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Nineteen red deer areas in a densely populated region with a huge network of fenced motorways and the division into administrative management units (AMUs) with restricted ecological connectivity were investigated. In the season 2018/2019, a total of 1291 red deer samples (on average 68 per area) were collected and genotyped using 16 microsatellite markers. The results show a clear genetic differentiation between most of the AMUs. Fourteen AMUs may be combined into four regions with a considerable internal genetic exchange. Five areas were largely isolated or showed only a limited gene flow with neighbouring areas. Ten of the 19 AMUs had an effective population size below 100. Effective population sizes greater than 500–1000, required to maintain the evolutionary potential and a long-term adaptation potential, were not achieved by any of the studied AMUs, even when AMUs with an appreciable genetic exchange were aggregated. Substantial genetic differentiation between areas can be associated with the presence of landscape barriers hindering gene flow, but also with the maintenance of ‘red deer–free’ areas. Efforts to sustainably preserve the genetic diversity of the entire region should therefore focus on measures ensuring genetic connectivity. Opportunities for this goal arise from the establishment of game bridges over motorways and from the protection of young male stags migrating through the statutory ‘red deer–free’ areas.

Actinobacillus (A.) pleuropneumoniae is one of the most important respiratory pathogens in global pig production. Antimicrobial treatment and vaccination provide only limited protection, but genetic disease resistance is a very promising alternative for sustainable prophylaxis. Previous studies have discovered multiple QTL that may explain up to 30% of phenotypic variance. Based on these findings, the aim of the present study was to use genomic sequencing to identify genetic markers for resistance to pleuropneumonia in a segregating commercial German Landrace line. 163 pigs were infected with A. pleuropneumoniae Serotype 7 through a standardized aerosol infection method. Phenotypes were accurately defined on a clinical, pathological and microbiological basis. The 58 pigs with the most extreme phenotypes were genotyped by sequencing (next-generation sequencing). SNPs were used in a genome-wide association study. The study identified genome-wide associated SNPs on three chromosomes, two of which were chromosomes of QTL which had been mapped in a recent experiment. Each variant explained up to 20% of the total phenotypic variance. Combined, the three variants explained 52.8% of the variance. The SNPs are located in genes involved in the pathomechanism of pleuropneumonia. This study confirms the genetic background for the host’s resistance to pleuropneumonia and indicates a potential role of three candidates on SSC2, SSC12 and SSC15. Favorable gene variants are segregating in commercial populations. Further work is needed to verify the results in a controlled study and to identify the functional QTN. Electronic supplementary material The online version of this article (10.1007/s00335-019-09825-0) contains supplementary material, which is available to authorized users.

During routine electron microscopy of fecal samples from diarrheic dogs dated from 2000 virus particles resembling circovirus in shape and size were detected in two samples (V2177/00; V3374/00). Polymerase chain reaction (PCR) using primers specific for porcine circovirus type 2 (PCV2) amplified DNA recovered from both samples. Sequencing of PCR amplificates (V2177/00) obtained with PCV2-specific primer pairs revealed a genome size of 1768 bp. The nucleotide sequence was highly similar (98% nucleotide identity) to the PCV2a reference sequence., Highlights • Electron microscopy has revealed circoviruses in two fecal samples of diarrheic dogs. • PCV2-specific PCR amplified DNA extracted from the samples. • By sequencing one sample a genome size of 1768 bp and a nucleotide sequence identity of 98% to PCV2a were achieved. • This is the first time that PCV2 has been reported in dogs.

