Serotypes, virulence genes and polymorphism of capsule gene cluster in Lactococcus garvieae isolated from diseased rainbow trout (Oncorhynchus mykiss) and mugger crocodile (Crocodylus palustris) in Iran

Document Type: Full paper (Original article)

Authors

1 Resident of Aquatic Animal Health, Department of Clinical Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran

2 Department of Clinical Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran

Abstract

Background: Lactococcus garvieae causes lactococcosis in rainbow trout in many parts of the world. Aims: This study was conducted for the existence of the virulent factors and differentiation of the two serotypes in L. garvieae. Methods: Twenty-two strains of L. garvieae isolated from diseased rainbow trout from farms in different regions and mugger crocodile of Iran, were investigated. In order to rapidly detect the presence of the hly1, hly2, hly3, NADH oxidase, sod, pgm, adhPsaA, eno, LPxTG-3, adhCI, and adhCII virulence genes, two multiplex polymerase chain reaction (PCR) assays were developed. Also, simplex PCR method was used to identify the bacterial serotypes, CGC, LPxTG-2, Adhesion, and adhPav virulence genes using the specific primer. Results: All varieties of L. garvieae contained the hly1, hly2, hly3, NADH oxidase, pgm, adhPav, LPxTG-3, sod, eno, adhPsaA, adhCI, and CGC virulence genes. Also, adhCII gene was present in all strains except one of the isolates originated from mugger crocodile. In addition, LPxTG-2 gene was onlypresent in one of the isolates belonging to mugger crocodile. Adhesion gene was not present in all the strains. Interestingly, all the 22 strains originated from both hosts were identified as belonging to the serotype I. Based on the phylogenetic sequences of the capsule gene cluster, group all fish isolates into a cluster together with one isolate obtained from mugger crocodile. Conclusion: Further studies are recommended to investigate the role of virulence genes in L. garvieae and evaluate their pathogenicity to rainbow trout.

Keywords

Main Subjects


Akhlaghi, M and Keshavarzi, M (2002). The occurrence of streptococcosis in the cultured rainbow trout of Fars province. Iran. J. Vet. Res., 2: 183-189.

Coutte, L; Alonso, S; Reveneau, N; Willery, E; Quatannens, B; Locht, C and Jacob-Dubuisson, F (2003). Role of adhesin release for mucosal colonization by a bacterial pathogen. J. Exp. Med., 197: 735-742. https://doi.org/10. 1084/jem.20021153.

Englen, MD and Kelley, LC (2000). A rapid DNA isolation procedure for the identification of Campylobacter jejuni by the polymerase chain reaction. Lett. Appl. Microbiol., 31: 421-426. https://doi.org/10.1046/j.1365-2672.2000.00841. x.

Erfanmanesh, A; Soltani, M; Pirali, E; Mohammadian, S and Taherimirghaed, A (2012). Genetic characterization of Streptococcus iniae in diseased farmed rainbow trout (Onchorhynchus mykiss) in Iran. Sci. World J., 2012: 1-6. http://dx.doi.org/10.1100/2012/594073.

Fukuda, Y; Tue, Y; Oinaka, D; Wada, Y; Yamashita, A; Urasaki, S; Yoshioka, S; Kimoto, K and Yoshida, T (2015). Pathogenicity and immunogenicity of non-agglutinating Lactococcus garvieae with Anti-KG (-) phenotype rabbit serum in Seriola spp. Fish. Pathol., 50: 200-206. https://doi.org/10.3147/jsfp.50.200.

Karsidani, SH; Soltani, M; Nikbakhat-Brojeni, G; Ghasemi, M and Skall, HF (2010). Molecular epidemiology of zoonotic streptococcosis/lactococcosis in rainbow trout (Oncorhynchus mykiss) aquaculture in Iran. Iran. J. Microbiol., 2: 198-209.

Mata, AI; Gibello, A; Casamayor, A; Blanco, MM; Domínguez, L and Fernández-Garayzábal, JF (2004). Multiplex PCR assay for detection of bacterial pathogens associated with warm-water streptococcosis in fish. Appl. Environ. Microbiol., 70: 3183-3187. https://doi.org/10.1128 /AEM.70.5.3183-3187.2004.

Meyburgh, CM; Bragg, RR and Boucher, CE (2017). Lactococcus garvieae: an emerging bacterial pathogen of fish. Dis. Aquat. Organ., 123: 67-79. https://doi.org/10. 3354/dao03083.

