Yersinia ruckeri is the causative agent of Yersiniosis or Enteric Red mouth Disease that causes huge amount of economic loss in mariculture industry worldwide. Definitive detection and effective treatment for septicemia like Y. ruckeri that outbreak critically, requires rapid and specific diagnosis. Despite the advantages of both PCR and Histopathology techniques in the terms of diagnosis and monitoring they have some defects, the aim of this study is the comparison of PCR and histopathology methods for detection of Yersiniosis in trout fish in Iran.
In PCR-based detection, Extracted DNA from suspected Rainbow trout tissue utilized in PCR reaction and polymerase chain products were visualized by gel electrophoresis. In pathological detection, all tissues from live or moribund fish were fixed in 10% formalin saline. Then Samples were embedded in paraffin block and sectioned with digital microtome at 5-7 micrometer thickness. Slides were stained by haematoxillin & eosin, and then treated with mounting media.
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In this study, 20 suspected samples were tested by both techniques that 2 samples were positive in PCR, and 5 samples were positive in pathologic technique with especially clinical signs such as acute septicemia in internal tissues and superficial hemorrhage with bleeding in the base of fish or around the mouth. Differences in the number of positive samples in this comparison demonstrated that each mentioned technique has its advantages so if the advantages of each method exploit and one method supplements the other one, the efficiency and accuracy of monitoring the experiments would boost.
Yersiniosis or enteric red mouth disease (ERM) is one of the most common and fatal bacterial diseases in salmonid farms, the first isolation of the disease were reported in rainbow trout (Onchorhynchus mikiss) in the USA in the 1950s (Bullock et al., 1978; Rucker, 1966; Tobback et al., 2007). Yersinia is a gram negative coccobacillus, non-spore-forming bacterium that belongs to the Enterobacteriaceae family that often has flagella, actively growing cell are approximately 0.75 Î¼m in diameter and 1.0-3.0 Î¼m in length (Rucker, 1966; Tobback et al., 2007).
Infection may result in a septicemia condition with hemorrhages on the body surface and in the internal organs. It is clinically characterized by bilateral exophthalmia, hemorrhages of mouth and gills, abdominal distension because of fluid accumulation, general septicemia with inflammation of gut, diffuse hemorrhages with in a swim bladder, enlarged black color spleen and the intestine is inflamed and filled with purulent material. Hemorrhagic inflammation in hindgut, accumulation of fluid in stomach and intestines, enlargement of hematopoietic organs, kidneys and splenomegaly are among the pathologic landmarks (Rucker, 1966; Gibello et al., 1999; Avci and Birincioglu, 2005; Tobback et al., 2007). various PCR methods have been suggested for diagnosis of Yersinia ruckeri (Argenton et al., 1996; Gibello et al., 1999; Altinok et al., 2001; Lejeune and Rurangirwa, 2000; DelCerro et al., 2002; Roozbahani et al., 2009) .Pathologic technique is one of the beneficial methods in detection of specialized clinical and typical microscopic signs in Yersiniosis, (Haghighi 2008) therefore we had a standard method of Histopathology to provide samples in research (Roberts.2001) .The aim of this study was to compare PCR and histopathology technique in diagnosis of yersinia rockery in rainbow trout in Iran.
MATERIALS AND METHODS
This study was conducted from September 2008 to September 2009 in Tehran, Iran. With the contribution of Iranian Veterinary Organization, Infected trout samples were collected from farms around Iran and transferred to the laboratory in 20% ethylic alcohol or in frozen form for PCR experiments and in 10% saline formalin for histopathology experiments.
DNA was extracted from the last 1/3 intestine tissue (25-50mg) of suspected infectious rainbow trout using the Roche Genomic DNA isolation kit (Roche Company, USA) according to the manufacturer's DNA extraction instruction.
To avoid false negative result in PCR technique two pairs of primers (table 1) were selected, one pair based on 16S rRNA sequence of Yersinia ruckeri and another pair based on 18S rRNA of Oncorhynchus mykiss in a Multiplex - PCR as described earlier by Roozbahani et al (2009)..
Table 1: The primer sequences, expected size of products and PCR condition used in diagnostic test.
PCR Primer name Primer sequence (5Â´-3Â´) Expected size PCR condition
Multiplex YER3 5`- CGAÂ Â GGAÂ GGAÂ Â AGGÂ GTTÂ AAGÂ T-3
YER4 5`- AAGÂ Â GCAÂ Â CCA Â Â AGGÂ CATÂ CTCÂ T-3 573bp 94ËšC 2min, 30 cycles (94ËšC 30s, 55ËšC
Always on Time
Marked to Standard
Omny F 5`- CTGÂ TGGÂ CAAÂ TTCÂ TAGÂ AGC -3` 30 sec, 72ËšC 30 sec) 72ËšC 5min
Omny R 5`- CGT CCCÂ TCTÂ TAAÂ TCAÂ TGG -3` 725 bp
PCR amplification was carried out in Corbet Research thermal cycler in a final volume 20Î¼l. The reaction mixture contained 1 Î¼g DNA (host DNA or host and bacterial DNA), 1X PCR buffer, 20 pico mol of each primer, 1.5 mM MgCl2, 0.15 mM dNTPs, 1.25 units of Taq DNA polymerase enzyme (CinnaGen, Iran) and distilled water up to 20 Î¼L final volume. Amplification conducted after an initial denaturation step of 2 min at 94ËšC, followed by 30 cycles of denaturation for 30 sec at 94Â°C, annealing for 30 sec 55ËšC and extension for 30 sec at 72Â°C and finally the reaction lasted 5 min at 72Â°C (McPherson et al., 2000).
