Casualty Agent (Yersiniosis) Detection in Reared Rainbow Trout (Oncorhynchus Mykiss) in Kohgiluyeh-Va-Boyerahmad Province in Iran

Nowadays, Rainbow trout culturing has been developed in Kohgiloyeh-&-Boyer Ahmad province of Islamic Republic of Iran. It is a province in the southwest of Iran. There are many water resources in the province which originated from Zagross mountains so aquaculture is a lucrative profession in the province. Rainbow trout cultivated lonely. In 2013 AD about 13400 tons fish (Oncorhynchus mykiss) cultivate in the province. It cost is about 38 million dollars [1]. Infectious diseases are being emerged due to developing fish culture at artificial condition especially at intensive systems. On the other hand circulated water and intensive system increase disease incidence [2]. Fish are depressed by low water flow in warm seasons and infectious agents may release in ponds by recirculated water. Depression decrease immunity in fish and water recirculation spread pathogens in fish flock horizontally especially while circulated water wasn’t treated properly. Yersinia ruckeri causes Enteric-red-mouth disease (ERM) in rainbow trout. It is an important disease of salmonid fish and was first described by Ross et al. [3] and Rucker [4]. The disease has been called in other word such as: Yersiniosis, Yersinic septicemia, Hagerman-redmouth-disease, etc. ERM produces high mortalities and severe economic losses on fish farms [5].


Introduction
Nowadays, Rainbow trout culturing has been developed in Kohgiloyeh-&-Boyer Ahmad province of Islamic Republic of Iran. It is a province in the southwest of Iran. There are many water resources in the province which originated from Zagross mountains so aquaculture is a lucrative profession in the province. Rainbow trout cultivated lonely. In 2013 AD about 13400 tons fish (Oncorhynchus mykiss) cultivate in the province. It cost is about 38 million dollars [1]. Infectious diseases are being emerged due to developing fish culture at artificial condition especially at intensive systems. On the other hand circulated water and intensive system increase disease incidence [2]. Fish are depressed by low water flow in warm seasons and infectious agents may release in ponds by recirculated water. Depression decrease immunity in fish and water recirculation spread pathogens in fish flock horizontally especially while circulated water wasn't treated properly.
Yersinia ruckeri causes Enteric-red-mouth disease (ERM) in rainbow trout. It is an important disease of salmonid fish and was first described by Ross et al. [3] and Rucker [4]. The disease has been called in other word such as: Yersiniosis, Yersinic septicemia, Hagerman-redmouth-disease, etc. ERM produces high mortalities and severe economic losses on fish farms [5].
Enteric red mouth disease (ERM) has been one of the most significant diseases in salmonid aquaculture. Even though the disease has been diagnosed more than 25 years ago and commercial vaccines are available [6,7]. ERM have reported from several countries such as turkey, USA, Romania and Iran [8][9][10][11]. ERM is being reported since last few years from some provinces (Tehran, Fars, Hamadan and Chaharmohalvabakhtiyari) in Iran [12][13][14][15].
Signs of ERM particularly in infected rainbow trout may include hemorrhages in various tissue and organs, particularly

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around the mouth, mouth cavity, in the gills, in the musculature, the peritoneum, the body fat, viscera and in intestine. Clinical infection can also be characterized by a yellow discharge from the vent. Furthermore, severe inflammations of the distal portion of intestine and vent are frequently observed [16,17].

Fish
Three ponds from eight ponds have more mortality. It estimated that each pond contained about 4000 fish. 45 moribund rainbow trout with abnormal signs such as skin darkness, bad swimming, hemorrhagic were sampled from the ponds and examined for the presence of the etiological agent of the disease by dissection and bacteriological procedures.

Bacteriological examination
Plate culture is commonly used to isolate Y. ruckeri, so it used to isolation, then identification done by biochemical tests and staining [20,21].
Kidneys, Livers and Spleen of fish were cultured aseptically by streaking a loop onto Blood Agar (BA), Tryptic Soy Agar (TSA), Thiosulphate Citrate Bile Sucrosr Agar (TCBS) and MacConkey Agar plates and incubated at 30 °C for 48 hours. Isolated colonies were subcultured onto TSA and identified using conventional biochemical tests [22]. Characteristics of our isolated bacterium were compared with the reported biochemical properties of the Yersinia ruckeri in the literature [2,6,21,22].

