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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.

SummaryObjective: Anaerobic spirochetes of the genus Brachyspira are important pathogens causing swine dysentery (Brachyspira [B.] hyodysenteriae) and porcine intestinal spirochetosis (B. pilosicoli, PIS). In addition, avian intestinal spirochetosis (AIS) is caused by B. pilosicoli, B. intermedia and B. alvinipulli. Despite the economic impact of AIS, the disease has not received appropriate attention in Germany. This study was aimed at identifying Brachyspira spp. in Germany and determining their antimicrobial susceptibility. Material and methods: From 2009 to 2013, a total of 71 fecal swabs were obtained from clinically healthy layer hens from eight different commercial flocks. Brachyspira spp. culture was performed in trypticase soybean agar added with 5% sheep blood. Species determination was conducted by PCRs targeting the NADH-gen and the 16S rDNA or by nox-gene sequencing. Antimicrobial susceptibility to macrolides, lincosamides and pleuromutilins was tested by a microdilution assay. Results: Brachyspira spp. were isolated from 40 (56.3%) swabs distributed over all eight flocks. In 26 cases, the following species were determined by PCR: B. pilosicoli (n = 16), B. intermedia (2), B. innocens (3), B. murdochii (1), mixtures of B. pilosicoli/B. intermedia (2), B. innocens/B. intermedia (1), B. innocens/B. murdochii (1). Remaining isolates were characterized by noxgene sequencing as B. “pulli” (n = 9), B. alvinipulli (3), B. intermedia (1) and as not identifiable (1). Antimicrobial susceptibility testing of 37 isolates revealed minimal inhibitory concentrations 90 (MIC90) of > 128 mg/l (tylosin), 64 mg/l (lincomycin), 8 mg/l (tiamulin) and 4 mg/l (valnemulin), respectively. Comparing to breakpoints applied to pigs, these values lie within the range of resistance. Conclusion The demonstration of different Brachyspira spp., particularly B. pilosicoli, intermedia and alvinipulli in commercial layers, indicates the need of further research to assess their potential role in causing AIS in German poultry flocks. The increased antimicrobial resistance of Brachyspira spp. isolates to tylosin and pleuromutilins is likely associated with extensive use of these drugs in poultry medicine.

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.

Population genetic parameters from different studies might be significantly influenced by differences in sample size, fraction of males and females, marker number, and sets of markers used, reducing the comparability between studies. This hypothesis was tested on a red deer population of 205 individuals with an estimated size of 1000 animals. Four tests were performed: (1) the population was subdivided into 10 populations each with 10 to 150 individuals and genotyped with 16 markers, (2) the total population was genotyped 10 times with different panels of microsatellite loci containing 2 to 14 markers, (3) a subset of 8 microsatellite loci was used to genotype the total population; markers of this subset were replaced one by one with a different marker set and genotyping results were compared to the results of the original subset and (4) the effect of sex was estimated. Additionally, 24 references from literature, including 256 European red deer populations, were analyzed. A median of 25 individuals per population was investigated in published studies using 11 microsatellite markers (5 to 22). Sixty-eight percent of possible study comparisons matched with less than 10% of microsatellite loci. Our results show that the factors investigated, except for the factor gender, lead to significant deviations in the population genetic results, especially with sample sizes below 30, with less than 6 to 8 microsatellite markers and with the use of different panels of microsatellite loci. This is also true with respect to population genetic structure and the use of Bayesian methods. Therefore, populations from different studies should be compared with each other with caution.

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.

