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\def\RunningAuthor{Kolli Guna Ranjan et al}
\firstPage{797}
\articleType{Original Article}
\receivedDate{02.08.2019}
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\title{Isolation and Characterization of Vibrio parahaemolyticus in the hepatopancreas of cultured white pacific shrimp - Litopenaeus vannamei}
\author{Kolli~Guna~Ranjan\textsuperscript{*}\textsuperscript{1},  
            Girija~Sankar~G\textsuperscript{2}, 
            Satyanarayana~Raju~DVV\textsuperscript{3}~\\[5pt]{\textsuperscript{1}GITAM Institute of Pharmacy, GITAM University, Visakhapatnam-530017, AndhraPradesh, India }~\\{\textsuperscript{2}College of Pharmaceutical Science, Andhra University, Visakhapatnam-530003, AndhraPradesh, India}~\\{\textsuperscript{3}FOR U~International Private Limited, East point colony, Visakhapatnam-530003, AndhraPradesh, India}}

\begin{abstract}
 Vibrio parahaemolyticus is the most common species among crustaceans, often causing various diseases and significant losses in aquaculture. Acute hepatopancreatic necrosis disease (AHPND) is a newly emerging shrimp disease that has severely damaged the global shrimp industry. This species of bacteria is associated with gastrointestinal illness in humans and has been implicated in foodborne disease. The present study carried out, isolation and characterization of pathogenic bacterial flora isolated from the infected hepatopancreas of vannamei, obtained from various aquafarms in Andhra Pradesh, India, on 11\ensuremath{^{th}} June 2018. The collected samples were plated on TCBS- (Thiosulfate-Citrate-Bile salt-Sucrose) agar medium and Hi -Chrome vibrio, as described in Bergey's manual of systematic bacteriology. Isolated colonies were subjected to the following tests- microscopic examination, growth at different temperatures, growth at different NaCl concentrations, and biochemical tests. Further purity, maintenance, and propagation of purified cultures were done. The microbial culture was identified using 16s rRNA molecular technique. Phylogenetic Evolutionary analyses and distance matrix were conducted in MEGA7.In the present study, different samples were screened, a total of three green colonies (V44, V45, V46) were isolated, identified by biochemical tests and genetic identification as Vibrio parahaemolyticus. A systematic methodology has been developed to isolate and characterize Vibrio sp. from diseased shrimp and identify them by genetic analysis.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Vibrio parahaemolyticus,\newline vannamei,\newline TCBS agar,\newline Sample no V44,\newline Sample no V45,\newline Sample no V46
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Kolli~Guna~Ranjan~\\ Phone:\ +91-8978973377~\\  Email:\ gunaranjankolli@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
 According to the Food and Agriculture Organization (FAO),'' Aquaculture means farming of fish, crustaceans, molluscs, aquatic plants. Aquaculture involves cultivating freshwater and saltwater populations under controlled conditions. Particular techniques include aquaponics and integrated multi-trophic aquaculture, both of which integrate fish farming and aquatic plant farming\unskip~\citep{639420:15001764}. Fishing is a major industry in India, with the country exporting 13,77,244 metric tonnes of seafood worth US\$7.08 billion  (45,106.89  crores) in 2017-18, with shrimp being the top item for export worth US\$4.84 billion. (30,868.17 crore). The state of Andhra Pradesh accounts for 42\% of total shrimp farming in India, with 64,283 hectares \unskip~\citep{639420:15001765}. \ensuremath{^{}}P. vannamei and P. monodon are the primary species of farm-raised shrimp cultivated in both the Eastern and the Western hemispheres. \unskip~\citep{639420:15001766} Cultivation of shrimp is faced with problems of water quality, unsatisfactory practices, and shrimp diseases caused by bacteria and viruses, resulting in great economic losses\unskip~\citep{639420:15001767}. Pathogenic bacteria of Vibrio sp. such as V. harveyi, V. anguillarum, V. parahaemolyticus, and V. vulnificus have been involved in mortality of cultured shrimp in hatcheries \unskip~\citep{639420:15001768,639420:15001769}.\ensuremath{^{}}In shrimp cultures, Vibrio parahaemolyticus is the major pathogen isolated from the infected hepatopancreas of shrimp \unskip~\citep{639420:15001771}. Hence the present investigation is done on isolation and identification of Vibrio parahaemolyticus from various sources of shrimp aquaculture farms.
    
