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23 Págs. Pictures., The understanding of the pathogenic mechanisms and the clinicopathological forms caused by currently circulating African swine fever virus (ASFV) isolates is incomplete. So far, most of the studies have been focused on isolates classified within genotypes I and II, the only genotypes that have circulated outside of Africa. However, less is known about the clinical presentations and lesions induced by isolates belonging to the other twenty-two genotypes. Therefore, the early clinicopathological identification of disease outbreaks caused by isolates belonging to, as yet, not well-characterised ASFV genotypes may be compromised, which might cause a delay in the implementation of control measures to halt the virus spread. To improve the pathological characterisation of disease caused by diverse isolates, we have refined the macroscopic and histopathological evaluation protocols to standardise the scoring of lesions. Domestic pigs were inoculated intranasally with different doses (high, medium and low) of ASFV isolate Ken05/Tk1 (genotype X). To complement previous studies, the distribution and severity of macroscopic and histopathological lesions, along with the amount and distribution of viral antigen in tissues, were characterised by applying the new scoring protocols. The intranasal inoculation of domestic pigs with high doses of the Ken05/Tk1 isolate induced acute forms of ASF in most of the animals. Inoculation with medium doses mainly induced acute forms of disease. A less severe but longer clinical course, typical of subacute forms, characterised by the presence of more widespread and severe haemorrhages and oedema, was observed in one pig inoculated with the medium dose. The severity of vascular lesions (haemorrhages and oedema) induced by high and medium doses was not associated with the amount of virus antigen detected in tissues, therefore these might be attributed to indirect mechanisms not evaluated in the present study. The absence of clinical signs, lesions and detectable levels of virus genome or antigen in blood from the animals inoculated with the lowest dose ruled out the existence of possible asymptomatic carriers or persistently infected pigs, at least for the 21 days period of the study. The results corroborate the moderate virulence of the Ken05/Tk1 isolate, as well as its capacity to induce both the acute and, occasionally, subacute forms of ASF when high and medium doses were administered intranasally., The animal study was funded under an Innovate UK and Genus funded sub-contract from the Roslin Institute and APHA (CSKN0019). This research was also financially supported by the Department for Environment, Food and Rural Affairs (DEFRA), the Scottish Government and the Welsh Government. The development and refinement of the scoring systems was supported by the Swedish Environmental Protection Agency

African swine fever vaccines could pose risk of causing disease and spreading the virus further

Review. et al., Many infectious diseases originating from, or carried by, wildlife affect wildlife conservation and biodiversity, livestock health, or human health. We provide an update on changes in the epidemiology of 25 selected infectious, wildlife-related diseases in Europe (from 2010–16) that had an impact, or may have a future impact, on the health of wildlife, livestock, and humans. These pathogens were selected based on their: 1) identification in recent Europe-wide projects as important surveillance targets, 2) inclusion in European Union legislation as pathogens requiring obligatory surveillance, 3) presence in recent literature on wildlife-related diseases in Europe since 2010, 4) inclusion in key pathogen lists released by the Office International des Epizooties, 5) identification in conference presentations and informal discussions on a group email list by a European network of wildlife disease scientists from the European Wildlife Disease Association, or 6) identification as pathogens with changes in their epidemiology during 2010–16. The wildlife pathogens or diseases included in this review are: avian influenza virus, seal influenza virus, lagoviruses, rabies virus, bat lyssaviruses, filoviruses, canine distemper virus, morbilliviruses in aquatic mammals, bluetongue virus, West Nile virus, hantaviruses, Schmallenberg virus, Crimean-Congo hemorrhagic fever virus, African swine fever virus, amphibian ranavirus, hepatitis E virus, bovine tuberculosis (Mycobacterium bovis), tularemia (Francisella tularensis), brucellosis (Brucella spp.), salmonellosis (Salmonella spp.), Coxiella burnetii, chytridiomycosis, Echinococcus multilocularis, Leishmania infantum, and chronic wasting disease. Further work is needed to identify all of the key drivers of disease change and emergence, as they appear to be influencing the incidence and spread of these pathogens in Europe. We present a summary of these recent changes during 2010–16 to discuss possible commonalities and drivers of disease change and to identify directions for future work on wildlife-related diseases in Europe. Many of the pathogens are entering Europe from other continents while others are expanding their ranges inside and beyond Europe. Surveillance for these wildlife-related diseases at a continental scale is therefore important for planet-wide assessment, awareness of, and preparedness for the risks they may pose to wildlife, domestic animal, and human health., F.R.F. was funded by the Spanish Ministry for the Economy, Industry and Competitiveness (MINECO) through the ‘Ramon y Cajal program.

Wildlife health is important for conservation, healthy ecosystems, sustainable development and biosecurity. It presents unique challenges for national programme governance and delivery because wildlife health not only crosses jurisdictional responsibilities and authorities but also inherently spans multiple sectors of expertise. The World Organisation for Animal Health (OIE) encourages its Members to have wildlife disease monitoring and notification systems. Where national wildlife health surveillance programmes do exist, they vary in scope and size. Evidence-based guidance is lacking on the critical functions and roles needed to meet the OIE's recommendations and other expectations of a national programme. A literature review and consultation with national wildlife health programme leaders identified five key attributes of national programmes: 1) being knowledge and science based; 2) fostering cross-nation equivalence and harmonisation; 3) developing partnerships and national coordination; 4) providing leadership and administration of national efforts; and 5) capacity development. Proposed core purposes include: 1) establishment and communication of the national wildlife health status; 2) leading national planning; 3) centralising information and expertise; 4) developing national networks leading to harmonisation and collaborations; 5) developing wildlife health workforces; and 6) centralising administration and management of national programmes. A national wildlife health programme should aim to identify, effectively communicate and manage the risk to or from a country's wildlife populations. It should generate the appropriate knowledge required to improve the effectiveness of wildlife policies and systems, including identifying and assessing emerging priorities, thus facilitating early warning, preparedness and preventive actions.

