Biochemical and molecular characterization of pathogenic vibrios from hatcheries and aquaculture farms

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BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF PATHOGENIC VIBRIOS FROM HATCHERIES AND

AQUACULTURE FARMS

THESIS SUBMl'l‘TF.D 'I‘() Tm:

COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY

IN PARTIAL FlJLFIL1\’lEN'l‘ OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

IN

MICROBIOLOGY

(Under the FACULTY or MARINE SCIENCES)

BY

B. MADHUSUDANA RAO Rm}. NO. 2827

MICROBIOLOGY, FERMENTATION AND BIOTECHNOLOGY DIVISION CENTRAL INSTITUTE OF FISHERIES TECHNOLOGY

(INDIAN COUNCII; 01-“ AGRICIJI ,'l'URAl .L RESEARCH)

COCHIN-682 029, INDLA

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2666845, 266846, 2666847, 2666848, 2668145 www.cift.res.in

Telephone } 2666763. 2666764, 2666765, 2666766 Fax : 0091-484 -2668212 2668576, 2668577, 2668578, 2668579, 2668580 E-mail : enk_mciftaris@sancharnetin

cift@ciﬂmai|.org

I gill HEEHI ingm Hi {E “I ;<~%l7i;\

Q§:/f//' CENTRAL msmurr or nsnzmrs mnuoioov

[CAR (Indian COUl'1Cl| oi Agricultural Research)

anwgfr Iii; an, n#ir%i=r - 682 029

Wtllingdon island, Matsyapuri P. 0., Cochin - 682 O29

CERTIFICATE

Dr P. K. SURENDRAN, Ph.D.,

Principal Scientist (Retd.) and Former Head of Division, Microbiology, Fennentation and Biotechnology Division, Central Institute of Fisheries Technology,

Cochin-682029

This is to certify that this thesis entitled ‘Biochemical and molecular characterization of pathogenic vibrios from hatcheries and aquaculture fanns’ embodies the result of original work conducted by Badireddy Madhusudana Rao under my supervision and guidance from November 2004 to December 2008. I further certify that no part of this thesis has previously formed the basis for the award to the candidate, of any degree, diploma, associateship, fellowship or other

similar title of this or any other University or society. He has passed the Ph.D qualifying

examination of the Cochin University of Science and Technology, held in April 2006.

Place ; Cochin J0 /

oategjg -ol - 9,0 lo (Dr P. K. s REND N)

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DECLARATION

l hereby declare that thesis is a record of bonafide research carried out by me under the supervision and guidance of Dr P.K. Surendran, my supervising guide, and it has not previously formed the basis of award of any degree, diploma, associateship, fellowship or other similar title or recognition to me, from this or any other University or society.

Place : Cochin {\/L}"' %/

Date: 2% [01 /lvtv (B. MADHUSUDANA RAO)

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A CKNO WLED GEMENTS

I wish to express my heartfelt gratitude and deep sense ofindebtedness to

my supervising guide and teacher Dr P. K. Surendran, Ph.D., Principal

Scientist (Retd.) and former Head of Division, Microbiology, Fermentation and Biotechnology Division, Central Institute of Fisheries Tech.nology, for his motivation, excellent guidance and constructive criticism throughout the study period; right from planning of research to the preparation of this thesis. It is indeed my good fortune for having the opportunity to work under him. He has been my source of inspiration throughout my career and it is from him I tried to learn the skills for planning and executing research in food microbiology. I express my sincere thanks for his magnanimity.

I am sincerely thankful to Dr B. Meenakumari, Director, Central Institute of Fisheries Technology for her encouragement and for providing excellent facilities to carry out my research work.

I am gratefully obliged to Dr K.V.Lalitha, Head of Division,

Microbiology, Fermentation and Biotechnology Division, Central Institute of Fisheries Technology for her help, constant support and advise rendered during my period of research.

I express my sincere thanks to Dr Nirmala Thampuran, former Head of Division, Microbiology, Fermentation and Biotechnology Division, Central

Institute of Fisheries Technology and an expert on Vibrios for her encouragement and guidance during my work on Vibrios. Nothing could adequately repay the help rendered by her.

I express my thanks to Dr Toms C Joseph, Scientist (SG) and Dr Rakesh Kumar, Scientist (SS), Microbiology, Fermentation and

Biotechnology Division, Central Institute of Fisheries Technology for timely and valuable suggestions during my work on real time PCR and PCR ﬁngerprinting.

My reverence is due, to Dr K. Devadasan, Former Director, Central

Institute of Fisheries Technology, Sri Sibsankar Gupta, Former, SIC,

Visakhapatnam Research Centre of CIFT, Dr D. Imam Khasim, SIC,

Visakhapatnam Research Centre of CIFT, Sri A.K. Chattopadhyay, Principal

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Scientist (Retired), Sri V. Narayanan Nambiar, Principal Scientist

(Retired), Dr R. Chakrabarti, Principal Scientist, Dr M.M. Prasad, Principal Scientist for their words of support and encouragement during the period of study. I also wish to thank Dr G. Rajeswari, Dr R. Raghu Prakash, Dr

U_.Sreedhar, Dr Sanjoy Das, Senior Scientists, CIFT for their earnest support

I also express my thanks to Mr N.M. Vasu, Mr Raman

Namboothiri, Mrs K.S. Mythri, Mrs Ammini of MFB Division, CIFT, Cochin and Mr V.V. Ramakrishna, Mr N. Venkata Rao, Mr P.

Radhakrishna, Mr B.K. Panda and suppoting staﬂ‘ of Visakhapatnam

Research Centre of CIFI‘ for their technical help and support during the period

of study.

The assistance and cooperation rendered by the library staff of CIFT, Mrs T. Silaja and Mr Bhaskaran and Mr M.M.Devasya are gratefully

acknowledged.

The love, affection and care shown by my father Dr

B.Ch.Suryanarayana (the late), my mother B. Lakshmi Kanthamma, my wife Swarna, my son Abhinav and my father-in-law Sri G. Gnana

Prakasam are beyond words and for which I will remain indebted forever. I am thankful to my sisters, mother~in~law, brothers-in-law, sisters-in-law, nieces and nephews for their affection. My warmest thanks are due, to Md.

Yousuj, J .K., and Ravi Chochipatala for beings my friend in need.

I thank all my teachers during my school, college, graduate and post

graduate studies, for imparting the knowledge and instilling the conﬁdence in

me to meet the challenges of life.

It gives me utmost pleasure to place on record my deep sense of gratitude to my beloved institute the ‘Central Institute of Fisheries Technology’.

Last but not the least, I thank all the people who have directly or indirectly helped me in carrying out the research work and in preparing this

thesis.

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CONTENTS Hg

RSI N0

Title 7 _

^{Page N0.}

l. INTRODUCTION g g

¹

REVIEW’ OF LITERATURE 3__. _ ..___.

12.1_2.2.

g\(ibriosvis-a-vis Humandisease vines ^{1 13}

¹³

1-2-.1.. V. <_rh.01eru@ pg

1 1 5 1

Serotypes of V.

.3/I0/(W08 W__

^{1 5 H 2}

1 2.2.1.1

1 2.2.1.2;

2.2.1.3

.._ ( . .

V. <.'h0lerac_+ “O 1 39

lﬂon O1 V. cholerae g_

16 17

2.2.1.4

Sources of V. cholwae g

¹⁸

2.2.1.5 Cholera disease and predisposing factors 3.3.- -._

2.2.2.

V. parahuemolyricus g g

²³

2.2.3. V. r=z1lrziﬁcr1.s' 26

2.2.4.

V. algin0Iyticu.s' g g

^23937”

2.2.5. Other pathogenic Vibijio spp.

. 30

2.3. Vibrios vis-a-vis Fish/Shrimp Disease ³⁰

2.4. 2

Vibriosvisia-vis post-harvestquality

2.5. ldentitication of\/ibrios p

⁴²

2.6. PCR (Standard, Multiplex and Real Time PCR) methods for

the d.etectionofVibrios X {M

45

2.6.1. PCR methods for pathogenic vibrio species 4.6._

2.6.2. Multiplex methods targeting more thanﬁone organism 48 2.§.3.

7741

Real-Tim em PC R g

⁴⁹

2.7. Growth Kinetics and Enzymatic activities of Vibrios ⁵¹

1

2.8.