10 Î¼l of PCR products were analyzed by electrophoresis on 1.5% agarose gel containing ethidium bromide and viewed using 260 nm ultraviolet waves in an UV trans-illuminator. 100bp DNA Ladder marker (Fermentas, Lithuania) was used as a molecular weight marker (Boffy, 1984).
The specimens for histopathology were sent to the pathology section and a standard tissue preparation was applied, where all tissues from live or moribund fish were fixed in 10% formalin saline for 24 hours. Samples were then embedded in paraffin block and sectioned with digital microtome at 5-7 micrometer thickness. Slides were stained by haematoxillin & eosin, and then treated with mounting media (Roberts, 2001). the study in addition to other diagnoses of pathological microorganisms in terms of Y.ruckeri positive or negative sign were recorded notation.
Figures 1and 2 demonstrate electrophoresis of Multiplex PCR product on 1.5% agarose gel, which in positive samples (725 and 573 bp as PCR product of Oncorhynchus mykiss 18S rRNA genes as Yersinia ruckeri 16S rRNA, respectively) and in negative samples (only 725 bp of PCR product of Oncorhynchus mykiss 18S rRNA gene as internal control of PCR reaction) are seen. As it is shown in Fig. 1, there is only host PCR product in negative samples, but in Fig. 2 there are both host and pathogen PCR product in positive samples.
As it showed in haemorrhaging can be found in periocular tissue, liver (Fig 3), skeletal muscle, and viscera with haemorrhagic and ascitic fluids in abdomen cavity. Out of twenty suspected samples in PCR technique, two of them were positive and other eighteen remain samples were negative but in Histopathology tests on mentioned samples, there were 5 positive and 15 negative.
Fig. 1: Electrophoresis of PCR product on 1.5% agarose gels. PCR product of negative sample. Lane 1: Host (trout) PCR product, lane 2: 100 bp DNA ladder marker
Fig. 2: 1.5% agarose gel electrophoresis Lane 1: PCR product of positive sample with host and pathogen PCR product, lane 2: 100 bp DNA ladder marker
Fig. 3: Histopathology section of liver, sever focal haemorrhagic and necrotic area within the liver resulting from infection by Yersinia ruckeri (H&E staining)
Table 1- PCR and histopathology results
Number of susceptible samples
2 PCR 5 Histopathology
18 PCR 15 Histopathology
20 PCR 20 Histopathology
It seems that Yersiniosis has a worldwide distribution and it is considered endemic in most trout producing countries and in natural hosts in marine habitats. According to British Trout Association in 1998, annual loss related to yersiniosis assuming costs related loss, decreased growth, reduced food conversion ratio, increased use of antibiotic and delayed harvest equals 10% of all the costs in the industry. Regarding to the importance of fish production in country's agriculture and the diversity of natural and artificial habitats for salmonid and natural hosts of Yersinia in Iran, monitoring and definitive diagnosis of Yersiniosis is significantly important. The results of this study indicate that Yersiniosis infection can be found in rainbow trout hatcheries and brood stock sites in Iran. The severity of Yersiniosis is dependent on fish resistance, stress factors and environmental condition related to season, temperature and PH changes. An increased awareness of on-site hygiene and bio-security rules, screening of fish population for this pathogen and isolation and quarantine of infected fish or fish with abnormal behaviors has played a major role in limiting the spread of Yersiniosis in Iran.
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In both mentioned methods, all positive results confirmed the existence of yersiniosis infection in Iran, which their comparison is very useful for monitoring and diagnosis of pathogen in the country. Differences in the number of positive samples in this comparison demonstrate that although PCR technique would provide a more rapid and sensitive alternative to traditional diagnosis in detection of harmful pathogens but in some cases such as necrotic and degenerated cells in tissue, PCR ability collapses in the comparison of other detection technique like histopathology.
Both PCR and Histopathology techniques are more useful for the diagnosis of disease and these methods have almost perfect agreement with their unique advantages so if the advantages of each method exploited and one supplemented the other one, the efficiency and accuracy of monitoring experiments would rise up.
This study was supported by Shahid Beheshti University, M.C., Tehran, Islamic Republic of Iran and Iranian Veterinary Organization (IVO). The authors wish to thank directors. Iranian Veterinary Organization (IVO) gifted the bacterial colony used for initial test.