\section{Materials and Methods}
\textbf{Collection of samples }

In the present study, infected shrimp were collected on 11\ensuremath{^{th}} June 2018 from shrimp aquaculture farms (Kallanka, Vendra, and Juvvalapalem), which are located in East and West Godavari Dist. of Andhra Pradesh, India ,Tables~\ref{tw-d6e9668fcc2d}, \ref{tw-6c2a2ada16e4} and~\ref{tw-f59b1914cd3d}.

\textbf{Isolation of colonies from infected shrimp}

The hepatopancreas of infected shrimp was dissected and isolated for pathogenic bacteria. The samples were serially diluted 10\ensuremath{^{-1}} to 10\ensuremath{^{-9}} factor, and 10\ensuremath{^{-5}} to 10\ensuremath{^{-7 }} dilutions are spread over sterilized TCBS Petri plates (Thiosulphate Citrate Bile salt Sucrose). The Petri dish is placed at 37-45\ensuremath{^{o}}C overnight in an incubator. The incubation period was maintained at 37\ensuremath{^{o}}C for 24-48 hours. For vibrio species identification, the colonies isolated were then purified and stored in TCBS agar slants for further studies. 

\textbf{ Identification of selected Vibrio isolates}

Identification and further characterization of vibrio sp.(V44, V45, V46) grown on TCBS agar and Hi -Chrome vibrio agar was done by the procedure described in Bergey's manual of systematic bacteriology with following tests carried out: microscopic examination, growth at different temperatures, growth at different NaCl concentration and biochemical tests\unskip~\citep{639420:15001772,639420:15001773,639420:15001774,639420:15001775,639420:15001776,639420:15001777}.

\textbf{Microscopic examination }

 Purity, morphology, and microscopic examination of Vibrio isolates was confirmed by performing gram staining for which a single colony of each isolate was stained as per standard protocol and viewed under oil immersion for a similar type of cells. 

\textbf{\space }\textbf{Physiological Characteri} \textbf{zation of Isolates}

Once the purity of culture was confirmed, each isolates were further assessed for growth at different temperatures and salt concentration. 

\textbf{Growth of isolates at (10\ensuremath{^{o}}C, 30\ensuremath{^{o}}C, 40\ensuremath{^{o}}C)}

The isolates were examined for growth in nutrient agar plates at 10\ensuremath{^{o}}C for 7 days and 30\ensuremath{^{o}}C,40\ensuremath{^{o}}C for 24-48h. 1 \% of vibrio isolates culture were mixed with 20ml nutrient agar, and growth was observed at different temperatures was noted as positive or negative.

\textbf{Effect of NaCl concentration on isolates growth }

The isolates were inoculated with different NaCl concentrations (0\%,3\%,6\%, 7\% and 8\%) in nutrient agar plates and incubated at 37\ensuremath{^{o}}C for 24-48h. Culture plates were observed for the presence or absence of growth. 

\textbf{Biochemical characterization of isolates }

 \textbf{Oxidase test}

The test is used to identify bacteria that produce cytochrome C oxidase, an enzyme of bacterial electron transport chain. The culture was placed on slide using inoculation needle, and drop of N, N, N\ensuremath{'}, N\ensuremath{'}-tetramethyl-p-phenylenediamine solution was added on to the culture and observed for the oxidized purple colour end product which indicates a positive result.

 \textbf{Catalase test}

The test was performed on isolated cultures to determine the degradation of hydrogen peroxide by producing enzyme catalase. The culture was placed on slide using inoculation needle, and a drop of 3\% hydrogen peroxide solution was added on to the culture and observed for the evolution of bubbles, which indicates a positive result.

\textbf{KOH String Test}

It relies on the differential resistance to 3\% potassium hydroxide between gram-positive and negative cells, where a portion of a colony is mixed with a small volume of 3\% KOH. If the cells lyse, the liberated cellular DNA makes the mixture viscous or "stringy."

\textbf{Arginine hydrolysis}

Arginine hydrolysis broth was inoculated with isolated cultures and incubated at 37\ensuremath{^{o}}C for 48h. After incubation, 3-5 drops of Nessler's reagent were added to each tube and observed for change in colour (yellow to orange), indicating a positive result. 

\textbf{Lysine hydrolysis }

It is used to detect specific enzyme production. Medium is inoculated with bacteria which ferments dextrose, causing a lowering of pH in the medium due to acid production and color of the indicator changes from purple to yellow. Acidic condition stimulates decarboxylase activity.

