Bull. Eur. Ass. Fish Pathol., 27(1) 2007, 2 Comparative histopathology of iniae and Streptococcus agalactiae-infected tilapia

C.-Y. Chen1, C.-B. Chao2 and P.R. Bowser1*

1 Aquatic Animal Health Program, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 USA; 2 Institute for Animal Disease Prevention and Control, Kaohsiung, Taiwan.

Abstract In this study, the microscopic pathology resulting from of tilapia with or S. agalactiae was compared. Pathological changes associated with infection of tilapia by either pathogen include: pericarditis, epicarditis, myocarditis, endocarditis, and . Large numbers of cocci were present in tissues and in the circulation of S. agalactiae-infected, but not in S. iniae-infected fish. Lesions similar to those caused by S. agalactiae, including the presence of large numbers of intralesional , can be reproduced by intraperitoneal injection of a relatively high dose in of S. iniae. This suggests that the pathogenesis of different streptococcal in tilapia may be similar. However, tilapia may control natural S. iniae infections more effectively, resulting in a more chronic form of disease compared to that caused by S. agalactiae.

Introduction 2002). All of these fish-pathogenic streptococci The genus Streptococcus includes many are closely related and may share similar important human and animal pathogens and pathogenic determinants with each other and was first reported in fish in 1957 (Hoshina et the human pathogens S. pyogenes and al., 1958). Streptococcus iniae, the most S. agalactiae. commonly reported streptococcal pathogen of fish, having been documented in tilapia in The pathology of S. iniae infection generally Japan, Israel, the US, and Taiwan (Bowser et includes meningitis, epi/myocarditis and al., 1998; Eldar et al., 1994; Kitao et al., 1981; septicemia, with enlargement or necrosis in Perera et al., 1994; Plumb, 1999; Tung et al., spleen and kidney. Interestingly, infections 1985) and in rainbow trout and tilapia in Israel caused by other streptococci have similar (Eldar et al., 1994; 1995). Phylogenetically, pathology, despite the different species of host S. iniae is closely related to S. parauberis, both or pathogen involved. In this study we being clustered with S. agalactiae and examined the pathology caused by natural S. dysgalactiae, and the whole group clustering S. iniae or S. agalactiae infections in tilapia and with S. pyogenes according to sequence compared those observations to the pathology similarity in their 16S rRNA gene (Facklam, associated with experimental infection of tilapia with S. iniae.

* Corresponding author’s E-mail: [email protected] Bull. Eur. Ass. Fish Pathol., 27(1) 2007, 3

Materials and methods 222, Sigma, St. Louis, Missouri, USA), and Sample collection processed for histological evaluation as Naturally infected tilapia were collected from previously described. For intraperitoneal and different outbreaks in the United States and intravascular infection trials, fish were placed southern Taiwan. The tilapia in the United under light anesthesia with MS-222 and a States were pure strain Oreochromis niloticus. 100 μl suspension containing 107 CFU/mL S. Tilapia collected in Taiwan are referred to as iniae strain 97057 cells was injected into the hybrid tilapia because of extensive cross peritoneum or the heart of each test fish. The breeding in that commercial environment. bacteria used to produce the experimental Several fish with apparent signs of disease, infection were cultured in either 100 mL including lethargy, corneal opacity, and dark (immersion infection) or 3 mL (intraperitoneal coloration were submitted for disease and intravascular infections) Brain Heart diagnosis. Standard bacterial was Infusion Broth (BHI broth, Becton Dickinson performed from kidney or spleen on Brain and Company, Sparks, Maryland, USA) for Heart Infusion (BHI) agar or Blood Agar 48 h, centrifuged, and washed twice in tilapia (Trypticase Soy Agar plus 5 % sheep blood, phosphate buffer saline (TPBS, 0.01 M pH 7.3 Becton Dickinson and Company, Sparks, phosphate buffer supplemented with 150 mM Maryland, USA). Internal organs were fixed NaCl), and then re-suspended in TPBS for use. in 10 % neutral buffered formalin, embedded Identification of pathogens in paraffin, sectioned and stained with Bacterial isolation from diagnostic cases was hematoxylin and eosin stains for histological attempted from spleen or trunk kidney of evaluation (Luna, 1968). moribund fish. All bacterial isolates meeting Artificial infections the following requirements were included in Experimental infection trials were conducted this study. Bacterial growth consisted of with S. iniae to compare the resulting white, pinpoint raised colonies on BHI agar pathology with that observed in natural or Blood Agar after 24 to 48 h of incubation. S. iniae or S. agalactiae outbreaks. Streptococcus Further, microscopic examination revealed iniae strain 97057 (Cornell University) isolated gram-positive chained cocci after Gram’s stain from infected tilapia (Bowser et al., 1998) was procedure. Twenty isolates that met these used as the model pathogen. In immersion requirements were then subjected to two infections, pure strain O. niloticus, approxi- polymerase chain reaction (PCR) iden- mately 200 g, were immersed in S. iniae strain tifications. 97057 suspension at the concentration of 107 DNA was extracted and purified following CFU/mL in tank water for 30 min, and then procedures previously described (Chao et al., the fish were transferred to a 100 L 2002). A set of primers was used to amplify experimental tank for observation. Fish the bacterial 16S ribosomal RNA gene showing dark coloration and lethargy within sequence according to Weinsburg et al. (1991). 1 week were collected, euthanized with an PCR products were recovered from the gel, overdose of tricaine methanesulfonate (MS- Bull. Eur. Ass. Fish Pathol., 27(1) 2007, 4

