3R-Project 116-09

Organotypic brain slice cultures derived from regularly slaughtered animals as in vitro alternative for the investigation of neuroinfectious diseases in ruminants

Anna Oevermann and Torsten Seuberlich
Neurocenter, Department of Clinical Research and Veterinary Public Health, Vetsuisse Faculty Bern, 3001 Bern, Switzerland
anna.oevermann@itn.unibe.ch, torsten.seuberlich@itn.unibe.ch

Keywords: live stock; brain; cns, brain disorders; veterinary disease; cell cultures: organ-specific; reduction; replacement

Duration: 3 years Project Completion: 2013

Background and Aim
Infectious disorders of the central nervous system in livestock may have severe economic and public health implications and are therefore of major concern. This was demonstrated dramatically in the mid 1990s, when it became evident during the upsurge of bovine spongiform encephalopathy (BSE) that the disease was transmissible from cattle to humans. Recently, atypical variants of transmissible spongiform encephalopathies (TSEs) have been detected in ruminants, whose potential to cross over to other species including humans is not known at present. Listeriosis, caused by Listeria monocytogenes (LM), is another infectious and zoonotic CNS disease of high impact on livestock and humans.
Despite intense research activities in the field of TSEs and listeriosis during the past decades, very few in-vitro models for their neuropathogenesis, host-pathogen interactions and strain-typing exist. Studies largely depend on bioassays either in laboratory rodents or in the natural ruminant host because they reflect best the intricate pathogenesis of CNS infections. Such experiments raise fundamental ethical concerns, considering the highly invasive inoculation routes and the resulting severe disease, because of which most of these experiments are classified into the highest severity degree (Schweregrad 3). In the case of TSEs, experiments may last up to several years until the animals are sacrificed at the end. In addition, is often not clear to what extent the results of rodent models can be extrapolated to the natural hosts’ situation. On the other hand, pertinent cell models of ruminant neuroinfectious diseases do not exist. Our aim is, therefore, to develop an ethically sustainable, host specific, organotypic brain slice culture of regularly slaughtered ruminants as an in-vitro system for the identification and investigation of neuroinfectious diseases using the examples of prion diseases and listeric encephalitis.

Method and Results
in progress (present status)
The validation of bovine organotypic brain slice cultures derived from the slaugtherhouse has been accomplished (Guldimann et al., 2012, International Journal of Experimental Pathology).

Figure 1: Hippocampal and cerebellar brain slice culture from a bovine. Anatomical architecture is maintained and at the edges cells grow out.

We assessed viability of brain slices by determining the difference between the number of dead cells and the total number of cells present in a slice (Figure 2).

Figure 2: Hippocampal slice, combined fixable-viability (blue, nuclei of dead cells) and PI (red, all nuclei) stain.

Furthermore, the presence of brain cell-types (neurons, microglia, astrocytes, oligodendrocytes) was assessed by double-immunofluorescence. We could show that bovine brain slices can be maintained in culture up to 49 days in culture and that all endogenous brain cell populations are present (Figure 2-4). Viability results of hippocampal slices are consistently better than those of cerebellar slices.

Figure 3: The average proportion of viable cells as estimated from dead cell and total cell counts are indicated at different time points for hippocampal and cerebellar tissue-slice cultures from six different calves (error bars represent standard deviations).

Because viability of brainstem and cortical slices was low, experiments with these brain regions were discontinued and infection assays were performed with hippocampal slices.

Figure 4: a) Immunofluorescence of the hippocampal dentate gyrus (day 7 in vitro). Neurons are stained in green (NeuN), nuclei are stained in blue (TOTO-3).
b) Immunofluorescence of the hippocampal dentate gyrus (day 28 in vitro). Neurons are stained in green (NeuN), nuclei are stained in blue (TOTO-3).
c) Immunofluorescence of the hippocampal dentate gyrus (day 49 in vitro). Neurons are stained in green (NeuN), nuclei are stained in blue (TOTO-3).

This in vitro system is susceptible to Listeria monocytogenes (LM) infection and replicates features of natural rhombencephalitis in ruminants (Figure 5). Forty-seven LM strains from various clinical origin and various genetic complexes were inoculated in hippocampal brain slices in order to investigate whether these strains differ in neuropathogenicity. The infection assays were performed according to the protocol published in the first manuscript (Guldimann et al, 2012) with slight modifications. Briefly, a micromanipulator with a 0.5 microliter Hamilton syringe was used to inject 0.1ul of the bacterial solution coresponding to approximately 100 colony forming units (CFU) into the dentate gyrus of the hippocampal brain slice. Slices were incubated without antibiotics for 3 hours to allow the infection to establish. The medium was then changed to a gentamicin-containing formula at 3h to kill extracellular bacteria. Infection assays were analyzed for bacterial replication (cfu number), bacterial spread (size of bacterial foci) and cytopathic effect (percentage of dead cells). Results of the experiments were normalized to an internal control strain (L104).

