Low levels of classical BSE infectivity in rendered fat tissue
BSE infectivity in mesentery fat is most likely associated with embedded nervous tissue. To prove this mesentery containing celiac ganglion was taken from oral BSE infected cattle in different stages of the disease and from one control animal. Fat was rendered according to standard tallow production methods and the prion infectivity therein analysed in transgenic mouse bioassay. Rendered fat of the clinical animal revealed low infectivity levels, whereas preclinical and control animals remained negative. This study, although not representative, provides a proof of principle, indicating the potential contamination of melted mesenteric fat by embedded nervous structures during standard tallow production.
bovine spongiform encephalopathy
transmissible spongiform encephalopathies
pathological prion protein
celiac and mesenteric ganglion complex
chronic wasting disease
specified risk material
Introduction, methods and results
Bovine spongiform encephalopathy (BSE) is a zoonotic , fatal neurodegenerative disease, belonging to the group of transmissible spongiform encephalopathies (TSE). All TSEs are associated with the accumulation of the pathological prion protein (PrPSc), which results by the conversion of the normal host-encoded cellular prion protein and is the marker of the disease . Among other TSEs classical BSE is transmitted via the oral uptake of infectious material [3, 4]. Besides cattle, cats , goats  and humans  are susceptible to the disease by oral uptake of infectious foodstuff. During the incubation period the BSE agent spreads from the gut, in particular the ileum, via sympathetic and parasympathetic nerve fibers and ganglia to spinal cord and brain stem [7, 8]. In orally BSE infected cattle first traces of infectivity can be detected in splanchnic nerves and in the celiac and mesenteric ganglion complex (CMGC) at 16 and 20 months post-infection (mpi), followed by an increase of infectivity until to the clinical stage when even a PrPSc accumulation can be seen in the ganglion . Therefore, it is widely accepted that infectivity in mesentery, if present at all, is most probably associated with fat embedded nerve fibers and autonomic ganglia . However, studies using fat from BSE affected cattle failed to detect any infectivity in RIII wild type mice . More recently infectivity in adipose tissues has been shown for murine adapted scrapie and for chronic wasting disease (CWD) [11, 12].
The BSE exposure risk to humans and animals is currently minimized by the removal of specified risk material (SRM) from the food and feed chains. SRMs, as defined in Regulation (EC) No. 999/2001 as amended, are tissues containing the highest level of infectivity in BSE respectively TSE incubating animals. These include, among others, intestine and mesentery of animals of certain age/species from animals whose origin is in Member States or third countries with controlled or undetermined BSE risk. In this regard it has to be bear in mind that mesentery contains a network of nerves, ganglia, blood and lymph vessels as well as lymph nodes to support the gut . However, up to now, it remains unclear whether a BSE contamination of rendered fat could occur during tallow production. Therefore, the study presented here intended to clarify, whether BSE infectivity can be found in fat rendered from mesentery fat embedded CMGCs of BSE infected cattle at different stages of the disease. Obtained data are important for qualitative and quantitative risk assessments and for the still ongoing discussion regarding the further usage of ruminant adipose tissue.
All infection experiments in cattle (LVL-MV 310-4/7221.3-1.1-019/02) and mice (LALLF 7221.3-1.1-25/09) described in this manuscript were approved by the competent authority of the Federal State of Mecklenburg-Western Pomerania, Germany, on the basis of national and European legislation, namely the directive 2010/63/EU on the protection of animals used for scientific purposes.
Anamnestic data of the celiac and mesenteric ganglion complex used and results of the mouse bioassay done with rendered mesentery fat
Results CMGC 
Results rendered fat
Mouse-bioassay (Tgbov XV)
Mouse-bioassay (Tgbov XV)
> 629 dpi
> 664 dpi
> 592 dpi
> 633 dpi
The results presented here indicate that a certain amount of BSE infectivity must be present in the mesentery to contaminate the rendered fat in a detectable level during tallow production methods. As shown by  BSE infectivity increases in the CMGC of orally BSE infected cattle from early to late preclinical up to the highest levels at clinical stage. Hence, it comes without surprise that the infectious fat presented here originates from the CMGC of the clinical animal, which also showed a clear PrPSc accumulation pattern . Additionally these results are in accordance with the infectivity data reported for fat tissue of CWD infected deer at later stages of the disease .
In our hands only low level of infectivity were found. However, the experimental adipose/CMGC tissue dilution factor needed for the fat rendering should also be considered, so the actual infectious load of the samples might be higher. However, the low infectivity load found here might explain why earlier studies failed to detect BSE infectivity in adipose tissue. For one reason this studies did not definitely regard nerves and ganglia which are involved in the pathogenesis of BSE and for another reason less susceptible conventional wild type mice were used [10, 15, 16]. Interestingly omental fat of deer infected with CWD at a clinical stage revealed a much higher infectivity level as compared to our results . Therefore, it is tempting to speculate that these differences might be due to the qualitative and quantitative more widespread CWD distribution in bodily tissues  as compared to cattle BSE. However, it has to bear in mind that the two TSE strains are different, different mouse line were used and neither for CWD samples nor for our BSE sample a calibration curve for infectivity exist. Nevertheless, it would be of interest to what extent rendered fat could be contaminated by using mesentery from sheep and goats infected with classical scrapie or BSE, all entities showing in most cases a higher PrPSc accumulation in the autonomous nervous system than BSE infected cattle [18, 19, 20, 21].
