Distribution, prevalence, and intensity of Sarcocystis infections in sika deer (Cervus nippon) of free-ranging populations in Germany and Austria.

Sika deer (Cervus nippon), which are native to the Japanese islands and the adjacent mainland of eastern Asia, have been introduced into Europe and established free-ranging populations in several countries. Various Sarcocystis species have been identified recently from farmed "mainland sika" deer in Lithuania and native "Japanese sika" deer in Japan. To study the distribution, prevalence and intensity of Sarcocystis infection in free-ranging sika deer outside of their natural range heart and/or diaphragm samples of 311 animals from nine populations in Germany and Austria were examined by histology.Overall, sarcocysts were detected in either heart or diaphragm of 107/311 deer (34.4%) with prevalence ranging roughly from 5 to 50% among the populations. Considering the 263 animals with both heart and diaphragm available, prevalence varied significantly (p < 0.0001) among calves (20.2%), yearlings (40.3%), and adult deer (49.1%) but did not differ between male and female deer (48.3% vs. 43.7%; p = 0.6483). Occurrence of sarcocysts in heart vs. diaphragm indicated a marginal difference (27.8% vs. 20.9%; p = 0.0839). Intensity of infection in all but one heart positive and all diaphragm positive animals was low (< 10 sarcocysts per square centimeter muscle cut). While heart sarcocyst counts of yearlings and adult deer were higher than those of calves and were higher in male compared to female sika deer, diaphragm sarcocyst counts did not differ significantly between age groups and sexes. Sarcocystis infection was demonstrated at variable prevalence in sika deer in all populations but intensity is apparently low. Further studies are needed to identify the species of Sarcocystis infecting sika deer naturalized outside their natural range.

Anaplasma phagocytophilum and Babesia spp. in roe deer (Capreolus capreolus), fallow deer (Dama dama) and mouflon (Ovis musimon) in Germany.

Infections with the tick-borne pathogens Anaplasma phagocytophilum and Babesia spp. can cause febrile disease in several mammalian species, including humans. Wild ruminants in Europe are suggested to serve as reservoir hosts for particular strains or species of these pathogens. The aims of this study were to investigate the occurrence of A. phagocytophilum and Babesia spp. in roe deer (Capreolus capreolus), fallow deer (Dama dama) and mouflon (Ovis musimon orientalis) in Germany, and the diversity and host association of genetic variants of A. phagocytophilum and Babesia species. From 2009 to 2010, 364 spleen samples from 153 roe deer, 43 fallow deer and 168 mouflon from 13 locations in Germany were tested for DNA of A. phagocytophilum and Babesia spp. by real-time PCR or conventional PCR, respectively. Variants of A. phagocytophilum were investigated with a nested PCR targeting the partial 16S rRNA gene, and species of piroplasms were identified by sequencing. DNA of A. phagocytophilum was detected in 303 (83.2%) samples: roe deer, 96.1% (147/153); fallow deer, 72.1% (31/43); and mouflon, 74.4% (125/168). Sequence analysis of 16S rRNA-PCR products revealed the presence of nine different genetic variants. DNA of Babesia spp. was found in 113 (31.0%) samples: roe deer, 62.8% (96/153); fallow deer, 16.3% (6/43); and mouflon, 6.5% (11/168). Babesia capreoli, Babesia sp. EU1 (referred to also as B. venatorum), B. odocoilei-like and a Theileria species were identified. Co-infections with A. phagocytophilum and Babesia spp. were detected in 30.0% of the animals which were tested positive for A. phagocytophilum and/or Babesia spp. Roe deer had a significantly higher percentage of co-infections (60.8%), followed by fallow deer (14.0%) and mouflon (6.5%). Thus, the results suggest that roe deer plays a key role in the endemic cycles of the pathogens investigated.

Schmallenberg virus in Germany 2011-2014: searching for the vectors.

Following the emergence of Schmallenberg virus (SBV) in 2011, 21,397 culicoid biting midges (Diptera: Ceratopogonidae) from targeted and non-targeted sampling activities carried out during the summer months of 2011 to 2013 and in late 2014 in various regions in Germany were analyzed for the virus by real-time RT-PCR. While no SBV was found in biting midges collected during 2011 and 2013, 2 out of 334 pools including 20 and 22 non-engorged females of the Obsoletus complex sampled in 2012 tested positive for the SBV S-segment with C(t) values of 42.46 and 35.45. In addition, 673 black flies (Diptera: Simuliidae) captured during the same studies were screened for the presence of SBV and proved negative. In late autumn 2014, biting midges were collected again in a limited study in eastern Germany after some cases of SBV infection had occurred in a quarantine station for cattle. Due to the unfavorable seasonal weather conditions, only few specimens were caught, and these were also negative for SBV. The German experience suggests that biting midge collections launched only after an outbreak and are not locally targeted may be ineffective as to virus detection. It rather might be advisable to collect biting midges at sentinel farms on a permanent basis so to have material available to be examined in the case of a disease outbreak.

