Disease Diagnosis Of Hepatobacter penaei

(Necrotizing Hepatopancreatitis)
Introduction  Diagnosis
Outbreak LEVEL 1
Taxonomic position LEVEL 2
Description LEVEL 3
Host range and Transmission Prevention and control
measures
Environmental factors and
prevalence References
Shrimp diseases are among the main constraints to the sustainable
growth of the shrimp industry
In 2010, according to the Office of International Epizootics (OIE),
the diseases of penaeid shrimp that required official reporting by
member country veterinary authorities were diseases caused by:
WSSV, TSV, IHHNV, IMNV, YHV/GAV and NHP-B (O.I.E 2010).
All of these diseases have caused estimated economic losses of
more than 10 billion dollars to the worldwide shrimp farming industry
(Lightner 2011).
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.
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)
Infection with Hepatobacter penaei means infection with the
pathological agent Candidatus Hepatobacter penaei – an obligate
intracellular bacterium.
Gram negative, pleomorphic intracytoplasmic alpha proteobacterium.
Initially 3 morphological variants are observed (Krol et al. 1991).
The two morphological variants of this bacterium (Frelier et al. 1992,
Lightner et al. 1992)
 Rickettsial like organism (RLO): Rod shaped and predominant form
(0.25 by 0.9), non flagellated.
 Helical form: eight flagella at the basal apex (0.25 by 2 to 3.5)
and 1 or 2 on the helix crest
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)
Western hemisphere – United states, Mexico, Panama, Belize,
Guatemala, Colombia, Ecuador, Peru, Brazil.
The replication of the NHPB increase with long periods of high
temperature (>29C) and salinity changes (20-38%) (Frelier et al. 1992).
High prevalence and mortality in September, October, November
(Morales-Covarrubias, 2008).
Horizontal Transmission: (Aranguren et al., 2006; Lightner, 2005)

Fecal Direct
cannibalism
contamination ingestion
 CONFIRMATIVE
PRESUMPTIVE DIAGNOSIS DIAGNOSIS LEVEL 3
LEVEL 1 AND 2
HISTOPATHOLOGY
ELECTRON
MICROSCOPY

GROSS LEVEL 2 WET MOUNT

LEVEL 1 LEVEL 3 PCR
SIGNS
INSITU
HYBRIDIZATION
BIO ASSAY
Disease signs at the farm, tank or Gross pathological signs are:
pond level are:
soft shell
lethargy
flaccid body
emaciation
black gills
heavy protozoan or bacterial
fouling empty intestinal tract
reduced growth rate. degenerated or atrophied digestive
gland (hepatopancreas), which
appears pale to white
black (melanised) streaks in the
hepatopancreas.
Darkening at base of swimmerets, giving a Marked reduction in size of
fouled, ‘dirty’ appearance hepatopancreas

SOURCE: D.V. LIGHTNER

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
.
CHRONIC PHASE:

The tubule epithelial cells show no

cytoplasmic lipid droplets, but instead
contain masses of the tiny, non–
membrane bound, intracytoplasmic NHP
bacteria (arrow).

SOURCE: D.V.LIGHTNER
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
Two distinct versions of NHP bacterium can be visualized in infected
hepatopancreatic cells – rod shaped rickettsial like form and helical form.

Profiles of intracellular rod-

shaped forms (large arrow)
and helical forms (small
arrow) of the NHP bacterium
are abundant in the
cytoplasm.

SOURCE: D.V.LIGHTNER
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.

