BACTERIAL INFECTIONS IN SOME RED SEA FISHES

Assiut University web-site: www.aun.edu.eg

BACTERIAL INFECTIONS IN SOME RED SEA FISHES

MAHMOUD MOSTAFA MAHMOUD ¹; EBTSAM SAYED HASSAN¹; ESSAM ALI NOUR EL-DEEN ²; MOHIE HARIDY ³; FATMA ABD EL-MOGHNY SALEM ⁴ and MAHMOUD ABDEL-RAHMAN MOHAMED ⁴

¹ Fish Diseases and Management, Faculty of Veterinary Medicine, Assiut University, Egypt

² Fish Diseases, Faculty of Veterinary Medicine, South Valley University, Egypt

³ Pathology and Clinical Pathology, Faculty of Veterinary Medicine, South Valley University, Egypt

⁴ Fish Diseases Lab., National Institute of Oceanography and Fisheries, Egypt

Received: 28 September 2017       Accepted: 22 October 2017

INTRODUCTION

Fisheries in the Red Sea are of considerable socio-economic importance to the Red Sea countries in terms of national food security and source of income for rural communities. In Egypt, the Red Sea has a major importance as a natural resource of fish (Sanders and Morgan, 1989; Azab et al., 1998). The marine environment encompasses a wide variety of biological, chemical and physical parameters, which if altered beyond acceptable limits, such as any stress inducer, may modulate the fish immunity leading to disease outbreaks (Roberts, 2012). Bacterial diseases represent the most influential category of disease problems that has a massive impact on fish health and production (Austin and Austin, 2016).

Many species belonging to genus Vibrio induce disease problems with subsequent mortalities in marine fish. Interestingly, Vibrios are present everywhere in marine environment, but clinical disease outbreaks only occur when stressed fish are exposed   to   the     pathogenic      infectious     agent.

Corresponding author: Dr. Mahmoud Mostafa Mahmoud

E-mail address: mahmoud88@hotmail.com;

                          mahmoud88@aun.edu.eg

Present address: Fish Diseases and Management, Faculty of Veterinary Medicine, Assiut University, Egypt

Septicemia caused by vibriosis is characterized by hemorrhages at the bases of fins, exophthalmia, and ulcers on the body surface (Abdel-Aziz et al., 2013).

The objectives of current study were to investigate bacterial infections of some Red Sea fishes at the area of Hurghada, Egypt; to examine the pathogenicity of the dominant bacterial isolate; to study the histopathological alterations induced by the pathogen and to determine the most effective antibiotic(s) against the dominant isolate.

MATERIALS AND METHODS

Fish sampling:

A total of 120 marine fish (Hipposcarus harid, Scarus ferrugineus and Scarus niger; n=100, 15 and 5 respectively) with average body weight of 200 ± 20 g were collected from Red Sea at Hurghada, Red Sea governorate, Egypt. The collected fishes were transmitted immediately to the fish diseases laboratory at the National Institute of Oceanography and Fisheries (Hurghada branch, Red Sea governorate).

Bacterial isolation and identification:

Following their clinical examination, fish necropsy has been carried out as described by Noga (2010). Thereafter, fish were subjected to bacterial isolation and identification according to Austin and Austin (2016). Briefly, loopfuls from kidney, liver and/or spleen were inoculated onto trypticase soy agar (TSA; Oxoid, UK) supplemented with 1.5% (w/v) sodium chloride. The inoculated media were incubated at 25ºC for up to 72 hrs.

Phenotypic characterization of the isolates recovered was performed followingthe techniques previously reported by Buller (2004). Table 1 shows the different biophysical and biochemical characters examined. Motility has been examined using a semi solid TSA supplemented with of 1.5% (w/v) sodium chloride. Isolates were identified biochemically using API-20NE (Oxoid, UK) according to the instructions of manufacturer.

