Pathogenicity of streptococcosis in rainbow trout

Bull. Eur. Ass. Fish Pathol. 19(3),114, 1999.

PATHOGENICITY OF STREPTOCOCCOSIS IN RAINBOW TROUT(ONCORHYNCHUS MYKISS): DEPENDENCE ON AGE OF DISEASED FISH. J.L. MUZQUIZ, F.M. ROYO, C.

ORTEGA,

l

DE

BLAS, I. RUIZ, J.L. ALONSO.

Unidad de Enfermedades Infecciosas, Departamento de Patologia lnfecciosa y Epidemiologia de la Facullad de Veteri­ naria de la Universidad de Zaragoza.

Abstract

The su·eptococcosis produced by Lactococcus garvieae is a major world wide disease present in Spain. In this study are described the different courses of disease, depending on age of diseased fish. While SO g fish have more sensitivity, and

suffer longer acute period of disease, IOOg fish are able to eliminate bacterial agent from internal organs. However, bacte­ ria is maintained in organs related to bacterial elimination, such gills and intestine. Bacteria could also be isolated from fish aquarium water.

Introduction

Streptococcosis is a major bacterial disease, with a world-wide distribution, which affects both freshwater and marine species of fish. In spite of a wide range of genera being in­ volved, all of them were included in the ge­ nus Streptococcus before it was divided. The first su·eptococcal septicemia affecting rainbow trout was diagnosed by Hoshina et al. in 1958, in Japan. Some years later, Ku­ suda et al. described it affecting yellowtail (Serio/a quinqueriadata) and eel (Angila japonica), in 1976, and Kitao et al. reported its occurrence in tilapias (Oreochromis sp.) and ayu (Plecoglossus altivelis), in 1981 (Austin & Austin, 1987). In USA, the first case was reported by Robinson & Meyer in 1966, who described infected golden shiners (Notemignus cryso­ leucas) by Streptococcus sp. The disease also appeared in South Africa, in 1979, re­ ported by Barham et al. (Austin & Austin, 1987). The disease has also been diagnosed in Aus­ tralia (Carson et al., 1990), U.K., Norway (Austin & Austin, 1987), and France (Michel et al., l 997). Nowadays, Mediterranean countries are suf­ fering several streptococcal outbreaks, the reason being probably that water reaches high temperatures. Some of these counU-ies are Italy (Chesia et al., 1992, Ghittino et al., 1992), Spain, where streptococcosis has oc­ curred in rainbow trout (Prieta et al., 1993) and turbot (Scophthalmus maximus) (To-

ranzo et al., 1994), and Israel (Eldar et al., 1994). Although this infection has not produced high mortality in Spain, around 1 0% (Prieta et al., 1993), it has given rise to mass mor­ tality in Israel, 50%, (Eldar et al., 1995a) and Australia, 60% (Carson et al., 1993). In addition, there are economical losses be­ cause growing rates decrease, and the ap­ pearance of these fish make them unmarket­ able (Toranzo et al., l 994). Some years ago, bacterial agents could only be identified as Streptococcus sp. But devel­ opment of genetic probes made possible a better classification of this agent. At this moment, causative agents could be differen­ tiated into two groups, according to the virulence they show at both high and low temperatures (Ghittino & Muzquiz, 1998). Thus, Lactococcus garvieae, (Ptieta et al., 1 993) formerly Enterococcus seriolicida (Kusuda et al., 1991; Domenech et al. 1993, Eldar et al., 1996), Streptococcus iniae (El­ dar et al., 1995b), Streptococcus difficile (Eldar et al., 1994), and Streptococcus parauberis (Domenech et al., 1996) could be included in one group which presents high virulence with temperatures of water hotter than l 7°C. The second group is composed by Lactococ­ cus piscium (Williams et al., 1990) and Va­ gococcus salmoninarum, (Wallbanks et al., 1990) both agents with high virulence at l 4°C. Typical external signs of affected fish are exophthalmia and swollen abdomen, and

Bull. Eur. Ass. Fish Pathol., 19(3),115, 1999. presence of haemorrhages in periorbital area, base of fins and perianal region. At necropsy, haemorrhages in liver, swim bladder and intestine could be found. Brain could also be affected (Prieta et al. 1 993; Nieto et al., 1 995). These kinds of lesions were observed in a Spanish rainbow trout farm in 1997 where two bacterial strains, TTL1 and MTLC-33 , were isolated from diseased fishes. Isolates were identified as L. garvieae as will be described below. This bacte­ ria was used in an experimental disease in order to compare sensitivity to the agent and infection development of fishes of different ages. Material and Methods

