Reoccurring Bovine Anthrax in Germany on the Same Pasture after 12 Years.

The zoonotic disease anthrax, caused by the endospore-forming bacterium Bacillus anthracis, is very rare in Germany. In the state of Bavaria, the last case occurred in July of 2009, resulting in four dead cows. In August of 2021, the disease reemerged after heavy rains, killing one gestating cow. Notably, both outbreaks affected the same pasture, suggesting a close epidemiological connection. B. anthracis could be grown from blood culture, and the presence of both virulence plasmids (pXO1 and pXO2) was confirmed by PCR. Also, recently developed diagnostic tools enabled rapid detection of B. anthracis cells and nucleic acids directly in clinical samples. The complete genome of the strain isolated from blood, designated BF-5, was DNA sequenced and phylogenetically grouped within the B.Br.CNEVA clade, which is typical for European B. anthracis strains. The genome was almost identical to BF-1, the isolate from 2009, separated only by three single nucleotide polymorphisms (SNPs) on the chromosome, one on plasmid pXO2 and three indel regions. Further, B. anthracis DNA was detected by PCR from soil samples taken from spots in the pasture where the cow had fallen. New tools based on phage receptor-binding proteins enabled the microscopic detection and isolation of B. anthracis directly from soil samples. These environmental isolates were genotyped and found to be identical to BF-5 in terms of SNPs. Therefore, it seems that the BF-5 genotype is currently the prevalent one at the affected premises. The area contaminated by the cadaver was subsequently disinfected with formaldehyde.

Epidemiologically non-feasible singleton reactors at the final stage of BoHV1 eradication: serological evidence of BoHV2 cross-reactivity.

A voluntary marker-independent Bovine Herpesvirus 1 (BoHV1) eradication program started in 1986; in 1998 it changed to a compulsory one. Certification of free regions in European member states is based on Article 10 of directive 64/432/EEC. According to this rule Bavaria is listed as free of BoHV1 since October 2011. Surveillance of BoHV1-free dairy cattle farms is currently performed with quarterly bulk-milk testing. Non-negative bulk-milk results must be confirmed by blood tests in cattle older than nine months. An increased regional rate of non-negative bulk-milk samples and the subsequent detection of epidemiologically non-feasible singleton BoHV1-reactors by analysis of blood were observed at the final stage of eradication in southwest Bavaria. Nineteen case farms (734 animals) defined by singleton reactors born at least two years after certification of the farms as BoHV1-free, 23 negative control (NC) farms (NC I: 321 animals) from the same region, 11 NC-farms (NC II: 423 animals) from an already-certified Article 10 region in northeast Bavaria and two BoHV1-infected farms (264 animals) were analysed using BoHV1-, BoHV2- and Feline Herpesvirus 1 (FeHV1)-neutralisation tests (NTs), and three commercially available ELISAs supplied by Idexx Laboratories, B.V., The Netherlands: the CHEKIT™ Trachitest 2nd Gen. test for milk or serum (Trachitest), Herdchek™ gB- (gB-ELISA) and Herdchek™ gE-ELISA (gE-ELISA). Significantly increased levels of BoHV2 antibodies were observed on case farms compared to NC I or II farms. Additionally, reactivity by gB-ELISA and the Trachitest was significantly increased for animals with BoHV2 neutralising antibodies. Singleton BoHV1-reactors tested negative by gE-ELISA even if an elevated cut-off of 0.95±0.05 was applied. At this cut-off, the gE-ELISA was as sensitive and specific as the gB-ELISA. Comparative titration of milk samples from seropositive animals from a BoHV1-infected dairy cattle farm and from singleton BoHV1-reactors performed in CHEKIT™ Trachitest 2nd Gen. Milk revealed that the slopes of both groups were distinct; therefore, optimised cut-offs for bulk-milk testing to exclude singleton BoHV1-reactors are proposed.

Production of monoclonal antibodies and development of an antigen capture ELISA directed against the envelope glycoprotein GP of Ebola virus.

Ebola virus (EBOV) causes severe outbreaks of Ebola hemorrhagic fever in endemic regions of Africa and is considered to be of impact for other parts of the world as an imported viral disease. To develop a new diagnostic test, monoclonal antibodies to EBOV were produced from mice immunized with inactivated EBOV species Zaire. Antibodies directed against the viral glycoprotein GP were characterized by ELISA, Western blot and immunofluorescence analyses. An antigen capture ELISA was established, which is specific for EBOV-Zaire and shows a sensitivity of approximately 10(3) plaque-forming units/ml. Since the ELISA is able to detect even SDS-inactivated EBOV in spiked human sera, it could complement the existing diagnostic tools in the field and in routine laboratories where high containment facilities are not available.