UDK 619:579.843.2:639.212 DETECTION OF AEROLYSIN (aerA) GENE IN AEROMONAS HYDROPHILA STRAINS ISOLATED FROM DISEASED CARP

Bacterial septicemia caused by motile aeromonads is common infection in the intensive fish production. Aeromonas (A.) hydrophila is often present in fish populations. Ubiquitous distribution of these bacteria in the aquatic environment, and the stress caused by intensive breeding are predisposing factors for the occurence of the disease. A. hydrophila is considered a major cause of septicaemia caused by motile aeromonads. Several A. hydrophila extracellular products (ECP) are considered as important factors in pathogenesis, primarily aerolysin (aerA), the extracellular lipase, cytolytic enterotoxin, hemolytic toxin and extracellular proteases. PCR detection of aerolysin (aerA) is considered a reliable method of identifying potentially pathogenic Aeromonas strains. In spring 2012, after a sudden increase in water temperature, disease occured in common carp population in one fish farm in Serbia. Five specimens of the one-year-old carp with clinical symptoms of motile aeromonas septicaemia were used for isolation of the bacteria. Identification of A. hydrophila was done on the basis of morphological, physiological, cultural and biochemical characteristics. PCR amplification of DNA from A. hydrophila isolates revealed presence of aerolysin (aerA) gene in all examined A. hydrophila isolates from carp with motile aeromonas septicaemia.


INTRODUCTION
Aeromonas hydrophila, a Gram-negative, motile rod that is a member of the family Aeromonadaceae (Joseph & Carnahan 2000;Abbott et al. 2003), has been widely studied and is regarded as the most important bacterium causing "aeromonosis or haemorrhagic septicaemia or motile aeromonas septicaemia" in fish (Rhaman et al. 2001) and other aquatic animals (Hill et al. 2010;Pearson et al.2000).There have been a number of epidemiological studies indicating Aeromonas species as a cause of diarrheal disease in children, elderly people and immunocompromised patients (Figueras, 2005;von Gravaenitz, 2007).
The widespread of the bacteria in the aquatic environment and the stress caused by intensive breeding are predisposing factors for the disease.Stressful environmental factors, especially high water temperature, high levels of ammonia and nitrite, sudden changes in pH, and low concentrations of oxygen increases the possibility of disease occurence (Jeremic et al. 2005).Several extracellular products (ECP) of A. hydrophila are considered important virulence factors, primarily aerolysin, extracellular lipase, cytolytic enterotoxin, hemolytic toxin and extracellular protease (John et al., 1997, Shome et al., 2005).Detection of virulence genes by PCR is very useful for the identification of pathogenic isolates of aeromonads (Uzbas et al., 2000).
Detection of aerolysin (aerA) using PCR and RFLP is considered a reliable for identification of virulent strains of A. hydrophila (Kingombe et al., 1999).The aim of this study was to determine the presence of aerA gene in A. hydrophila strains isolated from carp with motile aeromonad septicaemia.

MATERIAL AND METHODS
Five samples of the one-year-old carp with clinical symptoms of bacterial infection were used for isolation.From each fish, samples were collected from the kidney, liver, spleen and gills.The samples were streaked on tryptic soy agar (TSA, HiMedia), Mueller-Hinton agar (HiMedia) containing 5% defibrinated sheep blood erythrocytes (BA), selective Rimler-Shotts (RS) media (HiMedia), and incubated at 30 o C for 24 to 48 h .Following incubation, one typical colony (entire circular, convex, white to greyish, semitranslucent, size 2 to 3 mm, haemolytic) was selected from each plate with a pure culture and subcultivated in order to test the purity of isolates.The isolates were preliminary grouped according to colony morphology, haemolysis, and pigmentation before they were stored at -80 o C in 15% glycerol until further characterization.The type strain of Aeromonas hydrophila ATCC 7966 (American Type Culture Collection) was included in the phenotypic characterization.
Isolates were classified as Aeromonas hydrophila according to their reactions in the API 20E (Biomerieux) and following conventional tests, based on standard bacterial taxonomic procedures (Holt et al 1994;Austin and Austin, 2007).
For identification of virulence genes, five colonies of A. hydrophila were selected and 24 hours old cultures were used for extraction of genomic DNA.DNA extraction was performed using commercial kit (QIAamp DNA Mini Kit, Qiagen) according to manufacturer's protocol.Detection of the aerolysin gene was performed using polymerase chain reaction (PCR) (Chu et al., 2005).Primers were used to detect 462bp aerolysin gene fragment (Aero1: 5'-CTCAGTCCGTGCGACCGACT-3' and Aero2: 5'-GATCTCCAGCCTCAGGCCTT-3').Amplification was performed by 35 cycles of denaturation at 95 o C for 1 min, annealing at 56 o C for 2 min and extension at 72 o C for 2 min.After amplification, PCR products were characterized by 1.5% agarose gel electrophoresis in Tris-borate-EDTA buffer.

