臺灣博碩士論文加值系統

此實驗是以1998到2000年間於台灣沿海擱淺之鯨豚新鮮樣本進行檢查以得知台灣鯨豚擱淺與morbillivirus之感染的關係。於二十例樣本中，僅有一隻於1999年在南台灣擱淺的野生小抹香鯨 (Kogia breviceps, KH9901) 以聚合酶連鎖反應 (PCR) 可增幅出morbillivirus之P與N基因片段。該動物有亞急性多發性支氣管間質性肺炎，並可於肺臟、淋巴結及脾臟中見到少量核內或質內嗜伊紅性包涵體與融合細胞。以免疫組織化學染色法可於其肺臟、淋巴結、脾臟、及肝臟偵測到morbillivirus之抗原。KH9901之部分P基因片段經定序後可與其他已知的morbillivirus之P基因片段進行比對。結果發現該片段與海豚、鼠海豚以及領航鯨之morbillivirus非常相近。進一步利用原位反轉錄酶聚合酶連鎖反應 (in situ RT-PCR) 可於肺臟之肺泡與支氣管腔的細胞、肺泡之單核細胞、以及肺泡上皮細胞偵測到morbillivirus的核酸。其他的病例並未偵測到morbillivirus的RNA或是抗原。根據組織病理學、免疫組織化學染色、PCR、及in situ RT-PCR的發現，我們可確定該鯨魚因morbillivirus感染而生病。以此初步資料我們證明morbillivirus之感染於台灣鯨豚具有影響。

Tissues from freshly dead cetaceans stranded along the Taiwan coast from 1998 through mid-2000 were examined to determine the relationship between strandings and morbillivirus infection. Of 20 carcasses, only a free-living adult male pygmy sperm whale (Kogia breviceps, KH9901) found stranded on the coast of Southern Taiwan in 1999 was identified that morbillivirus P and N genes were amplified from its lung and lymph nodes by polymerase chain reaction (PCR). None of morbilliviral RNA was detected in other cases. The whale had subacute multifocal bronchiolo-interstitial pneumonia, rare syncytia and intranuclear and intracytoplasmic eosinophilic inclusions within lung, lymph nodes, and spleen. Morbilliviral antigen was detected in its lung, lymph node and spleen by immunohistochemistry. Sequence analysis of the partial P gene of KH9901 revealed its close relationship to it of dolphin and pilot whale morbilliviruses. Based on a in situ reverse transcriptase PCR (in situ RT-PCR) detection, morbilliviral nucleic acid was usually found in the cells in alveolar and bronchiolar lumina, but mononuclear cells within alveolar septae and alveolar epithelial cells also stained. Histopathologic, immunohistochemical, PCR, and in situ RT-PCR results provide evidence that the whale had morbillivirus-induced disease. We do possess rudimentary information to state that morbillivirus infection appears to be having an effect on Taiwan’s cetaceans.

English abstract……………………………………………………………………….1
Chinese abstract………………………………………………………………………3
1. Introduction………………………………………………………………………...4
2. Paper review
2.1. Introduction of cetacean morbillivirus……………………………………….5
2.2. Morbilliviral disease of cetaceans……………………………………………9
2.3. Epidemiology of cetacean morbillivirus…………………………………….11
2.4. Taiwan cetacean strandings………………………………………………….19
3. Materials and methods
3.1. Necropsy and sampling……………………………………………………...22
3.2. Reverse transcription-polymerase chain reaction (RT-PCR)………………..22
3.3. Direct in situ RT-PCR……………………………………………………….24
3.4. Immunohistochemistry………………………………………………………26
3.5. Virus isolation………………………………………………………………..27
4. Results
4.1. Pathologic findings…………………………………………………………..28
4.2. RT-PCR and sequence analysis……………………………………………...31
4.3.In situ RT-PCR……………………………………………………………….32
4.4. Immunohistochemistry………………………………………………………32
4.5. Virus isolation………………………………………………………………..32
5. Discussion
5.1. Pathologic findings…………………………………………………………..33
5.2. RT-PCR and sequence analysis……………………………………………...35
5.3. In situ RT-PCR and immunohistochemistry…………………………………37
5.4. Morbillivirus infection in cetaceans in Taiwan……………………………....41
6. Conclusion…………………………………………………………………………45
7. References………………………………………………………………………….46

Aguilar, A., Raga, J.A., 1993. The striped dolphin epizootic in the Mediterranean Sea. Ambio. 22, 524-528.