A broad spectrum screening of Schmallenberg virus antibodies in wildlife animals in Germany.

To identify native wildlife species possibly susceptible to infection with Schmallenberg virus (SBV), a midge-transmitted orthobunyavirus that predominantly infects domestic ruminants, samples from various free-living ruminants, but also carnivores, small mammals and wild boar were analyzed serologically. Before 2011, no SBV-specific antibodies were detectable in any of the tested species, thereafter, a large proportion of the ruminant population became seropositive, while every sample taken from carnivores or small mammals tested negative. Surprisingly, SBV-specific-antibodies were also present in a large number of blood samples from wild boar during the 2011/2012 and 2012/2013 hunting seasons. Hence, free-ranging artiodactyls may play a role as wildlife host.

Reintroductions and genetic introgression from domestic pigs have shaped the genetic population structure of Northwest European wild boar.

BACKGROUND: Population genetic studies focus on natural dispersal and isolation by landscape barriers as the main drivers of genetic population structure. However, anthropogenic factors such as reintroductions, translocations and wild x domestic hybridization may also have strong effects on genetic population structure. In this study we genotyped 351 Single Nucleotide Polymorphism markers evenly spread across the genome in 645 wild boar (Sus scrofa) from Northwest Europe to evaluate determinants of genetic population structure. RESULTS: We show that wild boar genetic population structure is influenced by historical reintroductions and by genetic introgression from domestic pigs. Six genetically distinct and geographically coherent wild boar clusters were identified in the Netherlands and Western Germany. The Dutch Veluwe cluster is known to be reintroduced, and three adjacent Dutch and German clusters are suspected to be a result of reintroduction, based on clustering results, low levels of heterozygosity and relatively high genetic distances to nearby populations. Recent wild x domestic hybrids were found geographically widespread across clusters and at low frequencies (average 3.9%). The relationship between pairwise kinship coefficients and geographic distance showed male-biased dispersal at the population genetic level. CONCLUSIONS: Our results demonstrate that wildlife and landscape management by humans are shaping the genetic diversity of an iconic wildlife species. Historical reintroductions, translocation and recent restocking activities with farmed wild boar have all influenced wild boar genetic population structure. The current trend of wild boar population growth and range expansion has recently led to a number of contact zones between clusters, and further admixture between the different wild boar clusters is to be expected.

Suspicion of Mycobacterium avium subsp. paratuberculosis transmission between cattle and wild-living red deer (Cervus elaphus) by multitarget genotyping.

Multitarget genotyping of the etiologic agent Mycobacterium avium subsp. paratuberculosis is necessary for epidemiological tracing of paratuberculosis (Johne's disease). The study was undertaken to assess the informative value of different typing techniques and individual genome markers by investigation of M. avium subsp. paratuberculosis transmission between wild-living red deer and farmed cattle with known shared habitats. Fifty-three M. avium subsp. paratuberculosis type II isolates were differentiated by short sequence repeat analysis (SSR; 4 loci), mycobacterial interspersed repetitive-unit-variable-number tandem-repeat analysis (MIRU-VNTR; 8 loci), and restriction fragment length polymorphism analysis based on IS900 (IS900-RFLP) using BstEII and PstI digestion. Isolates originated from free-living red deer (Cervus elaphus) from Eifel National Park (n = 13), six cattle herds living in the area of this park (n = 23), and five cattle herds without any contact with these red deer (n = 17). Data based on individual herds and genotypes verified that SSR G2 repeats did not exhibit sufficient stability for epidemiological studies. Two common SSR profiles (without G2 repeats), nine MIRU-VNTR patterns, and nine IS900-RFLP patterns were detected, resulting in 17 genotypes when combined. A high genetic variability was found for red deer and cattle isolates within and outside Eifel National Park, but it was revealed only by combination of different typing techniques. Results imply that within this restricted area, wild-living and farmed animals maintain a reservoir for specific M. avium subsp. paratuberculosis genotypes. No host relation of genotypes was obtained. Results suggested that four genotypes had been transmitted between and within species and that one genotype had been transmitted between cattle herds only. Use of multitarget genotyping for M. avium subsp. paratuberculosis type II strains and sufficiently stable genetic markers is essential for reliable interpretations of epidemiological studies on paratuberculosis.

An Agouti-Signaling-Protein Mutation is Strongly Associated with Melanism in European Roe Deer (Capreolus capreolus).

