Dept. of Animal Medicine, Fish Diseases and Management,

Fac. of Vet. Med., Assiut University, Assiut, Egypt.

Columnaris Disease in Sharptooth Catfish, Clarias gariepinus

(With 5 Tables and 2 Figures)

By

Sh.M. Ahmed; A.I. Ahmed*; A.A. Elkamel

and Walaa F. Emeish**

*: Dept. of Poultry Diseases, Fac. of Vet. Med., South Valley University,

Qena, Egypt.

**: Dept. of Fish Diseases and Management, Fac. of Vet. Med.,

South Valley University, Qena, Egypt.

(Received at 6/3/2007)

مرض الکولمنارس فى الأسماک القطية النيلية (القراميط)

شعبان محمد احمد ، أحمد إبراهيم أحمد ، احمد عبد الهادى الکامل ،

ولاء فتحى عميش

الهدف من هذه البحث هو اجراء دراسة ميدانية لتسجيل إصابة الأسماک القطية النيلية (القراميط) بمرض الکولمنارس وکذلک التغيرات المرضية نتيجة الإصابة. تم فحص عدد 144 سمکة على مدار عام  بمعدل 12 أسماک شهريا ذات الأوزان المختلفة والتى تم جمعها بشکل عشوائي من روافد نهر النيل المختلفة بمحافظه قنا کشف الفحص الظاهري، البکتريولوجى بان نسبه الاصابه بمرض الکولمنارس 4.86%. کما تم مقارنة معدل الإصابة للأوزان المختلفة وفى فصول السنة المختلفة. الأعراض الظاهرية التي شوهدت على خياشيم الأسماک المصابة هى ظهور الخياشيم باللون الشاحب وفى بعض الأسماک شوهد زيادة في نمو طبقات الخياشيم بينما في البعض الآخر شوهد تهتک في طبقات الخياشيم.أيضا شوهد وجود تقرحات على الجلد في المنطقة تحت الزعنفة الزيليه مع تآکل في الزعنفة الزيليه في بعض من الأسماک المفحوصة. الأعراض الظاهرية لأسماک القرموط المصابة تجريبيا بعتره الفلافوبکتريم کولمنارس توضح انه يوجد التهابات في الجلد, تقصف في الزعانف والخياشيم شاحبه في اللون مع تهتک في طبقات الخياشيم الذي يبدا من الخارج. أيضا توجد منطقه باهته عند قاعدة الزعنفة الظهرية تسمى اصابه السدل التي تبدأ صغيره ثم تصبح کبيره. الاصابه بالفلافوبکتريم کولمنارس تنتقل تجريبيا بواسطة الغمر مع جرح أو خدش السمک سواء الخياشيم أو الجلد عند قاعدة الزعنفة الظهرية. بينما العدوى بواسطة الغمر فقط فشلت في ظهور المرض تجريبيا. توضح نتيجة عمل اختبار حساسية دوائية لميکروب الفلافوبکتريم کلومينارس هى انه حساس جدا الى السيبروفلوکساسين, تارفيد (اوفلوکساسين) والسيفوتاکزيم (کلافوران), ولکن مقاوم الى السيفرادين (فيلوسيف).

Summary

The aim of this study was to investigate the prevalence of columnaris disease in wild sharptooth catfish, Clarias gariepinus, in Upper Egypt. Columnaris was detected in 7 (4.86%) fish out of the 144 fish collected indicating light infection. The main signs observed on fish were paleness and sloughing of gill filaments, in addition to skin erosions and fin rot that were seen on some specimens. No specific pattern was detected in weight susceptibility of sharptooth catfish to columnaris. Prevalence of the disease was highest in autumn than in other seasons of the year. Pathogenicity of Flavobacterium columnare isolated in the present study was investigated through an immersion challenge. Fish groups to be challenged were either subjected to skin or gill scarification or remained un-scarified. All challenged fish were immersed in 3.5X10⁷ colony forming units/ml of F. columnare challenge suspension. Clinical signs as loss of appetite and sluggish movement began to appear on fish 48 hours post challenge, while respiratory manifestations and skin erosions appeared later. Two fish died out of the group that had skin scarification. It was interesting to notice that fish challenged through immersion without scarification did not develop typical signs of infection. The antibiogram of F. columnare was also investigated where it was highly sensitive to cefotaxim, ciprofloxacin and ofloxacin, but resistant to cephradine, while moderately to less sensitive to trimethoprim-sulfamethoxazole, E-moxclav, colistin, and flummox.

