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Examination of entry portal and pathogenesis of Edwardsiella ictaluri infection in striped catfish, Pangasianodon hypophthalmus
Aquaculture 464 (2016) 279–285
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Examination of entry portal and pathogenesis of Edwardsiella ictaluri infection in striped catfish, Pangasianodon hypophthalmus Nopadon Pirarat a,⁎, Ei Lin Ooi b, Kim D. Thompson c, Nguyen Huu Thinh d, Masashi Maita e, Takayuki Katagiri e a
Wildlife, Exotic and Aquatic Pathology - Special Task Force for Activating Research, Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand, DSM Nutritional Products, Aquaculture Center Asia Pacific, Bangkok, Thailand, c Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Edinburgh, United Kingdom d Department of Fish Pathology, Faculty of Fisheries, Nong Lam University, Ho Chi Minh City, Viet Nam e Laboratory of Fish Health Management, Tokyo University of Marine Science and Technology, Japan b
a r t i c l e
i n f o
Article history: Received 3 June 2016 Received in revised form 29 June 2016 Accepted 30 June 2016 Available online 01 July 2016 Keywords: BNP Edwardsiella ictaluri Immunohistochemistry Necrotic dermatitis Striped catfish
a b s t r a c t The host-bacterium interaction between striped pangasius catfish (Pangasianodon hypophthalmus) and Edwardsiella ictaluri was examined during experimentally induced bacillary necrosis of pangasius (BNP). After infection by immersion challenge, fish samples were taken over the course of the infection. Necrotic dermatitis, associated with vasculitis-perivasculitis changes, was the most notable gross and histopathological change observed, similar to that described during channel catfish enteric septicemia. Typical histopathological change associated with BNP were initially observed by 96 h post-infection (hpi) and these lasted until 14 days post infection (dpi) with no granulomatous formation evident. The presence of the bacterium was observed in various organs using immunohistochemistry (IHC), except for the brain, with positive staining observed within differing cell types, including phagocytic cells, myocardia, gill epithelia and capillary endothelia. No extracellular deposition of bacterial antigen was observed. Bacterial antigen persisted up to 1 month in the independent phagocytic cells, necrotic-associated phagocytic cells and melano-macrophage centers, confirming the intracellular nature of E. ictaluri disseminated in the tissues during septicemia. The bacterium was evident in the gills early in the infection (6–12 hpi) and in necrotic skin at 72 hpi. With the histopathological changes observed, this suggests that skin is another possible natural route for disseminating E. ictaluri in pangasius catfish during infection. Statement of relevance: This information will improve our understanding of the pathogenesis of BNP, and establish more effective methods of control for this important disease, including methods for quantifying sub-lethal infections and assessing response against the invading pathogen. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Over the past decade, striped catfish, Pangasianodon hypophthalmus, has been increasingly important as a new aquaculture whitefish product on the world market, with production increasing dramatically to meet global demand (Globefish, 2015). However, the need for intensive culture to meet this demand has been hampered by a number of problems, especially poor husbandry and water quality, and increase in infectious disease episodes in culture systems. Bacillary necrosis of Pangasianodon (BNP), a recently recognized disease caused by Edwardsiella ictaluri, has been highlighted as one of the most important infectious disease problems in intensive striped catfish production (Ferguson et al., 2001). BNP is characterized by multifocal irregular white lesions of varying size located on different organs, including liver, spleen and kidney. E. ictaluri is also the etiological agent of enteric ⁎ Corresponding author. E-mail address: [email protected] (N. Pirarat).
http://dx.doi.org/10.1016/j.aquaculture.2016.06.043 0044-8486/© 2016 Elsevier B.V. All rights reserved.
septicemia in channel catfish (Ictalurus punctatus) (ESC), causing enteritis and septicemia, with red and white ulcers covering the skin and petechial hemorrhages around the mouth and fin bases (Areechon and Plumb, 1983; Miyazaki and Plumb, 1985). Assessment of the inflammatory response is often used to examine the host's immune response to an infectious agent. Many authors have described the inflammatory response that occurs in channel catfish in response to E. ictaluri during outbreaks of ESC. An effusive form of the disease is found in channel catfish, characterized by the production of ascites in the abdominal cavity of the fish, swollen kidneys and spleens, mottled livers, hemorrhage intestines and generalized septicemia (Areechon and Plumb, 1983; Jacrboe et al., 1984; Miyazaki and Plumb, 1985; Newton et al., 1989). BNP in pangasius catfish, by contrast is a non-effusive form of the disease, characterized by necrotic changes in different organs, sometimes including the brain (Ferguson et al., 2001; Crumlish et al., 2010). Both types of presentations can be observed in other fish species, including green knife fish (Eigenmannia virescens) (Kent and Lyons, 1982), zebra danio (Danio rerio) (Waltman et al.,
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1985) rosy barbs (Pethia conchonius) (Humphrey et al., 1986) and walking catfish (Clarias batrachus) (Kasornchandra et al., 1987). Several studies have been published on entry portal and pathogenesis of E. ictaluri in ESC, as well as treatment and control of the disease via vaccination and antibiotics (Areechon and Plumb, 1983, Miyazaki and Plumb, 1985, Ciembor et al., 1995; Booth et al., 2009; Crumlish et al., 2010; Dung et al., 2012). However, there are few studies examining pathogenesis and entry portal of E. ictaluri in BNP outbreaks in striped catfish. Dung et al. (2012) was the first to describe the pathogenesis of E. ictaluri infections in Pangasianodon catfish, and there are a few studies focusing on the immune response against this bacterium (Sirimanapong et al., 2015a, 2015b). The objectives of the current study were: 1) to characterize the early histopathological events in freshwater striped catfish after experimentally infecting them with E. ictaluri using a simulated natural route of exposure, and 2) to examine the distribution of the bacterium in the fish during infection using immunohistochemistry (IHC). This information will improve our understanding of the pathogenesis of BNP, and establish more effective methods of control for this important disease, including methods for quantifying sub-lethal infections and assessing response against the invading pathogen. 2. Materials and methods 2.1. Experimental design One hundred and ninety four striped catfish, P. hypophthalmus, (40 ± 5 g), were purchased from a local hatchery in Vietnam and transported, quarantined and maintained according to the standard operating procedures of the National Agriculture Research Center (NARC) for fish husbandry. Fish with uniform weights were selected and randomly allocated to two 1000 L tanks (97 fish each) in Nong Lam University's challenge facility (Ho Chi Min City, Vietnam) and acclimated for N 1 week before initiating the trial. Two treatment groups were used in the study, including an uninfected control group and a group experimentally infected with E. ictaluri by immersion challenge. Before exposing the fish to the bacteria, ten fish from each of the two tanks were checked for the presence of external parasites and sub-clinical E. ictaluri infection. Wet mount preparations were made from the skin and gills and examined microscopically. Samples of the liver and posterior kidney were taken and streaked onto brain-heart infusion agar (BHIA), and plates incubated at 28 °C for 48 h. The presence of external parasite was not observed by gill and skin scraping. There was no growth of E. ictaluri in any of swabs taken from the ten fish. A diet (32% crude protein; 6% fat) was formulated to meet the nutritional requirements of striped catfish (Tran et al., 2010). The diets were fed once daily at 3% body weight. Water temperature was maintained at 29 ± 2 °C prior to the challenge and at 26 ± 1 °C post-infection. The duration of the trial lasted one month, and the experiment was performed in triplicate. 2.2. Infection of fish with Edwardsiella ictaluri A subclinical dose of Edwardsiella ictaluri, strain NLF33 (courtesy of Nong Lam University) was used as the immersion challenge (Sirimanapong et al., 2015a, 2015b). The bacteria were first streaked onto BHIA and cultured at 30 °C for 24 h, and then three to five colonies of E. ictaluri were placed in sterile brain heart infusion broth (BHIB) and cultured at 28 °C for 18 h. The bacterial concentration was determined from CFUs using a plate counting method and adjusted to 106 CFU/mL with sterile saline. The fish were experimentally infected with E. ictaluri by immersing the fish in static, well-aerated water to which the bacteria has been added at a final concentration of 8 × 104 CFU/mL. The fish were held in the bacterial suspension for 30 min after which fifteen fish were randomly selected and placed in three replicate 80 L tanks at 10.48 kg m3. Uninfected fish used as negative controls were immersed in BHIB instead of the bacterial suspension. The fish were monitored
