By using fresh brain tissue, the results of RT-PCR (Fig. 3) and subsequent 303
sequence analysis of the N and G genes confirmed that TWFB No.1-3 were 304
RABV-infected. By using paraffin-embedded brain tissues, the RT-PCR also 305
demonstrated specific amplicons of N gene with the anticipated sizes of 150 and 236 306
bp in TWFB No.1-3, but failed to demonstrate any detectable amplicons in the six 307
archival cases which were run parallel (data not shown). The strain of RABV of 308
TWFBs (RABV-TWFB) belongs to lyssavirus genotype 1 and the nucleotide identity 309
of the N gene within the 3 isolates of TWFB No.1-3 was 97-99%. The N gene of the 3 310
18
RABV-TWFB isolates showed an 89-90% identity to the CNFB isolates (F02, F04, 311
JX08-45, JX08-48, JX09-18), 91% to the dog-related RABV within the China I 312
lineage (HN10, GD-SH-01, CTN-1, CTN-181), 88% to the Southeast Asia dog isolate 313
(QS-05), 88% to the dog-related RABV isolates within the China II lineage (JX09-17, 314
SH06), 86-87% to the cosmopolitan isolates (serotype 1, FluryLEP, DRV-NG11, 315
9147FRA, SAD B19), 87% to the golden palm civet and human India isolates 316
(H-08-1320, H-1413-09), and 84% to the North American bat-related RABV 317
(SHBRV-18).
318 319
Discussion
320Aside from China, Taiwan is the second region in the world where FB-associated 321
rabies has been diagnosed. The TWFB-associated rabies was diagnosed based on 1) 322
characteristic pathological findings of non-suppurative meningoencephalomyelitis, 323
ganglionitis, and Negri bodies; 2) positive IHC staining and DFA/IFA test for RABV 324
antigens; and 3) positive viral nucleic acid detection by RT-PCR followed by genomic 325
sequencing (GenBank accession numbers KF620487-KF620489) (Chiou et al. 2014) 326
in TWFB No.1-3 collected during 2012 and 2013. The most recent phylogeographic 327
study has shown that the RABV-TWFB is a distinct lineage within the Asian group 328
and has been differentiated from the RABV of CNFBs 158-210 years before present 329
19
with the most recent common ancestor of RABV-TWFB originating 91-113 years ago;
330
and the data suggest that the RABV-TWFB could have been cryptically circulating in 331
the environment without being recognized for a long period of time (Chiou et al.
332
2014). The result of retrospective study, which could trace the occurrence of 333
TWFB-associated rabies back to more than 10 years ago, further supports this 334
speculation. However, the postmortem autolysis and limited paraffin-blocks of 335
archival brain tissues preclude satisfactory morphological and molecular evaluation.
336
Following the diagnosis of rabies in TWFB No.1-3, rabies has been diagnosed by 337
FAT in additional 459 TWFBs, 5 gem-faced civets (Paguma larvata), 1 shrew, and 1 338
FB-bitten puppy by May 7, 2015 (data not shown).
339
In rabid TWFBs, lesions and RABV antigens were widely distributed in the 340
brainstem, cerebrum, cerebellum, anterior cervical spinal cord, peri-adrenal, peri-renal, 341
and myenteric plexus ganglia, and adrenal gland. Similar extensive lesion and/or 342
RABV antigen distribution in the central nervous system (CNS) has also been seen in 343
naturally acquired rabid raccoons and skunks (Stein et al. 2010; Hamir 2011).
344
However, hippocampus, brainstem/cerebellum, and cervical spinal cord/adjacent 345
brainstem were the best site for RABV antigen detection in dogs/cats, cattle, and 346
horses, respectively (Stein et al. 2010). In carnivores and herbivores/humans, 347
hippocampus and Purkinje cells of cerebellum are the common sites for finding Negri 348
20
bodies, respectively (Jackson et al. 2001; Maxie and Yousset 2007). On the contrary, 349
TWFBs, similar to raccoons, have widely distributed, large numbers of Negri bodies 350
readily observed in both central and peripheral neural tissues, although brainstem has 351
a better detection rate.
352
The formation of Negri bodies in the neural tissue of a rabid animal seems 353
related to the type of viral isolate to which the animal is exposed. A previous study 354
showed that raccoons infected with the raccoon isolate developed numerous neuronal 355
Negri bodies in the CNS and ganglion cells of non-CNS tissues; however, dog or bat 356
isolate resulted in the formation of either no or only occasional Negri bodies in 357
raccoons (Hamir 2011). Suspicious natural canine isolate-induced rabies cases in 358
CNFBs have been reported in China (Zhang et al. 2009). If the RABV 359
strain-dependent phenomenon observed in raccoons also occurs in FBs, the presence 360
of large numbers of Negri body in the neural tissue of the rabid TWFBs along with 361
the results of nucleotide identity of the N gene between RABV-TWFB isolates and 362
other known RABV isolates may further support that the RABV-TWFB is a distinct 363
lineage and has been circulating in TWFB population for some times, instead of a 364
recent spill-over from a canine or bat source. However, this assumption requires 365
further clarification.