 \textbf{Ornithine hydrolysis}

 It is done to detect the production of ornithine decarboxylase. Glucose the fermentable carbohydrate, which during the early stages of incubation, is fermented by microbe with acid production, resulting in a colour change to yellow due to change in pH.

 \textbf{Nitrate reduction test}

\textbf{\space }Isolates were added in trypticase nitrate broth and incubated at 37\ensuremath{^{o}}C for 24h. After incubation, 0.5 mL of sulphanilic acid and \ensuremath{\alpha }-naphthylamine were added into the tubes. The appearance of red or pink colour indicates a positive result for nitrate reduction. 

 \textbf{Indole test}

\textbf{\space }Isolates were inoculated in tryptophan broth, incubated at 37\ensuremath{^{o}}C for 24h. After incubation, 0.5 ml of Kovac's reagent was added. Pink colour ring in the top layer indicates a positive result.

\textbf{ONPG Test}

The microbe is selected from a medium with high lactose concentration and is then inoculated into the ONPG Broth. If the microbe contains beta-galactosidase, the enzyme will then split the beta-galactoside bond, releasing o-nitrophenol, which is a yellow-colored compound.

\textbf{Citrate utilization test}

The isolates were inoculated in Simmons citrate agar, incubated at 37\ensuremath{^{o}}C for 24h. After incubation, the appearance of blue color indicates a positive result for citrate utilization.

\textbf{Hydrogen Sulfide} (\textbf{H2S}) \textbf{Production Test}

 In this test, sulfur-containing compounds reduced to sulfides during metabolism is tested. The presence of sulphide, on reaction with iron compounds, produces FeS a black precipitate.

\textbf{Urease test}

Microbe producing an exoenzyme urease in a differential medium of Urease broth that hydrolyzes urea to ammonia and carbon dioxide is tested. Urea broth is designed to test urease positive microbes. 

\textbf{VP ( Voges Proskauer) test}

Isolates were added to MRVP broth and incubated for 37\ensuremath{^{o}}c for 48 h. For VP test Barrit's reagent was added to the tube. The pink colour indicates VP positive\textbf{.}

\textbf{Gelatin liquefaction}

In this test, microbes producing gelatinase\textbf{\space }that liquefy gelatin are detected. Gelatin hydrolysis indicates the presence of gelatinases. 