Experimental infection (Challenge):

Twenty five apparently healthy longnose parrotfish, H. harid, with an average body weight of 100 ± 12 g were used for challenge test. Fish were acclimated in 200-liters glass aquaria, for 2 weeks and feed was discontinued two days before injection as previously described (Ellsaesser and Clem, 1986). The dominant isolate (V. logei) was grown on trypticase soy broth (TSB; Oxoid, UK) supplemented with 1.5% (w/v) sodium chloride. Fishes were divided into 5 groups with 5 fish per group. The first 3 groups (representing 3 replicates of challenge) were intraperitoneally injected (I/P) with 0.3 ml bacterial culture suspension in saline (1×10⁷ CFU/ ml), according to our preliminary challenge trials. The fourth (sham control) group was injected with 0.3 ml sterile normal saline. The fifth group was kept un-injected as an absolute control. Fish were observed daily for 15 days to record the clinical signs and mortalities. Moribund fish demonstrating lesions were subjected to V. logei re-isolation on a Vibrio spp. selective medium; Thiosulfate Citrate Bile Salts (TCBS) agar (Oxoid, UK). After 15 days, cumulative mortalities were recorded, then the surviving fish were sacrificed, post-mortem findings were recorded and the pathogenwas isolated from kidney, liver and spleen on TCBS.

Antibiotic sensitivity:

The antibiotic sensitivity of the dominant isolate (V. logei) was examined following the Kirby Bauer disk diffusion method (Bauer et al., 1966) usingMueller-Hinton Agar (MHA; Oxoid, UK) supplemented with 1.5% (w/v) sodium chloride. Antibiotics used are listed in Table 2. At the end of incubation, inhibition zones were measured using measuring caliber. The interpretation of the results as sensitive, moderately sensitive and resistant was done according to the interpretive standards of Clinical and Laboratory Standards Institute (2007).

Histopathological examination:

Specimens from kidney, liver and spleen of experimentally infected fish were trimmed, fixed in 10% phosphate-buffered formalin and processed for histopathological examination as describedbyDrury and Wallington (1980).

RESULTS

Bacterial isolation and identification:

No apparent specific clinical signs were observed on the fish samples either externally or internally. The bacterial isolation resulted in recovery of 35 isolates from kidney, liver and spleen of the examined fish. Phenotypic (biophysical and biochemical) characterization and identification of the different Gram negative isolates are shown in Table 1. The identification resulted in 33 Gram negative isolates representing 94.3% of the total isolates. Two isolates were Gram positive (5.7%) and displayed small and colorless colonies on TSA, alpha hemolysis on blood agar (containing 5% sheep blood) and cocci arranged in chains when examined microscopically after staining with Gram stain. Of the Gram negative, 30 isolates belonged to genus Vibrio (~ 91.0%), 1 isolate of Moritella marina, Photobacterium damselae subsp. piscicida, and Edwardsiella tarda (~ 3.0% each). On the other hand, the two Gram positive isolates obtained were of the genus Streptococcus (5.7%). V. logei was the dominant pathogen with 28 isolates representing 80% of total isolated pathogens; 84.8% of the Gram negative isolates and 93.3% among the isolated Vibrio spp. The other species of Vibrio in the present study were V. ichthyoenteri and V. fischeri (1 isolate for each species; approx. 2.9%). It is noteworthy that out of the 30 Vibrio spp. isolates, 18 were isolated from liver; 8 from kidney and 4 from spleen (60%, 26.7% and 13.3% isolation rates respectively).

Antibiotic sensitivity:

V. logei, as the dominant isolate in the current study, was sensitive to Norfloxacin, Ciprofloxacin and Ofloxacin, moderately sensitive to Amikacin and Gentamicin and resistant to Cefotaxime (Table 2).

Experimental infection (Challenge):

Challenge confirmed V. logei pathogenicity to H. harid with cumulative mortality of 86.7% ± 11.5 (Mean ± standard deviation). No mortalities have been recorded in control group. Data are shown in Fig. 1. The external signs appeared as haemorrhagic areas on skin and at the base of fins, skin ulceration and abdominal distension (Fig. 2 and 3) and corneal opacity (Fig. 4). Meanwhile, internal examination revealed paleness of the liver with peticheal hemorrhages, fibrinous gas bladder, intestine filled with white serous fluid, presence of caseous material in the abdominal cavity (Fig. 4), congestion and enlargement of spleen and kidney. Bacteria were recovered from kidney, liver and spleen of the experimentally infected fish and confirmed as V. logei.