sequences used include S. agalactiae JCM5671 (Genbank accession number AB023574), S. bovis ATCC43143 (AF104114), S. dysgalactiae (AB002513), S. equi ATCC 43079 (AB002516), S. iniae (X58316), S. parauberis (X89967), S. pneumoniae (AF003930), and S. suis (AF009509). (AF015929) was also included as an outgroup. ClustalX software (Thompson et al., 1997) version 1.81 was used for multiple lignment of the sequences and generation of the bootstrap neighborhood-joint tree. The phylogenetic tree was displayed using TreeView version 1.6.5, available from R. D. M. Page (Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK), at http:// .zoology.gla.ac.uk/rod/rod.html.

Results Identification of pathogens Figure 1. Phylogenetic tree of selected members of Sequence analysis of the 16S rRNA gene was Streptococcus indicating relationship between tilapia performed on one typical isolate from each isolates and other streptococci. Tilapia-pathogenic S. iniae and S. agalactiae were sequenced and outbreak. The obtained sequences were compared to other sequences available in Genbank compared to the sequences available in using a 1356-bp sequence of 16S rRNA gene Genbank (Figure 1). One group of our isolates sequence. The Staphylococcus aureus (Sta. aureus) was used as an outgroup. had very high similarity to S. agalactiae, and another group clustered with S. iniae. The cloned into pGEM-T, and transfected into similarities were approximately 99 %. All Escherichia coli strain DH5a using Transform isolates in the first group were Lancefield Aid Bacterial Transformation System (MBI group B; while none of the isolates in the Fermentas Inc., Hanover, New Hampshire, second group were typable. Therefore, the USA). DNA sequences were then determined first group was identified as S. iniae and the by an ABI Prism 377-96 DNA Sequencer second group S. agalactiae. Interestingly, all the (Perkin-Elmer, Wellesley, Massachusetts, important fish pathogenic streptococci USA) with an ABI Prism Big Dye Terminator (S. dysgalactiae, S. difficile, S. parauberis, S. iniae) Cycle Sequencing Ready Reaction Kit. The form a separated cluster from other obtained sequences were compared with streptococci. This suggests that these species available sequences from other streptococci are evolutionarily closely related and may in Genbank, and selected sequences were apply a similar strategy in pathogenesis. used to construct a phylogenetic tree. The Bull. Eur. Ass. Fish Pathol., 27(1) 2007, 5

