YERSINIOSIS OF FRESHWATER FISHES IN SOHAG, UPPER EGYPT

YERSINIOSISOF FRESHWATER FISHES IN SOHAG, UPPER EGYPT

EL-SEEDY, F.R.^*; RADWAN, I.A.^*; ABD EL-GALIL, M.A. ^** and SAYED, H.H. ^***

^* Department of Bacteriology, Mycology and Immunology, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef, Egypt.

^** Department of Fish Diseases and Management, Faculty of Veterinary Medicine, SohagUniversity, Sohag, Egypt.

^*** Department of Microbiology, Faculty of Veterinary Medicine, SohagUniversity, Sohag, Egypt.

Email: vet_haitham@yahoo.com                                                                         AssiutUniversity web-site: www.aun.edu.eg

INTRODUCTION

In Egypt, O. niloticus and C. gariepinus are the two main species reared in aquaculture (Ibrahem et al., 2011). Y. ruckeri causes enteric redmouth disease which is a serious bacterial septicaemia in salmonids and other fish species of commercial importance worldwide (Furones et al., 1993). Y. ruckeri presents in a carrier state in many fish species and remains undetected until stressors particularly associated with intensive culture and poor water quality where may result in heavy losses requiring immediate intervention(Horne and Barnes, 1999). Y. ruckeri strains can be divided into two biotypes, biotype 1 and 2 based on motility, Lipase activity, VP reaction, and the ability to hydrolyse Tween (Tinsley et al., 2010) and they are serologically diverse (Austin and Austin, 2007). There is little phenotypic variation between Y. ruckeri strains other than that present in the two biotypes within serogroup O1 (Davies and Frerichs, 1989).

Y. ruckeri were isolated from freshwater fishes in some regions in Egypt (Mosad et al., 1992; Hussein et al., 1997; Abd El-Latif et al., 2001 and Ali et al., 2008). It is well known that diagnosis of a particular infection depends on detection and identification of its causative agent(Das et al., 2014). The present study was carried out to isolate and identify Y.ruckeri from the diseased O. niloticus and C. gariepinus and to estimate the prevalence of yersiniosis (red mouth disease) in Sohag Governorate, Upper Egypt.

MATERIALS and METHODS

Fish samples and clinical examination

Ninety-three samples of the diseased O. niloticus and eighty-seven samples of the diseased C. gariepinus were subsequently collected from the different fish farms and markets of Sohag Governorate during the period from March 2014 to March 2015. These samples included live fishes, moribund and recently dead ones.The collected fishes were transported rapidly to the laboratory in insulated ice box containing ice. They were subjected to clinical and post-mortem examination according to Amlacher (1970)for recording the clinical abnormalities present externally and internally.

Isolation of Y. ruckeri

Aseptically, sterile bacteriological loops from the internal organs (liver, kidney and spleen) were taken and were immediately inoculated into tryptone soya broth (TSB) and incubated under aerobic condition at 25°C for up to 48 hours. The incubated broth was streaked onto Tryptone Soya agar and incubated at 25°C for up to 48 hours (Ross et al., 1966). The isolates were preserved frozen at -80°C in TSB supplemented with 15% glycerol till further use (Bastardo et al., 2011).

Identification on Y. ruckeri

This was done on basis of cultural and morphological characters, conventional biochemical tests plus API 20E system (bioMerieux, France) according to manufacturer's instructions. The isolate was identified according to criteria of Ross et al. (1966), Ewing      et al. (1978), Austin and Austin (2007) and Tinsely   et al. (2010).

RESULTS

Clinical and post-mortem examination

Examination of O.  niloticusinfected with Y. ruckerirevealed presence of extensive hemorrhages on the skin, congestion of the lips, isthmus region, oral cavity and the fins. Erosions of the oral cavity were detected. Internally, there were haemorrhages in the muscles, hyperemia and severe congestion of the spleen and kidney. Hemorrhagic gastroenteritis (Figs. 1, 2, 3 and 4).

Concerning C. gariepinus infected with Y. ruckerirevealed the presence of hemorrhages on the skin, lips, isthmus region & in the eyes and congestion of the fins. There were hyperemia and congestion of the spleen, kidneys, and intestine (Figs. 5, 6, 7, 8 and 9).

Y. ruckeri isolation from the examined fishes

Y. ruckeri were isolated from the examined O. niloticus (3.2%) and C. gariepinus (2.3%).

Phenotypic identification of Y. ruckeri isolates

On Tryptone Soya agar, colonies of Y. ruckeri were small, round, white and creamy.Microscopical examination for Gram-stained smears prepared from these colonies, revealed Gram-ngative short bacilli to coccobacilli. They were motile Biochemical characterization of the isolates revealed that all the 5 isolates were biochemically homogeneous except in Voges-Proskauer test and gelatine liquifaction (Table 1). Based on results of phenotypic characterization for Y. ruckeri isolates, these isolates were belong to Biotype 1 of Y. ruckeri and similarity percentage of their phenotypic characteristicswas ranged from 92.9 to 100%.

Fig. 9: C. gariepinus infected with Y. ruckeri showing congestion in the liver and intestine.