RESULTS
On a carp pond, localized in the northeastern part of the Republic of Serbia, in the spring, during a sudden increase in water temperature, increased mortality of young carp occured.The main signs of the diseased fish were anorexia, exophthalmus, redding due to haemorrhage of the skin and swimming at the surface of the pond, near fresh water supply.In scaled fish, scale pockets become edematous, causing lepidorthosis (Figure 1).Internal organs were edematous with hemorrhage and erythema on liver and kidney.
Figure 1.The pathological symptoms of the common carp suffering from motile aeromonad septicemia.
Radosavljević V. i dr.: Detection of Aerolysin … Pure cultures of Aeromonas hydrophila were obtained from all samples (Figure 2). Figure 2. Growth of Aeromonas hydrophila on Rimler-Shotts medium.
A PCR amplification revealed that all A. hydrophila isolates were PCR positive for the aerA gene.

DISCUSSION
Motile aeromonads cause different pathologic conditions that include acute, chronic and latent infection.Severity depends on a number of factors including bacterial virulence, type and level of stress, resistance and physiological state of the host.In the acute phase, this condition is characterized by rapid fatal septicemia with little macroscopic evidence of disease.When present, the most important symptoms are exophthalmos, skin redness and fluid collection in the scale pockets (Faktorovich, 1969).Arhiv veterinarske medicine, vol.6, br. 2, 55-63, 2013.
Radosavljević V. i dr.: Detection of Aerolysin … There is evidence that the motile Aeromonas complex involves secondary and opportunistic pathogens, but ability of A. hydrophila to cause disease and death of fish should not be overlooked because occasionally highly virulent strains emerge.Regardless of whether or not the organism serves as a primary or secondary invader of stressed fish, it is often the final insult that leads to death (Plumb and Hanson 2011).
In attempt to explain the pathogenesis of infection caused by A. hydrophila several virulence factors were investigated.Toxins with haemolytic, cytotoxic and enterotoxic activities have been described in many Aeromonas spp.(Chopra et al., 1990).Although rare, A. hydrophila may cause high mortality among cultured fish without presence of severe external (stressful) influences.This inconsistency may result from the presence of A. hydrophila strains that possess specific virulent or pathogenic characteristics (Plumb and Hanson 2011).
In our study, PCR was performed to detect aerolysin (aerA) gene as a genetic marker for the determination of virulence.Role of aerolysin (aerA) gene in the pathogenicity of Aeromonas genus was previously demonstrated (Kozaki et al., 1989, Shaw, 2003).In present study, PCR amplification of A.hydrophila isolates for the 426-bp aerA gene, in samples from diseased fish, showed the presence aerolysin gene, which is an essential A. hydrophila virulence gene.Similar results were obtained in previous studies with A. jandaei (Chacón, 2003).It is well known that the screening of specific cytotoxin and hemolysin genes is the most effective way of detecting and characterizing Aeromonas virulence factors (Yousr et al., 2007).

CONCLUSIONS
Despite disagreement among scientists regarding significance of A. hydrophila infection, the frequency of its appearance in aquaculture environment, together with a high potential for stress, shows that this problem sholud not be ignored, because it is often what kills the fish.PCR test for the detection of aerolysin gene proved to be a useful tool for the detection of virulent strains of Aeromonas hydrophila.