\begin{table*}[!htbp]
\caption{\boldmath {Sample no -V44 } }
\label{tw-d6e9668fcc2d}
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\tbltoprule \rowcolor{kwdboxcolor}Date & Name & Place & Pond size & DOC & Length\\
\tblmidrule 
06-09-2018 &
  Anil Kumar &
  Kallanka &
  4AC &
  45 Days &
  14cm\\
Weight &
  HP Weight &
  WSSV &
  Harvest days &
  Severity &
  \\
19 gm &
   650 mg &
  negative(physical) &
   still culture &
   + &
  \\
\tblbottomrule 
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\begin{table*}[!htbp]
\caption{\boldmath {Sample no-V45 } }
\label{tw-6c2a2ada16e4}
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\tbltoprule \rowcolor{kwdboxcolor}Date &  Name & Place & Pond size & DOC & Length\\
\tblmidrule 
 11/6/2018 &
  SR Raju &
  Vendra &
  4AC &
  43 Days &
  9cm\\
Weight &
  HP Weight &
  WSSV &
  Harvest days &
  Severity &
  \\
10.40gm &
  500mg &
  ehp negative (RT-PCR) &
  still culture &
   ++ &
  \\
\tblbottomrule 
\end{tabulary}\par 
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\caption{\boldmath {Sample no-V46 } }
\label{tw-f59b1914cd3d}
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\tbltoprule \rowcolor{kwdboxcolor}Date & Name & Place & Pond size & DOC & Length\\
\tblmidrule 
13/6/2018 &
  V Bhaskar Raju  &
  Juvvalapalem &
  7AC &
  46 Days &
  10.5cm\\
Weight &
  HP Weight &
  WSSV &
  Harvest days &
  Severity &
  \\
7.60gm &
  350mg &
  positive (physical) &
  still culture &
   ++ &
  \\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{ Doc- date of collection, HP-hepatopancreas, WSSV-white spotsyndrome virus}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Morphological, physiological and biochemicalcharacterization of isolates} }
\label{tw-0d7bbb0ae064}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.45129999999999995\linewidth-2\tabcolsep}p{\dimexpr.20840000000000003\linewidth-2\tabcolsep}p{\dimexpr.18160000000000004\linewidth-2\tabcolsep}p{\dimexpr.1587\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Characteristics & V44 & V45 & V46\\
\tblmidrule 
Morphological characterization &
  \multicolumn{3}{p{\dimexpr(.54870000000000005\linewidth-2\tabcolsep)}}{}\\
Colour  &
  Green  &
   Green  &
   Green \\
Shape  &
  Rod  &
  Rod  &
  Rod \\
Colony size (\ensuremath{\mu}m) &
   3 &
   3 &
   3\\
Colony Form  &
  Circular  &
  Circular  &
  Circular \\
Motility  &
   Motile  &
  Motile  &
   Motile \\
Grams reaction  &
  Negative  &
  Negative  &
  Negative \\
Physiological characterization &
  \multicolumn{3}{p{\dimexpr(.54870000000000005\linewidth-2\tabcolsep)}}{}\\
Growth at different temperature &
   &
   &
  \\
10\ensuremath{^{o}}C &
  - &
  - &
  -\\
30\ensuremath{^{o}}C &
  + &
  + &
  +\\
40\ensuremath{^{o}}C &
  + &
  + &
  +\\
Growth at different NaCl concentration &
   &
   &
  \\
0\% &
  - &
  - &
  -\\
3\% &
  + &
  + &
  +\\
6\% &
  + &
  + &
  +\\
7\% &
  + &
  + &
  +\\
8\% &
  + &
  + &
  +\\
Biochemical characterization  &
  \multicolumn{3}{p{\dimexpr(.54870000000000005\linewidth-2\tabcolsep)}}{}\\
Growth on TCBS &
  Green  &
  Green  &
  Green \\
Growth on Hichrome vibrio agar &
  Green  &
  Green  &
  Green \\
Cytochrome oxidase &
  + &
  + &
  +\\
Catalase  &
  + &
  + &
  +\\
KOH string &
  + &
  + &
  +\\
Arginine dihydrolase &
  - &
  - &
  -\\
Lysine decarboxylase &
  + &
  + &
  +\\
Ornithine decarboxylase &
  + &
  + &
  +\\
Nitrate reduction &
  + &
  + &
  +\\
Indole  &
  + &
  + &
  +\\
\ensuremath{\beta }- galactosidase (ONPG) &
  - &
  - &
  -\\
Citrate utilization &
  - &
  + &
  +\\
Production of H2S &
  - &
  - &
  -\\
Urease  &
   + &
  + &
  +\\
VP test &
  - &
  - &
  -\\
Gelatin liquification &
  + &
  + &
  +\\
Acid from &
  \multicolumn{3}{p{\dimexpr(.54870000000000005\linewidth-2\tabcolsep)}}{}\\
Glucose  &
  + &
  + &
  +\\
Mannitol  &
  + &
  + &
  +\\
Arabinose  &
  + &
  + &
  +\\
Sucrose  &
  - &
  - &
  -\\
Sorbital  &
  - &
  - &
  -\\
Cellobiose  &
  - &
  - &
  -\\
Inositol  &
  - &
  - &
  -\\
Mannose  &
