68
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Figure 10. (a) In the ISA reaction, amyloid deposits were stained blackish-brown and formed a leopard-print pattern (arrows). (b) Amyloid deposits were present in the perifollicular area. H&E.
The deposited amyloid was determined to be AA amyloid. (c) Amyloid deposits were not found with H&E, but a small amount of amyloid deposits was detected with anti-human AA amyloid mAb.
Naturally, the amount of amyloid deposits could not be observed by macroscopic observation under ISA reactants. (d) Deposited amyloid was observed in the interstitium of the medulla in kidney. H&E.
(e) Amyloid deposits were not found with H&E, but a small amount of amyloid deposits was detected with anti-human AA amyloid mAb. (f) Amyloid deposits were present in the glomeruli (arrows).
Insets in b to f: Immunohistological staining with a 1:200 dilution of anti-human AA amyloid mouse monoclonal antibody. The degree of amyloid deposits in the tissues in indicated by +++: severe deposits, ++: moderate deposits and +: mild deposits.
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Figure 11. Serum SAA concentrations in each group rabbits. The bars indicate the median values.
“A, D or F (+)” indicates rabbits with amyloid deposits in group A, D or F, and “A, D or F (-)”
indicates rabbits without amyloid deposits in group A, D or F. The SAA concentrations collected from healthy rabbits (control) and SH rabbits (control SH) before the first inflammatory stimulation were 0.065 and 0.162 at a median, respectively. In group C, serum of rabbits couldn't be collected in this experiment.
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Table 6. Experimental groups and number of rabbits with amyloidosis.
Group Inflammatory
stimulations State of foot Bovine amyloid†† No. of rabbits used
No. of rabbits with amyloidosis ISA* reaction Histological examination
A FCA** SH† + 27 7 (26%) 9 (33%)
B FCA SH-like + 11 0 0
C FCA Normal + 6 0 0
D StA*** SH + 9 5 (56%) 7 (78%)
E StA SH-like + 7 2 (29%) 3 (43%)
F StA Normal + 7 0 0
*ISA: iodine sulfuric acid, **FCA: Freund’s complete adjuvant. ***StA: Staphylococcus spp. adjuvant.
†SH: Sore Hocks. SH-like: artificially developed pododermatitis.
††Bovine amyloid: administration of 1ml bovine amyloid fibril solution.
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Table 7. The severity of foot lesions and degree of amyloid deposits.
Group (SH or SH-like)
No. of rabbit
Grading of foot lesion*
Degree of amyloid deposits**
renal medulla renal cortex spleen
A (SH)
1 + ++ - ++
6 + - + +
17 ++ ++ + -
10 ++ - - +++
3 +++ - - +
7 +++ ++ - +
12 +++ ++ ++ +
24 +++ + + ++
25 +++ - - +++
D (SH)
3 + + + -
5 ++ + + +
7 ++ + + ++
2 +++ + ++ +++
8 +++ - + ++
9 +++ - - ++
4 +++ + + +++
E (SH-like)
2 + - - ++
7 + - - ++
6 +++ + - -
*SH: +,mild legion; ++,moderate legion frequently associated with abscesses in subcutaneous tissue;
+++,severe legion with multiple abscess formations. SH-like: +,mild legion; ++,moderate legion;
+++,severe legion
** +, mild amyloid deposits; ++, moderate amyloid deposits; +++, severe amyloid deposits
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CONCLUSION
In chapter 1, the author demonstrated that atypical bovine amyloidosis does not produce clinical symptoms due to the absence of amyloid deposition in glomeruli; even though considerable amyloid deposition occurred in extra-renal organs. Although bovine AA amyloidosis is a rare disease in clinical practice, amyloid deposition in slaughtered cattle were observed at a rate of 0.8 to 5%. Analysis of the distribution of amyloid deposition revealed that it occurred in animal parts that are frequently consumed, including the liver, tongue, intestine, and skeletal muscle.
