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Lesions Induced in Seriola dumerili Following Exposure to Streptococcus dysgalactiae by Oral Treatment or Immersion
J. Comp. Path. 2010, Vol. 143, 262e267
Available online at www.sciencedirect.com
www.elsevier.com/locate/jcpa
Lesions Induced in Seriola dumerili Following Exposure to Streptococcus dysgalactiae by Oral Treatment or Immersion H. Hagiwara, R. Takano, N. Noguchi and M. Narita Advanced Technology Development Center, Kyoritsu Seiyaku Corporation, Takamihara, Tsukuba, Ibaraki 300-1252, Japan
Summary Seriola dumerili were infected experimentally with Streptococcus dysgalactiae by oral dosing or immersion. There was moderate mortality after immersion in water containing defined numbers of bacteria, regardless of the dose, whereas the effect on the oral challenge groups depended on the dose of bacteria administered. The characteristic lesions were microabscesses and/or pyogranulomatous inflammation of the caudal peduncle, pectoral and/or dorsal fin, heart and olfactory region. S. dysgalactiae antigen was found within necrotic foci at these sites. There was no difference in distribution of S. dysgalactiae antigen in fish that were exposed by oral or immersion challenge. There was no difference in antigen distribution when fish that died were compared with those that survived and were killed. Immersion exposure is therefore a more effective natural route of infection than oral challenge. Ó 2010 Elsevier Ltd. All rights reserved. Keywords: fish; immunohistochemistry; Seriola dumerili; Streptococcus dysgalactiae
Introduction Economic losses caused by streptococcal infections have been reported worldwide in many intensively cultured fish species. In 2002, an infection characterized by severe focal necrosis of the caudal peduncle and high mortality in cultured fish of the species Seriola dumerili and Seriola quinqueradiata was reported from fish farms in southern Japan. Bacterial strains isolated from diseased fish were identified as Lancefield group C Streptococcus dysgalactiae (Nomoto et al., 2004, 2006). From 2003 to 2008 the outbreak spread to involve other fish farms. A potential means of controlling S. dysgalactiae infection would be through the development of an effective vaccine. Unfortunately, this has been hindered by the lack of an adequate challenge system for the infection. Intraperitoneal, intramuscular and intradermal injections are the most widely used methods (Nomoto et al., 2004; Hagiwara et al., 2009); however, these routes bypass the natural skin and
mucosal barriers of the fish, leading to a faster and less natural progression of the disease. The aim of the present study was to characterize the pathology in S. dumerili experimentally infected with S. dysgalactiae by the oral route and by immersion challenge.
Materials and Methods Fish
S. dumerili were obtained from Kinki University, Japan. Fifty-two fish, each weighing approximately 142 g, were used in this study. The fish were divided into seven groups, each containing seven individuals, except for the non-infected control group (group 7), which comprised 10 fish. The fish were held in separate 200 l acryl tanks with a continuous flow of sea water (29 C) and fed a commercial dry pellet (2% body weight, Hayashikane Sangyo, Yamaguti, Japan) once daily throughout the experiment. Experimental Procedure
Correspondence to: H. Hagiwara (e-mail: h_hagiwara@kyoritsuseiyaku. com). 0021-9975/$ - see front matter doi:10.1016/j.jcpa.2010.04.005
Lancefield group C S. dysgalactiae strain 04K01, isolated from ulcers on the caudal peduncle of diseased Ó 2010 Elsevier Ltd. All rights reserved.
263
Streptococcal Infection in Seriola dumerili
S. dumerili in Kagoshima Prefecture, Japan, was used in all experimental infections (Hagiwara et al., 2009). Oral challenge was carried out with three groups of seven fish. Ground commercial pellet feed was mixed with an appropriate volume of the bacterial suspension at concentrations of 109 (group 1), 108 (group 2) and 107 (group 3). Immersion challenge was carried out with three groups of seven fish. Fish were anaesthetized in 0.015% FA-100 (10% 4-allyl-2-methoxyphenol, Tanabe Seiyaku Co., Japan). Bacteria were suspended in 5 l of sea water at concentrations of 107 (group 4), 106 (group 5) and 105 (group 6) CFU/ml, and the fish were immersed for 10 min. All fish were examined at least three times daily and mortality and any clinical signs were recorded. Dead and moribund fish were removed from the tank as soon as they were identified. All fish were subject to gross necropsy examination and sampled for bacteriology. The experiment was terminated after 14 days, at which point all of the remaining fish were anaesthetized and killed by decapitation before sampling for bacteriological and histopathological studies. Ten untreated fish were used as non-infected controls (group 7): three were anesthetized and killed on day 0 of the experiment, and seven were killed on day 14.
rabbit polyclonal antibody against S. dysgalactiae isolated from S. dumerili was used at a dilution of 1 in 15,000 in 0.05 M Tris HCl buffer (DAKO, Carpinteria, California). 3-amino-9-ethylcabazole (AEC) was used as chromogen and sections were counterstained with Mayer’s haematoxylin. Tissue sections from the uninfected control fish (group 7) and serum from a non-immunized rabbit were used as controls.