Control ofVibrios pg

^-54

2.9. 2 DNA Fingerprinting of pathogenic “Vibrios with special

reference to Vibrio cholerae

57 A

'12.9.1. ERIC-PCR: Enterobacterial repetitive intergenic consensus

(QERICR) g pg W

⁶²

J

I‘

2.9.2. REP-PCR (Repetitive Extragenic Pa1"ind1'omes)

1 64.- MD

2.9.3. RS-PCR : Ribosomal Gene Spacer Sequenc-e( RS) 65

2.10.

3.

Qholera toxin andgc-tx genes W

MATERIALS AND METHODS

1 66

70

23.1.

Materials g

⁷⁰

3.1.1.

Hatchery Samples p

3.1.2.

Aquaculture samples U

1 3.1.3. Bacteriological Media and cultures g__

70

70 20 3.1.4. Chemicals / preservatives used for special studies ⁷¹

3.1.5.

Polymerase Chain Reaction chemicals pg J

⁷¹

3.1.5.1

Chemicals for DNA extraction WM g _g 7 ^{71 7'}

3.1.5.2. PCR_Con1ponents Kg

⁷²

L_3_.l .5.3. Agarose gel electrophoresis components g 72

3.1.5.4. ; Oligonucleotide primers used in the detection of pathogenic ; 73 Vibrios g

i

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Equipment and software it 7 K

⁷⁵

3.-2.

Methods H

⁷⁶

73.2.1.

Estimation of abiotic factors in water samples from’

hatcheries and aquaculture farms.

76

3.2.2. A Bacteriological analysis ⁷⁶

3.2.2.1. Quantitative analysi s ⁷⁶

3.2.2.111.

Preparation ofsample ,

^-76 ^{7 l}^l¹^I

l

32.2.1.2.

Total Plate Counts (TPC) / Aerobic Plate Count (APC) ⁷⁷

3.2.2.l.3. ¹¹ ^{E. 6011'} 77

_3.2.2.1.4. Total Vibrio Counts (_:[7\/C) 77

3.2.2.2.

Qualitative analysis ofVibrios 7

⁷⁸

3.2.2.2.l.

Identiﬁcation of pathogenic_Vibr1'0s spp. l

H’ 3.2.2.2.2. Scheme ot‘Alsina and Blanch (1994)

73 78

3.2.2.23. I§loguero1a and Blancht2(_)O8) scheme 7

⁸⁰

l 3.2.2.2.4. Biochemical reactions of pathogenic Vibrios ⁸² 32.2.2.5.

l

Biochemical tests for identiﬁcation of pathogenic Vibrios l isolatedfjom shrimp hatcheries and aquaculture farms

83

1 3.2.2.2.5.1. Grams staining ₈₃

L3.2.2.2.5.2.3.2.2.2.5.3.

Cytochrome Oxidase test _ M l.\/l.O'[llllfy A é

⁸³⁸² ¹

3.2.2.2.5.4.

Nitrate reduction test My Wm Ml" A it

⁸⁴

3.2.2.255. Hugh and Leifson’s oxidative / fermentative metabolisml ('H&L O/F) test

l

84

_;1.2.2.2.5.6.

Urease test” _*__

⁸⁴

3.2.2.2.5.7. Indole test ⁸⁴

3.2.2.2.5.s.

Vo7ges-Proskauer (VP) test 1

y 3.2.2.2.5.9.i

Methyl Red (MR) 16$?" g__

⁸⁵⁸⁵

l 3.2.2.2.5.10

7Cwitrate utilization test i

⁸⁵

3.2.2.2.5.11

reactions Triple Sugar Iron (TST) and Kligler lron Agar (KIA)

⁸⁵

l3.2.2,2g.5. 12

Sugar fermentation tests K it __n W p

⁸⁵ ^l

3.2.2.2.5.13

Amino Acid decarboxylase / dihydrolase testm W

⁸⁶

3.2.2.2.5.14

pteri dine) 1

Sensitivity to O/ 129 vibriostat (2.4.-diamino-6,7-diisopropyl ⁸⁶

3.2.2.2.s.15

Salt tolerancetest 7

⁸⁶

3.2.3. Slide agglutination tests for identiiying V. cholerue O1 and V. c'l1()ler1.1e O l 39 and V. c/iolerue Non O land Non _Qgl 39

86

l

. 3.2.4.

Biochemical characterization of Vibrios isolated from

shrimp hatcheries and farms_

87

_3_.2.4.l .

Utilization ofsugars _ 1

^is?

(8)

it 3.2.5.2.

3.2.41.1.

Utilization of pentoses 3.2.41.2. Utilization of hexoses

1

3.2.41.3. i Utilizationofdisaccharides N

‘y 3.2.4.l.4. 5 Utilization of sugarderivatives

3.2.4.15. 5

Utilization ofGlyeogeni jg 2

^l

53.2.4.2. J Utilization of amino acids g V i g

^I

3.2.4.3. 1 Enzyme activities of pathogenic Vibrios isolated from

shrimp hatcheries and farms A gg

-J

l, 3.2.43.1. Determination ofAmylolytic activity 3._2.4.3.2. _y Determination of proteolytic activity 3.2.4.3.2.1. Gelatin liquefaction

3.2.4.322. Gelatin hydrolysis in agar medium J

3 3.2.4.3.2.3. 1 Proteolytic activity against fish protein

'”3.2.4.3.2.4.

Proteolytic activity against shrimp protein

3.24.3.3. Determination of DNAse activity W g

^l ^l

_, 32.4.3.4.

Determination of lipolytic activity

3.2.4.3.4.1. 1 Tributyrin hydrolysis by pathogenic Vibrios g

3.2.4.342. Phospholipase activity of pathogenic Vibrios 5 g _ 7 L3.2.4.3.5. . Determination of phosphatase activity

^l^{i .}

3.2.5.

Studies on growth kinetics of pathogenic Vibrios isolated.

from shrimp hatcheries and farms g

3.2.5.1. A

Effect of temperature on the growth of Vibrios isolated from shrimp hatcheries and farms

32.5.1.1.

1 _ _l Effect of temperature on the utilization

pathoge_nic\/ibrios 7 g of sugars by 3.2.5.1.2. l

Effect of temperature on the utilization of amino acids by

Vibrios

I

[_...

Effect of pH on the growth of Vibrios isolated from shrimp hatcheries and farms

3.2.5.3. Effect of salt on the growth of Vibrios isolated from shrimp hatcheries and farms

3.2.5.4. Effect of salt on the enzymatic activities of pathogenic M A Vibrios isolated from shrimp hatcheries and farms H

ⁱ

. 32.5.4.1. , Effect of salt on the amylolytic activity of pathogenic

Vibrios g g

32.5.4.2.

Effect of salt on the proteolytic activity of pathogenic

Vibrios

3.2.5.4.3. 5

Effect of salt on the DNAse activity of pathogenic Vibrios g ^F

11 3.2.5.5. V

Effect of salt on the swarming behaviour of V.ct1gin0lyri<:z15

3.2.5.6. .

l _ . Effect of salt on the utilization of sugars by pathogenic

Vibrios i

3.2.5.7. Effect of salt on the utilization of amino acids by pathogenic Vibrios

r_ .

i11 l

i

l

iii

1

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E96

3.2.6.1.

Effect of potassium chloride (KC1) on the growth of

pathogenicvibrios

96 3.2.6.2. Effect of potassium sorbate (C(,1'1702K) on the growth of

pathogenicVibrios

97 3.2.6.3.

Effect of sodium citrate (Na;C(,H5O7) on the growth of

pgathogenic Vihrios

973 3.2.6.4. Effect of sodium tri polyphosphate (Na5P;,O10, STPP) on the

growth gofpathgogenic Vibrios

97 3.2.7.”

3.2.7.1.

