A new S. suis serotype 3 infection model in pigs: lack of effect of buprenorphine treatment to reduce distress.

BACKGROUND: Streptoccocus suis (S. suis) is a major porcine pathogen causing meningitis, septicemia, arthritis and endocarditis. These diseases severely impair welfare of pigs. Experimental studies in pigs are important to better understand the pathogenesis and to identify protective antigens, as so far there is no vaccine available protecting against various serotypes (cps). Due to the severity of disease, application of appropriate refinement strategies in experimental S. suis infections is essential to reduce distress imposed on the piglets without jeopardizing the scientific output. The objectives of this study were to evaluate buprenorphine treatment as a refinement measure and serum cortisol levels as a distress read out parameter in a new S. suis cps3 infection model in pigs. RESULTS: Intravenous application of 2 × 108 CFU of S. suis cps3 (sly+, mrp+) to 6-week-old piglets led to severe morbidity in approximately 50% of the animals. Main pathological findings included suppurative meningoencephalitis and arthritis as well as fibrinosuppurative endocarditis. Buprenorphine treatment (0.05 mg/kg every 8 h) did not prevent signs of severe pain, high clinical scores, moderate to severe pathologies or high levels of serum cortisol in single severely affected piglets. Significant differences in the course of leukocytosis, induction of specific antibodies and bactericidal immunity were not recorded between groups with or w/o buprenorphine treatment. Of note, clinically unobtrusive piglets showed serum cortisol levels at 2 and 5 days post infectionem (dpi) comparable to the levels prior to infection with cps3. Cortisol levels in serum were significantly increased in piglets euthanized due to severe disease in comparison to clinically unobtrusive pigs. CONCLUSIONS: Different clinical courses and pathologies are induced after intravenous challenge of piglets with 2 × 108 CFU of this S. suis cps3 strain. The chosen protocol of buprenorphine application does not prevent severe distress in this infection model. Important parameters of the humoral immune response, such as the level of IgM binding to S. suis cps3, do not appear to be affected by buprenorphine treatment. Serum cortisol is a meaningful parameter to measure distress in piglets experimentally infected with S. suis and to evaluate refinement strategies. In this intravenous model, which includes close clinical monitoring and different humane endpoints, clinics and cortisol levels suggest convalescence in surviving piglets within 5 days following experimental infection.

Expanding the host range: infection of a reptilian host (Furcifer pardalis) by an atypical Brucella strain.

Atypical brucellae show deviant phenotypes and/or genotypes. Besides Brucella inopinata, B. microti and B. vulpis, atypical strains have been described infecting humans, rodents, amphibians and fish. They represent potential zoonotic agents. Here, we provide evidence that reptiles as the remaining poikilothermic vertebrate class also represent susceptible hosts for atypical Brucella.

Chronic wasting associated with Chlamydia pneumoniae in three ex situ breeding facilities for tropical frogs.

A number of different Chlamydia spp. have been detected in the class Amphibia with C. pneumoniae being the predominant species involved. Chlamydiae have been linked to mass mortality events, thereby representing significant pathogens that deserve attention with respect to worldwide amphibian decline. We here present six cases of chlamydiosis and asymptomatic chlamydial infections in different frog species from three ex situ amphibian conservation facilities. Clinical signs predominantly characterised by regurgitation, chronic wasting, lethargy and suspended breeding were associated with C. pneumoniae infection. Despite various treatment regimens, it was not possible to clear infections. However, intra vitam diagnostics succeeded from skin, faeces and urine for the first time.

Displaying high-affinity ligands on adeno-associated viral vectors enables tumor cell-specific and safe gene transfer.

Gene transfer vectors derived from the adeno-associated virus (AAV) have recently received increasing attention due to substantial therapeutic benefit in several clinical trials. Nevertheless, their great potential for in vivo gene therapy can only be partially exploited owing to their broad tropism. Current cell surface targeting strategies expanded vector tropism towards transduction of cell types that are inefficiently infected naturally, but failed to restrict or fully re-direct AAV's tropism. Hypothesizing that this limitation can be overcome by equipping natural receptor-blinded AAV vectors with high-affinity ligands, we displayed designed ankyrin repeat proteins (DARPin) as VP2 fusion proteins on AAV capsids ablated for natural primary receptor binding. These second generation targeting vectors demonstrated an as of yet unachieved efficiency to discriminate between target and non-target cells in mono- and mixed cultures. Moreover, DARPin-AAV vectors delivered a suicide gene precisely to tumor tissue and substantially reduced tumor growth without causing fatal liver toxicity. The latter caused death in animals treated with conventional AAV vectors with unmodified capsids, which accumulated in liver tissue and failed to affect tumor growth. This novel targeting platform will be key to translational approaches requiring restricted and cell type-specific in vivo gene delivery.

DARPin-targeting of measles virus: unique bispecificity, effective oncolysis, and enhanced safety.

Oncolytic virotherapy is an emerging treatment modality that uses replication-competent viruses to destroy cancers. Many naturally occurring viruses have a preferential, although nonexclusive, tropism for tumors and tumor cells. In addition, specific targeting of cancer cells can be achieved at the virus entry level. We optimized retargeting of cell entry by elongating the measles virus attachment protein with designed ankyrin repeat proteins (DARPins), while simultaneously ablating entry through the natural receptors. DARPin-targeted viruses were strongly attenuated in off-target tissue, thereby enhancing safety, but completely eliminated tumor xenografts. Taking advantage of the unique properties of DARPins of being fused without generating folding problems, we generated a virus simultaneous targeting two different tumor markers. The bispecific virus retained the original oncolytic efficacy, while providing proof of concept for a strategy to counteract issues of resistance development. Thus, DARPin-targeting opens new prospects for the development of personalized, targeted therapeutics.