for 30 days for clinical signs and gross pathology, which was recorded twice daily. 2.3. Histopathology Ten tissues (i.e. brain, heart, gill, spleen, head and trunk kidney, liver, skin, pancreas and intestine) were collected at 0, 1, 3, 6, 12, 24, 48, 72, 96 h post-infection (hpi), and 5, 10 and 30 days post-infection (dpi) from six fish per group (i.e. two fish per tank) and fixed in 10% buffered formalin for histopathology. Fixed tissues were processed according to standard histological techniques, and tissue sections were stained with hematoxylin and eosin (H&E). Tissue sections were examined under a light microscope for pathological changes (Pirarat et al., 2006). 2.4. Immunohistochemistry Microscopic localization of E. ictaluri antigens and phenotypic characterization of associated cells was performed as previously described (Pirarat et al., 2006). Briefly, after placing 5 μm tissue sections on glass microscope slides and encircling them with a wax ring, the tissue sections were dehydrated through an alcohol series. The tissues were then incubated with 3% (v/v) hydrogen peroxide in methanol for 20 min to block non-specific endogenous peroxidase activity, and sections were washed in 0.02 M phosphate buffered saline (PBS) pH 7.3 and incubated with 1% (w/v) bovine serum albumin for 30 min. Rabbit polyclonal anti-E. ictaluri antibody (1:250 dilution in PBS) developed inhouse, was applied to the tissues, and the slides were incubated at 4 °C overnight. The tissues were then rinsed with PBS and incubated with a secondary antibody conjugated with a universal immuno-enzyme polymer using a Histofine MAX PO kit (Nichirei, Japan) for 45 min at 22 °C. The tissues were again washed in PBS and incubated with either diaminobenzidine (DAB) or 3-amino, 9-ethyl-carbazole (AEC) substrate for 10 min. The tissues were counterstained with hematoxylin, rinsed in serial graded alcohols and xylene and mounted with mounting media. Normal, healthy striped catfish tissues were used as a negative control. 3. Results 3.1. Clinical signs and gross pathology After 72 hpi infection, fish exhibited clinical signs of lethargy, slowed swimming and gathering at the water surface. Patchy and ecchymotic hemorrhages were initially observed at the pectoral fin bases of one affected fish at 96 hpi. Multiple, raised pimple-like lesions, or a “buckshot” appearance, (Fig. 1A & B) were apparent 96 hpi. Bacillary necrosis of the spleen, kidney and liver were noted after 96 hpi in four of the six fish examined, and bacteria were observed until the end of the trial at 30 dpi in two of the six fish examined. Abdominal distension, or ascites, was not observed. No mortalities occurred during the one month period of the challenge trial. No abnormal clinical signs were noted in the control group throughout the trial period. 3.2. Histopathology The most notable histopathological changes in infected fish were bacillary necrosis, vasculitis and perivasculitis. An inflammatory response was obvious at 72 hpi with widespread diffusion in organs, generating the septicemic condition (Fig. 2). At this early stage, the necrotic lesion was characterized by a central necrotic core composed of phagocytic cells loaded with necrotic debris and haemolyzed red blood cells. The higher aggregation of phagocytic cells at the center of the core, surrounded by mononuclear inflammatory leukocytes was noted later in the infection (10 dpi). There was no evidence of any well-organized fibrosing granulomas developing during the infection. Necrotic lesions developed early and were quite widespread by 5–10 dpi in the head kidney (Fig. 2, C & D), and in trunk kidney, spleen, liver, skin and gill,
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within phagocytic cells, gill epithelia and capillary endothelia. Immuno-labelling of the bacteria was predominantly distributed in the head kidney, spleen, trunk kidney, skin, liver, gills, and muscle respectively (Table 2). No positive reaction was observed in the lamina propria or the mucosal epithelium of intestinal tissues throughout the experiment. Bacteria were localized within the necrotic skin (Fig. 3A) and the perivasculitis-associated endothelial cells (Fig. 3B) below the skin by 96 hpi. A positive reaction was clearly seen in glomeruli and interstitial tissues of the trunk kidney, and bacteria also occasionally presented in the renal tubular epithelium. Immuno-labelling of bacteria in the liver was also occasionally seen on the serosal surface. Early in the infection (1–3 dpi), stained bacteria could be clearly seen in the brush border of primary and secondary lamellar epithelium of the gills (Fig. 3C). After 3 dpi, stained bacteria could also be observed in necrotic epithelium with hyperplastic changes evident (Fig. 3D). Bacteria could also be seen in the heart myocardium. By 3 dpi, immuno-labelling of bacteria was present in the spleen, which was diffusely distributed within independent-circulating phagocytic cells, especially located in the perisinusoidal and ellipsoidal spaces. By 4 dpi, the pattern of positive staining in the spleen could be seen within the necrotic-participating phagocytic cells (Fig. 3E). Positive staining was initially also observed within independent phagocytic cells in the head kidney by 3 dpi, and this also gradually spread to the necrotic-participating phagocytic cells (Fig. 3F) and later to the melano-macrophage centers. Positive signals were clearly observed within necrotic-participating phagocytic cells between 10 dpi and the end of the trial at 30 dpi. Fig. 1. Macroscopic finding of striped catfish experimentally infected with Edwardsiella ictaluri. (A) Gross pathology; (B) Multiple red skin nodules observed at 6 days postinfection. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
respectively, as indicated in Table 1. Raised nodular lesions of necrotic skin and underlying myositis were obvious by 5 dpi and could still be seen by 30 dpi. Perivasculitis, characterized by an infiltration of inflammatory leukocytes into the wall of blood vessels, surrounded by degenerated muscle fibers, as shown in Fig. 2B, was observed from 48 hpi until the end of the trial (Table 1). Thrombosis and vasculitis, evidenced by inflammatory leukocyte aggregation into the tunica media, was present in the liver (Fig. 2E). Endothelial cell hypertrophy and loss of endothelial cell lining were also noted. Fibrinoid degeneration of vessel walls was not apparent. Single cell necrosis of hepatocytes was seen at 3 dpi before developing necrotic foci (Fig. 2F). Multiple foci of hepatic necrosis were abundant adjacent to areas of vasculitis, from 96 hpi. In trunk kidney, interstitial glomerulonephritis, composed of small mononuclear leukocyte infiltration in the glomeruli and tubular interstitial tissues, and multiple phagocytic cells loaded with bacteria within necrotic areas, was present at 96 hpi. Vasculitis lesions (Fig. 2H) with thickening of vessel walls was also noted, with evidence of fibrinoid degeneration and fibrin thrombi. Thickening of glomerular basement membrane, with infiltration of inflammatory leukocytes into glomeruli, was also present in the later stages of the infection, i.e. from 10 dpi. Hyaline degeneration of renal tubules was occasionally seen. In gills, necrotic changes were seen earlier (72 hpi) than in other tissues (Fig. 2A). Goblet cell hyperplasia was observed as early as 12 to 48 hpi in primary lamellar gill. Distorted and congested gill lamellae (aneurism) were also noted at this early stage. Secondary lamellar hyperplasia and fusion were evident by 3 dpi. Myocarditis with mononuclear cell infiltration was noted in the heart by 10 dpi (Fig. 2G). Brain tissues showed congestion without any inflammatory cell response throughout the course of the infection. 3.3. Immunohistochemistry Edwardsiella ictaluri antigen was diffusely detected in various organs, except the brain, using IHC. Positive IHC signals were identified
4. Discussion According to previous reports, both BNP and ESC differ in clinical manifestation. In infected striped catfish, few external signs are evident, including pale gills and skin BNP (Ferguson et al., 2001; Crumlish et al., 2010; Dung et al., 2012). With catfish ESC, on the other hand, fish exhibit petechial hemorrhaging and ulcerative skin lesions (Areechon and Plumb, 1983; Hawke et al., 1998). In our study, fish had macroscopic skin lesions resembling red pimples, or with a “buckshot” appearance, and ulcerative skin lesions with patchy hemorrhages at the fin bases were also present, which are typically described in channel catfish. A necrotic response was present in various tissues of the striped catfish 3 dpi with E. ictaluri by immersion bath, and this response was evident at least up to 30 dpi. The difference in clinical manifestation and macroscopic finding between fish species may be due to an intraspecific diversity of E. ictaluri strains (Bartie et al., 2012; Dung et al., 2012). The nature of host-pathogen inflammatory response and the susceptibility of the fish to the bacterium may differ between fish species, as suggested by Dung et al. (2012). ESC and BNP histopathological lesions share some microscopic similarities in channel catfish and striped catfish, including multifocal necrosis in multiple organs. However, lesions representing ulcerative dermatitis, necrotic myositis and vasculitis-perivasculitis have never been recorded in pangasius BNP (Crumlish et al., 2010; Dung et al., 2012). Our study revealed marked necrosis and ulcerative lesions on the skin of infected fish, and the necrotic myositis of underlying musculature was associated with vasculitis and perivasculitis lesions. The diffuse to multifocal necrosis in internal organs were also related to vasculitis-perivasculitis changes, as seen in catfish ESC. It is important to examine the host-pathogen interaction to enable understanding of how the bacillary necrotic changes develop in the striped catfish. Bacteria are distributed by and replicate within circulating macrophages and necrotic-phagocytic cells that are scattered throughout the body of the fish. The vasculitis-perivasculitis changes associated with the BNP during our study suggest that bacteria can directly damage the endothelial cells resulting in vasculitis and perivasculitis, and eventual tissue hypoxia and necrosis as evidenced by the positive IHC staining seen within the perivasculitis-endothelial cells in the skin
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Fig. 2. Histopathology of striped catfish experimentally infected with Edwardsiella ictaluri (A) gill necrosis (arrowhead) and gill epithelial hyperplasia (arrow) at 72 h post-infection (hpi); (B) myositis (arrow) and perivasculitis change (arrowhead) at 6 days post-infection (dpi); (C and D) bacillary necrotic lesion observed in head kidney at 10 dpi; (E) necrotic hepatitis and (F) perivasculitis changes were multifocal in the liver at 10 dpi. (G) Myocarditis with mononuclear cell infiltration observed at 14 dpi; (H) vasculitis-perivasculitis changes in trunk kidney.