366
21
In some animal species, care must be taken to differentiate Negri bodies from 367
nonspecific inclusions such as Hirano, Pick, Lewy, Lafora, Bunina, and motor neuron 368
disease inclusion bodies that have been found in the pyramidal cells of the 369
hippocampus or the lateral geniculate nucleus neurons in cats, skunks, dogs, and 370
horses and in the larger neurons of the medulla and spinal cord of old sheep and cattle 371
(Maxie and Youssef 2007). Owing to the fact that TWFBs are susceptible to canine 372
distemper virus (CDV) infection (Chen et al. 2008), intracytoplasmic CDV inclusion 373
bodies should also be considered as a possible differentiation. However, aside from 374
non-suppurative meningoencephalitis and neuronal intracytoplasmic inclusions as 375
seen in rabid TWFBs, CDV-infected TWFBs may also have neuronal and/or glial 376
intranuclear inclusions (Chen et al. 2008). Based on the results of pathological 377
findings and RABV antigen detection, brainstem seems an ideal place for sampling 378
and diagnosing FB-associated RABV infection, although cerebral cortex, 379
hippocampus, thalamus or hypothalamus is also suitable.
380
The spongiform change seen in the brain of rabid TWFBs is similar to that 381
reported in RABV-infected skunks and foxes (Charlton 1984; Charlton et al. 1987) 382
and a heifer (Foley and Zachary 1995). The pathogenesis of such change in the rabid 383
animals is unclear. Electron-microscopic findings suggested the lesion development 384
starting gradationally from small to large membrane-bound vacuoles in the neuronal 385
22
processes, primarily dendrites, with no need of incorporation of viral components 386
(Charlton et al. 1987).
387
The changes seen in the submandibular salivary gland of the rabid TWFB No.3 388
are similar to those seen in the parotid salivary gland of those experimentally 389
RABV-infected skunks and foxes, characterized as an interstitial sialoadenitis 390
(Balachandran and Charlton 1994). They also share a similar scattered distribution 391
pattern of RABV antigens in the ganglia, nerve bundles, and acinar epithelial cells 392
(Balachandran and Charlton 1994). The positive result of the RT-PCR of saliva swab 393
obtained from the rabid TWFB No.3 further demonstrated that RABV nucleic acid 394
was also produced and released (unpublished data).
395
To our knowledge, adrenal necrosis has not been described as a rabies-associated 396
histopathological change in rabid animals so far, although Negri bodies have been 397
found in the ganglion cells of the adrenal medulla (Hamir et al. 1992). Scattered to 398
significant amounts of IHC staining-positive signals for RABV antigens were seen in 399
the remaining parenchymal cells and some macrophages located in the necrotic areas 400
of both cortex and medulla, but not in the neuron and/or nerve fibers within the 401
parenchyma. Although RABV antigens were detected in the remaining adrenal 402
parenchymal cells in the necrotic regions, whether the necrosis is directly 403
RABV-induced requires further elucidation. Aside from the adrenal gland, sporadic 404
23
IHC staining-positive macrophages could also be seen in a variety of tissues (Table 2).
405
A recent study has shown that although macrophages are less likely to support RABV 406
production, they can act as a source of infectious virus upon transfer in mice (Naze et 407
al. 2013). This may explain why IHC-positive RABV antigens could be detected in 408
the macrophages widely distributed in various tissues in the present study.
409
The currently accumulated data show that the FB-associated RABV infection in 410
Taiwan still occurs mainly in TWFBs, although sporadic spill-over to other animal 411
species occurs. The gradual increase in the population of TWFBs in the past 10 years 412
has been considered possibly playing certain role on the recent outbreak; however, 413
whether there is (are) other co-factor(s) or other cause(s) is not known and requires 414
further investigation. The origin of the TWFB-associated RABV is an interesting but 415
remaining answered question. The earliest written record of rabies in Taiwan was in 416
the early nineteen century when Taiwan was under Japanese colonization.
417
Human-mediated animal translocation and animal self-movement are considered to 418
play the major role on the dispersal of RABV (Fèvre et al. 2006; Bourhy et al. 2008).
419
The dog has been suggested to serve as the main vector for inter-species RABV 420
transmission, through which viral lineages are generated and then spread to other taxa 421
(Bourhy et al. 2008). Taiwan is an island and, aside from being ruled by Chinese, it 422
has also been colonized by Spanish, Dutch, and Japanese for various time periods.
423
24
Thus, it is reasonable to speculate that human-mediated animal translocation, 424
especially dogs, was one of the most likely sources of RABV in TWFBs.
425
This first reported pathological changes and viral antigen distribution in FBs 426
naturally infected with the strain of RABV-TWFB may provide some bases for future 427
studies such as cross-species disease spreading and vaccine development. The 428
diagnosis of rabies in TWFBs has clearly demonstrated that some known or unknown 429
diseases may cryptically circulate in the wild-ranging wildlife sharing the same 430
habitat with us. These underlying diseases may have serious impacts on our and other 431
animal’s health, wildlife conservation, and/or economy. This incident also reminds us 432
once again the essentiality of systematic wildlife disease surveillance.
433 434
Acknowledgements
435We thank Jen-Tzu Yang, Ying-Hui Wu, and staffs of the Taiwan Endemic Species 436
Research Institute, Council of Agriculture, for the collection and submission of 437
carcasses of the free-range wildlife. Special thanks to Dr. Tsung-Chou Chang from 438
National Pingtung University of Science and Technology for his kind help in the 439
study and in memory of his recent passing away. The study was supported in part by 440
grants 102AS-10.1.1-BQ-B1(3), 103AS-10.1.1-BQ-B1(1), 104AS-10.1.2-BQ-B3(1), 441
MOST 103-3114-Y-518-001, and MOST 104-3114-Y-518-002 from the Bureau of 442
25
Animal and Plant Health Inspection and Quarantine, Council of Agriculture, 443
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