Introduction
Nocardiosis is well known as a systemic bacterial infection caused by a Grampositive, partially acid-fast, aerobic, and
filamentous bacterium in farmed, and ornamental freshwater and marine fishes (Wang et al., 2007). The clinical signs of this disease are sluggish or flashing behavior, emaciation, exophthalmia, and distended abdomen. In later stages, nodular and diffuse granulomatous lesions can be found in skeletal muscle, gills, and any of the visceral organs (Lee et al., 2007). Three species of Nocardia including Nocardia salmonicida, N. seriolae (formerly N. kampachi), and N. asteroides have been previously identified from diseased fish. N. asteroides, one of the most virulent species, has been detected from humans, cats, dogs, goats, cattle, marine mammals, and fishes (Chen et al., 2000). In humans, clinical forms consist of pulmonary implication, skin or soft tissue involvement, and diffused forms with the brain and pulmonary infections. Primary nocardial infection presents by pulmonary or cutaneous and/or subcutaneous lesions. Disseminated disease is determined by the detection of nocardial infection in two or more organs. The lung is the most frequently implicated organ, followed by skin and brain. Of note, nocardiosis has been demonstrated in human receiving immunosuppressive therapy, subjects with chronic pulmonary diseases, organ transplant recipients, and patients infected with the human immunodeficiency virus (HIV) (Corti and Fioti, 2003). Because of the zoonotic character of nocardial infection, enhancing importance of aquariology, and lack of any information about the presence of Nocardiosis in fish
species in Iran, the present study was undertaken to detect Nocardia genus and zoonotic N. asteroids in diseased and clinically healthy ornamental fish by polymerase chain reaction (PCR) assay.
 
Materials and methods

Fish sampling One hundred freshwater aquarium fishes of 22 species were examined from April to September 2010. These fishes were purchased from some local shops in four cities in Iran (including Tehran, Tabriz, Zanjan, and Shahindezh). Among 100 fish, 22 samples were apparently healthy. Seventy-eight moribund fish with clinical signs including emaciation, clamped fin, fin rot, lethargy, and anorexia were selected. Live fishes were transported in oxygenated insulated coolers to the bacteriology laboratory of veterinary faculty, University of Tabriz. Live fishes were killed by immersion in clove oil (50 μL L-1). PCR assay DNA was extracted from internal organs, including the digestive system and kidney using isopropanol method, according to the manufacturer’s instructions. Extracted DNA was verified by electrophoresis on 1% agarose gel. The concentration and purity of DNA was assessed with a NanoDrop spectrophotometer (Thermo Fisher ScientificTM). Gene encoding 16S ribosomal RNA was used as a target gene in Nocardia identification. NO1 and NO2 primers were conducted according to the published sequences of the genes (Accession No JX484797.1). Gene encoding DNA gyrase subtype B was used to detect N.asteroides. NAD1 and NAD2 primers were designed according to the published sequences of the genes (Accession No JN041222.1). Primer synthesis was performed by Bioneer Company (Korea). The PCR was conducted by Master Cycler Gradient-Eppendorf (Germany). The PCR reagents included 1 µL of each primer, 1 µL of extracted DNA, 12.5 µL of master mix (consisting buffer, MgCl2, dNTP, Taq DNA polymerase) with a final volume of 25 μL. The PCR thermal cycling parameters were: 1 cycle at 95°C for 1 min, 35 cycles by 94°C for 60s, 57°C for 60s and 72°C for 60s followed with final extension for 10 min at 72°C. The sequences of the primers used in this study are displayed in Table 1. Amplicons were analyzed by the agarose gel electrophoresis, stained with fluoro dye, visualized under UV light, and photographed at final with 700 and 406-bp for identification of Nocardia and N. asteroides, respectively.

Results

In this study, the Nocardia genus was detected in diseased and apparently healthy ornamental fish by polymerase chain reaction (PCR) assay.  Results of the PCR reaction for 16S ribosomal RNA of sampled fishes are shown in Fig. 1. Four out of 100 extracted DNA for
16S ribosomal RNA genes were Nocardia positive. Identification for DNA gyrase B subunit gene of N. asteroids was also performed that no positive sample was found. Among four fish samples infected with Nocardia, one green neon tetra (Paracheirodon simulans) was apparently healthy. In contrast, three remaining infected fishes, including one shark (Myxocyprinus asiaticus), one oscar (Astronotus ocellatus) and one goldfish (Carassius auratus) showed some signs of the clamped fin, lethargy, and slow motion. However, no macroscopic nodules were seen in fish samples.  
 