  + &
  + &
  +\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{\ensuremath{^{V,variable; ONPG, ortho-nitrophenyl-\ensuremath{\beta }-galactoside; VP, Voges-Proskauer test; +,positive; {-}, negative}}}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Estimates of Evolutionary Divergence between Sequences} }
\label{tw-1a3252ff0ced}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.04149999999999998\linewidth-2\tabcolsep}p{\dimexpr.12529999999999998\linewidth-2\tabcolsep}p{\dimexpr.06340000000000001\linewidth-2\tabcolsep}p{\dimexpr.0666\linewidth-2\tabcolsep}p{\dimexpr.06659999999999999\linewidth-2\tabcolsep}p{\dimexpr.063\linewidth-2\tabcolsep}p{\dimexpr.0705\linewidth-2\tabcolsep}p{\dimexpr.071\linewidth-2\tabcolsep}p{\dimexpr.07750000000000001\linewidth-2\tabcolsep}p{\dimexpr.076899999999999995\linewidth-2\tabcolsep}p{\dimexpr.07189999999999998\linewidth-2\tabcolsep}p{\dimexpr.07490000000000002\linewidth-2\tabcolsep}p{\dimexpr.0805\linewidth-2\tabcolsep}p{\dimexpr.0504\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor} & Distance matrix & 1 & 2 & 3 & 4 & 5 & 6 & 7 & 8 & 9 & 10 & 11 & \\
\tblmidrule 
1 &
  V44 &
   &
  58.869 &
  66.398 &
  49.177 &
  58.869 &
  49.537 &
  66.398 &
  49.177 &
  58.869 &
  58.869 &
  49.537 &
  1\\
2 &
  NR\_118569.1 &
  0.000 &
   &
  0.002 &
  0.001 &
  0.000 &
  0.001 &
  0.002 &
  0.001 &
  0.000 &
  0.000 &
  0.001 &
  2\\
3 &
  NR\_113791.1 &
  0.000 &
  0.002 &
   &
  0.003 &
  0.002 &
  0.002 &
  0.000 &
  0.003 &
  0.002 &
  0.002 &
  0.002 &
  3\\
4 &
  NR\_113782.1 &
  0.000 &
  0.001 &
  0.004 &
   &
  0.001 &
  0.002 &
  0.003 &
  0.000 &
  0.001 &
  0.001 &
  0.002 &
  4\\
5 &
  NR\_113604.1 &
  0.000 &
  0.000 &
  0.002 &
  0.001 &
   &
  0.001 &
  0.002 &
  0.001 &
  0.000 &
  0.000 &
  0.001 &
  5\\
6 &
  NR\_118258.1 &
  0.000 &
  0.001 &
  0.001 &
  0.002 &
  0.001 &
   &
  0.002 &
  0.002 &
  0.001 &
  0.001 &
  0.000 &
  6\\
7 &
  NR\_118093.1 &
  0.000 &
  0.002 &
  0.000 &
  0.004 &
  0.002 &
  0.001 &
   &
  0.003 &
  0.002 &
  0.002 &
  0.002 &
  7\\
8 &
  NR\_118091.1 &
  0.000 &
  0.001 &
  0.004 &
  0.000 &
  0.001 &
  0.002 &
  0.004 &
   &
  0.001 &
  0.001 &
  0.002 &
  8\\
9 &
  NR\_117893.1 &
  0.000 &
  0.000 &
  0.002 &
  0.001 &
  0.000 &
  0.001 &
  0.002 &
  0.001 &
   &
  0.000 &
  0.001 &
  9\\
10 &
  NR\_114630.1 &
  0.000 &
  0.000 &
  0.002 &
  0.001 &
  0.000 &
  0.001 &
  0.002 &
  0.001 &
  0.000 &
   &
  0.001 &
  10\\
11 &
  NR\_025491.1 &
  0.000 &
  0.001 &
  0.001 &
  0.002 &
  0.001 &
  0.000 &
  0.001 &
  0.002 &
  0.001 &
  0.001 &
   &
  11\\
 &
   &
  1 &
  2 &
  3 &
  4 &
  5 &
  6 &
  7 &
  8 &
  9 &
  10 &
  11 &
  \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Estimates of Evolutionary Divergence between Sequences} }
\label{tw-c87d3d560df1}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.0415\linewidth-2\tabcolsep}p{\dimexpr.12540000000000001\linewidth-2\tabcolsep}p{\dimexpr.06789999999999998\linewidth-2\tabcolsep}p{\dimexpr.0635\linewidth-2\tabcolsep}p{\dimexpr.07339999999999999\linewidth-2\tabcolsep}p{\dimexpr.07050000000000001\linewidth-2\tabcolsep}p{\dimexpr.07539999999999999\linewidth-2\tabcolsep}p{\dimexpr.06619999999999999\linewidth-2\tabcolsep}p{\dimexpr.0695\linewidth-2\tabcolsep}p{\dimexpr.07339999999999999\linewidth-2\tabcolsep}p{\dimexpr.071499999999999995\linewidth-2\tabcolsep}p{\dimexpr.075\linewidth-2\tabcolsep}p{\dimexpr.0749\linewidth-2\tabcolsep}p{\dimexpr.051899999999999995\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor} & Distance matrix & 1 & 2 & 3 & 4 & 5 & 6 & 7 & 8 & 9 & 10 & 11 & \\
\tblmidrule 
1 &
  V45 &
   &
  58.869 &
  66.398 &
  49.177 &
  58.869 &
  49.537 &
  66.398 &
  49.177 &
  58.869 &
  58.869 &
  49.537 &
  1\\
2 &
  NR\_118569.1 &
  0.000 &
   &
  0.002 &
  0.001 &
  0.000 &
  0.001 &
  0.002 &
  0.001 &
  0.000 &
  0.000 &
  0.001 &
  2\\
3 &
  NR\_113791.1 &
  0.000 &
  0.002 &
   &
  0.003 &
  0.002 &
  0.002 &
  0.000 &
  0.003 &
  0.002 &
  0.002 &