We report the first detection of chronic wasting disease (CWD) in Sweden, in three old female moose (Alces alces). Prions (PrPCWD) were detected in brain but not in lymph nodes. The findings are similar to previously described CWD cases in old moose in Norway, where a spontaneous origin is hypothesized.

In France, tularemia is caused by Francisella tularensis subsp. holarctica and is a sporadic disease affecting mainly wildlife animals and humans. F. tularensis species presents low genetic diversity that remains poorly described in France, as only a few genomes of isolates from the country are available so far. The objective of this study was to characterize the genetic diversity of F. tularensis in France and describe the phylogenetic distribution of isolates through whole-genome sequencing and molecular typing. Whole genomes of 350 strains of human or animal origin, collected from 1947 to 2018 in France and neighboring countries, were sequenced. A preliminary classification using the established canonical single nucleotide polymorphism (canSNP) nomenclature was performed. All isolates from France (except four) belonged to clade B.44, previously described in Western Europe. To increase the resolution power, a whole-genome SNP analysis was carried out. We were able to accurately reconstruct the population structure according to the global phylogenetic framework, and highlight numerous novel subclades. Whole-genome SNP analysis identified 87 new canSNPs specific to these subclades, among which 82 belonged to clade B.44. Identifying genomic features that are specific to sublineages is highly relevant in epidemiology and public health. We highlighted a large number of clusters among a single clade (B.44), which shows for the first time some genetic diversity among F. tularensis isolates from France, and the star phylogeny observed in clade B.44-subclades revealed that F. tularensis biodiversity in the country is relatively recent and resulted from clonal expansion of a single population. No association between clades and hosts or clinical forms of the disease was detected, but spatiotemporal clusters were identified for the first time in France. This is consistent with the hypothesis of persistence of F. tularensis strains found in Western Europe in the environment, associated with slow replication rates. Moreover, the presence of identical genotypes across long periods of time, and across long distances, supports this hypothesis but also suggests long-distance dispersal of the bacterium. This work was supported by the French National Research Agency (ANR) and the Direction Générale de l’Armement (DGA) (No. ANR-15-ASTR-0021-01). MK is a Ph.D. student co-supported by Université Paris-Est and DGA grants.

SUMMARYTularemia caused by the bacteriumFrancisella tularensisis a zoonotic disease. Tularemia is a common disease in the hare, and as a game species can be an important source of infection for humans. In this study, hares diagnosed with tularemia were examined with the aim to investigate whether the muscle (meat) had any pathological changes and/or containedF. tularensis. Real-time PCR and/or immunohistochemistry (IHC) detected the bacteria in muscle samples from 40 out of 43 investigated hares. IHC showed that bacteria were few and most commonly located in the peri- and endomysium. Histopathology showed occasional perimysial necroses and mild inflammation in association to the bacteria. Attempts to culture from 14 muscle samples were successful in two cases, both stored in the freezer F. tularensisis present in the muscle of infected hares, there is a risk for human infection when consuming undercooked hare meat. The risk is enhanced by the fact that some hares do not have easily detected gross lesions. The study contributes to a better understanding of sources of infection and risk factors for humans to contract tularemia.

Lagoviruses belong to the Caliciviridae family. They were first recognized as highly pathogenic viruses of the European rabbit (Oryctolagus cuniculus) and European brown hare (Lepus europaeus) that emerged in the 1970–1980s, namely, rabbit haemorrhagic disease virus (RHDV) and European brown hare syndrome virus (EBHSV), according to the host species from which they had been first detected. However, the diversity of lagoviruses has recently expanded to include new related viruses with varying pathogenicity, geographic distribution and host ranges. Together with the frequent recombination observed amongst circulating viruses, there is a clear need to establish precise guidelines for classifying and naming lagovirus strains. Therefore, here we propose a new nomenclature based on phylogenetic relationships. In this new nomenclature, a single species of lagovirus would be recognized and called Lagovirus europaeus. The species would be divided into two genogroups that correspond to RHDV- and EBHSV-related viruses, respectively. Genogroups could be subdivided into genotypes, which could themselves be subdivided into phylogenetically well-supported variants. Based on available sequences, pairwise distance cutoffs have been defined, but with the accumulation of new sequences these cutoffs may need to be revised. We propose that an international working group could coordinate the nomenclature of lagoviruses and any proposals for revision., This work was supported in part by a grant from the Agence Nationale de la Recherche (France), Calilago and by a grant from the Région des Pays de la Loire (France) Armina to JLP. It was performed within the framework of the ECALEP project selected during the 2nd joint call of the Animal Health and Welfare ERA-Net (Anihwa) initiative, a Coordination Action funded under the European Commission’s ERA-Net scheme within the Seventh Framework Programme (Contract No. 291815). The ECALEP project is funded by the Agence Nationale de la Recherche (France), the Ministry of Health, Depa for Veterinary Public Health, Nutrition and Food Safety (Italy) and the Research council FORMAS (Sweden). FCT-Foundation for Science and Technology, Portugal, supported the FCT Investigator grant of JA (ref. IF/01396/2013) and the Post-doc grant of AML (SFRH/BPD/115211/2016).