Bacterial Isolation
Gross Pathology
Samples for bacterial isolation were taken from the liver, spleen, kidney, heart, brain and caudal peduncle with a plastic loop and spread on soya bean casein digest broth (SCDB) agar. Plates were incubated at 30 C for 2 days.
The main gross pathological findings in the fish that died after oral or immersion challenges were erosions and/or abscesses on the caudal peduncle and pectoral and/or dorsal fin, epicarditis with small nodules (Fig. 1) and mild swelling of the spleen (Table 1). At the end of the experiment, there were only moderate erosions and/or abscesses on the caudal peduncle. No abnormalities were recorded in control fish (group 7).
Pathological Examination
Samples of liver, spleen, kidney, heart, gill, stomach, pyloric caecum, intestine, brain, caudal peduncle, pectoral and dorsal fin, skin, skeletal muscle and olfactory region were collected for microscopical examination. Tissues were fixed in 10% neutral-buffered formalin, embedded in paraffin wax and processed by routine methods. Sections were stained with haematoxylin and eosin (HE). Immunohistochemistry
Immunohistochemistry (IHC) was used to detect the presence of S. dysgalactiae antigen in the heart, caudal peduncle, pectoral and dorsal fins and olfactory region by the streptavidinebiotin complex immunoperoxidase (SAB-IP) method using an SAB kit (Nichirei Corp., Tokyo, Japan). Endogenous peroxidase activity was quenched with 3% H2O2 for 5 min. A
Results Mortality and Clinical Features
The mortality rates within the experimental groups are given in Table 1. Following oral challenge the fish in groups 1 and 2 began to die after 3e6 days and the cumulative mortality rates reached 71% (group 1) and 29% (group 2). No fish in group 3 died. Following immersion challenge the fish in groups 4e6 began to die after 5e10 days and cumulative mortality reached 29% (group 4), 71% (group 5) and 43% (group 6). No mortality occurred in the control fish (group 7). The clinical signs in the infected fish included lethargy, anorexia, and erratic swimming. Infected fish ate only half as much feed as the control group for 2e4 days after oral challenge and for 3e6 days after immersion challenge.
Bacterial Isolation
No pathogenic bacteria were cultured from fish of the control group (group 7). S. dysgalactiae was isolated from the heart, caudal peduncles, liver, spleen, kidney and brain of all dead fish infected via the oral route or by immersion. At the end of the experiment (day 14), S. dysgalactiae was isolated from the caudal peduncle of two orally infected and two immersion infected fish, from the heart of one orally infected fish and from the spleen of one orally infected fish (Table 2). Microscopical Features
The most consistently observed microscopical change in infected fish that died during the experiment was
264
Table 1 Gross and microscopical lesions, bacterial isolation and immunohistochemistry for fish that died during the experiment Group
1 2 3 4 5 1 2 1 2 1 2 3 4 5 1 2 3
Day of death
3 5 5 5 6 5 5 7 8 5 5 6 7 10 7 7 8
Liver
Spleen
Kidney
Heart
Brain
Peduncle
Fin
Gill
Olfactory
GL/HL/IB
GL/HL/IH/IB
GL/HL/IB
GL/HL/IH/IB
GL/HL/IB
GL/HL/IH/IB
GL/HL/IH
GL/HL/IH
GL/HL/IH
N/e/+ N/e/+++ N/e/+ N/e/+++ N/e/++ N/e/++ N/e/+++ N/e/+ N/e/++ N/e/++ N/e/++ N/e/++ N/e/++ N/e/e N/e/++ N/e/e N/e/++
P/3+/+2/+++ P/3+/+3/++ P/e/e/+ P/3+/+3/+++ P/3+/+2/++ P/3+/+3/++ P/e/e/+++ P/3+/+3/+++ P/3+/+2/+++ P/3+/+3/+++ P/e/e/e P/3+/+3/+++ N/3+/+3/+++ P/e/e/e P/3+/+3/+++ P/1+/+1/+ P/1+/e/+++
N/3+/+++ N/e/++ N/e/+ N/1+/++ N/e/++ N/e/++ N/e/+++ N/1+/++ N/e/++ N/1+/++ N/e/+ N/e/++ N/e/++ N/e/e N/e/+++ N/e/+ N/e/++
P/3+/+3/ND N/3+/+3/+++ N/3+/+2/++ N/3+/+3/+++ N/3+/+3/++ N/3+/+3/+++ N/1+/e/ND N/3+/+3/+++ P/3+/+3/++ N/3+/+3/+++ N/ND/ND/e N/3+/+3/ND N/3+/+3/+++ N/1+/e/e P/3+/+3/ND P/3+/+3/+ N/3+/+3/++
N/e/+++ N/e/++ N/e/ND N/e/++ N/e/++ N/e/ND N/e/ND N/e/+ N/e/++ N/e/+++ N/e/e N/e/++ N/e/+++ N/e/e N/e/+++ N/e/+ N/e/++
P/3+/+2/++ P/3+/+3/+++ P/3+/+3/+++ P/3+/+3/+++ P/3+/+3/+++ P/3+/+3/+++ P/3+/+3/+++ P/3+/+3/+++ P/3+/+3/+++ P/3+/+3/+++ P/3+/+3/+++ P/3+/+3/+++ P/3+/+3/+++ P/3+/+2/+ P/3+/+3/+++ P/3+/+3/+++ P/3+/+3/+++
N/3+/+2 N/3+/+3 N/3+/+3 N/3+/+3 N/3+/+3 P/3+/+3 N/3+/+3 P/3+/+3 P/3+/+3 N/3+/+3 P/3+/+3 N/3+/+3 P/3+/+3 P/2+/+2 P/3+/+3 P/3+/+3 N/3+/+3
N/3+/+3 N/e/e N/e/e N/e/e N/e/e N/e/e N/e/e N/e/e N/e/e N/e/e N/e/e N/e/e N/e/e N/e/e N/e/e N/e/e N/e/e
N/3+/3+ N/3+/2+ N/3+/+3 N/3+/+3 N/3+/+3 N/3+/3+ N/3+/3+ N/3+/3+ N/3+/3+ N/3+/3+ N/3+/3+ N/3+/3+ N/3+/3+ N/3+/3+ N/3+/3+ N/3+/3+ N/3+/3+
GL, detection of gross lesions scored as (N) negative, (P) positive; HL, severity of histological lesion scored as (e) no lesion, (1+) slight, (2+) moderate, (3+) severe; IH, identification of bacterial antigen by IHC scored as (e) no lesion, (1+) slight, (2+) moderate, (3+), severe; IB, isolation of bacteria scored as (e) no colonies, (+) 1e10 colonies, (++) 10e100 colonies, (+++) over 101 colonies.
