest, indicating that serotypes of both moderate and high virulence at least in vitro are reacting almost identical to iron restriction. One notable exception, however, is the haemoglobin-haptoglobin binding protein cluster which merits further investigation.

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.

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.

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.

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 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.

Evidence of multiple Treponema phylotypes involved in bovine digital dermatitis as shown by 16S rRNA gene analysis and fluorescence in situ hybridization.

The etiopathogenesis of the skin disease digital dermatitis (DD), an important cause of lameness in cattle, remains uncertain. Microscopically, the disease appears to be polymicrobial, with spirochetes as the predominant bacteria. The objective of this study was to identify the main part of the bacteria involved in DD lesions of cattle by using culture-independent molecular methods. Ten different phylotypes of Treponema were identified either by 16S rRNA gene sequencing of bacteria from DD lesions or by fluorescence in situ hybridization (FISH) analysis using phylotype-specific 16S rRNA-directed oligonucleotide probes. Two phylotypes, phylotype 1 (PT1) and PT2, were not closely related to any characterized treponemal species. PT7 was 99.3% identical to Treponema denticola, while PT9 resembled T. vincentii by 96%. The remaining phylotypes, PT3, PT4, PT5, PT6, and PT8, and Treponema brennaborense had previously been isolated from DD lesions. Forty DD biopsy specimens were examined for Treponema by FISH. With one exception, all of the biopsy specimens revealed epidermotropic, intermingled infection with three or more different phylotypes (mean, 4.7). The most prevalent species were PT1 (95%), PT6 (93%), and PT3 (85%). While colonization by PT3 was confined to the surface of the epidermis, both PT1 and PT6 invaded deep into the stratum spinosum and were seen in ulcerated dermal papillae. In two cases, all 10 phylotypes were demonstrated. Furthermore, FISH with a Treponema group-specific probe showed that Treponema accounted for more than 90% of the total bacterial population in the biopsy specimens. These data strongly suggest that a group of apparently symbiotic Treponema species are involved as primary bacterial pathogens in DD.

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.