and kidneys of infected fish. However, there were no pin-point petechial hemorrhages in internal organs correlating with vasculitis lesions as seen in channel catfish enteric septicemia (Areechon and Plumb, 1983; Jacrboe et al., 1984; Hawke et al. 1998). This might be explained by differences in the inflammatory response elicited by these two different host species, possibly through the release of vasoactive amines, which cause endothelial cell retraction, increased vascular permeability and development of characteristic protein-rich exudates. The positive correlation between the vasculitis-perivasculitis change and the positive stained bacteria within the vascular endothelial cells, suggests
that an immune-complex mediated vasculitis or immune-complex mediated glomerulonephritis, a type III hypersensitivity reaction, cannot be ruled out as a cause of the pathogenesis resulting from the host response. The circulating, complement-fixing immune complexes, recognized by phagocytic cells lodged within blood vessel walls, lead to development of the bacillary necrotic changes. The presence of fibrinoid degeneration and thickening of vascular walls is similar to the Schwartzman reaction resulting in a thrombohaemorrhagic response to bacterial toxin as described in rabbits and humans (Starzl et al., 1968; Nordstoga, 1977). Unfortunately, there was no evidence of
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Table 1 Histopathology of striped catfish challenged with E. ictaluri. Time post-infection
1 h (h)
Group
Cont
Infect
3h Cont
Infect
6h Cont
Infect
Cont
Infect
Cont
Infect
Cont
Infect
Head kidney Bacillary necrosis Melanomacrophage center
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (4/6)
(0/6) (1/6)
(0/6) (5/6)
Skin Myositis Perivasculitis/vasculitis Bacillary necrosis
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(2/6) (2/6) (0/6)
Spleen Ellipsoidal enlargement Bacillary necrosis Melanomacrophage center
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (4/6)
(0/6) (0/6) (1/6)
(0/6) (0/6) (5/6)
Trunk kidney Vasculitis/perivasculitis Bacillary necrosis
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
Heart Myocarditis Vasculitis/perivasculitis Bacillary necrosis
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(1/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
Liver Vasculitis/perivasculitis Bacillary necrosis
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
Gill Goblet cell hyperplasia Lamella hyperplasia Bacillary necrosis
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(2/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(4/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(5/6) (0/6) (0/6)
Swim bladder Aerocystitis
(0/6)
(0/6)
(0/6)
(2/6)
(0/6)
(0/6)
(0/6)
(1/6)
(0/6)
(3/6)
(0/6)
(2/6)
Intestine Lamina propria inflammation
(0/6)
(0/6)
(0/6)
(0/6)
(0/6)
(0/6)
(1/6)
(0/6)
(0/6)
(0/6)
(0/6)
(0/6)
96 h
12 h
5 days (d)
24 h
48 h
Time post-infection
72 h
10 d
30 d
Group
Cont
Infect
Cont
Infect
Cont
Infect
Cont
Infect
Cont
Infect
Head kidney Bacillary necrosis Melanomacrophage center
(0/6) (0/6)
(0/6) (6/6)
(0/6) (2/6)
(4/6) (4/6)
(0/6) (4/6)
(5/6) (4/6)
(0/6) (3/6)
(4/6) (3/6)
(0/6) (2/6)
(4/6) (6/6)
Skin Myositis Perivascultitis/vasculitis Bacillary necrosis
(0/6) (0/6) (0/6)
(3/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(5/6) (5/6) (2/6)
(0/6) (0/6) (0/6)
(2/6) (1/6) (5/6)
(0/6) (0/6) (0/6)
(3/6) (0/6) (3/6)
(0/6) (0/6) (0/6)
(5/6) (5/6) (3/6)
Spleen Ellipsoidal enlargement Bacillary necrosis Melanomacrophage center
(0/6) (0/6) (0/6)
(2/6) (0/6) (6/6)
(0/6) (0/6) (0/6)
(1/6) (4/6) (6/6)
(0/6) (0/6) (3/6)
(3/6) (5/6) (2/6)
(0/6) (0/6) (2/6)
(0/6) (5/6) (1/6)
(0/6) (0/6) (4/6)
(0/6) (2/6) (1/6)
Trunk kidney Vasculitis/perivasculitis Bacillary necrosis
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(3/6) (0/6)
(0/6) (0/6)
(3/6) (3/6)
(0/6) (0/6)
(4/6) (5/6)
(0/6) (0/6)
(2/6) (2/6)
Heart Myocarditis Vasculitis/perivasculitis Bacillary necrosis
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(1/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(3/6) (2/6) (0/6)
(0/6) (0/6) (0/6)
(2/6) (0/6) (0/6)
Liver Vasculitis/perivasculitis Bacillary necrosis
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(2/6) (1/6)
(0/6) (0/6)
(2/6) (2/6)
(0/6) (0/6)
(3/6) (3/6)
(0/6) (0/6)
(1/6) (0/6)
Gill Goblet cell hyperplasia Lamella hyperplasia Bacillary necrosis
(0/6) (0/6) (0/6)
(3/6) (3/6) (2/6)
(0/6) (0/6) (0/6)
(0/6) (4/6) (5/6)
(0/6) (0/6) (0/6)
(0/6) (5/6) (2/6)
(0/6) (0/6) (0/6)
(0/6) (3/6) (3/6)
(0/6) (0/6) (0/6)
(0/6) (4/6) (5/6)
Swim bladder Aerocystitis
(0/6)
(2/6)
(0/6)
(2/6)
(2/6)
(3/6)
(0/6)
(4/6)
(0/6)
(0/6)
Intestine Lamina propria inflammation
(0/6)
(1/6)
(0/6)
(0/6)
(0/6)
(0/6)
(0/6)
(0/6)
(0/6)
(0/6)
284
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Table 2 Distribution of Edwardsiella ictaluri in striped catfish tissues after immersion infection, detected by immunohistochemistry. Time post-infection
1h 3h 6h 12 h 24 h 48 h 72 h 96 h 5d 10 d 30 d
Tissue positive for the bacterium/number of tissues examined Head kidney
Skin
Brain
Spleen
Trunk kidney
Heart
Liver
Gill
Swim bladder
Intestine
0/6 0/6 0/6 0/6 0/6 0/6 (3/6) (4/6) (3/6) (5/6) (2/6)
0/6 0/6 0/6 0/6 0/6 (2/6) (3/6) (5/6) (2/6) (3/6) (3/6)
0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6
0/6 0/6 0/6 0/6 0/6 0/6 (5/6) (4/6) (3/6) (5/6) (2/6)
0/6 0/6 0/6 0/6 0/6 0/6 0/6 (1/6) (5/6) (4/6) (3/6)
0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 (1/6) 0/6
0/6 0/6 0/6 0/6 0/6 0/6 (2/6) (2/6) (3/6) (3/6) (1/6)
0/6 0/6 0/6 0/6 (3/6) (4/6) (3/6) (5/6) (5/6) (3/6) (4/6)
0/6 0/6 0/6 0/6 (3/6) (2/6) (3/6) (1/6) (1/6) 0/6 0/6
0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6
h - hours; d - days.
immunolabelling of bacterial antigen associated within the vascular endothelial cells of glomerulitis-pathological changes. Several studies have reported the gastrointestinal tract and gills are important portals of entry for bacteria into channel catfish ESC (Newton et al., 1989; Ciembor et al., 1995). According to Dung et al. (2012), the gill epithelium and gastrointestinal tract also are entry portals in
pangasius BNP. Based on our immunohistochemical results, positive signals were observed in gills early in the infection (6–12 hpi) and in ulcerative skin at 72 hpi, suggesting skin may be another natural route of disseminating the E. ictaluri infection in pangasius catfish. A similar situation is described for channel catfish ESC, where bacteria can enter the body via skin abrasions (Menanteau-Ledouble et al., 2011).