Key words: Columnaris, sharptooth catfish, flavobacterium, saddle-back,     gill rot

Introduction

Columnaris disease is one of the oldest known bacterial diseases of warm water fish. This disease was first described in 1922 as the cause of a fish kill in The Mississippi River, USA. The causative agent was named Bacillus columnaris because wet mounts prepared from diseased fish showed characteristic column-like masses of bacterial cells (Davis 1922). The nomenclature of the bacterium has changed many times and included the synonyms Chondrococcus columnaris, Cytophaga columnaris and Flexibacter columnaris (Bernardet and Grimont 1989), but in 1996 it was transferred to the genus Flavobacterium and named Flavobacterium columnare (Bernardet et al., 1996).

Columnaris is distributed world wide in aquatic environments, affecting wild and cultured fish as well as ornamental fish, and may result in acute or chronic infections in both coldwater and warm water fishes (Austin and Austin 1999). It occurs both as external or systemic infections that result in significant losses of fish, particularly at warm summer temperatures (Pacha and Ordal 1970 and Becker and Fujihara 1978). Columnaris disease is enhanced by stress factors such as unfavorable water temperature, crowding, injury, another disease, or improper fish husbandry.

Columnaris disease usually begins as an external infection of fins, body surface or gills (Austin and Austin 1999). In temperate fish, columnaris disease is recognized by the appearance of grayish white or yellow areas of erosion, usually surrounded by a reddish hyperemic zone on the body surfaces or the gills of fish. When these lesions occur around the dorsal fin, they are called saddle-back lesions, which are typical for columnaris disease (Austin and Austin, 1987). 

The ubiquitous distribution of the organism in fresh water environments and the tendency for fish to acquire the disease after mechanical and/or environmental stress makes F. columnare among the most detected pathogens in cultured, ornamental, and wild fish populations (Shamsudin, 1994 and Shotts and Starliper, 1999). There are few literature in hand about columnaris in Egypt. Thus, the aim of this study was to investigate the prevalence of columnaris disease in wild sharptooth catfish Clarias gariepinus, in Upper Egypt and detect the pathogenicity of isolated F. columnare strains to apparently healthy fish.  Also, weight and seasonal susceptibility to the disease were investigated.  This study is a component of a comprehensive research project on all Flavobacterium spp. in sharptooth catfish.

MATERIALS and METHODS

Fish collectio:

A total of 144 freshly caught sharptooth catfish, Clarias gariepinus, were collected from different localities of The River Nile, Qena Governorate, over a calendar year (12 fish/month). Collected fish were divided according to their body weight into three groups (Table 1).  The body weight of examined fish ranged from 100 to 500g with mean length of 27.7 to 37.5cm. Fish were transported to The Aquatic Animals Diagnostic Laboratory, Faculty of Veterinary Medicine, South Valley University, Qena, where clinical and bacteriological examination had been conducted.

Table 1: Clarias gariepinus groups according to body weight and length

Clinical and Bacteriological Examination of fish:

Fish were examined for clinical signs or external lesions according to Stoskopf (1993). Opercula were removed to expose the gill tissues and samples were cultured into cytophaga agar medium (Anacker and Ordal, 1959) and incubated at 25º ± 2ºC for 48 hours. Samples from fins were also, cultured directly on cytophaga agar medium. Suspected colonies were picked up and subcultured for purification and identification according to Austin and Austin (1987).

Identification of bacterial isolates:

            Bacterial isolates were identified based on colony morphology and ability to produce flexirubin pigment, growth on selective media, microscopic examination (Gram stain and motility test), biochemical characters that include oxidase, catalase, ability to produce H₂S, methyl red, and sugar fermentation ability (glucose, maltose, and sucrose) according to Austin and Austin (1987).

Pathogenicity of Flavobacterium columnare to Clarias gariepinus:

Fish: Apparently healthy sharptooth catfish with an average body weight of 150-200g and mean length of 27.7-28.8cm were obtained from the River Nile. Fish were acclimated to laboratory conditions for two weeks according to the protocol of maintaining bioassay fish described by Ellsaesser and Clem (1986).

Bacterial challenge suspension and counts: A preliminary growth curve study was conducted to determine counts of colony forming units (cfu) of F. columnare in cytophaga broth at various growth phases using standard plate count method (Elkamel et al., 2003). F. columnare was grown in cytophaga broth at 25ºC to reach an optical density of 0.3 at 600nm, which was found to be equivalent to 7.0X10⁷cfu/ml.  Challenge suspension was prepared by diluting the above F. columnare culture in sterile water (1:1) to reach 3.5X10⁷cfu/ml.

Experimental infection: Acclimated sharptooth catfish were divided into groups of 7 fish each. Fish groups to be challenged were either subjected to skin or gill scarification (Fish and Rucker 1943) or remained un-scarified. All challenged fish were immersed for 30 min in the challenge suspension prepared, while the control group was un-scarified and immersed in a solution of sterile cytophaga broth and sterile water (1:1) as shown in Table 2. Clinical signs and mortalities were recorded daily over 21 days. Moribund fish were examined to record clinical signs and isolate the bacteria from lesion sites.

