Geographic Distribution of Raccoon Roundworm, Baylisascaris procyonis, Germany and Luxembourg.

Infestation with Baylisascaris procyonis, a gastrointestinal nematode of the raccoon, can cause fatal disease in humans. We found that the parasite is widespread in central Germany and can pose a public health risk. The spread of B. procyonis roundworms into nematode-free raccoon populations needs to be monitored.

Spread of the Zoonotic Nematode Baylisascaris procyonis into a Naive Raccoon Population.

The raccoon roundworm (Baylisascaris procyonis), a gastrointestinal nematode of the raccoon (Procyon lotor), may cause a severe form of larva migrans in humans, which can lead to death or permanent neurological damage. Although roundworms were inadvertently introduced to Europe alongside their raccoon hosts, the parasite is not present in every raccoon population. It is important to understand the geographic distribution of B. procyonis, as early and rapid treatment can prevent severe pathologies in humans. We present evidence for the roundworm spreading into a naive raccoon population through natural dispersal of infected raccoons. We sampled 181 raccoons from Saxony-Anhalt, a German federal state containing contact zones of different raccoon populations, two of which were previously free of the parasite. We screened the raccoons for roundworms and used microsatellite-based assignment tests to determine the genetic origin of the raccoons and their parasites. We detected roundworms in 16 of 45 raccoons sampled in a previously roundworm-free area in the northern part of the state. The largest proportion of the genetic ancestry (≥ 0.5) of the 16 raccoon hosts was assigned to the previously naive raccoon population. Conversely, the genetic ancestry of almost all the roundworms was assigned to the nearest roundworm population in the southern part of the state. Infected raccoons have, therefore, spread to the north of the state, where they interbred with and infected local raccoons. It seems likely that the roundworms will continue to spread. Health authorities should consider continuous surveillance programmes of naive populations and raise public awareness.

Re-Introduction of Bovine Viral Diarrhea Virus in a Disease-Free Region: Impact on the Affected Cattle Herd and Diagnostic Implications.

Bovine viral diarrhea (BVD) is one of the most important infectious cattle diseases worldwide. The major source of virus transmission is immunotolerant, persistently infected (PI) calves, which makes them the key target of control programs. In the German federal state of Saxony-Anhalt, a very low prevalence was achieved, with more than 99.8% of the cattle herds being free from PI animals since the year 2013. In 2017, BVD virus was detected in a previously disease-free holding (herd size of ~380 cows, their offspring, and fattening bulls). The purchase of two so-called Trojan cows, i.e., dams pregnant with a PI calf, was identified as the source of infection. The births of the PI animals resulted in transient infections of in-contact dams, accompanied by vertical virus transmission to their fetuses within the critical timeframe for the induction of PI calves. Forty-eight days after the birth of the first PI calf, all animals in close contact with the Trojan cows during their parturition period were blood-sampled and serologically examined by a neutralization test and several commercial ELISAs. The resulting seroprevalence strongly depended on the applied test system. The outbreak could be stopped by the immediate elimination of every newborn PI calf and vaccination, and since 2018, no BVD cases have occurred.

Serological and Molecular Investigation of Batai Virus Infections in Ruminants from the State of Saxony-Anhalt, Germany, 2018.

Arthropod-borne Batai virus (BATV) is an Orthobunyavirus widely distributed throughout European livestock and has, in the past, been linked to febrile diseases in humans. In Germany, BATV was found in mosquitoes and in one captive harbor seal, and antibodies were recently detected in various ruminant species. We have, therefore, conducted a follow-up study in ruminants from Saxony-Anhalt, the most affected region in Eastern Germany. A total of 325 blood samples from apparently healthy sheep, goats, and cattle were tested using a BATV-specific qRT-PCR and SNT. Even though viral RNA was not detected, the presence of antibodies was confirmed in the sera of all three species: sheep (16.5%), goats (18.3%), and cattle (41.4%). Sera were further analyzed by a glycoprotein Gc-based indirect ELISA to evaluate Gc-derived antibodies as a basis for a new serological test for BATV infections. Interestingly, the presence of neutralizing antibodies was not directly linked to the presence of BATV Gc antibodies. Overall, our results illustrate the high frequency of BATV infections in ruminants in Eastern Germany.