Background Transarterial particle embolization is a common treatment of uterine fibroids, aiming to obtain ischemia resulting in shrinking of the fibroid with preservation of normal uterine tissue. Embolization with non-degradable microspheres is established, but causes permanent occlusion of the arteries, affecting both the uterus as well as the fibroids. Purpose To evaluate in vivo degradation, local tissue effects, and possible recanalization following intra-arterial deposition of the new, degradable starch microspheres (DSM), in a short-term experimental pilot study. Material and Methods Under general anesthesia, unilateral transarterial embolization of the uterine artery (UA) with DSM 500–700 µm was performed in five female sheep. The animals underwent renewed angiography at different intervals after embolization (19–65 h) and were subsequently sacrificed. Histological examination was performed. Results Embolization with absent flow in the UA could be completed in five of six animals. At final angiographic evaluation, recanalization of the embolized arteries was evident in three sheep. At the gross postmortem examination, edema and discoloration indicating ischemia of the uterus at the embolized side, was observed in all the sheep. At histopathological examination, different stages of DSM degradation in the arterial branches were observed in both endometrium and myometrium. Mild-to-moderate vasculitis and mild-to-extensive ischemic changes were present along with degeneration of the uterine glands. Conclusion This short-term pilot study proved efficacy of embolization with DSM causing ischemic changes in the embolized organ, but also degradation of the DSM with subsequent recanalization of the embolized arteries.

Background Due to increasing popularity in Sweden during the last decade, alpacas are frequently encountered by practising veterinarians and pathologists. Knowledge regarding their health and diseases under Swedish conditions is, however, limited. Objectives To improve knowledge about the health of alpacas in Sweden by collecting information on diseases and health status. Design A retrospective study was made of 93 necropsies conducted on alpacas in Sweden during the period 2001–2013. Setting Data were obtained from the two major veterinary pathology centres in Sweden. The alpacas were hobby or farm animals and they were submitted by veterinarians in local practices or at a national animal healthcare organisation. Results The digestive system was most frequently affected (29 per cent), with parasitic gastroenteritis (17 per cent) and hepatic disease being especially prevalent (15 per cent fascioliasis and 7 per cent hepatitis). Cardiovascular conditions (9 per cent), systemic diseases (7 per cent) and perinatal deaths were also common, including abortions (10 per cent) and fatal septicaemia (4 per cent). Wasting/emaciation was a frequent finding (26 per cent). Other diagnoses included dermatitis (8 per cent), diseases of the central nervous system (8 per cent), traumatic injuries (7 per cent), neoplasia (5 per cent), pneumonia (5 per cent) and nephritis (3 per cent). Conclusions This study identified areas of concern regarding diagnostic and pathological procedures, for which specific measures have been recommended. One particular cause for concern was the number of deaths from emaciation in weanling alpacas during late winter or early spring. For adult alpacas, infectious and non-infectious causes of death were approximately equally frequent. Many of the diseases were considered clinically acute but pathology often showed them to be chronic conditions that had eventually deteriorated and presented as acute cases in the late stages. This study revealed similarities in the health/disease status reported in other European countries and in North America. The results can be used by alpaca keepers and veterinary practitioners to improve management, diagnosis and treatment of alpacas.

International audience; AbstractLagovirus europaeus GI.2, also known as RHDV2 or RHDVb, is an emerging virus that causes rabbit haemorrhagic disease (RHD) in European rabbits (Oryctolagus cuniculus). In contrast to L. europaeus GI.1 (or RHDV/RHDVa) viruses that are only pathogenic for adults, GI.2 causes clinical disease in both adults and kittens. However, detailed descriptions of the pathology of this virus that may provide insight into its pathogenicity and emergence are lacking. Using an Australian GI.2 field strain isolated in 2015, we provide the first detailed description of pathology, viral antigen distribution and tissue load of GI.2 in adult and 5-week old New Zealand white rabbits using histology, immunohistochemistry and RT-qPCR. Liver was the target organ, but in contrast to GI.1 viruses, lesions and inflammatory responses did not differ between adults and kittens. Lymphocytic inflammation, proposed to be protective in kittens infected with GI.1, was notably absent. We also present the first descriptions of bone marrow changes in RHD, including decreased myeloid-to-erythroid ratio. Consistent with other pathogenic lagoviruses, intracellular viral antigen was demonstrated in hepatocytes and cells of the mononuclear phagocytic system. In terminal stages of disease, viral loads were highest in liver, serum and spleen. Despite the small sample size, our data suggest that unlike early European GI.2 strains, the pathogenicity of the Australian GI.2 virus is similar to GI.1 viruses. Additionally, GI.2 was fatal for all (n = 5) inoculated kittens in this study. This may significantly alter RHD epidemiology in the field, and may impact biocontrol programs for invasive rabbits in Australia where GI.1 viruses are intentionally released.