H. Hagiwara et al.
1 1 1 1 1 2 2 4 4 5 5 5 5 5 6 6 6
Fish number
265
Streptococcal Infection in Seriola dumerili
comprised severe arterial thrombosis, granulomatous valvular endocarditis, and epicarditis with macrophage and lymphocyte infiltration and proliferation of endocardial cells. The 14 orally infected and the 11 immersion infected fish that were killed on day 14 of the experiment had granulomas with caseous centres encapsulated by an attenuated epithelial cell layer. These lesions sometimes extended into the overlying dermis and epidermis. Some foci in the peduncle and fin displayed proliferation of fibroblasts and collagen fibres and lymphoid cell infiltration. The fish that were killed also had thrombosis, with vascular congestion, bacterial colonies in the peduncle, necrotizing vasculitis and purulent meningitis of the spinal cord. Fig. 1. Epicarditis with small nodules in a fish that died following immersion challenge with 107 CFU/ml of S. dysgalactiae.
the development of severe septicaemia (Table 1). In contrast, the fish killed on day 14 had systemic microabscessation and/or multifocal granulomatous inflammation consistent with bacterial embolism (Table 2). These inflammatory infiltrates were predominantly localized to the heart, caudal peduncle (Fig. 2), pectoral and/or dorsal fin and olfactory region. No microscopical changes were present in the tissues derived from the control fish. Seven orally infected (from groups 1e3) and 10 immersion infected fish (from groups 4e6) that died during the experiment had severe microabscessation of the aortic bulb and heart, caudal peduncle, pectoral and/or dorsal fin and olfactory region, as well as severe necrotizing splenitis (Fig. 3). The abscesses comprised a central core, with coagulative necrosis and/or gram-positive cocci. These fish also had bacterial emboli in the capillary and lymphatic vessels of the heart, spleen, kidney, liver, intestine (Fig. 4), caudal peduncle, fin, gill and olfactory region. Characteristic lesions in the atrium, myocardium and arterial cone
Immunohistochemistry
All fish had similar distribution of S. dysgalactiae antigen within tissues, but no bacterial antigen was detected in the tissues from the control fish. There were no major differences in the distribution of S. dysgalactiae antigen in fish from the orally infected and immersion infected groups that died, were moribund or survived until the end of the experiment. Bacterial antigen was found in the abscesses (Fig. 5), blood vessels (Fig. 6), lymphatic vessels and phagocytes (macrophages) in the loose connective tissue in the heart, caudal peduncle, fins and olfactory region. Bacterial antigen was also observed in the spleen of many of the dead fish. The location of bacterial antigen was closely associated with the distribution of inflammatory foci within tissues and bacterial isolation from tissues in the infected fish (Tables 1 and 2).
Discussion The virulence of S. dysgalactiae in cultured S. dumerili has previously been demonstrated by intramuscular,
Table 2 Gross and microscopical lesions, bacterial isolation and immunohistochemistry for fish that were killed at the conclusion of the experiment Group
1 2 3 4 5 6 7
Number of fish
2 5 7 5 2 4 7
Heart
Spleen
Peduncle
Fin
Gill
Olfactory
GL/HL/IH/IB
GL/HL/IH/IB
GL/HL/IH/IB
GL/HL/IH
GL/HL/IH
GL/HL/IH
0/0/0/1 0/2/1/0 0/2/0/0 0/1/1/0 0/1/0/0 0/1/0/0 0/0/0/0
2/0/0/1 0/1/1/0 0/0/0/0 3/0/0/0 2/0/0/0 4/0/0/0 0/0/0/0
2/2/2/1 1/3/3/1 0/0/0/0 0/4/4/0 0/1/1/0 3/3/3/2 0/0/0/0
0/1/1 0/2/2 0/0/0 0/2/2 0/0/0 0/0/0 0/0/0
0/0/0 0/0/0 0/0/0 0/0/0 0/0/0 0/0/0 0/0/0
0/2/2 0/3/2 0/0/0 0/0/0 0/0/0 0/2/2 0/0/0
GL, gross lesion; HL, histological lesion; IH, immunohistochemical detection of bacterial antigen in tissue; IB, isolation of bacteria; in each case the number represents the number of fish positive for that parameter.