In chapter 2, AA amyloidosis was suspected of being induced in growing chickens by vaccinations, and also that chicken meat was contaminated by amyloid fibrils. In addition to broiler chickens, some parts of culled layer chickens are consumed as a meat source. Follow-up surveillance revealed the existence of minute amyloid deposits in culled layer chicken that had been exposed to multiple vaccinations, and amyloid deposits were observed mainly in the muscle tissue where the vaccines were administered (data not shown).
In chapter 3, chicken AA amyloidosis was shown to be transmitted within a species after oral administration of chicken AA fibrils. During epidemics of AA amyloidosis in wild and captive birds, the horizontal spread of AA amyloidosis among birds has been suspected but never demonstrated. However, the results of the present study showed that the horizontal spread of amyloidosis by ingestion of amyloid-contaminated feed or feces occurs among avian species.
Chapter 4 described how bovine AA amyloidosis was transmitted to rabbits with SH and SH-like lesions. In these animal systems, the author found that the persistent S.
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aureus infection associated with SH facilitates the development of experimental AA amyloidosis in rabbits, and that the inflammatory stimulation provided by SH promotes the onset of experimental AA amyloidosis in rabbits.
The results show that amyloid fibrils from domestic animals appear to have penetrated the human food chain deeper than was suspected. In Japan, human AA amyloidosis is most frequently observed in patients with rheumatoid arthritis, which itself is associated with high and sustained plasma SAA concentrations. Among patients exhibiting these symptoms, ingestion of amyloid-contaminated food is suspected of causing the development of AA amyloidosis. Since the publication of a recent study, which revealed that bovine and avian AA amyloidosis can be transmitted between different species, the presence of amyloid in meat for consumption may be a major public health hazard. Although the potential risk of food animal amyloidosis cannot be accessed at present, the author considers that cross-species transmission of AA amyloidosis into humans is difficult, primarily because of the existence of a species-barrier in cross-species transmission of AA amyloidosis. Furthermore, as shown in chapter 4, successful transmission of AA amyloidosis between species requires the existence of special pathological conditions in the host animal.
Despite having been discovered a long time ago, the transmissibility of AA amyloidosis has only received attention in the literature relatively recently. Indeed, numerous questions related to the cross-species transmission of AA amyloidosis remain to be resolved. The concept of transmission via self-propagating protein structures or by seeding is unique, and it is important to understand the pathogenesis of protein misfolding diseases. The present study will provide valuable information for further investigations on the transmission of amyloidosis.
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ACKNOWLEDGEMENTS
The author is deeply appreciative and grateful to my chief supervisor, Prof. Dr.
Naotaka Ishiguro, Laboratory of Food and Environmental Hygiene, Department of Veterinary Medicine, Gifu University, for his instructions, kind direction, valuable suggestions and criticisms.
The author is also greatly indebted to my other supervisors, Prof. Dr. Yoshiyasu Kobayashi, Laboratory of Veterinary Pathology, Department of Basic Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, Prof. Dr.
Masanobu Goryo, Department of Veterinary Pathology, Faculty of Agriculture, Iwate University, Prof. Dr. Makoto Shibutani, Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology and Prof. Dr. Tokuma Yanai, Laboratory of Veterinary Pathology, Department of Veterinary Medicine, Gifu University, for their careful reading of this manuscript, helpful suggestions and kind supports in my experiments.
The author is very grateful to Associate Prof. Dr. Yasuo Inoshima, Laboratory of Food and Environmental Hygiene, Department of Veterinary Medicine, Gifu University, for his valuable advice and support during my study time in Gifu University.
The author is very much indebted to former Prof. Dr. Takane Matsui, Laboratory of Veterinary Pathology, Department of Basic Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, for his cooperation and valuable advice throughout the experimental stage and my meaningful time in Obihiro.
The author would also like to thank all my labmates of Laboratory of Food and Environmental Hygiene and Laboratory of Veterinary Pathology, Department of
76
Veterinary Medicine, Gifu University and Laboratory of Veterinary Pathology, Department of Basic Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, for their valuable help during my research and private life in Gifu and Obihiro.
Finally, the author is extremely thankful to my parents and my sisters for their overwhelming and constant financial and other supports, as well as their unwavering faith in me throughout my career. They always gave me the feeling that there was a place where I could fall back on, something that enable me to freely make the choices about my career.
77
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