Shmeem Ummey, Ph.D, Shrmil Khn, Ph.D, Reserch Scholr, P.P.N Vijykumr, Ph.D,Reserch Scholr, Adnki Rmy, M.Sc. Zoology, Reserch Scholr

a Department of Zoology, Andhra University, Visakhapatnam, 530003, India

b Advanced Analytical Laboratory, Andhra University, Visakhapatnam, 530003, India

Keywords:

ABSTRACT

1. Introduction

India is the second largest fish producing country and hold second position as aquaculture producing nation in the world (DADF (2016).The sector has shown significant growth from traditional culture practices to commercial methods of culture, enhancing fish production from mere 0.75 million tons in 1950-51 to 10.795 million tons during 2015-2016, showing an earning of rupees 334.41 billion in 2014-15 (US$5.51 billion) through export of fish to different countries (DADF, 2016;FAO, 2014, 2016).The state of Andhra Pradesh is well known for its fish culture activities, especially involving Labeo rohita and Catla catla which are being extensively cultured both in Fresh water monoculture and polyculture systems. But, for the past few years the aquaculture production has witnessed a gradual downfall due to outbreak of various diseases, especially those caused by bacterial infections like Haemorrhagic septicemia (HS), Bacterial Gill Disease (BGD), Columnaris Disease (CD), Epizootic Ulcerative Syndrome (EUS) and Enteric Red mouth Disease (ERMD).

Enteric Red mouth disease is a bacterial disease of fresh water fishes.It was first reported in early 1950’s in a rainbow trout, Oncorhynchus mykiss from a trout farm at Idaho, USA by Rucker (1966). Later, it was found throughout USA, Europe, Australia, Africa and Asia (Zorriehzahra, Adel, & Torabidelshad, 2017). Ross, Rucker, and Ewing (1966)gave a description of the bacterium associated with Red mouth disease of rainbow trout (Salmo gairdneri) and reported it as “RM bacterium”. In recognition of that first description, the causative organism was named as “Yersinia ruckeri” by Ewing, Ross, Brenner, and Fanning (1978). Later studies were carried out on 18 strains of Y. ruckeri from a variety of hosts covering a wide geographic range (Bullock, Stuckey, & Shotts, 1978). A PCR-based method was developed for the specific detection of Y. ruckeri from tissues of experimental and naturally infected trout’s by (Gibello et al., 1999).

In India, very few reports are available on ERMD in freshwater fishes.It was first reported by Sharma, Katoch, Nagal, Sambhyal, and Asrani(1995) in rainbow trout from Himachal Pradesh. Manna et al., (2003)biochemically characterized Y. ruckeri isolated from tissues of IndianMajor Carps (L. rohita, C. catla and C. mrigala) from West Bengal. Surandraraj et al., (2009) detected high number of enteric bacterial counts,in farmed freshwater fish and its cultural environment in Kerala consisting of Salmonella, Shigella and Yersinia sps. Recently Kumar et al.,(2015) reviewed the current status on ERMD and discussed various control methods.

Table 1 Reports on Enteric Redmouth disease in freshwater fishes from all over the world.

Table 1 (continued)

Table 1 (continued)

Table 2 Reports on Enteric Red Mouth disease in freshwater fishes from the Indian region.

The present study reports for the first time the occurrence of ERMD from culture ponds of Andhra Pradesh, in L. rohita and C. catla. The bacterium was identified using both conventional microbiological and biochemical methods and molecular PCR based techniques.

Reports on Y. ruckeri from the Indian region are provided in Table 1.

2. Material and methods

The present investigation has been undertaken for a period of two years from July 2013-June 2015.

2.1. Study area

The study area covered culture ponds situated at Chinnapuram,Chakalipeta villages from Vizinagaram district, Padmanabham from Visakhapatnam district, Akiveedu, Bhimadolu and Eluru from West Godavari district and Kaikuluru and Mandavalli village from Krishna district.

2.2. Host

Two species of Indian Major Carps Labeo rohita Hamilton, 1822 and Catla catla Hamilton, 1822, which are cultured widely in the kolleru and non-kolleru regions of Krishna and West Godavari districts were used to collect the samples.

Fish showing symptoms of ERMD were brought to a nearby scientific laboratory owned by State Fishery Department in live or moribund condition and autopsied under aseptic conditions to collect tissue samples. Blood, liver, kidney, spleen, muscle and gill samples were inoculated aseptically into nutrient broth, labelled and incubated for 24 h at 37C to check the growth of bacteria. Positive samples were used for microbiological studies.