Miyauchi, E; Toh, H; Nakano, A; Tanabe, S and Morita, H (2012). Comparative genomic analysis of Lactococcus garvieae strains isolated from different sources reveals candidate virulence genes. Int. J. Microbiol., 2012: 1-6. http://dx.doi.org/10.1155/2012/728276.

Morita, H; Toh, H; Oshima, K; Yoshizaki, M; Kawanishi, M; Nakaya, K; Suzuki, T; Miyauchi, E; Ishii, Y; Tanabe, S and Murakami, M (2011). Complete genome sequence and comparative analysis of the fish pathogen Lactococcus garvieae. PLoS ONE. 6: e23184. https://doi. org/10.1371/journal.pone.0023184.

Ohbayashi, K; Oinaka, D; Hoai, TD; Yoshida, T and Nishiki, I (2017). PCR-mediated identification of the newly emerging pathogen Lactococcus garvieae serotype II from Seriola quinqueradiata and S. dumerili. Fish. Pathol., 52: 46-49. https://doi.org/10.3147/jsfp.52.46.

Ooyama, T; Hirokawa, Y; Minami, T; Yasuda, H; Nakai, T; Endo, M; Ruangpan, L and Yoshida, T (2002). Cell-surface properties of Lactococcus garvieae strains and their immunogenicity in the yellowtail Seriola quinqueradiata. Dis. Aquat. Org., 51: 169-177. https://doi.org/10.3354/ dao051169.

Raissy, M; Sarshoughi, M and Moumeni, M (2016). Molecular identification of some causative agents of warm-water streptococcosis by M-PCR in cultured rainbow trout, Chaharmahal-Bakhtiari Province, Iran. Iran. J. Fish. Sci., 15: 836-845. http://jifro.ir/article-1-2224-en.html.

Sharifiyazdi, H; Akhlaghi, M; Tabatabaei, M and Mostafavi Zadeh, SM (2010). Isolation and characterization of Lactococcus garvieae from diseased rainbow trout (Oncorhynchus mykiss, Walbaum) cultured in Iran. Iran. J. Vet. Res., 11: 342-350.

Shin, GW; Nho, SW; Park, SB; Jang, HB; Cha, IS; Ha, MA; Kim, YR; Dalvi, RS; Joh, SJ and Jung, TS (2009). Comparison of antigenic proteins from Lactococcus garvieae KG (−) and KG (+) strains that are recognized by olive flounder (Paralichthys olivaceus) antibodies. Vet. Microbiol., 139: 113-120. https://doi.org/10.1016/j.vetmic. 2009.05.007.

Sneath, PH and Sokal, RR (1973). Numerical taxonomy. The principles and practice of numerical classification. 1st Edn., San Francisco, USA, W. H. Freeman & Company. P: 573.

Stipcevic, T; Piljac, T and Isseroff, RR (2005). Di-rhamnolipid from Pseudomonas aeruginosa displays differential effects on human keratinocyte and fibroblast cultures. J. Dermatol. Sci., 40: 141-143. https://doi.org/10. 1016/j.jdermsci.2005.08.005.

Tamura, K; Dudley, J; Nei, M and Kumar, S (2007). MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol., 24: 1596-1599. https://doi.org/10.1093/molbev/msm092.

Ture, M and Altinok, I (2016). Detection of putative virulence genes of Lactococcus garvieae. Dis. Aquat. Org., 119: 59-66. https://doi.org/10.3354/dao02981.

Türe, M; Haliloğlu, Hİ; Altuntaş, C; Boran, H and Kutlu, İ (2014). Comparison of experimental susceptibility of Rainbow Trout (Oncorhynchus mykiss), Turbot (Psetta maxima), Black Sea Trout (Salmo trutta labrax) and Sea Bass (Dicentrarchus labrax) to Lactococcus garvieae. Turk. J. Fish. Aquat. Sci., 14: 507-513. https://doi.org/10. 4194/1303-2712-v14_2_22.

Vanaporn, M; Wand, M; Michell, SL; Sarkar-Tyson, M; Ireland, P; Goldman, S; Kewcharoenwong, C; Rinchai, D; Lertmemongkolchai, G and Titball, RW (2011). Superoxide dismutase C is required for intracellular survival and virulence of Burkholderia pseudomallei. Microbiology. 157: 2392-2400. https://doi.org/10.1099/ mic.0.050823-0.

Yoshida, T; Endo, M; Sakai, M and Inglis, V (1997). A cell capsule with possible involvement in resistance to opsonophagocytosis in Enterococcus seriolicida isolated from yellowtail Seriola quinqueradiata. Dis. Aquat. Org., 29: 233-235. https://doi.org/10.3354/dao029233.