  0.002 &
  3\\
4 &
  NR\_113782.1 &
  0.000 &
  0.001 &
  0.004 &
   &
  0.001 &
  0.002 &
  0.003 &
  0.000 &
  0.001 &
  0.001 &
  0.002 &
  4\\
5 &
  NR\_113604.1 &
  0.000 &
  0.000 &
  0.002 &
  0.001 &
   &
  0.001 &
  0.002 &
  0.001 &
  0.000 &
  0.000 &
  0.001 &
  5\\
6 &
  NR\_118258.1 &
  0.000 &
  0.001 &
  0.001 &
  0.002 &
  0.001 &
   &
  0.002 &
  0.002 &
  0.001 &
  0.001 &
  0.000 &
  6\\
7 &
  NR\_118093.1 &
  0.000 &
  0.002 &
  0.000 &
  0.004 &
  0.002 &
  0.001 &
   &
  0.003 &
  0.002 &
  0.002 &
  0.002 &
  7\\
8 &
  NR\_118091.1 &
  0.000 &
  0.001 &
  0.004 &
  0.000 &
  0.001 &
  0.002 &
  0.004 &
   &
  0.001 &
  0.001 &
  0.002 &
  8\\
9 &
  NR\_117893.1 &
  0.000 &
  0.000 &
  0.002 &
  0.001 &
  0.000 &
  0.001 &
  0.002 &
  0.001 &
   &
  0.000 &
  0.001 &
  9\\
10 &
  NR\_114630.1 &
  0.000 &
  0.000 &
  0.002 &
  0.001 &
  0.000 &
  0.001 &
  0.002 &
  0.001 &
  0.000 &
   &
  0.001 &
  10\\
11 &
  NR\_025491.1 &
  0.000 &
  0.001 &
  0.001 &
  0.002 &
  0.001 &
  0.000 &
  0.001 &
  0.002 &
  0.001 &
  0.001 &
   &
  11\\
 &
   &
  1 &
  2 &
  3 &
  4 &
  5 &
  6 &
  7 &
  8 &
  9 &
  10 &
  11 &
  \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Estimates of Evolutionary Divergence between Sequences} }
\label{tw-39bf360a22fc}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.0409\linewidth-2\tabcolsep}p{\dimexpr.12860000000000001\linewidth-2\tabcolsep}p{\dimexpr.07409999999999999\linewidth-2\tabcolsep}p{\dimexpr.06850000000000001\linewidth-2\tabcolsep}p{\dimexpr.0661\linewidth-2\tabcolsep}p{\dimexpr.07\linewidth-2\tabcolsep}p{\dimexpr.0715\linewidth-2\tabcolsep}p{\dimexpr.071\linewidth-2\tabcolsep}p{\dimexpr.06949999999999999\linewidth-2\tabcolsep}p{\dimexpr.0715\linewidth-2\tabcolsep}p{\dimexpr.07390000000000001\linewidth-2\tabcolsep}p{\dimexpr.071\linewidth-2\tabcolsep}p{\dimexpr.0735\linewidth-2\tabcolsep}p{\dimexpr.0499\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor} & Distance matrix & 1 & 2 & 3 & 4 & 5 & 6 & 7 & 8 & 9 & 10 & 11 & \\
\tblmidrule 
1 &
  V46 &
   &
  58.869 &
  66.398 &
  49.177 &
  58.869 &
  49.537 &
  66.398 &
  49.177 &
  58.869 &
  58.869 &
  49.537 &
  1\\
2 &
  NR\_118569.1 &
  0.000 &
   &
  0.002 &
  0.001 &
  0.000 &
  0.001 &
  0.002 &
  0.001 &
  0.000 &
  0.000 &
  0.001 &
  2\\
3 &
  NR\_113791.1 &
  0.000 &
  0.002 &
   &
  0.003 &
  0.002 &
  0.002 &
  0.000 &
  0.003 &
  0.002 &
  0.002 &
  0.002 &
  3\\
4 &
  NR\_113782.1 &
  0.000 &
  0.001 &
  0.004 &
   &
  0.001 &
  0.002 &
  0.003 &
  0.000 &
  0.001 &
  0.001 &
  0.002 &
  4\\
5 &
  NR\_113604.1 &
  0.000 &
  0.000 &
  0.002 &
  0.001 &
   &
  0.001 &
  0.002 &
  0.001 &
  0.000 &
  0.000 &
  0.001 &
  5\\
6 &
  NR\_118258.1 &
  0.000 &
  0.001 &
  0.001 &
  0.002 &
  0.001 &
   &
  0.002 &
  0.002 &
  0.001 &
  0.001 &
  0.000 &
  6\\
7 &
  NR\_118093.1 &
  0.000 &
  0.002 &
  0.000 &
  0.004 &
  0.002 &
  0.001 &
   &
  0.003 &
  0.002 &
  0.002 &
  0.002 &
  7\\
8 &
  NR\_118091.1 &
  0.000 &
  0.001 &
  0.004 &
  0.000 &
  0.001 &
  0.002 &
  0.004 &
   &
  0.001 &
  0.001 &
  0.002 &
  8\\
9 &
  NR\_117893.1 &
  0.000 &
  0.000 &
  0.002 &
  0.001 &
  0.000 &
  0.001 &
  0.002 &
  0.001 &
   &
  0.000 &
  0.001 &
  9\\
10 &
  NR\_114630.1 &
  0.000 &
  0.000 &
  0.002 &
  0.001 &
  0.000 &
  0.001 &
  0.002 &
  0.001 &
  0.000 &
   &
  0.001 &
  10\\
11 &
  NR\_025491.1 &
  0.000 &
  0.001 &
  0.001 &
  0.002 &
  0.001 &
  0.000 &
  0.001 &
  0.002 &
  0.001 &
  0.001 &
   &
  11\\
 &
   &
  1 &
  2 &
  3 &
  4 &
  5 &
  6 &
  7 &
  8 &
  9 &
  10 &
  11 &
  \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\bgroup
\fixFloatSize{images/8e910b0c-09e6-4588-a507-13cdbde58a7e-upicture1.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/8e910b0c-09e6-4588-a507-13cdbde58a7e-upicture1.png}{\includegraphics{images/8e910b0c-09e6-4588-a507-13cdbde58a7e-upicture1.png}}{}
\makeatother 
\caption{\boldmath {Phylogenetic Tree\ensuremath{^{}}}}
\label{f-7686f35bb768}
\end{figure*}
\egroup

