Baculovirus penaei (BP) infection in shrimp

EZHIL. S. AAH-06

 Write down about morphological and

genomic characteristics of BP. About clinical sign and symptoms and histopathological lesion. Abt dignostic techniques. Host range and geographic range and disease transmission Control and preventive measures Target organs

Introduction
BP was first discovered in P. duorarum in

USA (couch 1974) Fam: baculoviridae Rod shaped enveloped virus Envelope 75-300nm Nucleocapsid -270nm Cyclic, dsDNA, Type A intranuclear tetrahedral occlusion bodies

Baculovirus penaei (BP)

Name of the Disease and Etiological Agent
Tetrahedral baculovirosis is the name given to the

disease caused by infection with Baculovirus penaei

The ICTV name for the virus is PvSNPV (singly

enveloped nucleopolyhedrovirus from Penaeus vannamei).

Baculovirus disease, nuclear polyhedrosis disease,

PIB ("polyhedral inclusion body") virus disease, Baculovirus penaei (BP) virus disease.

Agent
BP is an occluded baculovirus (formerly a type-A baculovirus), and

was originally named Baculovirus penaei (Couch).

Bonami et al. (1995) gave BP the designation PvSNPV (for the most

characterized variant, from P. vannamei, in the singly enveloped nuclear polyhedrosis virus group) according to the guidelines for virus nomenclature published by the International Committee on Nomenclature of Viruses (Franki et al. 1991).
Several strains of BP are likely to exist. Different morphological

characteristics, especially in virion size, have been noted for BP as a function of geographic source. Enveloped virions from P. marginatus from Oahu, Hawaii average 56 by 286 nm, while virions from Ecuadorian P. vannamei are 79 by 337 nm, and from Florida derived P. aztecus and P. duorarum measure 75 by about 330 nm.

Geographic Range

BP is widespread in its distribution in cultured and wild penaeids in the Americas, ranging from the Northern Gulf of Mexico south through the Caribbean and reaching at least as far south as the State of Bahia in Central Brazil. On the Pacific Coast, BP ranges from Peru to Mexico, and it has been observed in wild shrimp in Hawaii. BP has not yet been reported outside of the Americas and Hawaii

Presence in AsiaPacific Tetrahedral baculovirosis is not officially reported under the NACAFAOOIE quarterly aquatic animal disease reporting program, it is known to be present in the region.

Host range
Crustaceans known to be susceptible to BP:

aloha prawn (Penaeus marginatus) blue shrimp (Penaeus stylirostrus) giant black tiger prawn (Penaeus monodon) northern brown shrimp (Penaeus aztecus) northern pink shrimp(Penaeus duorarum) northern white shrimp (Penaeus setiferus) Pacific white shrimp(Penaeus vannamei) Pomada prawn (Protrachypene precipua) red-spotted shrimp (Penaeus brasiliensis) redtail prawn (Penaeus pencillatus) roughback shrimp (Trachypenaeus similis) San Paulo shrimp (Penaeus paulensis) southern brown shrimp (Penaeus subtilis) southern white shrimp (Penaeus schmitti)

Susceptible stages of the host

All life stages, except eggs and nauplii, are susceptible to

infection by BP.

BP infection is typically most severe (and most readily diagnosed with simple wet-mount methods) in the larval and early postlarval (PL) stages and in adult females in spawning condition.

Diagnosis is accomplished by demonstration of prominent

tetrahedral occlusion bodies in simple wet-mounts of whole larvae, in the hepatopancreas excised from PLs, or in faecal strands shed from spawning adult female shrimp and collected from spawning tanks.