Summary Rabbit haemorrhagic disease virus (RHDV) is a lagovirus that can cause fatal hepatitis (rabbit haemorrhagic disease, RHD) with mortality of 80–90% in farmed and wild rabbits. Since 1986, RHDV has caused outbreaks in rabbits (Oryctolagus cuniculus) in Europe, but never in European brown hares (Lepus europaeus, EBH). In 2010, a new RHDV‐related virus, called RHDV2, emerged in Europe, causing extended epidemics because it largely overcame the immunity to RHDV present in most rabbit populations. RHDV2 also was identified in Cape hare (Lepus capensis subsp. mediterraneus) and in Italian hare (Lepus corsicanus). Here, we describe two distinct incidents of RHDV2 infection in EBH that occurred in Italy (2012) and Spain (2014). The two RHDV2 strains caused macroscopic and microscopic lesions similar to European brown hare syndrome (EBHS) in hares, and they were genetically related to other RHDV2 strains in Europe. EBHs are common in Europe, often sharing habitat with rabbits. They likely have been exposed to high levels of RHDV2 during outbreaks in rabbits in recent years, yet only two incidents of RHDV2 in EBHs have been found in Italy and Spain, suggesting that EBHs are not a primary host. Instead, they may act as spillover hosts in situations when infection pressure is high and barriers between rabbits and hares are limited, resulting in occasional infections causing EBHS‐like lesions. The serological survey of stocked hare sera taken from Italian and Spanish hare populations provided an understanding of naturally occurring RHDV2 infection in the field confirming its sporadic occurrence in EBH. Our findings increase the knowledge on distribution, host range and epidemiology of RHDV2.

Ceftaroline is a new cephalosporin with activity against methicillin-resistant Staphylococcus aureus (MRSA). A collection of 17 clinical and veterinary mecC-positive MRSA isolates was tested to evaluate the in vitro efficacy of ceftaroline against recently emerged mecC-MRSA isolates. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of ceftaroline for the 17 isolates were determined by broth microdilution using the methodology and interpretive criteria of the Clinical and Laboratory Standards Institute (CLSI). Additional susceptibility tests were performed using ceftaroline M.I.C.Evaluator (M.I.C.E.™) strips. All isolates showed susceptibility according to CLSI breakpoints, with MICs of ceftaroline ranging from 0.125mg/L to 0.25mg/L. MBCs were identical or up to a twofold dilution step higher. In conclusion, all tested isolates, from various sources and belonging to several clonal complexes (CCs), but predominantly to CC130, were found to be susceptible to ceftaroline. Ceftaroline could thus be an option for the treatment of mecC-MRSA infections.

1. Wildlife disease surveillance is the first line of defence against infectious disease. Fluctuations in host populations and disease prevalence are a known feature of wildlife disease systems. However, the impact of such heterogeneities on the performance of surveillance is currently poorly understood. 2. We present the first systematic exploration of the effects of fluctuations prevalence and host population size on the efficacy of wildlife disease surveillance systems. In this study efficacy is measured in terms of ability to estimate long term prevalence and detect disease risk. 3. Our results suggest that for many wildlife disease systems fluctuations in population size and disease lead to bias in surveillance-based estimates of prevalence and over-confidence in assessments of both the precision of prevalence estimates and the power to detect disease. 4. Neglecting such ecological effects may lead to poorly designed surveillance and ultimately to incorrect assessments of the risks posed by disease in wildlife. This will be most problematic in systems where prevalence fluctuations are large and disease fade-outs occur. Such fluctuations are determined by the interaction of demography and disease dynamics and although particularly likely in highly fluctuating populations typical of fecund short lived hosts, can’t be ruled out in more stable populations of longer lived hosts. 5. Synthesis and Applications: Fluctuations in population size and disease prevalence should be considered in the design and implementation of wildlife disease surveillance and the framework presented here provides a template for conducting suitable power calculations. Ultimately understanding the impact of fluctuations in demographic and epidemiological processes will enable improvements to wildlife disease surveillance systems leading to better characterisation of, and protection against endemic, emerging and re-emerging disease threats.

Background Prior to 2010, the lagoviruses that cause rabbit hemorrhagic disease (RHD) in European rabbits (Oryctolagus cuniculus) and European brown hare syndrome (EBHS) in hares (Lepus spp.) were generally genus-specific. However, in 2010, rabbit hemorrhagic disease virus 2 (RHDV2), also known as Lagovirus europaeus GI.2, emerged and had the distinguishing ability to cause disease in both rabbits and certain hare species. The mountain hare (Lepus timidus) is native to Sweden and is susceptible to European brown hare syndrome virus (EBHSV), also called Lagovirus europaeus GII.1. While most mountain hare populations are found on the mainland, isolated populations also exist on islands. Here we investigate a mortality event in mountain hares on the small island of Hallands Väderö where other leporid species, including rabbits, are absent. Results Post-mortem and microscopic examination of three mountain hare carcasses collected from early November 2016 to mid-March 2017 revealed acute hepatic necrosis consistent with pathogenic lagovirus infection. Using immunohistochemistry, lagoviral capsid antigen was visualized within lesions, both in hepatocytes and macrophages. Genotyping and immunotyping of the virus independently confirmed infection with L. europaeus GI.2, not GII.1. Phylogenetic analyses of the vp60 gene grouped mountain hare strains together with a rabbit strain from an outbreak of GI.2 in July 2016, collected approximately 50 km away on the mainland. Conclusions This is the first documented infection of GI.2 in mountain hares and further expands the host range of GI.2. Lesions and tissue distribution mimic those of GII.1 in mountain hares. The virus was most likely initially introduced from a concurrent, large-scale GI.2 outbreak in rabbits on the adjacent mainland, providing another example of how readily this virus can spread. The mortality event in mountain hares lasted for at least 4.5 months in the absence of rabbits, which would have required virus circulation among mountain hares, environmental persistence and/or multiple introductions. This marks the fourth Lepus species that can succumb to GI.2 infection, suggesting that susceptibility to GI.2 may be common in Lepus species. Measures to minimize the spread of GI.2 to vulnerable Lepus populations therefore are prudent. Electronic supplementary material The online version of this article (10.1186/s12917-018-1694-7) contains supplementary material, which is available to authorized users.