Molecular characterization of pathogenic Vibrios isolated

from shrimp hatcheries and farms _ %___ g_ ___

PCR for the detection of enterotoxigenic V .ch01erae

98

3.2.7.2. PCR. for the detection of V.ch0l@rae using species speciﬁc primers

98

99 3.2.7.3. Duplex PCR for the simultaneous detection of V. cholerae

and differentiation of cholera toxin producing V. cholerae

isolates (W V. ch0Ierue~dup1ex PCR)

100

3.2.7.3.1. End point dilution of V. cholerae-duplex PCR ¹⁰¹

3.2.v.zi1Wig_i PCR for and_V._ch01erae O1 and V. cholerae 0139 ¹⁰¹

3.2.7.5. PCR for the detection of V. ulg1'n0Iyt1'cu.\' ¹⁰¹ 3.2.7.5.1. End point dilution of V. a1

“0lyn'cus PC R W

3.2.7.6. ¹11

8U1@ gs gr or or

Duplex PCR for the V. a1gm0Iyr:'cu.s' using species speciﬁc and genus speciﬁc primers ( V. algin0lyn'cu.s'-duplex PCR)

192

1 02

3237.7. PCR for detection of V. vulng'ﬁcu.s' 103

3.2.7.8. PCR for d@te<>ti9_n 9!‘ V-11Mtraevwlym?mi, . ^__l9_3, 3.2.7.9.

3.2.7.9.1.

Multiplex PCR for the detection of pathogenic Vibrios viz., I/.c.:/tolerue, V. cholerae (cor), V. algz'n0lyricu.s-, V. vulng'ﬁcu.s' agndgl/g.pa:'ahaemolyticzm ("Path ogenic Vibrio-multiplex PCR)

Multiplex PCR for pathogenic Vibrios using unknown

cultures. H_

1 104

105 3.2.7.10.

PCR fingerprinting of I/.ch01erae isolates M 105

3.2.7.11.

4.

4.1. ¹

1

Real time PCR for Vgchglegqe

RESULTS AND msCUssION _____ U

Estimation” of abiotic factors in water samples from

hatcheries andaqtiaculture farms.

107 109 109

4.2.

Bacteriological analysis of hatchery and farm samples ¹⁰⁹

4-3.-1--2.

Quantitative analysis g g g

¹⁰⁹

4.2.1.1. Total Plate Counts (TPC) / (Aerobic Plate Count APC) of

hatchery and farm S_£1lT1p16S L 7 g

109 4.2.1.2. E.s"chericln'a 0011' (E.c01ii) in P. monodon hatcheries and

aquaculture farms

111

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aquaculture farms p W p

¹¹²

14.2.2.

Qualitative analysis of Vibrios isolated from shrimp

hatcheries and aquaculture farms.

118 1 4.2.2.1. identiﬁcation ofpathogenic VibI'i() app. isolated from shrimp

hatcheriesuand aquaculture farms. up p

“ii 1 8 1 4.2.2.1.1 Incidence of pathogenic Vpib1"ios in shrimp hatcheries W ¹¹⁸

42.2.1.2. B

Incidence of pathogenic-XX/ib1"ios in aquaculture farms K 120

4.2.3. Slide agglutination tests for identification of V. crholerae O1 and V. cliolerae O139 and V clzolei-‘ac Non O1 and Non 0139

I26 4.2.4.

Biochemical characterization of Vibiios isolated from

aquaculture fa1msand.hatcheries p p

126 4.2.4.1. Biochemical reactions of pathogenic Vibrios isolated from

shrimp hatcheries and farms used for biochemical

studies

128

4.2.4.1.1

_l

Biochemical reactions of V.ch0lerue cultures isolated. from aquaculture farms

128 4.2.4.1 .2 Biochemical reactions of I/.vu11-uficus isolated from shrimp

hatcheries

131 1 42.4.1.3 Biochemical reactions of V.algir1.0lytic.'u.s‘ isolated from

shrimp hatcheries and aquaculture farms

133 l 4.2.4.l.4 Biochemical reactions of V.puru/iaeniolyricus isolated. from

shrimp hatcheries _ p

¹⁹⁷⁵

4.2.4.1 .5

1

Biochemical reactions of V./iuweyi isolated ﬁ'om shrimp hatcheries.

137 4.2.4.2. Utilization of sugars by vibrio cultures isolated from shrimp

hatcheries and aquaculture farms

139 4.2.42.1

Utilization of pentoses by Vibrio cultures isolated from

shrimp culture system H _

¹⁴⁰

4.2.4.2.2

l

Utilization of hexoses by Vibrio cultures isolated from

shrimp culture system

140 42.4.2.3 Utilization of disaccharides by Vibrio cultures isolated from

shrimp culture system _H_

¹⁴¹

1 4.24.2.4 Utilization of sugar derivatives by Vibrio cultures isolated

ﬁ'om shrimp culture system pp M

1 42 A 4.24.2.5 Utilization of Glycogen by Vibrio cultures isolated from

shrimp culture system p_W

¹⁴³

4.2.4.4. Utilization of amino acids by Vibrio cultures isolated from

shrimp culture system p 1 ﬂy H

143 4.2.4.5.

Enzyme activities of pathogenic Vibrios isolated from

hatcheries and farms

144

\/

(11)

Determination of proteolytic activity by Vibrio cultures

isolated from shrimp culture system

146 1 4.2.4.5.2.l. Gelatin liquefaction by Vibrio cultures isolated from shrimp

culture system 77 7 77 7 7 7 7

146 . 4.2.4.5.2.2. Proteolytic activity (gelatin) by Vibrio cultures isolated from

shrimp culture system77 7 7 7 _

l47

4.2.4.523.

Proteolytic activity of pathogenic Vibrio cultures on ﬁsh

protein _7 _____ 77

¹⁴⁸

l

!

4.2.4.5.2.4.

4.2.4.5.3.

Prqeolytic activityQ.tvath.0s@ni¢ .Y1l?_11l9§°_'1§hl‘imHP1.‘.Q1¢lll

Determination of DNAse activity of Vibrio cultures isolated from shrimp culture system

17750

152 4.2.4.5.4. Determination of lipolytic activity of Vibrio cultures isolated

ﬁ‘om shrimp culture system

153 4.2.4.5753. Determination of phosphatase activity of Vibrio cultures

isolated ﬁ'om shrimp culture system

156

4.2.4.6. Growth kinetics of pathogenic Vibrios cultures isolated from

shrimp culture system 7_ 7 77

¹⁵⁶

i4.2.4.6.1.

1

4

Effect of temperature on the growth of pathogenic Vibrio cultures isolated from shrimp culture system

157 i 4.2.4.6.2.

3

l

Effect of pH on the growth of pathogenic Vibrio cultures isolated from shrimp culture system

158 4.2.4.6.3. Effect of salt on the growth of pathogenic Vibrio cultures

isolated t‘ifom shrimp culture system

159 4.2.4.6.3.l. Effect of salt on the growth of V.c/zolerue isolated from

shrimp fa1'm7777

159 i 4.2.4.6.3.2. Effect of salt on the growth of ctx toxigenic V.<:h0/erue

isolated from farm water WM

161 4.2.4.6.3.3.

4

Effect of salt on the growth of I/.i>u1n.1'ficus isolated from shrimp_hatche1y _777__7 _

163 1 4.2.4.6.3.4. Effect of salt on the growth of V.purahuem01yticu.s' isolated

from Shrimp hatchet);

164

4.2.4.635.

Effect of salt on the growth of V.ulg1'n0lyricfz1.s* isolated from shrimp farm

167 1 4.2.4.7. Effect of salt on the enzymatic activity pathogenic Vibrio

cultures isolated from shrimp culture 7system 7

168 1 4.24.7.1 if

4.2.4.7.2

Effect of salt on the amylolytic activity of pathogenic Vibrio cultures isolated f1‘o1_nsht‘i111pculture system 7 7 Effect ofsalt on the proteolytic activity of pathogenic Vibrio cultures isolated from shri_mp77cu7lture system 7 7 7 77

169 170 l4.2.4.7.3.

Effect of salt on the DNAse activity pathogenic Vibrio

cultures isolated from shrimp culture system

171

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isolated from shrimp culture systemr M g

I72 4.2.4.9.

Effect of salt on the utilization of sugars by pathogenic

Vibrio cultures isolatedpfroni shrimp culture system

174 4.2.4.9.l. Effect of salt on the utilization of sucrose by pathogenic

Vibrios

174 4.2.4.9.2. Effect of salt on the utilization of Mannitol by pathogenic

Vibrios

I77 y 42.4.9.3. Effect ofsalt on the utilization of Cellobiose lJy7V.1--‘Zl[727{'ﬁCZl.S‘

isolated fromshrimp hatchery pp W _ g_

I79 ^l

5 4.2.4.10. Effect of salt on the utilization of amino acids by pathogenic Vibrio cultures isolated from shrimpgculture system g

179 _4.2.4.10.i Effect of salt on the arginine dihydrolase activity of Vibrios

4.2.4.l0.2 Effect of salt on thelysine decarboxylaseactivity of Vibrios

iso

1s2 4.2.4.102»

i Effect of salt on the omithine decarboxylase activity of

Vibrios g_ p g_ My ^is4

” 4.2.4.1 l.

