Bacterial Fish Pathogens

Brian Austin • Dawn A. Austin

Bacterial Fish Pathogens Disease of Farmed and Wild Fish

Sixth Edition Brian Austin Dawn A. Austin Institute of Aquaculture School of Life Sciences University of Stirling Heriot-Watt University Stirling , UK Edinburgh , Scotland , UK

ISBN 978-3-319-32673-3 ISBN 978-3-319-32674-0 (eBook) DOI 10.1007/978-3-319-32674-0

Library of Congress Control Number: 2016952087

4th edition: © Praxis Publishing Ltd, Chichester, UK, 2007 5th edition: © Springer Science+Business Media Dordrecht 2012 © Springer International Publishing Switzerland 2016 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made.

Printed on acid-free paper

This Springer imprint is published by Springer Nature The registered company is Springer International Publishing AG Switzerland To Aurelia Jean

Pref ace

This sixth edition of Bacterial Fish Pathogens is the successor to the original ver- sion, fi rst published by Ellis Horwood Limited in 1987, which was planned to fi ll the need for an up-to-date comprehensive text on the biological aspects of the bacte- rial taxa which cause disease in fi sh. In those days, interest focused largely on bacte- rial kidney disease, the Cytophaga -like (CLBs), furunculosis and vibriosis (caused by Vibrio anguillarum). Over the intervening years, we have witnessed changes in impetus as new methodologies, notably involving molecular biology, have been developed. Interest in vaccines has been overtaken by probiotics and plant products. In terms of the number of publications, is the pathogen of the moment. The impetus to prepare a sixth edition stemmed initially from discussion with the publisher when it became apparent that the book was par- ticularly well used (there have been >19,000 downloads since the publication of the fi fth edition in 2012). Since publishing the fi fth edition in 2012, there has been only a comparatively few new bacterial fi sh pathogens described, but numerous publica- tions deal with diagnosis, pathogenicity and especially control. Consequently with all the new publications, we considered that it is timely to prepare a new edition. Thinking back to the preparation of the fi rst edition, it is mind numbing how the mechanics of publication have changed. For the fi rst edition, we spent ages in librar- ies, using interlibrary loans; pouring over paper copies of journals, books and monographs; making handwritten notes; and preparing text for typing [on a type- writer – corrections led to use of white-out or the typing of replacement pages]. Line drawings were done using a drawing board, Rotring pens, set squares/French curves and stencils. Photographs were developed in a dark room and could be hit or miss in terms of quality. Now, we have access to an even wider range of journals electroni- cally, which have been accessed from the laptop computer via a wireless Internet connection. Weeks of waiting for interlibrary loans from around the globe did not even feature during the initial research phase of the fi fth or sixth editions. Because of the extensive new information published since the start of the millennium, we have been selective and, in particular, have once again condensed details of the pathology of the diseases, because there are excellent texts already available, which cover detailed aspects of the pathological conditions. For some diseases that have

vii viii Preface occurred only occasionally and mostly in the distant past, we have condensed the text. Nevertheless, this sixth edition will hopefully meet the needs of the readership. As with all the preceding editions, it is emphasised that most of the information still appertains to diseases of farmed, rather than wild, fi sh. The scope of the book covers all of the bacterial taxa, which have at one time or another been reported as fi sh pathogens. Of course, it is realised that some taxa are merely secondary invaders of already damaged tissues, whereas others comprise serious, primary pathogens. Shortcomings in the literature or gaps in the overall understanding of the subject have been highlighted, sometimes with subtlety. As a general comment, it is still apparent that little is known about anaerobic or micro- aerophilic pathogens – does this mean that they do not exist, or are scientists ignor- ing them? Also, it is mostly unclear whether organisms act by themselves or with others to cause a disease manifestation. Uncultured pathogens have been recognised in the form of Candidatus whereby the organisms may be visualised in pathological material, but not grown in the laboratory. The cause of some diseases is uncertain, although in the case of red mark syndrome/strawberry disease of , there is serological and molecular evidence for the involvement of rickettsial-like organisms, but which are still yet to be cultured. One wonders how many times the presence of uncultured or diffi cult-to-culture organisms is masked by culturable contaminants or secondary invaders. A fi nal comment is that the overwhelming bulk of publications deal with diseases caused by single species of pathogen. Is this really true in that disease is a pure culture phenomenon, or is science missing micro- bial consortia that could work synergistically, simultaneously or sequentially to cause disease? In preparing the text, we have sought both advice and material from colleagues. We are especially grateful to the following for the supply of photographs: Dr. J.W. Brunt Dr. G. Dear Dr. M. Crumlish Professor H. Daskolov Dr. T. Itano Professor V. Jencic Dr. D.-H. Kim Professor C. Mothersill Dr. A. Newaj-Fyzul Dr. P. Orozova Dr. N. Pieters Dr. S.M. Sharifuzzaman Professor J.F. Turnbull Professor X.-H. Zhang

Stirling, UK Brian Austin Scotland, UK Dawn A. Austin 2016 Contents

1 Introduction ...... 1 Conclusions ...... 19 References ...... 19 2 Gram-Positive Bacteria (Anaerobes and ‘Lactic Acid’ Bacteria) . . . . 21 Anaerobes ...... 21 Clostridiaceae Representative ...... 22 Clostridium botulinum ...... 22 Eubacteriaceae Representative ...... 25 Eubacterium tarantellae ...... 25 ...... 27 Carnobacteriaceae Representative ...... 27 Carnobacterium piscicola (and the Lactobacilli) ...... 28 Leuconostocacceae Representative ...... 31 Weissella sp...... 31 Weissella ceti ...... 33 Gram-Positive Cocci in Chains: The Early Literature ...... 34 General Comments ...... 34 Isolation ...... 36 Epizootiology ...... 36 Development of Vaccines ...... 37 Use of Inhibitory Compounds ...... 38 Representatives ...... 38 Enterococcus (Streptococcus) faecalis subsp. liquefaciens ...... 38 salmoninarum ...... 39 Streptococcaceae Representatives ...... 41 Lactococcus garvieae (= Enterococcus seriolicida) ...... 41 Lactococcus lactis subsp. lactis ...... 46 Lactococcus piscium ...... 47 Streptococcus agalactiae (= Str. diffi cilis) ...... 48 Streptococcus dysgalactiae subsp. dysgalactiae ...... 53 Streptococcus ictaluri ...... 55