Simple Summary Swine inflammation and necrosis syndrome (SINS) is a newly identified syndrome associated with inflammatory and necrotic signs in different body parts of suckling piglets, weaners, and fatteners. While the proven inflammatory basis of the disease and the development of signs in even newborns indicate a primarily endogenous etiology, the mechanisms underlying SINS development are largely unknown. In this study, the hypothesis was tested that SINS is indirectly triggered by the translocation of gut-derived microbial components into the liver, thereby causing derangements of liver metabolism, by comparing the hepatic transcriptome and plasma metabolome of SINS-affected and unaffected piglets. Apart from demonstrating that hepatic inflammation occurs in SINS-affected piglets, the study clearly shows for the first time that several metabolic derangements are induced in SINS-affected piglets that may contribute to the clinical and histopathological signs of SINS. Abstract Swine Inflammation and Necrosis Syndrome can lead to severe clinical signs, especially in tails, ears, teats, and claws in pigs. Clinical and histopathological findings in newborn piglets with intact epidermis indicate a primarily endogenous etiology, and microbial-associated molecular patterns (MAMPs), such as lipopolysaccharide (LPS) are assumed to play a central role in the development of the syndrome. We hypothesized that swine inflammation and necrosis syndrome (SINS) is indirectly triggered by gut-derived MAMPs entering the circulatory system via the liver and thereby causing derangements on liver metabolism. To test this hypothesis, metabolomes, candidate genes of the liver and liver transcriptomes of 6 piglets with high-grade clinical signs of SINS (SINS high) were examined and compared with 6 piglets without significant signs of SINS (SINS low). Several hepatic pro-inflammatory genes and genes involved in stress response were induced in piglets of the SINS high group. The most striking finding from hepatic transcript profiling and bioinformatic enrichment was that the most enriched biological processes associated with the approximately 220 genes induced in the liver of the SINS high group were exclusively related to metabolic pathways, such as fatty acid metabolic process. Within the genes (≈390) repressed in the liver of the SINS high group, enriched pathways were ribosome biogenesis, RNA processing, RNA splicing, spliceosome, and RNA transport. The transcriptomic findings were supported by the results of the metabolome analyses. These results provide the first evidence for the induction of an inflammatory process in the liver of piglets suffering from SINS, accompanied by lipid metabolic derangement.

Background Avian Haemosporida are vector-borne parasites that commonly infect Passeriformes. Molecular analyses revealed a high number of different lineages and lineage specific traits like prevalence and host-specificity, but knowledge of parasite prevalence and lineage diversity in wild birds in Central Germany is still lacking. Results Blood samples from a total of 238 adult and 122 nestling songbirds belonging to six species were investigated for infections with avian haemosporidian genera and lineages (Haemoproteus spp., Plasmodium spp., Leucocytozoon spp.) and Trypanosoma avium using PCR, targeting the parasite mitochondrial cytochrome b gene and 18S ribosomal RNA. In total, the prevalence in adult birds was 31.3% infected with Haemoproteus, 12.5% with Plasmodium and 71.0% with Leucocytozoon (nestlings excluded). None of the tested birds was infected with Trypanosoma avium. Only in two nestling birds, aged 12–17 days, a Leucocytozoon spp. infection was proven. Among 225 successfully sequenced samples, we found four Haemoproteus, three Plasmodium and 19 Leucocytozoon lineages, including two new Leucocytozoon lineages. Furthermore, we report two new host-lineage associations. Conclusions As first study investigating avian haemosporidian parasites in Central Germany, we provide new information on genetic diversity of Haemosporida infecting Passeriformes. We show that even with a small sample size new lineages as well as previously unknown linkages between certain lineages and host species can be detected. This may help to elucidate the diversity of lineages as well as lineage-host-connections of avian Haemosporida.