Both strains, TTL-I , recovered at the beginning of the outbreak, and MTLC33 , recovered at the end, were identi­ fied by testing several biochemical probes referenced on Bergey' s Manual (Holt et al., 1994), as growth on pH 9.6, growth on 6.5% NaCl medium, range of temperatures and carbohy­ drates and amino acids degradation (Table I). The second part of this study was an experimental disease study, to carry out a challenge with two groups of fish of different weight. In natural outbreak no mortality or clinical signs were founded in fish lighter than 80g (Prieta et al., 1993 ). So we placed 24 rainbow trout (average weight 50g) in an 1 OOL aquarium, ref­ erenced as G50, and 20 rainbow trout (average weight l OOg) in a 200L aquarium (Initially both grups included 25 fish, but some of them died by jour­ ney stress), as Gl OO. Aquarium equip­ ment also includes recirculating filter system and constant aeration of water. Temperature of water was maintained at 18±1°C by refrigerator equipments. In addition there were two groups of fish maintained as control. Fish came from a government rainbow

Table 1: Results of biochemical probes tested to . Iates and b" hem1caI propertJ.es of L. garvieae. 1so IOC Strain

MTLC33

TTL-I

L.garvieae*

Gram

+

+

+

Coco

Coco

Coco

4°C

+

+

+

20°c

+

+

+

3 7°C

+

+

+

44°C

+

+

-

PH 9.6

+

+

+

BAA

+

+

+

Haemolysis

-

-

.

Catalase

-

-

-

Oxidase

-

-

-

A/A-

A/A-

A/A-

Cell form Growth

TSI ADH

+

+

+

N03

-

-

-

Citrate

-

-

ND

O/F

F

F

F

Motility

-

-

-

Glycerol

-

-

-

Raffinose

-

-

-

Arabinose

-

-

-

Sorbitol

-

-

+/-

Manito!

+

+

+

Acid from

Resistance/Sensitivity to Ampicillin

s

s

s

Enrofloxacin

R

R

s

Erythromicyn

I

s

s

Nitrofurantoin

R

R

s

Oxytetracycline

R

R

s

*L.garvieae NCDO 2155 (dates from Toranzo et al., 1994). NA-: Acidification of medium TSI and H,S not produced. ADH: Arginine dihydrolase; N03: Nitrate reduction S: Sensitive; I: lntern1ediate; R: Resistant +/-: Discrepancy on the results depending of the method used (conventional or API test). ND: unstated results.

Bull. Eur. Ass. Fish Pathol. 19(3),116, 1999. trout fann (Monasterio de Piedra. Zaragoza, Spain) where there has never occurred a case of streptococcosis, and any streptococcal species have been recovered at routine con­ trols made by Ictiopathology Laboratory. Prior to any inoculation, some fish were sac­ rificed and analysed to confirm that they were free from any bacterial pathogens. Moreover, the fish were acclimatised to the experimental conditions during seven days between their arrival to the laboratory and the start of inoculations. Only MTLC-33 was used for infection stud­ ies, because both MTLC-33 and TTL-I were the same species, and MTLC-33 had been isolated later. The virulence of strain MTLC-33 was in­ creased after passage in vivo, by intraperito­ neal (i.p.) inoculation of three fish. After three days a fish was sacrificed and samples from kidney were cultured on Trypticase Soy Agar. A colony was cultured onto Brain Hea1t Infusion, and used in experimental disease. In order to reduce effect of handling stress, all fish were anaesthetised with 2-phenoxi­ ethanol before the inoculations started. Both G50 and G 100 were inoculated with 105 cfu/fish, suspended in O .lml sterile saline solution. Mortality was monitored for 20 days, according to previous results published (Toranzo et al., 1994; Chang & Plumb, 1996). From dead fishes, samples from kidney, liver, spleen, muscle, blood, eye, brain, in­ testine, and mucus from gills were inocu­ lated onto TSA at 20°C and Bile Aesculin Azide Agar (BAA) at 37°C. MTLC-33 was recovered and identified when a colony of gram positive cocci, cata­ lase and oxidase negative, and black colonies grow on BAA (produced by degradation of Aesculin).