ix x Contents

Streptococcus iniae ...... 56 Streptococcus milleri ...... 63 Streptococcus parauberis ...... 64 Streptococcus phocae ...... 66 References ...... 68 3 Aerobic Gram-Positive Rods and Cocci ...... 83 Erysipelothrix rhusiopathiae ...... 83 Characteristics of the Disease ...... 83 Isolation ...... 83 Characteristics of the Pathogen ...... 84 Pathogenicity ...... 84 Disease Control ...... 84 Renibacterium salmoninarum ...... 84 Characteristics of the Disease ...... 84 Isolation ...... 86 Characteristics of the Pathogen ...... 89 Diagnosis ...... 93 Epizootiology ...... 98 Pathogenicity ...... 101 Disease Control ...... 103 Aerococcaceae Representative ...... 106 Bacillaceae Representatives ...... 107 Corynebacteriaceae Representatives ...... 110 Coryneform Bacteria ...... 112 Microbacteriaceae Representative ...... 113 Micrococcaceae Representative ...... 113 Mycobacteriaceae Representatives ...... 115 Nocardiaceae Representatives ...... 125 Planococcaceae Representative ...... 136 Staphylococcaceae Representatives ...... 138 References ...... 141 4 Aeromonadaceae Representatives (Motile Aeromonads) ...... 161 Aeromonas allosaccharophila ...... 162 Characteristics of the Disease ...... 162 Isolation ...... 162 Characteristics of the Pathogen ...... 162 Diagnosis ...... 163 Pathogenicity ...... 164 Aeromonas bestiarum ...... 164 Aeromonas caviae ...... 165 Characteristics of the Disease ...... 165 Isolation ...... 165 Characteristics of the Pathogen ...... 165 Contents xi

Aeromonas dhakensis ...... 165 Characteristics of the Disease ...... 166 Isolation ...... 166 Characteristics of the Pathogen ...... 166 Diagnosis ...... 166 Epizootiology ...... 167 Pathogenicity ...... 167 Disease Control ...... 167 Aeromonas hydrophila ...... 167 Characteristics of the Diseases ...... 167 Isolation ...... 170 Characteristics of the Pathogen ...... 170 Diagnosis ...... 172 Pathogenicity ...... 173 Outer Membrane Proteins (OMPs) ...... 176 Extracellular Products (ECPs) ...... 176 Quorum Sensing ...... 179 Disease Control ...... 179 Immunostimulants/Dietary Supplements ...... 183 Aeromonas jandaei ...... 193 Characteristics of the Disease ...... 193 Characteristics of the Pathogen ...... 193 Pathogenicity ...... 194 Aeromonas piscicola ...... 194 Characteristics of the Pathogen ...... 194 Aeromonas schubertii ...... 194 Characteristics of the Disease ...... 194 Isolation ...... 194 Characteristics of the Pathogen ...... 195 Pathogenicity ...... 195 Disease Control ...... 196 Aeromonas sobria ...... 196 Characteristics of the Disease ...... 196 Isolation ...... 196 Characteristics of the Pathogen ...... 196 Epizootiology ...... 197 Pathogenicity ...... 197 Disease Control ...... 198 biovar sobria...... 198 Characteristics of the Disease ...... 198 Isolation ...... 198 Characteristics of the Pathogen ...... 198 Diagnosis ...... 199 Pathogenicity ...... 199 Control ...... 199 xii Contents

Aeromonas veronii biovar veronii ...... 200 References ...... 200 5 Aeromonadaceae Representative ( ) ...... 215 Aeromonas salmonicida ...... 215 Characteristics of the Diseases ...... 215 Isolation ...... 220 Characteristics of the Pathogen ...... 223 Atypical Isolates of Aeromonas salmonicida ...... 230 Plasmid Profi les of Aeromonas salmonicida ...... 233 The Taxonomic Dilemma ...... 234 Serology ...... 236 Bacteriophage Typing ...... 238 Haemophilus piscium ...... 239 Diagnosis ...... 239 Epizootiology ...... 242 Pathogenicity ...... 262 Disease Control ...... 281 References ...... 298 6 Representatives ...... 323 ...... 323 Characteristics of the Disease ...... 323 Isolation ...... 324 Characteristics of the Pathogen ...... 324 Epizootiology ...... 324 Pathogenicity ...... 324 Disease Control ...... 325 Edwardsiella anguillarum ...... 325 Characteristics of the Pathogen ...... 325 Pathogenicity ...... 325 Edwardsiella ictaluri ...... 326 Characteristics of the Disease ...... 326 Isolation ...... 327 Characteristics of the Pathogen ...... 327 Diagnosis ...... 328 Epizootiology ...... 328 Pathogenicity ...... 329 Disease Control ...... 331 Edwardsiella piscicida ...... 333 Characteristics of the Disease ...... 333 Isolation ...... 333 Characteristics of the Pathogen ...... 333 Diagnosis ...... 334 Pathogenicity ...... 334 Edwardsiella tarda (=Paracolobactrum anguillimortiferum, Edw.anguillimortifera) ...... 334 Contents xiii

