Seroprevalence of tick-borne encephalitis virus in wild and domestic animals in northern Germany.

Tick-borne encephalitis virus (TBEV) is a tick-transmitted flavivirus, which can infect humans and animals, sometimes even with a fatal outcome. Since many decades, TBEV is endemic in southern Germany, while only sporadic occurrence has been noted in northern parts of the country so far. Nevertheless, autochthonous human clinical cases are increasing in the federal state of Lower Saxony in north-western Germany, and several natural foci of TBEV transmission have recently been detected in this federal state. In order to shed more light on the current distribution of TBEV in Lower Saxony, the present study examined blood samples from wild and domestic animals for antibodies against TBEV. Overall, samples from 4,085 animals were tested by ELISA, including wild boar (N = 1,208), roe deer (N = 149), red deer (N = 61), fallow deer (N = 18), red foxes (N = 9), nutria (N = 9), raccoon dogs (N = 3), raccoons (N = 3), badgers (N = 1), European pine martens (N = 1), horses (N = 574), sheep (N = 266), goats (N = 67), dogs (N = 1,317) and cats (N = 399). Samples with an ELISA result of ≥60 Vienna units (VIEU)/ml were subjected to confirmatory serum neutralization tests (SNT). In total, 343 of 4,085 (8.4%) animals tested positive for anti-TBEV-IgG by ELISA, of which 60 samples were confirmed by SNT. Samples of 89 animals showed a cytotoxic effect in the SNT and were excluded from seroprevalence calculation, resulting in an overall seroprevalence of 1.5% (60/3,996). Seroprevalence was higher among wild animals (wild boar: 2.9% [34/1,190], roe deer: 2.7% [4/149], red deer: 1.7% [1/60], fallow deer: 5.6% [1/18]) than among domestic animals (dogs: 1.1% [15/1,317], horses: 0.8% [4/505], sheep: 0.4% [1/266]). No anti-TBEV-antibodies were detected in the other wild animal species as well as goats and cats. A notable clustering of positive samples was observed in districts where TBEV transmission foci have been described. Further clusters in other districts suggest the existence of so far undetected transmission foci, underlining the fact that both wild and domestic animals are useful sentinels for monitoring the spread of TBEV.

Detection and Characterization of Alongshan Virus in Ticks and Tick Saliva from Lower Saxony, Germany with Serological Evidence for Viral Transmission to Game and Domestic Animals.

The newly discovered group of Jingmenviruses has been shown to infect a wide range of hosts and has been associated with febrile illness in humans. During a survey for Jingmenviruses in ticks from Lower Saxony, Germany, Alongshan virus (ALSV) was identified in Ixodes spp. ticks. Additional virus screenings revealed the presence of ALSV in the bodies and saliva of ticks collected at several locations in Lower Saxony. Vector competence studies that included Ixodes ricinus and Dermacentor reticulatus validated the replication of ALSV within those tick species. In vitro feeding experiments with ALSV-injected Ixodes ricinus demonstrated effective viral transmission during blood feeding. To evaluate the potential viral transmission during a natural blood meal, sera from wild game and domestic animals were investigated. One serum sample from a red deer was found to be positive for ALSV RNA, while serological screenings in game and domestic animals revealed the presence of ALSV-specific antibodies at different locations in Lower Saxony. Overall, those results demonstrate the broad distribution of ALSV in ticks in Lower Saxony and hypothesize frequent exposure to animals based on serological investigations. Hence, its potential risk to human and animal health requires further investigation.

Genetic Characterization of Taenia saginata Cyst Isolates from Germany.

The beef tapeworm Taenia saginata, which causes taeniosis in humans and cysticercosis in cattle, is of medical and economic importance. Understanding the parasite's genetic population structure may help to analyze transmission patterns and aid in the development of control measures. As information on sequence variability is scarce for European isolates, this study aimed to elucidate the intraspecific genetic variability of T. saginata cysts from German cattle by sequence comparison of the mitochondrial cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 5 (nad5) genes, in relationship to sequences from other geographical origins. Cysts were collected from northern German, Swiss, and Belgian cattle. Moreover, proglottids from an adult T. saginata specimen from Palestine were included. Amplification and Sanger sequencing of the cox1 gene was successful for 57 samples (45 German, 9 Swiss, 2 Belgian, 1 Palestinian), whereas 32 sequences were obtained for the nad5 gene (21 German, 10 Swiss, 1 Palestinian). For German isolates, sequence comparison revealed minor genetic variability with two polymorphic sites and mutations in both genes. Three haplotypes with haplotype diversity of 0.088 for cox1 and 0.186 for nad5, as well as nucleotide diversities of 0.00028 and 0.00095, respectively, were observed. Comparison of the cox1 gene sequence of German isolates with other European, African, American, and Asian isolates obtained from National Center for Biotechnology Information (total of 71 sequences) raised 11 polymorphic sites and mutations as well as 10 haplotypes (haplotype diversity: 0.239; nucleotide diversity: 0.00097). Although nad5 sequence comparison comprised less sequences (N = 33), analyses revealed 11 polymorphic sites, 12 mutation sites, and 7 haplotypes (haplotype diversity: 0.335, nucleotide diversity: 0.00391), indicating a better resolution of genetic variability compared to cox1. Thus, nad5 may be particularly useful for in-depth studies on genetic divergence of T. saginata.