Survival outside the host

BP produces a specialised tetrahedral occlusion body in which virions are embedded in a crystalline protein (polyhedron) matrix

The occlusion bodys function is to protect the occluded virions from the environment until ingested by a suitable host.
The occlusion body of BP is expected to serve the same function. It is assumed that virions in occlusion bodies remain infectious for months to years in the sediments (e.g. of estuaries or culture ponds) and that they are essential to the infection cycle of BP indifferent generations occupying the same nursery areas. Free (non-occluded) virions are more subject to degradation by sunlight (UV), desiccation, microbial degradation, etc., than are occluded virions and they are believed to remain infectious from no more than a few hours to a few days outside a host.

Epidemiology
Transmission is horizontal, directly from the water column or

through cannibalism.
Eggs and newly hatched nauplii may be exposed to the virus

through faeces of infected adult spawners taken from the wild.

Infection is restricted to the hepatopancreas and anterior midgut. Disease is not known to occur in wild populations infected with

Baculovirus penaei.
Crowding, chemical stress or environmental stress may increase

pathogenicity and the prevalence of disease.

Transmission typically occurs via the oral route, with cannibalism

and faecaloral contamination the principal mechanisms.

Disease patterns
Transmission mechanisms Transmission of BP is horizontal by ingestion of infected tissue (cannibalism), faeces, occlusion bodies, or contaminated detritus or water Prevalence Prevalence is highly variable: from <1% in wild and cultured populations up to 100% in cultured populations in larval-rearing tanks and nursery ponds

Mortality and morbidity

The larval stages (specifically protozoea and mysis) and early PL stages are the most easily

infected in laboratory challenge studies and are the stages where the highest mortalities are likely to occur in penaeid shrimp hatcheries.
High mortality rates are unusual as a consequence of BP infection in the juvenile or adult stages, but infection may cause poor growth and reduced

survival in nursery or grow-out ponds at shrimp farms

Economic and production impact of the disease

BP has caused serious disease with high

mortality rates in hatcheries and significant production losses in farms in the Americas.
While not usually causing high mortality rates

in infected juvenile or adult stages, BP has been documented to cause reduced growth and lower harvest production in populations that are persistently infected with the virus

 Target organs and infected tissue BP is strictly enteric infecting mucosal epithelial

cells of the hepatopancreas tubules and the anterior midgut .

Persistent infection with lifelong carriers

Persistent infection occurs commonly in penaeid hosts of BP. Wild adult L. vannamei females that are heavily infected with BP have been shown to excrete BP-contaminated faeces when spawning, thereby contaminating the eggs and passing the virus to the next generation

Vectors

None is known in natural infections,but the rotifer Brachionus plicatilis and Artemia salina nauplii were used as passive carriers of BP to deliver the virus to larval stages of L.vannamei in experimental infections

Clinical signs of disease:

high mortality in larval, post larval and juvenile prawns reduced growth rates in surviving juveniles and adults increased fouling with exo parasites 100% mortality within 24-48 hrs

Gross signs of disease :

milky-white midgut

There are few visible signs indicating infection with this disease other than rapid high mortality of hatchery prawns in the early life stages. Therefore, diagnosis is usually based on microscopic and histological examination.

Current Diagnostic Method(s) of Choice

Direct microscopy of squash preparations of

feces, HPs, or midgut and demonstration of pathognostic BP PIBs.

Routine H&E histopathology. In situ hybridization with DIG-labeled

genomic probes to BP.

Diagnostic Procedures for Disease Situations

Presumptive Diagnosis

The presence of clinical signs in Protozoea, mysis and early PL stages with severe BP infections may present a whitish midgut (due to the presence of occlusion bodies and cell debris in the faecal material). Juveniles and adults present no gross signs of diagnostic value, nor do larvae with less severe infections

A history of the culture facility, region, or species cultured

that indicates a likelihood of BP infection

Definitive Diagnosis
Direct microscopy/wet-mount method
Diagnosis is made by demonstration of single or multiple tetrahedral occlusion (inclusion) bodies (= PIBs) in epithelial cell nuclei in squash preparations of hepatopancreas, midgut, or feces examined by phase or bright field microscopy.
PIBs are tetrahedral or pyramidal in three dimensional form, and range in size from less

than 0.1 m to nearly 20 m from pyramidal base to peak, with a modal, vertical length of 8-10 m.