Characteristics of the Disease ...... 334 Isolation ...... 335 Characteristics of the Pathogen ...... 336 Diagnosis ...... 338 Epizootiology ...... 338 Pathogenicity ...... 339 Disease Control ...... 342 ...... 347 Characteristics of the Disease ...... 347 Isolation ...... 347 Characteristics of the Pathogen ...... 348 Pathogenicity ...... 348 Escherichia vulneris ...... 348 Characteristics of the Disease ...... 348 Isolation ...... 348 Characteristics of the Pathogen ...... 349 Pathogenicity ...... 349 Hafnia alvei ...... 349 Characteristics of the Disease ...... 349 Isolation ...... 350 Characteristics of the Pathogen ...... 350 Pathogenicity ...... 351 ...... 351 Characteristics of the Disease ...... 351 Isolation ...... 351 Characteristics of the Pathogen ...... 351 Pathogenicity ...... 352 Pantoea agglomerans ...... 352 Characteristics of the Disease ...... 352 Isolation ...... 352 Characteristics of the Pathogen ...... 353 Epizootiology ...... 353 Disease Control ...... 353 ...... 354 Characteristics of the Disease ...... 354 Isolation ...... 354 Characteristics of the Pathogen ...... 354 Pathogenicity ...... 355 Epizootiology ...... 355 Disease Control ...... 355 ...... 355 Characteristics of the Disease ...... 355 Isolation ...... 356 Characteristics of the Pathogen ...... 356 Epizootiology ...... 356 Pathogenicity ...... 356 xiv Contents

Providencia vermicola ...... 357 Characteristics of the Disease ...... 357 Isolation ...... 357 Characteristics of the Pathogen ...... 357 Epizootiology ...... 357 Pathogenicity ...... 357 subsp. arizonae ...... 358 Characteristics of the Disease ...... 358 Isolation ...... 358 Characteristics of the Pathogen ...... 358 Epizootiology ...... 359 Pathogenicity ...... 359 Disease Control ...... 359 Serratia liquefaciens ...... 359 Characteristics of the Disease ...... 359 Isolation ...... 360 Characteristics of the Pathogen ...... 360 Epizootiology ...... 361 Pathogenicity ...... 361 Disease Control ...... 361 Serratia marcescens ...... 361 Characteristics of the Disease ...... 361 Isolation ...... 362 Characteristics of the Pathogen ...... 362 Epizootiology ...... 362 Pathogenicity ...... 363 Disease Control ...... 363 Serratia plymuthica ...... 363 Characteristics of the Disease ...... 363 Isolation ...... 363 Characteristics of the Pathogen ...... 364 Epizootiology ...... 364 Pathogenicity ...... 364 Disease Control ...... 364 ...... 365 Characteristics of the Disease ...... 365 Isolation ...... 365 Characteristics of the Pathogen ...... 365 Epizootiology ...... 366 Pathogenicity ...... 366 ...... 366 Characteristics of the Diseases ...... 366 Isolation ...... 368 Characteristics of the Pathogen ...... 368 Diagnosis ...... 370 Epizootiology ...... 371 Contents xv

Pathogenicity ...... 373 Disease Control ...... 375 References ...... 379 7 Flavobacteria and Cytophagas ...... 397 Chryseobacterium spp...... 401 Chryseobacterium aahli ...... 401 Characteristics of the Disease ...... 401 Isolation ...... 401 Characteristics of the Pathogen ...... 402 Pathogenicity ...... 402 Chryseobacterium balustinum (= balustinum)...... 402 Isolation ...... 402 Characteristics of the Pathogen ...... 403 Chryseobacterium indologenes ...... 403 Characteristics of the Disease ...... 403 Isolation ...... 403 Characteristics of the Pathogen ...... 404 Pathogenicity ...... 404 Chryseobacterium piscicola ...... 404 Characteristics of the Disease ...... 404 Characteristics of the Pathogen ...... 404 Pathogenicity ...... 405 Chryseobacterium scophthalmum (= Flavobacterium scophthalmum) . . 405 Characteristics of the Disease ...... 405 Isolation ...... 405 Characteristics of the Pathogen ...... 406 Epizootiology ...... 406 Pathogenicity ...... 406 Disease Control ...... 406 Flavobacterium sp...... 407 Characteristics of the Diseases ...... 407 Isolation ...... 407 Characteristics of the Pathogen ...... 407 Diagnosis ...... 407 Disease Control ...... 408 Flavobacterium branchiophilum ...... 408 Isolation ...... 408 Characteristics of the Pathogen ...... 408 Diagnosis ...... 409 Epizootiology ...... 409 Pathogenicity ...... 409 (=Flexibacter/Cytophaga columnaris) . . . . . 410 Characteristics of the Disease ...... 410 Isolation ...... 410 xvi Contents

Characteristics of the Pathogen ...... 411 Diagnosis ...... 412 Epizootiology ...... 413 Pathogenicity ...... 414 Disease Control ...... 416 Flavobacterium hydatis (= Cytophaga aquatilis) ...... 419 Isolation ...... 419 Characteristics of the Pathogen ...... 419 Pathogenicity ...... 420 Disease Control ...... 420 Flavobacterium johnsoniae (= Cytophaga johnsonae) ...... 420 Characteristics of the Disease ...... 420 Isolation ...... 420 Characteristics of the Pathogen ...... 421 Disease Control ...... 421 Flavobacterium oncorhynchi ...... 421 Isolation ...... 422 Characteristics of the Pathogen ...... 422 Flavobacterium psychrophilum (=Cytophaga psychrophila = Flexibacter psychrophilum) ...... 422 Characteristics of the Diseases ...... 422 Isolation ...... 423 Characteristics of the Pathogen ...... 423 Diagnosis ...... 426 Epizootiology ...... 428 Pathogenicity ...... 429 Disease Control ...... 431 Flavobacterium piscicida ...... 434 Flavobacterium spartansii ...... 434 Characteristics of the Disease ...... 434 Isolation ...... 435 Characteristics of the Pathogen ...... 435 Flavobacterium succinicans ...... 436 Flectobacillus roseus ...... 436 Characteristics of the Disease ...... 436 Isolation ...... 436 Characteristics of the Pathogen ...... 436 Pathogenicity ...... 437 Flexibacter spp...... 437 Myroides odoratimimus ...... 437 Characteristics of the Disease ...... 437 Isolation ...... 437 Characteristics of the Pathogen ...... 438 Epizootiology ...... 438 Pathogenicity ...... 438 Contents xvii