Figure 5: a) Natural case of listeric rhombencephalitis, double-immunofluorescence for L. monocytogenes (red) and microglia cells (CD68 in green). Focal replication of L. monocytogenes.
b) Brain slice inoculated with L. monocytogenes (red). Microglia cells are stained with CD68 (green). Focal replication of L. monocytogenes.

All but one LM strains were able to infect and replicate in bovine hippocampal slices. Although infection assays in brain slice cultures do not clearly discriminate encephalitogenic from non-encephalitogenic and environmental LM strains, they show interstrain differences in neurovirulence linked to the genotype. LM strains from MLVA complex A (as determined by Multilocus Variable Number of Tandem Repeats Analysis, MLVA; L. Balandyte et al. 2011, Applied and Environmental Microbiology) replicate and spread more efficiently in hippocampal slices than strains belonging to MLVA complex C (Figures 6 and 7).

Figure 6: CFU’s recovered from lysed hippocampal slices inoculated with Listeria monocytogenes strains (n=47) from MLVA complexes A and C. Values are expressed as % of an internal reference strain (L104). Complex A strains have significantly increased CFU’s compared to complex C strains indicating they replicate more efficiently in hippocampal slices.

Figure 7: µm² area of hippocampal slices invaded by Listeria monocytogenes strains from MLVA complexes A and C. Values are expressed as % of an internal reference strain (L104). Areas occupied by complex A strains are significantly larger than areas occupied by complex C strains indicating complex A strains spread more efficiently between cells in hippocampal slices.

Conclusions and Relevance for 3R
Slaughterhouse-derived brain-slices showed sufficient viability and could be successfully infected with L. monocytogenes (Guldimann et al. 2012, International Journal of Experimental Pathology). Results from our infection assays with LM indicate that strains from all three genetic complexes (L. Balandyte et al. 2011, Applied and Environmental Microbiology) are able to cause encephalitis and that all LM strains have to be considered as potentially neurovirulent. However, together with our MLVA data (L. Balandyte et al. 2011), according to which most encephalitis strains from ruminants strongly cluster in the genetically homogenous complex A, our infection assays indicate that complex A strains are particularly neurovirulent. Organotypic brain slices from the slaughterhouse appear to be an adequate in vitro model to study pathogenesis of the intracerebral phase of LM infection and to screen LM strains for neurovirulence. We now have a well characterized in vitro model available that we trust to be a valuable tool for infection experiments with L. monocytogenes strains from different sources and genotypes. In the future, ruminant brain-slices will offer us the possibility to study bacterial determinants of neurovirulence and intracerebral spread, which is crucial for the understanding of the neuropathogenesis.
We are confident that organotypic brain slices cultures derived from the slaughterhouse are an ethically sustainable and host specific in vitro system for the identification and investigation of neuroinfectious agents. The suitability of the system for long culture times lends itself to the investigation of slow-growing agents like prions and indeed we have first indications (that need to be confirmed) that brain slices are susceptible to prion infection. Such cultures not only help to spare the life of animals, but also offer the combined advantages of reflecting the organ-specific microarchitecture of the brain similar to the in vivo situation and at the same time of working under controlled conditions.
Our results on CNS listeriosis might stimulate other scientists to consider our in vitro approach as an alternative to animal experiments for their own research. This system has the potential to significantly replace the use of animals in models of prion diseases and other neuroinfectious diseases with impact on veterinary and public health.

References
Guldimann C, Lejeune B, Hofer S, Leib SL, Frey J, Zurbriggen A, Seuberlich T, Oevermann A. Ruminant organotypic brain-slice cultures as a model for the investigation of CNS listeriosis. Int J Exp Pathol. 2012 Aug;93(4):259-68. doi:10.1111/j.1365-2613.2012.00821.x.

Lina Balandyté, Isabelle Brodard, Joachim Frey, Anna Oevermann and Carlos Abril. Ruminant Rhombencephalitis-Associated Listeria monocytogenes Alleles Linked to a Multilocus Variable-Number Tandem-Repeat Analysis Complex. Appl. Environ. Microbiol. December 2011 vol. 77 no. 23 8325-8335. doi:10.1128/AEM.06507-11