Presence in water was also studied by filter­ ing water through a sterile 45µm filter, and placing it onto BAA medium. Skin samples were also cultured in the same conditions. Results a) Bacterial identification As result of bio­ chemical probes, both isolates TTL-1 and MTLC-33 were identified as Lactococcus garvieae.(forrnerly Enterococcus seriolicida

(Domenech et al. 1 993). They showed a dif­ ferent sensitivity to erythromycin, maybe due to treatment during the outbreak. The isolates were gram positive cocci, with­ out mobility, catalase and oxidase negative, which grew at pH 9.6 and in medium with 6.5 % NaCl, and that produced hydrolysis of arginine and aesculin (Table 1). b) Experimental study High mortality was observed as a result of experimental disease, so we can conclude that MTLC-33 LDSO (Median Lethal Dose) was lower than l 05 cfu/fish, because mortal­ ity observed was 92% in G50, and 75% in G100 (Fig. 1 ). These results are similar to another published data (Bercovier et al., 1997). G50 showed a high mortality that started day 3. This mortality was observed until day 9 post-inoculation {p.i.). After this one death occurred day 13 (Table 2). In GlOO former m01tality was observed days 3 and 4 p.i. In that case, mortality stopped until day 9. From 'day 9 to day 1 6, 5 fish died (Table 2). We could not appreciate differences in clini­ cal signs between the two groups. Diseased fish showed lethargy and anorexia, exoph­ thalmia and petechial haemorrhages in the skin of the mouth, base of fins and around the eyes. Internal lesions as abdominal liquid, haemor-

Table 2. Mortality studies on 50g and 1OO g rainbow trout infected with L.garvieae. I 2 3 4 5 6 7 8 9 1 0 11 12 1 3 14 15 16 17- 20

Days

GIOO

0

0

4

6

0

0

0

G50

0

0

2

8

5

2

2

0

l

0

0

0

0

0

0

I

1

1

0

0

0

0

0

Bull. Eur. Ass. Fish Pathol., 19(3),117, 1999. Figure 1 Mortality of 050 and 0100 groups infected with L. garvieae 100 80 Mortality

(%)

60

-+-G100

40

�G50

20 0 1

3

5

7

9

11 13 15 17 19 days

rhages in lowest part of intestine and swim bladder, and yellowish liquid filling cranial cavity. However, we could appreciate some differ­ ences between 050 and 0100 in the isolation of the bacteria throughout duration of the experimental study. In 0100, we could recovered the agent from all samples of dead fish in days 3 and 4, but isolation failed from cultured samples taken from fish who died day 9 next days, in this animals we could only recover L. garvieae from intestine and gills (Table 3). All samples from dead fish of 050 showed, after culture, growing of L. garvieae. Sur­ vival animals were sacrificed after day 20 of the study. In this animals the agent was not present in samples of organs. At this mo­ ment, only gills and intestine of a fish from 0I 00 enable us to isolate the bacterial agent. L. garvieae could be isolated from water of both 0 l 00 and 050 aquru.iums, in a concen­ tration of 360 ufc/ml, and could also be de­ tected from skin of fish from both groups. No mortality, clinical signs or streptococcal bacteria could be recovered from control groups.

Table GSO

3

Discussion

Both TTL-1 and MTLC-33 isolates were classified as L. garvieae, a true pathogen for rainbow trout. The later was selected for an experimental study, because it had been iso­ lated more recently than TTL-1. We observed an acute septicaemia, and we could isolate bacterial agent from blood, so haematic distribution is confirmed, such as previously had been suggested (Chang & Plum, 1996). Typical signs of st:reptococcosis were repro­ duced on experimental infection, and dis­ eased fish showed exophthalmia, petechial haemorrhages on skin of mouth, eye, anus, and base of fins. Also liquid in abdominal and cranial cavities was observed. Faecal elimination occuned in less than 72h p.i., at the same time as former signs of dis­ ease and death. In Addition, bacteria could be isolated from mucus of skin, which should be considered as a transmission factor. L. garvieae was also present in water, so it could explain experi­ mental diseases reproduced by bath exposi­ tion (Munday et al., 1993). Younger fish (050) suffered higher mortality than older (0100), and the acute period of disease was also longer, while lOOg fish

Isolation of MTLC-33 throu h ex erimental eriod.

