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Photobacterium damselae subsp. piscicida and Photobacterium damselae subsp. damselae:

Fish Pasteurellosis (photobacteriosis) - Molecular diagnosis (PCR and sequencing)

 

Information 09-16-2018.

 

Photobacterium damselae species are one of the most devastating bacterial pathogens in marine aquaculture worldwide and some of them are pathogenic to marine and human animals. These species are the causative agents of photobacteriosis (formerly known as pasteurelosis) one of the bacterial diseases that most affect both wild marine species and species grown in Europe, Japan and the Mediterranean region.

 

Photobacterium damselae and its impact on aquaculture

 

P. damselae is a Gram negative bacterium that includes two subspecies P. damselae subsp. damselae and P. damselae subsp. piscicida. Both subspecies include the species formerly known as Vibrio damsela and Pasteurella piscicida, respectively, and which were therefore classified into two different families Vibrionaceae and Pasteurellaceae, an aspect that was consistent with the wide range of phenotypic differences between the two. However, studies performed from the sequencing of the 16S ribosomal RNA gene and DNA-DNA hybridization assays demonstrated that Vibrio damsela and Pasteurella piscicida were members of a single species.

Photobacterium damselae subsp. piscicida

Pasteurellosis or photobacteriosis is caused by P. damselae subsp. piscicida, and is one of the diseases of bacterial etiology with greater economic impact on marine fish cultures worldwide. Probably, it is the most devastating pathology in the production of snapper (Pagrus pagrus) in the Mediterranean area. This disease is also known as pseudotuberculosis, because chronically ill fish have prominent white granules in internal organs such as spleen, kidney or liver. Other external signs of this disease are a slight darkening of the skin and abdominal distension sometimes accompanied by hemorrhagic areas on the head and gills.

Pasteurellosis seems to be a disease of greater prevalence in the summer months where the water temperature is higher (higher than 23ºC). P. damselae subsp. piscicida survives in viable state in water for 4 or 5 days, and is able to enter a viable non-culturable state for at least one month, using marine sediments and seawater as a reservoir and transmission vehicle. The hosts of P. damselae subsp. piscicida include different species of marine fish, both wild and farmed.

The disease was initially described in 1963 in wild populations of white perch (Morone americanus) and to a lesser extent in those of American seabass (Morone saxatilis) in an epizootic in Chesapeake Bay (Maryland, USA). Since 1969, pasteurellosis has been one of the most important fish diseases in Japan, mainly affecting Serola (Seriola quinqueradiata), although it also affects other species such as the red spotted grouper (Epinephelus akaara) and a species of blennium (Pictiblennius yatabei). Since 1990, it has caused great economic losses in gilthead seabream (Sparus aurata), sea bass (Dicentrarchus labrax) and sole (Solea senegalensis and Solea solea) farmed in European countries in the Mediterranean area and also in perch (Morone americanus) in the United States. Recently it has been isolated from cobia (Rachycentron canadum) and paradise fish (Macropodus opercularis) in Taiwan and also from mullet (Mullus spp.) In Italy.

Photobacterium damselae subsp. damselae

In 1981, P. damselae subsp. damselae was isolated from ulcers on the skin of the angelfish (Chromis punctipinnis). P. damselae subsp. damselae can be isolated from different aquatic ecosystems, since this organism is able to survive in seawater and sediments for long periods of time. In addition, it is capable of transmitting through water, being responsible for causing pathological processes in various marine species. Normally the infectious outbreaks produced by P. damselae subsp. damselae follow a seasonal distribution, coinciding with more temperate water temperatures and a decrease in the resistance of the host organism due to physiological changes during sexual maturity. In addition, the increase in the average temperature of seawater, which is occurring in a generalized manner during these last decades as a consequence of global warming, will possibly cause an increase in the number of infections produced by this organism.

The most remarkable external signs caused by P. damselae subsp. damselae in fish are: abdominal distension, presence of hemorrhagic areas around the eyes, mouth and anus and sometimes skin ulcers. These signs are similar to those produced by Yersinia ruckeri in salmonids. Internally there is an accumulation of hemorrhagic fluid inside the peritoneal cavity.

The hosts of P. damselae subsp. damselae include different species of marine animals, both wild and farmed. P. damselae subsp. damselae is a pathogen for reptiles such as leatherback turtles (Dermochelys coriacea), mollusks such as octopus (Octopus joubini), crustaceans such as shrimp (Penaeus monodon); mammals such as the dolphin (Tursiops truncatus, Delphinus delphis) or the whale (Balaenoptera edeni), wild fish such as the shark (Carcharhinus plumbeus, Squalus acanthias), angelfish (Chromis punctipinnis), amberjack of Japan (Seriola quinqueradiata) or ray (Dasiatis stingray). But it is also a pathogen of fish of economic interest such as turbot (Psetta maxima), rainbow trout (Oncorhynchus mykiss), white pompano (Trachinotus ovatus), a species of sole (Cynoglossus semilaevis) or giant perch (Lates calcarifer). used for aquaculture in China and Thailand, respectively.

