Streptococcus Infections in Cultured Pond Loach Paramisgurnus

Short communication Bureau, 2017). Loach diseases have been increasingly observed Streptococcus Infections with the continuous expansion of loach aquaculture and are now a major threat to the industry (Qin et al., 2014). in Cultured Pond Loach Some pathogens co-occurring with cultured loach have Paramisgurnus dabryanus been reported, and are mainly bacterial pathogens, including Flavobacterium columnare (Triyanto and in China Wakabayashi, 1999), Vibrio parahaemolyticus (Xu et al., 2016), and members of the genus Aeromonas, such as Zhang Luo1,a, Xiao Hui Bai1,a, Aeromonas hydrophila and Aeromonas veronii (Jun et al., 1 1 2013; Zhu et al., 2016), as well as Shewanella sp. and Zhen Guo Zhang , Shuang Hao , Listonella sp. (Qin et al., 2014). The reported patho- Wen Ping Jia1, Jin Lan Liu2, gens are mostly Gram-negative bacteria. Recently, Peng Shao2, Wen Li Zhou2 Gram-positive bacteria, Strepococcus dysgalactiae, 1,3* was reported in loach by Nguyen et al. (2017). In the and Shou Ming Feng current study, Streptococcus dysgalactiae and Streptococcus agalactiae were isolated from diseased 1Tianjin Fishery Research Institute, Tianjin 300221, loach and were considered to be the cause of the dis- P. R. China ease. To the best of our knowledge, this is the first 2College of Fisheries, Tianjin Agricultural University, report of Streptococcus agalactiae in loach. Also, our Tianjin 300384, P. R. China result proves again that loach is a susceptible host for 3Prevention and Cure Center for Aquatic Animal Disease streptococcosis. in Tianjin, Tianjin 300221, P. R. China In this study, All the animal experiments were per- formed according to local government regulations and (Received October 22, 2018) the protocols were approved by the Institutional Animal Care & Use Committee (IACUC) of the Tianjin Fishery Research Institute. ABSTRACT―In 2017, high mortalities were observed in In June and July 2017, two loach farms in the cultured pond loach Paramisgurnus dabryanus in Tianjin, Jinghai and Baodi districts of the Tianjin municipality China. The symptoms observed in the moribund loach reported diseased fish, with cumulative mortality rates of included hemorrhage on the body surface and head, exoph- 32% and 45%, respectively. The water temperatures thalmos, and erratic swimming around the water surface. recorded during the outbreaks were 28°C and 30°C. Two different Gram-positive bacteria were isolated from the The main symptoms of the diseased loach were diseased loach in two different fish farms. They were iden- anorexia, swimming abnormalities, and loss of balance tified as Streptococcus dysgalactiae and Streptococcus in the water. Six samples of fish (body weight 22.3– agalactiae, using 16S rRNA gene sequencing and bio- 28.1 g) with symptoms typical of the disease were sent chemical characterization. The median lethal doses of the from each farm to our laboratory for diagnosis. The dis- 5 5 bacterial strains in the loach were 3.2 × 10 and 5.0 × 10 eased loach showed hemorrhagia in the body and head CFU/fish, respectively. (Fig. 1A), prominent cloudy eyeballs (Fig. 1B), swollen and pale liver, swollen spleen, and eroded kidneys (Fig. Key words: Paramisgurnus dabryanus, pond loach, 1C). No other parasites were observed, except for a streptococcosis, Streptococcus dysgalactiae, few Dactylogyrus flukes in the gills. Streptococcus agalactiae The samples of the spleen, kidney, and liver from the diseased fish were sliced and stained with hema- toxylin and eosin (H&E). Pathological sections showed Loach Paramisgurnus dabryanus is an impor- increased leukocytosis in the kidney from diseased tant freshwater aquaculture species in Asia (Qin et al., loach, necrosis and disintegration of renal tubular epi- 2014). Given its appealing taste, and rich nutritional thelial cells, accumulation of red blood cells in renal and medicinal values, it is also known as ‘ginseng in tubules (Fig. 2A), and increased leukocytes, with a large water’ and is popular among consumers (Zhu et al., number of red blood cells in the spleen. Some of the 2016). Furthermore, loach mucus can be processed to cells in the spleen were enlarged and necrotic (Fig 2C). produce cosmetics. With increasing consumer needs, Hepatocyte necrosis was severe, and the cell mem- the demand for loach is increasing, prompting the rapid branes had disappeared. A large number of hepato- development of loach aquaculture. In 2016, the annual cytes were also replaced by adipocytes (Fig. 2E). production of loach was 400,209 tons in China (Fisheries After being anesthetized with MS222, the liver, spleen, kidney, and brain of six loach were removed * Corresponding author under aseptic conditions. Each organ was scored on E-mail: [email protected] Trypticase Soy Agar (TSA) containing 5% sheep blood a These authors contributed equally to the work. and cultured at 28°C for 48 h. Dominant colonies were Streptococcosis in Paramisgurnus dabryanus 13

