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(Riemerellosis) - Kasetsart Universitythaifarmer.lib.ku.ac.th/f/44bf6d24d150f19ac5fd82a1c5cf01...1932 New duck disease Duck Graham et al. 1938 Duck septicemia Duck Dougherty 1955 Infectious

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Page 1: (Riemerellosis) - Kasetsart Universitythaifarmer.lib.ku.ac.th/f/44bf6d24d150f19ac5fd82a1c5cf01...1932 New duck disease Duck Graham et al. 1938 Duck septicemia Duck Dougherty 1955 Infectious
Page 2: (Riemerellosis) - Kasetsart Universitythaifarmer.lib.ku.ac.th/f/44bf6d24d150f19ac5fd82a1c5cf01...1932 New duck disease Duck Graham et al. 1938 Duck septicemia Duck Dougherty 1955 Infectious

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(Riemerellosis)

���� � ��������

�������������������� !�� ก��#$������

ก�%&�� �ก'����%��ก���

Page 3: (Riemerellosis) - Kasetsart Universitythaifarmer.lib.ku.ac.th/f/44bf6d24d150f19ac5fd82a1c5cf01...1932 New duck disease Duck Graham et al. 1938 Duck septicemia Duck Dougherty 1955 Infectious

������

�������������� � ������������������������ก��� ���������� ���� �� �ก�ก�!�"#� 8 �� ���% ���������������� �ก�� �&��'(������ �� ) 1982 ����!�ก������ก �&��-�.� )!#�������ก���&/�����0ก� �� #����1��� �ก�"�/���234�5����% ���1��5/�16�����(�����5/��ก��� �&(�����/���� ���16��ก�������6�(�� �5��&(�������"5�0!�%��ก��ก7-%�16��(�.���� ���16��1���� !#��5��� ���ก8/�16��(�. ����!�ก����ก8��ก��5/�16����ก7-&�����ก�����"��3#����6���9 �&ก�/ก�/��3-&�����"�/��:��;0.�1����1��1����� 2<��&�&����(�. ก� Dr Tetsuo Morozumi �3.���1�����ก�2�� /������� �&�16���� !#�(�.����ก��'#ก7��16�����ก���� ���"#�ก�������I���16���3.��0ก� ���.�� !������"5�0!�%(�.���� ���16�� Riemerella (Pasteurella, Moraxella) anatipesifer �16������������#ก��/������!��ก�5��&� ���16�����(���� Mก����������� �&1������������%�/(N��������1.����/����( �.�������5��������������������16���.�� �16��!#������"�5�&3#��(�. �� Mก�����������ก8���� ���/ !#���ก���!& �ก!�ก�16������� Mก����/1���6��9 ������������ก��'#ก7��!����6����16�� Riemerella anatipestifer //�����6�������ก�������0�(����ก �&ก�/�.�����!�ก����O����ก� ���!�ก�2�� �&���!�ก����� �I5�&���!�ก���1������0���� �&��'���������6����� �% �& �&��'(�� ������/�� �&��'(����;0.�3��������������������16�����������������6��� #�����%�����0.���(�.'#ก7��!�� �&���ก��������������������/�/ ) !�(�.��5�5%���������1�ก�������� �&��' �&���� �&��' ��ก��������5�5%������ �&��'(�."0ก���( �.��������ก����1�ก�����9����ก��ก���!�"#� P!!2/�� ����I5�&������� Disease of Poultry 11th edition ) 2003 /���6��� Riemerellosis ;0.�3���!#������!"���������%�����0.���ก�������������6��������

;0.�3���(�.���#ก������&�����"#�ก����������(�.'#ก7�(�.�.������� �&������(�.�������������� �&���� �&��'�56��!2� �&���%��.� �� �����������/ก��'#ก7��16�����(������0.!�ก#����!5/(�.������� ;0.�3���������������6��������!&� �� �&��1�%���;0.���!&'#ก7���6����������ก�����9( ��ก5/3.�/ก5������9;0.�3���3��.����/ �&!&���( ก.(3����ก�����9( ��ก�����6������������������0�/.��3��2�'��. �.�5.���� 55]ก2�;0.� ��;0./����//��1��� �ก �&� ��;0.���1�ก�����.3.�5�!.��3.��0�"���1�ก��������

5��58� 5�̂ ���4- 3 �2���� 2551

Page 4: (Riemerellosis) - Kasetsart Universitythaifarmer.lib.ku.ac.th/f/44bf6d24d150f19ac5fd82a1c5cf01...1932 New duck disease Duck Graham et al. 1938 Duck septicemia Duck Dougherty 1955 Infectious

i

������

��� 1 �� ���������������������� �����ก ����

1.1 ���� 1 1.2 �� ����ก�� ��������� 1 1.3 ���������ก����� 2 1.4 ก�����ก� �!��� 2 1.5 �#�$%&!'�� (!)* ก�����ก� �!�� (! R. anatipestier 2 1.6 ก�%��,-����.��.%��/�&!' R. anatipestifer ก��

Pasteurella )* Moraxella 3

��� 2 ก�����"����"#�$�� %��&"�'��ก��ก()'�����

2.1 �,��;'&!'���*����!<��*�(�'�� (! 6 2.2 �,��;'&!')���������=* 7 2.3 ก��% �*�(�')* ก��ก?���ก@ 7 2.4 �AB�.�����'��������)* �����.� 8 2.5 ก����-����-D�������E PCR ������ 9

��� 3 ก���ก�+��� ��ก�� �� ��$���

3.1 %�/�ก����� 14 3.2 ��<�A��E.�� 14 3.3 ก������ (!)* %< 14 3.4 !ก� 15 3.5 ก���*����)�*'�'%�/����� 16

��� 4 -��.��/ ���'�� ก������������ �����01����

4.1 =���I��J 20 4.2 ก����-<=���I��J 21

Page 5: (Riemerellosis) - Kasetsart Universitythaifarmer.lib.ku.ac.th/f/44bf6d24d150f19ac5fd82a1c5cf01...1932 New duck disease Duck Graham et al. 1938 Duck septicemia Duck Dougherty 1955 Infectious

ii

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4.3 ������!'���� ก!�&!'�� (! R.anatipestifer 23 4.4 !'���� ก!�&!'������<E��.�AE.��� 27

��� 5 ก���+���3������-� ก�����"4��������+ �� ก����ก(����

5.1 ก���*!'L*�����=�������=**���.�(��*�(�'�� (! 29 5.2 ก���*!'���=������<�;���;!� (sub-unit vaccine) 31 5.3 ก���*!'���=�������.-ก�(��*�(�'�� (! 32 5.4 ก����-<)!����!���E����/� ELISA 36 5.5 ก���ก@��� 36

����A� 37

ก����ก��.�� กY 39

�!ก��!E'!�' 40

Page 6: (Riemerellosis) - Kasetsart Universitythaifarmer.lib.ku.ac.th/f/44bf6d24d150f19ac5fd82a1c5cf01...1932 New duck disease Duck Graham et al. 1938 Duck septicemia Duck Dougherty 1955 Infectious

iii

�����������

��'��� 1 ก�����ก� �!�����<���ก������ (! R. anatipestifer ��������Zก

3

��'��� 2 �AB�.�����'��������)* �����.�&!'�� (! R. anatipestifer

10

��'��� 3 ก�)�กก*A;.&!'�� (! R. anatipestider ���!Y���AB�.�����'�����.���!<��*�(�'�� (! buffer single substrate test

11

��'��� 4 �]�ก�����;!�!?�=�.�&!'�� (! R. anatipestifer ��%��/A��E!'#��� 20 ��%��/A������������ก�������!E'!�'.��^� ���APIZYM

12

��'��� 5 �����!E'!�'�����E��<��������.)!���=���.���-< =���I��J

23

��'��� 6 ก���*!'<!'���� ก!�&!'������<E��.�AE.��� ���ก��<E��.�E!�)* �;!��E���!?�=�.��;'a ����b�%��/A�กก�)�.�J��*

28

��'��� 7 ก�<��.�&E.&E�&!'�� (!����<. �.��<������=�� ����� 1081 ����b�

31

��'��� 8 ก�<��.�AE.���&!'���=��<�;���;!� (Subunit vaccine) �����.-ก�� (! R. anatipestifer ����� 1081 ����b��;!ก�D��%�@����E����������.�����E�����.���=������b�%��/A�กก�)�.�J��*

32

��'��� 9 ก�<��.�AE.���&!'���=�������.-ก�(��*�(�'�� (! )*E�D��%�@����E���� (!����� 1081 �����E�����=�� )* ����� 328 =���I��J 19

34

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iv

�������&�

�,���� 1 ก�#!��<����� 16S r RNA &!'�� (! R. anatipestifer �����.��^� ATCC 11845 �����������ก���� (!�;'a 14 �������.���.�ก*E���ก��

5

�,���� 2 A ���*��&!'�� (! Riemerella anatipestifer �� 5% blood agar

6

�,���� 2 B ���*��&!'�� (! Pasteurella multocida �� 5% blood agar

6

�,���� 3 !A�ก�B���<������-<�!?�=�.�!'���� ก!�&!'�� (!�E�� APIZYM

13

�,���� 4 �]�ก�����;!�!?�=�.�&!'�� (! R. anatipestifer ������E!'#���

13

�,���� 5 ��b�)��'!ก�I! -. .��(�.,กI<* 17 �,���� 6 ��b�.�!ก��E!'���� &E!!�ก��� ����กE���� 17 �,���� 7 ��b�)��'!ก��'�� ��.�<������ &��(�.ก-ก

ก��E!'���� L��<��')<E'������'ก� �,ก 17

�,���� 8 ��b��'���)��'!ก��'� ���� �� .���ก �!��� 18 �,���� 9 *�ก@B -��% &!'��� Riemerellosis �. �!��i�L;-

%��� �!<AE.<���-<� (Pericarditis) �'���('%��(����;!'!ก)* �;!'�E!'