\bgroup
\fixFloatSize{images/38b53df1-6b0b-46ed-b969-06578cb271c8-upicture2.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/38b53df1-6b0b-46ed-b969-06578cb271c8-upicture2.png}{\includegraphics{images/38b53df1-6b0b-46ed-b969-06578cb271c8-upicture2.png}}{}
\makeatother 
\caption{\boldmath {PhylogeneticTree\ensuremath{^{}}}}
\label{f-6084cd80c9f4}
\end{figure*}
\egroup

\bgroup
\fixFloatSize{images/48d90a23-145b-4419-a3bd-2dc9434b2762-upicture3.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/48d90a23-145b-4419-a3bd-2dc9434b2762-upicture3.png}{\includegraphics{images/48d90a23-145b-4419-a3bd-2dc9434b2762-upicture3.png}}{}
\makeatother 
\caption{\boldmath {Phylogenetic Tree}}
\label{f-be29f4fe1430}
\end{figure*}
\egroup
\textbf{Carbohydrates fermentation by isolates}

Sugars were used to determine the fermentation profile and further characterization of \textit{Vibrio isolates}. Carbohydrates basal media was prepared, and pH adjusted to 7- 7.4. Phenol red was added as an indicator. Sugars were mixed to the basal media (500mg of sugar in 50mL of basal media) and autoclaved. Each tube was inoculated with 50\ensuremath{\mu}L of inoculum in 250\ensuremath{\mu}L of sugar basal media. Control using sterile water was prepared to compare colour change.

\textbf{Maintenance and propagation of cultures}

Isolates of \textit{Vibrio sp.}were maintained at -70\ensuremath{^{o}}C in glycerol stocks in triplicate for use in an experiment at different stages.