4.2.4.1 1.1

Effect of temperature on the utilization of sugars by

pathogenic Vibrio cultures isolated. from shrimp culture

System

Effect of temperature on the utilization of sucrose by

pathogenic Vibrios "W My ”_

186

iss

l 4.2.4T11.2

Effect of temperature on the utilization of mannitol by

pathogenic Vibrios g g__

¹⁸⁷

4.2.4.12.

_i _

Effect of temperature on the utilization of amino acids by

pathogenic Vibrio cultures isolated from shrimp culture

system

^{1 89}

‘4.2.4.l2.l

Effect of temperature on the utilization of arginine by

pathogenic\/ibrios

189 4.2.4.l2.2. Effect of temperature on lysine decarboxylase activity by

pa*t_hogenic\/ibrios g_ p_

190 4.2.4.123 Effect of temperature on ornithine decarboxylase activity by

pathogenic Vibrios

192 4.2.5.

Effect of preservatives/ chemicals on the growth of

pathogenic Vibrio cultures isolated from shrimp culture

H M“ system A g __ H

194

l4.2.5.l. Effect of potassium chloride (KCl) on the growth of

pathogenic Vibrio cultures isolated from shrimp culture

system W W pp

i 94

4.2.5.2. Effect of potassium sorbate (C'<.H;O2Ki) on the growth of pathogenic Vibrio cultures isolated from shrimp culture

! p .._ S)’$t§m pg _ 2 2 __. it

I95

L

4.2.5.3. 4 Effect of sodium citrate (Na;C6H_<O;) on the growth of pathogenic Vibrio cultures isolated from shrimp culture

system p g

^l96

I

l|

vii

(13)

<>u1w1'¢SySt@m .. .

197

4.3.

Molecular characterization of pathogenic Vibrios isolated

from shrimp hatchery and aquaculture farms g __

199

4.3.1. PCR for the detection of enterotoxigenicHV.ch0Iei'ae _ ¹⁹⁹

4.3.2.

14.3.3.

14.3.3.1.

PCR for the detection of V.<..'h0lerae using species specific

primers g

V.¢-hoierue-duplex PCR for the simultaneous detection of V.ch01erae and differentiation of cholera toxin producing

lg/_.ql10le:'ue isolates g W_

End point dilution of V.(.th0le1'ae-duplex PCR _

201 203

209 94.3.4.

4.3.4.1

PCR for the detection of V.ulgi141.0lyti<:us H End point dilution of V.algir1QZyriczt.s' PCR _ 7

210 212 4.3.5. V.algiizoIyt1cu.s'-duplex PCR roﬁhé using species speciﬁc

and genusspecificp1'ime1"s pg _

²¹³

1 4.3.6.

PCR for detection of I/.1-;{ulr11'ﬁcus' M

214

I

1

1 4.3.7.

PCR for detection of V.pa1fglz.aéi1-zolyrtcfzis M

²¹⁵

4.3.8.

Pathogenic Vibrio-Multiplex PCR for the detection of

pathogenic Vibrios viz., V.c/10lerae_._ V.t.?h0Ie1'ae (cor),

V.algir10Iyn'c:us,g I/.1->ulm'ﬁcu.v and V.pa:'alz¢wm()lyti(.-us

216

4.3.8.1. Multiplex PCR for pathogenic Vibrios using unknown culture if

ⁱ¹¹¹¹ ²¹⁸

4.3.9. RCR fingerprinting of V.crlt0leme isolates using RS-PCR.

REP-PCR and ERIC-PCR

218 4.3.9.1. RES-PCR targeting Ribosomal GeneS_pacer Sequence if if ²¹⁹ 4.3.9.2. REP-PCR targeting Repetitive Extragenic_Palindromes ₂₃₂

4.3.9.3. ERIC-PCR targeting Enterobacterial repetitive intergenic consensus

225 4.3.9.4. Similarity among patterns of the V. cholerue isolates from

shrimp aquaculture farms obtained by three different PCR

fingerprint_ingmethods. W M W pg

228

4.3.10.

Real time PCR for V.c/zolerae

²²⁹

4.3.10.1 Real time PCRgfo1'gV.cl1.0lerue using species specific primers ¹ 230

5

SUMMARY g_g _ g

²³⁴

1‘

1

BIBL-I0GRAiI3HY

²⁴⁶

1,.

ANNEXURE g g

²⁸⁸

1 LINST OF F11 BLICATIONS BY THE AUTHOR ²⁹⁷

(14)

LIST OF TABLES

p Table N 0 TITLETOF THE TABLE Page N0.

Table 1.1 State wise details of shrimp production (2007-08)

2

Table l .2. RASFF notiﬁcations regarding

processed ﬁsh and shrimp

EU countries I

the detection of Vibrio in

products imported into

8

Table 1.3. RASFF notiﬁcations vis-a-vis

the detection of Vibrios

9 Table 2.1

1 _.._

from processed seafood from lndia Worldwide occurrence of Vibrio

specimens g

species in human clinical ^{it W14} Table 2.2.

Diseases in marine ﬁsh and i

associated with Vibrio species

nyeitebrates caused byor

³¹

Table 2.3. PCR methods for V.ch0lerue, V.purahaemolyriciis.

V."i-'irlri{ﬁc¢r1.s'. l’.a{gii'i.0l};ri§§zi.s' and 1/.ltc'ii'i-‘ey1T ¹

471 Table 2.4.

Multiplex-PCR methods targeting V. c/iolerae.

⁴⁸

|'—

1 Table 2.5. Real-Time PCR methods for V. (hole; aa

V.purah.aem01yti<:u.s', V.vzilrzi'fi“<ri1.sj pp g K

V. pui‘ahaem0lyriczi.s', V. i-=-ulni‘ﬁ *(.1IS and V.algirzolytic-its N

51

Table 3.1 5.4 Details of the primers used in t

Vibrios by PCR p _

he detection of pathogenic ⁷³ Table 3.1.5.5. Details of the primers used for PCR typing of V.(T/I()/efllclé? 74 1 Table 3.1.5.6.

Details of the primers used

V.ch0Ierae

in Real Time PCR for

74 1 Table 3.1.5.7. Composition of SYBR greenju^m

for quantitative PCR (Sigma)? g

p stait rag ready mix ⁷⁵ Table 3.2.22.3. Details of the remaining clust s (ﬁgures) of Noguerola

'- ‘ __ - er

and Blanch (2008) scheme for identification of Vibrio

82

1 Table 3.22.2.4. List of biochemical tests for Vib Berg_ey’s manual of Systematic

ii'0 spp mentioned in the

acteriology g

⁸²

Table 4.1

species M g_

' B

Total Plate Counts in Penaei(.5

aquaculture farms g

' monodon hatcheries and

iio

1 Table4.2^O E.c0li' counts in P. monodon

samples g

hatchery and aquacultureui ti 2 Table 4.3

J

Mean Vibrio loads in P. monodon hatcheries and

113

Table 4.4

Relative occurrence ot suci

fermenting Vibrios in P. m0n.0d<

samples

se fermenting and non

1'3. hatchery and aquaculture

116

Tablel4.5 Incidence ot pathogenic Vibriosin P.H'lOI‘I()(i()I‘I_1'l£1'[C1'l6l'l6S 1.19..

Table 4.6 The incidence of pathogenic Vi

post-laiyae W

brios in hatchery water and ¹¹⁹ TTable 4.7 The incidence of total pathog 1

Aquaculture farms p 7 7

. V _p . _O

)

eitic Vibrios in pond water. ¹²¹

ix

(15)

1 Table 4.28. Tributyrin hydrolysis by pathogenic Vibrios A _g

3 sediment and shrimp U

7 Table 4.8. Incidence of pathogenic Vibrio spp in paquaculture farms ^{1 it}

1 Table 4.9. 1 The distribution of pathogenic Vibrios .s-pp in pond water. 122

sediment and shrimp“ 1

Table 4.10. 1 Pathogenic Vibrio cultures isolated from aquaculture farms 127 and hatcheries used for biochemical characterization

My My studies g H W g

Table 4.ll. “Biochemical reactions of V.ch01erue isolated from

1 aquaculture farms _ _

Table 4.12. Biochemical reactions of V.1-*z1ln(ﬁt__'u.s" isolated from shrimp

. s . hatchelles . . ..