Table 2: Experimental infection of sharptooth catfish, Clarias gariepinus, with Flavobacterium columnare.

Antibiogram:

            Antimicrobial susceptibility test was investigated on cytophaga agar medium against 8 antimicrobial agents using the disc diffusion technique as described by Finegold and Martin (1982). Antibiotic sensitivity was determined based on the diameter of clearance zone around the discs. Tested antimicrobial agents were cefotaxim (30 μg), cephradine (30 μg), Ciprofloxacin (5 μg), colistin (25 μg), E-moxclav (30 μg), flumox (10 μg), Ofloxacin (10 μg) and Trimethoprim-sulfamethoxazole (25 μg).

Results

Clinical Examination:

Examined wild fish showed a wide variety of gill and skin lesions. Nearly one third of the fish had pale gills, while few showed sloughing of gill filaments (Fig. 1) or hyperplasia in gills. On the other hand, some fish had erosions at the caudal peduncle and fin while others had caudal fin rot.

Bacteriological isolation and identification:

Bacteriological examination of the collected fish resulted in isolation of 59 isolates. According to morphological and cultural characters, 34 isolates were suspected to be Flavobacterium species.  According to biochemical characters, however, the isolates were identified as F. columnare (7 isolates), F. psychrophilum (21isoaltes) and F. branchiophilum (6 isolates).  F. columnare colonies were mucoid, yellow or orange in colour with irregular edges and showed gliding movement on cytophaga agar media.

Table 3: Phenotypic characteristics of suspected Flavobacterium columnare isolates.

Fig. 1: Sharptooth catfish, Clarias gariepinus, naturally infected with Flavobacterium columnare showing paleness and sloughing of gill filaments (arrows).

Survey and prevalence:

Body weight and seasonal susceptibility of sharptooth catfish to F. columnare infections are presented in Tables 4 and 5, respectively.

Table 4: Body weight susceptibility of Clarias gariepinus to Flavobacterium columnare infections

Table 5: Seasonal susceptibility of Clarias gariepinus to Flavobacterium columnare infections

Experimental infection

Clinical signs as loss of appetite and sluggish movement began to appear on fish 48 hours post challenge. Three days later, respiratory manifestations were evident on the fish that had gill scarification. Some fish were found near the surface with open mouth gasping air, while others found at the bottom with respiratory distress. Fin rot of the dorsal and caudal fins were evident almost on all fish. Also, erosions and ulceration of the skin and pale areas were found at the base of the dorsal fin that starts small then enlarge giving the characteristic appearance of “saddle-back lesion” (Fig. 2). Out the group that had skin scarification, two fish died within 2 weeks post challenge showing large pale area and erosions at the base of dorsal fin and paleness and sloughing at the periphery of gill filaments. By the 21^st day, all fish showed signs of advanced infections. Fish that had gill scarification did not show mortalities till the end of the experiment.  Interestingly, fish challenged through immersion without scarification did not develop typical signs of infection described above and survived to the 21^st day.

Antibiogram:

            Flavobacterium columnare in the present study was highly sensitive to cefotaxim, ciprofloxacin and ofloxacin, but resistant to cephradine, and moderately to less sensitive to trimethoprim-sulfamethoxazole, E-moxclav, colistin, and flummox.

Fig. 2: Sharptooth catfish, Clarias gariepinus, challenged after skin scarification with 3.5X10⁷ colony forming units/ml of Flavobacterium columnare showing “saddle-back lesion”.

Discussion

Results of the present study showed that F. columnare causes light infections among wild sharptooth catfish in Qena, Egypt. Out of 144 fish examined over a year, 7 (4.86%) F. columnare strains were isolated from wild sharptooth catfish. Low prevalence of columnaris in the present study may be due to the fact that outbreaks of columnaris are rarely spontaneous and influenced by a combination of environmental and other factors that are stressful to the host (Wakabayashi, 1991) that may not occur frequently in the wild.  Furthermore, Shotts and Starliper (1999) concluded that most species of fish are susceptible to columnaris following some type of environmental stress and when water temperatures are in the upper part of their preferred temperature range.  Columnaris epizootics in natural populations of fish have been, however, repeatedly reported from fish in lakes, rivers and reservoirs across the US, and in anadromous salmonids in the Pacific North-West (Becker and Fujihara 1978). Also, Schachte (1983) stated that epizootics of columnaris disease frequently occur in natural populations of fish and high losses may be observed.