Fig. 3. Immunohistochemistry of tissues from striped catfish experimentally infected with Edwardsiella ictaluri. Positive staining of bacteria in (A) necrotic skin; (B) perivasculitisassociated endothelial cells (arrow) beneath the skin; (C) the surface of the secondary lamellae of the gill (24 h); (D) within the necrotic gill; (E) bacteria widely distributed in the necrotic area in spleen; and (F) head kidney.
N. Pirarat et al. / Aquaculture 464 (2016) 279–285
In spite of many studies examining the effects of E. ictaluri on channel catfish immune cells, insights into the fate of E. ictaluri in vivo has been limited in striped catfish (Dung et al., 2012). The ability of striped catfish to eliminate E. ictaluri may determine how long this bacterium can affect the fish's immune system. Several authors suggest that E. ictaluri has the ability to survive and grow within catfish macrophages, based on histopathological observation, although bacteria were also observed within interstitial spaces (Miyazaki and Plumb, 1985; Shotts et al., 1986; Baldwin and Newton, 1993). In the present study, the bacteria can persist up to 1 month in necrotic-participating phagocytic cells and in melano-macrophage centers, and no extra-cellular positive reaction was found throughout the experiment. The study confirmed the intracellular nature of E. ictaluri, disseminated in striped catfish tissues during septicemia. The reticuloendothelial system, including phagocytic macrophages and endothelial cells allowed E. ictaluri replication. The granuloma-participating phagocytic cells and the melano-macrophage centers were the site of E. ictaluri persistence in pangasius catfish, as seen in channel catfish ESC (Miyazaki and Plumb, 1985) and E. tarda infection (Pirarat et al., 2007). However, activating phagocytic cells seem to play a major protective inflammatory response against this intracellular bacterium. Bacterial antigen was largely reduced and limited within necrotic-participating phagocytic cells and melano-macrophage centers at the late stage of infection. In conclusion, the use of IHC allows the visualization of E. ictaluri in situ and the distribution of the bacteria correlates with the BNP histopathological changes observed. Necrotic-participating phagocytic cells acts as an early defensive mechanism, localizing this intracellular pathogen before generating septicemic death. The control of and protection against BNP by the pangasius catfish industry is urgently needed. Understanding the time course of infection and the fundamentals of disease pathogenesis using an immersion model helps meet this need. Acknowledgements We would like to thank Research Fellowship program JASSO 2014 for financial support. This work was partially supported by Ratchadapiseksompoj grant from Chulalongkorn University (grant number: GSTAR 59-004-31-003). References Areechon, N., Plumb, J.A., 1983. Pathogenesis of Edwardsiella ictaluri in channel catfish, Ictalurus punctatus. J. World Maricult. Soc. 14, 249–260. Baldwin, T., Newton, J., 1993. Pathogenesis of enteric septicemia of channel catfish, caused by Edwardsiella ictaluri: bacteriologic and light and electron microscopic findings. J. Aquat. Anim. Health 5, 189–198. Bartie, K.L., Austin, F.W., Diab, A., Dickson, C., Dung, T.T., Giacomini, M., Crumlish, M., 2012. Intraspecific diversity of Edwardsiella ictaluri isolates from diseased freshwater catfish, Pangasianodon hypophthalmus (Sauvage), cultured in the Mekong Delta, Vietnam. J. Fish. Dis. 35, 671–682. Booth, N.J., Beekman, J.B., Thune, R.L., 2009. Edwardsiella ictaluri encodes an acid-activated urease that is required for intracellular replication in channel catfish (Ictalurus punctatus) macrophages. Appl. Environ. Microbiol. 75, 6712–6720.
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Ciembor, P., Blazer, V., Dawe, D., Shotts, E., 1995. Susceptibility of channel catfish to infection with Edwardsiella ictaluri: effect of exposure method. J. Aquat. Anim. Health 7, 132–140. Crumlish, M., Thanh, P.C., Koesling, J., Tung, V.T., Gravningen, K., 2010. Experimental challenge studies in Vietnamese catfish, Pangasianodon hypophthalmus, exposed to Edwardsiella ictaluri and Aeromonas hydrophila. J. Fish Dis. 33, 717–722. Dung, T.T., Chiers, K., Tuan, N.A., Sorgeloos, P., Haesebrouck, F., Decostere, A., 2012. Early interactions of Edwardsiella ictaluri, with Pangasianodon catfish and its invasive ability in cell lines. Vet. Res. Commun. 36, 119–127. Ferguson, H.W., Turnbull, J.F., Shinn, A., Thompson, K., Dung, T.T., Crumlish, M., 2001. Bacillary necrosis of farmed Pangasianodon hypophthalmus (Sauvage) from the Mekong Delta Vietnam. J. Fish Dis. 24, 509–513. Globefish, F., 2015. Pangasius market report. March. [online] Available at: http://www. globefish.org/pangasius-march-2015.html (accessed 03.03.16). Hawke, J.P., Durborow, R.M., Thune, R.L. and Camus, A.C., 1998. Enteric Septicemia of Catfish. SRAC Publication no. 477. College Station, TX: Texas A & M University. Humphrey, J.D., Lancaster, C., Gudkovs, N., McDonald, W., 1986. Exotic bacterial pathogens Edwardsiella tarda and Edwardsiella ictaluri from imported ornamental fish Beta splendens and Puntius conchonius, respectively: isolation and quarantine significance. Aust. Vet. J. 63, 369–371. Jacrboe, H.H., Bowser, P.R., Robinette, H.R., 1984. Pathology associated with natural Edwardsiella ictaluri infection in channel catfish (Ictalurus punctatus (Rafinesque)). J. Wildl. Dis. 20, 352–354. Kasornchandra, J., Rogers, W.A., Plumb, J.A., 1987. Edwardsiella ictaluri from walking catfish, Clarias batrachus L., in Thailand. J. Fish Dis. 10, 137–138. Kent, M.L., Lyons, J.M., 1982. Edwardsiella ictaluri in the green knife fish, Eigemannia virescens. Fish Health News 2 (2). Menanteau-Ledouble, S., Attila Karsi, A., Lawrence, M.L., 2011. Importance of skin abrasion as a primary site of adhesion for Edwardsiella ictaluri and impact on invasion and systematic infection in channel catfish Ictalurus punctatus. Vet. Microbiol. 148, 425–430. Miyazaki, T., Plumb, J.A., 1985. Histopathology of Edwardsiella ictaluri in channel catfish, Ictalurus punctatus (Rafinesque). J. Fish Dis. 8, 389–392. Newton, J., Wolfe, L., Grizzle, J., Plumb, J., 1989. Pathology of experimental enteric septicemia in channel catfish, Ictalurus punctatus, following immersion-exposure to Edwardsiella ictaluri. J. Fish Dis. 12, 335–347. Pirarat, N., Kobayashi, T., Katagiri, T., Maita, M., Endo, M., 2006. Protective effects and mechanisms of a probiotic bacterium Lactobacillus rhamnosus against experimentally Edwardsiella tarda infection in tilapia (Oreochromis niloticus). Vet. Immunol. Immunopathol. 113, 339–347. Pirarat, N., Maita, M., Endo, M., Katagiri, T., 2007. Lymphoid apoptosis in Edwardsiella tarda septicemia in tilapia, Oreochromis niloticus. Fish Shellfish Immunol. 22, 608–616. Shotts, E.B., Blazer, V.S., Waltman, W.D., 1986. Pathogenesis of experimental Edwardsiella ictaluri infections in channel catfish (Ictalurus punctatus). Can. J. Fish. Aquat. Sci. 43, 36–42. Sirimanapong, W., Adams, A., Ooi, E.L., Green, D.M., Nguyen, D.K., Browdy, C.L., Collet, B., Thompson, K.D., 2015a. The effects of feeding immunostimulant β-glucan on the immune response of Pangasianodon hypophthalmus. Fish Shellfish Immunol. 45 (2), 357–366. Sirimanapong, W., Thompson, K.D., Ooi, E.L., Bekaert, M., Collet, B., Taggart, J.B., Bron, J.E., Green, D.M., Shinn, A.P., Adams, A., Leaver, M.J., 2015b. The effects of feeding β-glucan to Pangasianodon hypophthalmus on immune gene expression and resistance to Edwardsiella ictaluri. Fish Shellfish Immunol. 47 (1), 595–605. Starzl, T.E., Lerner, R.A., Frank, J., Dixon, F.J., Groth, C.G., Lawrence Brettschneider, L., Terasaki, P.I., 1968. Shwartzman reaction after human renal homotransplantation. N. Engl. J. Med. 278, 642–648. Tran, T.T.H., Nguyen, T.P., Tran, L.C.T., Glencross, B., 2010. Assessment of methods for the determination of digestibilities of feed ingredients for tra catfish, Pangasianodon hypophthalmus. Aquac. Nutr. 16, 351–358. Waltman, W.D., Shotts, E.B., Blazer, V.S., 1985. Recovery of Edwardsiella ictaluri from danio (Danio devario). Aquaculture 46, 63–66.