266
H. Hagiwara et al.
Fig. 2. Inflammatory infiltration of the caudal peduncle of a fish that died following immersion challenge with 106 CFU/ ml of S. dysgalactiae. HE. Bar, 400 mm.
Fig. 4. A bacterial embolus and cellular infiltration of a blood vessel within the intestine of a fish that died following immersion challenge with 107 CFU/ml of S. dysgalactiae. HE. Bar, 200 mm.
intradermal and intraperitoneal injections of the bacteria. Infection with S. dysgalactiae has also been established in S. quinqueradiata following immersion exposure. Such infections lead to moderate mortality associated with severe necrotic lesions of the caudal peduncle (Nomoto et al., 2004; Hagiwara et al., 2009). In the present study, immersion exposure of S. dumerili to S. dysgalactiae led to moderate mortality associated with development of necrotic foci in the caudal peduncle, regardless of the dose of bacteria. In contrast, the outcome of oral infection depended greatly on the concentration of bacteria used. The clinical and pathological findings following immersion infection closely resembled those induced by intradermal or intraperitoneal inoculations of the bacteria (Hagiwara et al., 2009). Therefore, immersion infection of S. dumerili with S. dysgalactiae might
be considered a more effective natural infection route than via the oral route. The most prominent pathological change noted in fish infected experimentally with S. dysgalactiae was the development of severe necrotic lesions in the caudal peduncle, similar to those seen in naturally infected fish (Nomoto et al., 2004). These lesions consisted of microabscesses and/or pyogranulomatous inflammation of the caudal peduncle, pectoral and/ or dorsal fin, heart and olfactory region. The characteristics of these lesions and their distribution in dead and killed fish were similar to those described following intraperitoneal or intradermal injection of S. dysgalactiae (Hagiwara et al., 2009). Therefore, granulomatous inflammation of the caudal peduncle, fin and arterial cone might be considered
Fig. 3. Necrosis and numerous bacteria within the spleen of a fish that died following oral challenge with 108 CFU/ml of S. dysgalactiae. HE. Bar, 200 mm.
Fig. 5. Bacterial antigen in abscesses and macrophages in the caudal peduncle of a fish that died following immersion challenge with 106 CFU/ml of S. dysgalactiae. IHC. Bar, 200 mm.
267
Streptococcal Infection in Seriola dumerili
References
Fig. 6. Bacterial antigen in blood vessels in the caudal peduncle of a fish that died following immersion challenge with 107 CFU/ml of S. dysgalactiae. IHC. Bar, 100 mm.
pathognomonic lesions in cultured S. dumerili infected with S. dysgalactiae. Streptococci are well known causative agents of bacterial septicaemia (Vasconcelos et al., 1994; Katsumi et al., 1997; Nguyen et al., 2001; Madsen et al., 2002a, b; Benga et al., 2008). In the present study, S. dysgalactiae antigen was detected in the cytoplasm of macrophages in peripheral lymphatic and blood vessels, and the presence of bacterial antigen in the granulomatous lesion was closely correlated with the results of bacterial isolation. Therefore, bacterial septicaemia accompanied by necrosis of the spleen might have induced the death of fish, whereas in the surviving fish S. dysgalactiae multiplied and spread throughout the body, forming multifocal granulomatous foci. There were no major differences in the distribution of S. dysgalactiae antigen in the fish infected via the oral, immersion, intraperitoneal or intradermal routes (Hagiwara et al., 2009) S. dysgalactiae infections established by immersion or intradermal injection of S. dumerili would therefore appear to be better experimental models than those involving oral or intraperitoneal exposure. These model systems will be useful for the development of vaccines, immune stimulants and chemotherapeutic agents to treat or prevent S. dysgalactiae infection in cultured fish.