Use of competition ELISA for monitoring of West Nile virus infections in horses in Germany.

West Nile virus (WNV) is a mosquito-borne viral pathogen of global importance and is considered to be the most widespread flavivirus in the World. Horses, as dead-end hosts, can be infected by bridge mosquito vectors and undergo either subclinical infections or develop severe neurological diseases. The aim of this study was to detect WNV specific antibodies in horses in Germany as an indicator for an endemic circulation of WNV. Sera from more than 5,000 horses (primarily fallen stock animals) were collected in eight different federal states of Germany from 2010 to 2012. Sera were screened by a competitive ELISA and positive reactions were verified by an indirect IgM ELISA and/or by virus neutralization tests (VNT) for WNV and Tick-borne encephalitis virus (TBEV) in order to exclude cross-reacting antibody reactions. In essence WNV specific antibodies could not be detected in any of the horse sera. Not surprisingly, a small number of sera contained antibodies against TBEV. It is noteworthy that equine sera were often collected from horse carcasses and therefore were of poor quality. Nonetheless, these sera were still suitable for WNV ELISA testing, i.e., they did not produce a high background reaction which is a frequently observed phenomenon. According to these data there is no evidence for indigenous WNV infections in horses in Germany at present.

An immunoblot for detection of Taenia saginata cysticercosis.

Control measures to prevent human infections with the food-borne zoonotic helminth Taenia saginata are currently based on meat inspection, which shows rather low diagnostic sensitivity. To develop an immunoblot for detection of T. saginata-infected cattle, crude proteins of T. saginata cysts were extracted and separated with SDS-PAGE. The cyst antigens showed ten protein bands ranging from 260 to 14 kDa. T. saginata cyst proteins 260, 150, 130, 67, 60, 55, 50, and 23 kDa were immunoreactive with known positive sera of T. saginata-infected cattle but cross-reacted with sera from Echinocccus granulosus-infected ruminants. By contrast, 14- and 18-kDa cyst proteins reacted specifically with T. saginata-positive sera and thus might be potential candidates for the development of a T. saginata-specific immunoassay. Proteins of E. granulosus cysts and Taenia hydatigena cysts were also extracted and separated with SDS-PAGE. E. granulosus cysts revealed 11 protein bands ranging from 260 to 23 kDa. E. granulosus protein 60 kDa was immunoreactive with E. granulosus-positive sera only. The cyst of T. hydatigena showed 11 protein bands ranging from 290 to 14 kDa. The protein band 35 kDa showed cross-reaction with positive sera from both T. saginata- and E. granulosus-infected animals. A protein of 67 kDa was present in all three tested cestode species and was the major antigenic protein detected by sera of T. saginata- and E. granulosus-infected animals. Therefore, this protein represents a potential vaccine candidate against both cysticercosis and cystic echinococcosis in cattle.

Seroprevalence of Taenia saginata cysticercosis in the federal state of Lower Saxony in Germany.

Based on ELISA results from randomly selected serum samples taken from 128 cattle from different administrative and urban districts in the federal state of Lower Saxony in Germany a seroprevalence estimate of Taenia saginata cysticercosis in this area was derived. This estimate was subsequently used to calculate the sample size required in an epidemiological study to determine the actual prevalence of this infection in the cattle population (n = 2 604 767) in this federal state. The sample size was calculated as 1518 and the samples were collected according to the distribution of cattle among the 48 administrative and urban districts in Lower Saxony. The samples were tested with an evaluated antibody ELISA. The results showed a positive antibody titre rate of 8.83% from the total tested samples.

Meat juice as diagnostic sample for virological and serological diagnosis of classical swine fever.

The objective of this paper was to assess if meat juice is a suitable substrate for virological and serological diagnosis of classical swine fever (CSF). Fifty-six domestic pigs and 21 wild boars experimentally vaccinated and/or infected as well as 129 field samples from wild boars were involved in this study. Meat juice from diaphragm, forequarter and hindquarter was used for investigations. CSFV and viral RNA were detected in meat juice between days 5 and 21 post infection (pi). Animals which had survived the infection were diagnosed virologically negative and antibody-positive in muscle fluid. After vaccination or vaccination and subsequent infection of animals (n = 42), meat juice samples scored serologically positive. The antibody titres of these samples were significantly lower than in serum. Serological investigations of field samples derived from wild boars (n = 75) shot in Mecklenburg-Western Pomerania showed a clear correlation between the antibody-positive samples in serum and in meat juice, whereas the serological results of meat juice samples (n = 54) from wild boars collected in Lower Saxony were slightly different. The reasons for these differences are discussed. Nevertheless, meat juice seems to be a suitable substrate for CSF diagnosis, especially for wild boars.