Discussion

Nocardiosis was frequently observed in the aquarium industry in recent years (Itano et al., 2006; Wang et al., 2009; Cornwell et al., 2011; Elkesh et al., 2013). In the present study, nocardiosis was found in some diseased and apparently healthy ornamental fishes in Iran; however, N. asteroids was negative by PCR method. Noteworthy, regarding the similar clinical signs and pathology with fish Mycobacterium, some mistakes in detection might happen. Therefore, some sensitive methods for differentiation are needed. PCR has higher sensitivity and specificity compared to the histological and bacteriological methods in fish nocardiosis diagnosis (Wang et al., 2014). In this regard, Alfaresi and Elkosh (2006) reported 90% sensitivity and 100% specificity for PCR. It commonly takes about two weeks for microbiological tests and antibiotic susceptibility assays for nocardiosis diagnosis, but real-time PCR analysis of 16S rDNA can be performed in a few hours (Alfaresi and Elkosh, 2006). In previous studies, PCR was used for the identification of the Nocardia genus and species. For example, Itano et al (2006) found N. seriolae in some infected fishes by Loop-mediated isothermal amplification (LAMP) and PCR methods. In fact, LAMP could demonstrate N. seriolae in four unknown states of N. seriolae infectivity. However, PCR could identify only one sample from these fishes. According to literature, the identification of N. seriolae by LAMP is more sensitive than that of PCR, but the PCR method is still a sensitive way for Nocardia confirmation. In another study by Wang et al (2009), in cultured three striped tiger-fish, Terapon jarbua, with clinical signs including lethargy, hemorrhages, and ulcers on the skin, varying degrees of ascites associated with the enlargement of the spleen, liver, kidney, and obvious white nodules in these organs, N. seriolae was positive by PCR method. Meanwhile, Cornwell et al (2011) detected nocardial infection on cultured weakfish, Cynoscion regalis (Bloch and Schneider) using pathological and molecular analysis.These researchers believed that Nocardia spp. are opportunistic infectious agents, and it is probable that the stress of capture and higher stocking density, coupled with the slightly raised levels of ammonia and nitrites in the system, allowed the infection to cause mortalities in cultured fishes (Cornwell et al., 2011). In recent years, some studies were mainly focused on the use of immunostimulnats or efficient vaccines against Nocardia. For example, Kato et al. (2012) found that Japanese flounder (Paralichthys olivaceus) were partially protected against nocardiosis with Freund´s complete adjuvant. Meanwhile, inactivated N. seriolae could increase the non-specific immunity of Channa argus, producing remarkable protection against the pathogen
bacteria (Xie et al., 2013). On the other hands, there was a significant protective impact for Nocardia infected Channa argus with the whole peptidoglycan from N. seriolae intraperitoneal injection more than 21 days (Peng et al., 2013). In recent studies, it was shown that Interleukin-12 and recombinant interferon gamma elicited effective protective immunity, which will help in development of a suitable vaccine and/or adjunct therapy against N. seriolae infection in fish (Nayak et al., 2014; Matsomoto et al., 2017). In this study, detection of Nocardia in diseased ornamental fishes could have a similar reason to the previous study (Cornwell et al., 2011). On the other hand, identification of Nocardia in one healthy fish, green neon tetra, shows the low level of bacterial particles in the PCR method, which is an important point in disease transmission, especially in zoonotic N.asteroids. In conclusion, the current study proved the presence of Nocardia in diseased and even apparently healthy aquarium fishes in Iran by PCR assay; however, zoonotic N. asteroids was not identified in similar fish samples. Considering Nocardia infection as an occupational hazard for certain professionals like pet shop workers and possible transmission of bacterium from infected fishes to humans with the suppressed immune system, more attention is needed to recognize fish nocardiosis in different regions in Iran.
 
Acknowledgments

The authors are grateful to the Research affairs of Tabriz University for financial support.