Background Transarterial embolization with particles is a relatively common treatment method in both malignant and benign disorders. Permanent occlusion of the uterine arteries may sometimes be disadvantageous. Purpose To compare the local tissue effects, possible side effects, and extent of recanalization following uterine artery embolization, using either degradable or non-degradable microspheres in a sheep model. Material and Methods In 22 female sheep, the uterine artery (UA) was unilaterally, superselectively embolized, with either degradable starch microspheres-DSM (group A) or calibrated gelatin coated spherical shape tris-acryl microspheres-TGMS (group B). The completion of embolization was confirmed by angiography. The animals were kept in the animal research facilities for 14 days and sacrificed following new angiographic evaluation. Gross and histological examination of the uterus and other organs was performed. Results The procedure was successful in all animals. At final angiographic evaluation recanalization was found in 82% of the ewes in group A and in 18% in group B. At histopathological examination, tissue impairment was similar in both groups, whereas vascular changes were more pronounced in the TGMS-group. Conclusion Embolization with DSM was associated with significantly higher degree of recanalization, than after embolization with TGMS.

STAT3 is a master regulator of the immune responses. Here we show that M. tuberculosis-infected stat3fl/fl lysm cre mice, defective for STAT3 in myeloid cells, contained lower bacterial load in lungs and spleens, reduced granuloma extension but higher levels of pulmonary neutrophils. STAT3-deficient macrophages showed no improved control of intracellular mycobacterial growth. Instead, protection associated to elevated ability of stat3fl/fl lysm cre antigen-presenting cells (APCs) to release IL-6 and IL-23 and to stimulate IL-17 secretion by mycobacteria-specific T cells. The increased IL-17 secretion accounted for the improved control of infection since neutralization of IL-17 receptor A in stat3fl/fl lysm cre mice hampered bacterial control. APCs lacking SOCS3, which inhibits STAT3 activation via several cytokine receptors, were poor inducers of priming and of the IL-17 production by mycobacteria-specific T cells. In agreement, socs3fl/fl cd11c cre mice deficient of SOCS3 in DCs showed increased susceptibility to M. tuberculosis infection. While STAT3 in APCs hampered IL-17 responses, STAT3 in mycobacteria-specific T cells was critical for IL-17 secretion, while SOCS3 in T cells impeded IL-17 secretion. Altogether, STAT3 signalling in myeloid cells is deleterious in the control of infection with M. tuberculosis., Author summary We studied the role of STAT3, a major regulator of immunity, in the control of the infection with M. tuberculosis. Stat3fl/fl lysm cre mice, deficient in STAT3 in myeloid cells, showed lower bacterial levels in organs and reduced extension of lung granulomas after infection with M. tuberculosis. STAT3-deficient APCs stimulated with innate receptor agonists released high levels of IL-6 and IL-23, and promoted IL-17 production by mycobacteria-specific CD4+ T cells. Increased IL-17 levels accounted for the increased resistance to M. tuberculosis of the STAT3-deficient mice. Instead, stat3fl/fl lysm cre macrophages showed no improved control of mycobacterial growth. SOCS3 is a negative regulator of STAT3 activation. The ability of socs3fl/fl lysm cre APCs to secrete IL-6 and IL-23 and to stimulate IL-17 production by antigen-specific T cells was reduced. In agreement, mice lacking SOCS3 in DCs showed increased susceptibility to M. tuberculosis infection. Different to a role in myeloid cells, STAT3 expression by mycobacteria-specific T cells was required for IL-17 secretion while SOCS3 in T cells hampered IL-17 production. Therefore, despite STAT3 expression in T cells is required for Th17 differentiation, STAT3 in APCs hampers secretion of Th17 promoting cytokines and the secretion of IL-17 by mycobacteria-specific T cells and reduces the resistance of mice to infection with M. tuberculosis.