This study investigates the genetic development of the German Krofdorfer Forst red deer population, located in the federal state of Hesse, by genotyping red deer hunted between 1960 and 1984 (“ancient” population) and between 2002 and 2012 (“extant” population). Krofdorfer Forst belongs to the smaller red deer areas in Hesse, with a census size in spring of 200 animals and is surrounded by areas designated as free of red deer. Because of this “island” position, it should be evaluated whether there was a significant decrease in genetic diversity over time. DNA extracted from bone shavings of antlers and skulls was genotyped with 16 unlinked microsatellites. In the “ancient” population, 131 alleles were detected, from which 39 were private alleles which disappeared from the “extant” population over time. On the other hand, the “extant” population had 112 alleles, from which 20 were new alleles acquired between 1984 and 2012. This means a net loss of 19 alleles over time in the “extant” population. Despite this decrease in genetic diversity, there was a high number of effective alleles as compared to red deer populations in Bavaria. Furthermore, the low F value of 0.032 indicates low (or no) inbreeding and thus a likewise extremely low probability of deleterious alleles to become fixed. Using demographic data obtained from the red deer association of the Krofdorfer Forst, the effective population size was calculated to be 73, which reflects an increase in inbreeding of only 0.7 % per generation which confirms the low F value.

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.

Porcine circovirus 2 (PCV2) is responsible for a wide range of associated diseases (PCVD) affecting swine production worldwide. Highly efficient commercial vaccines induce protective immunity, but PCV2 is still circulating in vaccinated farms. Thus, and because of the viruś high mutation rate, recent findings provide concerns about PCV2 strains capable to escape vaccination. Based on 2156 samples from individual pigs of 315 herds from Germany we describe a high effectivity of vaccination between 2008 and the third quarter of 2011. In this period, virus load dropped continuously and at the end of this period it hardly reached the limit of quantification. Thereafter, virus loads re-increased, although most of the herds were still vaccinated. Sixty-two randomly selected samples from vaccinated (n=28) and non-vaccinated (n=26) herds between 2008 and 2012 were completely sequenced. As compared to the PCV2b reference sequence 259 polymorphisms were detected. Polymorhisms were analysed for associations to vaccination status, genotype (PCV2a/PCV2b), and virus load. PCV2a sequences were significantly repelled by PCV2b. One SNP at position 1182 (g.1182G>T), involved in capsid epitope formation, was significantly associated with the PCV2 genotype (2a/2b). Moreover, this SNP was affected by vaccination, with effects on allele frequencies and viral load, independent from the PCV2 genotype (2a/2b). We conclude that there is indeed evidence for a selectional impact of vaccination on the PCV2 sequence, especially on nucleotides involved in epitope formation. Such variation might be responsible for the observed re-increase of PCV2-loads in samples from the end of 2011 in Germany.

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.

International audience; We describe a novel high-resolution melt assay that clearly differentiates Chlamydia abortus live vaccine strain 1B from field C. abortus strains and field wild-type isolates based on previously described single nucleotide polymorphisms. This modern genotyping technique is inexpensive, easy to use, and less time-consuming than PCR-RFLP.

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.

Interferon regulatory factor 7 (IRF7), as a key regulator of type I interferon response, plays an important role during innate response against viral infection. Although well conserved across species, the structure of IRF7 and its function during parasite infection are not well documented in farm animals, such as the pig. To bridge this gap, we have determined the porcine IRF7 gene structure and identified two intronic single nucleotide polymorphisms (SNPs), SNP g.748G>C and SNP g.761A>G, in commercial pig breeds. The distribution of SNP g.761A>G in multiple breeds suggested that it was in Hardy-Weinberg equilibrium and allowed us to map it at the top of SSC2. We found that during Sarcocystis miescheriana infection, the G allele was associated with high lymphocyte levels (P G enhances the transactivation activity of IRF7, possibly by improving IRF7 transcript splicing of intron-3. These findings would suggest that IRF7, as a transcriptional regulator, is involved in the defence mechanism against a larger spectrum of pathogens, and in more host species, than initially anticipated.

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.