Background: Avian trichomonosis is known as a widespread disease in columbids and passerines, and recent findings have highlighted the pathogenic character of some lineages found in wild birds. Trichomonosis can affect wild bird populations including endangered species, as has been shown for Mauritian pink pigeons Nesoenas mayeri in Mauritius and suggested for European turtle doves Streptopelia turtur in the UK. However, the disease trichomonosis is caused only by pathogenic lineages of the parasite Trichomonas gallinae. Therefore, understanding the prevalence and distribution of both potentially pathogenic and non-pathogenic T. gallinae lineages in turtle doves and other columbids across Europe is relevant to estimate the potential impact of the disease on a continental scale.Results: We examined 281 samples from four wild columbid species for Trichomonas infection and determined the genetic lineages. The overall prevalence was 74%. There were significant differences between the species (P = 0.007). The highest prevalence was found in stock doves Columba oenas (86%, n = 79) followed by wood pigeons Columba palumbus (70%, n = 61) and turtle doves (67%, n = 65), while three of five collared doves Streptopelia decaocto (60%) were infected. We found seven different lineages, including four lineages present in columbids in the UK, one lineage already described from Spain and three new lineages, one of those found in a single turtle dove migrating through Italy and another one found in a breeding stock dove. Stock doves from Germany and collared doves from Malta were infected with a potentially pathogenic lineage (lineage A/B), which is known to cause lesions and mortality in columbids, raptors and finches.Conclusions: Generally, turtle doves showed high prevalence of Trichomonas infection. Furthermore, the potentially pathogenic lineage A/B (or genotype B according to previous literature) was found in a recovering stock dove population. Both findings are worrying for these columbid species due to the occasional epidemic character of trichomonosis, which can have severe negative effects on populations. Fil: Marx, Melanie. Justus-Liebig-University. Department of Animal Ecology & Systematics,; Alemania Fil: Reiner, Gerald. Justus-Liebig-University. Department of Clinical Veterinary Sciences; Alemania Fil: Willems, Hermann. Justus-Liebig-University. Department of Clinical Veterinary Sciences; Alemania Fil: Rocha, Gregorio. Universidad de Extremadura. Departamento de Ingeniería Agro-Forestal; España Fil: Hillerich, Klaus. Röntgenstraße; Alemania Fil: Masello, Juan Francisco. Justus-Liebig-University. Department of Animal Ecology & Systematics,; Alemania Fil: Mayr, Sylvia L.. Justus-Liebig-University. Department of Clinical Veterinary Sciences; Alemania Fil: Moussa, Sarah. Justus-Liebig-University. Department of Animal Ecology & Systematics,; Alemania Fil: Dunn, Jenny C.. University of Lincoln. School of Life Sciences, ; Reino Unido Fil: Thomas, Rebecca C.. University of Leeds. School of Biology; Reino Unido Fil: Goodman, Simon J.. University of Leeds. School of Biology; Reino Unido Fil: Hamer, Keith C.. University of Leeds. School of Biology; Reino Unido Fil: Metzger, Benjamin. Birdlife Malta. Xemxija Waterfront Apartments; Malta Fil: Cecere, Jacopo G.. Institute for Environmental Protection and Research; Alemania Fil: Spina, Fernando. Institute for Environmental Protection and Research; Alemania Fil: Koschkar, Steffen. Dorfstraße; Alemania Fil: Calderón, Pablo Luciano Sebastian. Justus-Liebig-University. Department of Animal Ecology & Systematics,; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentina Fil: Romeike, Tanja. Justus-Liebig-University. Department of Animal Ecology & Systematics,; Alemania Fil: Quillfeldt, Petra. Justus-Liebig-University. Department of Animal Ecology & Systematics,; Alemania

Actinobacillus pleuropneumoniae is among the most important pathogens worldwide in pig production. The agent can cause severe economic losses due to decreased performance, acute or chronic pleuropneumonia and an increased incidence of death. Therapeutics cannot be used in a sustainable manner, and vaccination is not always available, but discovering more about host defence and disease mechanisms might lead to new methods of prophylaxis. The aim of the present study was to detect quantitative trait loci (QTL) associated with resistance/susceptibility to A. pleuropneumoniae. Under controlled conditions, 170 F2 animals of a Hampshire/Landrace family, with known differences in founder populations regarding A. pleuropneumoniae resistance, were challenged with an A. pleuropneumoniae serotype 7 aerosol followed by a detailed clinical, radiographic, ultrasonographic, pathological and bacteriological examination. F2 pigs were genotyped with 159 microsatellite markers. Significant QTL were identified on Sus scrofa chromosomes (SSC) 2, 6, 12, 13, 16, 17 and 18. They explained 6–22 % of phenotypic variance. One QTL on SSC2 reached significance on a genome-wide level for five associated phenotypic traits. A multiple regression analysis revealed a combinatory effect of markers SWR345 (SSC2) and S0143 (SSC12) on Respiratory Health Score, Clinical Score and the occurrence of death. The results indicate the genetic background of A. pleuropneumoniae resistance in swine and provide new insights into the genetic architecture of resistance/susceptibility to porcine pleuropneumonia. The results will be helpful in identifying the underlying genes and mechanisms.