18

�,���� 10 �!�.��* !����' #A'<AE.<���-<� 18 �,���� 11 $%-A*%�/�����)��' �� �!<AE.<���-<����.�ก�

)��ก���&!' inflammatory cells )* fibrin 19

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v

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�,���� 12 $%-A*%�/�����)��' fibrinous pneumonia .�ก�)��ก���&!' polymorphonuclear cells, hyperemia )* fibrin

19

�,���� 13 $%-A*%�/�����)��' fibrinous perihepatitis, .�ก�<����&!'�� �!<AE.��� (Glissonos capsule) .�ก�)��ก���&!' inflammatory cells )* fibrin

19

�,���� 14 Precipitin line ����ก��� <�;'<*A.&!')!����-� ก��)!���=���.

22

�,���� 15 ������!'���� ก!�&!'�� (! R. anatipestifer �����/� SDS-PAGE

25

�,���� 16 ������!'���� ก!����� �!<AE.�=**��;���!ก (outer membrane protein, Omp)

26

�,���� 17 Immunoblotting 26

Page 9: (Riemerellosis) - Kasetsart Universitythaifarmer.lib.ku.ac.th/f/44bf6d24d150f19ac5fd82a1c5cf01...1932 New duck disease Duck Graham et al. 1938 Duck septicemia Duck Dougherty 1955 Infectious

1

����� 1 ���� ����������������������ก�����

�����

������������ (Riemerellosis) ����������� �!�"#$%กก%����'�() �����*+%�����+�� (New Duck Syndrome) �� '��67�'$'ก�8�9:;������! Riemerella (Pasteurella, moraxella) anatipestifer (R. anatipestifer) ��=������;'+��> ?'�%@����=+ A;�%>* ���������6B')C�%>*�ก �?'�*'� "@� �!��#ก%;76 '�ก���ก'���9!)��=+��=�!B')�'ก ���>)$'ก��%6�'ก'�6'! ") �9?'��%ก+ %6*D6%*��>�+8�*�6กE$��6�;�6 �B �;"�FD :���:ก�E� �7FG'A�'ก6>?' �?'��#%6�'ก'�H"ก�%+��9) ") �ก�%9)!%) �9����)�B'�8#$B'!�'ก��ก'���#!'�%กI':�*%����

���� ก������������ Riemer (1904) ��=�M"#�'!)'������������� ��=���%9):�ก���B'�

���!ก8�>�8�9��>��=� '��67��9*B' Pfeiferella anatipestifer :����!ก8�>�����>��;'+��96'�%กIF�()�����>A;*B' septicemia anserum exudativa ������ก'���'!)'�ก'���;'+��%9):�ก��"ก��=+�+!���!ก8�>*B' S��*+%�+��� T (new duck disease) �+! Hendrickson and Hilbert (1932) 6B�'��� +#�ก�+��Y'�D���=+��>���! D ���!ก8�>*B' +%���A6������! (duck septicemia) (Graham et al., 1938) $'ก�%9�กE���'!)'�ก'���;'+!B')6B���>)��������6B')C +#:กB :��'+' (Taylor, 1955) �% ���! (Goroshko and Prokofera, 1958) �6���! (Grimes and Rosenfield, 1972) :��'!��������!7��� (Brogden, 1989) ������!���'!)'�ก'���;'+����!��*'�ก#���!)ก%���> �)����D (Singh et al., 1982) �! (Mahitanan et al., 1982) ;%)ก'��� (Mustafa et al., 1985) ���+�! (Sarma et al., 1985) :�@�>�7f� (Baba et al., 1987)

Page 10: (Riemerellosis) - Kasetsart Universitythaifarmer.lib.ku.ac.th/f/44bf6d24d150f19ac5fd82a1c5cf01...1932 New duck disease Duck Graham et al. 1938 Duck septicemia Duck Dougherty 1955 Infectious

2

��������ก ����

%6*D�gก�ก�;�7ก8��+ '�'�H��=������������� +# ��>���'!)'�:#* +#:กB ��=+ (Ziedler et al., 1984) �B'� (Miklovichnè-Kis Csatári, 1965) กB)*) (Zehr and Ostendorf, 1970) กB (Rosenfeld, 1973) กBYp' (Bruner et al., 1970) �ก��q���8'6� +#:กB ��=+�f' �B'� :��) D (Munday et al., 1970; Karstad et al., 1970; Wobeser et al., 1982; Wobeser and Ward, 1974)

ก������ก������� ������������ ก'����!ก8�>��ก'����>!�:�) ��+!?'+%;�ก$'ก��>

กB'*�':#* 6'�6'�')��> 1 Dougherty, et al. (1955) ���!ก8�>�����9*B' � �Y��8�! ���� �6� (infectious serositis) ��#8�>�+!A�$'�F'Ht)ก'����>!�:�)�')A!'q� G'A 6B�'����ก�+���B'� Levine (1965) ���!ก*B'��� ก"v ��Y"�E��B' (goose influenza) B*�8�> R. anatipestifer infection � �:���+! Liebovitz (1972) �A�>��# '�'�H:!ก�����9$'ก�����>��A!'q� G'A�#'!Cก%� B' 7+ Ryll et al. (2000) ���!ก�����9*B' Riemerellosis �t>) +�#)6'�:;;:M�ก'����!ก8�>�����>�ก�+$'ก�8�9:;������!�%>* � �8B��+�!*ก%; Pasteurellosis, Salmonellosis, Streptococosis xx �������� �!�"#$%กก%����'�() T�����*+%�����+��T

��������� ���!�"#ก������ก�������!� R. anatipestier

ก'����!ก8�>()�8�9 R. anatipestier ��ก'����>!�:�)�'�+!?'+%;�8B��+�!*ก%;ก'����!ก8�>��� ���>�$'ก Hendrickson and Hilbert (1932) ��#8�>*B' Pfeiferella anatipestifer �+!A�$'�F'$'ก�"��B') ก'�!#�6�+ � :�%กIF� ก'��$��@;�'�'���9!)�8�9 ���> Pfeiferella H"ก+8%9�) ��+! Bergeyys manual (1934) +%)�%9� H'�G'A()�8�9กE���>!� ��+!H"ก$%+��#!"B�� genus Haemophilus (Gerriets, 1953) 6B�' Bruner and Fabricant (1954) +#�tกI'����!;���!;�7F%กIF�() R. anatipestier ก%;�8�9 Brucella, Pasteuella,

Page 11: (Riemerellosis) - Kasetsart Universitythaifarmer.lib.ku.ac.th/f/44bf6d24d150f19ac5fd82a1c5cf01...1932 New duck disease Duck Graham et al. 1938 Duck septicemia Duck Dougherty 1955 Infectious

3

Moraxella, Actinobacillus, :� Haemophilus :#* �7�*B' R. anatipestier ���*'������ก%;�8�9 Moraxella �'ก��> 7+ $t):���?'��#���!ก8�>*B' Moraxella anatipestifer :6B���g 1957 R. anatipestier ��'ก|�� Bergeyys Manual of Determinative Bacteriology A��AD��%9)��>�$E+*B' Pasteurella anatipestifer (Breed et al., 1957) �+!��67��>!%) �B���*'�:�B����ก'�6%9)8�> R.

anatipestier $t)H"ก$%+!"B�� species incertae sedis �� Bergeyys Manual of Determinative Bacteriology A��AD��%9)��>:�+ (Smith, 1974) :��ก#'

6'�')��> 1 ก'����!ก8�>�����>�ก�+$'กก'�6�+�8�9 R. anatipestifer �� %6*D�gก

Reporter Year Names of the disease Poultries

Riemer 1904 Septicemia anserum exudativa

Goose

Hendrickson and Hilbert

1932 New duck disease Duck

Graham et al. 1938 Duck septicemia Duck Dougherty 1955 Infectious serositis Duck Levine 1965 Goose influenza Goose Leibovitz 1972 R. anatipestifer infection Duck Ryll et al. 2000 Riemerellosis Duck

ก��� �./����0��0���1��� R. anatipestifer ก�� Pasteurella �� Moraxella ��ก'��tกI'�')+#'��*'� +�#)()!�� (DNA homology) A;*B' %6�' B*�() guanine + cytosine (G + C) () R. anatipestifer !"B��> 32-35% ��(F���> Pasteurella :� Moraxella !"B��> 40 � 45% (Bangun et al., 1987) :� DNA homology ���*B')�8�9 R. anatipestifer ��>:!ก +#���#)��>!"B��> 52

Page 12: (Riemerellosis) - Kasetsart Universitythaifarmer.lib.ku.ac.th/f/44bf6d24d150f19ac5fd82a1c5cf01...1932 New duck disease Duck Graham et al. 1938 Duck septicemia Duck Dougherty 1955 Infectious

4

� 100 % :6B���*B') R. anatipestifer ก%; Pasteurella :� Moraxella ���*'������ก%�:�B 3 �17% 6B�' Piechulla et al. (1986) � ���#$%+!"B��ก7B� Flavobacterium / Cytophaga ���>)$'ก�8�9��9�� DNA binding :�#$��#!:6BกE���%! ?'�%@ :���ก'�M�6 menaquinones :� branched-chain fatty acid ��ก'�*����'��D cellular fatty acid ()�8�9 R. anatipestifer, Pasteurella :� Moraxella �+!*�q� gas chromatography A;*B' R.

anatipestifer ���"�:;;() cellular fatty acid ��>6B')$'ก Pasteurella :� Moraxella (Bangun et al., 1987) ���>�*��7F �;%6�()�8�9�t>)���ก;+#*! fatty acid ��>�� branch �'ก :��� menaquinones ��=� respiratory quinines �A�!)!B')�+�!* �B���|�ก��!'ก%;�'�D�; ��+�� �*�Ht)�"�:;;()�|�ก��!' 6B�E��'!�D6B')C �?'��#�8�9 R. anatipestifer H"ก$%+!"B��ก7B� Flavobacterium-Cytophaga (Rossau et al., 1991) 6B�'��%;��7)��=� family Flavobacteriacae :�$'กM()ก'�*����'��D DNA � DNA :� DNA � rRNA hybridization R. anatipestifer $t)H"ก$%+!"B��8%9� superfamily V (Bangun et al., 1987; Bernardet et al., 1996; Vandamme et al., 1994)

