The "Friedrich-Loeffler-Institut": past, present and future of research in infectious diseases of animals

The Friedrich-Loeffler-Institut, founded in 1910 by Friedrich Loeffler, the discoverer of the first animal virus, foot-and-mouth disease virus, is the oldest virological research facility in the world. Beyond viruses, its area of competence has significantly expanded since its foundation and now also covers bacterial, parasitic and prion diseases of livestock, poultry and aquatic animals. Presently located at four sites within Germany (Insel Riems, Jena,Tübingen,Wusterhausen) the tasks of the institute as delineated in the Animal Disease Act encompass research on infectious animal diseases including zoonoses, import/export examinations, epidemiological studies in case of outbreaks of notifiable animal diseases, acting as reference laboratory for notifiable animal diseases and nationwide quality management of diagnosis of notifiable animal diseases. It is obliged to publish and maintain up-to-date diagnostic regimes for notifiable animal diseases, and it publishes a yearly report on animal health in Germany. With the increasing importance of infectious diseases of animals, in particular those potentially harmful to man (zoonoses), the Friedrich-Loeffler-Institut will be moving into new facilities including laboratories and animal facilities up to the highest biosafety level at its main site Insel Riems on the occasion of its 100th anniversary.     

Experimental alveolar echinococcosis in pigs, lesion development and serological follow up

Liver lesions were found in 6/6 pigs 7 months after oral inoculation with 5000 or 35,000 Echinococcus multilocularis eggs. However, lesion morphology differed considerably among the animals. The largest lesions (3–8 mm in diameter) were found in a single pig and smaller lesions (1.5–3 mm) in three pigs. These lesions were clearly circumscribed and had pronounced central necroses and dystrophic calcifications. In contrast, most of the smallest (usually <1.5 mm in diameter) found in two other pigs, had small compact fibrotic areas and blurred borders with obvious fibrous infiltrations into the interlobular tissues. E. multilocularis specific DNA was detected by PCR in all lesion types, but metacestode viability, as assessed by in vivo intraperitoneal inoculations in jirds, could not be demonstrated. Within 1 month post inoculation, all pigs developed specific IgG antibody responses against a battery of different antigens (metacestode, cyst fluid, and protoscoleces-derived native E. multilocularis and E. granulosus antigens, affinity purified Em2G11 antigen, antigen B, recombinant Em II/3–10 antigen). Two different reaction patterns were recorded. In the two pigs with the small lesions, pronounced reactions against all crude antigens with peaks 3–5 months p.i. and clearly elevated levels until the end of the experiment were noted. In all other pigs, antibody reactions remained low in all cases. In conclusion, we demonstrated two types of E. multilocularis metacestode development in pigs with distinct immunological response patterns.     

Development and validation of species-specific primers that provide a molecular diagnostic for virus-vector longidorid nematodes and related species in German viticulture

The nematode species Longidorus attenuatus, L. elongatus, L. macrosoma and Paralongidorus maximusare economically important pests to the viticulture industry due to their ability to vector two nepoviruses (Raspberry Ringspot Virus and Tomato Black Ring Virus) to grapevines. In Germany, these species occur in vineyard soil with other non-vector but morphologically similar longidorid species, L. helveticus, L.profundorum and L. sturhani. Species-specific primers were designed from ribosomal DNA for all seven species to facilitate taxonomic identification for non-specialists. Primers were assessed for their reliability by screening, where possible, a number of populations of each species. Furthermore, their selectivity and sensitivity were determined when challenged with closely related longidorid species and general nematode communities typical of vineyard soil. A multiplex approach using a common forward primer combined with species-specific reverse primers enabled three target nematode species to be detected in the same PCR reaction. All primers were highly specific, detecting all nematode developmental forms from disparate populations and were sufficiently sensitive to detect a single target nematode within a whole nematode community typical of a vineyard soil comprising of a range of non-target species. Given their specificity, sensitivity and reliability, these diagnostic primers should be of great benefit to both phytosanitary/quarantine services related to the viticulture industry and also as a decision management tool for growers.     

Improved arsenic speciation analysis for extracts of commercially available edible marine algae using HPLC-ES-MS/MS

Their increasing commercial availability and their rather high total arsenic contents necessitate a more detailed arsenic speciation analysis of marine algal products. Compared to current HPLC-ICPMS methods, HPLC on-line with electrospray tandem mass spectrometry in the selected reaction monitoring mode (HPLC-ES-SRM) offers much higher detection selectivity with similarly high sensitivity for most known organoarsenic species. This study demonstrates the advantages of HPLC-ES-SRM for the detection of the main as well as trace arsenic species in extracts of 12 commercially available marine algal powders. The main focus was the unambiguous identification of the detected arsenic species. Four quality control tools were applied for this purpose, including matching chromatographic retention times, comparing SRM transition ratios, recording product ion spectra, and determining accurate masses. As a result, evidence was obtained for the presence of 19 organoarsenic species in the analyzed algal extracts. The method of standard addition was used for quantification. Estimated matrix effects on the analyte signal were similar for most of the investigated arsenic species in extracts of different types of brown algae. This allowed the comparison of the contents of the arsenic species present in the 12 algal extracts on the basis of normalized peak areas. A partial correlation of the arsenic speciation pattern with the algal family or algal order, respectively, was found.     