West Nile virus epizootic in Germany, 2018.

The summer of 2018 in Germany was the second hottest and driest on record. These generally extremely favorable climatic conditions most likely triggered the further expansion and the efficient propagation of the zoonotic arthropod-borne West Nile virus in many Southern/Southeastern and even Central European countries. WNV infections were detected for the first time in resident wild and aviary birds, such as common blackbirds, northern goshawks and great grey owls in Eastern and Southeastern Germany. The causative WNV strain belonged to the central European subclade II. Phylogeographic analysis indicated a single introduction event of WNV into Germany, most likely in 2016 from Czech Republic, and also a unique non-synonymous mutation in the NS3 gene. Extraordinary high temperatures in 2018 presumably led to decreased averaged extrinsic incubation period values for WNV in mosquitoes, leading to rapid virus amplification and greater transmission risk for vertebrates in Germany. Blood transfusion services and clinicians in Germany should be aware of these possible WNV infection risks in humans especially during late summer.

Seroprevalance of Batai virus in ruminants from East Germany.

Batai virus (BATV), a mosquito-transmitted Orthobunyavirus, was first detected in Southwest Germany in anopheline and culicine mosquitoes in 2009. However, little is known about the exposure to BATV infections for farm animals and humans in Germany as almost no systematic surveillance or infection studies have been carried out to date. This may explain why clinical symptoms in animals or humans have not been reported so far. Therefore and since BATV has meanwhile been detected repeatedly in different mosquito species in several regions of Germany, we performed a surveillance study by assaying more than 1300 blood samples from ruminants (goats, bovines, sheep) from six different federal states covering the years 2013 to 2016. Samples were investigated by BATV-specific real-time polymerase chain reaction as well as by virus neutralisation test. BATV-specific RNA was not detected, whereas BATV-specific antibodies were found in livestock from various geographic regions. We have determined the seroprevalence of 38.8% for goats, 44.7% for sheep and 36.4% for bovines in Saxony-Anhalt. The seroprevalence of goats from Brandenburg was 38.6% and of goats from Saxony 28.4%. These results confirm the levels of seroprevalence to BATV, suggesting endemic circulation, in different regions and indicate that ruminants are potential hosts of BATV in East Germany. Furthermore, the role of BATV as segment donor in disease emergence events should not be overlooked.

High-Quality Genome Sequence of Bacillus anthracis Strain 14RA5914 Isolated during an Outbreak in Germany in 2014.

Bacillus anthracis is a zoonotic agent causing anthrax, a notifiable disease in animals. The last anthrax outbreak among cattle in Germany occurred in April 2014 in Saxony-Anhalt. Here we report a high-quality genome sequence of the Bacillus anthracis strain 14RA5914 Dobichau isolated from the spleen of a dead cow.

Molecular detection and analysis of Sheeppox and Orf viruses isolated from sheep from Qalubia, Egypt.

In this study an outbreak with Sheeppox virus (SPPV) and Orf virus (ORFV) in one sheep herd in the Qalubia province, Egypt, was investigated. Both, SPPV and ORFV caused clinically manifest infections among sheep. The affected sheep showed skin lesions around the mouth or all over the body. Therefore, reliable diagnosis should confirm the aetiology of the infection and then reduce spread of the diseases in the affected areas. Clinical samples were investigated by virus isolation, PCR and real-time PCR assays. Furthermore, PCR-products of SPPV and ORFV isolates were sequenced and alignment to reference isolates was performed for phylogenetic analyses. The laboratory diagnosis showed that real-time PCR assay was more accurate and sensitive than conventional PCR and virus isolation. In phylogenetic analysis of the A29L gene genetic differences between SPPV field strains were not observed and the strains showed 100% homology with two SPPV isolates from Kazakhstan and one isolate from Turkey. The ORFV field strains are in the P55 gene genetically distinct from another and from other published isolates from Egypt 2006 and 2009.