PCR method
Isolated colonies of bacteria evaluated by PCR assay. The bacteria cultured on TSB (Trypticase soy broth) medium (Merck, Germany) and was incubated at 30 °C for 48 hours, Then DNA isolated and extracted from it. Therefore, One milliliter of culture was centrifuged for 5min at 12,000 × g, the pellet was resuspended in 50ml of distilled water, and cells were lysed by boiling for 10min. Cell debris was removed by centrifugation for 30s at 12,000 × g. Aliquots of 5 µl of supernatant were removed and was used as template DNA for the PCR assay. Thermal cycling was done with the following conditions: an initial denaturation cycle at 94 °C for 2min, followed by 35 cycles of amplification (denaturation at 94°C for 40s, annealing at 60 °C for 40s, and extension at 72 °C for 60s), and a final 5min elongation period at 72 °C. Finally, PCR product were analyzed by electrophoresis on 1.5% agarose gels and stained with ethidium bromide [24].

Anti biogram test
Anti biogram test achieved based on disk diffusion method [23]. Gross clinical signs were characterized in infected fish by general lethargy, swimming difficulties and swimming close to the surface of the water or bottom of the pond. Often, hemorrhages were observed in the gills, around eye, around mouth, and in oral cavity. Dark pigmentation was commonly. Dissection shows some petechial hemorrhages on the surface of the liver. Furthermore, the peritoneum was occasionally filled with a yellowish fluid. Infected fish have lethargy and low appetite. Exophthalmos was sometimes accompanied by hemorrhages around the ocular cavity in some cases. The liver was slightly pale discoloration. A Gram's(-) negative bacterium isolated from all samples. All sampled fish have infected and a bacterium isolated from their livers, kidneys and spleens. So, the septicemia has been confirmed. Diagnosis of the causative agent of disease is important to specify a preventive strategy. Identity biochemical tests did to detection of the bacterium clearly so PCR-assay done. The bacterium did not growth on TCBS media therefore it was not member of Vibrio genus.It was a gram's negative bacteria which was able to growth on MacConkey agar medium. Its catalase test was positive while its oxidase test was negative. It wasn't Streptococcus bacteria based on catalase test. Other test which achieved to detection of the bacterium summarized in Table 1.

Results
In the current study, some isolated colonies (10 colonies) were diagnosed from 45 moribund fish and compared with the reported results of biochemical characteristics of Y. ruckeri by Austin and Austin [22] and Akhlaghi [2]. The isolated bacteria in this study found to be very similar with Y. ruckeri. After biochemical tests some isolated colonies (10 colonies) evaluated by PCR assay. However, PCR procedure confirmed that the isolates are belong to the Yersinia genus as Yersinia ruckeri species.

Discussion
All samples were contaminated and a type of bacterium isolated from Liver, Kidney and spleen of all the moribund fish. Only a kind of colony has been grow on culture media, so mix infection didn't confirm. On the other hand, Gram's staining and growing on MacConkey agar show that the septicemia caused by a type of gram negative bacterium. The bacterium has been seen in form of cocco-rod shape. Therefore, according to the issue and also according to the examining results of catalase was inference such that mentioned bacterium couldn't be Streptococcus and Lactococcus. Also, the lack of growing on Vibrios'-selective medium (TCBS agar) caused to was rejected the possibility of existence of vibriosis illness. It should be acknowledge that the separated bacterium was similar to Yersinia ruckeri from the view point of characteristic features, according to clinical signs and performed biochemical tests. It confirmed by PCR procedure.
It stated that yersiniosis was observed in rainbow trout at acute and chronic form and the bacteria may cause infection in all of ages of the fish [2]. Our observation also shows that some infected fish indicate clinical signs obviously, and the others have fewer signs with more activity and affected by pathogen later. Also, this matter is the reason of casualty's stairs. It founded that weaker fish become ill too early and stronger fish subsequently. The total mortality was about 1.87 to 7.5 percent in the 3 pounds.
Although 14 biochemical tests achieved but identification was not valid so there is need to a procedure to complete detection. Bacterial detection performed by PCR-assay. It confirmed the identity of the bacteria. PCR results shows that the bacteria were belong to the Yersina ruckeri species clearly.
Also, in current research both of fry fish and adult fish were infected and pathogen found in both of them. Therefore, the bacterium is able to cause infection in fish at different ages. But, we ought to mention that infection was acute in fry fish compare with adults. Guguianu also has been declared fry fish are more susceptible with the yersiniosis [9]. The application of Tetracycline or Enrofloxacine for 7 days as food additive show that both of the antibiotics are effective against this disease and cause to reduce mortalities but wasn't enough to eradicate of the disease. So, it recommended that the length of therapy should select more than 7 days.