2.3. Biochemical analysis

Aliquots of serially diluted samples were inoculated on nutrient agar,and morphologically similar colonies were isolated again and again on non-selective media like Nutrient agar and Trypton Soya Agar (TSA) and also on selective media like Blood agar, Mac-Conkey agar and Commasie Blue-TSA-SDS-Agar, until pure cultures were obtained and details on colony morphology were recorded (Table 2). Biochemical characteristics were analyzed for cultures suspected as isolates of Y. ruckeri following the standard procedures of Bergey’s Manual of Bacteriology(1984) and Cowman’s and Steel’s Manual for the Identification of Medical Bacteria (1993).

3. Molecular studies

3.1. DNA extraction

DNA isolation and purification has been carried out by following the Phenol - Chloroform method (Sambrook & Russell, 1985). Extracted DNA was visualized through gel electrophoresis on 1.5% agarose before being stored at 4C.

Fig.1. Symptoms of ERMD collected in C. catla, (a). Hemorrhageson mouth & eyes, (b). Hemorrhaging of mouth on lower side, (c). Blackening of skin and hemorrhagesin mouth, (d).Accumulation of mucoid liquid with blood clots, (e). Hemorrhages on the ventral side of the mouth region, (f). Reddening around the mouth, operculum and oral cavity.

3.2. Primers and PCR assay

A forward primer YER8 (422F) (5-GCGAGGAGGAAGGGTTAAGTG-3) and a reverse primer YER10 (1010R) (5-GAAGGCACCAAGGCATCTCTG-3) (Gibello et al.,1999) available from the GenBank database were selected for PCR amplification. PCR was performed by using Kapa readymade reaction 94 mix purchased from KapaBio systems, US.The reaction mix was composed of approximately 100 ng total genomic DNA as template, 2.5 μl reaction buffer, 2 mM Mgcl2, 0.2 mM of each dNTP,0.5 μm of each primer, 1U Taq DNA Polymerase and filled up to 25 μl with sterile double-distilled water. PCR was performed in Master cycler personal (Eppendorf) thermal cycler, using 25 cycles of denaturation for 1 min at 92C, annealing for 1 min at (57-60C), extension for 1 min at 72C, followed by a final extension of 5 min at 720C.PCR-generated products were detected by using 1.5% agarose gel.

Table 3 Growth of bacterial pathogen on different culture media.

4. Results

Symptoms of ERM disease, viz sub-cutaneous hemorrhages surrounding the mouth, fins and eyes, blacking of skin, bleeding at the base of fins, inside and around mouth, bilateral exophthalmia with or without haemorrhages and periocular and perioral haemorrhages in severe cases were recorded in fishes collected from culture ponds along the coast of Andhra Pradesh, more common in those from West Godavari and Krishna districts (Fig.1 a-f). Data on prevalence of infection was recorded for a period of 24 months from July 2013-June 2015. The assessment of prevalence was based on externally visible disease specific symptoms and internal examination of fish to confirm the type of infection. Out of 1440 fish examined, from all districts 922 fish were found to be infected, recording a prevalence of 64%. Among different varieties of bacterial diseases identified, 67 fish were found to be showing symptoms of with ERMD, recording a prevalence of 7.26%.

4.1. Microbiological studies

Altogether 402 tissue samples were tested from various organs and tissues of L. rohita and C. catla, and only 22 samples were identified as those belonging to Y. ruckeri after carrying out biochemical characterization (Table 3).

4.2. Colony characteristics

Colonies appeared white, cream coloured colonies, translucent or opaque, smooth, round with somewhat irregular edges. Coloured colonies were noticed on specific media like Blood Agar and Mac-Conkey Agar, and also on Tryptone Soya Agar and Nutrient Agar whereas on Commassie Blue- TSA-SDS agar colourless colonies were noticed (Fig.2,a -d).

4.3. Biochemical characterization

Pink coloured, motile, straight or slightly curved rods were observed under the microscope. The bacteria are gram negative, oxidase negative and Catalase positive in reaction.

The results of various determinative tests, indicated the bacteria to be negative for indole, positive for Methyl red, negative for Voges Proskauer’s, positive for Citrate Utilization, Nitrate Reduction, ONPG,negative for H2S production, negative Urease, positive for Salt Toleranceat0%, 3% but negative at 6.5%, positive for OF showing fermentative reaction (Oxidative Fermentative), positive for Gelatin Hydrolysis test showing the presence of the gelatinase enzyme, positive for Starch hydrolysis test, Esculin Hydrolysis test, negative for Arginine Dihydrolase, positive for Lysine Decarboxylase, Hemolysis test showing the presence of hemolysin protein and negative for Phenyl alanine deaminase. Carbohydrate fermentation reaction showed positive reaction for glucose, xylose, maltose, fructose, dextrose, galactose, raffinose,trehalose, melibiose, sucrose, l-arabinose, mannose, sodium gluconate,glycerol, salicin, mannitol, rhamnose, celliobiose, xylitol and malonate sugars and negative reaction for lactose, insulin, dulcitol, 139 inositol,sorbitol, adonitol, arabitol, erythritol, sodium gluconate, rhamnose,methyl-d-glucoside, melezitose, methyl-d-mannoside, d arabinose and sorbose (Fig.2 e, f).