Fig. 1. Loach Paramisgurnus dabryanus showing typical symptoms of disease. (A) Hemorrhage on the body surface (→); (B) exophthalmia (→); (C) pale liver (☆), swollen spleen (▲), and eroded kidney (→).

Fig. 2. Histological sections of the visceral organs of diseased loach Paramisgurnus dabryanus. (A) Kidney, showing necrosis and disintegration of renal tubular epithelial cells (→) and accumulated red blood cells in renal tubules (☆). (C) Spleen, showing enlarged and necrotic cells (→). (E) Liver, showing severe hepatocyte necrosis, and fatty degeneration of hepatocytes (→). (B, D, F) Kidney, spleen, and liver of disease-free loach for comparison. selected for purification. Four bacterial strains were four strains were non-motile and non-spore-forming isolated from the spleen and kidney of diseased loach Gram-positive cocci. They were arranged in chains or from the two different farms: strain NQ-2017-3 and pairs, with a bacterial diameter of 0.5–0.8 μm. NQ-2017-4 were from the ﬁrst farm, and NQ-2017-5 and The strains were subjected to physiological and bio- NQ-2017-6 were from the second farm. After incuba- chemical tests using methods developed by Dong and tion, all four bacteria formed white, round, protuberant, Cai (2001). The four isolated strains were all negative moist colonies with a diameter of 0.8–1.0 mm. Strains for oxidase and catalase. Based on 43 biochemical NQ-2017-3 and NQ-2017-4 exhibited α hemolysis, reactions tested by VITEK® 2 Compact (Biomerieux), whereas strains NQ-2017-5 and NQ-2017-6 did not. All strains NQ-2017-3 and NQ-2017-4 were classiﬁed as S. 14 Z. Luo, X. H. Bai, Z. G. Zhang, S. Hao, W. P. Jia, J. L. Liu, P. Shao, W. L. Zhou and S. M. Feng

Streptococcus dysgalactiae (AP018726, source: amberjack) Streptococcus dysgalactiae (AB537916, source: amberjack) 100 Streptococcus dysgalactiae (AB102730, source: swine) NQ-2017-4

87 NQ-2017-3 NQ-2017-5 NQ-2017-6

71 Streptococcus agalactiae (EF092913, source: tilapia) 100 Streptococcus agalactiae (AP018935, source: dace) Streptococcus agalactiae (KP294526, source: human) 37 Streptococcus porcinus (EF121439) 86 Streptococcus pseudoporcinus (DQ303206) Streptococcus ictaluri (DQ462421) Streptococcus ratti (NR025516) 61 Streptococcus uberis (AB370974) 18 59 Streptococcus equi (NR025609) 51 Streptococcus pasteurianus (EU163502) 90 99 Streptococcus lutetiensis (EU163503) 59 99 Streptococcus infantarius (EU163504) Streptococcus manure (AY167955) 100 Streptococcus parauberis (FJ009631) Streptococcus iniae (DQ303187) Streptococcus entericus (NR025500) Streptococcus sanguinis (FJ655817)