����> 7+ Segers et al. (1993) A;*B' �8�9��9���*'�:6ก6B')ก%;�8�9 Flavobacterium :� Weeksella :�#*B'$����*'��ก#8�+�') genotype $t)� ���#:!ก�8�9��9ก�'6%9)��=� genus ���B:�� ���#���!ก8�> Riemerella

anatipestifer �A�>��=��ก�!�6�:กB Riemer �t>)��=�M"#�'!)'�ก'��ก�+����������-�� ���B'���=���%9):�ก���g 1904 �%9)��9�+!A�$'�F'$'ก DNA-ribosomal RNA hybridization analysis, %กIF�)�D���ก;:�:;;:M�() protein, fatty acid, methylester (FAME) :��7F%กIF��')Yg�� ��� (phenotype) �8B� �B��ก'�M�6 � :� �B��'ก|*B'�� respiratory quinine SMenaquinone 7T

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5

�"���> 1 ก'�H+��% !�� 16S r RNA ()�8�9 R. anatipestifer �6���'6��'� ATCC 11845 ����!;���!;ก%;�8�96B')C 14 8��+��>���*'��ก#8�+ก%� :��8�9��>���*'� %�A%�qD�B')ก � �8B� Bacteroides putredenis :� Blattabacterium sp. �8�9 R. anatipestifer �6���'6��'� ?'��%;���� ��� 6, 15 :� 19 !"B��6?':��B)�+�!*ก%; �6�� ATCC 11845 (Subramaniam et al., 1997)

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����� 2 ก����+ � +,�-��. /0+�1��ก��ก231�����

�.��3� �� ���#������4���#�!� ���!� �%)$'ก�A'���9!)��> 37oC 24 8%>*��) �����()�8�9 R. anatipestifer ��

(�'+ 2.0 � 2.5 mm ���' ก� �"� *'* (Brogden et al, 1982; Heddleston, 1975) �B!B! '!��E+��+:+) ���>�A'���9!)��'ก'���9!)�8�9��>: )MB'� +# �8B� dextrose starch agar ��� blood agar base ��>�6�� 5% bovine serum $���E� �()��������>!� � ��'�#'! ��7#) (iridescent)

�"���> 2 A �����()�8�9 Riemerella anatipestifer ;� 5% blood agar ;B� 24 8%>*��)�� candle jar $���E�*B'�������%กIF���#)�"� :����*'��!�9� �"���> 2 B �����()�8�9 Pasteurella multocida ;� 5% blood agar $� :;��';ก*B' ���'$')ก*B' :��6��E*ก*B' ���>;B�����!��*' 24 8%>*��)��>��B'ก%�

A B

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7

�.��3� �� "���������5##� �8�9 R. anatipestifer 6�+ �:ก��; �B �#') ��D �"�:�B)(�'+ 0.2 � 0.4 um Ht) 1.0 � 5.0 µm ��'ก|��=���D�+�>!* �����=��"B ���;')��%9)A;6Bก%���=� '! (Heddleston, 1975) �*'�:6ก6B')��9 �%ก�ก�+$'ก'�'���9!)�8�9��>�8#�A'���9!) :�'!7()�8�9;�'�'���9!)�8�9 ��ก'��A'�:!ก�8�9��%9):�ก$'ก���9�!�> %6*D�f*!-6'! $�A;ก'�6�+ �6�)�%*:��#'!()��D �t>)�(#�ก*B' B*��>� (bipolar) ���>!#�+#*! � Wrightys stain (Eleazer et al., 1973) ก'�6�*$:�E��" '�'�H+" +#�+!!#� ++#*! India ink (Heddleston, 1978) ก������#�!� "#ก���ก6���ก7� �8�9 R. anatipestifer '�'�H�A'���9!)��'�'���9!)�8�9 �8B� chocolate agar ��� trypic soy agar ��>�� 5% defribrinated blood ��� 0.5% !� 6D ก%+ ��� 5% ���%�"ก*%* �t>):���?'��#�8#�A'�:!ก�8�9$'ก���9�!�> ��� '��%+�%>)$'ก %6*D�f*!-6'! (Heddleston, 1975) ;B���;��!'ก'���>��กv'� �'�D;� +ก �+D 5 � 10% :��A�>��*'�8�9�$�8B*!��#�8�9��>�'$'ก���9�!�> %6*D�f*!-6'!�$��@ +#+� :6B���>MB'��8�9�+!ก'��A'���9!)��'�'���9!)�8�9�'!C��%9) �8�9กE$� '�'�H��%;6%*�(#'ก%;'�'���9!)�8�9�>�C +#+�(t9� �+!'$ �B6#)�6�����%� ���กv'��'�D;� +ก �+D (Heddleston, 1978) 7F�G"����>���'� ���ก'��$��@�6�;�6�� 37oC (Heddleston, 1978) ") 7+��> 45oC H#'�กE;��>7F�G"�� 55 oC �A�!) 12-16 8%>*��)�8�9กE6'!:#* ���>�8�9�A'�(t9�;�*7#���9!)�8�9:#*$� '�'�H��8�*�6!"B +# �B�ก�� 3-4 *%� ��> 37oC ���7F�G"���#) :6B��'�'���9!)�8�9��*'$!"B +#�'� 2-3 %�+'�D ��> 4 oC (Bangun et al., 1981) $'ก��� ;ก'�FD()M"#�(�!� '�'�H�กE;�� mist desiccans ��> -70 Ht) -80 oC +#�'��%; �;�g�+!�7F �;%6� �B���>!�:�) ���'$�กE;���"�() lyophilization ��> 4 oC กE '�'�H�?' +# �+!�%>* ��%ก�กE;�'!�"�:;;�A�>�p)ก%�ก'� "@� �!

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�89�0��� �� ���� ���"#����0� R. anatipestifer �ก$'ก%กIF������ �"��B')()��D ก'�!#�6�+

�:ก��; ��=�:�B) %9�C �����)ก�:#* R. anatipestifer !%)���7F �;%6��#'!ก%;�8�9��ก7B� Pasteurella :� Moraxella 6�)��>��#M;*ก6B oxidase :� catalase test �8�9 R. anatipestifer $%+!"B��A*ก non-fermentative metabolism �B(t9��� MacConkey agar �B�?'�|�ก��!'ก%;�'�D�; ��+�� 20 8��+ (Brogden et al. 1982; Bangun et al., 1981; Hendrickson and Hilbert, 1932) !B') �กE6'����>�8#'�'���9!)�8�9��>���'� ��8B� cystine tryptic agar (CTA) ��>�� 5% horse serum :#*�6�� 1% �'�D�; ��+�� �8�9 R. anatipestifer กE '�'�H��#M;*ก6B�'�D�; ��+��;')8��+ +# :6Bก'�B'�M6#)�8#�*' 7 Ht) 14 *%� (6'�')��> 2 , Pathanasophon et al. 1994) ���'$�8# buffer single substrate test (BSS) �t>)���ก;+#*! 0.1% (w/v) KH2PO4, 0.1% (w/v) K2HPO4, 0.5% (w/v) NaCl, phenol red 1:50,000 :��6���'�D�; ��+�� 2% �� '��'! 7+�#'! (Hinz et al., 1998) :6B6#)� B�8�9��>*%+6���g!ก�9?'��%ก ")Ht) 400 mg. �+!�A'��8�9;� blood agar 18 8%>*��) :#*$t)�'!�8�9�%9�)�� '��'! 2 ml. ��>���ก;+#*! 0.15 M NaCl pH 7 '�'�HB'�M�%);B���B')�9?'��> 37 oC ��=��*' 6 - 8 8%>*��)

Hinz et al. (1998) '�'�H:;B)�8�9 R. anatipestifer ก��=� 8 ;�*'�D 6'�6'�')��> 3 +#*!��67��>���*'�!7B)!'ก��ก'�A� "$�D�8�9�+!�8#�'�D�; ��+�� Pathanasophon et al. (1994) +#�'!)'�ก'�A� "$�D�8�9�+!�8#�E��'!�D (APIZYM, API System-La Balme Les Grottes, France) �t>)��=��ก�')��ก��t>) �?'��# '�'�H��';M +#G'!�� 5 8%>*��) �+!��>�8�9 R.

anatipestifer '�'�H���|�ก���!'ก%;�E��'!�D +#�'!8��+ :����"�:;;��>��=��ก%กIFD ���*'��(#����B�()�|�ก��!'6B�E��'!�D:6B�8��+ �B��B'ก%� ���>���!; �ก%;:MB����!; ���>M"#M�6��#�' A;*B'��#M;*ก 100% 6B�E��'!�D 7 8��+ +#:กB phostase alkaline, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, phosphatase acid :�

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9

phosphoamidase (Pathanasophon et al., 1991) 6'�6'�')��> 4 :��"���> 3 :� 4 ก����/ � /:�������; PCR ���� �

��$$7;%�*�*%��'ก'��')+#'�8�*���ก7 +#A%��'(t9�!B')�'ก�'! �t>) '�'�H6�*$*���$�%!�8�9:;������! +#!B')����� ��q�G'A B' 7+ Kardos et al. (2007) +#�'!)'�ก'��8# PCR ?'��%;6�*$*���$�%!�8�9 R. anatipestifer ��=�M ?'��E$�+!�8# A����D��>ก:;;(t9����ก;+#*!

669AF (5y-TTACCGACTGATTGCCTTCTAG-3y) 669AR (5y-AGAGGAAGACCGAGGACATC-3y) +# PCR product (�'+ 546 bp '�'�H6�*$*���$�%!�8�9��>�A'���9!)

;�'�'���9!)�8�9:�$'ก���9�!�>() %6*D�f*!�+!6�) �%;��=�A%��'ก'�()ก'�8%� "6������>������!8�D!�>) ���>)$'ก�?'��#ก'�6�*$*���$�%!�����=� �+#*!�*'��*+��E* H"ก6#):�:�B�!?'