Tenacibaculum spp...... 438 Characteristics of the Disease ...... 438 Characteristics of the Pathogen ...... 438 Tenacibaculum dicentrarchi ...... 439 Characteristics of the Disease ...... 439 Isolation ...... 439 Characteristics of the Pathogen ...... 439 Tenacibaculum discolor ...... 440 Characteristics of the Disease ...... 440 Isolation ...... 440 Characteristics of the Pathogen ...... 440 Pathogenicity ...... 441 Tenacibaculum gallaicum ...... 441 Characteristics of the Disease ...... 441 Isolation ...... 441 Characteristics of the Pathogen ...... 441 Pathogenicity ...... 442 Tenacibaculum maritimum (= Flexibacter maritimus = Cytophaga marina) ...... 442 Characteristics of the Diseases ...... 442 Isolation ...... 442 Characteristics of the Pathogen ...... 443 Diagnosis ...... 444 Epizootiology ...... 444 Pathogenicity ...... 444 Disease Control ...... 445 Tenacibaculum ovolyticum (=Flexibacter ovolyticus) ...... 446 Characteristics of the Disease ...... 446 Isolation ...... 446 Characteristics of the Pathogen ...... 446 Tenacibaculum soleae ...... 447 Characteristics of the Disease ...... 447 Characteristics of the Pathogen ...... 447 Identifi cation/Diagnosis ...... 448 Pathogenicity ...... 448 Sporocytophaga spp...... 448 References ...... 448 8 Francisellaceae Representatives ...... 467 Isolation ...... 467 Francisella noatunensis ...... 467 Characteristics of the Disease ...... 467 Characteristics of the Pathogen ...... 468 Diagnosis ...... 469 Epizootiology ...... 469 xviii Contents

Pathogenicity ...... 469 Control ...... 470 Francisella noatunensis subsp. orientalis ...... 470 Characteristics of the Disease ...... 470 Characteristics of the Pathogen ...... 470 Pathogenicity ...... 471 Disease Control ...... 471 Francisella spp...... 471 References ...... 471 9 Pseudomonads ...... 475 ...... 475 Pseudomonas alcaligenes ...... 476 Characteristics of the Disease ...... 476 Isolation ...... 476 Characteristics of the Pathogen ...... 476 Pathogenicity ...... 477 Pseudomonas anguilliseptica ...... 477 Characteristics of the Disease ...... 477 Isolation ...... 478 Characteristics of the Pathogen ...... 478 Diagnosis ...... 479 Epizootiology ...... 480 Pathogenicity ...... 481 Disease Control ...... 481 Pseudomonas baetica ...... 482 Characteristics of the Disease ...... 482 Isolation ...... 482 Characteristics of the Pathogen ...... 482 Pathogenicity ...... 483 Pseudomonas chlororaphis ...... 483 Characteristics of the Disease ...... 483 Isolation ...... 483 Characteristics of the Pathogen ...... 484 Epizootiology ...... 484 Pathogenicity ...... 484 Pseudomonas fl uorescens ...... 484 Characteristics of the Diseases ...... 484 Isolation ...... 485 Characteristics of the Pathogen ...... 486 Epizootiology ...... 486 Pathogenicity ...... 486 Disease Control ...... 487 Pseudomonas koreensis ...... 487 Characteristics of the Pathogen ...... 487 Contents xix

Pseudomonas luteola ...... 488 Characteristics of the Disease ...... 488 Isolation ...... 488 Characteristics of the Pathogen ...... 488 Pathogenicity ...... 488 Pseudomonas mosselii ...... 489 Pseudomonas plecoglossicida ...... 489 Characteristics of the Disease ...... 489 Characteristics of the Pathogen ...... 489 Epizootiology ...... 490 Pathogenicity ...... 490 Disease Control ...... 491 Pseudomonas pseudoalcaligenes ...... 491 Characteristics of the Disease ...... 491 Isolation ...... 491 Characteristics of the Pathogen ...... 492 Epizootiology ...... 492 Pathogenicity ...... 492 Disease Control ...... 492 Pseudomonas putida ...... 493 Characteristics of the Disease ...... 493 Characteristics of the Pathogen ...... 493 Stenotrophomonas maltophilia ...... 493 Characteristics of the Disease ...... 494 Isolation ...... 494 Characteristics of the Pathogen ...... 494 Pathogenicity ...... 494 References ...... 495 10 Vibrios ...... 499 Photobacterium damselae subsp. damselae ...... 501 Characteristics of the Disease ...... 501 Isolation ...... 501 Characteristics of the Pathogen ...... 502 Diagnosis ...... 502 Epizootiology ...... 503 Pathogenicity ...... 503 Photobacterium damselae subsp. piscicida (= Pasteurella piscicida) . . . 504 Characteristics of the Disease ...... 504 Isolation ...... 505 Characteristics of the Pathogen ...... 506 Diagnosis ...... 507 Epizootiology ...... 508 Pathogenicity ...... 509 Disease Control ...... 511 xx Contents

Aliivibrio (= Vibrio) fi scheri ...... 513 Aliivibrio logei ...... 513 Characteristics of the Disease ...... 513 Isolation ...... 513 Characteristics of the Pathogen ...... 514 Epizootiology ...... 514 Aliivibrio salmonicida ...... 514 Characteristics of the Disease ...... 514 Isolation ...... 515 Characteristics of the Pathogen ...... 515 Epizootiology ...... 516 Pathogenicity ...... 516 Disease Control ...... 517 Aliivibrio wodanis ...... 518 Characteristics of the Disease ...... 518 Characteristics of the Pathogen ...... 518 Vibrio aestuarianus ...... 519 Characteristics of the Disease ...... 519 Characteristics of the Pathogen ...... 519 Pathogenicity ...... 520 ...... 520 Characteristics of the Disease ...... 520 Isolation ...... 521 Characteristics of the Pathogen ...... 521 Diagnosis ...... 521 Epizootiology ...... 522 Pathogenicity ...... 522 Disease Control ...... 523 Vibrio anguillarum ...... 524 Characteristics of the Disease ...... 524 Isolation and Detection ...... 525 Characteristics of the Pathogen ...... 526 Diagnosis ...... 529 Epizootiology ...... 530 Pathogenicity ...... 532 Disease Control ...... 537 (non-O1) ...... 544 Characteristics of the Disease ...... 544 Isolation ...... 545 Characteristics of the Pathogen ...... 545 Epizootiology ...... 546 Pathogenicity ...... 546 Vibrio furnissii ...... 546 Vibrio harveyi (= V. carchariae = V. trachuri) ...... 547 Characteristics of the Diseases ...... 547 Isolation ...... 548 Contents xxi

