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Lactococcus Garvieae and Streptococcus Iniae Infections in Rainbow Trout Oncorhynchus Mykiss: Similar, but Different Diseases

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Lactococcus Garvieae and Streptococcus Iniae Infections in Rainbow Trout Oncorhynchus Mykiss: Similar, but Different Diseases DISEASES OF AQUATIC ORGANISMS Vol. 36: 227-231.1999 Published May 31 Dis Aquat Org NOTE Lactococcus garvieae and Streptococcus iniae infections in rainbow trout Oncorhynchus mykiss: similar, but different diseases A. Eldar', C. ~hittino~,' 'Department of Poultry and Fish Diseases. Kimron Veterinary Institute, POB 12, 50250 Bet-Dagan. Israel 2~ishDisease Laboratory, IZS - State Veterinary Institute. Via Bologna 148, 1-10154 Turin, Italy ABSTRACT. Chnical and macroscopic findings (anorexia, haemorrhage, ophthalmitis and congestion (Kusuda lethargy, loss of orientation and exophthalmia) indicate that et al. 1991, Domenech et al. 1996). Con~monsigns Streptococcus ~niaeand Lactococcus garvieae infections of (lethargy, dark pigmentation, erratic swimming and trout share some common features, but histopathology re- veals notable differences between the 2 diseases. Meningitis exophthalmos with clouding of the cornea) are also and panophthalmitis are the main lesions among S. iniae present in Lactococcus garvieae (Collins et al. 1984; infected trout, whereas L. garvieae infection results in a junior synonym: Enterococcus seriolicida IKusuda et hyperacute systemic disease. Differences in the LD,,s of al. 1991, Domenech et al. 1993, Eldar et al. 19961) and the 2 pathogens and the sudden onset of signs and death & correlate with the histopathological findings, indicating the Streptococcus iniae (Pier Madin 1976) infections of severity of L.garvieae infection of trout. rainbow trout Oncorhynchus mykiss reared above 15°C. Our findings now show that these are 2 defined KEY WORDS Trout . Streptococcus iniae . Lactococcus conditions. L. garvieae infection of trout produces a garvieae Pathology . Experimental disease generalized disease and rapid death, while the disease induced by S, iniae results in a more prolonged course with specific lesions. Materials and methods. Sampling procedure for During the last decade, Gram-positive cocci have pathology and bacteriological analysis: Specimens become important fish pathogens. Epidemic and spo- were collected from 2 farms. Farm 1, which has a his- radic diseases have been reported from different parts tory of production losses due to Lactococcus garvieae of the world, notably Japan (Kitao 1993), Singapore infection, is located in Northern Italy (POValley); at the (Foo et al. 1985). Australia (Carson et al. 1993), Israel time of sampling the water temperature was 18°C. (Eldar et al. 1995), Italy (Ghittino & Prearo 1992),Spain Farm 2 is located in Northern Israel (Upper Galilee) (Toranzo et al. 1995, Domenech et al. 1996), France and is supplied by water at a constant temperature of (Michel et al. 1997), South Africa (Braag & Broere 16°C; this farm has a similar history of Streptococcus 1986) and the United States (Perera et al. 1994). lniae infection. At the time of collection both farms Amended taxonomy reveals that at least 6 different were experiencing heavy mortality, and on bacterio- species of Gram-positive cocci, including streptococci logical examination (20 fish from each farm), pure cul- (Pier & Madin 1976, Domenech et al. 1996), lactococci tures of Gram-positive cocci were identified as S. iniae (Collins et al. 1984, Williams et al. 1990, Domenech et or L. garvieae (Eldar et al. 1995, 1996) when grown on al. 1993, Eldar et al. 1996), and vagococci (Wallbanks blood agar. Tissues (eye, brain, heart, spleen, kidney, et al. 1990), are responsible for such conditions. liver, intestine and muscle) from 15 diseased rainbow Despite progress in taxonomy and diagnosis (Zlotkin et trout were fixed in 10 % neutral buffered formalin and al. 1998a,b),the pathological basis of the diseases has stained by haematoxylin and eosin (HE). Gross lesions not been addressed. Infections with Gram-positive were recorded. cocci are followed by non-specific lesions, such as Fish and experimental procedures: Groups of 50 rainbow trout (100 g each) were maintained at 16°C in 300 1 tanks supplied with constant aeration and contin- 'Addressee for correspondence. E-mail: [email protected] uous water flow. Field isolates, Lactococcus garvieae O Inter-Research 1999 Resale of full article notpermitted 228 Dis Aquat Org 36: 227-231, 1999 Table 1 Streptococcus iniae and Lactococcus garvieae in- was monitored until no more dead fish were observed fecting Oncorhynchus mykiss. Gross pathology of naturally for 2 wk. Positive bacterial culture from dead fish was infected trout. Values show number of fish of 20 specimens the criterion for specific mortality. showing symptom Results. Pathology: Macroscopic findings could dif- ferentiate between the 2 diseases (Table 