Our results, in particular the close relationship with the infectivity/PrPSc data of the CMGC samples, clearly support the widespread accepted assumption that infectivity in the mesentery is most probably associated with nerves and autonomous ganglia , whereas the direct involvement of fatty cells is uncertain . Another source of infectivity could be in mesentery fat embedded lymph nodes. However, no infectivity has ever been detected in mesenterial lymph nodes of BSE infected cattle so far [13, 22] and all grossly evident lymphoreticular tissue was removed from our samples. Additionally a long term study showed that full blood transfusion from clinical BSE infected cattle to naïve calves did not transmit BSE . Therefore, a blood contamination of the sample as possible source of infectivity is highly unlikely. Furthermore, as all tissue remnants in the rendered fat were removed by a centrifugation step before inoculation, all infectivity must be bound to the liquid fat solely.
These results might not be representative due to the small sample size and therefore provide only a proof of principle. In particular with regard to the negative early and late preclinical samples, there still remain some uncertainties, which can only be resolved by a more extended, that say statistical profound study. However, the inocula were generated according to standard tallow production methods , therefore our results clearly show that such a contamination is conceivable. Rendered fat can been used for food (i.e. premier jus, frying agent), pet food and feed application , therefore BSE/TSE infectivity could enter both the food and feed chain. At the time of writing the current SRM legislation prevented the usage of mesentery fat from animals whose origin is from countries with controlled or undetermined BSE risk. However, this regulation is still under discussion and might be changed in near future (EU Commission, personal communication).
The authors declare that they have no competing interests.
CF did the scientific work with the support of MHG. The paper was written by CF and MHG. The animal experiments, necropsies and sampling were done by UZ, MKa, CF, MKe. MKe provided the Tgbov XV mice. All authors read and approved the final manuscript.
The authors would like to thank Gesine Kreplin for their excellent technical assistance and Kerstin Kerstel, Nicole Sinkwitz and Lukas Steimke for care and handling of the mice.
Ethics approval and consent to participate
All infection experiments in cattle (LALLF 7221.3-1.2-008/07) and mice (LALLF 7221.3-1.1-25/09) described in this manuscript were approved by the competent authority of the Federal State of Mecklenburg-Western Pomerania, Germany; on the basis of national and European legislation, namely the directive 2010/63/EU on the protection of animals used for scientific purposes.
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- 6.Eloit M, Adjou K, Coulpier M, Fontaine JJ, Hamel R, Lilin T, Messiaen S, Andreoletti O, Baron T, Bencsik A, Biacabe AG, Beringue V, Laude H, Le Dur A, Vilotte JL, Comoy E, Deslys JP, Grassi J, Simon S, Lantier F, Sarradin P (2005) BSE agent signatures in a goat. Vet Rec 156:523–524CrossRefGoogle Scholar
- 10.Fraser H, Foster J (1993) Transmission to mice, sheep and goats and bioassay of bovine tissues. In: Bradley R, Marchant B (eds) Transmissible spongiform encephalopathies. A consultation on BSE with the Scientific Veterinary Committee of the Commission of the European Communities. European Commission Agriculture, Brussels, pp 145–159Google Scholar
- 18.Van Keulen LJ, Schreuder BE, Vromans ME, Langeveld JP, Smits MA (2000) Pathogenesis of natural scrapie in sheep. Arch Virol 16:57–71Google Scholar
- 21.Tauscher K (2015) Studie zur Pathogenese der Bovinen Spongiformen Enzephalopathie der Ziege. Ph.D. Thesis, University of Veterinary Medicine Hannover, Foundation, GermanyGoogle Scholar
- 22.SSC (Scientific Steering Committee) (2002) Update of the Opinion on TSE Infectivity distribution in ruminant tissues. Scientific Steering Committee, ParisGoogle Scholar
- 23.Balkema-Buschmann A, Ziegler U, Tauscher K, Köster F, Fatola OI, Schinköthe J, Fast C, Ulrich R, Priemer G, Groschup MH (2018) Absence of classical and atypical (H- and L-type) BSE prions in the blood of clinical bovine cases as bioassayed by cattle to cattle blood transfusions. Proc Prion WA 11:41Google Scholar
- 24.Woodgate SL, van der Veen JT (2014) Fats and oils—animal based. In: Clark S, Jung S, Lamsal B (eds) Food processing: principles and applications, 2nd edn. Wiley, New YorkGoogle Scholar
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