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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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    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

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

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

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    �,���� 13 $%-A*%�/�����)��' fibrinous perihepatitis, .�ก�

  • 1

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    ������������ (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)

  • 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,

  • 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

  • 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

  • 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)

  • 6

    ����� 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

  • 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% !�6Dก%+ ��� 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'�"@��!

  • 8

    �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 :�

  • 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�!?'

  • 10

    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)

  • 11

    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 - - + + - - + -

  • 12

    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

  • 13

    �"���> 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%�

  • 14

    ����� 3 ก���ก5��� ��ก�� ����-���

    ���1ก����� ��� 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*D7(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��>

  • 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'�Yก (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%�

  • 16

    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)

  • 17

    �"���> 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

  • 18

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

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

    ����� 10 �����������

    ����������������

  • 19

    �"���> 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

  • 20

    ����� 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�'�

  • 21

    �� 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

  • 22

    (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%+

  • 23

    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 ��

  • 24

    �'!)'�*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#���

    DB*��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'��9B*���@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>)

  • 25

    �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

  • 26

    �"���> 16 ���6��)�D���ก;���!�>�7#���

    DB*��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

  • 27

    � ����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#��))�'

  • 28

    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 (P0.05 ) c PI : protective index f WC : whole culture

  • 29

    ����� 5 ก���5���

  • 30

    *�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#����

  • 31

    (#'����� �� +#�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 :!กB*����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#����

  • 32

    +# 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

  • 33

    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

  • 34

    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

  • 35

    �+!�%>* �:#* 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#)�?'

  • 36

    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 *%��%)$'กYก��=�6%* ก����ก7���� ก'��%กI'+#*!!'6#'�$78�A$?'��=�6#)��ก'��+;�*'� *6B!'6#'�$7

    8�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���

  • 37

    ��/�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'!�+$'ก:�B"B"กMB'��') (BYก �8�9'�'�H!"B���!�>���ก()��!��=+ +#�+!%6*D �B:+)'ก'��f*! %6*D��B'��9$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�'���

  • 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#���

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  • 39

    *%����$'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*%����

    ก'�6�*$�':�6�;+�����=+��>�+*%����'�'�H�?' +#+#*!*�q� ELISA �+!:�6��$��6��!�$'ก whole cell :6B:�6��$���>�6��!�$'ก Recombinant 41 KDa partial protein (P45Ny) '�'�H�8#��=�:�6��$�?'��%;6�*$�':�6�;+�6B�8�9 R. anatipestifer %��ก�+$'กก'�6�+�8�9 :�ก'� +#�%;*%���� +#

    ก'��*;�7������������� +#*!*%����!%)�)��=��@�'���>)$'ก(#$?'ก%+()*%������>�6��!�(t9�'�'�H��#�*'��7#����$?'�A'�6B���� ���+�!* :6B���*'���=�$��)��ก'���;'+()���Ht) 17 ���� ���������� �! ก'�A�$'�F'�?'*%����?'��%;�8#��Y'�D�$'ก�8�9��>��;'+��Y'�D��) (autogenous vaccine) กE'$��=��')��ก��t>):6B6#)��ก'��+)�%+��ก�6����>��#�*'��7#�+�?'��%;�8#�?'*%������!กB� �ก�%9)AB:�BA%�q7D �t>)�%9)%6*D��>:(E):�)'�'�H��=�A'��()��� +#�+!�กE;�8�9!"B��>�!�>���ก()��! :�:A�B��� �"B%6*D��>B�: ก'�ก?'$%+���ก �$'ก")'$�?' +#�+!ก?'$%+AB:�BA%�q7D��>��=�A'������ก � �A�> �B��#:A�B���MB'�"B (BYก �!%)"ก +# :� ��ก�?'�(#'"ก��=+$'กY'�D���>�+���

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    �%;�7���ก'��?')'�+#*!+����' :�((;�7F Dr. Tetsuo Morozumi, National Institute of Animal Health, Professor Dr Takuo Sawada, Nippon Veterinary and Animal Science University :� Professor Dr Ryozo Azuma, Tokyo University of Agriculture ������@�>�7f���> +#8B*!��#�?':���?' ��tกI' :�((;�7F ��. �.A. +�. �*��%ก+� B)���� �F�%6*:A�!�'6�D ��'*��!'%!�กI6��'6�D ��>��9�Y ¡ +DG'A�!����')$7A!'q�*��!'

  • 40

    A.@. +�. +�7F� �%�67*��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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