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Examination of entry portal and pathogenesis of Edwardsiella ictaluri infection in striped catfish, Pangasianodon hypophthalmus Nopadon Pirarat a,⁎, Ei Lin Ooi b, Kim D. Thompson c, Nguyen Huu Thinh d, Masashi Maita e, Takayuki Katagiri e a
Wildlife, Exotic and Aquatic Pathology - Special Task Force for Activating Research, Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand, DSM Nutritional Products, Aquaculture Center Asia Pacific, Bangkok, Thailand, c Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Edinburgh, United Kingdom d Department of Fish Pathology, Faculty of Fisheries, Nong Lam University, Ho Chi Minh City, Viet Nam e Laboratory of Fish Health Management, Tokyo University of Marine Science and Technology, Japan b
a r t i c l e
i n f o
Article history: Received 3 June 2016 Received in revised form 29 June 2016 Accepted 30 June 2016 Available online 01 July 2016 Keywords: BNP Edwardsiella ictaluri Immunohistochemistry Necrotic dermatitis Striped catfish
a b s t r a c t The host-bacterium interaction between striped pangasius catfish (Pangasianodon hypophthalmus) and Edwardsiella ictaluri was examined during experimentally induced bacillary necrosis of pangasius (BNP). After infection by immersion challenge, fish samples were taken over the course of the infection. Necrotic dermatitis, associated with vasculitis-perivasculitis changes, was the most notable gross and histopathological change observed, similar to that described during channel catfish enteric septicemia. Typical histopathological change associated with BNP were initially observed by 96 h post-infection (hpi) and these lasted until 14 days post infection (dpi) with no granulomatous formation evident. The presence of the bacterium was observed in various organs using immunohistochemistry (IHC), except for the brain, with positive staining observed within differing cell types, including phagocytic cells, myocardia, gill epithelia and capillary endothelia. No extracellular deposition of bacterial antigen was observed. Bacterial antigen persisted up to 1 month in the independent phagocytic cells, necrotic-associated phagocytic cells and melano-macrophage centers, confirming the intracellular nature of E. ictaluri disseminated in the tissues during septicemia. The bacterium was evident in the gills early in the infection (6–12 hpi) and in necrotic skin at 72 hpi. With the histopathological changes observed, this suggests that skin is another possible natural route for disseminating E. ictaluri in pangasius catfish during infection. Statement of relevance: This information will improve our understanding of the pathogenesis of BNP, and establish more effective methods of control for this important disease, including methods for quantifying sub-lethal infections and assessing response against the invading pathogen. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Over the past decade, striped catfish, Pangasianodon hypophthalmus, has been increasingly important as a new aquaculture whitefish product on the world market, with production increasing dramatically to meet global demand (Globefish, 2015). However, the need for intensive culture to meet this demand has been hampered by a number of problems, especially poor husbandry and water quality, and increase in infectious disease episodes in culture systems. Bacillary necrosis of Pangasianodon (BNP), a recently recognized disease caused by Edwardsiella ictaluri, has been highlighted as one of the most important infectious disease problems in intensive striped catfish production (Ferguson et al., 2001). BNP is characterized by multifocal irregular white lesions of varying size located on different organs, including liver, spleen and kidney. E. ictaluri is also the etiological agent of enteric ⁎ Corresponding author. E-mail address: [email protected] (N. Pirarat).
http://dx.doi.org/10.1016/j.aquaculture.2016.06.043 0044-8486/© 2016 Elsevier B.V. All rights reserved.
septicemia in channel catfish (Ictalurus punctatus) (ESC), causing enteritis and septicemia, with red and white ulcers covering the skin and petechial hemorrhages around the mouth and fin bases (Areechon and Plumb, 1983; Miyazaki and Plumb, 1985). Assessment of the inflammatory response is often used to examine the host's immune response to an infectious agent. Many authors have described the inflammatory response that occurs in channel catfish in response to E. ictaluri during outbreaks of ESC. An effusive form of the disease is found in channel catfish, characterized by the production of ascites in the abdominal cavity of the fish, swollen kidneys and spleens, mottled livers, hemorrhage intestines and generalized septicemia (Areechon and Plumb, 1983; Jacrboe et al., 1984; Miyazaki and Plumb, 1985; Newton et al., 1989). BNP in pangasius catfish, by contrast is a non-effusive form of the disease, characterized by necrotic changes in different organs, sometimes including the brain (Ferguson et al., 2001; Crumlish et al., 2010). Both types of presentations can be observed in other fish species, including green knife fish (Eigenmannia virescens) (Kent and Lyons, 1982), zebra danio (Danio rerio) (Waltman et al.,
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1985) rosy barbs (Pethia conchonius) (Humphrey et al., 1986) and walking catfish (Clarias batrachus) (Kasornchandra et al., 1987). Several studies have been published on entry portal and pathogenesis of E. ictaluri in ESC, as well as treatment and control of the disease via vaccination and antibiotics (Areechon and Plumb, 1983, Miyazaki and Plumb, 1985, Ciembor et al., 1995; Booth et al., 2009; Crumlish et al., 2010; Dung et al., 2012). However, there are few studies examining pathogenesis and entry portal of E. ictaluri in BNP outbreaks in striped catfish. Dung et al. (2012) was the first to describe the pathogenesis of E. ictaluri infections in Pangasianodon catfish, and there are a few studies focusing on the immune response against this bacterium (Sirimanapong et al., 2015a, 2015b). The objectives of the current study were: 1) to characterize the early histopathological events in freshwater striped catfish after experimentally infecting them with E. ictaluri using a simulated natural route of exposure, and 2) to examine the distribution of the bacterium in the fish during infection using immunohistochemistry (IHC). This information will improve our understanding of the pathogenesis of BNP, and establish more effective methods of control for this important disease, including methods for quantifying sub-lethal infections and assessing response against the invading pathogen. 2. Materials and methods 2.1. Experimental design One hundred and ninety four striped catfish, P. hypophthalmus, (40 ± 5 g), were purchased from a local hatchery in Vietnam and transported, quarantined and maintained according to the standard operating procedures of the National Agriculture Research Center (NARC) for fish husbandry. Fish with uniform weights were selected and randomly allocated to two 1000 L tanks (97 fish each) in Nong Lam University's challenge facility (Ho Chi Min City, Vietnam) and acclimated for N 1 week before initiating the trial. Two treatment groups were used in the study, including an uninfected control group and a group experimentally infected with E. ictaluri by immersion challenge. Before exposing the fish to the bacteria, ten fish from each of the two tanks were checked for the presence of external parasites and sub-clinical E. ictaluri infection. Wet mount preparations were made from the skin and gills and examined microscopically. Samples of the liver and posterior kidney were taken and streaked onto brain-heart infusion agar (BHIA), and plates incubated at 28 °C for 48 h. The presence of external parasite was not observed by gill and skin scraping. There was no growth of E. ictaluri in any of swabs taken from the ten fish. A diet (32% crude protein; 6% fat) was formulated to meet the nutritional requirements of striped catfish (Tran et al., 2010). The diets were fed once daily at 3% body weight. Water temperature was maintained at 29 ± 2 °C prior to the challenge and at 26 ± 1 °C post-infection. The duration of the trial lasted one month, and the experiment was performed in triplicate. 2.2. Infection of fish with Edwardsiella ictaluri A subclinical dose of Edwardsiella ictaluri, strain NLF33 (courtesy of Nong Lam University) was used as the immersion challenge (Sirimanapong et al., 2015a, 2015b). The bacteria were first streaked onto BHIA and cultured at 30 °C for 24 h, and then three to five colonies of E. ictaluri were placed in sterile brain heart infusion broth (BHIB) and cultured at 28 °C for 18 h. The bacterial concentration was determined from CFUs using a plate counting method and adjusted to 106 CFU/mL with sterile saline. The fish were experimentally infected with E. ictaluri by immersing the fish in static, well-aerated water to which the bacteria has been added at a final concentration of 8 × 104 CFU/mL. The fish were held in the bacterial suspension for 30 min after which fifteen fish were randomly selected and placed in three replicate 80 L tanks at 10.48 kg m3. Uninfected fish used as negative controls were immersed in BHIB instead of the bacterial suspension. The fish were monitored