First detection of autochthonous Lamanema chavezi infections in llamas (Lama glama) in Europe.

Lamanema chavezi is one of the most pathogenic nematode species of South American camelids (SAC), with a homoxenous life cycle involving enterohepatic migration of its larvae in the host. So far, it has been found in the Americas and New Zealand. The first autochthonous L. chavezi infections in SAC in Europe are reported here. On a SAC farm in Germany, a 15-month-old male llama with a short history of diarrhoea died in September 2017, followed nine months later by a three-year-old female llama with a history of emaciation, apathy, anorexia, anaemia and tetraparesis with retained sensorium. Both animals were born and raised on the farm, which had imported three llamas directly from Chile 4-14 years earlier. At necropsy, the main lesions in both cases were numerous white-yellow to dark red foci, up to 3 mm in size, close to the Glisson's capsule and deep in the parenchyma of the liver. Histologically, the livers showed haemorrhagic tracks by and with nematode larvae and a necro-haemorrhagic to fibrinous inflammation with a predominantly lymphohistiocytic infiltration. The larvae were 30-50 µm in diameter and had external longitudinal cuticular ridges. Larvae extracted from unfixed liver tissue were 1800-2000 µm long and about 80 µm in diameter, with a terminal spine at the posterior end, which is characteristic of female L. chavezi stages. The ribosomal DNA including the almost complete 18S rRNA gene, the first internal transcribed spacer, the 5.8S RNA gene, the second internal transcribed spacer (ITS) and the partial 28S rRNA gene from isolated larvae were amplified using nematode-specific oligonucleotide primers and then sequenced. The assembled nematode sequence of 3448 bp showed an identity of 99.4% to previously published L. chavezi sequences in the BLASTN search. Low numbers of L. chavezi-like eggs were found in the faeces of seven (29%) of 24 llamas and alpacas in the herd, including some farm-born crias, tested two years after the last fatal case. The results show for the first time that L. chavezi has not only been imported into Europe from South America, but has also completed its life cycle locally, resulting in autochthonous infections of SAC. This was also suspected to be the cause of the fatal disease in two llamas.

[Oligodendroglioma with neuronal differentiation in an 8-month-old African hedgehog (Atelerix albiventris)]. / Oligodendrogliom mit neuronaler Differenzierung bei einem 8 Monate alten Afrikanischen Weißbauchigel (Atelerix albiventris).

An 8-month-old, male African hedgehog clinically displayed a wobbly walk, anuria, inappetence and apathy, whereupon the suspected diagnosis wobbly hedgehog syndrome was made. After exacerbation, the hedgehog was euthanized. Histologically, a tumour mainly consisting of medium-sized, oval tumour cells and a smaller number of spindeloid cells was found in the cerebrum. The tumour contained neuropil islets and extracellular myxoid material. Immunohistochemically, the tumour cells expressed oligodendroglial (neurite outgrowth inhibitor, Nogo-A; oligodendrocyte transcription factor, Olig-2) and neuronal (neuron-specific enolase, NSE; microtubule-associated protein-2a, MAP-2a; synaptophysin) cell markers. Based on these findings, an oligodendroglioma with neuronal differentiation was diagnosed. Such a brain tumour has to date not been reported for African hedgehogs. At necropsy, a severely filled and dilated urinary bladder was observed, which was presumably caused by a central blockade of the micturition centre in the brain. In the case of neurological symptoms in young hedgehogs, a primary brain tumour should, as in adults, be considered as a differential diagnosis. As further differentials, inflammatory-infectious (rabies, herpes, baylisascariosis), degenerative (cardiomyopathy, intervertebral-disc disease), traumatic, alimentary (vitamin-B deficiency) and metabolic-toxic (heat-cold-torpor, hepatic encephalopathy) triggers have to be considered.

Intrahepatic application of suicide gene-armed measles virotherapeutics: a safety study in transgenic mice and rhesus macaques.

Abstract Oncolytic viruses such as measles virus (MV) represent a new class of therapeutic agents that might help to overcome current limitations in cancer therapy. Although MV-based virotherapeutics already have entered clinical testing for various tumor entities, the preclinical safety of MV virotherapeutics so far has not been elucidated for particular regimens with high medical need, such as (1) direct injection into hepatic tumor sites, (2) employing high doses ibidem, and (3) concurrent usage of arming with cytotoxic genes required to further enhance oncolytic efficiency. Here, we assessed the safety and pharmacokinetics of suicide gene-armed vector MV-SCD when administered intrahepatically in two animal models, IFNAR(tm)-CD46(Ge) (interferon-α receptor deficient and CD46 MV receptor knock-in) transgenic mice and rhesus macaques (Macaca mulatta). Clinically, singular direct intrahepatic applications of MV-SCD were found to be well tolerated. Quantitative RT-PCR demonstrated the transient presence of viral RNA in various organs, whereas no shedding of infectious virus particles was observed at any time point. Histological analyses of organs did not exhibit adverse effects attributable to the test article. Blood parameters including liver enzymes revealed no deviations from normal. In both species an antiviral humoral immune response was mounted shortly after virus administration. Surprisingly, daily repeated systemic applications of MV-SCD under concomitant prodrug administration resulted in side effects in IFNAR(tm)-CD46(Ge) mice, but were less pronounced than in a 5-fluorouracil standard therapy control cohort. Taken together, these data indicate that "single shot" direct intrahepatic injections of MV-SCD in conjunction with systemic prodrug administration are safe and could be used in future virotherapeutic treatments of liver cancers.