Necrotizing Hepatopancreatitis in shrimp, Slides of Fish Farming

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Disease Diagnosis Of Hepatobacter penaei

(Necrotizing Hepatopancreatitis)

By,

Subashini.V,

FBT-MA-09-

Course Teacher:

Dr.K.V.Rajendran,

HOD –AEHM

Contents

Contents

Introduction

Outbreak

Taxonomic position

Description

Host range and

Transmission

Environmental factors and

prevalence



Diagnosis

LEVEL 1

LEVEL 2

LEVEL 3

Prevention and control

measures

References

Outbreak of NHPOutbreak of NHP

Necrotizing Hepatopancreatitis (NHP), is also known as

Granulomatous Hepatopancreatitis, Texas necrotizing

Hepatopancreatitis (TNHP), Texas Pond Mortality Syndrome

(TPMS) and Peru Necrotizing Hepatopancreatitis (PNHP).

It is a lethal epizootic disease in farmed shrimp.

Causative agent: Hepatobacter penaei

NHP was first described in Texas in 1985 and was listed in the

list of crustacean diseases of world organization for animal

health (OIE) in 2010.

Causes up to 95% mortality in affected ponds (Johnson 1990)

within 30 days of outbreak.

Taxonomic positionTaxonomic position

Domain: Bacteria

Phylum: Proteobacteria

Class : Alphaproteobacteria

Order : Rickettsiales

Species : Candidatus Hepatobacter penaei

 Phylogenetic analysis, inferred from 16S rRNA and gyrase B gene

sequences, places this bacterium within the class Alphaproteobacteria.

(Nunan LM, Pantoja CR, Gomez-Jimenez S, Lightner DV, 2013)

Host Range And Vectors

Host Range And Vectors

species reported: Species affected by NHP are Litopenaeus

vannamei, L. setiferus, L. stylirostris, Farfantepenaeus aztecus

and F. californiensis (Lightner 1996)

Affected life stages: late postlarvae, juveniles and adults.

Species with incomplete evidence of susceptibility: Penaeus

duorarum, Penaeus stylirostris,Penaeus merguiensis, Penaeus

marginatus, Penaeus aztecus, Penaeus monodon and American

lobster ( Homarus americanus).

In recent years, rare non-specific amplifications have been

observed in the end-point PCR when screening for H.

penaei in Artemia cyst samples submitted to the UAZ-APL

(Aranguren et al 2018)

Geographic distributionGeographic distribution

Western hemisphere – United states, Mexico, Panama, Belize,

Guatemala, Colombia, Ecuador, Peru, Brazil.

Diagnostic Methods

Diagnostic Methods

LEVEL 1

GROSS

SIGNS

LEVEL 2

HISTOPATHOLOG

Y

WET MOUNT

BIO ASSAY

LEVEL 3LEVEL 3

ELECTRON

MICROSCOPY

ELECTRON

MICROSCOPY

PCRPCR

INSITU

HYBRIDIZATION

INSITU

HYBRIDIZATION

PRESUMPTIVE DIAGNOSIS

LEVEL 1 AND 2

PRESUMPTIVE DIAGNOSIS

LEVEL 1 AND 2

CONFIRMATIVE DIAGNOSIS

LEVEL 3

CONFIRMATIVE DIAGNOSIS

LEVEL 3

Clinical Signs (Level 1)Clinical Signs (Level 1)

Disease signs at the farm, tank

or pond level are:

lethargy

emaciation

heavy protozoan or bacterial

fouling

reduced growth rate.

Gross pathological signs are:

 soft shell

 flaccid body

 black gills

 empty intestinal tract

 degenerated or atrophied

digestive gland (hepatopancreas),

which appears pale to white

 black (melanised) streaks in the

hepatopancreas.

Histopathology (Level 2)Histopathology (Level 2)

ACUTE PHASE:

Severe haemocytic

inflammation (with some

melanised foci) of the

intratubular spaces (small

arrow) in response to necrosis,

cytolysis and sloughing of

hepatopancreas tubule

epithelial cells (large arrow)

Source: D.V.LIGHTNER

TRANSITION PHASE:

The hepatopancreas tubule

epithelium is markedly atrophied,

resulting in the formation of large

oedematous (fluid filled or

‘watery’) areas.

SOURCE: D.V.LIGHTNER

Wet Mounts ( Level 2)

Wet Mounts ( Level 2)

 Shrimp at intermoult stage is used and should not have undergone any

treatment.

 Wet mount analysis uses tubular deformation and atrophy mainly at the

apical region of hepatopancreas to detect early stages of infection.

Wet- mount of the HP of

infected shrimp with inflamed

hemocyte, melanized HP

tubules and absence of lipid

droplets.

No stain. 150x magnification

SOURCE: D.V.LIGHTNER

Cytoplasmic masses of

the NHP bacterium are

silver stained and appear

brown to black with the

modified Steiner stain.

Unaffected cells and

nuclei are pale brown

(1600×)

SOURCE: D.V.LIGHTNER

In Situ Hybridization (Level 3)In Situ Hybridization (Level 3)

Specific cDNA non radioactive probes used is digoxigenin-11-dUTP

labelled probes for NHPB.

Pathognomonic positive lesions give prominent blue to blue black

areas in cytoplasm of affected cells when reacted with probes.

Cytoplasmic

masses of the NHP

bacterium are

marked blue to

blue-black by the

probe. Unaffected

cells and host cell

nuclei take the

brown counter

stain.

Cytoplasmic

masses of the NHP

bacterium are

marked blue to

blue-black by the

probe. Unaffected

cells and host cell

nuclei take the

brown counter

stain.

SOURCE: D.V.LIGHTNER

PCRPCR

Polymerase Chain Reaction is the confirmation diagnosis criteria

for NHP bacterium.

PCR primers designated as

NHPF2: 5’-CGT-TGG-AGG-TTC-GTC-CTT-CAGT-3’

NHPR2: 5’-GCC-ATG-AGG-ACC-TGA-CAT-CAT-C-5’

It amplifies a 379 bp corresponding to 16S rRNA of NHPB

Candidatus Hepatobacter penaei.

The current PCR and quantitative PCR (qPCR) assays based on the

amplification of the 16S rRNA gene developed at the University of

Arizona Aquaculture Pathology Laboratory (UAZ-APL) are the only

techniques recommended in the World Organisation for Animal

Health (OIE) manual for H. penaei detection.