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6'�')��> 2 �7F �;%6��') ���*��!':�8�*����()�8�9 R. anatipestifer (Pathanasophon et al. 1994 :� Hinz et al, 1998)

Positive reaction Test Local isolates (n=20) ATCC 11845 (n=1)

Growth on Simmons citrate agar 0 0 Growth on MacConkey agar 0 0 Catalase test 20 1 Oxidase test 20 1 Gelatin liquefaction 20 1 Coagulase test 0 0 Coagulated serum proteolysis 6 1 Milk proteolysis 5 1 Nitrate reduction 0 0 Indole production 2 0 Urease production 0 1 Camp test 3* 0 Production of acid from carbohydrates Arabinose 1 0 Dextrin** 107/111 1 Dulcitol 0 0 Fructose 3 1 Glucose 18 1 Inositol 0 0 Lactose 0 0 Mannitol 0 0 Maltose 18 1 Mannose 1 0 Raffinose 0 0 Rhamnose 0 0 Saccharose** 0/111 0 Sorbitol 1 0 Sucrose 0 0 Trehalose 1 0 Xylose 0 0 *11 isolates were weakly positive, ** From (Hinz et al, 1998)

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6'�')��> 3 ก'�:!กก7B�()�8�9 R. anatipestider �+!'�%!�7F �;%6��') 8�*������'�'���9!)�8�9 buffer single substrate test (Hinz et al., 1998)

Biovars represent 106 (87%) of the 122 R. anatipestifer strains tested Acid from 1 2 3 4 5 6 7 8

(n=37) (n=34) (n=10) (n=8) (n=7) (n=4) (n=3) (n=3) D(+)-Glucose

+ + + + + - - +

D(-)-Fructose

- + + - - - - +

D-(+)Mannose

+ + + + - - - -

Maltose + + + + + + + - Dextrin + + + + + + + - Trehalose - - + + - - + -

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6'�')��> 4 �|�ก���!'6B�E��'!�D()�8�9 R. anatipestifer '!A%�q7D�#)H�>� 20 '!A%�q7D����!;���!;ก%; �6�� #')�)�'6��'� �+!APIZYM® bioMérieux, Inc. USA. (Pathanasophon, et al., 1991)

n Number of strains positive to enzyme substrates Strains

original 1* 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Thailand 20 0 20 20 20 0 20 20 19 13 19 20 20 0 0 0 18 0 2 0 0 NADC 7 0 7 7 7 0 7 7 7 2 5 7 7 0 0 0 7 0 0 0 0 ATCC11845 1 0 1 1 1 0 1 1 1 1 1 1 1 0 0 0 1 0 0 0 0 Total 28 0 28 28 28 0 28 28 27 16 25 28 28 0 0 0 26 0 2 0 0 *1 Control 2 Phosphatase alkaline 3 Esterase (C4) 4 Esterase lipase (C8) 5 Lipase (C14) 6 Leucine 7 Valine arylamidase 8 Cystine arylamidase 9 Trypsin 13 α-galactosidase 16 α-glucosidase

10 Chymotrypsin 14 ß-galactosidase 17 ß-glucosidase

11 Phosphatase acid 15 ß-gluculonidase 18 ß-glucosaminidase

12 naphtol-AS-Bl phosphohydrolase (Phosphoamidase)

19 α-mannosidase 20 α-fucosidase

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�"���> 3 7�ก�FD ?'��%;6�*$�'�E��'!�D)�D���ก;()�8�9+#*! APIZYM® bioMérieux, Inc. USA.

�"���> 4 �|�ก���!'6B�E��'!�D()�8�9 Riemerella anatipestifer �6���#)H�>����*'���=��กG'A $���E� +#*B'�*'��(#�B�() ��t>)�ก�+$'ก�|�ก���!'()�8�96B�E��'!�D:6B�8��+ �B��B'ก%�

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����� 3 ก���ก 5��� ��ก�� ����-���

���1 ก���� � ��� Riemerellosis �B�B!A;�ก�+;B! �����;'+ก*#')(*')���ก�f' :��ก�9?' :6B$���=�������$?'H�>� ?'��%;��=+ :� กB)*)��>��9!)�A�>ก'��#' %6�'ก'��f*!!"B���*B') 2 Ht) 30% (Heddleston, 1978) :�%6�'ก'�6'!'$!"B���*B') 5% - 75% (Sandhu, 2003) "ก��=+'!7 1 � 8 %�+'�D *6Bก'��ก�+����'ก ��=+'!76>?'ก*B' 5 %�+'�D �%ก$�6'!G'!�� 1 � 2 *%� ��=+��>'!7�'กก*B'กE$���8�*�6!"B +#�'�ก*B' �����9 �B�B!A;����=+AB:�BA%�q7D �� กB)*)��!���'!)'�ก'���;'+!B')�7�:�)������ก' (Helfer and Helmboldt, 1977; Zehr and Ostendorf, 1970) :��������>�C (Frommer et al., 1990; Pascucci et al., 1989; Smith et al., 1987) : +)*B' ��� Riemerellosis ���*'��7�:�) +#�%9)����=+:� กB)*) ���4�8��;0��� %6*D��>�ก�+�*'�����!+���>)$'ก'ก'����>!�:�) ก'���9!)!B'):%+ ���ก'����>�!#'! %6*D ����ก�+�B*�ก%;����>�C ��=� '��67��#��?'��#�ก�+ก'���;'+()��� Riemerellosis ก��� ����!�"#��4

ก'�:A�B�8�9 R. anatipestifer $'ก %6*D�f*! �!%) %6*D 7(G'A+�G'!���") '�'�H�ก�+ +#$'กก'�ก���9?'�B*�ก%� ����?' %6*D+��(#' ���9!)���#'��>��!�� %6*D�f*!+#*!�����9 (Bendheim and Even-Shoshan, 1975) ���6�+$'ก�9?'�"ก�9?''!() %6*D�f*! ก'��'!�$ :�6�+�8�9MB'�:M �ก$'ก��9!%) '�'�HA;�8�9 R. anatipestifer �� %6*D��> �B: +)'ก'��$E;�f*! �+! '�'�HA;�8�9��9!"B��>

Page 23: (Riemerellosis) - Kasetsart Universitythaifarmer.lib.ku.ac.th/f/44bf6d24d150f19ac5fd82a1c5cf01...1932 New duck disease Duck Graham et al. 1938 Duck septicemia Duck Dougherty 1955 Infectious

15

;���*F$�"ก6Bก%;��! (nasal pharynge) ()�B'� (Donahue and Olson, 1969), �) D (Munday, 1970) �8�9 '�'�H!"B���')�+���'!�$ B*�6#�()"ก��=+ 7(G'A�ก6� �+!'$!"B;��!�>���ก()��! (pharyngeal mucous membrane) �8�9��>���*'��7�:�) ")$�A;!"B������'F�#! ��(F���>�8�9��>���*'��7�:�)6>?''$:�)!"B��=��8�9���$?'H�>��!กE +# (Ryll et al., 2001) : +)*B' %6*D'$�f*!:;; �B: +)'ก'� �����=�A'��()��� ���>�(#' ����") %6*D!"B:;;:%+ ������*'� *6B����'กก*B' กE '�'�H�?'��#�ก�+ก'��$E;�f*! +# (Wobeser and Ward, 1974) �ก$'ก��9!%)���'!)'�*B'ก'�HB'!�+�8�9$'ก:�B "B"กกE��=� +# �+!���'!)'�*B' '�'�H�A'�:!ก�8�9 R. anatipestifer +#$'ก (B:+)() (B�B'�Y�ก (GlÜnder and Hinz, 1989) ��ก��

'ก'�()��� Riemerellosis ��#���#��E�*B'�8�9 R. anatipestifer �(#'�?''! %6*D�� '���;;+#*!ก%� �� ��;;�'!�$ ��;;!B!'�'� :���;;��� '� ��(F��+�!*ก%��8�9กE�(#' "Bก��: ���6+#*! 'ก'�$����>�6#���>�9?'6' � �9?'�"ก ���=��"ก� C �#)� �!�+!���>�$'ก��=��9?'7$$'�� �('*:#*���>!���=� ��(�!* $'� ���>���+?'����6B �กE$�A;('B� �+��ก#�6>?' 6%* %>� "@� �!ก'���)6%* �gก6ก �;�+ 8%ก ��+ 6� :�6'! 6%*��>�+8�*�6กE$�:���:ก�E� %6�'ก'�6'!'$!"B��8B*) 5 � 75% :6B%6�'ก'��f*!�+!�%>* �$��'กก*B'��9 (Heddleston, 1975; Bruner et al., 1970; Eleazer et al., 1973; Pickrell, 1966)

ก����#����"�# �� ���1 ���

�!�����>A;+#*!6'��B' $���%กIF���A'�()�����9�� �!�>��>�%ก� ; (fibrinous serositis) ��E� +#8%+����=+ ��A;()��*8��+ fibrinous �t>)��=� '��%+�%>)$'ก�!�>��>��+!�%>* ���'ก|��>G'!���!�>�7#��%*�$ �!�>�7#�6%; :�H7)� :�A; +#�8B��+�!*ก%��� กB)*):��ก�>�C A;H7)���':�ก'�%ก� ;�%>* ��%9)��8B)�#):�8B)ก �!�����>6%;���'!��>��=�:;;���!;A%�

Page 24: (Riemerellosis) - Kasetsart Universitythaifarmer.lib.ku.ac.th/f/44bf6d24d150f19ac5fd82a1c5cf01...1932 New duck disease Duck Graham et al. 1938 Duck septicemia Duck Dougherty 1955 Infectious

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A;;*� (7B� �#'�'$��ก'�(!'!��@B:���$7+�Eก �('* (mottled) ���9?'�"ก����)���A�)$�"ก:�%กIF��#'!��!:(E) (caseous) ��>�B�%) (B (Doughterty et al., 1955; Pickrell, 1966)

���'!��>�f*!:;;���9�%) A;�!�����>M�*��%)%กIF���=����96'!��>;���*F�%) B*��#'!:��;C�*'� A;()��* ����)!"B�����*B')M�*��%):�8%9� (�%� :�A;(#%ก� ;+#*!

�!����')+#'�$7A!'q�*��!' �>)�%+�%>)��>�%*�$���ก;+#*!��E+��+('* 8��+ mononuclear cells :� heterophil ����!���>�f*!:;;���!;A%��!�����>6%;$�A;��E+��+('*8��+ mononuclear �� B*� periportal A; cloudy swelling :� hydropic degeneration �� B*� parenchymal cells ���'!��>��=��f*! �B���!;A%�'$A;��E+��+('*8��+ lymphocyte �� B*� periportal B*�H7)�A; mononuclear cells ��=� B*���@B�� �>)�%+�%>) B*����'!��>�f*!:;;���9�%)'$A; multinuclear giant cells :� fibroblast �')�+���'!�$:��+ '$6�+�8�9�+! �B: +)'ก'� :6B$�A;��D��E+��+('*��6B��9?'���)��>6�+ก%; parabronchi (Pickrell, 1966) ���'$$�A;�+;*����!;A%�8��+ fibrino-purulent (Graham, 1938)

Page 25: (Riemerellosis) - Kasetsart Universitythaifarmer.lib.ku.ac.th/f/44bf6d24d150f19ac5fd82a1c5cf01...1932 New duck disease Duck Graham et al. 1938 Duck septicemia Duck Dougherty 1955 Infectious

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�"���> 5 "ก��=+�f*!$�: +)'ก'� $'� H#'ก+;���*F�A�)$�"ก$����9?'�"ก �ก�'

�"���> 6 ��=+��'ก'��#)� �! (#%ก� ; �+��ก#�6>?'