Porcine circovirus 2 (PCV2), the causative agent of postweaning multisystemic wasting syndrome (PMWS), has been detected in North American and European wild boars at prevalences arguing for high circulation rates among populations. Systematic data on the qualitative distribution of PCV2 infections and on PCVD (PCV2 diseases) in wild boars are rare, however, and quantitative data about viral loads are missing. To be able to judge the PCV2/PCVD situation in wild boars, evaluation of the nationwide qualitative and quantitative distribution of PCV2 and PCVD in Germany was the objective of the present study. Wild boar samples were compared with domestic pig samples of the same greater areas, including tonsils, lungs, spleen, Lnn. bronchiales and Lnn. mesenterici of 349 wild boars and 348 domestic pigs. All of the wild boars and 308 of the domestic pigs have been apparently free of PCVD, 40 of the domestic pigs had been rejected from slaughter due to health problems (i.e. wasting). Tissues were examined by pathohistology, immunohistology (IHC), nested PCR (nPCR and quantitative PCR (qPCR). One wild boar (0.3%) and 8.7% of the domestic pigs were classified as PCVD-affected, based on pathohistology and IHC. PCV2 DNA was detected in 63.1% and 45.4% of the wild boars by nPCR and qPCR, respectively, and in 100% and 98.8% of the domestic pigs. PCV2 loads differed significantly between wild boars (average: 10(2.8) PCV2 genomes/microg extracted sample DNA) and domestic pigs (average: 10(4.2) PCV2 genomes/microg of sample DNA). The qualitative detection of PCV2 DNA in tissues of wild boars and domestic pigs was abundant and not of any pathological relevance. The overall load of PCV2 in domestic pigs was relatively high and borderline with respect to PCVD, and there was no difference between apparently healthy pigs and pigs rejected from slaughter in this respect. Most of the wild boars were infected with PCV2 at loads less relevant for PCVD.

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.

Clinical-chemical traits are essential parameters to quantify the health status of individuals and herds, but the knowledge about their genetic architecture is sparse, especially in swine. We have recently described three QTL for serum aspartate aminotransferase activity (sAST), and one of these maps to a region on SSC14 where the aspartate aminotransferase coding gene GOT1 is located. This QTL was only apparent under the acute burden of a model disease. The aim of the present study was to characterize GOT1 as a candidate gene and to test the effects of different GOT1 SNPs as potential quantitative trait nucleotides (QTNs) for sAST. Nine SNPs within GOT1 were identified, and SNP c.-793C>G significantly increased the QTL effects and narrowed the confidence interval from 90 to 15 cM. Additionally, we found a significant association of SNP c.-793C>G in a commercial outbred line, but with reversed phase. We conclude that GOT1 is a putative candidate gene for the sAST QTL on SSC14, and that SNP c.-793C>G is close to the responsible QTN.

Summary Behavioural indices in vertebrates are under genetic control at least to some extent. In spite of significant behavioural problems in farm animals, information on the genetic background of behaviour is sparse. The aim of this study was to map QTL for behavioural indices in swine under healthy conditions and after infection with Sarcocystis miescheriana, as behaviour can be significantly influenced by disease. This well-described parasite model subsequently leads to acute (day 14 p.i.), subclinical (day 28 p.i.) and chronic disease (day 42 p.i.), allowing the study and comparison of the behaviour of pigs under four different states of health or disease. The study was based on a well-described Pietrain/Meishan F2 family that has recently allowed the detection of QTL for disease resistance. We have mapped six genome-wide significant and 24 chromosome-wide significant QTL for six behavioural indices in swine. Six of these QTL (i.e. 20% of total QTL) showed effects on behavioural traits of the healthy pigs (day 0). Some of them (QTL on SSC11 and 18) lost influence on behavioural activities during disease, while the effects of others (QTL on SSC5, SSC8) partly remained during the whole experiment, although with different effects on the distinct behavioural indices. The disease model has been of high relevance to detect effects of gene loci on behavioural indices. Considering the importance of segregating alleles and environmental conditions that allow the identification of the phenotype, we conclude that there are indeed QTL with interesting effects on behavioural indices in swine.

