Isolation and Molecular Detection of Pathogenic Vibrio Species among Economic Fish from Red Sea in Egypt

A total of 105 samples were collected from Siganus rivulatus, Mulloidichthys vanicolensis, and Lethrinus lentjan, freshly captured from the Red Sea along Hurghada City coastline zone, Egypt. Clinical and post mortem findings revealed the presence of characteristic clinical signs and lesions similar to those reported in vibriosis. Out of 43 putative Vibrio species isolates obtained by culturing; 30 isolates were presumptively discriminated into Vibrio cholera (n=11), Vibrio anguillarum (n=8), Vibrio fluvialis/ Vibrio furnissii (n=4), Vibrio harveyi (Vibrio carchariae) (n=4) and Vibrio alginolyticus (n=3), but it was not initially possible to approve or repudiate that the remaining 13 isolates were Vibrio species through phenotypic characterization. By using PCR, targeting Original Research Article Abdel-Azeem et al.; BMRJ, 12(6): 1-8, 2016; Article no.BMRJ.24016 2 Vibrio-specific 16S rRNA gene, the presumptive 30 Vibrio isolates and 9 out of the remaining 13 isolates were confirmed as Vibrio species. The prevalence of Vibrio species was 37.1% among the examined fish species; 47.1%, 34.3% and 30.6% in Mulloidichthys vanicolensis, Lethrinus lentjan and Siganus rivulatus, respectively. The occurrence of Vibrio species pathogenic for aquatic animals and humans was confirmed which possess public health concerns. Also, the utility of molecular technique to improve the identification of phenotypic Vibrio like species is recommended.


INTRODUCTION
Bacterial diseases outbreaks are a critical obstacle challenging aquaculture sector improvement worldwide [1,2]. The aquatic environment is a habitat for many bacterial pathogens, obligatory and opportunistic pathogens. Stresses exerted on fish due to the poor environmental conditions make them immunocompromised and, subsequently, more susceptible to pathogens infection [2,3].
Vibrio species are a normal part of the bacterial flora in aquatic environments and some species are commonplace in/on aquatic animals; considered to be mostly opportunistic pathogens [4,5]. Several Vibrio species; Vibrio anguillarum and Vibrio tapetis, associated with diseases of aquatic animals, whereas other species including Vibrio cholerae, comprise serious pathogens of human. A comparatively small number of Vibrio parahaemolyticus and Vibrio vulnificus, causes disease in both aquatic animals and human [5,6].
Vibrio species are responsible for vibriosis, one of the most devastating bacterial diseases in cultured fish and other aquatic animals globally [6,7]. The main pathogenic Vibrio species for marine fish are Vibrio alginolyticus, Vibrio anguillarum, Vibrio carchariae, Vibrio cholerae, Vibrio ordalii, Vibrio vulnificus and Vibrio parahaemolyticus, whereas, Vibrio mimicus and Vibrio cholerae are the main pathogenic Vibrio species for fresh water fish [8,9].
The 0/129 vibriostatic test is a useful as a primary screen for identification and differentiation of members of Vibrio species [12,13].
Hurghada coastline is exposed to a variety of stresses as a result of anthropogenic activities, sewage pollution, hypersaline water rejection of desalination plants, shipping operations, and landfilling [14]. Stressed aquatic animals are more susceptible to microbial infections [1][2][3]. Vibriosis in fish is accompanied with stress but some strains, especially of Vibrio anguillarum, Vibrio ordalii and Vibrio salmonicida appear to be highly infectious primary pathogens. Several pathogenic Vibrio species outbreaks are known to be commonly associated with water contaminated with human feces or sewage [15,16]. Vibrios are typically more common in geographic regions having temperate or tropical climates [17]. Salinity, specifically sodium salt (Na +) , is the most important factor governing the environmental distribution of vibrios [18].
The prevalence of bacterial pathogens has been confirmed in several cultured and wild freshwater fish species, however; only a few bacteriological surveys have involved the marine species disease outbreaks [19]. The present study is aimed to assess the prevalence Vibrio species, using molecular and conventional methods, among three economic marine fish species captured from the Red Sea along Hurghada City coastline, Egypt.

