Abisha.S.J

Pasteurellosis

Definition:It is a chronic to subacute systemic infectious disease of marine and brackish water fishes caused by Photobacterium damselae subsp. piscicida and characterized by white granulomatous lesions in the internal organs.

Aetiological agent; Photobacterium damselae subsp. piscicida Synonyms- Photobacteriosis Tuberculosis Pesudotuberculosis

Congested inflamed liver and splenomegaly with plenty of pseudotuberculi in the congested splenic parenchymacomprise the typical necropsy findings of pasteurellosis. The swim bladder is usually not distended, so the majority of the dying and dead fish sink to the bottom

Growing sea-bass suffering from pasteurellosis.

Epidemiology Photobacterium damselae subsp. piscicida (formerly known as Pasteurella piscicida) hence the term “ pasteurellosis ” still used as reference to the disease) causes one of the most serious bacterial diseases in warm and temperate marine aquaculture

“ Pasteurellosis ” is a common disease in cultured marine fish in Japan, including ayu, black sea bream, red sea bream, red grouper, oval fish, yellowtail has recently caused disease in Senegalese sole and Atlantic bluefin tuna

Photobacterium damselae subsp piscicida colonies isolated from Chaetodon semilarvatus

Scientific classificationDomain: BacteriaPhylum: Proteobacteria

Class: Gammaproteobacteria

Order: VibrionalesFamily: VibrionaceaeGenus: PhotobacteriumSpecies: P. damselaeSubspecies: P. d. piscicida

AetiologyPhotobacterium damsela organisms are stout, Gramnegative Rods varying length (0.8–1.3 × 1.4–4.0 µm) which tend to become coccobacillary in form in older cultures (0.7 × 1.0 µm).

Filamentous cells may be seen in very young cultures.

The organisms often show bipolar staining with methylene blue or Giemsa.

They are nonmotile.

Bipolar staining

oxidase and catalase-positive, facultatively anaerobic chemo-organotrophs with both respiratory and fermentative types of metabolism

They do not grow at 37°C, are halophilic are incapable of reducing nitrate.

Host and geographic distributionPasteurella piscicida has caused epidemics in White Perch and Striped Bass in Chasepeake BayStriped bass in Long island SoundStriped bass from culture ponds in coastal AlabamaGulf menhaden in Galveston bay

Common presence- Japan {widely distributed}Chasepeake bay

The bacteria spreads via infected phagocytes, mainly macrophages.

This spread can be rapid, and lethal effects may occur within a few days of challenge, affecting tissues containing large numbers of the pathogens

The discovery that certain bacterial traits, such as pili or capsules, could make a significant contribution to the ability of bacteria to cause disease opened a new era of disease research.

Virulence

VIRULENCE FACTORS

Iron uptake systemsPhotobacterium damselae subsp. piscicida can utilize heme, hemoglobin and ferric ammonium citrate as sole iron sources in vitro .

The presence of the heme receptor gene hutA was demonstrated in subsp.piscicida isolates from fish.

The identity at the DNA sequence level between the heme uptake clusters of subsp. piscicida strains was 97%

Adhesion Host cell adhesion molecules, machinery, and associated signaling systems are predominantly designed for cell adhesion to other neighboring cells or to the extracellular matrix.

Outer Membrane ProteinsAn essential part of the high affinity iron uptake systems (siderophres) is the production of iron-regulated outer membrane proteins (IROMP) which are either involved in the uptake or internalization of the siderophore/iron complex or act as receptors for transferrin or heme compounds as alternative sources of iron

When various strains of P. damselae are grown in iron-restricted media, three IROMP are expressed in all strains and a fourth one is expressed in some strains

AIP56, a protein abundantly secreted by virulent strains of phdp, as the apoptogenic factor responsible for the induction of apoptosis of sea bass macrophages and neutrophil.

AIP56 a novel plasmid encoded virulence factor of Photobacterium damselae subsp piscicida with apoptogenic activity against seabass macrophages and neutrophils.

SDS-PAGE profiles of culture supernants from Photobacterium damselae subsp piscicida(phdp) strains MT1415 (virulent)And ATCC29690 (non virulent)

Serum ResistanceVirulent P. damselae strains are serum resistant and can grow in fresh rainbow trout serum,

whereas non-virulent strains are sensitive to serum killing and their growth is totally inhibited in fresh serum.

This inhibitory effect of the serum on the non-virulent strains, however, is totally lost if the complement is inactivated by heating at 56°C for 1 hour.

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All the P. damselae isolates (originally capsulated and non-capsulated) were able to grow in normal serum . Photobacterium damselae was found to resist the bactericidal effects of normal HSB serum

Serum resistance is associated with capsule production, with capsulated strains resistant to serum killing, while non-capsulated strains were serum sensitive

Serum resistant

Serum positive

Capsule Capsule was first described for P. damselae as a rigid integral capsule that excludes Indian ink stain.