Tularaemia is an emerging zoonotic infectious disease caused by the bacterium Francisella tularensis. In Sweden, hares are considered to be key species in the epidemiology of tularaemia. The aim of this study was to characterize the pathology of natural tularaemia infection in European brown hares (EBHs) (Lepus europaeus) and mountain hares (MHs) (Lepus timidus) in Sweden, in order to better understand the presentation of disease and the routes of infection, body dissemination and shedding of F. tularensis. During 2000-2013, 49 EBHs and 37 MHs were diagnosed with tularaemia. Enlargement of the spleen was seen in 80% of EBHs and 62% of MHs. Necrosis was often obvious in the bone marrow, liver, lung and spleen, but 30% of the hares had no lesions or minimal gross lesions. On microscopical examination of tissues from 27 EBHs and three MHs, necrosis was seen in the majority of samples of liver, spleen, bone marrow, lymph node and adrenal glands and was common in the lungs and brain meninges. Immunohistochemistry for Francisella spp. detected bacteria in association with necrosis and inflammation. In several cases, Francisella spp. were also found inside blood vessels, in the renal pelvis, in lactating mammary glands, in bronchioles and in the skin, associated with tick bites. Using quantitative polymerase chain reaction, two genotypes of F. tularensis subsp. holarctica were found; canSNP group B.6, all belonging to subgroup B.7, and canSNP group B.12. There were no differences in pathology between the genotypes. Our results indicate that the urinary tract and mammary glands are important routes for the shedding of F. tularensis. Hunters may not be aware of the risks of contracting tularaemia while handling hares, since infected hares do not always show noticeable gross lesions.

International audience; European brown hare syndrome virus(EBHSV) is the aetiological agent of European brown hare syndrome (EBHS), a disease affecting Lepus europaeus and Lepus timidus first diagnosed in Sweden in 1980. To characterize EBHSV evolution we studied hare samples collected in Sweden between 1982 and2008. Our molecular clock dating is compatible with EBHSV emergence in the 1970s. Phylogenetic analysis revealed two lineages: Group A persisted until 1989 when it apparently suffered extinction; Group B emerged in the mid-1980s and contains the most recent strains. Antigenic differences exist between groups, with loss of reactivity of some MAbs overtime, which are associated with aminoacid substitutions in recognized epitopes. A role for immune selection is also supported by the presence of positively selected codons in exposed regions of the capsid. Hence, EBHSV evolution is characterized by replacement of Group A by Group B viruses, suggesting that the latter possess a selective advantage.

Seasonal variation in reproduction is common in mammals as an adaptation to annual changes in the habitat. In lynx, male reproduction activity is of special interest because female lynxes are monoestric with an unusual narrow (about 1 month) breeding season. In Eurasian lynx, mating occurs between January and April depending on the latitude. To characterize the seasonal pattern of sperm and testosterone production in free-ranging Eurasian lynxes, long-term frozen-stored testis material obtained postmortem from 74 hunted or road-killed lynxes in Sweden was used to analyze annual changes in testis mass, testicular testosterone content, and spermatogenetic activity. Values of most gonadal parameters obtained in subadult lynxes were significantly different from the values observed in adult males. In adult lynxes, a moderate annual fluctuation of gonadal parameters was found which was most profound for testis weight and testicular testosterone concentration reaching highest values in March (median of 2.18 g and 2.67 μg/g tissue respectively). Grouping the data of pre-/breeding (January–April) and postbreeding season (May–September) revealed significant changes in testis weight and testosterone concentration. The relative spermatogenetic activity remained high in postbreeding testes. However, net sperm production decreased according to reduction of testis mass and a tendency to lower cauda epididymal sperm numbers in the postbreeding period was observed. Our results demonstrate that it is possible to analyze the gonadal activity of frozen testis/epididymis tissue postmortem and that male Eurasian lynxes show—opposite to the females—only moderate seasonal changes in their reproductive capacity.

Incursion of rabbit haemorrhagic disease virus (RHDV) into Sweden was documented in 1990 and it is now considered endemic in wild rabbit (Oryctolagus cuniculus) populations. Rabbit haemorrhagic disease virus 2 (RHDV2), a new, related lagovirus was first detected in France in 2010, and has spread rapidly throughout Europe and beyond. However, knowledge of RHDV2 in northern Europe is sporadic and incomplete, and in Sweden, routinely available diagnostic methods to detect rabbit haemorrhagic disease (RHD) do not distinguish between types of virus causing disease. Using RHDV2-specific RT-qPCR, sequencing of the VP60 gene and immunological virus typing of archived and prospective case material from the National Veterinary Institute's (SVA) wildlife disease surveillance programme and diagnostic pathology service, we describe the emergence of RHDV2 in Sweden in both wild and domestic rabbits. The earliest documented outbreak occurred on 22 May 2013, and from May 2013 to May 2016, 10 separate incidents of RHDV2 were documented from six different municipalities in the southern half of Sweden. Phylogenetic analysis of the VP60 gene shows clear clustering of Swedish isolates into three separate clusters within two different clades according to geographic location and time, suggesting viral evolution, multiple introduction events or both. Almost all cases of RHD examined by SVA from May 2013 to May 2016 were caused by RHDV2, suggesting that RHDV2 may be replacing RHDV as the predominant cause of RHD in Sweden.