A 128

^{1 321}

Table 4.13. Biochemical reactions of V.algin0(yri(_?z¢.s- isolated from 133

1 shrimp gh_at_cher1'_es and aquaculture farms

‘Table 4.14. :Biochemical reactions of V.pmulruemolyriczls" isolated 135

1 U fromshrimphatcheries _gwggpW U U

Table 4.15. F Biochemical reactions of V.lza;'veyt isolated from shrimp A 137 hatcheries

Table 6. Utilization of pentoses by pathogenicggyibrios 140 p Table 4.17. 1 Utilization of hexoses by pathogenic Vibrios 141

. Table 4.1 8. 1 Utilization of disaccharides by p_a£lt_oggenicg\g/ibrios U pg gm“ 142

‘LTable 4.19. Utilization of sugar derivatives by pathogenic Vibrios" pg Table 4.20. } Utilization of Glycogen by pathogenic Vibrios 1_43

143 , Table 4.21. 1 Utilization of amino acids by pathogenic Vibrios 1

1 Table 4.22. i Amylolytic activity of pathogenic Vibrios 14L)

147 148

Table 4.23. _W_ Qelatigng1igquefactiigongbygpgathogenic Vibrios

Table 4.24. 1 Proteolytic activity (gelatin) by Vibrio cultures isolated

ﬁg from shrimp culture system g A H 1

lI1

1 Table 4.25. Proteolytic activity of pathogenic yjbrigopsggon ﬁsh protein 149

lTable4.26. Proteolytic activity of pathogenic Vibrios on shrimp 151

. protein is

' Table 4 2 D1\1Ase activity of pathogenic Vibi ios 2 2

¹⁵³

154

1 Table 4.32. Ef@ct of salt on the swa_rmingbehaviour of V.algiriig(}pZj»ti¢_fz1§ig

1 Table 4.29. Phospholipase activity of Vibrio cultures isolated from 155 A g sh ri mgpculture systemg g__

Table 4.30. 1 Effect of temperature on the growth of pathogenic Vibrio 157

cultures isolated from shrimp culture system

l Table 4.31. Effect ofpH on the growth ofpathogenicVibrios

^1152137

172

Table 4.33. Effect of salt on the utilization of Sucrose by pathogenic 175 Vibrio cultures isolated from shrimp culture system 1 7 Table 4.34. Effect of salt on the utilization of Mannitol by of: 177 pathogenic Vibrio cultures isolated from shrimp culture

3 system u g Table4.35. Effect of salt on the utilization of Cellobiose by 179

* 1/.1-=z1lnt'ficu.s"

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T Table 4.36

Effect of salt on the Arginine dihydrolase activity of

pathogenic Vibrio cultures isolated from shrimp culture system

Table 4.37 Effect of salt Lysine decarboxylase activity of pathogenic Vibrio cultures isolated from shrimp culture system

TTable 4.38 Effect of salt on the omithine decarboxylase activity of pathogenic Vibrio cultures isolated from shrimp culture

system g _ M

TTable 4.39

Effect of temperature on the utilization of Sucrose by

pathogenic Vibrio cultures isolated from shrimp culture

system g g g

Table 4.40 Effect of temperature on the utilization of mannitol by pathogenic Vibrio cultures isolated from shrimp culture

system g g ‘W

@FTable 4.41 Effect of temperature on the utilization of Arginine by pathogenic Vibrio cultures isolated from shrimp culture

system g ﬁg W

Table 4.42 Effect of temperature on Lysine decarboxylase activity by pathogenic Vibrio cultures isolated from shrimp culture

system g_

_Table 4.43

l

Effect of temperature on the utilization of Ornithine by pathogenic Vibrio cultures isolated from shrimp culture

5}’5t¢Fn . . ..

' Table 4.44 Quantity Calculations of Real time PCR of V.gt:lr_0ler'ae T Table 4.45

1 -»—:

Melting temperature calculation of the Real Time PCR products

xi

(17)

LIST OF FIGURES

Fig_|iife_l_\I_o. TITLE OF THE FIGUR ‘ iPa

^{ge E}^O

Fig 1Ii.

E

Gwhariadran W44 ism as

¹

_Fig 1.2a. lndianh/la1‘ine_Exp01ts -2007-08 (US $ realization) ⁶

Fig 1.21». 1ndian Marine Exports — 2007-08 (Quantity wise) ⁶

Fig 3.1a. Scheme of Alsina and Blanch (1994) for identification of V ibr '0 species _

79 Fig 3.11;.

I k . .

Scheme iofiAlsina and Blanch (1994) for identification of

V 1' bri 0 .s'pec:'e.s'

80 Fig 3.2. Initial key of the Noguerola and Blanch (2008) scheme for

identificationof Vibrio species Q

81

Fig 4.1. Vib1‘i@§@nI?§$ Agar ¹¹³ ^l¹¹

Fig 4.2. Sucrose fermenting and Non-fermenting Vibrios in hatchery and aquaculture samples

117 Fig 4.3. Distribution of V.algin01ytz'cu.s' in P.m0n0d0n Aquaculture

farms

123 Fig 4.4.

Fig 4.5.

Distribution of V .gp{zQ1¢{{'qgqWinpf.g;{1p()111()pi():'1 Aquaculture farms 1

Autoclavable 96 well plastic plates for Sugar fermentation tests

124“

139

Fi.g4-6.. Amylolyt_icActivityon Starch Agar Q W __

¹⁴⁵

I!

J Fig 4.7. Proteolytic activity on Gelatin Agar ¹⁴⁷

Fig 4.8. Proteolytic Activity of V.ch0lerae on Fish Protein agar ¹⁴⁹

_1=ig 4.9. Proteolytic Activity on Shrimp Protein agar ¹⁵⁰

Fig 10.

Proteolytic Activity of pathogenic Vibrios on different

protein substrates

152

Fig4.1l. DN Ase activity on DN Ase agar ¹⁵³

Fig 4.12. Phospholipaseactivity (lecithinase) onEgg Yolk agar ¹⁵⁵ Fig 4.13.

Fig 4.14.

Lipase and Phospholipase activity of pathogenic_\_/gi_briosp A Drop plate method on TCBS Agar for obtaining I/.0/zolerae counts

156 I59 Fig 4.15.

Effect of salt on the growth of V.ch0ler'ue isolated from

shrimp farm

160 Fig 4.16. Quantitative assessment of effect of salt on the growth of

V.ch0lerae by employing the Drop Plate method on TCBS

38111;. 1 is is is 1 1 is

160

4.17

Eff@¢t_@f$.a11-Qn 111$_1=l}T9W111_Qf_<?QF_¥Q.?€1g¢I15C V-<Yl1<>1@w@ 161

Fig.4.l8 Quantitative assessment of effect of salt on the growth of choleratoxigenic V.c.'h0lerae by employing the Drop Plate method on TCBS agar

162

Fig. 4.19. Effect of salt on the growth of V.w1ln_(ﬁcu.s' isolated from shrimp hatchery

163 Fig. 4.20

Drop plate method. on TCBS Agar for obtaining

V-W17¢?/1§*€{t1Q0i1i£?!lé[90U015-, 1

164

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Fig. 4.21 Quantitative assessment of effect of salt on the growth of V.pa:-uhuemolyri<:u.s' by employing the Drop Plate method on

TCBS agar g_ g

¹⁶⁵

Fig. 4.22 Effect of salt on the growth of V.puI‘uhaeni0lyi‘iCu.s' isolated.

from shrimp hatchery N g g g g

165 Trig. 4.23

Drop plate method on TCBS Agar for obtaining V.

u1gin0lyticu.s' counts g _g

¹⁶⁷

1 Fig. 4.24 Quantitative assessment of effect of salt on the growth of V.

algir-z0lyti<.'z1s by employing the Drop Plate method on TCBS agar

167

Fig. 4.25 Effect of salt on the growth of V.ulgm0Iyricu.s' isolated from shrimp farm

11 H,

¹⁶⁸

; Fig. 4.26

Comparison of amylolytic activity at different salt

concentrations of pathogenic Vibrios isolated from shrimp

culture system 7 A N 7

169

Fig. 4.27 Comparison of proteolytic activity of pathogenic Vibrios at

different saltconcentrations _ pg

D170

Fig. 4.28

Comparison of DNAse activity of pathogenic Vibrios at

differentsaltconcentrations g

171 Fig. 4.29 Effect of salt on the swarming behaviour of I/uigiitzt)/yricf‘z1.s"

(24 hours)

173 A Fig. 4.30.

Effect of potassium chloride (KC1) on the growth of

pathogenicvibrios

¹⁹⁴

1 Fig. 4.31 Effect of potassium sorbate (_C6H7O2Ki) on the growth of

pathogenic Vibrio cultures isolated from shrimp culture

system g

¹⁹⁵

Fig/1.32

I1

Effect of sodium citrate (Na;C(,H_<O7) on the growth of

pathogenic Vibrio cultures isolated from shrimp culture

system W g Z __

197

l Fig. 4.33 Effect of sodium tri polyphosphate (Na_<,P_~tOw, STPP) on the growth of pathogenic Vibrio cultures isolated from shrimp

culture system __ g

198

Fig. 4.34 PCR for e detection of enterotoxigenic V cholerae 9 if 199

Fig. 4.35

th 2 2 2 . . . .