0.02 Fig. 3. Phylogenetic tree constructed for strain NQ-2017-3, NQ-2017-4, NQ-2017-5 and NQ-2017-6 based on 16S rDNA sequences of related Streptococcus spp. dysgalactiae subsp. dysgalactiae (probability > 93%), Group 9 was injected with 0.1 mL 0.85% sterile saline as and strains NQ-2017-5 and NQ-2017-6 were classiﬁed a control. The test loach were continuously observed as S. agalactiae (probability > 93%). The 16S rRNA at 25°C–28°C for 14 days and any dead loach were genes of the four strains were cloned using the primers removed immediately. The mortalities of loach in (27F) 5′-AGAGTTTGATCCTGGCTCAG-3′ and (1492R) Groups 1–4 were 93%, 86.7%, 53.5%, and 20%, respec- 5′- TACGGCTACCTTGTTACGCTT-3′ (Lane, 1991). tively, and 100%, 83.3%, 37.7%, and 6.7% in Groups

The results showed that the sequences from NQ-2017-3 5–8, respectively. The LD50 of strain NQ-2017-3 and and NQ-2017-4 showed the highest homology (100%) NQ-2017-5 were approximately 3.2 × 105 CFU/fish and with S. dysgalactiae. By contrast, NQ-2017-5 and 5.0 × 105 CFU/fish, respectively. T he artificially infected NQ-2017-6 showed 100% homology with S. agalactiae. loach showed symptoms including abnormal swimming, In the phylogenetic tree, NQ-2017-3 (GenBank no. hemorrhage on the body surface, and swollen spleens. MH423901) and NQ-2017-4 (GenBank no. MH491050) It was possible to isolate samples of the injected bacte- clustered with S. dysgalactiae (GenBank no. AB102730, ria from the internal organs of the moribund loach. Fish source: swine; GenBank no. AP018726, source: amber- in the control group showed no morbidity or mortality. jack; AB537916, source: amberjack), whereas The result of the pathogenicity study suggests that S. NQ-2017-5 (GenBank no. MH423900 ) and NQ-2017-6 agalactiae and S. dysgalactiae were responsible for the (GenBank no. MH488994) clustered with S. agalactiae outbreak of loach disease. (GenBank no. EF092913, source: tilapia; GenBank no. Streptococcosis caused by S. agalactiae and S. KP294526, source: dace; GenBank no. AP018935, dysgalactiae frequently leads to high mortality and sig- source: human) (Fig. 3). Therefore, the results, com- nificant economic losses (Nomoto et al., 2004; Nomoto bined with those of the physiological and biochemical et al., 2006; Chen et al., 2012). S. dysgalactiae infects reactions, confirmed NQ-2017-3 and NQ-2017-4 to be S. a range of hosts, including amberjack Seriola dumerili, dysgalactiae and NQ-2017-5 and NQ-2017-6 to be S. yellowtail Seriola quinqueradiata (Nomoto et al., 2004), agalactiae. tilapia Oreochromis niloticus (Netto et al., 2011), cobia To verify the pathogenicity of the isolated strains Rachycentron canadum (Tsai et al., 2015; Nguyen et against loach, strains NQ-2017-3 and NQ-2017-5 were al., 2017; Nguyen et al., 2018) and so on. Similarly, a used to experimentally infect 270 disease-free loach variety of fish, including seabream Sparus aurata (Evans (15.3 ± 3.2 g); the fish were randomly divided into nine et al., 2002), silver pomfret Pampus argenteus groups. Groups 1–4 were injected intraperitoneally (Duremdez et al., 2004), gold pompano Trachinotus with 0.1 mL of 2.8 × 108, 107, 106, or 105 CFU/mL of ten- ovatus (Cai et al., 2016), tilapia O. niloticus (Li et al., fold dilution serials of an NQ-2017-3 suspension. 2013), barcoo grunter Scortum barcoo (Liu et al., 2014) Groups 5–8 were injected intraperitoneally with the could be infected by S. agalactiae. However, there same volumes and the same concentrations of an have been few reports on streptococcal disease in NQ-2017-5 suspension as used for Groups 1–4, and Cyprinidae, the most common aquaculture species in Streptococcosis in Paramisgurnus dabryanus 15

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