Table 1: Biochemical characteristics of Y. ruckeri isolates.

DISCUSSION

Y. ruckeri causes enteric redmouth disease which is a serious bacterial septicaemia in salmonids and other fish species worldwide (Furones et al., 1993). In this study, it was found that prevalence of Y.ruckeri infection among the examined O. niloticus was 3.2%. This finding nearly agreed with results of Ali et al. (2008) who reported that incidence of Y. ruckeri among the diseased O. niloticus at Beni-Suef Governorate (2.7%). Very higher rate of Y. ruckeri in O. niloticus was recorded by Essam (2004)whoisolated Y. ruckeri from the clinically infected, asymptomatic dead and apparently healthy O. niloticus from Egyptian farm with incidence 88%, 76% and 56% respectively. This disagreement may be attributed to the fluctuating climatic changes, faulty management practices and shedding of large number of bacteria in water under stress. Prevalence of Y. ruckeri infection among the examined C. gariepinus was 2.3%. Higher rate was recorded by Abd El-Latief et al. (2001) who recorded that prevalence of Y. ruckeri among the diseased C. gariepinus in Nile Delta region was 11.2%. This disagreement may be attributed to the fluctuating climatic changes, shedding of large number of bacteria in water under stress and location of the study.

Examination of O. niloticusinfected with Y. ruckerirevealed presence of extensive hemorrhages on the skin, congestion of fins, lips, snout region & oral cavity and erosions in tissues of oral cavity. Internally, there were haemorrhages in the muscles, hyperemia and severe congestion of the spleen, kidneys, and intestine (hemorrhagic gastroenteritis). These clinical signs and post-mortem lesions were similar to those reported by El-Gamal et al. (2005) and Eissa et al. (2008). While examination of C. gariepinus infected with Y. ruckeripointed number of the clinical abnormalities including hemorrhages on the skin, lips, snout region & in the eyes and congestion of the fins. Internally, there were hyperemia and congestion of the spleen, kidneys, and intestine. Similar signs and post-mortem lesions were reported by Abd El-Latief et al. (2001). The distinctive redmouth feature of ERM was recorded in some affected C. gariepinus and this agreed with Horne and Barnes (1999)who stated that the distinctive redmouth feature of ERM isn't invariably noticed.

Y. ruckeri suspectedcolonies on Tryptone Soya agar grown forming small, round, white-creamy and this result was similar to findings of Ross et al. (1966), Seker et al. (2011) and Seker et al. (2012). By microscopical examination, the isolates were Gram-negative short bacilli to coccobacilli and motile. These results were similar to findings of Abd El-Latif et al. (2001) who isolated Gram-negative, motile short coccobacilli or short-medium sized bacilli Y. ruckeri from O. niloticus, C. lazera and C. carpio and to some extent differed from findings of Bastardo et al. (2011) whoisolated five non-motile Y. ruckeri strains in Peru.

In regarding to the biochemical characteristics of Y. ruckeri isolates, results of the conventional biochemical tests and API 20E assay as shown in table (1) revealed that all Y. ruckeri isolates were biochemically homogeneous except in Voges-Proskauer and gelatine liquifaction tests. All the isolates were positive in lysine decarboxylase, ornithine decarboxylase, β-Galactosidase, citrate utilization, methyl red and utilization of glucose and mannitol and negative in indole production, H₂S production, urease, arginine dihydrolase, tryptophane deaminase, and utilization of inositol, sorbitol, rhaminose, sucrose, melibiose, amygdalin and arabinose while 60% and 80% of them were positive in Voges-Proskauer test and gelatine liquifaction test respectively. The biochemical characteristics of Y. ruckeri in this study were in agreement with findings ofEwing et al. (1978).

The variation among Y. ruckeri isolates in Voges-Proskauer test was reported by Abd El-Latif et al. (2001) and Bastardo et al. (2011)who found that 10.4% and 23.4% of Y. ruckeri isolates were positive in this test respectively. In contrast, Eissa et al. (2008) and Joh et al. (2010) found that Y. ruckeri isolates were homogenous and Voges-Proskauer positive while Şeker et al. (2012) and Altun et al. (2013) found that all Y. ruckeri isolates were Voges-Proskauer negative. Also, the variation among Y. ruckeri isolates in gelatine liquifaction test was reported by Altun et al. (2010) who reported that 23.8% of Y. ruckeri isolates were gelatine liquifaction test negative while Eissa et al. (2008) reported that all Y. ruckeri isolates were gelatine liquifaction test positive. The variation in any phenotypic characteristic may be attributed to presence or absence of plasmid (s) that controls its metabolic trait.

Based on results of the phenotypic characterization of Y. ruckeri isolates, it was concluded that the isolates were homogenous except in Voges-Proskauer and gelatine liquifaction tests, similarity of their phenotypic characteristicswas ranged from 92.9 to 100% as showed in table (19) and they were belonged to Biotype 1.

CONCLUSION

In this study, five Y. ruckeri isolates were isolated from O. niloticus and C. gariepinus at Sohag Governorate for the first time at this region, these isolates were homogeneous in their phenotypic characters except in Voges-Proskauer and gelatine liquifaction tests.

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