Characteristics of the Pathogen ...... 548 Diagnosis ...... 549 Pathogenicity ...... 550 Disease Control ...... 551 Probiotics ...... 553 Dietary Supplements ...... 553 Inhibitors of Quorum Sensing ...... 554 Antimicrobial Compounds ...... 554 Vibrio ichthyoenteri ...... 554 Characteristics of the Disease ...... 554 Characteristics of the Pathogen ...... 555 Control ...... 555 Vibrio mimicus ...... 555 Characteristics of the Disease ...... 555 Isolation ...... 556 Characteristics of the Pathogen ...... 556 Pathogenicity ...... 556 Disease Control ...... 556 Vibrio ordalii ...... 556 Characteristics of the Disease ...... 556 Isolation ...... 557 Characteristics of the Pathogen ...... 557 Diagnosis ...... 558 Epizootiology ...... 558 Pathogenicity ...... 559 Disease Control ...... 559 ...... 559 Disease Control ...... 559 Vibrio pelagius ...... 560 Characteristics of the Disease ...... 560 Isolation ...... 560 Characteristics of the Pathogen ...... 560 Epizootiology ...... 561 Pathogenicity ...... 561 Disease Control ...... 561 Vibrio ponticus ...... 561 Characteristics of the Disease ...... 561 Isolation ...... 562 Characteristics of the Pathogen ...... 562 Pathogenicity ...... 562 Vibrio scophthlalmi ...... 562 Characteristics of the Pathogen ...... 563 Pathogenicity ...... 563 Vibrio splendidus...... 563 Characteristics of the Disease ...... 563 Isolation ...... 564 xxii Contents

Characteristics of the Pathogen ...... 564 Epizootiology ...... 565 Pathogenicity ...... 565 Disease control ...... 565 Vibrio tapetis ...... 566 Characteristics of the Disease ...... 566 Isolation ...... 566 Characteristics of the Pathogen ...... 566 Disease Control ...... 567 Vibrio vulnifi cus ...... 567 Characteristics of the Disease ...... 567 Isolation ...... 567 Characteristics of the Pathogen ...... 568 Diagnosis ...... 569 Epizootiology ...... 569 Pathogenicity ...... 570 Disease Control ...... 570 References ...... 572 11 Miscellaneous Pathogens ...... 603 Representatives ...... 603 piscicida ...... 603 Pseudoalteromonas undina ...... 604 Shewanella putrefaciens ...... 605 Campylobacteriaceae Representative ...... 607 Arcobacter cryaerophilus ...... 607 Comamonadaceae Representative ...... 608 Delftia acidivorans ...... 608 Hahellaceae Representative ...... 608 Hahella chejuensis ...... 608 Halomonadaceae Representative ...... 610 Halomonas (=Deleya) cupida ...... 610 Moraxellaceae Representatives ...... 611 Acinetobacter sp...... 611 Acinetobacter johnsonii and Acinetobacter lwoffi i ...... 613 Moraxella sp...... 613 Moritellaceae Representatives ...... 615 Moritella marina (= V. marinus) ...... 615 Moritella viscosa (= V. viscosus) ...... 616 Mycoplasmataceae Representative ...... 618 Mycoplasma mobile ...... 618 Myxococcaceae Representative ...... 619 Myxococcus piscicola ...... 619 Representative ...... 620 sp...... 620 Oxalobacteraceae Representative ...... 620 Janthinobacterium lividum ...... 620 Contents xxiii

Pasteurellaceae Representative ...... 622 Pasteurella skyensis ...... 622 Piscirickettsiaceae Representative ...... 624 Piscirickettsia salmonis ...... 624 Rickettsia-Like Organisms ...... 628 Characteristics of the Disease ...... 628 Streptobacillus ...... 629 ‘ Candidatus ’ ...... 630 ‘ Candidatus Actinochlamydia clariae’ ...... 630 ‘ Candidatus Arthromitus’ ...... 630 ‘ Candidatus Branchiomonas cysticola’ ...... 631 ‘ Candidatus Clavochlamydia salmonicola’ ...... 631 ‘ Candidatus Piscichlamydia salmonis’ ...... 632 ‘ Candidatus Renichlamydia lutjani’ ...... 632 ‘ Candidatus Similichlamydia latridicola’ ...... 632 ‘ Candidatus Syngnamydia venezia’ ...... 632 Chlamydiales Representative ...... 632 Unidentifi ed Gram-Negative Bacteria ...... 633 References ...... 635 12 Isolation/Detection ...... 643 Appendix – Media Used for the Isolation and Growth of Bacterial Fish Pathogens ...... 647 Anderson and Conroy’s Medium for Sporocytophaga – Like Organisms ...... 647 Blood and Serum Free Medium for Piscirickettsia salmonis (Henriquez et al. 2013) ...... 647 Bootsma and Clerx’s Medium for Flavobacterium columnare ...... 648 Brewer’s Thioglycollate Medium ...... 648 Charcoal Agar; for the Growth of Renibacterium salmoninarum . . . . . 648 Columbia Agar ...... 649 Coomassie Brilliant Blue Agar (CBB) ...... 649 Cysteine Blood Agar ...... 649 Cytophaga Agar (Anacker and Ordal 1959) ...... 649 Improved Growth Medium for Fla. psychrophilum (Daskalov et al. 1999) ...... 650 LG Agar for Lactococcus garvieae (Chang et al. 2014a) ...... 650 Nutrient Agar Supplemented with Activated Charcoal for Fla. psychrophilum (Álvarez and Guijarro 2007) ...... 650 Dorset-Egg Medium ...... 650 Edwardsiella tarda (ET) Medium (Lindquist 1991; Castro et al. 2011) ...... 651 Emerson Agar ...... 651 Flavobacterium columnare Selective Medium (Fijan 1969) ...... 651 FLP Medium (for Fla. psychrophilum ; Cepeda et al. 2004) ...... 652 Kidney Disease Medium 2 (KDM2) ...... 652 L-F Medium ...... 652 xxiv Contents