�"���> 7 ��=+: +)'ก'��')��� '� �%* %>� ('+�9?'�'ก$'กก'��#)� �! M�*��%):�#)6�+���)ก��+"ก

Page 26: (Riemerellosis) - Kasetsart Universitythaifarmer.lib.ku.ac.th/f/44bf6d24d150f19ac5fd82a1c5cf01...1932 New duck disease Duck Graham et al. 1938 Duck septicemia Duck Dougherty 1955 Infectious

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�"���> 8 ��=+;')6%*: +)'ก'��')��;;��� '� �� 'ก'�8%ก �;�+

�"���> 9 %กIF�$?'�A'�()��� Riemerellosis ���>���+MB'$�A;�!�>�7#��%*�$��' (Pericarditis) ;')��%9)A;�9?'��8B)ก:�8B)�#)

����� 10 �� ������ ���

����������������

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�"���> 11 G'A$7A!'q�*��!': +) �!�>�7#��%*�$��'�+!��ก'�:��ก6%*() inflammatory cells :� fibrin

�"���> 12 G'A$7A!'q�*��!': +) fibrinous pneumonia �� hyperemia, ��ก'�:��ก6%*() inflammatory cells, :� fibrin

�"���> 13 G'A$7A!'q�*��!': +) fibrinous perihepatitis, ��ก'���'6%*()�!�>�7#�6%; (Glissonys capsule) ��ก'�:��ก6%*() inflammatory cells :� fibrin

��'!��67 �"���> 11 � 13 +#�%;�*'��7���'��D$'ก ��. �. A. +�. �*��%ก+�� B)� ����F� %6*:A�!�' 6�D ��'*��!'%!�กI6��' 6�D

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����� 3 6���7��8 ���1��ก����������������9:����

5���>��? Harry (1969) +#�?'ก'�:!ก���� ���()�8�9 R. anatipestifer :�$%+���!)��=�:;;:M� (profile) ��=���%9):�ก�*� 16 ���� ��� (A � P) �+!�8#�|�ก���!'ก'�$%;6%*��=�ก#� (plate and tube agglutination reactions) 6B�' �6����>��=�6%*:��()���� ��� 4 ���� ��� "@�'! �$'กก'��กE;�%กI' +#:กB ���� ��� E, F, J :� K :����� ��� G :� N �9?'ก%; I :� O 6'�?'+%; 6B�' Brogden et al. (1982) +#:!ก���� ���ก��=� 1 - 7 �+!*�q� agar gel precipitin test �t>)���� ����%9) ):;; '�'�H���!;���!)ก%� +# +%)6B ���9�� ���� ��� 1(A), 2(I/G), 3(L), 4(H), 5(M), 6(B), 7(O/N) �%9)��9�A�>�p)ก%�ก'� %; � :� �B��(#$?'ก%+�����>)6%*%กI� $t)��ก'�� ���#�8#6%*�('�';����=�6%*:��()���� ��� (Brogden et al., 1982) 6B�'กE��ก'��'!)'����� ������BC�A�>�(t9�!B')6B���>) ��(F��+�!*ก%�กE��ก'�!ก��ก;') �6����>��=�6%*:��();')���� ���ก � ���>)$'ก�*'��$��@�')*��!'�' 6�D�'ก(t9� �?'��# '�'�H�+ ;�7F �;%6�6B')C�A�>�(t9�$���E�(#:6ก6B') �8B� �6�� P-1785 (NADC) ���� ��� 4 '�'�H(t9� +#;� MacConkey agar (#')Ht)�+! Sandhu and Leister, 1991) �*��%9) �6�� 670/89 ���� ��� 20 �t>)��%กIF�:6ก6B')$'ก R.

anatipestifer ���7F �;%6�;')���ก'� +#:กB ����)()����� M6B ß-glucosidase, esculin hydrolysis, gelatinase activity :�ก'����!;�*'������() fatty acid profile ก%; �6���>�C (Ryll and Hinz, 2000) $�Ht)��$$7;%� R. anatipestifer ���'!)'�:#*�%9) �9� 21 ���� ��� �t>)���� ��� 20 :� 21 ��=����� ������B()�ก��>�'!)'�A;�������� �!�+! Pathanasophon et al., (1995) :� Pathanasophon et al. (2002) 6'�?'+%; �6��#')�)�'6��'�

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�� Moraxella (Riemerella) anatipestifer ATCC 11845 B*� �6���'6��'���>�8# ?'��%;�6��!�:�6����%�6�*$�'���� ��� 21 ���� ���6'�6'�')��> 5 ก����/4�5���>��?

�?' +# 3 *�q� �� slide agglutination test, tube agglutination test (Harry, 1969; Sandhu and Leister, 1991; Loh et al., 1992) :� agar - gel precipitin test (Brogden et al. 1982; Pathanasophon et al., 1995, 2002) :6B�*�q���(#+�:�(#� �!:6ก6B')ก%�;#')�Eก�#!

*�q� agglutination test :�6����%��6��!�$'กก��6B'! slide agglutination test �?'�|�ก���!'�+!6�)$'ก�����;� +DB'�M;*ก�+!�|�ก���!'$%;6%*��=�ก#����*B'):�6��$�ก%;:�6�;+��t>)��#M�*+��E* :6B'$�ก�+�|�ก���!' �B8%+�$� ��� �B$?'�A'��$'�$) (nonspecific reaction) (Sandhu and leister, 1991) ��ก�F�+%)กB'*$�6#)+?'����ก'�6B+#*! tube agglutination test �t>):�6��$���=�8��+�8�96'! (0.3% formalinised physiological saline)

*�q� agar gel precipitin test (AGPT) :�6����%��6��!�$'ก กB :�6��$��6��!�$'กก'� ก%++#*!�*'��#� B'�M;*ก�'กA;�|�ก���!' precipitin line ��>�ก�+(t9����*B')�7���>�!+:�6��$�:�:�6����%� �t>) precipitin line ��>�ก�+$'ก�8�9 R. anatipestifer $�8%+�$��'ก:��ก�+��E*G'!�� 18 -24 8%>*��) ���>���!;ก%; Pasteurella multocida �|�ก���!' precipitin line $�;'):��|�ก���!'�ก�+8#'6#)B'�M6%9):6B 24 8%>*��)����'กก*B' �?'��#6%+��@�'���>)ก'�B'�M�'+���>�ก � +# ก'�$?':�ก���� ������*'�$?'��=������>)ก'� ; *����:���;'+*��!' �8�9 R. anatipestifer '$��=����� �����>:6ก6B')��������ก%���ก'���;'+:6B���%9) :�#:6B��Y'�D��+�!*ก%�กE6'� ��ก'���;'+:6B���%9) '�'�HA;�8�9 +#�'กก*B'��t>)���� ��� :����� ���'$���*'�:6ก6B')��:6B�Y'�D� :6B��#)��> :6B��g :�:6ก6B')ก%���:6B��������ก+#*! ��8B*)�g 1980 - 1988 ���� �����>��;'+�������� ��%�����ก' B*���@B��=� ���� ��� 1 6'�+#*! 2 :� 5

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(Sandhu and Leister, 1991) �� �)����D8B*) 1982 � 1990 B*���@B��=����� ��� 1 6'�+#*! 14 :� 9 �������� �!8B*)�g 1988 � 1993 B*���@B ��=����� ��� 1 (56.3%) (Pathanasophon et al., 1995) 6B�'��8B*)�g 1994 �1999 ก'���;'+()�8�9��Ht) 17 ���� ��� �+!A;���� ��� 7 �'ก��> 7+ (27.5%) :�A;��=����� ��� 1 �A�!) 10% (Pathanasophon et al., 2002)

�"���> 14 Precipitin line ��>�ก�+���*B')�7�():�6��$� ก%;:�6����%� (�7���>���(ก?'ก%; ���� ���():�6����%�) ��E��(#�(#� :��+B�8%+

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6'�')��> 5 �6��#')�)��>�8# ?'��%;ก'��6��!�:�6����%�6�*$�'���� ��� Serotypes Strain Source Serotypes Strain Source

1 D-24105 DRLa 9 H-1785 HPRS P-1050 NADCb 10 H-2199 HPRS 2 D-24046 DRL 11 D-28020 DRL P-1645 NADC 12 8755 CCUGd 3 D-26338 DRL 13 11693 CCUG P-1667 NADC 14 D-664 CVLSe 4 H-2565 HPRSc 15 D-743 CVLS 5 D-24123 DRL 16 S-4801 DRL P-1641 NADC 17 K-1499 DRL 6 P-2123 NADC 18 540/86 CVLS 7 D-27179 DRL 19 30/90 CVLS P-2361 NADC 20 698/95 NIAHTf 8 D-26220 DRL 21 1062/91 NIAHT

aDRL: Duck Research Laboratory, New York, USA. bNADC: National Animal Disease Center, Ames, Iowa, USA. cHPRS: Houghton Poultry Research Station, Houghton, England. dCCUG: Culture Collection, University of Goteborg, Sweden. eCVLS: Central Veterinary Laboratory, Singapore. fNIAHT: National Institute of Animal Health, Thailand. ������� ����ก���� ���!� R. anatipestifer ���>)$'ก��� Riemerellosis ���>��ก'���;'+���")��=+:#*�%ก�?'�*'�� �!�'!�'ก +%)�%9�$t) +#���*'�A!'!'��+)M�6*%�����A�> �#')�*'��7#����:��p)ก%�ก'� "@� �! (Sandhu, 1979; Layton and Sandhu, 1984; Sandhu, 1991) !B') �กE6'�(#�"�ก�>!*ก%;�7F%กIF�():�6��$���>กB��#�ก�+�*'��7#������ %6*D!%) �B��ก'��'!)'��'กB� ?'��%;�8�9 Pasteurella multocida ��

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�'!)'�*B'��=� '�A*ก���6���B*�ก%;�'�D�; ��+�� (protein-polysaccharide complex) �t>)�8�>*B'��=� '���>กB��#�ก�+ก'� �#')�*'��7#���� (Lugtenberg, 1984) *�q��'6��'���>�8#��ก'�*����'��D�'���6��:��'�D�; ��+�� (lipopoly-saccharide, LPS) �� gel electrophoresis :� Western blotting '�'�H;ก�*'������:��*'�6B')()�8�9���*B') �6�� ������*B')8��+ (Lugtenberg et al., 1984; Buttenschon and Rosendal, 1990; Thirkell et al., 1990; Johnson et al., 1991) Pathanasophon et al. (1999) +#�'!)'��tกI'���6��)�D���ก;()�8�9 R. anatipestifer �+!*�q� sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) �� '!A%�q7D�#)H�>�() �! 28 '!A%�q7D :� �6��#')�)�'6��'� 23 �6�� �t>)��=�6%*:��()���� ��� 1-17 A;*B'�%9) '!A%�q7D�#)H�>� :� �6���'6��'���:;;:M����6���#'!�t)ก%��'ก �8�9 R. anatipestifer �7ก �6����� '!A%�q7D��>�tกI'���ก;+#*!:H;���6���%ก 5 :H; �t>)���9?'��%ก���ก7 70, 62, 51, 31, :� 27 ��� 25 + 4 Kd B*����6��)�D���ก;���!�>�7#���D B*��ก (outer membrane protein, Omp) �� 11 '!A%�q7D��>��=�6%*:�� '!A%�q7D�#)H�>� :� �6��#')�)�'6��'� ATCC 11845 A;*B'��:H;���6���%ก!"B 4 :H;��>�9?'��%ก���ก7 83, 78, 70, :� 62 + 4 Kd ��$?'�*�:H;���6�� 40 :H;��>��'ก|8%+���8�9��t>) �6����>:!ก +#�+!*�q� SDS-PAGE A;*B'��!B')�#! 11 :H;���6����>: +)��#��E�*B'���7F �;%6�����>!*�?'��#�ก�+ก'� �#'):�6�;+� +# �+!ก'��?' immunoblotting :H;���6����E��+B�8%+ +#:กB :H;��>���9?'��%ก���ก7 97, 92, 83, 78, 70, 62, 54, 48, 41, :� 33 + 4 Kd �t>):H;���6����B'��9 B*���@B��'ก|!"B;� Omp :��*�Ht):H;���6���%กC ��>A;*B'��=�)�D���ก;()�8�9+%)กB'* Mก'��tกI'��%9)��98�9��#��E�*B'�����6����>���7F �;%6�����>!*�?'��#�ก�+ก'� �#'):�6�;+�!"B�'ก�'!;�M�*() R. anatipestifer �+! �B���*'�:6ก6B')���*B')���� ��� !B') �กE6'�Mก'�6�*$�'���� ����%9) 3 *�q� �� slide agglutination test, tube agglutination test �t>)�8#�8�9�%9)��:� *�q� AGPT �t>)

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�8#:�6��$���> +#$'กก'� ก%++#*!�*'��#� �t>)�8�>*B'��=� '�A*ก LPS (Brogden et al., 1982) +%)�%9�$t)�8�> +#*B'�%9) Omp :� LPS '$��=� '���>��#�*'��7#���� +#�8B��+�!*ก%;��>���'!)'��ก�>!*ก%;�8�9 P. multocida ��>�8�>*B' '���>��#�*'��7#������ '����ก;()���6��ก%;ก%; LPS ��%กIF� protein polysaccharide complex (Lugtenberg et al., 1984).

�"���> 15 ���6��)�D���ก;()�8�9 R. anatipestifer �� *�q� SDS-PAGE �8�9 R. anatipestifer �7ก �6����� '!A%�q7D��>�tกI'���ก; +#*!:H;���6���%ก 5 :H; �t>)���9?'��%ก���ก7 70, 62, 51, 31, :� 27 ��� 25 + 4 Kd

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�"���> 16 ���6��)�D���ก;���!�>�7#���D B*��ก (outer membrane protein, Omp) �� 5 '!A%�q7D��>��=�6%*:�� '!A%�q7D�#)H�>� :� �6��#')�)�'6��'� ATCC 11845 A;*B'��:H;���6���%ก!"B 4 :H;��>�9?'��%ก���ก7 83, 78, 70, :� 62 + 4 Kd

�"���> 17 Immunoblotting :H;���6��!B')�#! 11 :H;��>: +)��#��E�*B'���7F �;%6�����>!*�?'��#�ก�+ก'� �#'):�6�;+� +# �+! :H;���6����E��+B�8%+ +#:กB :H;��>���9?'��%ก���ก7 97, 92, 83, 78, 70, 62, 54, 48, 41, :� 33 + 4 Kd

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� ����ก���� �������4;��0�8;0��� ก'��+)�A�>A� "$�D*B')�D���ก;() '���>��#�*'��7#�()*%����

��=� '�8��+�+ Pathanasophon et al. (1996c) +#�+)M�6*%�����8�96'!8��+���*��9?'��9!) �6�� 1081 ���� ��� 1 �+!��#�*'��#�*%������> 37, 56 ��� 100oC ��=��*'�'� 1 ��� 2 8%>*��)�� water bath :�!B!+#*!�E��'!�D8��+6B')C A;*B'*%������>�ก6� '�'�H��#�*'��7#� +# ")6Bก'���+A�I�%;+#*!�8�9 �6���+�!*ก%� :6B$���#�*'��7#�+)!B')���%! ?'�%@���>�?'��#*%�����#� 100oC �'� 1 8%>*��) ���!B!+#*!�E��'!�D trypsin �t>)ก��q����ก'�!B!���6�� ��(F���>�E��'!�D lipase �t>)ก��q����ก'�!B!A*ก (�%� +�*'��7#�()*%���� +#�'�ก')!B')���%! ?'�%@ (6'�')��> 6) $'กM+%)กB'*8�9��#��E�*B' ���6����� '����ก; peptide ��=�)�D���ก;��> ?'�%@��> 7+��ก'� �#')�*'��7#����6B�8�9 R. anatipestifer ��"ก��=+ ��(F���> lipid ��� '����ก;() lipid ��M6Bก'� �#')�*'��7#��))�'

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6'�')��> 6 ก'��+)�')�D���ก;() '�$'ก�8�9 R. anatipestifer ��>��# �*'��7#���� �+!ก'���#�*'��#�:�!B!+#*!�E��'!�D6B')C �� ��=+A%�q7Dก'ก�:�����; Treatment Protein content Mortality % mortality PIc (µg/ml) (No. dead/No.

tested)

Heata 37oC, 2h 1,312.5 5/18 27.8 72.2e 56oC, 1h 1,208.3 4/20 20 80e 100oC, 1h 750 13/20 65 35d Enzymeb Lipase 1,293 8/18 44.4 55.6d Muramidase 1,125 5/18 27.8 72.2e Trypsin 1,292 12/19 63.2 36.8d Control Untreated- vaccine (WC)f

1,395 4/20 20 80

Nonimmune 20/20 100 0

a Heat treatment was done in water-bath. d Significantly different (P<0.05) b Enzyme treatment was done at 37 oC e Significantly not different for 2 h in water-bath ( P>0.05 ) c PI : protective index f WC : whole culture

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����� 5 ก���5���<� ���6�� ก����+=����� ��5� ��

ก����ก2� ก����#� @# ���5���� ��5#�0�!���#�!� ���!� ก����;� ��0�8;0����� ���!� Riemerella anatipestifer

��� Riemerellosis (Anatipestifer infection) �������� �!A;�'ก����=+ ก'��f*!!"B���*B') 2 Ht) 30% (Heddleston, 1978) :�%6�'ก'�6'!'$!"B���*B') 5% - 75% (Sandhu, 2003) 95% !'6#'�$78�A�'!8��+��#M��ก'�+%6�'ก'�6'! :6B��(�+$?'ก%+��>�B'�8#$B'! "):�A;%6�'ก'�+�9!' ")(t9����>!C $�ก���%>) �B '�'�H�8# +#�ก6B ��8B�!'��ก7B��%Y'�t>)��!��8#ก%��'ก6B�'A;*B'%6�'ก'�+�9!' ") ก'��*;�7�����+!�8#*%�����B'$���=�*�q�ก'���>���'� � :6B*%����กE!%) �B��$?'��B'!!B'):A�B�'! :�#*B'$����'!)'�ก'��8#*%�����%9)�8�96'!:��8�9��=����"�():;������!�����9?'��9!) :����"�()*%����8��+�9?'�%���:H;!7��� :�����ก' *%���� B*���@B6#)��+:���#�'กก*B'��t>)��%9) (Sandhu, 1979; Layton and Sandhu, 1984; Sandhu, 1991) B*��������� �!�%9�!%) �B��ก'��+)M�6*%�����'กกB�

Pathanasophon et al. (1996a) +#�+)M�6*%�����8�96'!8��+���*��9?'��9!)�8�9 R. anatipestifer �6�� 1081 ���� ��� 1 �t>):!ก +#$'ก��=+�������� �! ��>�%กA;��ก'���;'+ B*���@B (Pathanasophon et al., 1995) ก'�*%+�B'�*'��7#����!ก*B' Protective index (PI) �8#�?'�*F6'�*�q�() Sandhu (1979) 6'� "6� %Mortality in controls - %Mortality in vaccinates

Protective index (PI) = ------------------------------------------------------------------ X 100