Staphylococcus aureus is a well-known colonizer and cause of infection among animals and it has been described from numerous domestic and wild animal species. The aim of the present study was to investigate the molecular epidemiology of S. aureus in a convenience sample of European wildlife and to review what previously has been observed in the subject field. 124 S. aureus isolates were collected from wildlife in Germany, Austria and Sweden; they were characterized by DNA microarray hybridization and, for isolates with novel hybridization patterns, by multilocus sequence typing (MLST). The isolates were assigned to 29 clonal complexes and singleton sequence types (CC1, CC5, CC6, CC7, CC8, CC9, CC12, CC15, CC22, CC25, CC30, CC49, CC59, CC88, CC97, CC130, CC133, CC398, ST425, CC599, CC692, CC707, ST890, CC1956, ST2425, CC2671, ST2691, CC2767 and ST2963), some of which (ST2425, ST2691, ST2963) were not described previously. Resistance rates in wildlife strains were rather low and mecA-MRSA isolates were rare (n = 6). mecC-MRSA (n = 8) were identified from a fox, a fallow deer, hares and hedgehogs. The common cattle- associated lineages CC479 and CC705 were not detected in wildlife in the present study while, in contrast, a third common cattle lineage, CC97, was found to be common among cervids. No Staphylococcus argenteus or Staphylococcus schweitzeri-like isolates were found. Systematic studies are required to monitor the possible transmission of human- and livestock- associated S. aureus/MRSA to wildlife and vice versa as well as the possible transmission, by unprotected contact to animals. The prevalence of S. aureus/MRSA in wildlife as well as its population structures in different wildlife host species warrants further investigation.

Tularaemia is a zoonotic disease, in Europe caused byFrancisella tularensissubsp.holarctica. Many lagomorphs and a variety of small rodents are wildlife species prone to develop clinical disease, while predators and scavengers are relatively resistant and may serve as sentinels. Blood samples from 656 Swedish wild predators and scavengers were serologically investigated using slide agglutination and microagglutination. In the slide agglutination test, 34 seropositive animals were detected, and they were found among all species investigated: brown bear (Ursus arctos), Eurasian lynx (Lynx lynx), raccoon dog (Nyctereutes procyonoides), red fox (Vulpes vulpes), wild boar (Sus scrofa), wolf (Canis lupus) and wolverine (Gulo gulo). Due to haemolysis the microagglutination test was more difficult to read at low titres, and only 12 animals were classified as seropositive.F. tularensissubsp.holarcticawas detected by a polymerase chain reaction in lymphatic tissues of the head in one brown bear, one red fox and one wolf. The significance of this finding regarding possible latency of infection is not clear. In conclusion, the results of this study indicate that all predator and scavenger species included in this study may serve as sentinels for tularaemia in Sweden. Their role as reservoirs is unclear.

European brown hare syndrome (EBHS) is characterised by high mortality of European brown hares (Lepus europaeus) and mountain hares (Lepus timidus). European brown hare syndrome virus (EBHSV) and the closely related rabbit haemorrhagic disease virus (RHDV) comprise the genus Lagovirus, family Caliciviridae. In contrast to RHDV, which is well studied, with more than 30 complete genome sequences available, the only complete genome sequence available for EBHSV was obtained from a strain isolated in 1989 in France. EBHS was originally diagnosed in Sweden in 1980. Here, we report the complete coding sequences of two EBHSV strains isolated from European brown hares that died with liver lesions characteristic of EBHS in Sweden in 1982. These sequences represent the oldest complete coding sequences of EBHSV isolated from the original area of virus diagnosis. The genomic organisation is similar to that of the published French sequence. Comparison with this sequence revealed several nucleotide substitutions, corresponding to 6 % divergence. At the amino acid level, the Swedish strains are 2 % different from the French strain. Most amino acid substitutions were located within the major capsid protein VP60, but when considering the amino acid sequence length of each protein, VP10 is the protein with the highest percentage of amino acid differences. The same result was obtained when Swedish strains were compared. This evolutionary pattern has not been described previously for members of the genus Lagovirus.

We have used humanized mice, in which human immune cells differentiate de novo from transplanted cord blood progenitor cells, to study the human immune responses to infection with Mycobacterium bovis bacillus Calmette–Guérin and Mycobacterium tuberculosis . Granulomas with a core containing giant cells, human CD68 + macrophages, and high bacilli numbers surrounded by a layer of CD3 + T cells and a fibrotic response encapsulating the lesions were observed in livers and lungs from bacillus Calmette–Guérin-infected humanized mice but not in nonhumanized infected controls. Paradoxically, humanized mice contained higher mycobacterial numbers in organs than nonhumanized controls. The enhancement of bacterial load was mediated by human CD4 + cells and associated to an increased expression of Programmed Death-1 protein and CD57 on T cells, molecules associated with inhibition and senescence. The lesions from mice depleted of CD4 + cells were scarcer, minimal, and irregular compared with those from mice depleted of CD8 + cells or nondepleted controls. Granulomas of bacillus Calmette–Guérin-infected humanized mice administered with a TNF-neutralizing TNF receptor fusion molecule preserved their structure, but contained higher levels of intracellular bacilli. Extended necrosis was observed in granulomas from M. tuberculosis - but not bacillus Calmette–Guérin-infected humanized mice. Our data indicate that humanized mice can be used as a model to study the formation and maintenance of human granuloma in tuberculosis and other infectious or noninfectious diseases.