Histopathological diagnostic method

Histological diagnosis of BP is dependent upon the demonstration

of BP PIBs in the target organs or tissues (hepatopancreas and anterior midgut), which stain eosinophilically with hematoxylin and eosin stains with or without added phloxine.

In addition to the presence of BP PIBs, the virus causes striking cytopathological changes that include marked nuclear hypertrophy, chromatin diminution and margination, nucleolar dissociation in infected hepatopancreatic epithelial cells, and the presence of a pronounced proliferation of the Golgi to form a conspicuous membranous labyrinth, which is visible in some light microscopic sections as highly vacuolated areas in the cytoplasm of cells late in the BP replication cycle (with stage 3 nuclei and mature occlusion bodies).

Wet-mount of faeces from a white shrimp (Penaeus vannamei) with tetrahedral baculovirosis. The tetrahedral occlusion bodies (TOBs; arrows) are diagnostic for infection of the shrimps hepatopancreas (HP) or midgut epithelial (MG) cells. TOBs are released into the gut contents by the necrosis and lysis of tetrahedral baculovirusinfected HP or MG epithelial cells. 700x Source: DV Lightner

Low (Fig 2, 350x) and mid (Fig 3, 700x) magnification views of mid-sagittal sections of postlarva white shrimp with severe (grade 34) tetrahedral baculovirus infections of the HP. Baculovirus-infected cells display multiple eosinophilic baculovirus TOBs within markedly hypertrophied HP cell nuclei (arrows) Source: DV Lightner

 High-magnification (1800x) photomicrograph of an HP tubule

showing several tetrahedral baculovirus-infected cells that illustrate well the diagnostic intranuclear, eosinophilic, tetrahedral (triangular or rhombohedral in section) occlusion bodies of baculovirus (arrows)

Low-magnification (150x) view of a tetrahedral baculovirus-infected postlarva white shrimp that is similar in age and infection severity to the postlarva shown in Fig 2, but reacted with a DIGlabelled DNA probe for tetrahedral baculovirus. Baculovirus-infected cells are stained dark blue by the probe. Note that infected cells are confined to the HP and MG, and that baculovirus-positive cells are not present in the surrounding non-enteric tissues. Some nonspecific staining of the cuticle by the probe is apparent

High-magnification (700x) photomicrograph of the HP of a juvenile white shrimp infected with tetrahedral baculovirus. The section was reacted with a DIG-labelled DNA probe. Intact infected HP cell nuclei provide an intense positive reaction for virus and viral DNA that is free within the nucleoplasm (large arrow). However, because the TOBs are not penetrated by the probe, the TOBs by themselves do not show a positive reaction for the virus despite their viral content (small arrow)

Genomic probe method for BP

Dot blots. The presence of endogenous HP enzymes limits the usefulness of BP probes with

fresh HP tissue samples when DIG-labeled probes are used.

In situ hybridization. DIG-labeled BP probes provide a very sensitive method for the detection of BP in fixed tissue sections.

Alternative diagnostic procedures to be considered

TEM method Diagnosis of BP may be further confirmed by transmission electron microscopy (TEM), which reveals the presence of rod-shaped virus particles associated with occlusion bodies (the PIBs) or free in the nucleus.

Bioassay method

Overstreet et al. (1988) reported a bioassay procedure for BPV in which Pz-3 P. vannamei were used as the experimental host for BP. The most useful protocol was reported to be the exposure of Pz-3 larvae to rotifers that had been fed virus-contaminated material. Infections were also achieved by feeding virus contaminated tissue to artemia nauplii, and, in turn, feeding these to mysis stage larvae and PLs. The earliest patent infections observed in test shrimp was in Pz-3 larvae within one or two days of exposure to rotifers fed other BP-infected bioassay larvae; 5 to 6 days was required for patent infections when the BP source was (previously frozen or live) adults or juveniles

ELISA method using rabbit polyclonal antibody

An ELISA test using rabbit antibody to BP occlusion bodies (PIBs) was reported by Lewis (1986).