%Mortality in controls

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*�q�ก'��6��!�*%���� �?'�+!�A'���9!)�8�9 R. anatipestifer �� tryptic

soy broth (Difco) ��>�6�� yeast extract (Difco) 0.3% ;B���6"#;B�:;;�(!B'��> 37oC ��=��*' 24 h 6�*$ ;�*'�;�� 7�q���+!ก'�!#� �:ก�� B*���t>)()*%�����?'�'�$�$'):#*�A'�)'�'���9!)�8�9:(E)�A�>�%;$?'�*��8�9 :�6�*$�*'�;�� 7�q���ก��%9)��t>) �%)$'ก�%9��?'��#�8�9��+��q���+!�6�� formalin ��(�'+�*'��(#�(#� 0.3% v/v ��%;�*'�(7B���> 540 nm +#*!����>) Spectronic 20 A (Shimatzu, Millon Roy Company) ��# +# OD 2 ��> 540 nm :#*$t)�6�� 2% aluminium hydroxide gel ��# +#�*'��(#�(#� 25% �+!����'6� �*'��(#�(#� 7+�#'!()*%����$� +#�8�9����'F 1.0 x 1010 colony forming unit/ml

$'กก'��+)*%����+%)กB'*A;*B'�*'��(#�(#�()*%������>���'� � ���ก;+#*!�� 1010 colony forming unit/ml. (CFU/ml.) �8#��+6%*� 0.5 ml. ���>��+*%�����A�!)��%9)�+�!*��#��=+A%�q7D Khaki Campbell ��>'!7 2 %�+'�D A;*B'�*'��(#�(#� 7+�#'!()*%����$� +#�8�9����'F 1.0 x 1010 colony forming unit/ml '�'�H��#�*'��7#���� +# 85.7 PI ���>�A�>��*'��(#�(#�()�8�9��=� 2 ��B'�*'��7#����กE �B:6ก6B') :6BH#'+�*'��(#�(#�()*%����) 10 ��B' :� 100 ��B' �*'��7#����$�+)!B')���%! ?'�%@ (67.7 PI) (6'�')��> 7)

��ก'��+)�'*B'*%����$���#�*'��7#����> ��%)$'ก��=+ +#�%;*%���� A;*B'�+!��'ก|*B'*%������#�*'��7#� +#+� (88 PI) 6Bก'���+A�I�%;+#*! �6���+����>�8#�6��!�*%���� (homologous strain) 7 *%��%)ก'���+*%���� �+!��#�*'��7#� ") 7+ (95.6 PI) ��> 2 %�+'�D�%)��+*%���� :���#�*'��7#� +#6+ 6 �+�����> �9� 7+ก'��+) ก'��+)��+*%���� 2 ��%9)����=+��>'!7 1 :� 5 %�+'�D ��#�*'��7#� ")�'ก!B')���%! ?'�%@ (95.8 PI) *%�������*'��+G%! ") '�'�H��+ +#����=+'!7 1 %�+'�D :�!%) '�'�H��#�*'��7#� +#+� 6Bก'���+A�I�%;+#*! �6���>�C ��>!"B������ ��� 1 �8B��+�!*ก%� (66.7 Ht) 100 PI) :6B �B '�'�H��#�*'��7#�6B ���� ��� 6, 11, 14 :� 19 ��@�'���>)ก'���>*%���� �B '�'�H��#�*'��7#����

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(#'����� ��� +#�t>)กE��=���>��';ก%�+��8B��+�!*ก%;*%������>�?'$'ก:;������!8��+�>�C 6'�')��> 7 ก'��'�*'��(#�(#�()�8�9��>���'� � ?'��%;*%���� �6�� 1081 ����=+ Vaccine concentrationa

No. dead / No. tested %mortality PIb

2 x 4/30 13.3 86.7 X 1 4/28 14.3 85.7 x / 10 10/31 32.3 67.7c x / 100 15/32 46.9 53.1d x / 1,000 15/34 44.1 55.9d Nonimmune 20/20 100 a x 1 : �*'��(#�(#�1010 CFU/ml., bProtective index ��5���� �4�3��3�� (sub-unit vaccine) Pathanasophon et al. (1996b) +#�tกI'�*'��7#�() sub-unit vaccine *%�����p)ก%���� Riemerellosis �t>)�6��!�$'ก�8�9 R. anatipestifer �6�� 1081 ���� ��� 1 ��>��*�q�ก'��6��!��+!!B+%)��9�� �?'�8�9��>�A'�)��'�'���9!)�8�9��*�'�?'��#��:6ก�+!�8#��>��*'� %>� ����� (sonication) �����*'���E* ")�A�>:!ก�'M�%)��ก :!ก B*����6��ก�+!6ก6�ก�+#*! ammonium sulfate $'ก�%9��?'��# B*����6����>6ก6�ก��+! ammonium sulfate ;�� 7�q��(t9��+! ก%++#*! Sephadex G-200 gel filtration �?' B*����6����> ก%+ +#�'*%+�*'��(#�(#�()���6�� ��%;�*'��(#�(#�()���6�� ��# +# 400, 200, 100 :� 50 µg 6B�+v :���"�����!� �+�ก �+D!"B 25% v/v �?'��#'��>��=� adjuvant :#*�+)��+*%�����%9) 4 8��+��"ก��=+ A;*B'*%������>�����6�� 100 µg '�'�H��#�*'��7#��������=+'!7 14 *%� +# 100 PI �+!ก'���+�(#'�6#��%)�A�!)��%9)�+�!* 6Bก'���+A�I�%;+#*!�8�9+%)กB'* 5x109 CFU �%)ก'���+*%���� 21 *%� :����>+����'F���6����*%����)��� 50 µg '�'�H�7#����

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+# 50 PI �B' median effective dose (ED50) ()���6����*%���� +#$'กก'��?'�*F��B'ก%; 43 µg (6'�')��> 8)

6'�')��> 8 ก'��'�*'��7#����()*%������B*!!B! (subunit vaccine) �6��!� $'ก�8�9 R. anatipestifer �6�� 1081 ����=+6Bก'���+A�I�%; +#*! �6���+����>�8#�6��!�*%��������=+A%�q7Dก'ก�:�����; (Pathanasophon et al., 1996b)

Protein antigen Mortality % mortality PI (µg/dose) (No. dead/No.tested) 50 5/10 50 50 100 0/10 0 100 200 2/10 20 80 400 1/7 14 86 Nonimmune 10/10 100 0 ก����#� ��5�������0/�ก�!���#�!� ���!�

���'!)'�*B':�6��$���>�'!!"B��'�'���9!)�8�98��+��* (culture filtrate, CF) '�'�H��#�*'��7#���� +# :�:�6�;+�6B CF '�'�H��#�*'��7#�(#'����� ��� +# (White and Verwey, 1970; Rothe, 1982 and Sawada et al., 1987) +%)�%9�$t)��=���>�B' ��$*B' CF ()�8�9 R. anantipestifer $� '�'�H��#�*'��7#�(#'����� ��� +#��� �B Pathanasophon et al. (1996b) +#�+)�6��!�*%����8��+ CF $'ก�8�9 R. anantipestifer �6�� 1081 ���� ��� 1 :� �6�� 328 ���� ��� 19

*�q�ก'��6��!�+%)��9���A'��8�9:6B� �6��)��'�'���9!)�8�9��* �6��� 3 �6� �B'�8�9+#*!�+!ก'��6�� 0.3 % formalin (v/v) ����:!ก�'6%*��ก �?' B*�� �ก�)�'����>'$���)���!"Bก � +#��=� CF $'ก�%9��?'��# CF �(#�(#�(t9���=� 30 ��B' (30xCF) �+!�8# ultrafiltration (Amicon standard

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cell 402, Amicon) �?' 30xCF �'�$�$')��#��=� 20xCF :� 10xCF :#*�6�� aluminium hydroxide gel ��# +#��=� 25% (v/v) +#��=�*%���� 3 8��+��>���*'��(#�(#�:6ก6B')ก%� �?' ���+��"ก��=+�A�>�'�*'��7#���� Mก'��tกI'A;*B'*%���� 10xCF, 20xCF :� 30xCF () R. anantipestifer �6�� 1081 ���� ��� 1 ��#�*'��7#� 81.8, 90.9 :� 91.3 PI 6'�?'+%; ��(F���> 10xCF, 20xCF :� 30xCF () R. anantipestifer �6�� 328 ���� ��� 19 ��#�*'��7#� 64, 82 :� 82 PI 6'�?'+%; ���>��+A�I�%;+#*! �6���+����>�8#�6��!�*%���� (homologous strain) :6B �B '�'�H��#�*'��7#�(#'�ก%����*B') �6���%9) ) (heterologous strain) 6'�6'�')��> 9

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6'�')��> 9 ก'��'�*'��7#����()*%�����6��!�$'ก�9?'��9!)�8�9 :#*��+A�I�%; +#*!�8�9 �6�� 1081 ��>�8#�?'*%���� :� �6�� 328 ���� ��� 19 Challenge strain ( serotype ) Immunogena 1081 ( 1 ) 328 ( 19 ) D/Tb %c PId D/T % PI Strain 1081 30xCF 1/24 4.17 92.4 13/25 52 <0 20xCF 1/21 4.8 91.3 6/25 24 <0 10xCF 1/20 5 90.9 8/25 32 <0 10xCF+ KC 2/20 10 81.8 5/25 20 9.9 WC 3/22 13.64 75.2 4/25 26.7 24.7 Strain 328 30xCF 5/17 29.4 46.5 1/25 4 82 20xCF 17/24 70.8 <0 1/25 4 82 10xCF 12/22 54.5 0.9 2/25 8 64 10xCF+ KC 14/23 60.9 <0 2/25 8 64 WC 12/23 52 5.5 1/25 4 82 Nonimmune 11/20 55 0 4/18 22.2 0 a CF = culture filtrate, KC = killed cells, WC = whole culture, x = time of concentration from the origin b No. dead bird at 14 days post-challenge / No. challenged c % mortality d Protective index

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�+!�%>* �:#* Omp ()�8�9:;������!กB�����=� B*���>��#�*'��7#� +#��=�!B')+� (Bremer, et al., 1982; Cheng et al., 1996) :���;�;'� ?'�%@��ก'�กB��� (Weiser and Gotschlich, 1991) Subramaniam et al. (2000) +#�?'ก'����!�� ompA �t>)��=�6%*�*;�7�ก'� �#')���6��(�'+ 42-kDa ;� Omp :��?'(!'!!����9���8�9 Escherichia coli �A�>��# �#') Recombinant OmpA :�A;*B'���*'���=� � +#��> OmpA ()�8�9 R. anatipestifer $���=� B*���>กB���:�!%)A;*B' OmpA ��9 '�'�HA; +#���7ก���� ��� +%)�%9�$t) '�'�H�8#��=�6%*6�*$$%;�':�6�;+��� %6*D��>6�+�8�9 R. anatipestifer �+! �B;B);ก*B'��=� ���� ����+ +#

