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Nocardiosis in some ornamental fishes in Iran | ||
Journal of Zoonotic Diseases | ||
مقاله 4، دوره 4، شماره 1، مرداد 2020، صفحه 28-34 اصل مقاله (204.27 K) | ||
نوع مقاله: Original Article | ||
شناسه دیجیتال (DOI): 10.22034/jzd.2020.10586 | ||
نویسندگان | ||
Najmeh Sheikhzadeh* 1؛ Tayebeh Fakheri1؛ Katayoon Nofouzi Nofouzi2 | ||
1Department of Food Hygiene and Aquatic Animals, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran | ||
2Department of Pathobiology, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran | ||
چکیده | ||
Nocardiosis is a systemic disease in fish in which lesions are frequently localized in the integument and several internal organs, with the nodular lesions characteristic of granulomata. The zoonotic species of Nocardia (N. asteroides) is a pathogen of humans and many animals. Considering the lack of information about the status of Nocardia species in fish species in Iran, the widespread use of aquariums, and possible transmission of zoonotic diseases from ornamental fishes to human, primary study for detecting this bacterium was performed. In this study, 100 freshwater aquarium fishes (apparently healthy and unhealthy) were purchased from some shops located in Tehran, Tabriz, Zanjan, and Shahindej cities completely randomly and transferred to the laboratory. Fish samples were examined for the Nocardia genus and N. asteroides using nested polymerase chain reaction (PCR) assay. In the macroscopic examination, no granulomatous lesions were detected. PCR examination showed that one healthy fish and three sick fish (with clamped fin, lethargy, and slow-motion sings) were infected by Nocardia. But PCR amplification of N. asteroids by specific primers did not amplify DNA gyrase gene sequences of N. asteroids. This study is the first known identification of Nocardia -infected ornamental fishes in Iran. | ||
کلیدواژهها | ||
Nocardiosis؛ N. asteroides؛ Aquarium fishes؛ PCR | ||
اصل مقاله | ||
Introduction 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 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 The authors are grateful to the Research affairs of Tabriz University for financial support. | ||
مراجع | ||
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