PCR for the detection of enterotoxigenic V.c/zolerae in

shrimp aquaculture farms g g

²⁰⁰

1 Figi4.36 Incidence of cm‘ positive V.ch0Iei'ae in shrimp aquaculture farms

201 .Fig. 4.37 PCR for the detection of I/.ch0lerae using species specific

primers H g

²⁰²

. Fig. 4.38 Duplex PCR for the simultaneous detection of Vcholerae and differentiation of cholera toxin producing V.c/zolerue isolates

206

Fig. 4.39

PCR for testing the O1 and O139 status of cm‘ bearing

V.ch0lerae isolated from farm water

207

xiii

(19)

Fig. 4.40. ’

_Fig. 4.41

End point dilution of V.(?/I-0l6I"‘U£>.-Elupl€X PCR “

PCR for the detectionofV.u!gin01yricu.s' g

210 211 Fig. 4.42 End point dilution of V.alg1Tn.0lyticu.s' PCR g 212

Fig. 4.43

V.ulgin0lyricus~dupIex PCR g _

²¹³

4.44 PCR for detection of V. vzllniﬁcus g

²¹⁴

l Fig. 4.45 Fig. 4.46

PCR for detection of V. par-'ahaem0l;t ticus Pathogenic Vibrio-multiplex PCR

215

_g W217

Pig. 4.47 DNA ﬁnger print pattern of I/.ch0ler'¢':e isolates from farms

using RS-PCR 7

²²⁰

Fig. 4.48a.

Fig. 4.48b.

Dendogram of V.(:h.0!erue isolatesfrom farms using RS-PCR Dendograms of V.(?/10/(?I"‘d0 isolates from farms using RS

PCR

221 222 Fig. 4.49 DNA ﬁnger print. pattern of V.ch0ler'ae isolates from farms

using REP-PCR

223 Fig. 4.50a.

Fig. 4.50b. 4

De-ndograms of V.ch0Iera.e isolates from farms using REP

PCR g___ g M”

Dendograms of V.ch.0lerae isolates from faims using REP

PCR *__ M

224 225 Fig. 4.51 DNA ﬁnger print pattem of V.ch0lerae isolates H551 farms

using ERIC-PC-R g

²²⁶

Fig. 4.52a. Dendograms of V.c/iolerae isolates ﬁ'orn?ifaims using ERIC

PCR

227

Fig. 4.52b.

Dendograms of V.c/zoleraie isolates from farms using ERIC

PCR

228‘

l

Fig. 4.53

Real time PCR data Sheet for V.ch0Ierae using species

specificprimers g g

²³¹

Fig.4.54. Melt curve analysis of the Real time PCR products obtained using V .ch0ler'ae species speciﬁcgp1'im_ers_ ___

232

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CHAPTER ' 1

INTRODUCTION

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1. Introduction

Vibrio are important during hatchery rearing. aquacu)ture phase and post-harvest quality of shrimps. Vibrio spp are of concern to shrimp fanners and hatchery operators because certain species can cause Vibriosis. Vibrio species are of concern to humans because certain species cause serious diseases. Bergey's manual of Systematic

Bacterioiogy(2005) lists 44 species under the genus Vibrio, of which 12 are pathogenic 10 humans viz., V.cholerae, V. vu/nijicus, V.parahaemolyticus. V jUrnissi, V.mersc/mi/wvii, VCincimwtiellsis, V.algillo1yticus, V.mimicllS, V jluvialis, V.lwllisae. V.damsela and V.harreyi. Vibrios considered pathogenic to shrimps include V.hwlleyi, V.algilloIYliC!lS, V.parahaemolyticus. V. vulnificus, V.proteo!yticus, V flSCherl. V.anguil/arum and

V.~plelldidlls. Vibrios related to post harvest shrimp qualities are mainly V.cholerae. V.parahaemolyticlts and V. vulllijiclls.

Indian marine exports witnessed impressive growth from 37,175 tons in 1970 to 5.41,701 tons in 2007-08 (Fig. 1.1). In tenns ofvalue .. the increase was from Rs. 35.54 erores in 1970 to Rs. 7620.92 crores in 2007-08. These exports have generated valuable foreign exchange which increased from US $ 47.38 millions (1970) to US $ 1899.09 miliions(2oo7-08) (MPEDA, 2005; MPEDA 2008).

-

Fig 1.1. Growth of Indian Marine ElI:ports

us • (mWlonl

AU_(~' _ _ l Qu .... "y (.00 ' _ l

(22)

Frozen shrimp constituted a signiﬁcant part of the marine exports. The quantity of frozen shrimp exported from India in 2007-08 was 1.36.223 tons which had realized US

$980.62 million in foreign exchange earning (Rs. 3941 .62crores_).

World wide, penaeid shrimps are considered a crustacean with high potential for intensive aquaculture. P£2H(l£’-21.8‘ mo:-todon (tiger shrimp) is the main shrimp product of Asia, with 50% of global shrimp production. Tiger shrimp is the largest shrimp with a fast growth rate in aquaculture conditions. They tolerate wide range of salinities but the hatchery survivals are low. During the year 2007-08. a total of 1.06.165 MT of shrimp was produced from a culture area of 1,22.078.80 ha. Andhra Pradesh was the leading state (Table 1.1), both in terms of area under culture (50,396 ha) and shrimp production (56,557 MT).

Table 1.1. State wise details of shrimp production (2007-08)*

State Area under culture Production

(ha) (MT)

Andhra Pradesh 50.396 56,557

West Bengal 49,236 28,000

Kerala 75 97.86 5902.5 7

Orissa 6286 5410.4

Kamataka 3.577 21 19

Tamil Nadu 2729.7 3437.74

Gujarat 1,659.84 3148.9

Goa 840 643

Maharastra 756.4 946 .37

Total 1,22,07 8.8 1 ,06,1 64.98

* Source : MPEDA Annual Report 2007-08

With the progress in aquaculture, intensive systems used for shrimp aquaculture

create an artiﬁcial environment that increases bacterial growth. To maintain the

productivity of such an intensive aquaculture, high inputs of ﬁsh protein have to be employed for feeding. together with high levels of water exchange and the massive use of antibiotics/ probiotics / chemicals. It seems that the combination of these conditions favours the proliferation of vibrios and enhances their virulence and disease prevalence.

2

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Bacteria take advantage of ecological changes introduced in the aquaculture practice and may cause periodic disease. Most of the bacterial species are part of the autochthonous ﬂora ofecosystems and therefore a constant source of possible infection for crustaceans.

The risk of a microbial infection is high, mainly at larval stages. The effect and severity are related to Vibrio species and dose ﬁr/ater, feed, shrimp quality and aquaculture

management.