Histopathological examination:

Liver displayed dissociation and vacuolation of hepatocytes around central vein. Focal areas of coagulative necrosis and fatty degeneration of hepatocytes were also observed. Large fat cells replaced the exocrine pancreas accompanied by congestion of the portal vein. Necrosis of pancreatic acini was also recorded (Fig.5 to Fig. 8).

Kidney exhibited degeneration and necrosis of renal tubules. Also, shrinkage of renal corpuscles, hemorrhage and necrosis of hematopoietic tissue associated with inflammatory cell infiltration had been noticed. Melanomacrophage centers were atrophied (Figs. 9 &10).

Table 1: Phenotypic (biophysical and biochemical) characterization of Gram negative isolated bacteria.

ONPG, o-nitrophenyl-b-d-galactopyranoside; V-P, Voges-Proskauer; TDA, Tryptophane deaminase.

Table 2: Antibiotic sensitivity of V. logei.

Fig. 1: Mortalities in longnose parrotfish, Hipposcarus harid, challenged with Vibrio logei. 1^st., 2^nd. and 3^rd. groups represent the 3 replicates of infected fish. Control is the group of fish injected with sterile normal saline only.

Fig. 10: Kidney of longnose parrotfish, Hipposcarus harid, challenged with Vibrio logei exhibits shrinkage of renal corpuscles (arrow) with hemorrhage in hematopoietic tissue and inflammatory cell infiltration (arrow head). (H&E, bar = 200µm)

DISCUSSION

Red Sea represents a major source of marine fish in Egypt, however, little is still known about the map of diseases affecting fishes in this area. The current study draws attention to the bacterial affections of some economic and famous fish species in the area of Hurghada, Red Sea governorate.

Throughout the period from March 2013 to February 2014, a total of 120 marine fish of different species were collected from Red Sea at Hurghada. The bacteriological investigations resulted in recovery of 35 isolates, only 2 of which were Gram positive (Streptococcus spp.; 5.7% of total isolated bacteria), while the majority were Gram negative (94.3% of total isolated pathogens). The dominant isolates were Vibrio spp., and other Gram negative bacteria as M. marina, P. damselae (subsp. piscicida) and E. tarda were also isolated. Balebona et al. (1998) declared that the main pathogenic microorganisms isolated from diseased gilt-head sea bream in the marine environment were; Vibrio spp., Pseudomonas spp., Photobacterium piscicida, Tenacibaculum maritimum (= Flexibacter maritimus), Aeromonas spp. and Gram positive bacteria were also isolated but in low prevalence. Also, Moustafa et al. (2010) isolated V. anguillarum, V. alginolyticus, P. damselae subsp. piscicida (Formerly Pasteurella piscicida),               P. fluorescens, S. fecalis, A. hydrophila, A. sobria and Staphylococcus aureus from fish samples from areas of Qarun Lake and Suez Gulf, Egypt.

Results of present study revealed that V. logei, V. ichthyoenteri and V. fischeri were identical in morphology, motility test, and cultural characteristics on TCBS, oxidase test, nitrate test and sodium chloride tolerance. However, they displayed different characters in utilization of carbohydrates and carbon sources. In the same way, many authors reported similar characteristics of Vibrio spp. (Matte et al., 2007; Korun and Timur, 2008; Austin, 2009; Al-Sunaiher et al., 2010 and Mahbub et al., 2011).While, Sankar et al. (2012) reported differentcharacteristics among various Vibrio spp. examined. This variation was reported in Bergey’s Manual of Systemic Bacteriology (Johannes, 2005) as Vibrios can ferment and utilize a wide variety of simple and complex carbohydrates and utilize a wide variety of carbon sources. Also, the difference in characters among various strains may be attributed to wide distribution of Vibrios around the world and the difference of characteristics of each location from others in the availability of carbohydrates and carbon sources.