Huang et al. (2002b) +#�+)M�6*%����$'ก Recombinant OmpA :� 41-kDa partial protein �+!�8# Seppic Montanide ISA 773 ��=� adjuvant :#*�+)�'�*'��7#�()*%������"ก��=+�+!��+ 2 ��%9)*%���> 7 :� 14 A;*B'*%����+%)กB'* �B��#�*'��7#�:�#$���+A�I�%;+#*! �6���+�!*ก%;��>�8#M�6*%���� �7�*B' Recombinant 41 KDa partial protein (P45Ny) �B '�'�H����>!*�?'��#��=+ �#')�*'��7#���� +#

M$'กก'��tกI'��B'��9;B)8�9*B'�ก$'ก6%*��()�8�9:#* :�6��$���>�8�9 �#')(t9������*B')ก'��$��@�6�;�6�A�>�$?'�*���'�'���9!)�8�98��+��*!%) '�'�H��#�*'��7#��������=+ +# :6B���>)$'ก���� ���$'กก'���;'+:6B���%9)'$���>!�:�)!"B6+�*' �8B�กB��g 1994 ก'���;'+()��� Riemerellosis �������� �!��=����� ��� 1 ��=� B*���@B (56.3) �+!���� �����>A;�%9)��+���A�!) 8 ���� �����B'�%9� B*���8B*)�g 1994 �1999 ก'���;'+()�����ก'�ก��$'! �Ht) 17 ���� ��� �+!A;���� ��� 7 �'ก��> 7+ (27.5%) :���=����� ��� 1 �A�!) (10%)(Pathanasophon et al., 2002) +%)�%9�ก'��8#*%�����*���>�6��!�(t9�$'ก���� �����t>)��� )���� ��� '$ �B '�'�H�p)ก%������>��;'+���#)��> +#!B')��;�7� +%)�%9�ก'��?'*%���� ?'��%;�8#��Y'�D�$'ก�8�9��>��;'+��Y'�D��) (autogenous vaccine) '$��=��')��ก��t>):6B6#)�?'

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ก'��%+��ก �6����>��#�*'��7#�+� ?'��%;�8#�?'*%����� �!กB� �A�'� �B�8B�8�9�7ก �6����>���7F �;%6�+%)กB'* ก����/4�"��� �����;� 1� ELISA ก'�A%��' Enzyme-linked immunosorbent assay (ELISA) ?'��%;6�*$�' humoral antibody �+!�8#:�6��$�8��+ whole cell :#*�?'��#��D:6ก�+!ก'�:8B:(E):#*+�*'��!E�)��#�9?':(E)�'! (freeze and thaw) '�'�H6�*$�':�6�;+� +#�%9)����=+��> +#�%;*%����:���>6�+�8�9 (Hatfield et al., 1987) 6B�' Huang et al. (2002a) +#A%��'*�q� ELISA �+!�8# Recombinant 41 KDa partial protein (P45Ny) ��=�:�6��$� ?'��%;6�*$�':�6�;+�6B�8�9 R. anatipestifer %��ก�+$'กก'�6�+�8�9 :�ก'� +#�%;*%�������� ��� 1, 10, 19, 19 :� ATCC 11845 +#:�!%)6�*$A;:�6�;+��� 25% ()��=+��>�?'�(#' �)����D$'กY'�D���=+ 3 Y'�D� �ก$'ก��9!%)��ก'�A%��'*�q� ELISA ?'��%;ก'�6�*$�':�6�;+� (IgY) 6B R. anatipestifer :�6��$��� (B:+)$'ก:�B��=+��> +#�%;*%���� :�$'ก���%�()"ก��=+:�ก�ก�+�t>)�ก�+$'ก:�B��> +#�%;*%���� :�A;*B':�B��=+��> +#�%;*%���� '�'�HHB'!�+�*'��7#����MB'� (B "B"ก��=+:�ก�ก�+ +# :6B:�6�;+���> +#�%;$'ก:�B$�+)!B')�*+��E*G'!�� 10 *%��%)$'กY�ก��=�6%* ก����ก7���� ก'��%กI'+#*!!'6#'�$78�A$?'��=�6#)��ก'��+ ;�*'� *6B!'6#'�$78�A:6B�8��+��=���!�C ���>��ก'��8#!'G'!��Y'�D�!B')6B���>) !'��>�8# +#M B*���@B!"B��ก7B� �A������ :�A����� ��Y'��Eก��� �:���Y��� ���q��%!��� :� ������ B*�!'��>�8# �B +#M +#:กB �6�E��6�%!��� �$�6'�%!��� ก'�'�%!��� :��%Y' +����Eก���

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��/�9� ������������ (Riemerellosis) ����������� �!�"#$%กก%����'�

() �����*+%�����+�� (New Duck Syndrome) �� '��67�'$'ก�8�9:;������! Riemerella (Pasteurella, Moraxella) anatipestifer (R. anatipestifer) ��=������;'+��>�ก�+(t9��%>* ���������6B')C�%>*�ก �?'�*'� "@� �!��#ก%;76 '�ก���ก'���9!)��=+��=�!B')�'ก ���>)$'ก��%6�'ก'�6'! ") �9?'��%ก+ %6*D6%*��>�+8�*�6กE$��6�;�6 �B �;"�FD :���:ก�E� �7FG'A�'ก6>?' �?'��#%6�'ก'�H"ก�%+��9) ") �ก�%9)!%) �9����)�B'�8#$B'!�'ก��ก'���#!'�%กI':�*%���� �ก$'ก��=+:� กB)*):#* %6*D�gก�ก�;�7ก8��+ '�'�H��=������������� +# ��>���'!)'�:#* +#:กB �B'� กB กBYp' �ก��q���8'6� +#:กB ��=+�f' :��) D

�8�9 R. anatipestifer ��=��8�9��>$%+!"B��ก7B���> �B���|�ก���!'ก%;�'�D�; ��+�� ก'�A� "$�D�ก%กIFD()�8�9$t)�?' +#!'ก 6#)�8#ก'�A� "$�D�'!*�q��(#'�'8B*!�ก����$'ก�7F �;%6��') ����*��!' 8�*����:#*ก'�A� "$�D�8�9�+!�|�ก���!'6B�E��'!�DกE��=��ก�')��ก��t>) �*��%9)%กIF�)�D���ก;:�:;;:M�()���6�� :�ก�+ (�%� 6+$��������')8�*���ก7 ���� ���()�8�9 R. anatipestifer ��>���'!)'�:#*���ก�� 21 ���� ��� �+!���� �����> 20 :� 21 ��=����� ������B��>�'!)'�A;�������� �!

"ก��=+'!7 1 � 8 %�+'�D *6Bก'��ก�+�����9�'ก ��=+'!76>?'ก*B' 5 %�+'�D �%ก$�6'!G'!�� 1 � 2 *%��%): +)'ก'��f*! ��=+��>'!7�'กก*B'กE$����*'�6#'��'�����A�>�(t9�:��%กA;ก'�6�+�8�9��A'���> ����=+AB:�BA%�q7D�%ก �BA;'ก'��f*!:6B�8�9 '�'�HHB'!�+$'ก:�B "B"กMB'��') (BY�ก �8�9 '�'�H!"B���!�>���ก()��!��=+ +#�+! %6*D �B: +)'ก'��f*! %6*D��B'��9$t) '�'�H��=�A'��()���:�:A�B�8�9 � "B %6*D��>B�: +#

ก'��tกI')�D���ก;()�8�9 R. anatipestifer �� '!A%�qD�#)H�>�() �! 28 '!A%�q7D :� �6��#')�)�'6��'� 23 �6�� �t>)��=�6%*:��()���� ��� 1-17 �+!*�q� sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) A;*B'�%9) '!A%�q7D�#)H�>� :� �6���'6��'���

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38

:;;:M����6���#'!�t)ก%��'ก �8�9 R. anatipestifer �7ก �6����� '!A%�q7D��>�tกI'���ก;+#*!:H;���6���%ก 5 :H; �t>)���9?'��%ก���ก7 70, 62, 51, 31, :� 27 ��� 25 + 4 Kd B*����6��)�D���ก;���!�>�7#���D B*��ก (outer membrane protein, Omp) �� 11 '!A%�q7D��>��=�6%*:�� '!A%�q7D�#)H�>� :� �6��#')�)�'6��'� ATCC 11845 A;*B'��:H;���6���%ก!"B 4 :H;��>�9?'��%ก���ก7 83, 78, 70, :� 62 + 4 Kd ��$?'�*�:H;���6�� 40 :H;��>��'ก|8%+���8�9��t>) �6����>:!ก +#�+!*�q� SDS-PAGE A;*B'��!B')�#! 11 :H;���6����>: +)��#��E�*B'���7F �;%6�����>!*�?'��#�ก�+ก'� �#'):�6�;+� +#�+!ก'��?' immunoblotting :H;���6����E��+B�8%+ +#:กB :H;��>���9?'��%ก���ก7 97, 92, 83, 78, 70, 62, 54, 48, 41, :� 33 + 4 Kd �t>):H;���6����B'��9 B*���@B��'ก|!"B;� Omp :��*�Ht):H;���6���%กC ��>A;*B'��=�)�D���ก;()�8�9+%)กB'*

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*%����$'ก Recombinant OmpA :� 41-kDa partial protein �+!�8# Seppic Montanide ISA 773 ��=� adjuvant :#*�+)�'�*'��7#�()*%������"ก��=+�+!��+ 2 ��%9)*%���> 7 :� 14 A;*B'*%����+%)กB'* �B��#�*'��7#�:�#$���+A�I�%;+#*! �6���+�!*ก%;��>�8#M�6*%����

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A.@. +�. +�7F� �%�6 7*��F ��> +#ก�7F'6�*$:ก# (6#��;%; 7+�#'!((;�7Fก7B�*�$%!:�A%��' %6*D�gก ก����7 %6*D��> +#��9�Y ¡G'A กB:$# ?'��%;�ก

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