Liu et al (1994) observed that in giant tiger prawn (_P.m0n0d0n) hatchery., at prior stages, the major bacterial flora were Gram positive strains, but after Zoea lll stage, the Gram negative bacteria become the main bacterial flora of which the Vibrio were the dominant species. The major species causing vibriosis in shrimp are V.algr'n.0lyticus, V.angu1'Hurzun, V.harveyi and I/.pcn'aha.emolyricus (‘Lightner I988; Jiravanichpaisal er a1., 1994; Lightner 1996). Yasuda and Kitao (1980) observed low growth of shrimp larvae at protozoal stage when Vibrio species were present in high level (107 cfu/gl) in water and shrimp gut. Nayyaraharned and Karunasagar (1994) studied the microbiology of cultured shrimps in India by analyzing the microbial load of water, sediment. and cultured shrimp (P. morrodon) and their results suggested that potential pathogens like V.ch.0lerae, V.parahaemolyriczis and V.)-‘ufnificrrs could be normal inhabitants of the gut of cultured.

shrimp. Selvin and Lipton (V2003) reported that V.algin0lyticus was associated with white spot disease of P.m0n0c10n. Ponnuraj et al (1995) studied the mortality of shrimp (P.

monodon.) in culture ponds in Vedaranyam (Tamil Nadu) and the microbiological results indicated that the causative pathogen was V. parahaem0lyn'cu.s'. J ayaprakash et al (2006a) studied Vibrios associated with Macrobrachi-zmrr rosenbergii (De Man) larvae from three hatcheries on the southwest coast of lndia and found that V.ch0lerae was the predominant species in the apparently healthy larval samples, whereas V.algz'n0Iyticzzs and I/.r>r1lrr.g'fic-us dominated. during disease and morbidity. Ni et al (1995) detected ﬁve species of Vibrio viz., V.u/ginolyricus, V.par'0haaerr-rolyricur. I/.r>~uIrrr')‘icu.s', V._ﬂm-=iali.s- and V.nu'micu.s- in

pond water and the prawn body with V.algin0lyricu.s' and l/.par'0h.aem0lyricus as the dominant species for all ponds. Wei and Hsu (2001) analysed water samples from P.m0n0d0n. pond in Taiwan and found that the dominant species (47.5%) belonged to the genus Vibrio. Li et al (2000) compared Vibrios isolated ﬁ'om shrimps in 5 different countries (China, Ecuador, Belgium, Mexico, Indonesia) and their results showed. that the

(24)

Vibrios in shrimps of different species from different countries are similar in distribution of the dominant species. V.a1gin0/)--'ricus and l/.lr¢m-'eyr‘ was detected in all the samples species. V.algz'ri0Iyncu.s' was found. in both healthy and diseased larvae. Hisbi et al (2000) noted that the dominant bacterial strains associated with shrimp P.m0n0d0n larvae in

Indonesia were identified as V.a!giri0lyric.?us, V.dum.s"ela, and V.lzar'1»'eyi and Vibrio species were found at different larval stages and in both diseased and healthy larvae- The study supported the idea that Vibrio species are part of the resident microﬂora in P.m0n0d0n larvae. Main pathogenic bacteria in shrimp larvae are mostly V.fzar1-teyi while in adults it is V.parahaemolymrzis (_l..i er 01., 2000). Sung et al (2001) studied the relationships between disease outbreak in cultured tiger shrimp (P.m0n0d0n) and the composition of Vibrio communities in pond water and shrimp hepatopancreas during cultivation. lt was

observed that for the initial 60 days after transfer, the composition of the Vibrio

community in the pond water remained fairly diverse but subsequently decreases in species diversity were observed in ponds.

V. cholerae was the predominant species in the apparently healthy larval species of M. rosenhergii (De Man) whereas V. algrr-z.0lyti<_:zzs' and V. 1-*u1nr'ﬁcu.s' dominated during disease and morbidity (J ayaprakash er 01., 2006a). Gomez-Gil et al.(l998) found a wealth

of vibrios, i.e., 105 cfu/g and l04 cfu/ml, respectively, in the hepatopancreas and

hemolymph of healthy Liropenaeus rwtmu-rrei. Wang and Chen (2005) concluded that the shrimp transferred from 25 ppt salinity water to low salinity levels (5 and l5 ppt) had reduced immune ability and decreased resistance against V. u1_gm0lyr1'cus infection. The

Vibrio spp. isolated ﬁom the digestive tract of a population of healthy juvenile L.

vannamei consisted of both sucrose and non-sucrose fermenters whereas the haemolymph contained only non-sucrose fermenters (Gomez-Gil er a!._, 1998).

Consumption of seafood can occasionally result in food-bome illnesses due to the proliferation of indigenous pathogens like Vibrio (Chen, I995). Of the l2 pathogenic Vibrio species. 8 species are known to be directly food associated (Oliver and Kaper, 2001). Dalsgraad et al (l995) isolated I43 I/'.c?/rolerue non O1 strains from shrimp farms

in Thailand. V.ch0!erue non OI strains are far more frequently isolated from the

environmental sources than Ol strains and appear to constitute part of the microﬂora of prawns (Nair er crl., 1991). leyasekaran and Ayyappan (2002) reported the presence of

4

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V.clr0lerae in farm reared tropical fresh water prawn (M. r0senber'gii). Aravindan and Sheeja (2000) isolated V.t1rh0lerue in P.m.0n0d0n during processing for export in Visakhapatnam region. ‘TDalsgaard er ul., 1990) reported the presence of Non O1 V.ch0lerae in cooked frozen shrimp products originating from shrimp, produced by aquaculture. DePaola et al (1994) isolated V. iwlni)‘icu.s- from seawater, crustacean and

estuarine ﬁsh ﬁom US waters in the Gulf of Mexico. The highest concentration of

V.vulnificz1.s' (in one studyi),was found in the intestinal contents of bottom-feeding estuarine fish (sea catfish, sheepshe_ad,,,Atlantic croaker) that consume mollusks and crustacean

(DePaola er‘ 01., l994)f is rarely recovered from offshore fish. The presence of

V.vulngT/irrus in shellfish may result from the constant filtering by these organisms of seawater containing Vibrios rather than the active multiplication of V.1'u1ng'ficu.s' in shellfish tissues (Kelly and Dinuzzo, 1985). V.pa1‘ahaemolyricus has caused numerous

cases of gastroenteritis, including many outbreaks. Cases are associated with the

consumption of raw or undercooked shellﬁsh such as oysters, shrimp, crabs and lobster.

V. para/memolyticus has been isolated from various parts of the water column, sediment, zooplankton, shellﬁsh and ﬁsh. V.parahaemolyricus has been isolated from a variety of marine animals including clam, oyster, lobster, scallop, sardine, squid, eel, crab and

shrimp (Joseph er‘ a1., 1982). Most outbreaks of gastroenteritis caused by

V.parahaemolyricus have been linked to the consumption of crabs, shrimp, lobsters and

oysters. In Japan, V.parahaemolyrictrs is a major cause of food poisoning and is

associated with the ingestion of raw fish such as sashimi and sushi (Chakraborty er 01., 1997). Pathogenic strains of V.i>u1m'ficus and V.par'ahaenr0lyri<rus which are natural inhabitants of estuarine environments world wide are often transmitted to humans through consumption of raw shellfish that flourish in the same estuaries (Andrews, 2004).

European Union (EU) was the largest market for Indian marine exports during the year 2007-08 (Fig. 1.2a) with a percentage share of 35% in US $ realization followed by Japan (l6.l%), USA (13.3%), China ( 13.3%), South East Asia (7.5%), Middle East (5%) and other countries (10%). Quantity wise, EU was the main destination for Indian marine exports (27%) in 2007-08 (Fig. |.2b) followed by China (26%), Japan (12%), South East Asia (12%), USA (7%), Middle East (5%) and other countries (1 1%) (MPEDA, 2008).

5

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Fig. 1.2a. Indian Marine Exports -2007-08 (US $ realization) Source : MPEDA, 2008

Fig. l.2b. Indian Marine Exports — 2007-08 (Quantity wise) Source : MPEDA, 2008

Consumers’ greatest concem is the quality of food they eat. Strict quality

guidelines have been laid by the importing nations, for the food products that enter their

6

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markets. The microbiological quality requirement for export of frozen shrimp products is that V.c:li()lerae, V.puiahaernolyticzrs and V. 1-*zilm'ﬁcu.s' should be absent in 25g of the processed shrimp (‘Export Inspection Council of India, 1995). The mere presence of these pathogenic Vibrios is sufficient for the rejection of the exported product.

Rapid Alert System for Food and Feed (RASFF) of the European Commission has issued alert notiﬁcations for the presence of I/.c?lu)le/we and V.cht0{e1-'ue Non Ol. and Non 0139, V.pur"uhaemolyricus. V.i=z1ln{ficu.s-, V.alginolyricus and V.fim-"iali.s" in shrimps imported by the EU countries (Table 1.2). During the period 1999 to 2008. a total of 210

alert notifications were issued vis-a.-vis shrimp and fish. The presence _) of

V.parah.aemolyric-us, V.<.rh0lerae, V."vuln.iﬁcu.s- was the sole reason for rejection in 1 13; 55!

and 3 instances, respectively. However, in many eases, the alert notiﬁcations were issued]

due to the presence two or more Vibr-‘i0 .s-patties in the imported product.

