Bacterial Strains and Culture Conditions

8/15/2019 Bacterial Strains and Culture Conditions

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Signifcance o the proposed study

Bacteria are the leading fish pathogens which cause high mortalities (Brock and

Lightner, 1990). Fish farming is a high-risk, capital-intensie industr! that is site-

specific and re"uires technical e#pertise. $t is clearl! more difficult to %e

financiall! successful in fish farming than in conentional farming of liestock or

horticulture %ecause of %acterial and fungal pathogens. $n $ndia south east to south

west coered %! costal areas and people are more dependent in fish farming. &he

deli%erate s!nthesis of nanomaterials using the potential %acterial strain is at %est a

recent phenomenon to get control oer drug resistant %acterial pathogens

('araanan and anda et.al 010).

*onstruction of a c+ li%rar! for the parasite would therefore represent a maor

research adance, and is the central goal of this stud!. &he li%rar! will esta%lish a

genetic information %ase for M. anguillicaudatus, containing protein-encoding

se"uences from the genome, which can then %e used %! researchers to anal!se

functions of specific genes. &he process of constructing the c+ li%rar!

encompasses seeral topics of research. &he li%rar! must %e screened for positie M.

anguillicaudatus clones, from which some are selected for in vivo e#cision of the

pB-*/ phagemid ector (containing the insert) from the 2 3#press ector.

2lasmid + containing the insert must %e isolated from the ector, and then

anal!sed to determine the se"uence of the cloned c+. full-length c+

se"uence can then %e constructed using 4apid mplification of c+ 3nds (4*3).

Finall!, %ioinformatics programs are used to anal!se the + se"uences in order to

predict corresponding protein se"uences, which can %e compared with other genes

whose functions are known.

on-specific immune s!stem or innate immune s!stem is the first line of defense

mechanism against pathogenic diseases %! arious infectious micro%ial organisms.

ccording to 'oderhall and *erenius （'oderhall， 199）。crustaceans are lacking in

adaptie immune s!stem, so the! completel! depend on their innate immune s!stem

that includes %oth cellular and humoral immune s!stems. *ellular defense s!stem

depends on cell immune s!stem, including encapsulation, nodule formation, and


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phagoc!tosis whereas humoral immune s!stem possesses prophenolo#idase actiating

mechanism, clotting mechanism and production of antimicro%ial peptides (/2s)

（ 5esu 016 ） . /2s or host defense peptides are an eolutionaril! consered

component of the innate immune mechanism and are present in all the liing things.

&he! are important immune genes with a capa%ilit! to normali7e and8or destro!

inading micro%ial pathogens :. &hese /2s are commonl! small cationic proteins

that hae antimicro%ial response and hae the a%ilit! to enhance immunit! %!

functioning as immunomodulators. /2s are er! suita%le candidate for deelopment

of noel anti%iotics due to their antimicro%ial nature against ;ram-negatie, ;ram-

positie, fungal, some iral and proto7oan pathogens 6: and can also sere as

templates in the deelopment of therapeutic agents


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enironmental isolates. &he presumed eromonas isolates were con>rmed %! o#idase

and catalase test and %! determining the sensitiit! to the i%riostatic reagent 0819

(10 g ml1 C 'igma, 't. Louis, /o.). &he isolates were identi>ed to the species leel

%! traditional %iochemical methods, including tests for esculin h!drol!sis, l!sine

decar%o#!lase, arginine dih!drolase, and ornithine decar%o#!laseC tests for acid production from ara%inose, glucose, sucrose, and mannitolC and tests for suscepti%ilit!

to ampicillin and cephalothinell isolates were stored in 60D gl!cerol in %rain heart

infusion (B?$) %roth at E0* until further use, su%se"uentl! recultured on B?$ agar

plates (Becton +ickinson /icro%iolog! '!stems), and then incu%ated oernight at

6E*.