Specimens and Clinical Findings of Captured Fish Species
Over a 4 months period (May through August, 2012), a total of 105 samples from three different fish species (Fig. 1) were freshly captured in Hurghada city coastline zone, the Red Sea, Egypt by using small fishing vessels and gill nets. Whole fish were transported in ice-cooled insulated box to the laboratory within few hours, where they were investigated, Table 1 and Fig. 1.
The clinical and post mortem (PM) examination were carried out according to the methods described by [13].

Isolation of Vibrio Species
Fish liver, spleen and kidneys were sampled and cultured on general and selective media; soya agar (TSA, Oxoid) supplemented with 2% (w/v) NaCl and thiosulfate-citrate sucrose agar (TCBS, Oxoid), the inoculated media were incubated in aerobic conditions at 30°C for 2-5 days.

Initial Phenotypic Screening Putative Vibrio Species
Pure cultures of putative Vibrio isolates were initially identified via analyzing phenotypic properties following the criteria described by [12,13].

Molecular Identification Species Using Polymerase Chain Reaction (PCR)
Presumptive Vibrio species isolates were grown in tryptone soya broth (TSB, Oxoid) supplemented with 2% (w/v) Na Cl and incubated at 30°C for 18-24 h. The broth cultures were then transferred into 1.5 ml Eppendorf tubes and centrifuged at 5000 xg at room temperature. The resulting pellet was used for nucleic acid extraction.

DNA extraction from pure culture
The DNA was extracted using the QIAamp® DNA Mini Kit (Qiagen, CA, USA) according to manufacturer's instructions. Briefly, the culture pellet was re-suspended in ATL buffer. Then, cell lysis was performed by proteinase K and incubation in a shacking water bath at 56 Fish liver, spleen and kidneys were sampled and cultured on general and selective media; tryptone soya agar (TSA, Oxoid) supplemented with 2% citrate-bile saltssucrose agar (TCBS, Oxoid), the inoculated media were incubated in aerobic conditions at

Screening for
isolates were initially identified via analyzing phenotypic following the criteria described by of Vibrio Species Using Polymerase Chain species isolates were grown in tryptone soya broth (TSB, Oxoid) supplemented with 2% (w/v) Na Cl and incubated h. The broth cultures were then transferred into 1.5 ml Eppendorf tubes and emperature. The resulting pellet was used for nucleic acid

DNA extraction from pure culture
The DNA was extracted using the QIAamp® , CA, USA) according to manufacturer's instructions. Briefly, the culture suspended in ATL buffer. Then, cell lysis was performed by proteinase K and incubation in a shacking water bath at 56°C/60-90 min. Then, AL buffer was incubated for a further 10 min at 70 precipitation was performed by ethanol (96%). The purified DNA was eluted by AE buffer. DNA concentration and purity were measured by spectrophotometer.

Vibrio-specific 16S rRNA gene
The eluted DNA was used as a template for PCR detection of Vibrio-specific 16S rRNA gene using two primers targeting 162 bp; forward primer 5 GTCAAAGCGATGCAGGTG-3′ and reverse primer 5′-CTTCGCCACCGGTATTCCTT PCR amplification was performed according to manufacturer's instructions. A total 25 µl volume contains 12.5 µl of HotStarTaq Master Mix (Qiagen, CA, USA), 1.5 µl of 10 µM of each primer and 3 µl of DNA template. The mixture was processed in a gradient th (LongGene, A200, Japan) with the following condition; 95°C/15 min as an initial activation step for HotStarTaq DNA polymerase. To minimize PCR products derived from mis events, the actual amplification was initiated with a "touchdown" PCR step consisting of 19 cycles at 94°C/30 sec, 60-51°C/30 sec (decreasing 0.5°C/ cycle) and 72°C/1 min followed by 11 cycles of 94°C/30 sec, 51°C/30 sec and 72 min. The process was finished with a single cycle at 72°C/5 min. The template-free included in the PCR setup as negative controls.