Genome Sequence of Bacillus anthracis Strain Stendal, Isolated from an Anthrax Outbreak in Cattle in Germany.

In July 2012, an anthrax outbreak occurred among cattle in northern Germany resulting in ten losses. Here, we report the draft genome sequence ofBacillus anthracisstrain Stendal, isolated from one of the diseased cows.

Development of multiplex real-time PCR assay for the detection of Brucella spp., Leptospira spp. and Campylobacter foetus.

Abortion among dairy cattle is one of the major causes of economic losses in the livestock industry. This study describes a 1-step multiplex real-time polymerase chain reaction (PCR) to detect Brucella spp., Leptospira spp. and Campylobacter foetus, these are significant bacteria commonly implicated in bovine abortion. ß-actin was added to the same PCR reaction as an internal control to detect any extraction failure or PCR inhibition. The detection limit of multiplex real-time PCR using purified DNA from cultured organisms was set to 5 fg for Leptospira spp. and C. foetus and to 50 fg for Brucella spp. The multiplex real-time PCR did not produce any non-specific amplification when tested with different strains of the 3 pathogens. This multiplex real-time PCR provides a valuable tool for diagnosis, simultaneous and rapid detection for the 3 pathogens causing abortion in bovine.

Simultaneous detection of Chlamydia spp., Coxiella burnetii, and Neospora caninum in abortion material of ruminants by multiplex real-time polymerase chain reaction.

Chlamydia spp., Coxiella burnetii, and Neospora caninum are responsible for reproductive diseases and are closely linked with high abortion rates in ruminants. Furthermore, C. burnetii and Chlamydia spp. have zoonotic potential. A real-time polymerase chain reaction (PCR) assay was developed for the simultaneous detection of Chlamydia spp., C. burnetii, and N. caninum. The detection of beta-actin as internal control in the same PCR reaction provides additional information about sample quality by detecting the presence of PCR inhibitors. The multiplex real-time PCR developed in the current study shows a greater sensitivity compared to previously used single-target PCR reactions with a reproducible detection limit of 0.13 plasmid copies per PCR for each target. Additional parallel amplification of all detectable pathogens did not adversely impact sensitivity. This new multiplex PCR allows the highly sensitive, cost-effective, and rapid detection of 3 important pathogens and has the potential to be a useful time-saving tool in the routine diagnosis of abortion cases in ruminants.

Genome Sequence of Chlamydia psittaci Strain 01DC12 Originating from Swine.

Chlamydia psittaci is the etiological agent of psittacosis and is a zoonotic pathogen infecting birds and a variety of mammalian hosts. Here we report the genome sequence of the porcine strain 01DC12 which is representative of a novel clade of C. psittaci belonging to ompA genotype E.

Influences on the sensitivity of real-time PCR for the detection of bovine DNA in heat-sterilised feedstuffs.

The present study evaluated two previously developed methods for amplification of bovine mtDNA segments of 109 and 271 base pairs (bp) by real-time polymerase chain reaction (PCR). Beef samples were sterilised experimentally at different temperatures (126 degrees C, 129 degrees C, 132 degrees C and 135 degrees C). These experimentally sterilised beef samples and nine commercial meat and bone meals (MBM) were mixed to a reference plant concentrate in strengths of 50%, 10%, 5%, and 1%. The results of the following PCR showed that the Bos-109 real-time PCR assay was able to detect all the experimental beef samples with exception of the mixtures of beef heated experimentally to 135 degrees C. In mixtures of industrial MBM bovine DNA were always found. Comparatively, the beef sterilised at 135 degrees C and 132 degrees C (and their respective mixtures) and the mixture containing 1% of beef sterilised at 129 degrees C were not detectable with the PCR assay amplifying a target of 271 bp. Using this PCR mixtures of industrial MBM were only weakly detected. The low concentrated mixtures of the extremely processed MBM-1 and MBM-2 even reported negative. These results indicate that the detectability of bovine DNA is strongly influenced by the degree of the thermal treatment. Only the PCR assay amplifying relatively short fragments of a multi-copy mitochondrial target was reliable for the detection of correctly heated MBM in mixed feed.