Benga L, Fulde M, Neis C, Goethe R, Valetin-Weigand P (2008) Polysaccharide capsule and suilysin contribute to extracellular survival of Streptococcus suis co-cultivated with primary porcine phagocytes. Veterinary Microbiology, 132, 211e219. Hagiwara H, Takano R, Noguchi M, Narita M (2009) A study of the lesions induced in Seriola dumerili by intradermal or intraperitoneal injection of Streptococcus dysgalactiae. Journal of Comparative Pathology, 140, 25e30. Katsumi M, Kataoka Y, Takahashi T, Kikuchi N, Hiramune T (1997) Bacterial isolation from slaughtered pigs associated with endocarditis, especially the isolation of Streptococcus suis. Journal of Veterinary Medical Science, 59, 75e78. Madsen LW, Bak H, Nielsen B, Jensen HE, Aalbaek B et al. (2002a) Bacterial colonization and invasion in pigs experimentally exposed to Streptococcus suis serotype 2 in aerosol. Journal of Veterinary Medicine Series B, 49, 211e215. Madsen LW, Svensmark B, Elvestad K, Aalbaek B, Jensen HE (2002b) Streptococcus suis serotype 2 infection in pig: new diagnostic and pathogenetic aspects. Journal of Comparative Pathology, 126, 57e65. Nguyen HT, Kanai K, Yoshikoshi K (2001) Immunohistochemical examination of experimental Streptococcus iniae infection in Japanese flounder Paralichthys olivaceus. Fish Pathology, 36, 169e178. Nomoto R, Munasinghe LI, Jin D-H, Shimahara Y, Yasuda H et al. (2004) Lancefield group C Streptococcus dysgalactiae infection responsible for fish mortalities in Japan. Journal of Fish Diseases, 27, 679e686. Nomoto R, Unose N, Shimahara Y, Nakamura A, Hirae T et al. (2006) Characterization of Lancefield group C Streptococcus dysgalactiae isolated from farmed fish. Journal of Fish Diseases, 29, 673e682. Vasconcelos D, Middleton DM, Chirino-Trejo JM (1994) Lesions caused by natural infection with Streptococcus suis type 9 in weaned pigs. Journal of Veterinary Diagnostic Investigation, 6, 335e341.
August 27th, 2009 ½ Received, Accepted, April 12th, 2010
Available online at www.sciencedirect.com
www.elsevier.com/locate/jcpa
Lesions Induced in Seriola dumerili Following Exposure to Streptococcus dysgalactiae by Oral Treatment or Immersion H. Hagiwara, R. Takano, N. Noguchi and M. Narita Advanced Technology Development Center, Kyoritsu Seiyaku Corporation, Takamihara, Tsukuba, Ibaraki 300-1252, Japan
Summary Seriola dumerili were infected experimentally with Streptococcus dysgalactiae by oral dosing or immersion. There was moderate mortality after immersion in water containing defined numbers of bacteria, regardless of the dose, whereas the effect on the oral challenge groups depended on the dose of bacteria administered. The characteristic lesions were microabscesses and/or pyogranulomatous inflammation of the caudal peduncle, pectoral and/or dorsal fin, heart and olfactory region. S. dysgalactiae antigen was found within necrotic foci at these sites. There was no difference in distribution of S. dysgalactiae antigen in fish that were exposed by oral or immersion challenge. There was no difference in antigen distribution when fish that died were compared with those that survived and were killed. Immersion exposure is therefore a more effective natural route of infection than oral challenge. Ó 2010 Elsevier Ltd. All rights reserved. Keywords: fish; immunohistochemistry; Seriola dumerili; Streptococcus dysgalactiae
Introduction Economic losses caused by streptococcal infections have been reported worldwide in many intensively cultured fish species. In 2002, an infection characterized by severe focal necrosis of the caudal peduncle and high mortality in cultured fish of the species Seriola dumerili and Seriola quinqueradiata was reported from fish farms in southern Japan. Bacterial strains isolated from diseased fish were identified as Lancefield group C Streptococcus dysgalactiae (Nomoto et al., 2004, 2006). From 2003 to 2008 the outbreak spread to involve other fish farms. A potential means of controlling S. dysgalactiae infection would be through the development of an effective vaccine. Unfortunately, this has been hindered by the lack of an adequate challenge system for the infection. Intraperitoneal, intramuscular and intradermal injections are the most widely used methods (Nomoto et al., 2004; Hagiwara et al., 2009); however, these routes bypass the natural skin and
mucosal barriers of the fish, leading to a faster and less natural progression of the disease. The aim of the present study was to characterize the pathology in S. dumerili experimentally infected with S. dysgalactiae by the oral route and by immersion challenge.
Materials and Methods Fish
S. dumerili were obtained from Kinki University, Japan. Fifty-two fish, each weighing approximately 142 g, were used in this study. The fish were divided into seven groups, each containing seven individuals, except for the non-infected control group (group 7), which comprised 10 fish. The fish were held in separate 200 l acryl tanks with a continuous flow of sea water (29 C) and fed a commercial dry pellet (2% body weight, Hayashikane Sangyo, Yamaguti, Japan) once daily throughout the experiment. Experimental Procedure
Correspondence to: H. Hagiwara (e-mail: h_hagiwara@kyoritsuseiyaku. com). 0021-9975/$ - see front matter doi:10.1016/j.jcpa.2010.04.005
Lancefield group C S. dysgalactiae strain 04K01, isolated from ulcers on the caudal peduncle of diseased Ó 2010 Elsevier Ltd. All rights reserved.