Phloxine/epifluorescence method
The sensitivity of detection of low grade BPV infections in

tissue or fecal smears and in histological sections may be enhanced by the use of phloxine staining and epifluorescence microscopy.
BP occlusion bodies in fecal or fresh tissue squashes, when

stained with approximately 0.001% aqueous phloxine, or with 0.005% phloxine (as a component of the eosin part of normal hematoxylin and eosin staining procedures) in histological sections, fluoresce a bright yellow-green against a pale green background (using an epifluorescent microscope fitted with high intensity mercury lamp, barrier filters of 0515 nm, and an 490 nm exciter filter).

 Antibody-based methods Polyclonal antibodies for detection of BP have been

developed and reported (26, 28), but none is available for routine diagnosis of BP infections.
Electron microscopy/cytopathology BP infection can be confirmed by demonstration of the virus (or pathognomonic occlusion bodies) in sections, or demonstration of the virus in semi-

purified virus preparation prepared from the hepatopancreas

Control and prevention

Vaccination: No effective vaccination methods for BP have been developed

Chemotherapy: No scientifically confirmed reports

Immunostimulation: No scientifically confirmed reports

Resistance breeding:The potential for selective breeding for BP resistance has been demonstrated
Restocking with resistant species: As BP has responded to routine sanitary measures in penaeid shrimp hatcheries, the disease is seldom seen. Therefore, there has been no incentive to develop BP resistant stocks

General husbandry practices

Pre-screening of broodstock for BP has been somewhat

effective in detecting heavily infected carriers of the virus and thereby reducing the transmission of the disease from parent to offspring.
With non-lethal testing methods, this is accomplished by simple

light microscopic examination of faecal strands (or by polymerase chain reaction [PCR] testing of faecal strands if PCR testing facilities are readily available).
Alternatively, spent brood stock may be killed after spawning

and simple light microscopic examination of a hepatopancreas squash can be run (or the excised hepatopancreas may be tested by PCR) to determine the spawners BP infection status.

Contd
Since BP is transmitted from adults to their offspring by faecal contamination of the spawned

eggs, prevention of infection in hatcheries may be achieved by taking additional steps to eliminate faecal contamination of spawned eggs and larvae by thoroughly washing nauplii or eggs with formalin, iodophores, and clean sea water .
The routine disinfection of spawned eggs from infected or potentially infected broodstock has

reduced the incidence of BP epizootics in hatcheries

References

Bonami, J.R., L.D. Bruce, B.T. Poulos, J. Mari, and D.V. Lightner. 1995. Partial characterization and cloning of the genome of PvSNPV (= BP-type virus) pathogenic for Penaeus vannamei. Diseases of Aquatic Organisms 23: 59-66.

Brock, J.A., D.V. Lightner, and T.A. Bell. 1983. A review of four virus (BP, MBV, BMN, and IHHNV) diseases of penaeid shrimp with particular reference to clinical significance, diagnosis and control in shrimp aquaculture. Proc. 71st Intl. Council for the Exploration of the Sea, C.M. 1983/Gen:10/1-18.

Brock, J.A., and D.V. Lightner. 1990. Diseases of Crustacea. Diseases Caused by Microorganisms. pp. 245-349. In:

 Brock, J.A., L. K. Nakagawa, H. Van Campen,

T.Hayashi, S. Teruya. 1986. A record of Baculovirus penaei from Penaeus marginatus Randall in Hawaii. J. Fish Diseases 9: 353-355.
Brock, J.A., and K. Main. 1994. A Guide to the

Common Problems and Diseases of Cultured Penaeus vannamei. Published by the Oceanic Institute,Honolulu, HI. 241 p.

Thank you