SOURCE: D.V.LIGHTNER
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.
Cycling parameters:
95°C for 5 min – 1 cycle
95°C for 30 sec
60°C for 30 sec 35 cycles
72°C for 30 sec
60° C for 1 min
72 °C for 2min 1 cycle
4 °C infinite hold
 Agarose gel electrophoresis analysis of PCR amplification of extracted DNA from lobster
feces. PCR was performed using Primers NHP/F2 and NHP/R2 [9]. Lane M indicates low-mass
DNA marker, Lane 1 feces from control lobster, Lanes 2, 3, and 5 feces from treatment
lobster, Lane 4 positive control NHPB of shrimp hepatopancreas.( source : The Scientific
world journal, 2012)
Unlike the current OIE methods, the new H. penaei PCR assay did
not provide any non-specific amplification, and the qPCR assay had a
detection limit of 100 copies and a log-linear range up to 108 copies.
Because the previous PCR-based assay using the 16S rRNA was
showing non-specific amplification, the new non-specific product of
around 400 bp was sequenced to determine its identity.
To avoid these non-specific amplifications, new end-point PCR and
qPCR assays were developed based on the H. penaei flagella
gene, flgE.
A schematic diagram of bacterial flagellar system including the flgE region
(Modified from, Liu et al., 2017).
Source: del Río-Rodríguez, R.E, 2006.
 TARGETED SURVEILLANCE PRESUMPTIVE CONFIRMATORY
METHOD DIAGNOSIS DIAGNOSIS
LARVAE PLs JUVENILE ADULTS
GROSS SIGNS D D C C B D
BIOASSAY D D D D C D
DIRECT LM D D C D C D
HISTOPATHOLOGY D B B C A B
ISH A A A A A A
TEM D D D D C C
ANTIBODY BASED D D C STANDARD
GOLDEN C B B
ASSAY
REAL TIME PCR A A A A A A
PCR A A A A A A
SEQUENCING D D D D D A
Early detection (initial phase) of clinical infections
Liming before stocking for pond preparation
Good pond management practices.
SPF broodstock should be used.
Use of antibiotics – oxytetracycline and florfenicol in medicated feeds
every 8 hours for 10 days is the best treatment at present only at initial
phase of treatment.
Disinfection of eggs and larvae reduce contamination in spawned eggs.
Application of PCR for prescreening of wild or pond reared broodstock.
Aranguren, L. F. and Dhar, A. K. (2018) Detection and quantification of Hepatobacter penaei bacteria (NHPB)
by new PCR and quantitative PCR assays. Dis. Aquat. Org., 131: 49-57.
Avila-Villa, L.A., Gollas-Galván, T., Martínez-Porchas, M., Mendoza-Cano, F. and Hernández-López, J., 2012.
Experimental infection and detection of necrotizing hepatopancreatitis bacterium in the American
lobster Homarus americanus. The Scientific World Journal, 2012.
Del Río-Rodríguez, R.E., Soto-Rodríguez, S., Lara-Flores, M., Cu-Escamilla, A.D. and Gomez-Solano, M.I., 2006. A
necrotizing hepatopancreatitis (NHP) outbreak in a shrimp farm in Campeche, Mexico: a first case
report. Aquaculture, 255(1-4), pp.606-609.
Frelier, P. F., Loy, J. K. and Kruppenbach, B. (1993). Transmission of necrotizing hepatopancreatitis in Penaeus
vannamei. J. Invertebr. Pathol., 61:44–48. Lightner, D. V. (1996). A handbook of shrimp pathology and
diagnostic procedures for diseases of cultured penaeid shrimp. World aquaculture society, baton rouge,
louisiana, USA, 304 pp.
Johnson, S.K. 1990. Digestive gland manifestations. In: S.K. Johnson (ed.) Handbook of Shrimp Diseases. Sea
Grant Publication No. TAMU-SG-90-601, Texas A&MUniversity, Galveston.
Lightner, D. V. (2005). Biosecurity in shrimp farming: pathogen exclusion through use of SPF stock and routine
surveillance. J. World aquacult. Soc., 36: 229–248.
 .
Morales-Covarrubias, M. S., Lozano-Olvera, R. Y. and Hernández-silva, A. J. (2010). Necrotizing
hepatopancreatitis in cultured shrimp caused by extracellular and intracellular bacteria. Tilapia &
camarones, 5: 33–39

Nunan LM, Pantoja CR, Gomez-Jimenez S, Lightner DV. “Candidatus hepatobacter penaei,” an intracellular
pathogenic enteric bacterium in the hepatopancreas of the marine shrimp Penaeus vannamei
(Crustacea: Decapoda). Appl. Environ. Microbiol.. 2013 Feb 15;79(4):1407-9.