Electron microscopy revealed the presence of a capsule on the external surface of the outer membrane of bacteria grown in glucose rich YPGS-2 media, but it was absent in cells grown in BHIA-1 media.

Photobacterium damselae grown under iron-limited conditions had a significantly reduced amount of capsular material on their surfaces than those grown under iron supplemented conditions

The amount of capsular material decreased with the age of the culture, irrespective to amount of iron in media.

Capsulation is a crucial virulence determinant for a number of bacterial species, providing protection from serum killing and phagocytosis

no significant difference was found in adhesion to cell lines between virulent, capsulated P. damselae and non-virulent, non-capsulated strains

Lipopolysaccharide The LPS of P. damselae has been isolated and subjected to morphological analysis by polyacrylamide gel elecrophoresis (PAGE), gas chromatography, spectrophotometry and immunoblotting techniques.

The LPS of six P. damselae strains from diverse geographic regions in Japan and one isolate from Chesapeake Bay, USA were all found to have identical LPS structure

Plasmids Plasmids have been found to confer a number of important attributes to bacterial fish pathogens including antibiotic resistance, iron sequestering Systems and resistance to phagocytosis

Strain ATCC 17911 from Chesapeake Bay carries plasmids of 20 and 7 MDa molecular weight,

while strain SB2-KK from Long Island Sound possesses plasmid bands of 65, 25, 18, 6.5, and 3 MDa

TransmissionThe mode of transmission of pasteurella piscicida is unknown, although fish to fish contact and an invertebrate vector have both both been suggested.

Oral transmission is likely.

The reservoir of infection is uncertain, although striped bass are belived to be the major source of the infection in the Chasepeake Bay.

The bacterium appears to be short - lived in the environment,

Risk factors includeshigh salinity (greater than 10ppt)High temperature (greater than 25 degree C)

MODE OF TRANSMISSION

The exact mechanism of infection is still unknown.

It is assumed that feral fishes may transmit the disease to yellowtails, and that once a population is infected the disease is spread by fish-to-fish contact.

The disease is also spread by movement of yellowtails, which can be asymptomatic carriers

INCUBATION PERIOD Under experimental conditions, white perch died within 72 h after receivlng an intraperitoneal injection of 107 bacteria .The temperature (not stated) was probably in the range of 20-25 C.

Sea bream showingpale enlarged liver, engorged gall bladder, enlargement of the spleen kidney with typical greyish-white granulomatous-like deposits

PERIOD OF COMMUNICABILITY age-resistance relationship. Young yellowtails and red sea bream (Chrysophyrs major) are more susceptible than are older fish.

Pasteurellosis of a young bream (4g) with splenomegaly and yellowish fluid in the gut.

IsolationPasteurella piscicida has been isolated from white perch, striped bass, yellowtails, red sea bream, and black sea bream (Mylio macrocephalus).

Pasteurella plecoglosacida has been isolated only from the ayu.

OccurenceOutbreaks are most severe in autumn and spring, when water temperature is 23 to 26 C and salinity 30 to 33%.

Ayu fish

Clinical pathology Generally, affected fish are dark and inappetant.

HistopathologyIt reveals characteristic granulomata in haemopoietic tissue of the spleen and kidney which in later stages release bacteria to produce a generalised haemorrhagic septicaemia.

The internal organs of a nursery stage sea-bream (0.3g) suffering from pasteurellosis (under a stereoscope). The spleen is enlarged pseudotuberculi are already evident. The liver is inflamed and the intestines swollen.

Clinical signsPasteurella piscicida causes a bacteremic septicemia that takes one of two forms.

Acute form Chronic form

Acute FormFew clinical signsSmall hemorrhages around the gill covers or the base of fins in White perchAbnormal skin pigmentationEnlarged spleen and kidney in Sea Bass

Gills of a sea-bass suffering pasteurellosis under low magnification. Wide area of gill epithelial tissue necrosis next to congested,inflamed tissue.

Histologically there is acute necrosis of the spleen , liver, pancrease with no inflammation.

In the acute form there are few gross pathological changes. Evidence of edema and darkening may occur in yellowtails just before death.White perch showed only slight hemorrhages around the gill covers or bases of the fins.

Histological section of spleen from gilthead seabream showing phagocytescontaining numerous bacteria ( arrow )

chronic formStriped bass shows 1-2mm miliary lesions in the kidney and spleen that represent a chronic inflammatory response cited by bacteria.Multiple, white foci on spleen and kidney(imp)

The appearance of the latter lesions has led to the names tuberculosis or pseudotuberculosis for the disease in yellow tail.

In both chronic and acute forms, Pasteurella piscicida occurs throughout internal organs.

Spleen of gilthead seabream with multiple white foci caused by P. damsela subsp.piscicida .

Multifocal hepatic necrosis

In chronic form collections of necrotic lymphoid and peripheral blood cells were present in the spleen, focal areas of hepatocytes undergoing coagulation necrosis were apparent in the liver, and there was a conspicuous lack of an inflammatory cell response

(C) Liver shows an area of necrosis surrounded by fibrous connective tissue and hemorrhagic areas.