Da s

Bacteria from all samples 1 2 3 4 5 6 7

GlOO

Bacteria from all sam Ies

8

n

BulJ. Eur. Ass. Fish Pathol. 19(3),118, 1999. could stop acute septicemia, and remove bacteria from internal organs. A stronger immunity response from older fish may ex­ plain this fact. Immune response has been observed involving lesions (Nieto et al., 1995). However, when fish died bacteria were not present in internal organs. It could suggest that toxins are present. Previous studies have described endotoxins in streptococcosis (Ku­ suda y cols., 1 989). On the other hand, young fish rarely showed streptococcosis on fish farms (Prieta et

al.,

1993), so a better epidemiological descrip­ tion of the disease would be necessary, and more studies should be carried out. The results on isolation suggested that older internal organs but it remains in spreading places, such gills or intestine. It may be un­ derstood as a natural tendency to an asymp­ tomatic carrier state. This kind of carrier has been described in healthy fish that came from fishponds which streptococcosis had been diagnosed the year before, applying Polymerase Chain Reaction (Zlotkin et al., 1998). Gills and intestine should be considered as representative points of bacterial isolation, in kidney

and

brain.

an

emerging

disease

Aquaculture, 5, 32-33.

in

aquaculture.

Austasia

Carson, J., Gudkovs, N. and Austin, B. (1993). Charac­ teristics of an Enterococcus-like bacterium from Australia and South Africa, pathogenic for rainbow trout, Oncorhynchus mykiss (Walbaum). J. Fish.

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Ceschia, G., Giorgctti, G., Giavenni, R. and Sarti, M.

(1992). A new problem for italian trout farms: Streptococcosis in rainbow trout, Oncorhy11c/111s mykiss. Bull. Eur. Ass. Fish Pathol. 12, 71-72.

Chang, P.H. and Plumb, J.A. (1996). Histopathology of experimental Streptococcus sp. infection in tilapia,

Oreochromis niloticus (L.), and channel catfish,

Jctalurus punctatus (Rafincsque). ./. Fish. Dis; 19,

235-241. Domenech, A., Prieta, J., Fernandcz-Garayzabal, J.F., Collins, M.D., Jones, D. and Domfnguez, L. (1993). Phenotypic and phylogenetic evidence for a close

fish could eliminate bacterial agent from

addition to

Carson, J. and Munday, B. L. (1990). Strcptococcosis­

Moreover,

BAA medium is a good selective medium for recovering the bacteria. In conclusion, this study indicates the de­ mand for further knowledge of streptococco­ sis, in order to make possible control and eradication of the disease in fish farms.

relationship

between u1ctococcus garvieae

and

E11terococcus seriolicida. Microbiologia; 9, 63-68.

Domenech, A., Femandez-Garayzabal, J.F., Pascual, C., Garcia, J.A., Cutuli, M.T., Moreno, M.A., Collins, M.D. and Dominguez, L. (1996). Streptococcosis in cultured turbot, Scophthalmus maximu�· (L.), asso­

ciated with Streptococcus parauberis. J. Fish. Dis,

19, 33-38.

Eldar, A., Bejerano, Y. and Bcrcovier H. (1994). Strep­ tococcus shiloi and Streptococcus difficile: two new streptococcal

species

causing

a

mcningo­

encephalitis in fish. Curr. Microbial., 28, 139-143.

Eldar, A., Bejerano, Y., Livoff, A., Horovitcz, A. and Bercovier, H. (1995a). Experimental streptococcal meningo-encephalitis in cultured fish. Vet. Micro­

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Eldar, A., Frelier, P.F., Asanta, L., Varner, P.W., Law­ hon, S. and Bercovier, H. (1995b). Streptococcus shiloi, the name for an agent causing septicemic in­ fection in fish, is a junior synonym of Streptococ­

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Eldar, A., Ghittino, C., Asanta, L., Bozzella, E., Coria, M., Prcaro, M. and Bercovier, H. (1996). Entero­ coccus seriolicida is a junior synonym of u1ctococ­ cus garvieae, a causative agent of septicemia and

meningo-encephalitis in fish. Curr. Microbial., 32,

85-88.

Acknowledgements: We want to thank Diputacion General de Aragon and staff of Monasterio de Piedra (Zarago..:a) for supplying fishes used in this study. We also want to thank Proaqua nutrition, for bacterial

Ghittino, C. and Prearo, M. (1992). Report of Strepto­ coccosis in rainbow trout (Oncorhync/ws mykiss) in

Italy: preliminary note. S.l.P.I. Bull., 8, 4-11.

Ghittino, C. and Muzquiz, J.L. (1998). La estreptococo­

supplies.

sis de la trucha arco iris en Espana. Resumen de lo

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