1).The Strep- Pathology S. iniae L. garvieae tococcus iniae infected fish were found with ocular infected trout infected trout abnormalities (clouding of cornea and exophthalmos) Darkening of the s!un 16/20 20/20 and intracranial oedema. The Lactococcus garvieae Lethargy 20/20 20/20 infected trout presented, in addition to the signs de- lntracranial oedema 17/20 18/20 scribed above, haemorrhage, congestion of the in- Protrusion of the eye 17/20 19/20 Clouding of the cornea 17/20 12/20 ternal organs and enteritis. Histologically, both L. gar- Intraocular haemorrhage 5/20 2/20 vieae and S. iniae infected trout showed acute Diarrhea 2/20 20/20 meningitis, consisting of an exudate covering the brain Gill pallor 7/20 2/20 surface. Within the exudate, colonies of Gram-positive External haemorrhage 0/20 10/20 cocci were widely distributed over the meningeal sur- Internal haemorrhage 2/20 18/20 Anus inflammation 2/20 14/20 face and within the Virchoff's spaces (Fig. 1). These Splenomegaly 5/20 20/20 findings correlate with clinical findings of lethargy and Hepatic lesions 0/20 15/20 loss of orientation. Congested kidneys 0/20 18/20 Ocular alterations resulted from a severe haemor- Dilated intestine 2/20 20/20 Pericardial leslons 0/20 15/20 rhagic panophthalmitis with destruction of the anterior and posterior chambers; the optic nerve papilla was heavily affected and inflammation progressed into the ITP 2001 and Streptococcus iniae Dan-l, were grown retrobulbar fat and striated muscle. at 24°C on Columbia agar base (Difco) supplemented These findings were identical in grading and distrib- with 5% defibrinated sheep blood. After 24 h the col- ution in both infections, contrasting with the following onies were scraped off, washed twice in PBS (pH 7.2) histological differences: and resuspended in the same buffer at various concen- (1) Serositis, sometimes extending to the myocar- trations, ranging from 1.25 X 102 to 2.5 X 108 CFU ml-l. dium, was present in the Lactococcus garvieae in- Each group of fish was infected by intraperitoneal fected fish (Fig. 2), whereas no similar lesion was injection of 100 p1 of bacterial suspension. Mortality detected among Streptococcus iniae infected trout. Fig. 1 Streptococcus iniae Infected trout brain. Acute meningitis consistent w~thfibrino-purulent exudate wldely distributed over the meningeal surface and within the Virchoff's spaces. The brains in trout infected by Lactococcus garvieae show a similar picture. HE x20 Eldar & Ghittino: Lactococcus garvieae and Streptococcus iniae infections in trout 229 Fig. 2. Lactococcus garvieae infected trout heart. Moderate pericarditis; note rnesothelial cell proliferation and fat tissue depletion. HE x200 The most notable difference was found in the celomic with unremarkable crypt density and distribution. A cavity. The L. garvieae infected fish suffered a severe mild acute inflammatory infiltrate was seen over the peritonitis with fat necrosis (Fig. 3), whereas S. iniae mesothelial peritonea1 layer. In contrast, Lactococcus infected trout had a mild peritonitis, according to the garvieae infected fish had extensive superficial ero- degree of pericardial involvement. sions with pseudomembrane-like formation (Fig. 3). (2) Intestine. The architecture of the mucosal layer (3) Liver. In both infections, portal spaces revealed was preserved in the Streptococcus iniae infected trout, unremarkable bile ducts, blood vessels and limiting Fig. 3. Lactococcus garvieae infected trout intestine. Severe peritonitis with fat necrosis; extensive superficial erosions with pseudomembrane-like formation and globet cell depletion in the rnucosa. HE X 100 230 Dis Aquat Org 36: 227-231. 1999 - Fig 4. Lactococcus garvieae ~nfectedtrout liver. Peliosis Hepatis. Note RBC lakes within liver parenchyma HE x100 plates. A notable contrast was found in the paren- marked reactive hyperplasia of the haematopoietic tis- chyma of trout infected by the different bacteria. sue was evident in Lactococcus garvieae infected fish Blood-filling spaces in a diffuse fashion were a con- (Fig. 5). stant feature of Lacfococcus garvieae infection, consis- Experimental infections: The LD50of Streptococcu~ tent with Peliosis Hepatis (Fig. 4). Hepatic tissue of iniae was 2.5 X 105 CFU fish-' at 21 d post challenge, Streptococcus iniae infected trout was unremarkable. while that of Lactococcus garvieae was 1.25 X 10' CFU (4) Kidney. The glomeruli had a well preserved uri- fish-', attained 7 d post challenge. The kinetics of the 2 nary space, with no adhesions or crescent-like struc- experimental diseases varied in: (1) the lag period (the tures. No hypercellularity or capillary wall thickening time required for the death of the second fish), which was seen. Tubular structure and cellularity were nor- was 8 d for S. iniae, but only 3 d for L. garvieae, and mal in Streptococcus iniae infected fish, whereas a (2) the average day of death (defined as
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