Maintaining pH and blood gases in a narrow range is essential to sustain normal biochemical reactions. Decreased oxygenation, poor tissue perfusion, disturbance to CO(2) expiration, and shortage of HCO(3)(-) can lead to metabolic acidosis. This is a common situation in swine, and originates from a broad range of medical conditions. pH and blood gases appear to be under genetic control, and populations with physiological traits closer to the pathological thresholds may be more susceptible to developing pathological conditions. However, little is known about the genetic basis of such traits. We have therefore estimated phenotypic and genetic variability and identified quantitative trait loci (QTL) for pH and blood gases in blood samples from 139 F(2) pigs from the Meishan/Pietrain family. Samples were taken before and after challenge with Sarcocystis miescheriana, a protozoan parasite of muscle. Twenty-seven QTL influencing pH and blood gases were identified on nine chromosomes. Five of the QTL were significant on a genome-wide level; 22 QTL were significant on a chromosome-wide level. QTL for pH-associated traits have been mapped to SSC3, 18 and X. QTL associated with CO(2) have been detected on SSC6, 7, 8 and 9, and QTL associated with O(2) on SSC2 and SSC8. QTL showed specific health/disease patterns that were related to the physiological state of the pigs from day 0, to acute disease (day 14), convalescence (day 28) and chronic disease (day 42). The results demonstrate that pH and blood gases are influenced by multiple chromosomal areas, each with relatively small effects.

Summary Haematological traits are essential diagnostic parameters in veterinary practice but knowledge on the genetic architecture controlling variability of erythroid traits is sparse, especially in swine. To identify QTL for erythroid traits in the pig, haematocrit (HCT), haemoglobin (HB), erythrocyte counts (RBC) and mean corpuscular haemoglobin content (MCHC) were measured in 139 F2 pigs from a Meishan/Pietrain family, before and after challenge with the protozoan pathogen Sarcocystis miescheriana. The pigs passed through three stages representing acute disease, reconvalescence and chronic disease. Forty-three single QTL controlling erythroid traits were identified on 16 chromosomes. Twelve of the QTL were significant at the genome-wide level while 31 were significant at a chromosomewide level. Because erythroid traits varied with health and disease status, QTL influencing the erythroid phenotypes showed specific health/disease patterns. Regions on SSC5, 7, 8, 12 and 13 contained QTL for baseline erythroid traits, while the other QTL regions affected distinct stages of the disease model. Single QTL explained 9‐17% of the phenotypic variance in the F2 animals. Related traits were partly under common genetic influence. Our analysis confirms that erythroid trait variation differs between Meishan and Pietrain breeds and that this variation is associated with multiple chromosomal regions.

Beside domestic pigs wild boars can also be affected by postweaning multisystemic wasting syndrome (PMWS). For the first time a nationwide survey of wild boars (n = 356) and domestic pigs (n = 340) was carried out in Germany by histopathology, immunohistochemistry (IHC) and quantitative PCR (qPCR). Whereas 102/340 domestic pigs were immunoreactive for PCV2 antigen in at least one examined tissue, only 8/356 wild boars reacted positively. Similar findings could be found in qPCR: all domestic pigs showed viral DNA in at least one tissue, while in the examined tissues of 170 wild boars PCV2-DNA was not detectable. The specimens were examined histologically for histiocytosis and depletion of lymphocytes, both typical for PMWS. Based on these findings, six wild boars and 69 domestic pigs were assumed to be affected by PMWS.

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.