for 30 days for clinical signs and gross pathology, which was recorded twice daily. 2.3. Histopathology Ten tissues (i.e. brain, heart, gill, spleen, head and trunk kidney, liver, skin, pancreas and intestine) were collected at 0, 1, 3, 6, 12, 24, 48, 72, 96 h post-infection (hpi), and 5, 10 and 30 days post-infection (dpi) from six fish per group (i.e. two fish per tank) and fixed in 10% buffered formalin for histopathology. Fixed tissues were processed according to standard histological techniques, and tissue sections were stained with hematoxylin and eosin (H&E). Tissue sections were examined under a light microscope for pathological changes (Pirarat et al., 2006). 2.4. Immunohistochemistry Microscopic localization of E. ictaluri antigens and phenotypic characterization of associated cells was performed as previously described (Pirarat et al., 2006). Briefly, after placing 5 μm tissue sections on glass microscope slides and encircling them with a wax ring, the tissue sections were dehydrated through an alcohol series. The tissues were then incubated with 3% (v/v) hydrogen peroxide in methanol for 20 min to block non-specific endogenous peroxidase activity, and sections were washed in 0.02 M phosphate buffered saline (PBS) pH 7.3 and incubated with 1% (w/v) bovine serum albumin for 30 min. Rabbit polyclonal anti-E. ictaluri antibody (1:250 dilution in PBS) developed inhouse, was applied to the tissues, and the slides were incubated at 4 °C overnight. The tissues were then rinsed with PBS and incubated with a secondary antibody conjugated with a universal immuno-enzyme polymer using a Histofine MAX PO kit (Nichirei, Japan) for 45 min at 22 °C. The tissues were again washed in PBS and incubated with either diaminobenzidine (DAB) or 3-amino, 9-ethyl-carbazole (AEC) substrate for 10 min. The tissues were counterstained with hematoxylin, rinsed in serial graded alcohols and xylene and mounted with mounting media. Normal, healthy striped catfish tissues were used as a negative control. 3. Results 3.1. Clinical signs and gross pathology After 72 hpi infection, fish exhibited clinical signs of lethargy, slowed swimming and gathering at the water surface. Patchy and ecchymotic hemorrhages were initially observed at the pectoral fin bases of one affected fish at 96 hpi. Multiple, raised pimple-like lesions, or a “buckshot” appearance, (Fig. 1A & B) were apparent 96 hpi. Bacillary necrosis of the spleen, kidney and liver were noted after 96 hpi in four of the six fish examined, and bacteria were observed until the end of the trial at 30 dpi in two of the six fish examined. Abdominal distension, or ascites, was not observed. No mortalities occurred during the one month period of the challenge trial. No abnormal clinical signs were noted in the control group throughout the trial period. 3.2. Histopathology The most notable histopathological changes in infected fish were bacillary necrosis, vasculitis and perivasculitis. An inflammatory response was obvious at 72 hpi with widespread diffusion in organs, generating the septicemic condition (Fig. 2). At this early stage, the necrotic lesion was characterized by a central necrotic core composed of phagocytic cells loaded with necrotic debris and haemolyzed red blood cells. The higher aggregation of phagocytic cells at the center of the core, surrounded by mononuclear inflammatory leukocytes was noted later in the infection (10 dpi). There was no evidence of any well-organized fibrosing granulomas developing during the infection. Necrotic lesions developed early and were quite widespread by 5–10 dpi in the head kidney (Fig. 2, C & D), and in trunk kidney, spleen, liver, skin and gill,
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within phagocytic cells, gill epithelia and capillary endothelia. Immuno-labelling of the bacteria was predominantly distributed in the head kidney, spleen, trunk kidney, skin, liver, gills, and muscle respectively (Table 2). No positive reaction was observed in the lamina propria or the mucosal epithelium of intestinal tissues throughout the experiment. Bacteria were localized within the necrotic skin (Fig. 3A) and the perivasculitis-associated endothelial cells (Fig. 3B) below the skin by 96 hpi. A positive reaction was clearly seen in glomeruli and interstitial tissues of the trunk kidney, and bacteria also occasionally presented in the renal tubular epithelium. Immuno-labelling of bacteria in the liver was also occasionally seen on the serosal surface. Early in the infection (1–3 dpi), stained bacteria could be clearly seen in the brush border of primary and secondary lamellar epithelium of the gills (Fig. 3C). After 3 dpi, stained bacteria could also be observed in necrotic epithelium with hyperplastic changes evident (Fig. 3D). Bacteria could also be seen in the heart myocardium. By 3 dpi, immuno-labelling of bacteria was present in the spleen, which was diffusely distributed within independent-circulating phagocytic cells, especially located in the perisinusoidal and ellipsoidal spaces. By 4 dpi, the pattern of positive staining in the spleen could be seen within the necrotic-participating phagocytic cells (Fig. 3E). Positive staining was initially also observed within independent phagocytic cells in the head kidney by 3 dpi, and this also gradually spread to the necrotic-participating phagocytic cells (Fig. 3F) and later to the melano-macrophage centers. Positive signals were clearly observed within necrotic-participating phagocytic cells between 10 dpi and the end of the trial at 30 dpi. Fig. 1. Macroscopic finding of striped catfish experimentally infected with Edwardsiella ictaluri. (A) Gross pathology; (B) Multiple red skin nodules observed at 6 days postinfection. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
respectively, as indicated in Table 1. Raised nodular lesions of necrotic skin and underlying myositis were obvious by 5 dpi and could still be seen by 30 dpi. Perivasculitis, characterized by an infiltration of inflammatory leukocytes into the wall of blood vessels, surrounded by degenerated muscle fibers, as shown in Fig. 2B, was observed from 48 hpi until the end of the trial (Table 1). Thrombosis and vasculitis, evidenced by inflammatory leukocyte aggregation into the tunica media, was present in the liver (Fig. 2E). Endothelial cell hypertrophy and loss of endothelial cell lining were also noted. Fibrinoid degeneration of vessel walls was not apparent. Single cell necrosis of hepatocytes was seen at 3 dpi before developing necrotic foci (Fig. 2F). Multiple foci of hepatic necrosis were abundant adjacent to areas of vasculitis, from 96 hpi. In trunk kidney, interstitial glomerulonephritis, composed of small mononuclear leukocyte infiltration in the glomeruli and tubular interstitial tissues, and multiple phagocytic cells loaded with bacteria within necrotic areas, was present at 96 hpi. Vasculitis lesions (Fig. 2H) with thickening of vessel walls was also noted, with evidence of fibrinoid degeneration and fibrin thrombi. Thickening of glomerular basement membrane, with infiltration of inflammatory leukocytes into glomeruli, was also present in the later stages of the infection, i.e. from 10 dpi. Hyaline degeneration of renal tubules was occasionally seen. In gills, necrotic changes were seen earlier (72 hpi) than in other tissues (Fig. 2A). Goblet cell hyperplasia was observed as early as 12 to 48 hpi in primary lamellar gill. Distorted and congested gill lamellae (aneurism) were also noted at this early stage. Secondary lamellar hyperplasia and fusion were evident by 3 dpi. Myocarditis with mononuclear cell infiltration was noted in the heart by 10 dpi (Fig. 2G). Brain tissues showed congestion without any inflammatory cell response throughout the course of the infection. 3.3. Immunohistochemistry Edwardsiella ictaluri antigen was diffusely detected in various organs, except the brain, using IHC. Positive IHC signals were identified
4. Discussion According to previous reports, both BNP and ESC differ in clinical manifestation. In infected striped catfish, few external signs are evident, including pale gills and skin BNP (Ferguson et al., 2001; Crumlish et al., 2010; Dung et al., 2012). With catfish ESC, on the other hand, fish exhibit petechial hemorrhaging and ulcerative skin lesions (Areechon and Plumb, 1983; Hawke et al., 1998). In our study, fish had macroscopic skin lesions resembling red pimples, or with a “buckshot” appearance, and ulcerative skin lesions with patchy hemorrhages at the fin bases were also present, which are typically described in channel catfish. A necrotic response was present in various tissues of the striped catfish 3 dpi with E. ictaluri by immersion bath, and this response was evident at least up to 30 dpi. The difference in clinical manifestation and macroscopic finding between fish species may be due to an intraspecific diversity of E. ictaluri strains (Bartie et al., 2012; Dung et al., 2012). The nature of host-pathogen inflammatory response and the susceptibility of the fish to the bacterium may differ between fish species, as suggested by Dung et al. (2012). ESC and BNP histopathological lesions share some microscopic similarities in channel catfish and striped catfish, including multifocal necrosis in multiple organs. However, lesions representing ulcerative dermatitis, necrotic myositis and vasculitis-perivasculitis have never been recorded in pangasius BNP (Crumlish et al., 2010; Dung et al., 2012). Our study revealed marked necrosis and ulcerative lesions on the skin of infected fish, and the necrotic myositis of underlying musculature was associated with vasculitis and perivasculitis lesions. The diffuse to multifocal necrosis in internal organs were also related to vasculitis-perivasculitis changes, as seen in catfish ESC. It is important to examine the host-pathogen interaction to enable understanding of how the bacillary necrotic changes develop in the striped catfish. Bacteria are distributed by and replicate within circulating macrophages and necrotic-phagocytic cells that are scattered throughout the body of the fish. The vasculitis-perivasculitis changes associated with the BNP during our study suggest that bacteria can directly damage the endothelial cells resulting in vasculitis and perivasculitis, and eventual tissue hypoxia and necrosis as evidenced by the positive IHC staining seen within the perivasculitis-endothelial cells in the skin
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Fig. 2. Histopathology of striped catfish experimentally infected with Edwardsiella ictaluri (A) gill necrosis (arrowhead) and gill epithelial hyperplasia (arrow) at 72 h post-infection (hpi); (B) myositis (arrow) and perivasculitis change (arrowhead) at 6 days post-infection (dpi); (C and D) bacillary necrotic lesion observed in head kidney at 10 dpi; (E) necrotic hepatitis and (F) perivasculitis changes were multifocal in the liver at 10 dpi. (G) Myocarditis with mononuclear cell infiltration observed at 14 dpi; (H) vasculitis-perivasculitis changes in trunk kidney.