Gills of sharptooth catfish naturally infected by columnaris disease were necrotic and sloughed as previously reported (Post, 1983; Inglis, et al. 1993 and Noga, 1996), while lesions at dorsal fin could not be detected in examined fish. Virulent strains of F. columnaris may attack gill tissue and cause a “gill rot” condition (Wood 1974); however, cutaneous infections seem to be more prevalent in most species of fish (Schachte, 1983).

Weight susceptibility of sharptooth catfish to F. columnare infections showed no specific pattern for a particular weight rendering all body weight groups examined susceptible to the diseases. This is supported by Pacha aand Ordal (1967), Schäperclaus (1992), Noga (1996), who concluded that all ages are susceptible to columnaris.  Younger fish, however, are more susceptible (Schäperclaus 1992) since induction of this disease depends on the immune status of the fish.

Seasonal susceptibility of sharptooth catfish to columnaris demonstrated that the disease was more prevalent in higher temperature as was reported by Wakabayashi and Egusa, (1972), Chen et al. (1982) and Wakabayashi (1991).  Morrison et al. (1981) recorded that outbreaks of saddle back disease caused by F. columnare occurred at 14.4-22.2°C being more severe at the higher end of the range. Columnaris outbreaks may result in high mortality during spring and autumn and are generally associated with poor environmental conditions (Shotts and Starliper 1999). Alos, Durborow et al. (1998) found columnaris disease to occur in channel catfish, Ictalurus punctatus, when temperatures are in the range of 25°C to 30°C in the spring, summer and fall. Furthermore, Decostere et al. (1999) mentioned that the adhesion of high virulence strain of F. columnaris to gills of common carp, Cyprinus carpio (L.), was enhanced in high temperature.

Despite its significance as a pathogen, relatively little is known about the pathogenicity of F. columnare and no reproducible challenge model has been developed. A widely used, but not well-documented challenge model for F. columnare utilizes the scarification or abrasion of the host prior to bath immersion. In contrast to the majority of fish pathogens, artificial infection by a highly virulent strain of this bacterium is more effective by contact exposure than by injection (Pacha and Ordal, 1970). Contact infection represents a more natural way of infection whereas intramuscular injection bypasses the natural defense mechanisms such as skin and mucus. In the current study, clinical signs of the induced infection and route of induction clearly show that columnaris disease does not usually occur as a spontaneous infection but results from abrasion or scarification to fish either to gills or skin as suggested by Post (1987), Noga (1996), Durborow et al. (1998) and Bader et al. (2006).  This may explain the difficulty to induce infections in the present study through immersion of non-scarified fish. In contrast, Decostere et al. (1999) reported that black mollies challenged via immersion in F. columnare bacterial suspension showed respiratory distress and behavioral changes as early as 4 hour post-immersion. Such findings could be due to difference in the virulence of strains used.  Wood (1968) reported that less virulent strains of F. columnare caused outbreaks only at temperatures above 20° C and produced extensive tissue damage, whereas more virulent strains killed fish at temperatures as low as 15° C with no apparent gross tissue damage.

Clinical signs of sharptooth catfish experimentally infected by    F. columnare were, mainly, skin ulcerations, fin rot that usually starts at the outer edges of dorsal and caudal fins and gills affection with sloughing of gill filaments tips. The

 obtained clinical signs were nearly similar to those reported by Pacha and Ordal, (1967); Austin and Austin, (1987); Hawke and Thune, (1992); Inglis, et al. (1993); Noga, (1996) and Stringer-Roth, et al. (2002). The extensive necrosis and tissue destruction associated with F. columnare infections suggests that these bacteria produce strong tissue-destroying enzymes. When attached onto host surfaces, F. columnare release proteases to break down proteins of the host’s extracellular matrix (Aumailley and Gayraud 1998 and Durborow, et al., 1998), thus causing necrotic lesions (Miyoshi and Shinoda 2000). 

Due to the ubiquitous presence of F. columnare in aquatic environments, eradication of the disease in fish farms is not likely to occur. Control and treatment of columnaris have primarily been directed towards the use of improved water-management practices to reduce physiological and environmental stress and use of surface-acting agents (Wakabayashi 1991) as F. columnare primarily attacks gills, skin and fins of fish. On the other hand, medicated feed are commonly used (Noga, 1996). In the present study, F. columnare were highly sensitive to cefotaxim, ciprofloxacin and ofloxacin, but less sensitive to colistin and flummox. These results are nearly similar to those reported by Decostere, et al. (1998) as they recorded that minimum inhibitory concentration (MIC) value is high for colistin.  The bacteria were, also, moderately sensitive to trimethoprim-sulfamethoxazole and E-moxclav as concluded by Decostere, et al. (1998) who stated that MIC values were high for sulfamethoxazole.

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