Pathology of natural infections Gross pathological signs of S. iniae and S. agalactiae infection in tilapia were similar and could not be used for differential disease diagnosis. Fish were lethargic with dark coloration in both infections and some fish would show sudden erratic swimming movements. Microscopically, proliferation of lymphoid tissue was observed in the kidneys of fish infected by either bacterium. Kidneys of S. iniae-infected tilapia showed accu- mulations of eosinophilic material in the cytoplasm of the tubular cells, with the nuclei displaced to the side. In S. agalactiae-infected tilapia, dissolution of some tubules could be found. Cocci were seen surrounding renal tubules, within interstitial cells, and within glomeruli. Fibrin precipitation and lympho- cyte infiltration were noted in necrotic foci. In addition, in fish infected with S. agalactiae, bacterial cells were distributed throughout the spleen. The head kidney of fish infected with S. agalactiae had similar necrotic changes with presence of bacteria as that found in the spleen. Melanomacrophage centres were more evident in head kidneys in both S. iniae and S. agalactiae-infected fish than in uninfected control fish. Figure 2. A. Histopathology of S. iniae infection in In contrast, bacterial cells were rarely tilapia liver demonstrating vacuolation of hepatocytes and numerous pycnotic nuclei in observed in the liver of S. iniae infected fish pancreatic tissue. B. Histopathology of S. agalactiae (Figure 2). Necrotic changes were observed infection in tilapia liver demonstrating vacuolation of hepatocytes, with colonies of cocci (arrows). along the hepatic arteries and near the Vacuolation is likely due to excess energy in the capsule. Fish infected by either bacterium had ration of these cultured fish and not associated with variable vacuolation of the hepatocytes, which the bacterial infections. Bars = 10 μm. is not uncommon in commercially raised Visceral pericarditis was a common finding tilapia and should not be considered in both infections. In S. iniae infection, the pathological changes due to infections but epicardium was congested and thickened, rather to feeding a diet with excessive caloric with an associated infiltration of lymphocytes content. and macrophages. Numerous cocci and Bull. Eur. Ass. Fish Pathol., 27(1) 2007, 6 necrotic debris were being seen in epicardium, Streptococcus agalactiae infection also resulted but were rarely distributed between cardiac in necrotic foci between the mucosa and muscle fibers. In S. agalactiae-infected tilapia, submucosa. Pathological changes in the bacterial cells were not limited to the intestine due to the infection by either epicardium but were also widely distributed bacterium were generally mild, with scattered among or attached to cardiac muscle fibers individual cell necrosis. or found in necrotic foci diffusely or as Although bacterial cells of S. iniae were rarely colonies. High numbers of bacterial cells were found in internal organs, they were present observed in the circulation, in fish infected in very high numbers in the peritoneal cavity. with S. agalactiae but not for fish infected with Peritonitis, with a thick layer of fibrin, necrotic S. iniae. Inflammatory cell infiltration with debris, and bacteria, was seen in both some associated necrosis of the epicardium infections. This finding correlates well with and wall of the bulbus arteriosus was common the fact that necrosis was always more severe in S. agalactiae-infected tilapia. Valvular in the superficial regions of liver or spleen. endocarditis with large intralesional colonies of cocci were seen in both infections. We observed a moderate infiltration of inflammatory cells in ovary of S. iniae-infected Both infections lead to meningitis in tilapia, tilapia. In the specimens examined, testis which correlates to clinical findings that fish appeared to be less affected. showed erratic behaviour and lethargy. Histologically, S. iniae infection caused a No significant pathological changes were thickening of the meninges with an associated found in the gills in S. iniae-infected tilapia, infiltration of lymphocytes and macrophages. with the exception of external parasites (e.g. Areas with ischemia-like lesions were seen in Trichodina), or lesions similar to epitheliocystis the brain; a typical lesion associated with on the gill lamella. Bacterial colonies were streptococcal meningitis in other animals. observed in the gill of S. agalactiae-infected Streptococcus agalactiae induced similar, but tilapia, especially near the tip of gill lamella. more severe lesions. Within the optic lobe, Lesions in the skeletal muscle were rare in the small colonies of S. agalactiae were sometimes S. iniae infection. In S. agalactiae infected fish, observed in the capillaries. bacterial cells were observed in the musculature and were associated with a Gastroenteritis was seen in both infections. moderate necrosis. Streptococcus iniae invaded the submucosa and serosa, causing local extensive inflammation Pathology in experimental infections with fibrin, edema, and distention of the Experimental infection trials resulted in submucosa. In the mucosa, bacterial cells variable results with respect to the severity aggregated on the luminal surface and of disease signs observed in the tilapia resulted in necrosis. Streptococcus agalactiae challenged with S. iniae. Although some fish cells were usually observed between died within 24 h and some developed glandular tissue, sometimes causing necrosis darkened body coloration within days, over and dissolution of individual glands. Bull. Eur. Ass. Fish Pathol., 27(1) 2007, 7 half of the fish challenged did not develop any Perera et al. (1998) investigated S. iniae sign of disease in either immersion or IP- infection in hybrid tilapia and reported injected groups. Even among those fish that meningitis, granulomas in liver, epicarditis, displayed dark coloration few showed and myocarditis. Those descriptions are histological changes. Pathological changes similar to our findings. Chang & Plumb (1996) were only significant in moribund fish, with infected tilapia with