Low-pathogenic avian influenza (LPAI) viruses in wild birds are important as they can constitute the basis for the development of highly pathogenic avian influenza viruses or form part of human-adapted strains with pandemic potential. However, the pathogenesis of LPAI viruses is not well characterized in dabbling ducks, one of the natural reservoirs of LPAI viruses. Between 21 September 2009 and 21 December 2009, we used real-time reverse transcriptase polymerase chain reaction (q-PCR), histopathology, and immunohistochemistry (IHC) to study Mallards (Anas platyrhynchos) infected with an influenza A/H1N1 virus isolated from a wild Mallard in Sweden. The ducks were either inoculated intraesophageally ("artificial infection") or infected by virus shed by other ducks in the experiment ("contact infection"). The ducks were subjected to three low concentrations (80 ng/L, 1 μg/L, and 80 μg/L) of the active metabolite of oseltamivir (Tamiflu(®)), oseltamivir carboxylate (OC), which resulted in the development of the viral resistance mutation H274Y at 1 and 80 μg/L. The LPAI virus infection was localized to the intestinal tract and cloacal bursa except in one Mallard. The exception was a duck euthanized 1 day postinoculation, whose infection was located solely in the lung, possibly due to intratracheal deposition of virus. The intestinal infection was characterized by occasional degenerating cells in the lamina propria and presence of viral antigen as detected by IHC, as well as positive q-PCR performed on samples from feces and intestinal contents. Histopathologic changes, IHC positivity, and viral shedding all indicated that the infection peaked early, around 2 days postinfection. Furthermore, more viral antigen and viral RNA were detected with IHC and q-PCR in the proximal parts early in the infection. There was no obvious difference in the course of the infection in artificial versus contact infection, when the level of OC was increased from 80 ng/L to 1 μg/L (based on IHC and q-PCR), when the level of OC was increased to 80 μg/L, or when the resistance mutation H274Y developed (based on q-PCR).

Infectious disease surveillance is key to limiting the consequences from infectious pathogens and maintaining animal and public health. Following the detection of a disease outbreak, a response in proportion to the severity of the outbreak is required. It is thus critical to obtain accurate information concerning the origin of the outbreak and its forward trajectory. However, there is often a lack of situational awareness that may lead to over- or under-reaction. There is a widening range of tests available for detecting pathogens, with typically different temporal characteristics, e.g. in terms of when peak test response occurs relative to time of exposure. We have developed a statistical framework that combines response level data from multiple diagnostic tests and is able to ‘hindcast’ (infer the historical trend of) an infectious disease epidemic. Assuming diagnostic test data from a cross-sectional sample of individuals infected with a pathogen during an outbreak, we use a Bayesian Markov Chain Monte Carlo (MCMC) approach to estimate time of exposure, and the overall epidemic trend in the population prior to the time of sampling. We evaluate the performance of this statistical framework on simulated data from epidemic trend curves and show that we can recover the parameter values of those trends. We also apply the framework to epidemic trend curves taken from two historical outbreaks: a bluetongue outbreak in cattle, and a whooping cough outbreak in humans. Together, these results show that hindcasting can estimate the time since infection for individuals and provide accurate estimates of epidemic trends, and can be used to distinguish whether an outbreak is increasing or past its peak. We conclude that if temporal characteristics of diagnostics are known, it is possible to recover epidemic trends of both human and animal pathogens from cross-sectional data collected at a single point in time., Author Summary We have developed a Bayesian approach that can estimate the historic trend of incidence from cross-sectional samples, without relying on ongoing surveillance. This could be used to evaluate changing disease trends, or to inform outbreak responses. We combine two or more diagnostic tests to estimate the time since infection for the individual, and the historic incidence trend in the population as a whole. We evaluate this procedure by applying it to simulated data from synthetic epidemics. Further, we evaluate its real-world applicability by applying it to two scenarios modelled after the UK 2007 bluetongue epidemic, and a small outbreak of whooping cough in Wisconsin, USA. We were able to recover the epidemic trends under a range of conditions using sample sizes of 30–100 individuals. In the scenarios modelled after real-world epidemics, the hindcasted epidemic curves would have provided valuable information about the distribution of infections. The described approach is generic, and applicable to a wide range of human, livestock and wildlife diseases. It can estimate trends in settings for which this is not possible using current methods, including for diseases or regions lacking in surveillance; recover the pattern of spread during the initial “silent” phase once an outbreak is detected; and can be used track emerging infections. Being able to estimate the past trends of diseases from single cross-sectional studies has far-reaching consequences for the design and practice of disease surveillance in all contexts.

Lynx presents a unique sexual cycle with persistentcorpora lutea(CLs) and elevated serum progesterone (P4) throughout parturition and lactation. In other mammals, CLs normally disintegrate after parturition, therefore the aim of our study was to characterise the annual life cycle of lynx CLs. Ovaries from Eurasian lynxes were obtained from the National Veterinary Institute in Sweden, where tissues from killed lynx were stored at −20 °C. Ovaries from 66 animals were weighed; eachcorpus luteumwas segmented for histology and hormone analysis. Ovary and CLs weights were constant throughout the year, peaking during pregnancy. In non-pregnant lynxes, the seasonal level of intraluteal steroids was steady for P4(3.2±1.9 s.d. μg/g,n=53) and total oestrogens (18.3±15.5 s.d. ng/g,n=53). Within histology slides, structurally intact luteal cells were found throughout the year with the highest incidence in March/April; evidence of luteal regression was predominantly found in post-breeding season. Ovaries from pregnant animals contained two types of CLs. Group A was bigger in size with large luteal cells (P4, 72.3±65.4 s.d. μg/g; oestrogen, 454.0±52.4 s.d. ng/g). In contrast, group B were smaller, with greater luteal regression and lower steroid concentrations (P4, 8.3±2.9 s.d. μg/g; oestrogen, 31.5±20.4 s.d. ng/g). Our results suggest that structural luteolysis proceeds throughout the year and into next breeding cycle, resulting in two CLs types on the same ovary.