and kidneys of infected fish. However, there were no pin-point petechial hemorrhages in internal organs correlating with vasculitis lesions as seen in channel catfish enteric septicemia (Areechon and Plumb, 1983; Jacrboe et al., 1984; Hawke et al. 1998). This might be explained by differences in the inflammatory response elicited by these two different host species, possibly through the release of vasoactive amines, which cause endothelial cell retraction, increased vascular permeability and development of characteristic protein-rich exudates. The positive correlation between the vasculitis-perivasculitis change and the positive stained bacteria within the vascular endothelial cells, suggests
that an immune-complex mediated vasculitis or immune-complex mediated glomerulonephritis, a type III hypersensitivity reaction, cannot be ruled out as a cause of the pathogenesis resulting from the host response. The circulating, complement-fixing immune complexes, recognized by phagocytic cells lodged within blood vessel walls, lead to development of the bacillary necrotic changes. The presence of fibrinoid degeneration and thickening of vascular walls is similar to the Schwartzman reaction resulting in a thrombohaemorrhagic response to bacterial toxin as described in rabbits and humans (Starzl et al., 1968; Nordstoga, 1977). Unfortunately, there was no evidence of
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Table 1 Histopathology of striped catfish challenged with E. ictaluri. Time post-infection
1 h (h)
Group
Cont
Infect
3h Cont
Infect
6h Cont
Infect
Cont
Infect
Cont
Infect
Cont
Infect
Head kidney Bacillary necrosis Melanomacrophage center
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (4/6)
(0/6) (1/6)
(0/6) (5/6)
Skin Myositis Perivasculitis/vasculitis Bacillary necrosis
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(2/6) (2/6) (0/6)
Spleen Ellipsoidal enlargement Bacillary necrosis Melanomacrophage center
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (4/6)
(0/6) (0/6) (1/6)
(0/6) (0/6) (5/6)
Trunk kidney Vasculitis/perivasculitis Bacillary necrosis
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
Heart Myocarditis Vasculitis/perivasculitis Bacillary necrosis
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(1/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
Liver Vasculitis/perivasculitis Bacillary necrosis
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
Gill Goblet cell hyperplasia Lamella hyperplasia Bacillary necrosis
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(2/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(4/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(5/6) (0/6) (0/6)
Swim bladder Aerocystitis
(0/6)
(0/6)
(0/6)
(2/6)
(0/6)
(0/6)
(0/6)
(1/6)
(0/6)
(3/6)
(0/6)
(2/6)
Intestine Lamina propria inflammation
(0/6)
(0/6)
(0/6)
(0/6)
(0/6)
(0/6)
(1/6)
(0/6)
(0/6)
(0/6)
(0/6)
(0/6)
96 h
12 h
5 days (d)
24 h
48 h
Time post-infection
72 h
10 d
30 d
Group
Cont
Infect
Cont
Infect
Cont
Infect
Cont
Infect
Cont
Infect
Head kidney Bacillary necrosis Melanomacrophage center
(0/6) (0/6)
(0/6) (6/6)
(0/6) (2/6)
(4/6) (4/6)
(0/6) (4/6)
(5/6) (4/6)
(0/6) (3/6)
(4/6) (3/6)
(0/6) (2/6)
(4/6) (6/6)
Skin Myositis Perivascultitis/vasculitis Bacillary necrosis
(0/6) (0/6) (0/6)
(3/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(5/6) (5/6) (2/6)
(0/6) (0/6) (0/6)
(2/6) (1/6) (5/6)
(0/6) (0/6) (0/6)
(3/6) (0/6) (3/6)
(0/6) (0/6) (0/6)
(5/6) (5/6) (3/6)
Spleen Ellipsoidal enlargement Bacillary necrosis Melanomacrophage center
(0/6) (0/6) (0/6)
(2/6) (0/6) (6/6)
(0/6) (0/6) (0/6)
(1/6) (4/6) (6/6)
(0/6) (0/6) (3/6)
(3/6) (5/6) (2/6)
(0/6) (0/6) (2/6)
(0/6) (5/6) (1/6)
(0/6) (0/6) (4/6)
(0/6) (2/6) (1/6)
Trunk kidney Vasculitis/perivasculitis Bacillary necrosis
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(3/6) (0/6)
(0/6) (0/6)
(3/6) (3/6)
(0/6) (0/6)
(4/6) (5/6)
(0/6) (0/6)
(2/6) (2/6)
Heart Myocarditis Vasculitis/perivasculitis Bacillary necrosis
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(1/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(0/6) (0/6) (0/6)
(3/6) (2/6) (0/6)
(0/6) (0/6) (0/6)
(2/6) (0/6) (0/6)
Liver Vasculitis/perivasculitis Bacillary necrosis
(0/6) (0/6)
(0/6) (0/6)
(0/6) (0/6)
(2/6) (1/6)
(0/6) (0/6)
(2/6) (2/6)
(0/6) (0/6)
(3/6) (3/6)
(0/6) (0/6)
(1/6) (0/6)
Gill Goblet cell hyperplasia Lamella hyperplasia Bacillary necrosis
(0/6) (0/6) (0/6)
(3/6) (3/6) (2/6)
(0/6) (0/6) (0/6)
(0/6) (4/6) (5/6)
(0/6) (0/6) (0/6)
(0/6) (5/6) (2/6)
(0/6) (0/6) (0/6)
(0/6) (3/6) (3/6)
(0/6) (0/6) (0/6)
(0/6) (4/6) (5/6)
Swim bladder Aerocystitis
(0/6)
(2/6)
(0/6)
(2/6)
(2/6)
(3/6)
(0/6)
(4/6)
(0/6)
(0/6)
Intestine Lamina propria inflammation
(0/6)
(1/6)
(0/6)
(0/6)
(0/6)
(0/6)
(0/6)
(0/6)
(0/6)
(0/6)
284
N. Pirarat et al. / Aquaculture 464 (2016) 279–285
Table 2 Distribution of Edwardsiella ictaluri in striped catfish tissues after immersion infection, detected by immunohistochemistry. Time post-infection
1h 3h 6h 12 h 24 h 48 h 72 h 96 h 5d 10 d 30 d
Tissue positive for the bacterium/number of tissues examined Head kidney
Skin
Brain
Spleen
Trunk kidney
Heart
Liver
Gill
Swim bladder
Intestine
0/6 0/6 0/6 0/6 0/6 0/6 (3/6) (4/6) (3/6) (5/6) (2/6)
0/6 0/6 0/6 0/6 0/6 (2/6) (3/6) (5/6) (2/6) (3/6) (3/6)
0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6
0/6 0/6 0/6 0/6 0/6 0/6 (5/6) (4/6) (3/6) (5/6) (2/6)
0/6 0/6 0/6 0/6 0/6 0/6 0/6 (1/6) (5/6) (4/6) (3/6)
0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 (1/6) 0/6
0/6 0/6 0/6 0/6 0/6 0/6 (2/6) (2/6) (3/6) (3/6) (1/6)
0/6 0/6 0/6 0/6 (3/6) (4/6) (3/6) (5/6) (5/6) (3/6) (4/6)
0/6 0/6 0/6 0/6 (3/6) (2/6) (3/6) (1/6) (1/6) 0/6 0/6
0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6
h - hours; d - days.
immunolabelling of bacterial antigen associated within the vascular endothelial cells of glomerulitis-pathological changes. Several studies have reported the gastrointestinal tract and gills are important portals of entry for bacteria into channel catfish ESC (Newton et al., 1989; Ciembor et al., 1995). According to Dung et al. (2012), the gill epithelium and gastrointestinal tract also are entry portals in
pangasius BNP. Based on our immunohistochemical results, positive signals were observed in gills early in the infection (6–12 hpi) and in ulcerative skin at 72 hpi, suggesting skin may be another natural route of disseminating the E. ictaluri infection in pangasius catfish. A similar situation is described for channel catfish ESC, where bacteria can enter the body via skin abrasions (Menanteau-Ledouble et al., 2011).