A comprehensive chart Comparing the biochemical characteristics of present isolate, those prepared from Bergy’s Manual of Bacteriology,1984, Cowman and Steel’s Manual for the Identification of Medical Bacteria, 1993, and report available on Y. ruckeri is furnished in Table 4.The present Y.ruckeri isolate showed results similar to those reported by Ewing et al. (1978), Manna, Samanta, Das, and Mishra (2003), Eissa,Moustafa, Abdelaziz, and Ezzeldeen (2008), S¸eker, Karahan, Sarıeyyüpo˘glu, and Çetinkaya (2011), Abdel 147 latief et al. (2014) and Mohammed saeed G (2017).

Fig.2. Growth of Yersinaruckerion different culture media, (a). On Nutrient Agar, (b) On Blood Agar, (c) On Mac Conkey Agar, (d) On Coomassie Blue-TSA-SDS Agar, (e) Biochemical test -results: indole, MR, VP, Ornithine, Lysine, Citrate(f) Biochemical test kit - results: Part ALactose,Xylose, Maltose, Fructose, Dextrose, Galactose,Raffinose, Trehalose, Melibiose, Sucrose, L-Arabinose,MannosePart BInsulin, Sodium gluconate,Glycerol, Salicin, Dulcitol, Inositol, Sorbitol,Mannitol, Adonitol, Arabitol, Erythritol, Alpa-Methyl-D-glucoside. Part CRhamnose, Cellobiose, Melezitose, Alpa-Methyl-D-Mannoside,Xylitol, ONPG, Esculin, D-Arabinose, Citrate,Malonate, Sorbose, and control. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Table 4 Comparative biochemical characterization of Y. ruckeri.

Table 4 (continued)

4.4. DNA extraction

DNA samples extracted from all the 22 isolates identified as Y. ruckerishowed clear bands on gel electrophoresis run on 1.5%agarose. DNA bands were noted against 100bp DNA ladder (100bp-3000bp) used as molecular marker (Fig.3).

4.5. PCR analysis

In the PCR analysis only ten DNA samples have shown significant amplification with specific primers (YER 8 & 10) in PCR analysis, producing a strong amplification product with a band at 500 bps (Fig.4).

5. Discussion

Very few reports are available from the Indian region so far on Enteric Redmouth disease viz.Manna et al., (2003), Sharma et al. (1995)and Surendraraj, Sabeena, Yathavamoorthi, and Thampuran (2009).The present study records first report of ERM from the state of Andhra Pradesh. The clinical signs of the disease like haemorrhagic inflammation in hindgut, fluid accumulation in stomach and intestine, enlargement of hematopoietic organs like kidneys and spleen are well reported by many from various regions and fish hosts viz. Rucker 162 (1966),Gibello et al., (1999), Avciet al.,(2005) and Tobback et al.,(2007)).Wobeser (1973) reported ERMD in rainbow trout, Salmogairdnerifrom Southern Saskatchewan lake with symptoms like congestion of blood vessels, petechial haemorrhages affecting liver, pancreas, swim bladder,lateral muscle and adipose tissue associated with pyloric caecae.Leselet al., (1983) reported subcutaneous haemorrhages in the mouth,under the tongue, in gills, around the eyes, under the skin, at the base offins and around the vent, on liver, pancreas, pyloric caeca, intestine and swim bladder and oedema ofstomach wall in rainbow trout, Salmogairdnericollected during an outbreak of enteric Redmouth disease.Meier (1986) reported hyperaemia around the mouth and in the isthmus in rainbow trout, Salmogairdnenicollected fromRichardson, Switzerland.Manna et al., (2003) recorded typical ERMD symptoms like raised subcutaneous haemorrhagic areas, haemorrhages at the base of fins,blackening of gills, superficial ulcers and tail and fin rot. Besides,petechial haemorrhages were noted in the liver, body fat and peritoneal cavity with enlarged and highly congested spleen, necrosed and fragile kidney in L. rohita, C. catla and C. mrigala from Naidia district, West Bengal, India. Bravo and Kojagura (2004) reported clinical symptoms like reddening around the mouth, operculum and oral cavity, haemorrhages around the ocular cavity, an inflamed and haemorrhagic vent,haemorrhagic lower intestine, an empty gut filled with yellowish fluid,and a dark and enlarged spleen in ERMD affected hatchery reared rainbow trout, Oncorhynchus mykiss in Peru. Abdel-Latif, Khalil, Saad,and El-bably (2014) reported septicemic signs of yersiniosis in Oreochromisniloticusat Kafr El-sheikh in Egypt with haemorrhages on all organs and skin. Kumar et al., (2015) reported exophthalmia, darkening of skin, splenomegaly and inflammation of lower intestine with accumulation of thick yellow fluid in salmonid fishes. Mohammed Saeed (2017)reported clinical symptoms like general lethargy, swimming difficulties and swimming close to the surface of the water or bottom of pond,haemorrhages in gills, around eye, mouth and in oral cavity, dark pigmentation, internally petechial haemorrhages on surface of the liver,occasionally peritoneum filled with yellowish fluid in rainbow trout from Kohgiluyeh-Va-Boyerahmad Province in Iran. During the present study also similar symptoms were noticed in infected fishes, however the severity of infection varied from host to host.