Arkush, K.D., van Bonn, W.G., Poynton, S.L., 1998. Identification of ciliated protozoans in the respiratory tract, skin and somatic lymph nodes of bottlenose dolphins (Tursiops truncatus) from California, USA. Proc. Int. Assoc. Aquat. Amin. Med. Conf. 29, 27-29.
Bagasra, O., Hauptman, S.P., Lischner, H.W., Sachs, M., Pomerantz, R.J., 1992. Detection of human immunodefeciency virus type I provirus in mononuclear cells by in situ polymerase chain reaction. N. Engl. J. Med. 326, 1385-1391.
Barnes, L.G., Domning, D.P., Ray, C.E., 1985. Status of studies on fossil marine mammals. Mar. Mamm. Sci. 1, 15-53.
Baron, M.D., Barrett, T., 1995. Sequencing and analysis of the nucleocapsid (N) and polymerase (L) genes and the terminal extragenic domains of the vaccine strain of rinderpest virus. J. Gen. Virol. 76, 593-602.
Barrett, T., Visser, I.K.G., Mamaev, L., Goatley, L., van Bressem, M.F., Osterhaus, A.D.M.E., 1993. Dolphin and porpoise morbilliviruses are genetically distinct from phocine distemper virus. Virology 193, 1010-1012.
Birkun, A., Kuiken, J.T., Kricokhizhin, S., Haines, D.M., Osterhaus, A.D.M.E., Bildt, M.W.G., Joiris, C.R., Siebert, U., 1999. Epizootic of morbilliviral disease in common dolphins (Delphinus delphis ponticus) from the Black Sea. Vet. Rec. 144, 85-92.
Black, F., 1991. Epidemiology of Paramyxovirdae. In: Kingsburry DW (editor) The Paramyxoviruses. Plenum Press, New York, p-509-536.
Blixenkrone-Møller, M., Bolt, G., Gottschalk, E., Kenter, M., 1994. Comparative analysis of the gene encoding the nucleocapsid protein of dolphin morbillivirus reveals its distant evolutionary relationship to measles virus and ruminant morbilliviruses. J. Gen. Virol. 75, 2829-2834.
Blixenkrone-Møller, M., Bolt, G., Jensen, T.D., Harder, T., Svansson, V., 1996. Comparative analysis of the attachment protein gene (H) of dolphin morbillivirus. Virus Res. 40, 47-56.
Bolt, G., Blixenkrone-Møller, M., Gottschalk, E., Wishaupt, R.G.A., Welsh, M.J., Earle, P.J.A., Rima, B.K., 1994. Nucleotide and deduced amino acid sequences of the matrix (M) and fusion (F) protein genes of cetacean morbilliviruses isolated from a porpoise and a dolphin. Virus Res. 34, 291-304.
Chou, L.S., Wang, M.C., Yao, C.J., Chen, Y.H., 2000. Taiwan cetacean stranding records. Workshop: Cetacean stranding in Taiwan. pp.22-28.
De Swart, R.L., Harder, T.C., Ross, P.S., Vos, H.w., Osterhaus, A.D.M.E., 1995. Morbilliviruses and morbillivirus disease of marine mammals. Infect. Agents Dis. 4, 125-130.
Di Guardo, G., Agrimi, U., Morelli, L., Gardeti, G., Terracciano, G., Kennedy, S., 1995. Postmortem investigation on cetaceans found stranded on the coasts of Italy between 1990 and 1993. Vet. Rec. 136, 439-442.
Domingo, M., Ferrer, L., Pumarola, M., Marco, A., Plana, J., Kennedy, S., Mc Aliskey, M., Rima, B.K., 1990. Morbillivirus in dolphins. Nature 348, 21.