Fig. 3. Immunohistochemistry of tissues from striped catfish experimentally infected with Edwardsiella ictaluri. Positive staining of bacteria in (A) necrotic skin; (B) perivasculitisassociated endothelial cells (arrow) beneath the skin; (C) the surface of the secondary lamellae of the gill (24 h); (D) within the necrotic gill; (E) bacteria widely distributed in the necrotic area in spleen; and (F) head kidney.
N. Pirarat et al. / Aquaculture 464 (2016) 279–285
In spite of many studies examining the effects of E. ictaluri on channel catfish immune cells, insights into the fate of E. ictaluri in vivo has been limited in striped catfish (Dung et al., 2012). The ability of striped catfish to eliminate E. ictaluri may determine how long this bacterium can affect the fish's immune system. Several authors suggest that E. ictaluri has the ability to survive and grow within catfish macrophages, based on histopathological observation, although bacteria were also observed within interstitial spaces (Miyazaki and Plumb, 1985; Shotts et al., 1986; Baldwin and Newton, 1993). In the present study, the bacteria can persist up to 1 month in necrotic-participating phagocytic cells and in melano-macrophage centers, and no extra-cellular positive reaction was found throughout the experiment. The study confirmed the intracellular nature of E. ictaluri, disseminated in striped catfish tissues during septicemia. The reticuloendothelial system, including phagocytic macrophages and endothelial cells allowed E. ictaluri replication. The granuloma-participating phagocytic cells and the melano-macrophage centers were the site of E. ictaluri persistence in pangasius catfish, as seen in channel catfish ESC (Miyazaki and Plumb, 1985) and E. tarda infection (Pirarat et al., 2007). However, activating phagocytic cells seem to play a major protective inflammatory response against this intracellular bacterium. Bacterial antigen was largely reduced and limited within necrotic-participating phagocytic cells and melano-macrophage centers at the late stage of infection. In conclusion, the use of IHC allows the visualization of E. ictaluri in situ and the distribution of the bacteria correlates with the BNP histopathological changes observed. Necrotic-participating phagocytic cells acts as an early defensive mechanism, localizing this intracellular pathogen before generating septicemic death. The control of and protection against BNP by the pangasius catfish industry is urgently needed. Understanding the time course of infection and the fundamentals of disease pathogenesis using an immersion model helps meet this need. Acknowledgements We would like to thank Research Fellowship program JASSO 2014 for financial support. This work was partially supported by Ratchadapiseksompoj grant from Chulalongkorn University (grant number: GSTAR 59-004-31-003). References Areechon, N., Plumb, J.A., 1983. Pathogenesis of Edwardsiella ictaluri in channel catfish, Ictalurus punctatus. J. World Maricult. Soc. 14, 249–260. Baldwin, T., Newton, J., 1993. Pathogenesis of enteric septicemia of channel catfish, caused by Edwardsiella ictaluri: bacteriologic and light and electron microscopic findings. J. Aquat. Anim. Health 5, 189–198. Bartie, K.L., Austin, F.W., Diab, A., Dickson, C., Dung, T.T., Giacomini, M., Crumlish, M., 2012. Intraspecific diversity of Edwardsiella ictaluri isolates from diseased freshwater catfish, Pangasianodon hypophthalmus (Sauvage), cultured in the Mekong Delta, Vietnam. J. Fish. Dis. 35, 671–682. Booth, N.J., Beekman, J.B., Thune, R.L., 2009. Edwardsiella ictaluri encodes an acid-activated urease that is required for intracellular replication in channel catfish (Ictalurus punctatus) macrophages. Appl. Environ. Microbiol. 75, 6712–6720.
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Ciembor, P., Blazer, V., Dawe, D., Shotts, E., 1995. Susceptibility of channel catfish to infection with Edwardsiella ictaluri: effect of exposure method. J. Aquat. Anim. Health 7, 132–140. Crumlish, M., Thanh, P.C., Koesling, J., Tung, V.T., Gravningen, K., 2010. Experimental challenge studies in Vietnamese catfish, Pangasianodon hypophthalmus, exposed to Edwardsiella ictaluri and Aeromonas hydrophila. J. Fish Dis. 33, 717–722. Dung, T.T., Chiers, K., Tuan, N.A., Sorgeloos, P., Haesebrouck, F., Decostere, A., 2012. Early interactions of Edwardsiella ictaluri, with Pangasianodon catfish and its invasive ability in cell lines. Vet. Res. Commun. 36, 119–127. Ferguson, H.W., Turnbull, J.F., Shinn, A., Thompson, K., Dung, T.T., Crumlish, M., 2001. Bacillary necrosis of farmed Pangasianodon hypophthalmus (Sauvage) from the Mekong Delta Vietnam. J. Fish Dis. 24, 509–513. Globefish, F., 2015. Pangasius market report. March. [online] Available at: http://www. globefish.org/pangasius-march-2015.html (accessed 03.03.16). Hawke, J.P., Durborow, R.M., Thune, R.L. and Camus, A.C., 1998. Enteric Septicemia of Catfish. SRAC Publication no. 477. College Station, TX: Texas A & M University. Humphrey, J.D., Lancaster, C., Gudkovs, N., McDonald, W., 1986. Exotic bacterial pathogens Edwardsiella tarda and Edwardsiella ictaluri from imported ornamental fish Beta splendens and Puntius conchonius, respectively: isolation and quarantine significance. Aust. Vet. J. 63, 369–371. Jacrboe, H.H., Bowser, P.R., Robinette, H.R., 1984. Pathology associated with natural Edwardsiella ictaluri infection in channel catfish (Ictalurus punctatus (Rafinesque)). J. Wildl. Dis. 20, 352–354. Kasornchandra, J., Rogers, W.A., Plumb, J.A., 1987. Edwardsiella ictaluri from walking catfish, Clarias batrachus L., in Thailand. J. Fish Dis. 10, 137–138. Kent, M.L., Lyons, J.M., 1982. Edwardsiella ictaluri in the green knife fish, Eigemannia virescens. Fish Health News 2 (2). Menanteau-Ledouble, S., Attila Karsi, A., Lawrence, M.L., 2011. Importance of skin abrasion as a primary site of adhesion for Edwardsiella ictaluri and impact on invasion and systematic infection in channel catfish Ictalurus punctatus. Vet. Microbiol. 148, 425–430. Miyazaki, T., Plumb, J.A., 1985. Histopathology of Edwardsiella ictaluri in channel catfish, Ictalurus punctatus (Rafinesque). J. Fish Dis. 8, 389–392. Newton, J., Wolfe, L., Grizzle, J., Plumb, J., 1989. Pathology of experimental enteric septicemia in channel catfish, Ictalurus punctatus, following immersion-exposure to Edwardsiella ictaluri. J. Fish Dis. 12, 335–347. Pirarat, N., Kobayashi, T., Katagiri, T., Maita, M., Endo, M., 2006. Protective effects and mechanisms of a probiotic bacterium Lactobacillus rhamnosus against experimentally Edwardsiella tarda infection in tilapia (Oreochromis niloticus). Vet. Immunol. Immunopathol. 113, 339–347. Pirarat, N., Maita, M., Endo, M., Katagiri, T., 2007. Lymphoid apoptosis in Edwardsiella tarda septicemia in tilapia, Oreochromis niloticus. Fish Shellfish Immunol. 22, 608–616. Shotts, E.B., Blazer, V.S., Waltman, W.D., 1986. Pathogenesis of experimental Edwardsiella ictaluri infections in channel catfish (Ictalurus punctatus). Can. J. Fish. Aquat. Sci. 43, 36–42. Sirimanapong, W., Adams, A., Ooi, E.L., Green, D.M., Nguyen, D.K., Browdy, C.L., Collet, B., Thompson, K.D., 2015a. The effects of feeding immunostimulant β-glucan on the immune response of Pangasianodon hypophthalmus. Fish Shellfish Immunol. 45 (2), 357–366. Sirimanapong, W., Thompson, K.D., Ooi, E.L., Bekaert, M., Collet, B., Taggart, J.B., Bron, J.E., Green, D.M., Shinn, A.P., Adams, A., Leaver, M.J., 2015b. The effects of feeding β-glucan to Pangasianodon hypophthalmus on immune gene expression and resistance to Edwardsiella ictaluri. Fish Shellfish Immunol. 47 (1), 595–605. Starzl, T.E., Lerner, R.A., Frank, J., Dixon, F.J., Groth, C.G., Lawrence Brettschneider, L., Terasaki, P.I., 1968. Shwartzman reaction after human renal homotransplantation. N. Engl. J. Med. 278, 642–648. Tran, T.T.H., Nguyen, T.P., Tran, L.C.T., Glencross, B., 2010. Assessment of methods for the determination of digestibilities of feed ingredients for tra catfish, Pangasianodon hypophthalmus. Aquac. Nutr. 16, 351–358. Waltman, W.D., Shotts, E.B., Blazer, V.S., 1985. Recovery of Edwardsiella ictaluri from danio (Danio devario). Aquaculture 46, 63–66.