PCR product visualization
The amplified products were analyzed by electrophoresis on a 1.5% agarose gel in 1× TAE The eluted DNA was used as a template for PCR specific 16S rRNA gene using two primers targeting 162 bp; forward primer 5′-′ and reverse CTTCGCCACCGGTATTCCTT-3′ [20].
PCR amplification was performed according to manufacturer's instructions. A total 25 µl volume contains 12.5 µl of HotStarTaq Master Mix (Qiagen, CA, USA), 1.5 µl of 10 µM of each primer and 3 µl of DNA template. The mixture was processed in a gradient thermal cycler (LongGene, A200, Japan) with the following min as an initial activation step for HotStarTaq DNA polymerase. To minimize PCR products derived from mis-priming events, the actual amplification was initiated with PCR step consisting of 19 cycles sec (decreasing followed by 11 C/30 sec and 72°C/1 The process was finished with a single cycle reactions were included in the PCR setup as negative controls. The amplified products were analyzed by electrophoresis on a 1.5% agarose gel in 1× TAE buffer at 100 V for 45 min and visualized by ethidium bromide staining and a UV transilluminator (ECX-F20. M, France) and photographed by MicroDoc (Cleaver Scientific, UK). A 100-bp DNA Ladder (Promega, G210A, USA), consisting of DNA fragments ranging in size from 1500 to 100 bp, was used as a molecular weight marker.