Physiologic VDD versus nonphysiologic VVI pacing in canine 3rd-degree atrioventricular block

Historically, ventricular demand, nonphysiologic (VVI) pacing has been the most commonly used modality to treat 3rd-degree atrioventricular (AV) block. The goal of this study was to determine the feasibility of using a commercial, single-lead, physiologic (VDD) pacemaker in dogs with 3rd-degree AV block. Furthermore, we hoped to characterize and identify differences in the radiographic, echocardiographic, neurohormonal, and quality of life consequences of physiologic versus nonphysiologic pacing. We evaluated 10 dogs during a 12-week crossover study. Acutely, rate-matched physiologic pacing reduced pulmonary capillary wedge pressure by 19% compared with nonphysiologic pacing. VDD pacing significantly reduced left atrial size normalized to body weight, left atrial-to-aortic root ratio, and left ventricular end-systolic dimension and increased fractional shortening, aortic Doppler velocity, cardiac output, and stroke volume compared with VVI pacing. Variable rate VDD pacing resulted in a significantly slower heart rate (HR) during echocardiography than fixed-rate (100 bpm) VVI pacing. AV synchronous pacing reduced circulating N-terminal proatrial natriuretic peptide (ANP), norepinephrine (NOR), and epinephrine (EPI) concentrations compared with asynchronous pacing. There were no significant differences in systemic blood pressure, thoracic radiographs, or owner-perceived quality of life. The median percentage of AV synchronous pacing during the VDD modality was 99.8% (range, 1.2 to 99.9%). This study confirms the potential to achieve physiologic pacing with a commercial, single-lead system in dogs. VDD pacing improved hemodynamics and neurohormonal profiles over asynchronous pacing although the long-term clinical benefits of these changes remain to be determined.     

Chlamydiales in guinea-pigs and their zoonotic potential

The aim was to detect and characterize chlamydial infections in guinea-pigs (GP) with ocular disease, study their pathogenicity and zoonotic potential and to test for the presence of Acanthamoebae spp. in GP eyes and to investigate whether they could act as vectors for Chlamydia-like organisms. Overall 126 GP, of which 77 were symptomatic, were screened by clinical examination, cytology, gross pathology, histology, immunohistochemistry, polymerase chain reaction (PCR) and bacteriology. A new Chlamydiaceae-specific intergenic spacer rRNA gene PCR, designed to amplify this segment linking the 16S and 23S regions, was performed. DNA samples were also received from one owner including samples of his cat and rabbit. Guinea-pigs: 48 of 75 symptomatic, but only 11 of 48 asymptomatic GP were positive by PCR for Chlamydophila caviae guinea-pig inclusion conjunctivitis (GPIC) (P < 0.0001). Eighteen of 75 or 15/48, respectively, were positive for DNA from Chlamydia-like organisms. Acanthamoebae-DNA could be found in two GP, of which one was symptomatic. Owner, cat and rabbit: Samples of all three species were positive by PCR for C. caviae GPIC and the owner's one-day disposable contact lenses showed a positive PCR result for the Chlamydia-like organism Parachlamydia acanthamoebae. No Acanthamoebae-DNA could be detected. This study is the first to describe Chlamydia-like organisms in GP and to detect C. caviae GPIC in human, cat and rabbit. Therefore, C. caviae GPIC could pose a zoonotic potential. We believe that the finding of C. caviae GPIC in species other than GP is probably not unique.     

BKCa-Cav channel complexes mediate rapid and localized Ca2+-activated K+ signaling

Large-conductance calcium- and voltage-activated potassium channels (BKCa) are dually activated by membrane depolarization and elevation of cytosolic calcium ions (Ca2+). Under normal cellular conditions, BKCa channel activation requires Ca2+ concentrations that typically occur in close proximity to Ca2+ sources. We show that BKCa channels affinity-purified from rat brain are assembled into macromolecular complexes with the voltage-gated calcium channels Cav1.2 (L-type), Cav2.1 (P/Q-type), and Cav2.2 (N-type). Heterologously expressed BKCa-Cav complexes reconstitute a functional "Ca2+ nanodomain" where Ca2+ influx through the Cav channel activates BKCa in the physiological voltage range with submillisecond kinetics. Complex formation with distinct Cav channels enables BKCa-mediated membrane hyperpolarization that controls neuronal firing pattern and release of hormones and transmitters in the central nervous system.     

Effects of different media on proliferation and differentiation capacity of canine, equine and porcine adipose derived stem cells

Adult stem cells are of particular interest for therapeutic use in the field of regenerative medicine. Adipose-derived mesenchymal stem cells (ASCs) are an attractive stem cell source for all fields of regenerative medicine because adipose tissue - and therewith cells - can easily be harvested from each donor. However, common expansion using fetal bovine serum (FBS) can not be used for clinical applications as xenogenic proteins must be avoided. Adipose tissue from equine, canine and porcine donors was digested with collagenase to isolate ASCs. ASCs were either expanded in a cell culture medium supplemented with FBS or in a serum-free medium (UltraCulture; UC) supplemented with a serum substitute (UltroserG). From all three animal species, the adipogenic and osteogenic differentiation potential of ASCs cultured with different media was analyzed in vitro. Cell proliferation analysis showed a population doubling time of 48-68 h for canine cells, 54-65 h for porcine cells and 54-70 h for equine cells, expanded in different media. Except for porcine ASCs, cells cultured in media supplemented with FBS grew faster than cells expanded in UC medium with UltroserG. Yet, all cells maintained their potential to differentiate into adipocytes and osteoblasts. UltraCulture medium containing UltroserG can for all examined species be recommended if FBS needs to be avoided in the expansion of donor-derived (stem) cells.