Although the European roe deer (Capreolus capreolus) population of North-West Germany has a remarkable number of melanistic specimens between 10% and 25%, the underlying genetic mutation-causing melanism is still unknown. We used a gene targeting approach focusing on MC1R and ASIP as important genes of coat coloration. Overall, 1384 bp of MC1R and 2039 bp of ASIP were sequenced in 24 specimens and several SNPs were detected. But only the ASIP-SNP c.33G>T completely segregated both phenotypes leading to the amino acid substitution p.Leu11Phe. The SNP was further evaluated in additional 471 samples. Generally, all black specimens (n = 33) were homozygous TT, whereas chestnut individuals were either homozygote GG (n = 436) or heterozygote GT (n = 26). Considering the fact that all melanistic animals shared two mutated alleles of the strongly associated SNP, we concluded that melanism is inherited in a recessive mode in European roe deer.

Dental Defects as a Potential Indicator of Chronic Malnutrition in a Population of Fallow Deer (Dama dama) from Northwestern Germany.

We studied enamel defects in mandibular cheek teeth of fallow deer from an enclosed population that had grown far beyond the carrying capacity of its habitat. Macroscopic inspection revealed a high frequency of pathological enamel alterations in the permanent premolars and the third molar, which form late during dental development, while earlier forming teeth (deciduous premolars and first molar) were generally not affected. Macroscopic enamel alterations comprised opacity and posteruptive discoloration, loss of occlusal enamel ridges, and presence of enamel surface lesions. Backscattered electron imaging in the SEM revealed a marked hypomineralization and related increased porosity of the outer compared to the central and inner enamel portions in the affected teeth. In contrast, the enamel of the unaffected first molars showed a homogeneous high degree of mineralization. Microindentation hardness testing demonstrated a significantly reduced and highly variable hardness of the outer enamel of the affected teeth. The recorded enamel alterations are indicative of a disturbance of enamel maturation. Based on findings of experimental studies by other authors that explored the effects of dietary deficiencies on the mineralization of dental hard tissues in different mammal species, we suggest a dietary calcium deficiency as the cause of the observed pathological enamel changes in the fallow deer. The supposed calcium deficiency only affected teeth whose crown mineralization takes place after weaning, while the deciduous premolars and the first molar, whose crown formation occurs prenatally or during the early postnatal period of intense milk feeding, were unaffected. Anat Rec, 299:1409-1423, 2016. © 2016 Wiley Periodicals, Inc.

Absence of antibodies to foot-and-mouth disease virus in free-ranging roe deer from selected areas of Germany (2001-2002).

Blood samples (n = 223) of free-ranging roe deer (Capreolus capreolus) were collected from selected hunting grounds in Germany between October 2001 and October 2002. Samples originated from Lower Saxony (n = 43) and North-Rhine Westphalia (n = 108) within a 20-km area ("cordon") cordoned off along the border of The Netherlands. This is adjacent to the area of a foot-and-mouth disease outbreak that occurred between 21 March and 22 April 2001 in The Netherlands. Negative control samples were taken from northern Germany (Schleswig-Holstein, n = 72). Two different enzyme-linked immunosorbent assays (ELISAs) were used for the detection of antibodies against foot-and-mouth disease virus (FMDV) serotype O strain Manisa. To confirm ELISA-positive results, a virus neutralization test was performed. All samples tested negative for antibodies against FMDV. These results suggest that FMDV was not transmitted to free-ranging roe deer living in parts of Germany adjacent to the area affected by the 2001 foot-and-mouth disease outbreak in The Netherlands.

Increased Mycoplasma hyopneumoniae Disease Prevalence in Domestic Hybrids Among Free-Living Wild Boar.

Wildlife immune genes are subject to natural selection exerted by pathogens. In contrast, domestic immune genes are largely protected from pathogen selection by veterinary care. Introgression of domestic alleles into the wild could lead to increased disease susceptibility, but observations are scarce due to low introgression rates, low disease prevalence and reduced survival of domestic hybrids. Here we report the first observation of a deleterious effect of domestic introgression on disease prevalence in a free-living large mammal. A fraction of 462 randomly sampled free-living European wild boar (Sus scrofa) was genetically identified as recent wild boar-domestic pig hybrids based on 351 SNP data. Analysis of antibody prevalence against the bacterial pathogen Mycoplasma hyopneumoniae (Mhyo) showed an increased Mhyo prevalence in wild-domestic hybrids. We argue that the most likely mechanism explaining the observed association between domestic hybrid status and Mhyo antibody prevalence would be introgression of deleterious domestic alleles. We hypothesise that large-scale use of antibiotics in the swine breeding sector may have played a role in shaping the relatively deleterious properties of domestic swine immune genes and that domestic introgression may also lead to increased wildlife disease susceptibility in the case of other species.