Porcine circovirus 2 (PCV-2), the essential infectious agent in PCVD (porcine circovirus diseases) circulates at high rates among domestic pig and wild boar populations. Wild boars may be viremic and shed the virus with excretions and secretions, and thus serve as a reservoir for domestic pig PCV-2 infection. We hypothesize that PCV-2 strains circulating in wild boars and in domestic pigs are significantly different and thus, partially independent. To prove this hypothesis, the present study investigated by sequence analysis the distribution of ORF2 and ORF3 genotypes of the PCV-2 genome within wild boars (n=40) and domestic pigs (n=60) from overlapping greater areas of Germany. The genotypes were compared with PCV-2 sequences from the Genbank database. The dominating genotype in domestic pigs was PCV-2b (98.4% of infected pigs), while only 4.8% of them were infected with PCV-2a. The corresponding prevalences of PCV-2a and -2b genotypes in wild boars were 58% and 70%, respectively. When also ORF3 genotypes were taken into account, more than 50% of wild boar PCV-2 genotypes were rare among German and European domestic pigs. In conclusion, these data provide evidence for a certain independence of PCV-2 infections in both species and a low chance for domestic pigs to be infected with PCV-2 of wild boar origin. On the other hand, PCV-2 genotypes specific for domestic pigs are also common in wild boars, although at lower frequencies, suggesting the spread of domestic pig PCV-2 to the wild boar population.

Lawsonia intracellularis, Brachyspira hyodysenteriae, and Brachyspira pilosicoli are important pathogens in domestic pig production, responsible for porcine intestinal adenomatosis, swine dysentery, and porcine intestinal spirochetosis, respectively. They are widely distributed among pig-producing units around the world, and transmission is accomplished by relatively weak immunity, long shedding intervals, sequential shedding, and actual environmental survival. Little information is available on occurrence, prevalence, and quantity of these pathogens in free-ranging wild boars. The aim of the present study was to evaluate L. intracellularis, B. hyodysenteriae, and B. pilosicoli infections in wild boars in Germany. Tissue samples from ileocaecal mucosa of 165 wild boars from 18 hunting grounds situated in 14 of the 16 federal states of Germany were examined by conventional PCR and quantified by multiplex real-time PCR. None of the wild boars did show any gross pathological signs of enteritis. The overall prevalence for L. intracellularis, B. hyodysenteriae, and B. pilosicoli was 20.6%, 2.4%, and 12.1%, respectively. None of the three agents was detected in 68.5% of the wild boars and in 11.1% of the hunting grounds. Numbers of bacteria per sample were below the limit of quantification (100 cells/PCR reaction). This is the first study on L. intracellularis and Brachyspira spp. in free-ranging wild boars. The study revealed colonised animals without signs of disease. The meaning of these findings remains unclear, and we do not know whether and to what extent these three pathogens are exchanged between wild boars and domestic pigs. Further research is needed to get insight into the epidemiological impact of the results.

Haemophilus parasuis is the etiological agent of Glasser’s disease, often involved in pneumonia, and also an early colonizer of the upper respiratory tract of healthy domestic pigs. Little information is available on H. parasuis in wild boars. The aim of the present study was to evaluate H. parasuis infection in wild boars in Germany. Tissue samples from the lungs and tonsils of 531 wild boars from 52 hunts during the hunting seasons 2004/2005 to 2006/2007 were examined independently for H. parasuis by PCR because H. parasuis is a fastidious organism, which hampers its isolation from clinical samples. The overall prevalence of H. parasuis in wild boars in Germany was 74.2%. H. parasuis was detected in 69.1% of tonsils and 40.4% of lungs. In conclusion, the present study demonstrates a wide distribution of H. parasuis in German wild boar populations and further research is required to understand the virulence of H. parasuis strains in wild boars, as well as the distribution and potential exchange of different strains between wild boars and domestic pigs.