Domingo, M., Visa, J., Pumarola, M., Marco, A.J., Ferrer, L., Rabanal, R., Kennedy, S., 1992. Pathologic and immunocytochemical studies of morbillivirus infection in striped dolphins (Stenella coeruleoalba). Vet. Pathol. 29, 1-10.
Domingo, M., Vilafranca, M., Visa, J., Prats, N., Trudgett, A.L., Visser, I., 1995. Evidence of chronic morbillivirus infection in the Mediterranean striped dolphin (Stenella coeruleoalba). Vet. Microbiol. 44, 229-239.
Duignan, P.J., Geraci, J.R., Raga, J.A., Calzada, N., 1992. Pathology of morbillivirus infection in striped dolphins (Stenella coeruleoalba) from Valencia and Murcia, Spain. Can. J. Vet. Res. 56, 242-248.
Duignan, P.J., House, C., Geraci, J.R., Duffy, N., Rima, B.K., Walsh, M.T., Early, G., St Aubin, D.J., Sadove, S., Koopman, H., 1995a. Morbillivirus infection in cetaceans of the western Atlantic. Vet. Microbiol. 44, 241-249.
Duignan, P.J., House, C., Geraci, J.R., Early, G., Copland, H., Walsh, M.T., Bossart, G.D., Cray, C., Sadove, S., St. Aubin, D.J., Moore, M., 1995b. Morbillivirus infection in two species of pilot whale (Globicephala sp.) from the western Atlantic. Mar. Mam. Sci. 11, 150-162.
Duignan, P.J., House, C., Odell, D.K., Wells, R.S., Hansen, L.J., Walsh, M.T., Aubin, D.J.S., Rima, B.K., Geraci, J.R., 1996. Morbillivirus infection in bottlenose dolphins: evidence for recurrent epizootics in the Western Atlantic and Gulf of Mexico. Mar. Mam. Sci. 12, 499-515.
Embretson, J., Zupancic, M., Ribas, J.L., Burke, A., Racz, P., Tenner-Racz, K., Haase, A.T., 1993. Massive covert infection of helper T-lymphocytes and macrophages by HIV during the incubation period of AIDS. Nature 362, 359-362.
Fields, B.N., Knips, D.M. (editors), 1991. Fundamental virology, 2nd ed. Raven Press, New York, pp. 21-22.
Forcada, J., Aguilar, A., Hammond, P.S., Pastor, X., Aguilar, R., 1994. Distribution and numbers of striped dolphins in the western Mediterranean Sea after the 1990 epizootic outbreak. Mar. Mam. Sci. 10, 137-150.
Howard, E.B., Britt, J.O., Matsumato, G.K., 1983. Parasitic diseases. In Pathobiology of Marine Mammal Diseases, ed. Howard, E.B., vol. 1, pp. 119-230. CRC Press, Boca Raton, FL.
Jauniaux, T., Charlier, G., Desmecht, M., Coignoul, F., 1998. Lesions of morbillivirus infection in a fin whale (Balaenoptera physalus) stranded along the Belgian coast. Vet. Rec. 143, 423-424. Kennedy, S., Smyth, J.A., Cush, McCullough, S.J., Allan, G.M., McQuaid, S., 1988. Viral distemper now found in porpoises. Nature 336, 21.
Kennedy, S., Smyth, J.A., Cush, McCullough, S.J., Allan, G.M., McQuaid, S., 1988. Viral distemper now found in porpoises. Nature 336, 21.
Kennedy, S., Smyth, J.A., Cush, P.F., McAliskey, M., McCullough, S.J., Rima, B.K., 1991. Histopathologic and immunocytochemical studies of distemper in harbor porpoises. Vet. Pathol. 28, 1-7.
Kennedy, S., Kuiken, T., Ross, H.M., McAliskey, M., Moffett, D., McNiven, C.M., Carole, M., 1992. Morbillivirus infection in two common porpoises (Phocoena phocoena) from the coasts of England and Scotland. Vet. Rec. 131, 286-290.
Komminoth, P., Long, A.A., Ray, R., Wolfe, H.J., 1992. In situ polymerase chain reaction detection of viral DNA, single copy genes and gene rearragements in cell suspensions and cytospins. Diagn. Mol. Pathol. 1, 85-97.