263
Streptococcal Infection in Seriola dumerili
S. dumerili in Kagoshima Prefecture, Japan, was used in all experimental infections (Hagiwara et al., 2009). Oral challenge was carried out with three groups of seven fish. Ground commercial pellet feed was mixed with an appropriate volume of the bacterial suspension at concentrations of 109 (group 1), 108 (group 2) and 107 (group 3). Immersion challenge was carried out with three groups of seven fish. Fish were anaesthetized in 0.015% FA-100 (10% 4-allyl-2-methoxyphenol, Tanabe Seiyaku Co., Japan). Bacteria were suspended in 5 l of sea water at concentrations of 107 (group 4), 106 (group 5) and 105 (group 6) CFU/ml, and the fish were immersed for 10 min. All fish were examined at least three times daily and mortality and any clinical signs were recorded. Dead and moribund fish were removed from the tank as soon as they were identified. All fish were subject to gross necropsy examination and sampled for bacteriology. The experiment was terminated after 14 days, at which point all of the remaining fish were anaesthetized and killed by decapitation before sampling for bacteriological and histopathological studies. Ten untreated fish were used as non-infected controls (group 7): three were anesthetized and killed on day 0 of the experiment, and seven were killed on day 14.
rabbit polyclonal antibody against S. dysgalactiae isolated from S. dumerili was used at a dilution of 1 in 15,000 in 0.05 M Tris HCl buffer (DAKO, Carpinteria, California). 3-amino-9-ethylcabazole (AEC) was used as chromogen and sections were counterstained with Mayer’s haematoxylin. Tissue sections from the uninfected control fish (group 7) and serum from a non-immunized rabbit were used as controls.
Bacterial Isolation
Gross Pathology
Samples for bacterial isolation were taken from the liver, spleen, kidney, heart, brain and caudal peduncle with a plastic loop and spread on soya bean casein digest broth (SCDB) agar. Plates were incubated at 30 C for 2 days.
The main gross pathological findings in the fish that died after oral or immersion challenges were erosions and/or abscesses on the caudal peduncle and pectoral and/or dorsal fin, epicarditis with small nodules (Fig. 1) and mild swelling of the spleen (Table 1). At the end of the experiment, there were only moderate erosions and/or abscesses on the caudal peduncle. No abnormalities were recorded in control fish (group 7).
Pathological Examination
Samples of liver, spleen, kidney, heart, gill, stomach, pyloric caecum, intestine, brain, caudal peduncle, pectoral and dorsal fin, skin, skeletal muscle and olfactory region were collected for microscopical examination. Tissues were fixed in 10% neutral-buffered formalin, embedded in paraffin wax and processed by routine methods. Sections were stained with haematoxylin and eosin (HE). Immunohistochemistry
Immunohistochemistry (IHC) was used to detect the presence of S. dysgalactiae antigen in the heart, caudal peduncle, pectoral and dorsal fins and olfactory region by the streptavidinebiotin complex immunoperoxidase (SAB-IP) method using an SAB kit (Nichirei Corp., Tokyo, Japan). Endogenous peroxidase activity was quenched with 3% H2O2 for 5 min. A
Results Mortality and Clinical Features
The mortality rates within the experimental groups are given in Table 1. Following oral challenge the fish in groups 1 and 2 began to die after 3e6 days and the cumulative mortality rates reached 71% (group 1) and 29% (group 2). No fish in group 3 died. Following immersion challenge the fish in groups 4e6 began to die after 5e10 days and cumulative mortality reached 29% (group 4), 71% (group 5) and 43% (group 6). No mortality occurred in the control fish (group 7). The clinical signs in the infected fish included lethargy, anorexia, and erratic swimming. Infected fish ate only half as much feed as the control group for 2e4 days after oral challenge and for 3e6 days after immersion challenge.
Bacterial Isolation
No pathogenic bacteria were cultured from fish of the control group (group 7). S. dysgalactiae was isolated from the heart, caudal peduncles, liver, spleen, kidney and brain of all dead fish infected via the oral route or by immersion. At the end of the experiment (day 14), S. dysgalactiae was isolated from the caudal peduncle of two orally infected and two immersion infected fish, from the heart of one orally infected fish and from the spleen of one orally infected fish (Table 2). Microscopical Features
The most consistently observed microscopical change in infected fish that died during the experiment was
264
Table 1 Gross and microscopical lesions, bacterial isolation and immunohistochemistry for fish that died during the experiment Group
1 2 3 4 5 1 2 1 2 1 2 3 4 5 1 2 3
Day of death
3 5 5 5 6 5 5 7 8 5 5 6 7 10 7 7 8
Liver
Spleen