PCV-2 is involved in “postweaning multisystemic wasting syndrome” (PMWS), “porcine dermatitis and nephropathy syndrome” (PDNS), respiratory and reproductive disorders, and thereby plays a crucial role in today's swine production world-wide. The virus is apparently ubiquitous in domestic pigs and has also been demonstrated in wild pigs. Up to now, a characterization of PCV-2 samples from wild pigs, which might help to estimate the possible role of wild pigs as sources of domestic pig infection, has not been carried out. Spleen samples from 16 PCV-2-positive wild pigs from hunting grounds of four regions in Germany were used for the analysis of the viral genome. In one sample, the complete sequence of the genome was determined. In the other, a 742 nucleotide fragment from the highly variable capsid sequence of the ORF2 was sequenced. Analysis of the sequences led to the identification of three PCV-2 strains. One strain, representing 14 of the 16 samples, was closely related with Chinese, but not with German strains. The genome of this strain was shortened by one nucleotide by a deletion close to the end of ORF2. The deletion led to a shift of the stop-codon and to the insertion of a further codon. Two further strains differed in up to 4.7% of nucleotides and up to 10.5% of amino acids (aa). These strains were aligned with clusters of PCV-2 samples from mainly French and German domestic origin.

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.

Brachyspira hyodysenteriae, the causative agent of swine dysentery, is responsible for severe mucohaemorrhagic colitis with considerable financial loss to worldwide swine production. Antimicrobial resistance against macrolides and lincosamides is widespread and the mechanisms are well known. Currently, the most common treatment for swine dysentery is the use of pleuromutilins and resistance to these drugs also is increasingly being reported. Although resistance mechanisms against pleuromutilins are less clear than for other drugs, they seem to involve alterations of the peptidyl transferase centre (PTC), including ribosomal RNA and the ribosomal protein L3. The present study was conducted to examine molecular mechanisms of resistance on a representative set of B. hyodysenteriae field strains with different resistance patterns. In total, we identified 24 single nucleotide polymorphisms (SNPs) in the 23S rRNA gene and genes of the ribosomal proteins L3, L4, L2 and L22. The SNP in the ribosomal protein gene L3 at position 443 led to an amino acid substitution of asparagine (Asn) by serine (Ser) at position 148, significantly associated with MICs for pleuromutilins. Based on this SNP a correct assignment of 71% of the strains with respect to a threshold of >0.625 μg tiamulin/ml was reached. Unexpectedly low MICs in some of the Asn-strains were explained by a second SNP at position 2535 of the 23S rRNA. Our results clearly show the associations between MICs for pleuromutilins and mutations in their binding site. A complete list of SNPs that influence MICs of B. hyodysenteriae strains is needed to enable the interpretation of future molecular susceptibility testing.

Different vaccination strategies against Mycoplasma hyopneumoniae have been adopted worldwide. Reports from the field indicate varying levels of protection among currently available vaccines. The goal of the present study was to compare the efficacies of three widespread commercial vaccination strategies against M. hyopneumoniae under field conditions. 20 farms were included. 14 farms used different single dose vaccines (vaccine 1 [V1], 8 herds; vaccine 2 [V2], 6 herds); another 6 farms (V3) used a two dose vaccination strategy. Gross lesions of 854 lungs and histopathology from 140 lungs were quantified, and a quantitative PCR was applied to detect M. hyopneumoniae and porcine circovirus 2 (PCV2) DNA in lung tissue (n=140). In addition, porcine reproductive and respiratory disease virus (PRRSV), swine influenza virus (SIV), Actinobacillus pleuropneumoniae, Haemophilus parasuis and Pasteurella multocida were tested by qualitative PCR. 53% of lungs were positive for M. hyopneumoniae. 55.9% of lungs showed macroscopic enzootic pneumonia (EP)-like lesions. Lung lesion scores (P

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.

Surgical castration of young male piglets is now a generally accepted cause of serious distress and impairment of animal welfare. Awareness of this problem has created the moral commitment to seek for practical and more humane alternatives. As one possible alternative, the application of analgesics has been installed in Germany as an interim solution by the QS system, thus mandatory for the majority of German pig producers.Two analgesics have been authorised for this purpose. Both have been shown a significant positive impact on cortisol levels if administered pre-operatively. However, their effects on pain, stress and discomfort during castration, and on the post-castration period are conflicting. Thus, the aim of the present study was to compare the effects of Meloxicam and Flunixin on cortisol levels, behavioural indices, vocalisation, and wound healing of surgical castrated piglets in the field. There was no difference in vocalisation during castration in analgesic treated and untreated piglets. Piglets castrated under analgesia still had significantly elevated serum cortisol levels 30 min post castration, when compared to the sham castrated group. Both analgesics led to a significant impairment of behavioural indices and wound healing. It is concluded that analgesics can improve the welfare of piglets during the first part of the post-castration period. However, the benefits may be considered small and may not meet the requirements of the EU. Hence it is of high importance to prevent the interim practice of surgical castration of male piglets under analgesics from becoming implemented as a permanent condition in pig production.