Growth recorded on Blood agar (BA) and Mac-Conkey Agar showedwhite and cream coloured colonies, but translucent to opaque and smooth, round colonies with somewhat irregular edges was noted on TSA. Sekeret al. (2011) reported growth of Y. ruckerion TSA and Shotts-Waltman(SW) showing small round, white and cream coloured colonies of 1-2 cm diameter. Zorriehzahraet al. (2017) after incubation for 48hrs recorded white, opaque colonies of approximately 2-3 mm in diameter on BA and TSA.

Fig.3. Agarose gel electrophoresis of Genomic DNA of Y. ruckeri isolates Lane M: 1- Kb DNA ladder, Lane NC-Negative controlLane 1-13: Y. ruckeri isolates.Molecular weight markers (bp) are indicated to the left of the gel.

Fig.4. PCR amplification of Y. ruckeri DNA samples using specific primers(YER 8 & YER 10). Lane M: 1 Kb DNA ladder, Lane 1-6: Y. ruckeri isolates.Molecular weight markers indicated to the left of the gel.

In the present study, 22 isolates of Y. ruckeri were identified as positive from 402 samples collected from various organs and tissues of fish showing symptoms of ERMD. Biochemical characterization of the present isolates showed negative reaction towards Voges-Proskauer and inability to produce arginine and rhamnose when compared with Y. ruckeristrains reported by Eissaet al. (2008) and Abdel-latiefet al.(2014). The present isolates differ from Y. ruckeristrains reported by Manna et al., (2003) in showing negative reaction towards Xylose,Salicin, Sorbitol, Mannitol and Celliobiose utilization. The present isolates were also compared with Savvidiset al. (1990), Austin and Austin(1999), Horne and Barnes (1999), Rodriguez, Castillo, Gallardo, and Nieto (1999), Romaldeet al. (2003), Akhlaghet al. (2008) where they showed some variation in a few test results.

The greater speed and sensitivity of PCR compared to bacteriological culture of Y. ruckeri were discussed by Argenton. et al., (1996) and Gibello. et al., (1999). Some authors have proposed that early diagnostics of yersiniosis may be achieved within 12 h by PCR analysis of a small amount of blood from the heart of a live fish (Altinoket al., (2001),211 Seker et al., (2011).Altinok et al. (2001) evaluated a PCR method for detecting Y. ruckeri, in the blood of rainbow trout. S¸eker. et al. (2011)extracted 212 DNA from blood samples and carried out PCR amplification based upon a pair of Y. ruckeri specific primers. Mohammed Saeed(2017) evaluated PCR assay by using YER3 and YER 4 primers and confirmed that the isolates belong to the yersinia genus as Yersina ruckeri. Cao et al. (2018) isolated and identified Y.ruckeriby physiochemical characteristics, 16S rRNA and gyrB gene sequences analyses in hybrid sturgeon (Acipenserschrenchi) from a farm of Qionglari, Siehuan Province, China. In the present study, primer set YER 8 - YER 10 designed by Gibelloet al., (1999) based on amplification of non - conservative rDNA regions were used to detect and identify Y. ruckeri.Amplification of DNA extracted from all isolates were confirmed with the specific primers as Y. ruckeri by producing the suspected size of 500 bp.

The present work stands out in the fact that, it is the first report of Yersiniosis from the state of Andhra Pradesh and adds to the scientific knowledge on ERM from the Indian region.

CRediT authorship contribution statement

Shameem Ummey: Project administration, Supervision, Writing -review & editing, Data curation. Sharmila Khan: Formal analysis, Data curation. P.P.N Vijayakumar: Formal analysis, Data curation. Adanki Ramya: Formal analysis, Data curation.

Acknowledgements

The authors express their sincere thanks to Advanced Analytical Laboratory, Andhra University, Visakhapatnam for providing necessary facilities. The financial support was provided by a fellowship Grant from“Maulana Azad National Fellowship Programme” New Delhi, India.