Kidney
Heart
Brain
Peduncle
Fin
Gill
Olfactory
GL/HL/IB
GL/HL/IH/IB
GL/HL/IB
GL/HL/IH/IB
GL/HL/IB
GL/HL/IH/IB
GL/HL/IH
GL/HL/IH
GL/HL/IH
N/e/+ N/e/+++ N/e/+ N/e/+++ N/e/++ N/e/++ N/e/+++ N/e/+ N/e/++ N/e/++ N/e/++ N/e/++ N/e/++ N/e/e N/e/++ N/e/e N/e/++
P/3+/+2/+++ P/3+/+3/++ P/e/e/+ P/3+/+3/+++ P/3+/+2/++ P/3+/+3/++ P/e/e/+++ P/3+/+3/+++ P/3+/+2/+++ P/3+/+3/+++ P/e/e/e P/3+/+3/+++ N/3+/+3/+++ P/e/e/e P/3+/+3/+++ P/1+/+1/+ P/1+/e/+++
N/3+/+++ N/e/++ N/e/+ N/1+/++ N/e/++ N/e/++ N/e/+++ N/1+/++ N/e/++ N/1+/++ N/e/+ N/e/++ N/e/++ N/e/e N/e/+++ N/e/+ N/e/++
P/3+/+3/ND N/3+/+3/+++ N/3+/+2/++ N/3+/+3/+++ N/3+/+3/++ N/3+/+3/+++ N/1+/e/ND N/3+/+3/+++ P/3+/+3/++ N/3+/+3/+++ N/ND/ND/e N/3+/+3/ND N/3+/+3/+++ N/1+/e/e P/3+/+3/ND P/3+/+3/+ N/3+/+3/++
N/e/+++ N/e/++ N/e/ND N/e/++ N/e/++ N/e/ND N/e/ND N/e/+ N/e/++ N/e/+++ N/e/e N/e/++ N/e/+++ N/e/e N/e/+++ N/e/+ N/e/++
P/3+/+2/++ P/3+/+3/+++ P/3+/+3/+++ P/3+/+3/+++ P/3+/+3/+++ P/3+/+3/+++ P/3+/+3/+++ P/3+/+3/+++ P/3+/+3/+++ P/3+/+3/+++ P/3+/+3/+++ P/3+/+3/+++ P/3+/+3/+++ P/3+/+2/+ P/3+/+3/+++ P/3+/+3/+++ P/3+/+3/+++
N/3+/+2 N/3+/+3 N/3+/+3 N/3+/+3 N/3+/+3 P/3+/+3 N/3+/+3 P/3+/+3 P/3+/+3 N/3+/+3 P/3+/+3 N/3+/+3 P/3+/+3 P/2+/+2 P/3+/+3 P/3+/+3 N/3+/+3
N/3+/+3 N/e/e N/e/e N/e/e N/e/e N/e/e N/e/e N/e/e N/e/e N/e/e N/e/e N/e/e N/e/e N/e/e N/e/e N/e/e N/e/e
N/3+/3+ N/3+/2+ N/3+/+3 N/3+/+3 N/3+/+3 N/3+/3+ N/3+/3+ N/3+/3+ N/3+/3+ N/3+/3+ N/3+/3+ N/3+/3+ N/3+/3+ N/3+/3+ N/3+/3+ N/3+/3+ N/3+/3+
GL, detection of gross lesions scored as (N) negative, (P) positive; HL, severity of histological lesion scored as (e) no lesion, (1+) slight, (2+) moderate, (3+) severe; IH, identification of bacterial antigen by IHC scored as (e) no lesion, (1+) slight, (2+) moderate, (3+), severe; IB, isolation of bacteria scored as (e) no colonies, (+) 1e10 colonies, (++) 10e100 colonies, (+++) over 101 colonies.
H. Hagiwara et al.
1 1 1 1 1 2 2 4 4 5 5 5 5 5 6 6 6
Fish number
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Streptococcal Infection in Seriola dumerili
comprised severe arterial thrombosis, granulomatous valvular endocarditis, and epicarditis with macrophage and lymphocyte infiltration and proliferation of endocardial cells. The 14 orally infected and the 11 immersion infected fish that were killed on day 14 of the experiment had granulomas with caseous centres encapsulated by an attenuated epithelial cell layer. These lesions sometimes extended into the overlying dermis and epidermis. Some foci in the peduncle and fin displayed proliferation of fibroblasts and collagen fibres and lymphoid cell infiltration. The fish that were killed also had thrombosis, with vascular congestion, bacterial colonies in the peduncle, necrotizing vasculitis and purulent meningitis of the spinal cord. Fig. 1. Epicarditis with small nodules in a fish that died following immersion challenge with 107 CFU/ml of S. dysgalactiae.
the development of severe septicaemia (Table 1). In contrast, the fish killed on day 14 had systemic microabscessation and/or multifocal granulomatous inflammation consistent with bacterial embolism (Table 2). These inflammatory infiltrates were predominantly localized to the heart, caudal peduncle (Fig. 2), pectoral and/or dorsal fin and olfactory region. No microscopical changes were present in the tissues derived from the control fish. Seven orally infected (from groups 1e3) and 10 immersion infected fish (from groups 4e6) that died during the experiment had severe microabscessation of the aortic bulb and heart, caudal peduncle, pectoral and/or dorsal fin and olfactory region, as well as severe necrotizing splenitis (Fig. 3). The abscesses comprised a central core, with coagulative necrosis and/or gram-positive cocci. These fish also had bacterial emboli in the capillary and lymphatic vessels of the heart, spleen, kidney, liver, intestine (Fig. 4), caudal peduncle, fin, gill and olfactory region. Characteristic lesions in the atrium, myocardium and arterial cone
Immunohistochemistry
All fish had similar distribution of S. dysgalactiae antigen within tissues, but no bacterial antigen was detected in the tissues from the control fish. There were no major differences in the distribution of S. dysgalactiae antigen in fish from the orally infected and immersion infected groups that died, were moribund or survived until the end of the experiment. Bacterial antigen was found in the abscesses (Fig. 5), blood vessels (Fig. 6), lymphatic vessels and phagocytes (macrophages) in the loose connective tissue in the heart, caudal peduncle, fins and olfactory region. Bacterial antigen was also observed in the spleen of many of the dead fish. The location of bacterial antigen was closely associated with the distribution of inflammatory foci within tissues and bacterial isolation from tissues in the infected fish (Tables 1 and 2).