Krafft, A., Lichy, J.H., Lipscomb, T.P., Klaunberg, B.A., Kennedy, S., Taubenberger, J.K., 1995. Postmortem diagnosis of morbillivirus infection in bottlenose dolphins (Tursiops truncatus) in the Atlantic and Gulf of Mexico epizootics by polymerase chain reaction-based assay. J. Wildl. Dis. 31, 410-415.
Lipscomb, T.P., Kennedy, S., Moffett, D., Ford, B.K., 1994a. Morbilliviral disease in an Atlantic bottlenose dolphin (Tursiops truncatus) from the Gulf of Mexico. J. Wildl. Dis. 30, 572-576.
Lipscomb, T.P., Schulman, F.Y., Moffett, D., Kennedy, S., 1994b. Morbilliviral disease in Atlantic bottlenose dolphins (Tursiops truncatus) from the 1987-1988 epizootic. J. Wildl. Dis. 30, 567-571.
McCullough, S.J., McNeilly, F., Allan, G.M., Kennedy, S., Smyth, J.A., Cosby, S.L., Mcquaid, S., Rima, B.K., 1991. Isolation and characterization of a porpoise morbillivirus. Arch. Virol. 118, 247-252.
Myers, T.W., Gelfand, D.H., 1991. Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase. Biochemistry 30, 7661-7666.
Nortobartolo di Sciara, G., Venturino, M.C., Zanardelli, M., Bearzi, G., Borsani, F.J., Cavalloni, B., 1993. Cetaceans in the central Mediterranean Sea: distribution and sighting frequencies. Boll. Zool. 60, 131-138.
Nuovo, G.J., Gallery, F., MacConnell, P., Becker, J., Bloch, W., 1991. An improved technique for the in situ detection of DNA after polymerase chain reaction amplification. Am. J. Pathol. 139, 1239-1244.
Nuovo, G.J., 1992. PCR in situ hybridization. Protocols and applications. New York, Raven Press, 157-177.
Nuovo, G.J., 1996. Detection of viral infections by in situ PCR: theoretical considerations and possible value in diagnostic pathology. J. Clin. Lab. Anal. 10, 335-349.
Osterhaus, A.D.M.E., Visser, I.K.G., de Swart, R.L., van Bressem, M.F., van de Bildt, M.W.G., Örvell, C., Barrett, T., Raga, J.A., 1992. Morbillivirus threat to Mediterranean monk seals? Vet. Rec. 130, 141-142.
Osterhaus, A.D.M.E., de Swart, R.L., Vos, H.W., Ross, P.S., Kenter, M.J.H., Barrett, T., 1995. Morbillivirus infections of aquatic mammals: newly identified members of the genus. Vet. Microbiol. 44, 219-227.
Parsons, E.C.M., Bossart, G.D., Kinoshita, R.E., 1999. Postmortem findings in a finless porpoise (Neophocaena phocaenoides) calf stranded in Hong Kong. Vet. Rec. 144, 75-76.
Parsons, E.C.M., Jefferson, T.A., 2000. Post-mortem investigations on stranded dolphins and porpoises from Hong Kong waters. J. Wildl. Dis. 36, 342-356.
Ray, R., Komminoth, P., Machado, M., Wolfe, H.J., 1991. Combined polymerase chain reaction and in situ hybridization for the detection of single copy genes and viral genomic sequences in intact cells. Mod. Pathol. 4, 124A.
Ray, R.A., Smith, M., Sim, R., Bruce, I., Wakefield, A.J., 1995. In situ hybridization detection of short viral amplicon sequences within cultured cells and body fluids after in situ polymerase chain reaction. J. Virol. Methods 52, 247-263.
Reidarson, T.H., McBain, J., House, C., King, D.P., Stott, J.L., Krafft, A., Taubenberger, J.K., Heyning, J., Lipscomb, T.P., 1998. Morbillivirus infection in stranded common dolphins from the Pacific Ocean. J. Wildl. Dis. 34, 771-776.
Reilly, S.B., Barlow, J., 1986. Rates of increase in dolphin population size. Fish. Bull. 84, 527-533.