Loeffl er (Serum) Medium ...... 653 Löwenstein-Jensen Medium ...... 653 MacConkey Agar ...... 653 Medium K (Mudarris and Austin 1988) ...... 653 Middlebrook 7H10 Agar ...... 654 Myxobacterium Selective Medium (Hsu et al. 1983) ...... 654 Peptone Beef Extract Glycogen Agar (PBG) ...... 655 Petragnani Medium ...... 655 Photobacterium damselae subsp. piscicida Medium (Hashimoto et al. 1989) ...... 655 Pseudomonas F Agar ...... 656 Ribose Ornithine Deoxycholate Medium for the Isolation of Yersinia ruckeri (Rodgers 1992) ...... 656 Rimler-Shotts Medium (Shotts and Rimler 1973) ...... 656 Robertsons Meat Broth (= Cooked Meat Medium) ...... 657 Selective Flexibacter Medium (Bullock et al. 1986) ...... 657 Selective Kidney Disease Medium (SKDM) ...... 657 Semi Defi ned Medium for Renibacterium salmoninarum ...... 658 Shotts and Waltman Medium for the Isolation of Edwardsiella ictaluri (Shotts and Waltman 1990) ...... 658 Skimmed Milk Agar ...... 659 TCY Medium (Hikida et al. 1979) ...... 659 Thioglycollate Broth ...... 659 Thiosulphate Citrate Bile Salt Sucrose Agar (TCBS) ...... 659 Todd-Hewitt Broth ...... 660 Vibrio alginolyticus Agar (VAL) ...... 660 Vibrio anguillarum Medium (VAM) ...... 660 Waltman-Shotts Medium for the Isolation of Yersinia ruckeri (Waltman and Shotts 1984) ...... 661 Yeast Extract Glucose Agar (Michel et al. 1997) ...... 661 Yersinia ruckeri Selective Medium (Furones et al. 1993) ...... 661 References ...... 661 13 Diagnosis ...... 663 Gross Clinical Signs of Disease ...... 664 Histopathological Examination of Diseased Tissues ...... 666 Bacteriological Examination ...... 667 Tissues to be Sampled ...... 667 Identifi cation of Bacterial Isolates ...... 667 Serology ...... 667 Immunohistochemistry ...... 671 Molecular Techniques ...... 671 Phenotypic Tests ...... 673 References ...... 689 Contents xxv

14 Control ...... 693 Wild Fish Stocks ...... 693 Farmed Fish ...... 694 Husbandry/Management ...... 695 Disinfection/Water Treatments ...... 696 Genetically Resistant Stock ...... 696 Adequate Diets/Dietary Supplements/Non-Specifi c Immunostimulants...... 696 Vaccines ...... 698 Composition of Bacterial Fish Vaccines ...... 698 Methods of Vaccine Inactivation ...... 699 Methods of Administering Vaccines to Fish ...... 699 Antimicrobial Compounds ...... 701 Preventing the Movement and/or Slaughtering of Infected Stock . . . . 704 Probiotics/Biological Control ...... 705 References ...... 706 15 Conclusions ...... 711 The Recognition of New and Emerging Conditions ...... 712 Co-Infections ...... 712 and Diagnosis ...... 715 Isolation and Selective Isolation of Pathogens ...... 715 Ecology (Epizootiology) ...... 715 Pathogenicity ...... 716 Control Measures ...... 716 The Effects of Environmental Stress Including Physical and Chemical Pollution ...... 716 Zoonoses ...... 718 References ...... 718

Index ...... 723

About the Authors

Brian Austin is Professor Emeritus of Microbiology at the University of Stirling. From 1975 to 1978, he was research associate at the University of Maryland, USA, and from 1978 to 1984 was head of bacteriology at the Fish Diseases Laboratory in Weymouth, UK. He joined Heriot-Watt University as a lecturer in aquatic microbi- ology in 1984 and was promoted to reader in 1989 and to professor in 1992, before moving to the University of Stirling in 2009 as director of the Institute of Aquaculture and Professor of Microbiology. At Heriot-Watt University, he held various positions including Head of Department, Head of School and Dean of the University. Professor Austin gained a B.Sc. (1972) in microbiology and a Ph.D. (1975) also in microbiology, both from the University of Newcastle upon Tyne, and a D.Sc. (1992) from Heriot-Watt University. He was elected FHEA, FRSA and Fellow of the American Academy of Microbiology and until retirement was a member of the American Society of Microbiology, Society of Applied Bacteriology, Society of General Microbiology and European Association of Fish Pathologists and was President of Bergey’s International Society for Microbial Systematics. He has written previous books on bacterial taxonomy, marine microbiology, methods in aquatic bacteriology, methods for the microbiological examination of fi sh and shellfi sh and pathogens in the environment.