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.

Lawsonia (L.) intracellularis, Brachyspira (B.) hyodysenteriae and B. pilosicoli are important pathogens in domestic pig production world-wide, responsible for porcine intestinal adenomatosis, swine dysentery, and porcine intestinal spirochetosis, respectively. Conventional PCR is the major diagnostic tool in the detection of the three pathogens, but the sole detection of bacterial DNA might lead to misinterpretations of results with respect to their clinical relevance, especially with mixed infections. Thus, the present study targeted the detection and quantification of the three pathogens in samples from herds with a case history of diarrhoea. Herds and samples were selected by the practitioners on a voluntary basis. Results were based on 1176 individual samples from 95 herds from Southern Germany. The pathogens were detected simultaneously by multiplex real-time PCR. The overall prevalence for L. intracellularis, B. hyodysenteriae and B. pilosicoli was 12.6%, 8.4% and 3.2% in faecal samples and 48.4%, 24.2% and 31.6% in herds, respectively. Sixty one percent, 82.6%, and 73.4% of herds positive for L. intracellularis, B. hyodysenteriae, and B. pilosicoli, respectively, had mixed infections. Median log values of DNA equivalents/g of faeces for L. intracellularis, B. hyodysenteriae and B. pilosicoli were 3.3, 5.9 and 3.2, with maxima of 8.3, 8.0 and 6.3, respectively. Within herd prevalence of B. hyodysenteriae and B. pilosicoli as well as the load of B. hyodysenteriae were significantly associated with the severity of diarrhoea.

The development of biomarkers for application in diagnostics, in the identification of resistance and virulence mechanisms and in breeding for disease resistance is well underway. Proteomics, transcriptomics, and genomics are the all-embracing tools to reach these goals, to compete the high complexity of regulation that complicates the search for suitable candidate genes and markers. Small RNA molecules, microRNA, visible since decades, however only recently discovered, add a new quality to the "omics"-family. Although still in the process of elucidation, they have been figured out as nucleic acids with outstanding impact on development and homoeostasis of cells and associated disorders. Their dysfunction that might be caused by endogenous and exogenous factors, can lead to developmental disorders, cancer, and also metabolic disorders, immunological disorders, and disease. Their role as a conductor in the orchestra of gene regulation and their stability and traceability in serum make them the ideal biomarkers. Deciphering of associations between miRNA and disease still have mainly been achieved in human medicine, however, miRNAs are on the way to be widely used in veterinary medicine and diagnostics too. This review presents an overview on the most significant findings, related to miRNA, including most comprehensive leading articles to deepen the topic.

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.

A multiplex real-time PCR assay was developed to detect and quantify B. hyodysenteriae, B. pilosicoli, and L. intracellularis in pig faeces. Specific probes and primers were directed against the NADH oxidase (nox) gene of Brachyspira and the aspartate ammonia lyase (aspA) gene of L. intracellularis, respectively. The analytical sensitivity for the real-time PCR assay, expressed as limit of detection (LOD) was below 10 DNA copies for L. intracellularis, 14 DNA copies for B. pilosicoli and 26 DNA copies per PCR reaction for B. hyodysenteriae. The experimental sensitivity, expressed as limit of quantitation (LOQ) for the real-time PCR assay was set to 100 DNA copies per PCR reaction which equals 8 x 10(3) cells per gram of faeces. The multiplex real-time PCR was tested in parallel to conventional PCR on 749 faecal samples from 121 farms. 73 (9.7%), 30 (4%), and 30 (4%) faecal samples were positive for L. intracellularis, B. hyodysenteriae, and B. pilosicoli, respectively by conventional PCR and 59 (7.9%), 27 (3.6%), and 7 (0.9%) by multiplex real-time PCR. From the real-time PCR positive results 34 (4.5%), 25 (3.3%), and 4 (0.5%) were above the LOQ. The multiplex real-time PCR will allow rapid and quantitative detection of clinical relevant amounts of the three key porcine enteric pathogens simultaneously.