Ross, P., de Swart, R., Addison, R., van Loveren, H., Vos, J., Osterhaus, A.D.M.E., 1996. Contaminant-induced immunotoxicity in harbour seals: wildlife at risk? Toxicology 112, 157-169.
Schulman, F.Y., Lipscomb, T.P., Moffett, D., Krafft, A.E., Lichy, J.H., Tsai, M.M., Taubenberger, J.K., Kennedy, S., 1997. Histologic, immunohistochemical, and polymerase chain reaction studies of bottlenose dolphins from the 1987-1988 United States Atlantic coast epizootic. Vet. Pathol. 34, 288-295.
Schulman, F.Y., Lipscomb, T.P., 1999. Dermatitis with invasive ciliated protozoa in dolphins that died during the 1987-1988 Atlantic bottlenose dolphin morbilliviral epizootic. Vet. Pathol. 36, 171-174.
Scott, M.D., Wells, R.S., Irvine, A.B., 1990. A long-term study of bottlenose dolphins on the west coast of Florida. Pages 235-244 in S. Leatherwood and R.R. Reeves eds. The bottlenose dolphin. Academic Press, Inc., San Diego, CA.
Shane, S.H., Wells, R.S., Würsig, B., 1986. Ecology, behavior and social organization of the bottlenose dolphin: A review. Mar. Mam. Sci. 2, 34-63.
Taubenberger, J.K., Tsai, M., Krafft, A.E., Lichy, J.H., Reid, A.H., Schulman, F.Y., Lipscomb, T.P., 1996. Two morbilliviruses implicated in bottlenose dolphin epizootics. Emerg. Infect. Dis. 2, 213-216.
Taubenberger, J.K., Tsai, M.M., Atkin, T.J., Fanning, T.G., Krafft, A.E., Moeller, R.B., Kodsi, S.E., Mense, M.G., Lipscomb, T.P., 2000. Molecular genetic evidence of a novel morbillivirus in a long-finned pilot whale (Globicephalus melas). Emerg. Infect. Dis. 6, 42-45.
Uchida, K., Muranaka, M., Horii, Y., Murakami, N., Yamaguchi, R., Tateyama, S., 1999. Non-purulent meningoencephalomyelitis of a Pacific striped dolphin (Lagenorhynchus obliquidens). The first evidence of morbillivirus infection in a dolphin at the Pacific Ocean around Japan. J. Vet. Med. Sci. 61, 159-162.
Van Bressem, M.F., Visser, I.K.G., Van de Bildt, M.W.G., Teppema, J.S., Raga, J.A., Osterhaus, A.D.M.E., 1991. Morbillivirus infection in Mediterranean striped dolphins (Stenella coeruleoalba). Vet. Rec. 129, 471-472.
Van Bressem, M.F., Visser, I.K.G., De Swart, R.L., Örvell, C., Stanzani, L., Androukaki, E., Siakavara, K., Osterhaus, A.D.M.E., 1993. Dolphin morbillivirus infection in different parts of the Mediterranean Sea. Arch. Virol. 129, 235-242.
Van Bressem, M.F., Jepson, P., Barrett, T., 1998a. Further insight on the epidemiology of cetacean morbillivirus in the Northeastern Atlantic. Mar. Mam. Sci. 14, 605-613.
Van Bressem, M.F., van Waerebeek, K., Fleming, M., Barrett, T., 1998b. Serological evidence of morbillivirus infection in small cetaceans from the Southeast Pacific. Vet. Microbiol. 59, 89-98.
Visser, I.K.G., Van Bressem, M.F., De Swart, R.L., van de Bildt, M.W.G., Vos, H.W., Van der Heijden, R.W.J., Salidi, J.T., Örvell, C., Kitching, P., Kuiken, T., Barrett, T., Osterhaus, A.D.M.E., 1993. Characterization of morbilliviruses isolated from dolphins and porpoises in Europe. J. Gen. Virol. 74, 631-641.
Winn, H.E., 1982. A characterization of marine mammals and turtles in the Mid- and North Atlantic areas of the U.S. Outer Continental Shelf. Final report of the Cetacean and Turtle Assessment Program. Unites States Department of the Interior, Bureau of Land Management, Washington, D.C. 437 pp.