RESULTS AND DISCUSSION
Fish were captured during May through August, 2012. Fish diseases including vibriosis outbreaks are more prevalent in spring and summer, high temperature is a major predisposing risk factor for most types of vibriosis, making it a summer disease in most, but not all cases [16].
Fish liver, spleen and kidneys are iron-rich filtering organs that make them predilection sites for Vibrio species localization [16,21,22].
The clinical examination of fish samples, revealed the presence of some clinical signs similar to those reported by [19] for vibriosis including, skin darkness and scales detachment, ulcers, small and large areas of hemorrhages distributed over many parts of the body, particularly at fins bases, mouth region, and abdomen area which varied in its severity from fish to another. The clinical signs were well marked in Mulloidichthys vanicolensis, mild in Siganus rivulatus and no clinical signs were observed in Lethrinus lentjan. Haemorrhages were most common in Mulloidichthys vanicolensis while only few Siganus rivulatus samples showed small areas of haemorrhages distributed over some parts of the bodies.
The PM examination of fish species, revealed the presence of lesions characteristic to vibriosis; varied degrees of congestion, enlargement of internal organs especially liver, spleen and kidneys, distention of gall bladder, congestion of intestines, and accumulation of body fluids in the abdominal cavity similar to those reported by [23,24]. The severity of PM lesions was in accordance with the clinical signs.
As shown in Fig. 2 and Table 3 when the 43 isolates were submitted for molecular identification using PCR via targeting Vibriospecific 16S rRNA gene, the 30 vibriostatic 0/129 agent sensitive isolates were found positive for 16S rRNA gene. As well, 9 out of the 13 vibriostatic 0/129 agent resistant isolates were found positive (Siganus rivulatus (1/3), Lethrinus lentjan (4/6) and Mulloidichthys vanicolensis (4/4) while the remaining 4 isolates were negative,. Subsequently, TCBS medium is associated with limitations and is not 100% selective for the isolation of Vibrio species where bacteria other than Vibrio such as Enterobacteriaceae could be grow on it [26,27].
Vibriostatic 0/129 agent sensitivity test is of a great value in differentiating some Vibrio species from other Gram-negative rods and particularly from aeromonads, which are characteristically resistant to vibriostatic 0/129 agent. The degree of sensitivity of vibrios to 0/129 agent can also be used as a diagnostic feature in differentiation of Vibrio species; Vibrio anguillarum is sensitive to 0/129 agent at both concentrations while Vibrio fluvialis is sensitive to 0/129 agent at 150 µg and resistant at 10 µg [12,13]. It is important to emphasis that negative 0/129 agent test must be interpreted with caution; 9 vibriostatic 0/129 agent resistant isolates were identified as Vibrio species using PCR.
The identity of putative Vibrio species isolates obtained from TCBS medium should therefore be confirmed employing a number of phenotypic and genetic techniques to accurately identify the atypical isolates [16,28,29]. PCR technique was definitely able to confirm 30 phenotypically presumptive Vibrio species and to assert the suspicions related to 13 vibriostatic 0/129 agent resistant isolates.
No Vibrio species were isolated from some Mullidicthes vanicolensis and Siganus rivulatus samples in spite of observing clinical signs, and vice versa. Consequently, the clinical signs might    The PCR product obtained using specific primers targeting 162 bp of Vibrio rRNA gene after agarose gel electrophoresis and staining by ethidium bromide (0.5 photographed by MicroDoc system using Canon digital camera The total prevalence of Vibrio species (37.1%) in naturally infected marine fish species which appears to be higher than that reported by [31] which could be due to that most of the collected fish samples were freshwater fish while most Vibrio species are halophilic what decreases the viability and variability of Vibrio species in freshwater fish; this fact is very clear with comparison between Vibrio species incidences in Siganus rivulatus (50%) and Oreochromis niloticus (12.8%) in the same study. Also, they only sampled fish fleshes but in the present study liver, kidneys and spleen of examined fish were sampled [16,21,22]. The different fish species under both studies could give rise to variations in their susceptibility to Vibrio species infections. The high prevalence of Vibrio species may be attributed to the stresses induced by abundant anthropogenic activities in Hurghada City coastline zone in contrary to cultured fish farms which could be under good sanitary and management conditions. One of the most strong possible causes is that [31] depended in their study only on bacteriological analysis of examined fish and did not apply any molecular technique for screening their bacterial isolates for additional Vibrio species isolates while during the present study, applying of PCR technique added 9 Vibrio species isolates plus 30 initially phenotypically identified and PCR confirmed Vibrio species isolates.
The total prevalence of Vibrio species (37.1%) appears to be lower than that reported by [32] who analyzed 150 samples from two types of freshwater fish (included, catfish "Pangasius hypophthalmus" and red tilapia "Oreochromis spp") commonly sold at hypermarkets in state of Selangor, Malaysia, for the presence of Vibriospecies applying PCR via targeting Vibriospecific 16S rRNA gene. Different fish species under both studies could give rise to variations in their susceptibility to Vibrio species also, they sampled the flesh and gills of fish which are in direct contact with aquatic environments bacteria

CONCLUSION
The pathogenic Vibrio species are prevalent among the examined economic fish in Red Sea, along Hurghada City coastline, Egypt which possess public health concerns, necessity need for applying hygienic measurements to avoid water contamination and spread of infection among aquatic animals and human. The usage of combination of molecular and conventional methods for Vibrio species identification is necessary.

SIGNIFICANCE AND IMPACT OF THE STUDY
Vibrio species are common inhabitants in/on the aquatic animals, some species are pathogenic for aquatic animals and others are pathogenic for humans, meanwhile other species are pathogenic for both aquatic animals and humans. So, this study identifies Vibrio species isolated from three economic fish species in the Red Sea, Egypt, and assesses their prevalence and distribution using the conventional and molecular methods. The pathogenic Vibrio species for aquatic animals and humans are prevalent among economic fish in Egypt which possess public health concerns. The usage of molecular and conventional methods, together, for Vibrio species identification is necessary.