International audience; AbstractCattle are the most important reservoir for enterohemorrhagic Escherichia coli (EHEC), a subset of shigatoxigenic E. coli (STEC) capable of causing life-threatening infectious diseases in humans. In cattle, Shiga toxins (Stx) suppress the immune system thereby promoting long-term STEC shedding. First infections of animals at calves’ age coincide with the lack of Stx-specific antibodies. We hypothesize that vaccination of calves against Shiga toxins prior to STEC infection may help to prevent the establishment of a persistent type of infection. The objectives of this study were to generate recombinant Shiga toxoids (rStx1mut & rStx2mut) by site-directed mutagenesis and to assess their immunomodulatory, antigenic, and immunogenic properties. Cultures of bovine primary immune cells were used as test systems. In ileal intraepithelial lymphocytes both, recombinant wild type Stx1 (rStx1WT) and rStx2WT significantly induced transcription of IL-4 mRNA. rStx1WT and rStx2WT reduced the expression of Stx-receptor CD77 (syn. Globotriaosylceramide, Gb3) on B and T cells from peripheral blood and of CD14 on monocyte-derived macrophages. At the same concentrations, rStx1mut and rStx2mut exhibited neither of these effects. Antibodies in sera of cattle naturally infected with STEC recognized the rStxmut toxoids equally well as the recombinant wild type toxins. Immunization of calves with rStx1mut plus rStx2mut led to induction of antibodies neutralizing Stx1 and Stx2. While keeping their antigenicity and immunogenicity recombinant Shiga toxoids are devoid of the immunosuppressive properties of the corresponding wild type toxins in cattle and candidate vaccines to mitigate long-term STEC shedding by the reservoir host.

Porcine circovirus type 2 (PCV-2) is the etiologic agent of porcine circovirus-associated disease (PCVAD). PCV-2 is classified into three genotypes. Here, we present the complete genomic sequences of two PCV-2 isolates (KM and H026) with an unusual sequence duplication in the rep gene coding for viral replicase proteins.

Actinobacillus (A.) pleuropneumoniae is among the most important pathogens in pig. The agent causes severe economic losses due to decreased performance, the occurrence of acute or chronic pleuropneumonia, and an increase in death incidence. Since therapeutics cannot be used in a sustainable manner, and vaccination is not always available, new prophylactic measures are urgently needed. Recent research has provided evidence for a genetic predisposition in susceptibility to A. pleuropneumoniae in a Hampshire × German Landrace F2 family with 170 animals. The aim of the present study is to characterize the expression response in this family in order to unravel resistance and susceptibility mechanisms and to prioritize candidate genes for future fine mapping approaches. F2 pigs differed distinctly in clinical, pathological, and microbiological parameters after challenge with A. pleuropneumoniae. We monitored genome-wide gene expression from the 50 most and 50 least susceptible F2 pigs and identified 171 genes differentially expressed between these extreme phenotypes. We combined expression QTL analyses with network analyses and functional characterization using gene set enrichment analysis and identified a functional hotspot on SSC13, including 55 eQTL. The integration of the different results provides a resource for candidate prioritization for fine mapping strategies, such as TF, TFRC, RUNX1, TCN1, HP, CD14, among others.

For PCR detection of Coxiella burnetii in various clinical specimens we developed a sample preparation method in which silica binding of DNA was used. This method was found to be fast, easily performed with large numbers of samples, and equally sensitive for all of the specimens tested (livers, spleens, placentas, heart valves, milk, blood). The DNA preparation method described here can also be used as an initial step in any PCR-based examination of specimens. The procedure was tested with more than 600 milk samples, which were taken from 21 cows that were seropositive for C. burnetii and reportedly had fertility problems (and therefore were suspected of shedding the agent through milk intermittently or continuously). Of the 21 cows tested, 6 were shedding C. burnetii through milk. Altogether, C. burnetii DNA was detected in 6% of the samples. There was no correlation between the shedding pattern and the serological results.