For %acterial challenge, the fish were inected intraperitoneal! with . h!drophila

( G 10A *FH8ml) suspended in 1= phosphate %uffer saline (100 ml8fish).. 'amples

were collected %efore (0 h), and after inection (6, A, 1, < and


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g for 15 minutes at 4 to ueous phase R8 remains e.lusi+el" in

the a>ueous phase Transfer upper a>ueous phase .arefull" without

,isturing the interphase into fresh tue )easure the +olume of thea>ueous phase (The +olume of the a>ueous phase is aout :0/ of

the +olume of TRIZOL Reagent use, for homogenization* !re.ipitate

the R8 from the a>ueous phase " miing with isoprop"l al.ohol

'se 05 ml of isoprop"l al.ohol per 1 ml of TRIZOL Reagent use, for

the initial homogenization In.uate samples at 15 to 20o; for 10

minutes an, .entrifuge at not more than 14#000 g for 10 minutes

at 4 to ?o; The R8 pre.ipitate# often in+isile efore

.entrifugation# forms a gel-li$e pellet on the si,e an, ottom of the

tue Remo+e the supernatant .ompletel" @ash the R8 pellet

on.e with A5/ ethanol# a,,ing at least 1 ml of A5/ ethanol per 1 ml

of TRIZOL Reagent use, for the initial homogenization )i the

samples " +orteing an, .entrifuge at no more than A#500 g for 5

minutes at 4 to < o; Repeat ao+e washing pro.e,ure on.e

Remo+e all lefto+er ethanol ir-,r" or +a.uum ,r" R8 pellet for 5-

10 minutes 7o not ,r" the R8 pellet " .entrifuge un,er +a.uum

It is important not to let the R8 pellet ,r" .ompletel" as this will

greatl" ,e.rease its soluilit" !artiall" ,issol+e, R8 samples ha+e

an 4:0B4


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pool of !our control c+ for this). 9ul of mi# added to each sample

and negatie controls。 ll samples heated to A* for minutes and

returned to ice。 1ul 4& en7!me added to all samples (@& added

to 4&-3) 。ll samples heated at 6E* for A0 minutes. 'amples fro7en

or 1J60 dilution made if running 9A well plates in which case '*, 1J0dilution made. For 6I< well plate follow other protocol.

Bioinformatics anal!sis

&he Basic Local lignment &ool (BL'&) program was used to search similar

nucleotide and protein se"uences. &he open reading frame (@4F) and amino acid

se"uence was o%tained %! using +ssit .. *haracteristic domains or motifs were

identified using the 24@'$&3 profile data%ase. $dentit!, similarit! and gap

percentages were calculated using F'& program. &he -terminal transmem%rane

se"uence was determined %! +' transmem%rane prediction program (httpJ88

www.s%c.su.se8wmiklos8+'). 'ignal peptide anal!sis was done using the 'ignal2

worldwide 2 serer (httpJ88www.c%s.dtu.dk ). 2airwise and multiple se"uence

alignment were anal!7ed using the *lustalK ersion program. &he ph!logenetic

relationship of the /r-1 was determined using the neigh%or-oining (5) method

and /3;


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profile was used for the internal control gene, %-actin. ll the primers used in

this stud! were designed %! 2rimer6 and BL'&programme(httpJ88www.nc%i.

nlm.nih.go8tools8primer-%last8). ;ene specific primers were designed using

highl! consered regions from /. anguillicaudatus gene. %-actin primers

(forward primerC 0-accaccgaaattgctccatcctct-60 and reerse primerC 0-acggtcacttgtt caccatcggcatt-60) were designe %ased on the 3'& of a 16E %ase

pairs se"uence (;enBank ccession o. MA191I) from /.

anguillicaudatus. fter the 2*4 program, data were anal!7e with B$ E00

'+' software (pplied Bios!stems). &o maintain consistenc!, the %aselinewas

set automaticall! %! the software. &he comparatie *& method (Gdd*&

method)was used to anal!7e the e#pression leel of $mmune gene :. ll

samples were o%tained and anal!7ed in three replicates, and the results are

e#pressed as relatie fold of one sample as mean G standard deiation. For

comparison of relatie $mmune gene m4 e#pression, statistical anal!sis is

performe using one-wa!@ and meancomparisons were performe %!

&uke!s /ultiple 4ange &est using '2'' 11. at the D significant leel.
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Bacterial Strains and Culture Conditions