two non-hemolytic and such changes as meningitis, infiltration of one β-hemolytic streptococci. Findings similar lymphocytes, and macrophages in internal to ours, including pericarditis, infiltration of organs. However, histopathology in acute macrophages and lymphocytes into internal S. iniae infections, either through intraperi- organs, hyaline deposition in tubular cells in toneal or intravascular injection of the kidney, and meningitis were documented. pathogen, was more similar to the They did report meningitis, and macrophages pathology in natural S. agalactiae than to with engulfed bacteria in the spleen, heart, S. iniae infections, and characterized by the and ovary, which we did not observe. presence of numerous cocci in most organs. Pericarditis, meningitis, and some macrophages with engulfed bacteria were Discussion found in cage-cultured tilapia, O. mossambicus β In this study, we found that S. iniae and infected with a -hemolytic streptococcal S. agalactiae lead to different microscopic infection in Taiwan (Tung et al., 1985). These lesions in infected fish, even though they have findings also agree with those of our S. iniae similar gross clinical signs and basic infection studies. Streptococcus iniae infection bacteriological characteristics. The histo- in juvenile yellowtail leads to infiltration of pathology of streptococci infection in tilapia cocci-laden macrophages, and granulomas in has been described in the literature (Miyazaki the third ventricle, meninges, cerebellum, et al., 1984; Tung et al., 1985; Eldar et al., 1995; hepatic capsule, and peritoneum (Kaige et al., Chang & Plumb 1996; Bowser et al., 1998; 1984). Eldar & Ghittino (1999) compared Perera et al., 1998). Unfortunately, many of the histopathology of rainbow trout infected with early studies did not provide identification Lactococcus garvieae and S. iniae, which to species of the pathogens under study, and resulted in similar histology in brain, liver, usually the pathogens were only roughly kidney, and heart. Their findings were also characterized by reaction and similar to findings in our study for tilapia Lancefield serotyping. Since variation in infected with S. iniae. hemolytic ability was reported within even a In S. agalactiae-infected tilapia, the high single streptococcal strain (Bowser et al., number of bacteria observed in the circulation 1998), the ability to make definitive and internal organs suggest that either the judgements from information found in early pathogen can interfere with the immune literature may be open to question due to lack system, or grow faster than the immune of pathogen identification. system can eliminate them. Recently Mn-de- pendent superoxide dismutase in S. agalactiae Bull. Eur. Ass. Fish Pathol., 27(1) 2007, 8 has been shown to enable this bacterium to intracellular cocci. Although there seems to evade hydrogen superoxide secreted by be a contradiction in generalization of macrophages (Poyart et al., 2001). This could pathological changes across different fish explain why tilapia could not exert an species, it does, in fact, reveal that the outcome effective immune defense mechanism to of infection depends not only on the pathogen, eliminate S. agalactiae. This suggests that, even but also on how well the host can react to that though useful in the diagnosis of natural pathogen. It is probable that, if the host has a infection, the different pathological changes more effective immune function or is we observed in natural infections were not generally in a better state of health, it will specific to each disease. We speculate that better control the infection and have tilapia innate immunity may control S. iniae granuloma formation. If the growth of the more effectively than S. agalactiae. However, pathogen cannot be controlled, ubiquitous S. agalactiae may have additional mechanisms presence of bacterial cells in various tissues to invade tilapia and successfully multiply in will be found. Further immunology-oriented tilapia tissues. study is required to clarify this point before we can understand the nature of streptococcus Even though we have observed distinct infection in fish. histological changes in tilapia for both S. iniae and S. agalactiae infections, the same may not References be applicable in other fish species. Ferguson Bowser PR, Wooster GA, Getchell RG & et al. (2000) described S. iniae infection in Timmons MB (1998). Streptococcus iniae several Caribbean reef fishes. Although infection of tilapia Oreochromis niloticus in a similar pericarditis/epicarditis and meningo- recirculation production facility. Journal of the encephalitis were observed, accumulation of World Aquaculture Society 29, 335-339. eosinophilic substance in renal tubular cells Chang P–H & Plumb JA (1996). Histo- and granuloma-like structures in internal pathology of experimental Streptococcus sp. organs were not reported. Large numbers of infection in tilapia, Oreochromis niloticus (L.), and channel catfish, Ictalurus punctatus Gram-positive cocci in gill and internal (Rafinesque). Journal of Fish Diseases 19, 235- organs, especially spleen, was also a common 241. finding in those fish examined. Similar Chao C-B, Yang S-C, Tsai h-Y, Chen C-Y, Lin findings of large numbers of cocci in the C-S & Huang H-T (2002). A nested PCR for spleen and kidney, and the absence of the detection of Grouper Iridovirus in Taiwan granulomas, were also reported in red drum (TGIV) in cultured hybrid grouper, giant (Sciaenops ocellatus) (Eldar et al., 1999). seaperch and largemouth bass. Journal of Aquatic Animal Health 14, 104-113. Stoffregen et al. (1996) reported that for hybrid striped bass (Morone saxatilis male X Eldar A & Ghittino C (1999). Lactococcus M. chrysops female), that S. iniae infection garvieae and Streptococcus iniae infections in rainbow trout Oncorhynchus mykiss: similar, resulted in severe, subacute, extensive but different diseases. Diseases of Aquatic epicarditis, meningoencephalitis, uveitis, and Organisms 36, 227-231. branchitis associated with large numbers of Bull. Eur. Ass. Fish Pathol., 27(1) 2007, 9

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