Dawn A. Austin is a research associate at Heriot-Watt University, a position she has held since 1986. Prior to this, she was research assistant at the University of Maryland (1977–1979); lecturer in microbiology, University of Surrey (1983– 1984); and research fellow of the Freshwater Biological Association, The River Laboratory, Dorset (1984–1985). Dr. Austin gained a B.S. (1974) from the City College of the City University of New York and an M.S. (1979) and a Ph.D. (1982) both from the University of Maryland.

xxvii

Abbreviations

ABC Adenosine triphosphate-binding-cassette Aer. Aeromonas AFLP Amplifi ed fragment length polymorphism AHL Acylated homoserine lactone A-layer The additional surface layer of Aer. salmonicida Ali. Aliivibrio Arc. Arcobacter ARISA Automated ribosome intergenic spacer analysis ATCC American Type Culture Collection, Rockville, Maryland, USA ATP Adenosine triphosphate Bac. Bacillus BCG Bacillus Calmette Guérin BHI Brain-heart infusion BHIA Brain-heart infusion agar BKD Bacterial kidney disease BLIS Bacteriocin-like substance BMA Basal marine agar Bp Base pair Car. Carnobacterium CBB Coomassie brilliant blue agar CDC Centers for Disease Control and Prevention, Atlanta, USA CE Carp erythrodermatitis CFU Colony-forming unit CgP Cytidine-phosphate-guanosine Chrys. Chryseobacterium CHSE-214 embryo 214 cell line Cit. Citrobacter Cl. Clostridium CLB Cytophaga- like bacteria

xxix xxx Abbreviations

CLED Cystine lactose electrolyte-defi cient agar Cor. Corynebacterium Cyt. Cytophaga DGGE Denaturing gradient gel electrophoresis DNA Deoxyribonucleic acid ECP Extracellular product EDTA Ethylenediaminetetraacetic acid Edw. Edwardsiella ELISA Enzyme-linked immunosorbent assay Ent. Enterobacter En. Enterococcus EPC Epithelioma papulosum cyprini (cell line) ERIC Repetitive intergenic consensus ERM Enteric redmouth Esch. Escherichia E T Edw. tarda medium Eu. Eubacterium EUS Epizootic ulcerative syndrome FAME Fatty acid methyl ester FAT Fluorescent antibody test Fc Crystallisable fragment FCA Freund’s complete adjuvant FIA Freund’s incomplete adjuvant Fla. Flavobacterium Fle. Flexibacter Fr. Francisella GAPDH Glyceraldehyde-3-phosphate dehydrogenase GCAT Glycerophospholipid:cholesterol acyltransferase GFP Green fl uorescent protein GMO Genetically manipulated organism GRP Glucose-regulated protein G+C Guanine plus cytosine H. Haemophilus Haf. Hafnia HG Hybridisation group h.i. Hyperosmotic infi ltration HSP Heat shock protein iFAT Indirect fl uorescent antibody test IgM Immunoglobulin M i.m. Intramuscular i.p. Intraperitoneal IROMP Iron-regulated outer membrane protein ISR Intergenic spacer region IU International unit J. Janthinobacterium HG Hybridisation group Abbreviations xxxi

kb Kilobase kDa Kilodalton KDM2 Kidney disease medium 2 LAMP Loop-mediated isothermal amplifi cation

LD50 Lethal dose 50 %, i.e., the dose needed to kill 50 % of the population LD100 Lethal dose 100 % Lis Listeria LPS Lipopolysaccharide MARTX Multifunctional, autoprocessing repeats-in-toxin mDa Megadalton MEM Minimal essential medium MHC Mueller-Hinton agar supplemented with 0.1 % (w/v) L-cysteine hydrochloride MIC Minimum inhibitory concentration MLD Median lethal dose MLSA Multilocus sequence analysis Mor. Moraxella mRNA Messenger RNA MRVP Methyl red Voges Proskauer msa Major soluble antigen (gene) MSS Marine salts solution Myc. Mycobacterium NCBV Non-culturable but viable NCFB National Collection of Food Bacteria, Reading, England NCIMB National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland NCTC National Collection of Type Cultures, Colindale, London, England Nec. Necromonas Noc. Nocardia ODN Oligodeoxynucleotide OMP Outer membrane protein ORF Open reading frame p57 57 kDa protein (of Ren. salmoninarum) Pa. Pasteurella PAGE Polyacrylamide gel electrophoresis PAP Peroxidase-antiperoxidase enzyme immunoassay PBG Peptone beef extract glycogen medium PBS Phosphate-buffered saline PCR Polymerase chain reaction PFGE Pulsed-fi eld gel electrophoresis PFU Plaque-forming unit Ph. Photobacterium PMSF Phenylmethyl-sulphonyl fl uoride Pr. Providencia Ps. Pseudomonas PFGE Pulsed-fi eld gel electrophoresis xxxii Abbreviations

qPCR Quantitative polymerase chain reaction RAPD Randomly amplifi ed polymorphic DNA Ren. Renibacterium REP Repetitive extragenic palindromic RFLP Restriction fragment length polymorphism RLO Rickettsia -like organism ROS Reactive oxygen species RPS Relative percent survival rRNA Ribosomal ribonucleic acid RTFS Rainbow trout fry syndrome RTG-2 Rainbow trout gonad-2 cell line RT-PCR Reverse transcriptase polymerase chain reaction SBL Striped bass larvae

SD Dice coeffi cient SEM Scanning electron microscopy S-layer Surface layer Sal. Salmonella SDS Sodium dodecyl sulphate Ser. Serratia SKDM Selective kidney disease medium SNP Single-nucleotide polymorphism SSCP Single-strand conformation polymorphism SSH Suppression subtractive hybridisation Sta. Staphylococcus Str. Streptococcus T. Tenacibaculum T3SS Type III secretion system T6SS Type VI secretion system TCBS Thiosulphate citrate bile salts sucrose agar TCID Tissue culture infectivity dose TEM Transmission electron microscopy TLR Toll-like receptor TSA Tryptone soya agar TSB Tryptone soya broth TYES Tryptone yeast extract salt medium UV Ultraviolet V. Vibrio Vag. Vagococcus VA L Vibrio alginolyticus medium VA M Vibrio anguillarum medium vapA Virulence array protein gene A VHH Vibrio harveyi haemolysin VHML Vibrio harveyi myovirus-like (bacteriophage) VNTR Variable number tandem repeats Y. Yersinia List of Figures