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RASSF notiﬁcations were issued with respect to Pmonodon and other shrimp exported to EU from lndia. Recent rejections vis-a-vis Vibrios in black tiger shrimps were mainly due to the presence of V.<r/z<_>leme. and V. partrhaenio/yriczis (Table l.3). The export rejections cause serious economic loss to the shrimp industry and might harm the brand image of the shrimp products from the countiy.

(http://ec.europaeu/food/food/rapidalert/rasff_poital_database_en.htm).

Table 1.3. RASFF notiﬁcations vis-£1-vis the detection of Vibrios from processed seafood from India*

Year Notiﬁcation Notiﬁcation Notifying Cause of rejection

g Date Number Country

2005 03/ll/2005 2005.778 Norway Vi/)i'r'() c/10/erae NON Ozl/NON 0:136

03/l 1/2005

29/01/2007

13/12/2007 30/03/2005

29/05/2008 20/05/2008

l 2/06/ 2008 03/03/2008

27/'06/2008 12/06/2008

30/0'7,/2008 22./0'7./2008

2005.772

200/AFZ 2005.ATL

2008.058?»

2008.AKB

2008./XXE

2008.BDH

Norway

Denmark

France

Denmark

Norway

(presence) in black tiger shrimps (Penueus monodon)

Vi/Jrio c/20/erae NON O: 1/N ON O: l 39 (presence) in headless shell on black tiger shrimps ('1’enaeu.s' mormdon)

V i/trio par‘a/zaem0/i'r1‘('t1.s' (presence /25 g) in head on black tiger shrimps

Vi/Jriopara/1aenm/i=ti0iis (presence of pathogenic strain) in frozen black tiger shrimps (1’enaeus' monodon)

V1'br'i0 vu/ni'/i(ru.s' and high number of aerobic plate counts (Pseudomonas

dominated) in chilled shrimps (fl//erapenae-us

SP1?)

Vibri'0 (f/20/erae, V ibrio (rho/erae NON Ozl/NON O:l39, Vihrio_//m-'ia/is, Vi/Jrio para/zaen~z(2/Wit":/.s' and Vi/trio vu/nq'ﬁ('-u.s* in

frozen raw black tiger shrimps (1 ’enaeus monodon)

Vibrio ct‘/10/erae NON O: l/NON O:l39 (presence in I/I0 samples) in frozen black tiger shrimps

Vi/irio cholerae NON O: I/NON O: 1 39 and

V 1'/trio ])6l!'G/M'J6!1'20/_1-‘T/(‘US in frozen black

tiger shrimps from India

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30/07/2008 24/07./2008

20/08/2008 06/08/2008

2008.BDQ_ Norway

2008.BF P Norway

Vibrio cholerae NON Ozl./NON O: I 39 and

Vibrio [J07‘(J/I6l€l‘7'?()l\-‘I'l("US in frozen black

tiger shrimps

l/l'[)I'l'(') r-/tolerate NON O: 1./N ON O:l 39 and prohibited substances nitrofuran

(metabolite) furazolidone (AOZ) (7.5 ugrkg - ppb) and nitrofuran (metabolite)

nitrofurazone (SEM) (0.65 pg/kg - ppb) in frozen black tiger shrimps

*S0urce: (http1//ec.europa.eulfood/food/rapidalerr/rasff_poital_database__en.htm ).

There is a need for an independent study on the incidence of different pathogenic vibrios in shrimp aquaculture and investigate their biochemical characteristics to have a better understanding about the growth and survival of these organisms in the shrimp aquaculture niche. PCR based methods (conventional PCR, duplex PCR, multiplex-PCR and Real Time PCR) for the detection of the pathogenic Vibrios is important for rapid post-harvest quality assessment. Studies on the genetic heterogeneity among the specific pathogenic vibrio species isolated from shrimp aquaculture system provide; valuable information on the extent of genetic diversity of the pathogenic v'il>1“ios":; the shrimp aquaculture system.

The present study was undertaken with tb.is._goal.-i_T he following aspects were imiesti-gated-in detail.

t / Study the incidence of pathogenic Vibrios spp. in Penueus monodon shrimp

' hatche1“ie:s"".and aquaculture farms, .

~/ Biochemical investigations of the pathogenic Vihr'i0 spp isolated from Pmonodonr hatchery and. aquaculture environments.

~/ Assess the effect of salt (NaC1_) on the growth and enzymatic activities of

pathogenic Vibrio spp.

~/ Study the effect of preservativesl chemicals on the growth of pathogenic Vibri'0 spp.

~/ Employ polymerase chain reaction (PCR) methods for the detection of pathogenic V ibrio spp.

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/ Develop a duplex-PCR for the simultaneous detection of V. cholerae and

differentiation of cholera toxin producing V. cf/zolerue isolates.

\/ Develop a pathogenic Vibrio-Multiplex PCR for the detection of pathogenic Vibrios viz., V.clz01erae, V. choir:-rue (cztr), V. a1gim_>1yricu.s', V. 1-'ulrii'ﬁ<.rus and V. pai‘ahaemolyricm.

/ Study the genetic diversity of V. cholerae using three PCR typing methods based on enterobacterial repetitive intergenic consensus (ERIC) sequences, ribosomal

gene spacer (RS) sequence and repetitive extragenic palindromic (REP)

sequences.

About this thesis:

In this thesis, the investigation has been dealt in the following manner.

A detailed study was made on the total bacterial counts, E.c0li' and total vibrio loads in water and post-larvae samples from P.m.0n.0d0n shrimp hatcheries and pond water, pond sediment and shrimp samples from aquaculture farms. Qualitative analysis of the Vibrios was performed to determine the incidence of pathogenic Vibrio spp in the

aquaculture system. Biochemical properties of the pathogenic Vibrio spp. were

investigated in detail and special stress was given to assess the inﬂuence of salt on the growth and enzymatic activities of the pathogenic Vz'bri0 spp.. as salt plays an important role in the distribution of Vibrios in the environment. The effect of certain chemicals on the growth of pathogenic Vibrio spp. was studied so as to devise strategies for their

control.

In the next part, PCR methods were employed for the rapid detection of

pathogenic Vibrio spp. A duplex-PCR was developed for the simultaneous detection of V.

cholerae and differentiation of cholera toxin producing V. cholerae isolates which can help in monitoring the incidence of choleratoxigenic strains of V.cholerae in food. and environmental samples. A pathogenic Vibrio-Multiplex PCR was developed for the detection of pathogenic Vibrios viz.. V.ch01erae, V. cholerue (trrx), V. ulgm0iyricz1.s". V.

vuln.zﬁcu.r and Vparuhacanto/)--'ricu.s" which can help in identiﬁ/in g these human pathogenic Vibrios in a single PCR reaction tube. The genetic diversity of V. clz(')lercie was studied using three PCR typing methods based ERIC-PCR, RS-PCR and REP-PCR as this

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information provides an insight into the extent of genetic heterogeneity in V.r?l1(.>lerae in the black tiger shrimp aquaculture system .

The thesis is presented in 4 chapters. ln chapter-l. introduction is given. ln

chapter-2, a review of literature is presented. A detailed review on the role of vibrios in human disease, shrimp disease and post-harvest quality is given initially followed by a review on the identiﬁcation of vibrios with special emphasis on PCR, multiplex and Real Time PCR methods. DNA ﬁngerprinting of pathogenic vibrios with special reference to

V. cholerue is also reviewed.

Chapter-3 is the Material and Methods section. Details pertaining to the samples analyzed, bacteriological media, type cultures, PCR components, primers, equipment used and all the methods employed are presented.

In chapter-4, results and discussion are presented. Results are presented in tables, by graphical representation of data and by use of relevant photographs. The results obtained in this study are discussed with those of previous relevant studies.

A summary of the work presented in the thesis is given in chapter 5. A detailed bibliography of all the citations made in the thesis is given at the end. An annexure giving the composition ofbacteriological media and test reagents is given. A list ofpublications by the author is also appended.

Biochemical and molecular characterization of pathogenic vibrios from hatcheries and aquaculture farms