Detection of Chlamydophila caviae and Streptococcus equi subsp. zooepidemicus in horses with signs of rhinitis and conjunctivitis.

At a stud farm of Trakehner horses, all 33 foals of a birth cohort developed conjunctivitis and serous to muco-purulent rhinitis, and 7 older horses showed recurrent signs of conjunctivitis. Examination of nasal and conjunctival swabs by bacterial and cell culture, as well as real-time PCR, ArrayTube microarray analysis and DNA sequencing led to the identification of Chlamydophila (C.) caviae (first description in horses) and Streptococcus (S.) equi subsp. zooepidemicus. We presume a synergistic effect associated with these two agents by hypothesising that primary lesions were set by C. caviae and subsequently aggravated by Streptococcus equi subsp. zooepidemicus. Indications supporting this assumption include (i) the conjunctivitis caused by mono-infection with C. caviae, (ii) recurrent clinical symptoms in the affected animals, and (iii) the absence of a sustained clinical effect of antibiotic therapy with trimethoprim-sulfonamide, enrofloxacin and amoxicillin. The detection of C. caviae in horses raises questions about the significance and natural host range of this agent.

[Detection and species-specific differentiation of pestiviruses using real-time RT-PCR]. / Nachweis und Spezies-spezifische Differenzierung von Pestiviren mit der real-time RT-PCR.

An important prerequisite for an efficient eradication of pestiviruses like bovine viral diarrhea virus or classical swine fever virus are sensitive and specific detection methods. Beside antigen detection with antigen capture ELISAs and virus isolation using cell culture, the detection of virus genomes by reverse transcriptase polymerase chain reaction (RT-PCR) becomes more and more important. By using real-time RT-PCR, the disadvantages of conventional PCR methods concerning the risk of contamination and missing specificity controls are minimized. In the following, the validation and application of different real-time RT-PCR systems for the detection of pestiviruses are presented. In addition, a new "panpesti" probe was tested, and the ability to differentiate between the major species of pestiviruses with known PCR protocols was determined using the Light Cycler PCR machine.

[Elimination of persistently BVDV infect animals: efficient herd screening using RT-PCR and antigen ELISA in milk and serum samples]. / BVD-Virämikereliminierung: Effektives Herdenscreening durch Kombination von RT-PCR und Antigen-ELISA in Blut- und Milchproben.

With the long-term perspective of the eradication of BVD/MD in the German federal state Saxony-Anhalt a voluntary control program was initiated in 2002 by an administrative regulation based on federal German guidelines. The short-term aim of this program is the establishment of BVDV-unsuspected herds achieved by elimination of persistently infected cattle. The diagnostic program and particularly the choice of the diagnostic tools was based on the consideration of laboratory experiences as well as on economic and logistic aspects. A combination of RT-PCR and antigen-ELISA was found to be the suitable diagnostic methods. The screening started with examinations of sera pools using Real Time RT-PCR in the Light-Cylcer system. After positive pool results the individual persistently infected animals were detected using various commercial ERNS-antigen-ELISAs. The RT-PCR revealed a high degree of sensitivity and robustness. With respect to highly specific ELISAs the producers have to ensure the detection of all currently important virus strains. The prevalence of persistently infected animals in the cattle population of Saxony-Anhalt was about 0.2% during the last 3 years. This is probably due to nearly 10 years of broad vaccination and will prove advantageous for BVDV elimination.