\textbf{Purity of cultures}

\textit{Vibrio} species isolates were regularly tested for microscopic examination and catalase tests for confirmation and presence of contamination, if any. 

 \textbf{Identification of microbial culture using 16s rRNA based molecular technique method}

Genomic DNA was isolated, and quantity was measured using a nanodrop spectrophotometer, and the quality was determined using 2\% agarose gel\textbf{. } A single band of high-molecular-weight DNA has been observed. 16S rRNA gene was amplified by 16SrRNAF and 16SrRNAR primers. A single discrete PCR amplicon band of 1500 bp was observed when resolved on an agarose gel. The PCR amplicon was purified to remove contaminants. Forward and reverse DNA sequencing reaction of PCR amplicon was carried out with forwarding primer and reverse primers using BDT v3.1. Cycle sequencing kit on ABI 3730xl genetic analyzer. A consensus sequence of the 16S rRNA gene was generated from forward and reverse sequences. 16S rRNA gene sequence was used to carry out BLAST with the database of the NCBI Genbank database. Based on maximum identity score first ten sequences were selected and aligned using multiple alignment software program Clustal W. Distance matrix was generated, and the phylogenetic tree was constructed by using MEGA7. 

\textbf{Phylogenetic tree}

The evolutionary history was inferred using the Neighbor-Joining method \unskip~\citep{639420:15001778} The optimal tree with the sum of branch length = 0.09337190 was shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches \unskip~\citep{639420:15001779}.  The evolutionary distances were computed using the Maximum Composite Likelihood method \unskip~\citep{639420:15001780} and are in the units of the number of base substitutions per site. The analysis involved 6 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 5851 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 \unskip~\citep{639420:15001781}. 

\textbf{Distance matrix}

 The number of base substitutions per site from between sequences is shown. Analyses were conducted using the Maximum Composite Likelihood model \unskip~\citep{639420:15001778}. The analysis involved 11 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 5851 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 \unskip~\citep{639420:15001779}.\textbf{\space }
    
\section{Results and Discussion}
\textbf{ Biochemical and genetic identification }

Table~\ref{tw-0d7bbb0ae064} describes the Morphological, physiological, and biochemical characterization of isolates. The identification of microbial culture using 16s rRNA based molecular technique was carried out, and the results are mentioned below(Figures~\ref{f-7686f35bb768}, \ref{f-6084cd80c9f4} and~\ref{f-be29f4fe1430} and Tables~\ref{tw-1a3252ff0ced}, \ref{tw-c87d3d560df1} and~\ref{tw-39bf360a22fc}).  Based on sequence homology and phylogenetic analysis, the microbial culture labeled V44, V45 \&V46 were found to be Vibrio parahaemolyticus\textbf{.} \textbf{\space }

Shrimp was harvested from different ponds of varying sizes. Its length, weight, and hepatopancreas weight were checked. Harvest was done from still culture, and severity of disease was assessed.the hepatopancreas were dissected, suitable dilutions made, and plating was done on TCBS agar for isolation of vibrio sp. Morphology, physiological, and biochemical characterization of isolates was done to identify specific tests, which is typical for Vibrio sp.  

Growth was observed at a mesophilic temperature of 30\ensuremath{^{0}}C, 40\ensuremath{^{0}}C, but not at 10\ensuremath{^{0}}Cand microbes grew at a salt concentration of 3-8 \% but not 0\% for all the isolates. Positive results on TCBS, HiChrome vibrio agar, cytochrome oxidase, catalase, KoH, lysine, ornithine, nitrate, indole urease, gelatin liquefaction, and few sugars was observed. Negative results were observed for arginine, ONPG, Citrate, VP, and few sugars tests. Identification of isolates was made using 16s rRNA based Molecular technique. The results of the technique identified bacteria from harvested shrimp as Vibrio parahaemolyticus. The research attempt is to identify bacteria, causing the disease for the shrimp and isolate and identify them for specific treatment.  
    
\section{Conclusions}
In summary, the shrimp harvested instill cultures were studied for pathogenic bacteria of Vibrio sp.The vibrios were extensively characterized and confirmed as vibrio paraheamolyticus by genetic evaluation. This species is a source of water contamination and can cause gastroenteritis if taken as part of a food chain in humans. However, further study is required to evaluate them in animal experiments and in-vitro cell cultures.
    

\bibliographystyle{pharmascope_apa-custom}

\bibliography{\jobname}
\end{document}