Comparative genomic analysis of a wild-type strain of the ovine pathogen Chlamydophila abortus and its nitrosoguanidine-induced, temperature-sensitive and virulence-attenuated live vaccine derivative identified point mutations unique to the mutant (Burall et al. [1]). Here, we evaluate the capacity of some of these mutations to either create or eliminate restriction sites using the wild-type strain C. abortus S26/3 as a reference. Three of eight genomic sites with confirmed point mutations (CAB153, CAB636 and CAB648) were retained for analysis as each resulted in the loss of a restriction site in the genome sequence of the vaccine strain. PCR-restriction fragment length polymorphism analysis using restriction enzymes chosen to specifically target the three genomic sites was then applied to a large number of C. abortus field isolates and reference strains. Our results indicate that the three mutations are uniquely present in the vaccine strain, and as such provide easy-to-use markers for the differential identification of the vaccine strain and wild-type isolates.

Presently, potential associations between PCV2 virulence and -sequence are discussed controversially. Practitioners are rattled, whether and to which extent spatio-temporal variations in PCV2 associated diseases (PCVD) on farms might be attributed to distinct PCV2 strains, differing in virulence, and if this factor should be taken into account regarding diagnosis and prophylaxis. The present paper provides a review on known and discussed functional elements of the PCV2 genome and their significance in pathogenesis is discussed. Furthermore, variability between and within PCV2 strains and discrepancy between the common phylogenetic considerations of the total genome or the open reading frame 2 (ORF 2) and single nucleotide variations are presented. Only incoherent associations between phylogeny and virulence determining sequences serve as an explanation for inconsistent results of different authors regarding sequence and virulence. The conclusion is that there are presently no molecular markers for PCV2 virulence. Thus, quantitative considerations still remain the major factor in diagnosis, prognosis, prophylaxis and control of PCVD.

Clinical-chemical traits are diagnostic parameters essential for characterization of health and disease in veterinary practice. The traits show significant variability and are under genetic control, but little is known about the fundamental genetic architecture of this variability, especially in swine. We have identified QTL for alkaline phosphatase (ALP), lactate (LAC), bilirubin (BIL), creatinine (CRE) and ionized sodium (Na(+)), potassium (K(+)) and calcium (Ca(++)) from the serum of 139 F(2) pigs from a Meishan/Pietrain family before and after challenge with Sarcocystis miescheriana, a protozoan parasite of muscle. After infection, the pigs passed through three stages representing acute disease, subclinical disease and chronic disease. Forty-two QTL influencing clinical-chemical traits during these different stages were identified on 15 chromosomes. Eleven of the QTL were significant on a genome-wide level; 31 QTL were chromosome-wide significant. QTL showed specific health/disease patterns with respect to the baseline values of the traits as well as the values obtained through the different stages of disease. QTL influencing different traits at different times were found primarily on chromosomes 1, 3, 7 and 14. The most prominent QTL for the investigated clinical-chemical traits mapped to SSC3 and 7. Baseline traits of ALP, LAC, BIL, Ca(++) and K(+) were influenced by QTL regions on SSC3, 6, 7, 8 and 13. Single QTL explained up to 21.7% of F(2) phenotypic variance. Our analysis confirms that variation of clinical-chemical traits is associated with multiple chromosomal regions.