Vibrios represented 85.7% from the total recovered isolates as the most prevalent bacterial species identified in our study. This may be because Vibrios are very common in marine environments due to their halophilic nature (Asplund, 2013). Also,Tanekhy (2013) found that the highest percentage from all bacteria isolated in his study was Vibrio species   (41.2 %) in marine fish species raised in cage culture. Among the Vibrios isolated in the present study, V. logei was the dominant isolate which may be due to the host specificity and availability of bacterial sources at the site of fish collection.

Regarding the incidence rate of bacterial isolation from the vital organs of the fish examined, 60% of isolates were from the liver followed by 26.7% from kidney and 13.3% from the spleen. Similarly, Tanekhy (2013) recorded the highest incidence rate of bacteria in liver (40.0%), followed by kidney       (24.8 %) then heart (19.6 %) and the lowest incidence was found in spleen (15.6 %). However, Balebona et al. (1998) stated that the bacterial isolation rate from sea bream was higher in spleen (49.5%), followed by liver (29.1%), then kidney (11.7%) and other organs (9.7%). This may be attributed to the difference in fish species, stage of infection and bacterial species.

Antibiogram of the dominant isolate (V. logei) has declared its susceptibility to quinolones (Norfloxacin, Ofloxacin and Ciprofloxacin) and moderate susceptibility to aminoglycosides (Amikacin and Gentamicin) while resistance to beta-lactams (Cefotaxime). In a previous study, Laganà et al. (2011) reported susceptibility of the majority of Vibrio strains examined to aminoglycosides and other quinolones such as enoxacin, ofloxacin, ciprofloxacin and norfloxacin. However, Han et al. (2007) reported that tetracycline, cefotaxime, ceftazidime, and fluoroquinolones were highly effective against V.  parahaemolyticus strains recovered from Louisiana gulf and retail oysters. The difference in susceptibility to antibiotics may be attributed to variance of the characters among different Vibrio strains investigated in the different studies.

Since H. harid represents an economic and important species among the fish examined in the current study, we used it in the challenge test. Intraperitoneal injection of the dominant isolate, V. logei, induced mortalities in the experimentally infected fish reaching 86.7% as a mean cumulative mortality among the challenged groups. Clinical signs observed on challenged fish were typical to signs of vibriosis reported by Actis et al. (2011). The external signs appeared as haemorrhagic areas on skin and at the base of fins, skin ulceration, abdominal distension and corneal opacity. Meanwhile, internal examination revealed paleness of the liver with peticheal hemorrhages, fibrinous gas bladder, intestine filled with white serous fluid, presence of caseous material in the abdominal cavity, congestion and enlargement of spleen and kidney. Similar lesions were recorded in V. ordalii infection in cultured fishes (El-Bassiony, 2001).

Histopathological examination of Liver displayed dissociation and vacuolation of hepatocytes around central vein, focal areas of coagulative necrosis and fatty degeneration of hepatocytes (El-Bassiony, 2001; Roberts, 2012). While, Enany et al. (2011) reported vacuolar degeneration of hepatocytes, hyperplasia in bile epithelial lining and budding of newly formed bile ductules in cultured Mugil capito infected with Vibrio species. Fatty degeneration in hepatic tissue has been recorded in acute and chronic infection of M. capito infected with V. alginolyticus (Khalil and Abd El-Latif, 2013). Kidneys exhibited degeneration and necrosis of renal tubules, shrinkage of renal corpuscles, hemorrhage and necrosis of hematopoietic tissue and atrophy of melanomacrophage centers (Roberts, 2012; Korun and Timur, 2008; Enany et al., 2011).

Though fish samples used in present work did not show any specific disease signs, many bacterial spp. have been isolated indicating the subclinical infection (or carrier state) of fish. These fish may act as a source of infection to other aquatic animals and human. Further studies are still needed to investigate other microbial pathogens inhabiting fish in the Red Sea.

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