In the Mediterranean area, the intensive cultivation of new species has caused several outbreaks with moderate mortality in which P. damselae subsp. damselae. Among the affected species are: common dentex (Dentex dentex), semolina snapper (Pagrus auriga), common bream (Diplodus sargus), golden bream (Sparus aurata) and to a lesser extent sea bass (Dicentrarchus labrax).

P. damselae subsp. damselae not only affects poikilothermal species, but due to its ability to grow at 37°C, it is potentially pathogenic for homeothermic animals, and even man. This organism was isolated for the first time as the causative agent of an infectious case recorded in humans in 1971. In man, it causes from external ulcerative infections to generalized septicemia that can lead to the death of the individual, so its importance in human it has already been recognized. Normally the infection derives from superficial skin lesions produced during the handling of infected marine fish, or through external wounds that come into contact with seawater. Although cases have been detected in completely healthy patients, a large part of the clinical symptoms caused by P. damselae subsp. damselae occur in patients who have some kind of underlying disease such as diabetes, cirrhosis or heart disease.

Photobacterium damselae: Taxonomic position

Pasteurellosis is a disease known for many years. However, the taxonomic position of its causal agent has been the subject of constant controversy. In 1964 Snieszko et al. placed this microorganism within the genus Pasteurella due to its morphological and biochemical characteristics, despite its inability to reduce nitrates, being the only marine bacterium belonging to this genus and presenting a tolerance to unusual pH and temperature values ​​for the members of the genus Pasteurella. Later in 1968, Janssen and Surgalla, when performing new tests, found sufficient differences in physiological and serological characteristics in relation to species of the same genus to propose a new species called Pasteurella piscicida.

Later phylogenetic studies confirmed the relationship of Pasteurella piscicida with the family Vibrionaceae and the association of this organism with Photobacterium damselae. Thus, it was determined that the sequences of the 16S rRNA gene of Pasteurella piscicida and Photobacterium damselae differ only in one nucleotide of the 1,434 sequenced positions. When performing DNA-DNA hybridization analysis, it was concluded that Pasteurella piscicida and Photobacterium damselae were members of the same species.

Morphological, biochemical and serological characteristics

Photobacterium damselae subsp. piscicida

P. damselae subsp. piscicida is a bacillus (0.8-1.3 x 1.4-4 μm) Gram negative, non-mobile, oxidase and catalase positive, fermentative and facultative anaerobe. Negative for indole, urease, gelatinase, amylase and citrate reactions, it does not produce gas from glucose and is unable to reduce nitrates. It presents a characteristic bipolar stain and can show pleomorphism, varying its shape according to the growing conditions or the growth phase, since they grow in chains, are longer and show a more bacillary shape than in later phases where their shape resembles more to cocobacilli. It has lipase and phospholipase activities, but lacks β-hemolytic activity, although a phospholipase with haemolytic activity against erythrocytes of different fish has recently been described.

It has an obligate halophilic character, showing a good growth in media with a NaCl concentration of 1.5-2%. This growth is completely inhibited at NaCl concentrations above 5%. It does not present bioluminescence and is able to produce capsular material with 96% carbohydrate and 0.4% protein. It has a narrow range of temperatures between 15 and 30ºC to which it is capable of growing, although its optimum growth is around 25ºC.

Serological studies with total cells and somatic O antigen show that different serogroups cannot be established within P. damselae subsp. piscicida, a fact corroborated when studying the lipopolysaccharide patterns and the outer membrane proteins. The immunoblot assays demonstrate a high level of antigenic similarity between the strains, since all lipopolysaccharides have a similar reaction. On the other hand, the analysis of the plasmid content of several strains of P. damselae subsp. piscicida from Japan, the USA and Europe show a similar pattern between the strains of the United States and Europe, but this one differs a lot from that shown by the strains of Japanese origin. 

Photobacterium damselae subsp. damselae

P. damselae subsp. damselae is a Gram negative bacillus, movile by monotric polar flagella with certain pleomorphism. It has catalase and arginine dehydrolase, ferments glucose with gas production, mannose and maltose, produces urease, lipase, amylase, chitinase, phospholipase, chondroitinase, hyaluronidase and hemolysis, although not all strains have the same values, and even some lack of such activities.

It shows strict requirements of NaCl, tolerating concentrations that reach 5%. Unlike P. damselae subsp. piscicida, has broad growth temperature ranges, from 12ºC to 37ºC. The analysis of lipopolysaccharide content showed the existence of an enormous variability of lipopolysaccharide profiles. This result correlates with that obtained in the serological tests, where the existence of four different serogroups was found. 

Molecular characterization of Photobacterium damselae

The PCR test is very useful for the rapid and confirmatory diagnosis of the presence of P. damselae. The specific detection of both subspecies of P. damselae is carried out using the conventional PCR test, with primers corresponding to the gene encoding the capsular polysaccharide present in the two subspecies P. damselae.

On the other hand, the UreC genes are currently the only PCR assay that can be successfully performed to discriminate between the subspecies of P. damselae since P. damselae subsp. damselae possesses the UreC gene, included in the Ure operon and P. damselae subsp. piscicida lacks this gene, which agrees with its negative urease phenotype.

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• Molecular diagnosis (PCR) of the species Photobacterium damselae subsp. piscicida and Photobacterium damselae subsp. damselae: Consult ivami@ivami.com