Discussion The virulence of S. dysgalactiae in cultured S. dumerili has previously been demonstrated by intramuscular,
Table 2 Gross and microscopical lesions, bacterial isolation and immunohistochemistry for fish that were killed at the conclusion of the experiment Group
1 2 3 4 5 6 7
Number of fish
2 5 7 5 2 4 7
Heart
Spleen
Peduncle
Fin
Gill
Olfactory
GL/HL/IH/IB
GL/HL/IH/IB
GL/HL/IH/IB
GL/HL/IH
GL/HL/IH
GL/HL/IH
0/0/0/1 0/2/1/0 0/2/0/0 0/1/1/0 0/1/0/0 0/1/0/0 0/0/0/0
2/0/0/1 0/1/1/0 0/0/0/0 3/0/0/0 2/0/0/0 4/0/0/0 0/0/0/0
2/2/2/1 1/3/3/1 0/0/0/0 0/4/4/0 0/1/1/0 3/3/3/2 0/0/0/0
0/1/1 0/2/2 0/0/0 0/2/2 0/0/0 0/0/0 0/0/0
0/0/0 0/0/0 0/0/0 0/0/0 0/0/0 0/0/0 0/0/0
0/2/2 0/3/2 0/0/0 0/0/0 0/0/0 0/2/2 0/0/0
GL, gross lesion; HL, histological lesion; IH, immunohistochemical detection of bacterial antigen in tissue; IB, isolation of bacteria; in each case the number represents the number of fish positive for that parameter.
266
H. Hagiwara et al.
Fig. 2. Inflammatory infiltration of the caudal peduncle of a fish that died following immersion challenge with 106 CFU/ ml of S. dysgalactiae. HE. Bar, 400 mm.
Fig. 4. A bacterial embolus and cellular infiltration of a blood vessel within the intestine of a fish that died following immersion challenge with 107 CFU/ml of S. dysgalactiae. HE. Bar, 200 mm.
intradermal and intraperitoneal injections of the bacteria. Infection with S. dysgalactiae has also been established in S. quinqueradiata following immersion exposure. Such infections lead to moderate mortality associated with severe necrotic lesions of the caudal peduncle (Nomoto et al., 2004; Hagiwara et al., 2009). In the present study, immersion exposure of S. dumerili to S. dysgalactiae led to moderate mortality associated with development of necrotic foci in the caudal peduncle, regardless of the dose of bacteria. In contrast, the outcome of oral infection depended greatly on the concentration of bacteria used. The clinical and pathological findings following immersion infection closely resembled those induced by intradermal or intraperitoneal inoculations of the bacteria (Hagiwara et al., 2009). Therefore, immersion infection of S. dumerili with S. dysgalactiae might
be considered a more effective natural infection route than via the oral route. The most prominent pathological change noted in fish infected experimentally with S. dysgalactiae was the development of severe necrotic lesions in the caudal peduncle, similar to those seen in naturally infected fish (Nomoto et al., 2004). These lesions consisted of microabscesses and/or pyogranulomatous inflammation of the caudal peduncle, pectoral and/ or dorsal fin, heart and olfactory region. The characteristics of these lesions and their distribution in dead and killed fish were similar to those described following intraperitoneal or intradermal injection of S. dysgalactiae (Hagiwara et al., 2009). Therefore, granulomatous inflammation of the caudal peduncle, fin and arterial cone might be considered
Fig. 3. Necrosis and numerous bacteria within the spleen of a fish that died following oral challenge with 108 CFU/ml of S. dysgalactiae. HE. Bar, 200 mm.
Fig. 5. Bacterial antigen in abscesses and macrophages in the caudal peduncle of a fish that died following immersion challenge with 106 CFU/ml of S. dysgalactiae. IHC. Bar, 200 mm.
267
Streptococcal Infection in Seriola dumerili
References
Fig. 6. Bacterial antigen in blood vessels in the caudal peduncle of a fish that died following immersion challenge with 107 CFU/ml of S. dysgalactiae. IHC. Bar, 100 mm.
pathognomonic lesions in cultured S. dumerili infected with S. dysgalactiae. Streptococci are well known causative agents of bacterial septicaemia (Vasconcelos et al., 1994; Katsumi et al., 1997; Nguyen et al., 2001; Madsen et al., 2002a, b; Benga et al., 2008). In the present study, S. dysgalactiae antigen was detected in the cytoplasm of macrophages in peripheral lymphatic and blood vessels, and the presence of bacterial antigen in the granulomatous lesion was closely correlated with the results of bacterial isolation. Therefore, bacterial septicaemia accompanied by necrosis of the spleen might have induced the death of fish, whereas in the surviving fish S. dysgalactiae multiplied and spread throughout the body, forming multifocal granulomatous foci. There were no major differences in the distribution of S. dysgalactiae antigen in the fish infected via the oral, immersion, intraperitoneal or intradermal routes (Hagiwara et al., 2009) S. dysgalactiae infections established by immersion or intradermal injection of S. dumerili would therefore appear to be better experimental models than those involving oral or intraperitoneal exposure. These model systems will be useful for the development of vaccines, immune stimulants and chemotherapeutic agents to treat or prevent S. dysgalactiae infection in cultured fish.
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August 27th, 2009 ½ Received, Accepted, April 12th, 2010