(D) Liver show severe necrosis, edema of hepatocytes, vacuolisation and pyknotic nucleus and damaged blood vessels.

Histopathological changes were noted in naturally and experimentally infected gilthead sea bream.

The spleen and kidney showed circumscribed, acute necrotic changes and infiltration, with blood cells and masses of bacteria plugging capillaries and interstitial spaces

Phagocytes containing bacteria and swollen into large globules were seen in the spleen and kidney, and it was theorized that swollen phagocytes blocked capillary blood flow in the organs resulting in ischemia

Sparus aurata infected with Pasteurella piscicida

Altered bacterium (arrow) inside a phagocytic vacuole and the marked degranulation (arrowheads). Peritoneal macrophages (M) containing large numbers of phagocytosed bacteria. Granulocyte (G).

Kidney of Sparus auratus

Transmission electron micrograph showing a macro- phage containing phagocytosed apparently Intact bacteria. A bacterium (arrowheads), which appeared to be dividing, is also noted. Nucleus (N).

Kidney of fish injected with bacteria

Transmission electron micrograph showing 2 macrophages containing phagocytosed bacteria (B).

Although macrophages have a different electron density, the plasma membrane is well preserved (arrowheads).

extracellular bacteria (arrow)

Bacteria were observed inside macrophages (arrows) or forming a colony (B). Cells surrounding the colony were apparently normal.

(E) Kidney shows a necrotic area encapsulated by an accumulation of fibrous connective tissue and inflammatory cells.

(F) Kidney shows sever aggregation of melanomacrophage and inflammatory cells within the epithelial cells of the renal tubules.

(G) Spleen shows the activation of melanomacrophage centers and congestion of blood vessels. (H) Spleen shows free melanomacrophages.

Spleen

(a)chloride cell showing autolysosomes containing cytoplasmic membranous tubules(arrowheads) disintegrating mitochondria (arrows).

Fig. 15. Sparus aurata. Gill arch of a fish injected with P.piscicida (arrowheads) or disintegrating mitochondria (arrows).

Gill arch of Sparus

Micrographs of histological sections from sea bass challenged by P. damselae subsp. piscicida.

(A) Gills show destructive changes in both primary and secondary lamellae, collapsed and curled secondary lamellae, edema in filaments, severe lamellar aneurism.

(B) Gills show hyper activation of goblet cells

Diagnosis Method of Diagnosis

Culture of Photobacterium damselae ssp.PiscicidaPresumptive diagnosisConfirmatory diagnosis

Culture of Photobacterium damselae ssp. piscicida from typical skin and/or internal lesions

Culture and identification The microorganism is readily isolated from the kidney, liver and spleen of diseased fish.

It is a nonfastidious organism which can be readily cultured on general purpose nutrient media containing 1–3% sodium chloride.

Small, dewdrop-like colonies develop after 48 hours incubation at 20–25°C.

Like the Vibrionaceae, it is resistant to the vibriostat 0/129, is oxidase-positive and metabolises glucose fermentatively, but it is otherwise biochemically comparatively unreactive.

Colonies formed areShiny grey yellow Entire convex1 to 2mm size

In culture Pasteurella piscicida most closely resembles non pigmenting strains of Aeromonas salmonicida.

Gelatinase is not produced and acid but no gas is produced with glucose media

Glucose fermentation test

Presumptive diagnosis is based on the observation of Gram negative, non pigmented, non motile rods(0.5 to 0.75× 1 to 2µm), which stain bipolarity. show pleomorphism and filament formation

Confirmatory diagnosis can be performed using slide agglutination or immunofluroscence.

Treatment and control

Infections in Japan have been treated with many differentantibiotics, such as ampicillin and potentiated sulfonamides.However, there are serious problems with resistance

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Prophylactic chemotherapy with sulfonamides, nitrofurans, or antibiotics have been

employed successfully, but dosages have not been published

Daily administration of antibiotics, mixed in the feed, for 10 days is usually effective to eliminate mortalities temporarily

Oxytetracycline was not very effective in controlling an outbreak of Pasturellosis in striped bass

This is due to inadequate tissue levels sustained in target organs, not resistance of the bacterial isolates.

Experimental infections in Yellow tail have been controlled by Ampicillin.

Experimental vaccines also shows promise

Therapy Sulfonamides at 200-400 mg per kilogram of body weight per day or chloramphenicol at 20-40 mg per kilogram of body weight per day, both fed for a minimum of 6 days, are used to control outbreaks.

The antibacterial activity of ampicillin was 8 to 16 times that of chloramphenicol.

They suggested that ampicillin would be useful in treatment of Pasteurella septicemia.

Prevention Good sanitation and management procedures should be used to avoid overcrowding and other stresses that may predispose fish to disease

vaccination by immersion (dip) has shown poor results in preventing pasteurellosis in the field

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