Fig. 2.1 A rainbow trout displaying haemorrhaging in the eye caused by infection with Lactococcus garvieae ...... 41 Fig. 3.1 The rainbow trout on the left has bilateral exophthalmia caused by Ren. salmoninarum. The second fi sh is a healthy specimen ...... 85 Fig. 3.2 A distended abdomen on a rainbow trout with BKD ...... 85 Fig. 3.3 A blood blister on the surface of a rainbow trout with BKD...... 86 Fig. 3.4 Swollen kidneys associated with BKD ...... 86 Fig. 3.5 A virtual pure culture of Ren. salmoninarum on SKDM (right). On KDM2 (KDM2), the pathogen has been outcompeted by contaminants ...... 88 Fig. 3.6 An API ZYM strip after inoculation, incubation, and the addition of reagents. The organism is the type strain of Ren. salmoninarum ...... 93 Fig. 3.7 Mycobacteriosis in yellowtail. Extensive granulomas are present on the liver and kidney ...... 115 Fig. 3.8 Nocardiosis in yellowtail. Extensive granulomas are present on the liver and kidney ...... 126 Fig. 4.1 Generalised liquefaction of rainbow trout tissues resulting from infection with Aer. aquariorum ...... 162 Fig. 4.2 Surface haemorrhaging and mouth erosion on a carp which was infected with Aer. bestiarum ...... 164 Fig. 4.3 Erosion and haemorrhaging of the mouth of a ghost carp. The aetiological causal agent was Aer. bestiarum ...... 164 Fig. 4.4 Extensive surface haemorrhaging on tilapia infected with Aeromonas sp., possibly Aer. hydrophila ...... 168 Fig. 4.5 A crucian carp displaying extensive surface haemorrhaging attributed to infection with Aer. hydrophila ...... 168

xxxiii xxxiv List of Figures

Fig. 4.6 An extensive abscess with associated muscle liquefaction in the musculature of rainbow trout. The aetiological agent was Aer. hydrophila ...... 169 Fig. 4.7 A dissected abscess on a rainbow trout revealing liquefaction of the muscle and haemorrhaging. The aetiological agent was Aer. hydrophila ...... 169 Fig. 4.8 in goldfi sh, which is also displaying abdominal swelling (dropsy) and some surface haemorrhaging ...... 170 Fig. 4.9 Generalised liquefaction of a rainbow trout associated with infection by Aeromonas ...... 170 Fig. 4.10 Extensive erosion of the tail and fi ns on a rainbow trout. Also, there is some evidence for the presence of gill disease. The aetiological agent was Aer. hydrophila ...... 171 Fig. 5.1 A furuncle, which is attributable to Aer. salmonicida subsp. salmonicida, on the surface of a rainbow trout...... 216 Fig. 5.2 A dissected furuncle on a rainbow trout revealing liquefaction of the muscle...... 216 Fig. 5.3 Carp erythrodermatitis. The aetiological agent is likely to be atypical Aer. salmonicida ...... 218 Fig. 5.4 Extensive skin and muscle haemorrhaging in black rockfi sh caused by Aer. salmonicida subsp. masoucida ...... 219 Fig. 5.5 A well developed ulcer on a koi carp. The aetiological agent was atypical Aer. salmonicida ...... 219 Fig. 5.6 An ulcerated goldfi sh on which the lesion has extended across the body wall, exposing the underlying organs. The aetiological agent was atypical Aer. salmonicida ...... 220 Fig. 5.7 Aer. salmonicida subsp. salmonicida producing brown diffusible pigment around the colonies on TSA. The smaller (= rough) and larger (= smooth) colonies are readily observable ...... 221 Fig. 5.8 The dark blue colonies of Aer. salmonicida subsp. salmonicida on CBB ...... 222 Fig. 5.9 An intensively brown pigmented L-form colony (~0.5 mm in diameter) of Aer. salmonicida subsp. salmonicida growing within L-F agar ...... 224 Fig. 5.10 An L-form colony (~0.5 mm in diameter) of Aer. salmonicida subsp. salmonicida growing within L-F agar and stained by Dienes method ...... 224 Fig. 6.1 Edw. ictaluri infection in Pangasianodon hypophthalmus. Pale areas of cellular necrosis are evident in the anterior (AK) and posterior (PK) kidney ...... 326 List of Figures xxxv

Fig. 6.2 A rainbow trout displaying extensive haemorrhaging in the mouth caused by ERM ...... 367 Fig. 6.3 A blind rainbow trout that survived infection with ERM ...... 367 Fig. 7.1 Hyperplasia, hypertrophy and fusion of the gill lamellae in rainbow trout ...... 398 Fig. 9.1 Petechial haemorrhages on the surface of an eel with Sekiten-byo ...... 477 Fig. 9.2 Clown fi sh with systemic ...... 485 Fig. 10.1 Extensive surface haemorrhaging on a turbot with vibriosis ...... 500 Fig. 10.2 An ulcer, caused by Vibrio sp., on the surface of olive fl ounder .... 500 Fig. 10.3 Surface haemorrhaging on a tongue sole (Cynoglossus semilaevis ) infected with Ph. damselae subsp. piscicida ...... 505 Fig. 10.4 Haemorrhaging on the fi ns and around the opercula of a sea bass . The aetiological agent was V. anguillarum ...... 525 Fig. 11.1 The reddened area associated with red mark disease syndrome (= winter strawberry disease) in >500 g rainbow trout ...... 629 Fig. 11.2 Epitheliocystis (see arrow ) in Sparus aurata ...... 630 Fig. 11.3 Limited tail erosion and an ulcer on the fl ank of rainbow trout. The casual agent was considered to be linked to ultramicrobacteria ...... 635 Fig. 13.1 Exophthalmia and surface haemorrhaging on a carp that was suspected to have been infected with Aer. hydrophila ...... 665 Fig. 13.2 Pronounced bilateral exophthalmia on a trout ...... 665 Fig. 13.3 Distended abdomen/dropsy on a fathead minnow for which the causal agent was considered to be Aeromonas ...... 666 Fig. 13.4 Surface haemorrhaging and ulceration on an eel...... 666 Fig. 15.1 Pronounced bloat in goldfi sh, which revealed the presence of multiple bacterial pathogens ...... 713