Mycobactin J-dependent mycobacterial isolates from sheep, goat, and cattle herds with Johne's disease in Morocco, South Africa, the United States, and Germany were tested for the repetitive insertion sequence IS900 of Mycobacterium paratuberculosis by PCR. The IS900 PCR target sequence was detected in 90 of 93 fecal culture isolates tested (96.8%). Restriction fragment length polymorphisms (RFLPs) and in vitro growth characteristics were studied in 46 of the IS900-positive isolates and in two bovine vaccine strains of M. paratuberculosis. Five different RFLP types were identified in PvuII digests of genomic DNA by Southern hybridization with a DNA probe specific for IS900. All isolates of M. paratuberculosis could be classified into two major clusters by their growth rates as well as the relatedness of their PvuII-RFLP hybridization patterns. All of the sheep isolates were classified into cluster I (extremely slow growth), while all cattle and goat isolates were members of cluster II (moderately slow growth). Different PvuII-RFLP patterns were detected in different sheep flocks from Morocco and South Africa. Our results demonstrate that genetically and phenotypically different strains of M. paratuberculosis were present in ruminant populations. The strains from sheep in Morocco and South Africa tested in the study appeared to belong to a unique group of M. paratuberculosis strains that might have adapted to this host species. The presence of several genetically distinct strains in different sheep flocks suggested that analysis of IS900-specific RFLP patterns may provide a useful tool for the epidemiologic investigation of ovine paratuberculosis outbreaks.

Chlamydia felis is an important ocular pathogen in cats worldwide. A multilocus variable-number tandem-repeat analysis (MLVA) system for the detection of tandem repeats across the whole genome of C. felis strain Fe/C-56 was developed. Nine selected genetic loci were tested by MLVA in 17 C. felis isolates, including the C. felis Baker vaccine strain, and 122 clinical samples from different geographic origins. Analysis of the results identified 25 distinct C. felis MLVA patterns. In parallel, a recently described multilocus sequence typing scheme for the typing of Chlamydia was applied to 13 clinical samples with 12 different C. felis MLVA patterns. Rare sequence differences were observed. Thus, the newly developed MLVA system provides a highly sensitive high-resolution test for the differentiation of C. felis isolates from different origins that is suitable for molecular epidemiological studies.

Actinobacillus pleuropneumoniae is an important pathogen of the porcine respiratory tract. Subclinically and chronically infected pigs may enter a permanent carrier state, harboring A. pleuropneumoniae in lungs or tonsils. Despite the enormous role of A. pleuropneumoniae in domestic swine production, no published data about prevalence and distribution of A. pleuropneumoniae infections in wild boars (Sus scrofa) are available. Antibodies against A. pleuropneumoniae have been detected in serum of wild boars from Slovenia. The aim of the present study was to evaluate A. pleuropneumoniae infection status in wild boars in Germany. Tissue samples from lungs and tonsils of 531 wild boars from 52 hunts during the hunting seasons 2004-05 to 2006-07 were examined for A. pleuropneumoniae by polymerase chain reaction (PCR). Although gross pathologic lesions typical for pleuropneumonia were not detected, 35.8% of the wild boars were PCR-positive. Tonsils were involved in 94.3% and lungs in 15.5%. Actinobacillus pleuropneumoniae infection prevalence increased with age and body weight. Further research is needed to understand the involvement of different A. pleuropneumoniae serotypes and to understand the interrelationship of A. pleuropneumoniae infection between wild boars and domestic pigs.

Restriction fragment length polymorphism (RFLP) was used for the differentiation of 80 Coxiella burnetii isolates derived from animals and humans in Europe, USA, Africa and Asia. After NotI restriction of total C. burnetii DNA and pulsed field gel electrophoresis (PFGE) 20 different restriction patterns were distinguished. The index of discrimination for this typing system was 0·86. Comparison and phylogenetic analysis of the different RFLP patterns revealed evolutionary relationships among groups that corresponded to the geographical origin of the isolates. This finding was confirmed by genetic mapping. No correlation between restriction group and virulence of isolates was detected.